Researcher Profile and Settings

Master

Affiliation (Master)

• Creative Research Institution　Institute for Vaccine Research and Development

Affiliation (Master)

• Creative Research Institution　Institute for Vaccine Research and Development

researchmap

Profile and Settings

Affiliation

• University of Zambia, School of Veterinary Medicine, Visiting Professor

Profile and Settings

• Name (Japanese)

  Sawa

• Name (Kana)

  Hirofumi

• Name

  200901034013234450

Alternate Names

http://www.czc.hokudai.ac.jp/pathobiol/

Affiliation

• University of Zambia, School of Veterinary Medicine, Visiting Professor

Achievement

Research Interests

• Vaccinology   感染症疫学   モデルマウス   細胞内輸送   ウイルス学   animal model   trafficking   Cellular Biology   Molecular Pathology   Virology   

Research Areas

• Life sciences / Molecular biology
• Life sciences / Experimental pathology
• Life sciences / Cell biology
• Life sciences / Virology

Research Experience

• 2023/04 - Today Hokkaido University Institute for Vaccine Research and Development: HU-IVReD Professor
• 2021/04 - 2023/03 international institute for Zoonosis Control, Hokkaido University Division of Molecular pathobiology professor
• 2005/04 - 2021/03 Research center for Zoonosis Control, Hokkaido University Division of Molecular Pathobiology Profesor

Awards

• 2024/02 Hokkaido University Hokkaido University Presidential Commendation for Education and Research Excellence Award
  
• 2016/02 Hokkaido University Hokkaido University Presidential Commendation for Education and Research Excellence Award
  

Published Papers

• X-206 exhibits broad-spectrum anti-β-coronavirus activity, covering SARS-CoV-2 variants and drug-resistant isolates.

  Jiei Sasaki, Akihiko Sato, Michihito Sasaki, Iori Okabe, Kota Kodama, Satoko Otsuguro, Kosuke Yasuda, Hirotatsu Kojima, Yasuko Orba, Hirofumi Sawa, Katsumi Maenaka, Yusuke Yanagi, Takao Hashiguchi

  Antiviral research 106039 - 106039 2024/11/19 

  Coronaviruses such as the Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, causing MERS, SARS, and Coronavirus disease-19, respectively, are highly pathogenic to humans. Notably, several antiviral drugs against SARS-CoV-2, such as nirmatrelvir and remdesivir, have been approved. However, no approved vaccines or antiviral agents are available for other highly pathogenic β-coronaviruses. In this study, we identified two compounds, thapsigargin and X-206, that exhibit antiviral activities against SARS-CoV, MERS-CoV, and SARS-CoV-2. Notably, both compounds effectively inhibited the cell-to-cell fusion mediated by the Spike proteins of all three β-coronaviruses. X-206 exhibited antiviral activity against nirmatrelvir- and remdesivir-resistant SARS-CoV-2 isolates and SARS-CoV-2 variants, including Delta, BA.5, and XBB.1. Consequently, the mechanism of action of these compounds with anti-β-coronavirus activities may differ from that of the approved direct-acting drugs for SARS-CoV-2, thereby offering potential use as a cocktail with other antivirals, and serving as a chemical basis for developing therapeutic agents against β-coronaviruses in preparation for the next spillover and pandemic.
• A first report of rotavirus B from Zambian pigs leading to the discovery of a novel VP4 genotype P[9].

  Hayato Harima, Yongjin Qiu, Michihito Sasaki, Joseph Ndebe, Kapila Penjaninge, Edgar Simulundu, Masahiro Kajihara, Aiko Ohnuma, Keita Matsuno, Naganori Nao, Yasuko Orba, Ayato Takada, Kanako Ishihara, William W Hall, Bernard M Hang'ombe, Hirofumi Sawa

  Virology journal 21 (1) 263 - 263 2024/10/24 

  BACKGROUND: Rotavirus B (RVB) causes diarrhea in humans and pigs. Although various RVB strains were identified in humans and various animals globally, little is known about the epidemiology RVB infection in Africa. In this study, we attempted to examine the prevalence of RVB infection in pig populations in Zambia. METHODS: Metagenomic analyses were conducted on pig feces collected in Zambia to detect double stranded RNA viruses, including RVB. To clarify the prevalence of RVB infection in pig populations in Zambia, 147 fecal samples were screened for the RVB detection by RT-qPCR. Full genome sequence of a detected RVB was determined by Sanger sequencing and genetically analyzed. RESULTS: The metagenomic analyses revealed that RVB sequence reads and contigs of RVB were detected from one fecal sample collected from pigs in Zambia. RT-qPCR screening detected RVB genomes in 36.7% (54/147) of fecal samples. Among 54 positive samples, 13 were positive in non-diarrheal samples (n = 48, 27.1%) and 41 in diarrheal samples (n = 99, 41.4%). Genetic analyses demonstrated that all the segments of ZP18-18, except for VP4, had high nucleotide sequence identities (80.6-92.6%) with all other known RVB strains detected in pigs. In contrast, the VP4 sequence of ZP18-18 was highly divergent from other RVB strains (< 64.6% identities) and formed a distinct lineage in the phylogenetic tree. Notably, the VP8 subunit of the VP4 showed remarkably low amino acid identities (33.3%) to those of known RVB strains, indicating that the VP8 subunit of ZP18-18 was unique among RVB strains. According to the whole genome classification for RVB, ZP18-18 was assigned to a genotype constellation, G18-P[9]-I12-R4-C4-M4-A8-N10-T5-E4-H7 with the newly established VP4 genotype P[9]. CONCLUSIONS: This current study updates the geographical distribution and the genetic diversity of RVB. Given the lack of information regarding RVB in Africa, further RVB surveillance is required to assess the potential risk to humans and animals.
• Structural basis for receptor-binding domain mobility of the spike in SARS-CoV-2 BA.2.86 and JN.1

  Hisano Yajima, Yuki Anraku, Yu Kaku, Kanako Terakado Kimura, Arnon Plianchaisuk, Kaho Okumura, Yoshiko Nakada-Nakura, Yusuke Atarashi, Takuya Hemmi, Daisuke Kuroda, Yoshimasa Takahashi, Shunsuke Kita, Jiei Sasaki, Hiromi Sumita, Keita Matsuno, Naganori Nao, Hirofumi Sawa, Keita Mizuma, Jingshu Li, Izumi Kida, Yume Mimura, Yuma Ohari, Shinya Tanaka, Masumi Tsuda, Lei Wang, Yoshikata Oda, Zannatul Ferdous, Kenji Shishido, Hiromi Mohri, Miki Iida, Takasuke Fukuhara, Tomokazu Tamura, Rigel Suzuki, Saori Suzuki, Shuhei Tsujino, Hayato Ito, Naoko Misawa, Ziyi Guo, Alfredo A. Hinay, Kaoru Usui, Wilaiporn Saikruang, Spyridon Lytras, Keiya Uriu, Ryo Yoshimura, Shusuke Kawakubo, Luca Nishumura, Yusuke Kosugi, Shigeru Fujita, Jarel Elgin M.Tolentino, Luo Chen, Lin Pan, Wenye Li, Maximilian Stanley Yo, Kio Horinaka, Mai Suganami, Mika Chiba, Kyoko Yasuda, Keiko Iida, Adam Patrick Strange, Naomi Ohsumi, Shiho Tanaka, Eiko Ogawa, Tsuki Fukuda, Rina Osujo, Kazuhisa Yoshimura, Kenji Sadamas, Mami Nagashima, Hiroyuki Asakura, Isao Yoshida, So Nakagawa, Kazuo Takayama, Rina Hashimoto, Sayaka Deguchi, Yukio Watanabe, Yoshitaka Nakata, Hiroki Futatsusako, Ayaka Sakamoto, Naoko Yasuhara, Tateki Suzuki, Yukari Nakajima, Takashi Irie, Ryoko Kawabata, Kaori Sasaki-Tabata, Terumasa Ikeda, Hesham Nasser, Ryo Shimizu, M. S. T. Monira Begum, Michael Jonathan, Yuka Mugita, Sharee Leong, Otowa Takahashi, Takamasa Ueno, Chihiro Motozono, Mako Toyoda, Akatsuki Saito, Anon Kosaka, Miki Kawano, Natsumi Matsubara, Tomoko Nishiuchi, Jiri Zahradnik, Prokopios Andrikopoulos, Miguel Padilla-Blanco, Aditi Konar, Jumpei Ito, Katsumi Maenaka, Kei Sato, Takao Hashiguchi

  Nature Communications 15 (1) 2024/10/07
• Characterization of a mammalian orthoreovirus isolated from the large flying fox, Pteropus vampyrus, in Indonesia.

  Kittiya Intaruck, Koshiro Tabata, Yukari Itakura, Nijiho Kawaguchi, Mai Kishimoto, Agus Setiyono, Ekowati Handharyani, Hayato Harima, Takashi Kimura, William W Hall, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki

  The Journal of general virology 105 (9) 2024/09 

  Fruit bats serve as an important reservoir for many zoonotic pathogens, including Nipah virus, Hendra virus, Marburg virus and Lyssavirus. To gain a deeper insight into the virological characteristics, pathogenicity and zoonotic potential of bat-borne viruses, recovery of infectious viruses from field samples is important. Here, we report the isolation and characterization of a mammalian orthoreovirus (MRV) from a large flying fox (Pteropus vampyrus) in Indonesia, which is the first detection of MRV in Southeast Asia. MRV was recovered from faecal samples of three different P. vampyrus in Central Java. Nucleotide sequence analysis revealed that the genome of the three MRV isolates shared more than 99% nucleotide sequence identity. We tentatively named one isolated strain as MRV12-52 for further analysis and characterization. Among 10 genome segments, MRV12-52 S1 and S4, which encode the cell-attachment protein and outer capsid protein, had 93.6 and 95.1% nucleotide sequence identities with known MRV strains, respectively. Meanwhile, the remaining genome segments of MRV12-52 were divergent with 72.9-80.7 % nucleotide sequence identities. Based on the nucleotide sequence of the S1 segment, MRV12-52 was grouped into serotype 2, and phylogenetic analysis demonstrated evidence of past reassortment events. In vitro characterization of MRV12-52 showed that the virus efficiently replicated in BHK-21, HEK293T and A549 cells. In addition, experimental infection of laboratory mice with MRV12-52 caused severe pneumonia with 75% mortality. This study highlights the presence of pathogenic MRV in Indonesia, which could serve as a potential animal and public health concern.
• β-d-N4-hydroxycytidine, a metabolite of molnupiravir, exhibits in vitro antiviral activity against rabies virus.

  Kei Konishi, Shinji Kusakabe, Nijiho Kawaguchi, Takao Shishido, Naoto Ito, Michiko Harada, Satoshi Inoue, Ken Maeda, William W Hall, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki, Akihiko Sato

  Antiviral research 229 105977 - 105977 2024/09 

  Rabies is a fatal neurological disorder caused by rabies virus (RABV) infection. Approximately 60,000 patients die from rabies annually, and there are no effective treatments for this disease. Nucleoside analogs are employed as antiviral drugs based on their broad antiviral spectrum, and certain nucleoside analogs have been reported to exhibit anti-RABV activity. The nucleoside analog β-d-N4-hydroxycytidine (NHC) has antiviral effects against a range of RNA viruses. Molnupiravir (MPV), a prodrug of NHC, is clinically used as an oral antiviral drug for coronavirus infections. Despite its broad-spectrum activity, the antiviral activity of NHC against RABV remains unclear. In this study, we reveal that NHC exhibits comparable in vitro anti-RABV activity as ribavirin and favipiravir (also known as T-705) with a 90% effective concentration of 6 μM in mouse neuroblastoma cells. NHC reduced viral loads in neuronal and nonneuronal cells in a dose-dependent manner. Both laboratory and field RABVs (fixed and street strains, respectively) were susceptible to NHC. However, no increase in survival or reduction in viral titers in the brain was observed in RABV-infected mice treated prophylactically with MPV. These findings highlight the potential and challenges of NHC in the treatment of RABV infection.
• Increased production of orthoflavivirus single-round infectious particles produced in mammalian cells at a suboptimal culture temperature of 28°C

  Koshiro Tabata, Shintaro Kobayashi, Yukari Itakura, Gabriel Gonzalez, Chilekwa F. Kabamba, Shinji Saito, Michihito Sasaki, William W. Hall, Hirofumi Sawa, Yasuko Orba

  Journal of Virological Methods 329 115007 - 115007 0166-0934 2024/09
• Reverse genetic approaches allowing the characterization of the rabies virus street strain belonging to the SEA4 subclade.

  Nijiho Kawaguchi, Yukari Itakura, Kittiya Intaruck, Takuma Ariizumi, Michiko Harada, Satoshi Inoue, Ken Maeda, Naoto Ito, William W Hall, Hirofumi Sawa, Yasuko Orba, Michihito Sasaki

  Scientific reports 14 (1) 18509 - 18509 2024/08/09 

  Rabies virus (RABV) is the causative agent of rabies, a lethal neurological disease in mammals. RABV strains can be classified into fixed strains (laboratory strains) and street strains (field/clinical strains), which have different properties including cell tropism and neuroinvasiveness. RABV Toyohashi strain is a street strain isolated in Japan from an imported case which had been bitten by rabid dog in the Philippines. In order to facilitate molecular studies of RABV, we established a reverse genetics (RG) system for the study of the Toyohashi strain. The recombinant virus was obtained from a cDNA clone of Toyohashi strain and exhibited similar growth efficiency as the original virus in cultured cell lines. Both the original and recombinant strains showed similar pathogenicity with high neuroinvasiveness in mice, and the infected mice developed a long and inconsistent incubation period, which is characteristic of street strains. We also generated a recombinant Toyohashi strain expressing viral phosphoprotein (P protein) fused with the fluorescent protein mCherry, and tracked the intracellular dynamics of the viral P protein using live-cell imaging. The presented reverse genetics system for Toyohashi strain will be a useful tool to explore the fundamental molecular mechanisms of the replication of RABV street strains.
• A wide distribution of Beiji nairoviruses and related viruses in Ixodes ticks in Japan.

  Mai Kishimoto, Yukari Itakura, Koshiro Tabata, Rika Komagome, Hiroki Yamaguchi, Kohei Ogasawara, Ryo Nakao, Yongjin Qiu, Kozue Sato, Hiroki Kawabata, Masahiro Kajihara, Naota Monma, Junji Seto, Asako Shigeno, Masayuki Horie, Michihito Sasaki, William W Hall, Hirofumi Sawa, Yasuko Orba, Keita Matsuno

  Ticks and tick-borne diseases 15 (6) 102380 - 102380 2024/07/11 

  Beiji nairovirus (BJNV), in the family Nairoviridae, the order Bunyavirales, was recently reported as a causative agent of an emerging tick-borne zoonotic infection in China. This study investigated the prevalence of BJNV in ticks in Japan. Screening of over 2,000 ticks from multiple regions revealed a widespread distribution of BJNV and BJNV-related viruses in Japan, particularly in the northern island, and in other high altitude areas with exclusive occurrence of Ixodes ticks. Phylogenetic analysis identified three distinct groups of nairoviruses in ticks in Japan: BJNV, Yichun nairovirus (YCNV) and a newly identified Mikuni nairovirus (MKNV). BJNV and YCNV variants identified in ticks in Japan exhibited high nucleotide sequence identities to those in China and Russia with evidence of non-monophyletic evolution among BJNVs, suggesting multiple cross-border transmission events of BJNV between the Eurasian continent and Japan. Whole genome sequencing of BJNV and MKNV revealed a unique GA-rich region in the S segment, the significance of which remains to be determined. In conclusion, the present study has shown a wide distribution and diversity of BJNV-related nairoviruses in Ixodes ticks in Japan and has identified unique genomic structures. The findings demonstrate the significance of BJNV as well as related viruses in Japan and highlight the necessity of monitoring emerging nairovirus infections and their potential risks to public health.
• Detection of Human Adenovirus and Rotavirus in Wastewater in Lusaka, Zambia: Demonstrating the Utility of Environmental Surveillance for the Community

  Ngonda Saasa, Ethel M’kandawire, Joseph Ndebe, Mulenga Mwenda, Fred Chimpukutu, Andrew Nalishuwa Mukubesa, Fred Njobvu, Doreen Mainza Shempela, Jay Sikalima, Carol Chiyesu, Bruce Muvwanga, Sarah M. Nampokolwe, Clement Sulwe, Thokozile Khondiwa, Todd Jennings, Ameck Kamanga, Edgar Simulundu, Conceptor Mulube, Wizaso Mwasinga, Jalaimo Mumeka, John Simwanza, Patrick Sakubita, Otridah Kapona, Chilufya Susan Aneta Mulenga, Musole Chipoya, Kunda Musonda, Nathan Kapata, Nyambe Sinyange, Muzala Kapina, Joyce Siwila, Misheck Shawa, Masahiro Kajihara, Ayato Takada, Hirofumi Sawa, Simulyamana A. Choonga, Roma Chilengi, Earnest Muyunda, King S. Nalubamba, Bernard M. Hang’ombe

  Pathogens 13 (6) 486 - 486 2024/06/07 

  Enteric infections due to viral pathogens are a major public health concern. Detecting the risk areas requires a strong surveillance system for pathogenic viruses in sources such as wastewater. Towards building an environmental surveillance system in Zambia, we aimed to identify group A rotavirus (RVA) and human adenovirus (HAdV) in wastewater. Convenient sampling was conducted at four study sites every Tuesday for five consecutive weeks. The research team focused on three different methods of viral concentration to determine the suitability in terms of cost and applicability for a regular surveillance system: the bag-mediated filtration system (BMFS), polyethylene glycol-based (PEG) precipitation, and skimmed milk (SM) flocculation. We screened 20 wastewater samples for HAdV and RVA using quantitative polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR). Of the 20 samples tested using qPCR, 18/20 (90%) tested positive for HAdV and 14/20 (70%) tested positive for RVA. For the genetic sequencing, qPCR positives were subjected to cPCR, of which 12 positives were successfully amplified. The human adenovirus was identified with a nucleotide identity range of 98.48% to 99.53% compared with the reference genome from GenBank. The BMFS and SM flocculation were the most consistent viral concentration methods for HAdV and RVA, respectively. A statistical analysis of the positives showed that viral positivity differed by site (p < 0.001). SM and PEG may be the most appropriate options in resource-limited settings such as Zambia due to the lower costs associated with these concentration methods. The demonstration of HAdV and RVA detection in wastewater suggests the presence of the pathogens in the communities under study and the need to establish a routine wastewater surveillance system for the identification of pathogens.
• Antimicrobial Use Survey and Detection of ESBL-Escherichia coli in Commercial and Medium-/Small-Scale Poultry Farms in Selected Districts of Zambia

  Taona Sinyawa, Misheck Shawa, Geoffrey M. Muuka, Fusya Goma, Paul Fandamu, Joseph Yamweka Chizimu, Cynthia Sipho Khumalo, Malala Mulavu, Masuzyo Ngoma, Herman Moses Chambaro, Harvey Kakoma Kamboyi, Masahiro Kajihara, Hirofumi Sawa, Yasuhiko Suzuki, Hideaki Higashi, Geoffrey Mainda, Musso Munyeme, John Bwalya Muma, Christian Owusu Nyantakyi, Beverly Egyir, Bernard Mudenda Hang’ombe

  Antibiotics 13 (5) 467 - 467 2024/05/20 

  Antimicrobial resistance (AMR) among Escherichia coli from food animals is a rising problem, and heavy antimicrobial use in poultry is a contributing factor. In Zambia, studies linking poultry-associated AMR and antibiotic use (AMU) are rare. This study aimed to investigate commercial and medium-/small-scale poultry farmers’ usage of antimicrobials based on a questionnaire survey in ten districts of Zambia. In addition, the study characterized extended-spectrum β-lactamase (ESBL)-producing E. coli isolates obtained from poultry in the same districts. Data regarding knowledge and usage of antimicrobials were collected from commercial and medium-/small-scale poultry farmers using a pre-tested structured questionnaire. At the same time, cloacal samples were collected and analyzed. One hundred and fifty E. coli isolates were tested for antimicrobial susceptibility using eight antibiotic classes. The isolates were further screened for ESBL production by streaking them on cefotaxime (CTX)-supplemented MacConkey agar, then subjecting them to sequencing on a NextSeq. The questionnaire survey showed that more medium-/small-scale than commercial poultry farmers used antimicrobials (OR = 7.70, 95% CI = 2.88–20.61) but less prescriptions (OR = 0.02, 95% CI = 0.00–0.08). Susceptibility testing revealed that resistance was highest to ampicillin (128/148, 86.5%) and tetracycline (101/136, 74.3%) and that the prevalence of multidrug resistance (MDR) (28/30, 93.3%) was high. Whole-genome sequencing (WGS) of eight (8/30, 26.7%) isolates with CTX Minimum Inhibitory Concentration (MIC) ≥ 4 µg/mL revealed the presence of ESBL-encoding genes blaCTX-M-14, blaCTX-M-55, and blaTEM. WGS also detected other AMR genes for quinolones, aminoglycosides, phenicols, tetracycline, macrolides, and folate-pathway antagonists. Altogether, the questionnaire survey results showed a higher proportion of AMU and lower prescription usage among medium-/small-scale farmers. In addition, our results emphasize the circulation of ESBL-producing E. coli strains with associated MDR. It is critical to educate farmers about AMR risks and to encourage responsible usage of antimicrobials. Furthermore, there is a need to strengthen regulations limiting access to antimicrobials. Finally, there is a need to establish a one health system to guide public health response.
• Rab27a promotes degradation of West Nile virus E protein in the lysosome.

  Shintaro Kobayashi, Seira Kawai, Yukine Fukuda, Haruto Eguchi, Keisuke Maezono, Passawat Thammahakin, Hirofumi Sawa, Hiroaki Kariwa

  iScience 27 (4) 109539 - 109539 2024/04/19 

  Rab27a, a Rab family small GTPases, plays an important role in the trafficking and secretion of the intracellular proteins and has been reported to promote various viral multiplication. However, whether Rab27a is involved in West Nile virus (WNV) multiplication is unknown. This study examined the ability of Rab27a to suppress WNV multiplication. The inhibition of Rab27a expression increased viral multiplication and the intracellular levels of WNV structural proteins, E and prM proteins. Rab27a partially colocalized with E protein, mainly in the perinuclear region, while inhibition of Rab27a expression resulted in diffuse subcellular localization of E protein. In addition, some of the perinuclear E protein colocalized with the lysosomal marker LAMP1, and inhibition of lysosomal acidification increased intracellular levels of Rab27a and E proteins. These observations suggested that Rab27a inhibits WNV multiplication by inducing the degradation of viral protein in lysosomes.
• Genes involved in the limited spread of SARS-CoV-2 in the lower respiratory airways of hamsters may be associated with adaptive evolution.

  Kosuke Takada, Yasuko Orba, Yurie Kida, Jiaqi Wu, Chikako Ono, Yoshiharu Matsuura, So Nakagawa, Hirofumi Sawa, Tokiko Watanabe

  Journal of virology e0178423  2024/04/16 

  Novel respiratory viruses can cause a pandemic and then evolve to coexist with humans. The Omicron strain of severe acute respiratory syndrome coronavirus 2 has spread worldwide since its emergence in late 2021, and its sub-lineages are now established in human society. Compared to previous strains, Omicron is markedly less invasive in the lungs and causes less severe disease. One reason for this is that humans are acquiring immunity through previous infection and vaccination, but the nature of the virus itself is also changing. Using our newly established low-volume inoculation system, which reflects natural human infection, we show that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain. Furthermore, by characterizing chimeric viruses with the Omicron gene in the Wuhan strain genetic background and vice versa, we found that viral genes downstream of ORF3a, but not the S gene, were responsible for the limited spread of the Omicron strain in the lower airways of the virus-infected hamsters. Moreover, molecular evolutionary analysis of SARS-CoV-2 revealed a positive selection of genes downstream of ORF3a (M and E genes). Our findings provide insight into the adaptive evolution of the virus in humans during the pandemic convergence phase.IMPORTANCEThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread worldwide since its emergence in late 2021, and its sub-lineages are established in human society. Compared to previous strains, the Omicron strain is less invasive in the lower respiratory tract, including the lungs, and causes less severe disease; however, the mechanistic basis for its restricted replication in the lower airways is poorly understood. In this study, using a newly established low-volume inoculation system that reflects natural human infection, we demonstrated that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain and found that viral genes downstream of ORF3a are responsible for replication restriction in the lower respiratory tract of Omicron-infected hamsters. Furthermore, we detected a positive selection of genes downstream of ORF3a (especially the M and E genes) in SARS-CoV-2, suggesting that these genes may undergo adaptive changes in humans.
• Combinatorial Approach with Mass Spectrometry and Lectin Microarray Dissected Site-Specific Glycostem and Glycoleaf Features of the Virion-Derived Spike Protein of Ancestral and γ Variant SARS-CoV-2 Strains.

  Takahiro Hiono, Hiroaki Sakaue, Azusa Tomioka, Hiroyuki Kaji, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Atsushi Kuno

  Journal of proteome research 23 (4) 1408 - 1419 2024/04/05 

  The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public health globally. As the glycosylation of viral envelope glycoproteins is strongly associated with their immunogenicity, intensive studies have been conducted on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and γ-variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarrays (LMA). Our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, identified 1448 (ancestral strain) and 1785 (γ-variant strain) site-specific glycan compositions, respectively. Asparagine at amino acid position 20 (N20) is mainly glycosylated within two successive potential glycosylation sites, N17 and N20, of the γ-variant S protein; however, we found low-frequency glycosylation at N17. Our novel approaches, glycostem mapping and glycoleaf scoring, also illustrate the moderately branched/extended, highly fucosylated, and less sialylated natures of the glycoforms of S proteins. Subsequent LMA analysis emphasized the intensive end-capping of glycans by Lewis fucoses, which complemented the glycoproteomic features. These results illustrate the high-resolution glycoproteomic features of the SARS-CoV-2 S protein, contributing to vaccine design and understanding of viral protein synthesis.
• Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Enterobacteriaceae from Diseased Broiler Chickens in Lusaka District, Zambia.

  Chikwanda Chileshe, Misheck Shawa, Nelson Phiri, Joseph Ndebe, Cynthia Sipho Khumalo, Chie Nakajima, Masahiro Kajihara, Hideaki Higashi, Hirofumi Sawa, Yasuhiko Suzuki, Walter Muleya, Bernard Mudenda Hang'ombe

  Antibiotics (Basel, Switzerland) 13 (3) 2024/03/15 

  Poultry products in Zambia form an integral part of the human diet in many households, as they are cheap and easy to produce. The burden of poultry diseases has, however, remained a major challenge. Growing consumer demand for poultry products in Zambia has resulted in non-prudent antimicrobial use on farms, intending to prevent and treat poultry diseases for growth optimisation and maximising profits. This cross-sectional study aimed to identify the different types of bacteria causing diseases in chickens in Lusaka and to detect the extended-spectrum lactamase (ESBL)-encoding genes. We collected 215 samples from 91 diseased chickens at three post-mortem facilities and screened them for Gram-negative bacteria. Of these samples, 103 tested positive for various clinically relevant Enterobacteriaceae, including Enterobacter (43/103, 41.7%), Escherichia coli (20/103, 19.4%), Salmonella (10/103, 9.7%), and Shigella (8/103, 7.8%). Other isolated bacteria included Yersinia, Morganella, Proteus, and Klebsiella, which accounted for 21.4%. E. coli, Enterobacter, Salmonella, and Shigella were subjected to antimicrobial susceptibility testing. The results revealed that E. coli, Enterobacter, and Shigella were highly resistant to tetracycline, ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole, while Salmonella showed complete susceptibility to all tested antibiotics. The observed resistance patterns correlated with antimicrobial usage estimated from sales data from a large-scale wholesale and retail company. Six (6/14, 42.9%) E. coli isolates tested positive for blaCTX-M, whilst eight (8/14, 57.1%) Enterobacter samples tested positive for blaTEM. Interestingly, four (4/6, 66.7%) of the E. coli isolates carrying blaCTX-M-positive strains were also positive for blaTEM. Sanger sequencing of the PCR products revealed that five (5/6, 83.3%) of the abovementioned isolates possessed the blaCTX-M-15 allele. The results suggest the presence of potentially pathogenic ESBL-producing Enterobacteriaceae in poultry, threatening public health.
• Establishment of a lethal mouse model of emerging tick-borne orthonairovirus infections.

  Takuma Ariizumi, Koshiro Tabata, Yukari Itakura, Hiroko Kobayashi, William W Hall, Michihito Sasaki, Hirofumi Sawa, Keita Matsuno, Yasuko Orba

  PLoS pathogens 20 (3) e1012101  2024/03 

  Emerging and reemerging tick-borne virus infections caused by orthonairoviruses (family Nairoviridae), which are genetically distinct from Crimean-Congo hemorrhagic fever virus, have been recently reported in East Asia. Here, we have established a mouse infection model using type-I/II interferon receptor-knockout mice (AG129 mice) both for a better understanding of the pathogenesis of these infections and validation of antiviral agents using Yezo virus (YEZV), a novel orthonairovirus causing febrile illnesses associated with tick bites in Japan and China. YEZV-inoculated AG129 mice developed hepatitis with body weight loss and died by 6 days post infection. Blood biochemistry tests showed elevated liver enzyme levels, similar to YEZV-infected human patients. AG129 mice treated with favipiravir survived lethal YEZV infection, demonstrating the anti-YEZV effect of this drug. The present mouse model will help us better understand the pathogenicity of the emerging tick-borne orthonairoviruses and the development of specific antiviral agents for their treatment.
• Immunogenicity and protective efficacy of a co-formulated two-in-one inactivated whole virus particle COVID-19/influenza vaccine.

  Chimuka Handabile, Marumi Ohno, Toshiki Sekiya, Naoki Nomura, Tomomi Kawakita, Mamiko Kawahara, Masafumi Endo, Tomohiro Nishimura, Minako Okumura, Shinsuke Toba, Michihito Sasaki, Yasuko Orba, Brendon Y Chua, Louise C Rowntree, Thi H O Nguyen, Masashi Shingai, Akihiko Sato, Hirofumi Sawa, Kazumasa Ogasawara, Katherine Kedzierska, Hiroshi Kida

  Scientific reports 14 (1) 4204 - 4204 2024/02/20 

  Due to the synchronous circulation of seasonal influenza viruses and severe acute respiratory coronavirus 2 (SARS-CoV-2) which causes coronavirus disease 2019 (COVID-19), there is need for routine vaccination for both COVID-19 and influenza to reduce disease severity. Here, we prepared individual WPVs composed of formalin-inactivated SARS-CoV-2 WK 521 (Ancestral strain; Co WPV) or influenza virus [A/California/07/2009 (X-179A) (H1N1) pdm; Flu WPV] to produce a two-in-one Co/Flu WPV. Serum analysis from vaccinated mice revealed that a single dose of Co/Flu WPV induced antigen-specific neutralizing antibodies against both viruses, similar to those induced by either type of WPV alone. Following infection with either virus, mice vaccinated with Co/Flu WPV showed no weight loss, reduced pneumonia and viral titers in the lung, and lower gene expression of proinflammatory cytokines, as observed with individual WPV-vaccinated. Furthermore, a pentavalent vaccine (Co/qFlu WPV) comprising of Co WPV and quadrivalent influenza vaccine (qFlu WPV) was immunogenic and protected animals from severe COVID-19. These results suggest that a single dose of the two-in-one WPV provides efficient protection against SARS-CoV-2 and influenza virus infections with no evidence of vaccine interference in mice. We propose that concomitant vaccination with the two-in-one WPV can be useful for controlling both diseases.
• Phylogenetic Analysis of Newcastle Disease Virus Isolated from Poultry in Live Bird Markets and Wild Waterfowl in Zambia.

  Annie Kalonda, Ngonda Saasa, Masahiro Kajihara, Naganori Nao, Ladslav Moonga, Joseph Ndebe, Akina Mori-Kajihara, Andrew Nalishuwa Mukubesa, Yoshihiro Sakoda, Hirofumi Sawa, Ayato Takada, Edgar Simulundu

  Microorganisms 12 (2) 2024/02/08 

  Poultry production is essential to the economy and livelihood of many rural Zambian households. However, the industry is threatened by infectious diseases, particularly Newcastle disease virus (NDV) infection. Therefore, this study employed next-generation sequencing to characterise six NDV isolates from poultry in Zambia's live bird markets (LBMs) and wild waterfowl. Four NDV isolates were detected from 410 faecal samples collected from chickens in LBMs in Lusaka and two from 2851 wild birds from Lochinvar National Park. Phylogenetic analysis revealed that the four NDVs from LBM clustered in genotype VII and sub-genotype VII.2 were closely related to viruses previously isolated in Zambia and other Southern African countries, suggesting possible local and regional transboundary circulation of the virus. In contrast, the two isolates from wild birds belonged to class I viruses, genotype 1, and were closely related to isolates from Europe and Asia, suggesting the possible introduction of these viruses from Eurasia, likely through wild bird migration. The fusion gene cleavage site motif for all LBM-associated isolates was 112RRQKR|F117, indicating that the viruses are virulent, while the isolates from wild waterfowl had the typical 112ERQER|L117 avirulent motif. This study demonstrates the circulation of virulent NDV strains in LBMs and has, for the first time, characterised NDV from wild birds in Zambia. The study further provides the first whole genomes of NDV sub-genotype VII.2 and genotype 1 from Zambia and stresses the importance of surveillance and molecular analysis for monitoring the circulation of NDV genotypes and viral evolution.
• 血管内皮細胞におけるSARS-CoV-2侵入機構の解析(Analysis of SARS-CoV-2 uptake mechanism in vascular endothelial cells)

  桜井 優弥, 間石 奈湖, 藤岡 容一郎, 大場 雄介, 武田 遼, 佐々木 道仁, 大場 靖子, 樋田 泰浩, 澤 洋文, 樋田 京子

  日本病理学会会誌 113 (1) 369 - 369 0300-9181 2024/02
• Trends, patterns and relationship of antimicrobial use and resistance in bacterial isolates tested between 2015-2020 in a national referral hospital of Zambia.

  Misheck Shawa, Atmika Paudel, Herman Chambaro, Harvey Kamboyi, Ruth Nakazwe, Luke Alutuli, Tuvshinzaya Zorigt, Taona Sinyawa, Mulemba Samutela, Joseph Chizimu, Manyando Simbotwe, Kyoko Hayashida, Naganori Nao, Masahiro Kajihara, Yoshikazu Furuta, Yasuhiko Suzuki, Hirofumi Sawa, Bernard Hang'ombe, Hideaki Higashi

  PloS one 19 (4) e0302053  2024 

  Increased antimicrobial resistance (AMR) among bacteria underscores the need to strengthen AMR surveillance and promote data-based prescribing. To evaluate trends and associations between antimicrobial usage (AMU) and AMR, we explored a dataset of 34,672 bacterial isolates collected between 2015 and 2020 from clinical samples at the University Teaching Hospital (UTH) in Lusaka, Zambia. The most frequently isolated species were Escherichia coli (4,986/34,672; 14.4%), Staphylococcus aureus (3,941/34,672; 11.4%), and Klebsiella pneumoniae (3,796/34,672; 10.9%). Of the 16 drugs (eight classes) tested, only amikacin and imipenem showed good (> 50%) antimicrobial activity against both E. coli and K. pneumoniae, while nitrofurantoin was effective only in E. coli. Furthermore, 38.8% (1,934/4,980) of E. coli and 52.4% (2,079/3,791) of K. pneumoniae isolates displayed multidrug resistance (MDR) patterns on antimicrobial susceptibility tests. Among S. aureus isolates, 44.6% (973/2,181) were classified as methicillin-resistant (MRSA). Notably, all the MRSA exhibited MDR patterns. The annual hospital AMR rates varied over time, while there was a weak positive relationship (r = 0.38, 95% CI = 0.11-0.60) between the monthly use of third-generation cephalosporins (3GCs) and 3GC resistance among Enterobacterales. Overall, the results revealed high AMR rates that fluctuated over time, with a weak positive relationship between 3GC use and resistance. To our knowledge, this is the first report to evaluate the association between AMU and AMR in Zambia. Our results highlight the need to strengthen antimicrobial stewardship programs and optimize AMU in hospital settings.
• Combination therapy with oral antiviral and anti-inflammatory drugs improves the efficacy of delayed treatment in a COVID-19 hamster model.

  Michihito Sasaki, Tatsuki Sugi, Shun Iida, Yuichiro Hirata, Shinji Kusakabe, Kei Konishi, Yukari Itakura, Koshiro Tabata, Mai Kishimoto, Hiroko Kobayashi, Takuma Ariizumi, Kittiya Intaruck, Haruaki Nobori, Shinsuke Toba, Akihiko Sato, Keita Matsuno, Junya Yamagishi, Tadaki Suzuki, William W Hall, Yasuko Orba, Hirofumi Sawa

  EBioMedicine 99 104950 - 104950 2023/12/29 

  BACKGROUND: Pulmonary infection with SARS-CoV-2 stimulates host immune responses and can also result in the progression of dysregulated and critical inflammation. Throughout the pandemic, the management and treatment of COVID-19 has been continuously updated with a range of antiviral drugs and immunomodulators. Monotherapy with oral antivirals has proven to be effective in the treatment of COVID-19. However, treatment should be initiated in the early stages of infection to ensure beneficial therapeutic outcomes, and there is still room for further consideration on therapeutic strategies using antivirals. METHODS: We studied the therapeutic effects of monotherapy with the oral antiviral ensitrelvir or the anti-inflammatory corticosteroid methylprednisolone and combination therapy with ensitrelvir and methylprednisolone in a delayed dosing model of hamsters infected with SARS-CoV-2. FINDINGS: Combination therapy with ensitrelvir and methylprednisolone improved respiratory conditions and reduced the development of pneumonia in hamsters even when the treatment was started after 2 days post-infection. The combination therapy led to a differential histological and transcriptomic pattern in comparison to either of the monotherapies, with reduced lung damage and down-regulation of expression of genes involved in the inflammatory response. Furthermore, we found that the combination treatment is effective in case of infection with either the highly pathogenic delta or circulating omicron variants. INTERPRETATION: Our results demonstrate the advantage of combination therapy with antiviral and corticosteroid drugs in COVID-19 treatment from the perspective of lung pathology and host inflammatory responses. FUNDING: Funding bodies are described in the Acknowledgments section.
• Viral uptake and pathophysiology of the lung endothelial cells in age‐associated severe SARS‐CoV‐2 infection models

  Takuya Tsumita, Ryo Takeda, Nako Maishi, Yasuhiro Hida, Michihito Sasaki, Yasuko Orba, Akihiko Sato, Shinsuke Toba, Wataru Ito, Takahito Teshirogi, Yuya Sakurai, Tomohiro Iba, Hisamichi Naito, Hitoshi Ando, Haruhisa Watanabe, Amane Mizuno, Toshiki Nakanishi, Aya Matsuda, Ren Zixiao, Ji‐Won Lee, Tadahiro Iimura, Hirofumi Sawa, Kyoko Hida

  Aging Cell 23 (2) e14050  1474-9718 2023/12/14 

  Abstract Thrombosis is the major cause of death in severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, and the pathology of vascular endothelial cells (ECs) has received much attention. Although there is evidence of the infection of ECs in human autopsy tissues, their detailed pathophysiology remains unclear due to the lack of animal model to study it. We used a mouse‐adapted SARS‐CoV‐2 virus strain in young and mid‐aged mice. Only mid‐aged mice developed fatal pneumonia with thrombosis. Pulmonary ECs were isolated from these infected mice and RNA‐Seq was performed. The pulmonary EC transcriptome revealed that significantly higher levels of viral genes were detected in ECs from mid‐aged mice with upregulation of viral response genes such as DDX58 and IRF7. In addition, the thrombogenesis‐related genes encoding PLAT, PF4, F3 PAI‐1, and P‐selectin were upregulated. In addition, the inflammation‐related molecules such as CXCL2 and CXCL10 were upregulated in the mid‐aged ECs upon viral infection. Our mouse model demonstrated that SARS‐CoV‐2 virus entry into aged vascular ECs upregulated thrombogenesis and inflammation‐related genes and led to fatal pneumonia with thrombosis. Current results of EC transcriptome showed that EC uptake virus and become thrombogenic by activating neutrophils and platelets in the aged mice, suggesting age‐associated EC response as a novel finding in human severe COVID‐19.
• Ubiquitin accumulation induced by the finger and palm sub-domains of NS5 modulates the replication of West Nile virus.

  Shintaro Kobayashi, Ryoko Kawakami, Chisaki Takeda, Keisuke Maezono, Passawat Thammahakin, Haruto Eguchi, Bernard M Hang'ombe, Yasuko Orba, Hirofumi Sawa, Kentaro Yoshii, Hiroaki Kariwa

  Virology 588 109902 - 109902 2023/11 

  West Nile virus (WNV) causes encephalitis in human and animals. WNV is phylogenetically classified into at least five distinct genetic lineages with different pathogenicity. The pathogenesis of West Nile encephalitis is affected by ubiquitin accumulation in infected cells, but the mechanism is unknown. In this study, the association between ubiquitin accumulation and WNV pathogenicity was investigated. Ubiquitin accumulation was detected in cells infected with NY99 strain belonging to lineage-1, but not FCG and Zmq16 strains belonging to lineage-2. Substitution of the Finger and Palm sub-domains of NS5 from lineage-1 to -2 decreased ubiquitin accumulation and viral replication. Furthermore, the survival rate was increased, and viral replication and ubiquitin accumulation in the brain were attenuated, in mice inoculated with the substituted WNV compared with lineage-1 WNV. Therefore, the intracellular ubiquitin accumulation induced by the Finger and Palm sub-domains of NS5 is linked to the differences in pathogenicity among WNV lineages.
• Determination of the factors responsible for the tropism of SARS-CoV-2-related bat coronaviruses to Rhinolophus bat ACE2

  Shigeru Fujita, Yusuke Kosugi, Izumi Kimura, Kenzo Tokunaga, Jumpei Ito, Kei Sato, Keita Matsuno, Naganori Nao, Hirofumi Sawa, Shinya Tanaka, Masumi Tsuda, Lei Wang, Yoshikata Oda, Zannatul Ferdous, Kenji Shishido, Takasuke Fukuhara, Tomokazu Tamura, Rigel Suzuki, Saori Suzuki, Hayato Ito, Yu Kaku, Naoko Misawa, Arnon Plianchaisuk, Ziyi Guo, Alfredo A. Hinay, Keiya Uriu, Jarel Elgin M. Tolentino, Luo Chen, Lin Pan, Mai Suganami, Mika Chiba, Ryo Yoshimura, Kyoko Yasuda, Keiko Iida, Naomi Ohsumi, Adam P. Strange, Shiho Tanaka, Kazuhisa Yoshimura, Kenji Sadamasu, Mami Nagashima, Hiroyuki Asakura, Isao Yoshida, So Nakagawa, Akifumi Takaori-Kondo, Kayoko Nagata, Ryosuke Nomura, Yoshihito Horisawa, Yusuke Tashiro, Yugo Kawai, Kazuo Takayama, Rina Hashimoto, Sayaka Deguchi, Yukio Watanabe, Ayaka Sakamoto, Naoko Yasuhara, Takao Hashiguchi, Tateki Suzuki, Kanako Kimura, Jiei Sasaki, Yukari Nakajima, Hisano Yajima, Takashi Irie, Ryoko Kawabata, Kaori Tabata, Terumasa Ikeda, Hesham Nasser, Ryo Shimizu, M. S. T. Monira Begum, Michael Jonathan, Yuka Mugita, Otowa Takahashi, Kimiko Ichihara, Chihiro Motozono, Takamasa Ueno, Mako Toyoda, Akatsuki Saito, Maya Shofa, Yuki Shibatani, Tomoko Nishiuchi, Kotaro Shirakawa

  Journal of Virology 97 (10) 0022-538X 2023/10/31 

  ABSTRACT Differences in host angiotensin converting enzyme 2 (ACE2) genes may affect the host range of SARS-CoV-2-related coronaviruses (SC2r-CoVs) and further determine the tropism of host ACE2 for the infection receptor. However, the factor(s) responsible for determining the host tropism of SC2r-CoVs, which may in part be determined by the tropism of host ACE2 usage, remains unclear. Here, we use the pseudoviruses with the spike proteins of two Laotian SC2r-CoVs, BANAL-20-236 and BANAL-20-52, and the cells expressing ACE2 proteins of eight different Rhinolophus bat species to show that these two spikes have different tropisms for Rhinolophus bat ACE2. Through structural analysis and cell culture experiments, we demonstrate that this tropism is determined by residue 493 of the spike and residues 31 and 35 of ACE2. Our results suggest that SC2r-CoVs exhibit differential ACE2 tropism, which may be driven by adaptation to different Rhinolophus bat ACE2 proteins. IMPORTANCE The efficiency of infection receptor use is the first step in determining the species tropism of viruses. After the coronavirus disease 2019 pandemic, a number of SARS-CoV-2-related coronaviruses (SC2r-CoVs) were identified in Rhinolophus bats, and some of them can use human angiotensin converting enzyme 2 (ACE2) for the infection receptor without acquiring additional mutations. This means that the potential of certain SC2r-CoVs to cause spillover from bats to humans is "off-the-shelf." However, both SC2r-CoVs and Rhinolophus bat species are highly diversified, and the host tropism of SC2r-CoVs remains unclear. Here, we focus on two Laotian SC2r-CoVs, BANAL-20-236 and BANAL-20-52, and determine how the tropism of SC2r-CoVs to Rhinolophus bat ACE2 is determined at the amino acid resolution level.
• Expanding diversity of bunyaviruses identified in mosquitoes.

  Yasuko Orba, Yusuf Eshimutu Abu, Herman M Chambaro, Tapiwa Lundu, Walter Muleya, Yuki Eshita, Yongjin Qiu, Hayato Harima, Masahiro Kajihara, Akina Mori-Kajihara, Keita Matsuno, Michihito Sasaki, William W Hall, Bernard M Hang'ombe, Hirofumi Sawa

  Scientific reports 13 (1) 18165 - 18165 2023/10/24 

  Mosquitoes interact with various organisms in the environment, and female mosquitoes in particular serve as vectors that directly transmit a number of microorganisms to humans and animals by blood-sucking. Comprehensive analysis of mosquito-borne viruses has led to the understanding of the existence of diverse viral species and to the identification of zoonotic arboviruses responsible for significant outbreaks and epidemics. In the present study on mosquito-borne bunyaviruses we employed a broad-spectrum RT-PCR approach and identified eighteen different additional species in the Phenuiviridae family and also a number of related but unclassified bunyaviruses in mosquitoes collected in Zambia. The entire RNA genome segments of the newly identified viruses were further analyzed by RNA sequencing with a ribonuclease R (RNase R) treatment to reduce host-derived RNAs and enrich viral RNAs, taking advantage of the dsRNA panhandle structure of the bunyavirus genome. All three or four genome segments were identified in eight bunyavirus species. Furthermore, L segments of three different novel viruses related to the Leishbunyaviridae were found in mosquitoes together with genes from the suspected host, the Crithidia parasite. In summary, our virus detection approach using a combination of broad-spectrum RT-PCR and RNA sequencing analysis with a simple virus enrichment method allowed the discovery of novel bunyaviruses. The diversity of bunyaviruses is still expanding and studies on this will allow a better understanding of the ecology of hematophagous mosquitoes.
• 2-thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses.

  Kentaro Uemura, Haruaki Nobori, Akihiko Sato, Shinsuke Toba, Shinji Kusakabe, Michihito Sasaki, Koshiro Tabata, Keita Matsuno, Naoyoshi Maeda, Shiori Ito, Mayu Tanaka, Yuki Anraku, Shunsuke Kita, Mayumi Ishii, Kayoko Kanamitsu, Yasuko Orba, Yoshiharu Matsuura, William W Hall, Hirofumi Sawa, Hiroshi Kida, Akira Matsuda, Katsumi Maenaka

  Proceedings of the National Academy of Sciences of the United States of America 120 (42) e2304139120  2023/10/17 

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are causing significant morbidity and mortality worldwide. Furthermore, over 1 million cases of newly emerging or re-emerging viral infections, specifically dengue virus (DENV), are known to occur annually. Because no virus-specific and fully effective treatments against these or many other viruses have been approved, there is an urgent need for novel, effective therapeutic agents. Here, we identified 2-thiouridine (s2U) as a broad-spectrum antiviral ribonucleoside analogue that exhibited antiviral activity against several positive-sense single-stranded RNA (ssRNA+) viruses, such as DENV, SARS-CoV-2, and its variants of concern, including the currently circulating Omicron subvariants. s2U inhibits RNA synthesis catalyzed by viral RNA-dependent RNA polymerase, thereby reducing viral RNA replication, which improved the survival rate of mice infected with DENV2 or SARS-CoV-2 in our animal models. Our findings demonstrate that s2U is a potential broad-spectrum antiviral agent not only against DENV and SARS-CoV-2 but other ssRNA+ viruses.
• Development of flavivirus subviral particles with low cross-reactivity by mutations of a distinct antigenic domain.

  Koshiro Tabata, Yukari Itakura, Takuma Ariizumi, Manabu Igarashi, Hiroko Kobayashi, Kittiya Intaruck, Mai Kishimoto, Shintaro Kobayashi, William W Hall, Michihito Sasaki, Hirofumi Sawa, Yasuko Orba

  Applied microbiology and biotechnology 2023/10/13 

  The most conserved fusion loop (FL) domain present in the flavivirus envelope protein has been reported as a dominant epitope for cross-reactive antibodies to mosquito-borne flaviviruses (MBFVs). As a result, establishing accurate serodiagnosis for MBFV infections has been difficult as anti-FL antibodies are induced by both natural infection and following vaccination. In this study, we modified the most conserved FL domain to overcome this cross-reactivity. We showed that the FL domain of lineage I insect-specific flavivirus (ISFV) has differences in antigenicity from those of MBFVs and lineage II ISFV and determined the key amino acid residues (G106, L107, or F108), which contribute to the antigenic difference. These mutations were subsequently introduced into subviral particles (SVPs) of dengue virus type 2 (DENV2), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). In indirect enzyme-linked immunosorbent assays (ELISAs), these SVP mutants when used as antigens reduced the binding of cross-reactive IgG and total Ig induced by infection of ZIKV, JEV, and WNV in mice and enabled the sensitive detection of virus-specific antibodies. Furthermore, immunization of ZIKV or JEV SVP mutants provoked the production of antibodies with lower cross-reactivity to heterologous MBFV antigens compared to immunization with the wild-type SVPs in mice. This study highlights the effectiveness of introducing mutations in the FL domain in MBFV SVPs with lineage I ISFV-derived amino acids to produce SVP antigens with low cross-reactivity and demonstrates an improvement in the accuracy of indirect ELISA-based serodiagnosis for MBFV infections. KEY POINTS: • The FL domain of Lineage I ISFV has a different antigenicity from that of MBFVs. • Mutated SVPs reduce the binding of cross-reactive antibodies in indirect ELISAs. • Inoculation of mutated SVPs induces antibodies with low cross-reactivity.
• Prevalence and Genomic Characterization of Rotavirus A from Domestic Pigs in Zambia: Evidence for Possible Porcine–Human Interspecies Transmission

  Joseph Ndebe, Hayato Harima, Herman Moses Chambaro, Michihito Sasaki, Junya Yamagishi, Annie Kalonda, Misheck Shawa, Yongjin Qiu, Masahiro Kajihara, Ayato Takada, Hirofumi Sawa, Ngonda Saasa, Edgar Simulundu

  Pathogens 12 (10) 1199 - 1199 2023/09/27 [Refereed]
   

  Rotavirus is a major cause of diarrhea globally in animals and young children under 5 years old. Here, molecular detection and genetic characterization of porcine rotavirus in smallholder and commercial pig farms in the Lusaka Province of Zambia were conducted. Screening of 148 stool samples by RT-PCR targeting the VP6 gene revealed a prevalence of 22.9% (34/148). Further testing of VP6-positive samples with VP7-specific primers produced 12 positives, which were then Sanger-sequenced. BLASTn of the VP7 positives showed sequence similarity to porcine and human rotavirus strains with identities ranging from 87.5% to 97.1%. By next-generation sequencing, the full-length genetic constellation of the representative strains RVA/pig-wt/ZMB/LSK0137 and RVA/pig-wt/ZMB/LSK0147 were determined. Genotyping of these strains revealed a known Wa-like genetic backbone, and their genetic constellations were G4-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1 and G9-P[13]-I5-R1-C1-M1-A8-N1-T1-E1-H1, respectively. Phylogenetic analysis revealed that these two viruses might have their ancestral origin from pigs, though some of their gene segments were related to human strains. The study shows evidence of reassortment and possible interspecies transmission between pigs and humans in Zambia. Therefore, the “One Health” surveillance approach for rotavirus A in animals and humans is recommended to inform the design of effective control measures.
• ザンビアにおけるブタインフルエンザの疫学調査

  播磨 勇人, 奥谷 公亮, 梶原 将大, 小川 寛人, Simulundu Edgar, Bwalya Eugene, 邱 永晋, 迫田 義博, 西藤 岳彦, Hang' ombe Bernard, 澤 洋文, Mweene Aaron, 高田 礼人

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 166回 154 - 154 1347-8621 2023/09
• Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5

  Tomokazu Tamura, Daichi Yamasoba, Yoshitaka Oda, Jumpei Ito, Tomoko Kamasaki, Naganori Nao, Rina Hashimoto, Yoichiro Fujioka, Rigel Suzuki, Lei Wang, Hayato Ito, Yukie Kashima, Izumi Kimura, Mai Kishimoto, Masumi Tsuda, Hirofumi Sawa, Kumiko Yoshimatsu, Yuki Yamamoto, Tetsuharu Nagamoto, Jun Kanamune, Yutaka Suzuki, Yusuke Ohba, Saori Suzuki, Marie Kato, Zannatul Ferdous, Hiromi Mouri, Kenji Shishido, Naoko Misawa, Keiya Uriu, Yusuke Kosugi, Shigeru Fujita, Mai Suganami, Mika Chiba, Ryo Yoshimura, So Nakagawa, Jiaqi Wu, Akifumi Takaori-Kondo, Kotaro Shirakawa, Kayoko Nagata, Yasuhiro Kazuma, Ryosuke Nomura, Yoshihito Horisawa, Yusuke Tashiro, Yugo Kawai, Takao Hashiguchi, Tateki Suzuki, Kanako Kimura, Jiei Sasaki, Yukari Nakajima, Ayaka Sakamoto, Naoko Yasuhara, Takashi Irie, Ryoko Kawabata, Terumasa Ikeda, Hesham Nasser, Ryo Shimizu, Monira Begum, Otowa Takahashi, Kimiko Ichihara, Takamasa Ueno, Chihiro Motozono, Mako Toyoda, Akatsuki Saito, Yuri L. Tanaka, Erika P. Butlertanaka, Maya Shofa, Kaori Tabata, Isao Yokota, Keita Matsuno, Kazuo Takayama, Shinya Tanaka, Kei Sato, Takasuke Fukuhara

  Communications Biology 6 (1) 2023/07/24 

  Abstract The unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates ongoing control measures. Given its rapid spread, the new Omicron subvariant BA.5 requires urgent characterization. Here, we comprehensively analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1. Although in vitro growth kinetics of BA.5 was comparable among the Omicron subvariants, BA.5 was much more fusogenic than BA.1 and BA.2. Airway-on-a-chip analysis showed that, among Omicron subvariants, BA.5 had enhanced ability to disrupt the respiratory epithelial and endothelial barriers. Furthermore, in our hamster model, in vivo pathogenicity of BA.5 was slightly higher than that of the other Omicron variants and less than that of ancestral B.1.1. Notably, BA.5 gains efficient virus spread compared with BA.1 and BA.2, leading to prompt immune responses. Our findings suggest that BA.5 has low pathogenicity compared with the ancestral strain but enhanced virus spread /inflammation compared with earlier Omicron subvariants.
• Generation and characterization of a genetically modified live rabies vaccine strain with attenuating mutations in multiple viral proteins and evaluation of its potency in dogs.

  Fumiki Izumi, Shoya Miyamoto, Tatsunori Masatani, Michihito Sasaki, Kazuo Kawakami, Tatsuki Takahashi, Takuro Fujiwara, Yuji Fujii, Misuzu Okajima, Shoko Nishiyama, Hirofumi Sawa, Makoto Sugiyama, Naoto Ito

  Vaccine 41 (33) 4907 - 4917 2023/07/01 

  Live rabies vaccines have advantageous features that can facilitate mass vaccination for dogs, the most important reservoirs/transmitters of rabies. However, some live vaccine strains have problems in their safety, namely, risks from the residual pathogenicity and the pathogenic reversion of live vaccine strains. The reverse genetics system of rabies virus provides a feasible option to improve the safety of a live vaccine strain by, for example, artificially introducing attenuating mutations into multiple viral proteins. It was previously demonstrated in separate studies that introduction of amino acid residues Leu at position 333 in the viral glycoprotein (G333), Ser at G194, and Leu/His at positions 273/394 in the nucleoprotein (N273/394) enhance the safety of a live vaccine strain. In this study, to test our hypothesis that combinational introduction of these residues would significantly increase the safety level of a vaccine strain, we generated a novel live vaccine candidate, ERA-NG2, that is attenuated by mutations at N273/394 and G194/333, and we examined its safety and immunogenicity in mice and dogs. ERA-NG2 did not cause any clinical signs in mice after intracerebral inoculation. After 10 passages in suckling mouse brains, ERA-NG2 retained all of the introduced mutations except the mutation at N394 and the highly attenuated phenotype. These findings indicate that the ERA-NG2 is highly and stably attenuated. After confirming that ERA-NG2 induced a virus-neutralizing antibody (VNA) response and protective immunity in mice, we immunized dogs intramuscularly with a single dose (105-7 focus-forming units) of ERA-NG2 and found that, at all of the tested doses, the strain induced a VNA response in dogs without inducing any clinical signs. These findings demonstrate that ERA-NG2 has a high level of safety and a substantial level of immunogenicity in dogs and thus is a promising live vaccine candidate that can facilitate vaccination in dogs.
• Impact of imprinted immunity induced by mRNA vaccination in an experimental animal model.

  Shigeru Fujita, Keiya Uriu, Lin Pan, Naganori Nao, Koshiro Tabata, Mai Kishimoto, Yukari Itakura, Hirofumi Sawa, Izumi Kida, Tomokazu Tamura, Takasuke Fukuhara, Jumpei Ito, Keita Matsuno, Kei Sato

  The Journal of infectious diseases 2023/06/27 

  The emergence of SARS-CoV-2 Omicron variants has led to concerns that ancestral SARS-CoV-2-based vaccines may not be effective against newly emerging Omicron subvariants. The concept of "imprinted immunity" suggests that individuals vaccinated with ancestral virus-based vaccines may not develop effective immunity against newly emerging Omicron subvariants, such as BQ.1.1 and XBB.1. Here, we investigated this possibility using hamsters. While natural infection induced effective antiviral immunity, breakthrough infections in hamsters with BQ.1.1 and XBB.1 Omicron subvariants after receiving the 3-dose mRNA-LNP vaccine resulted in only faintly induced humoral immunity, supporting the possibility of imprinted immunity.
• Surveillance, Isolation, and Genetic Characterization of Bat Herpesviruses in Zambia.

  Hayato Harima, Yongjin Qiu, Junya Yamagishi, Masahiro Kajihara, Katendi Changula, Kosuke Okuya, Mao Isono, Tomoyuki Yamaguchi, Hirohito Ogawa, Naganori Nao, Michihito Sasaki, Edgar Simulundu, Aaron S Mweene, Hirofumi Sawa, Kanako Ishihara, Bernard M Hang'ombe, Ayato Takada

  Viruses 15 (6) 2023/06/13 

  Bats are of significant interest as reservoirs for various zoonotic viruses with high diversity. During the past two decades, many herpesviruses have been identified in various bats worldwide by genetic approaches, whereas there have been few reports on the isolation of infectious herpesviruses. Herein, we report the prevalence of herpesvirus infection of bats captured in Zambia and genetic characterization of novel gammaherpesviruses isolated from striped leaf-nosed bats (Macronycteris vittatus). By our PCR screening, herpesvirus DNA polymerase (DPOL) genes were detected in 29.2% (7/24) of Egyptian fruit bats (Rousettus aegyptiacus), 78.1% (82/105) of Macronycteris vittatus, and one Sundevall's roundleaf bat (Hipposideros caffer) in Zambia. Phylogenetic analyses of the detected partial DPOL genes revealed that the Zambian bat herpesviruses were divided into seven betaherpesvirus groups and five gammaherpesvirus groups. Two infectious strains of a novel gammaherpesvirus, tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1), were successfully isolated from Macronycteris vittatus bats, and their complete genomes were sequenced. The genome of MaGHV1 encoded 79 open reading frames, and phylogenic analyses of the DNA polymerase and glycoprotein B demonstrated that MaGHV1 formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses. Our findings provide new information regarding the genetic diversity of herpesviruses maintained in African bats.
• Genome Sequence of a Clinical Isolate of the Human Pathogenic Strain "Candidatus Borrelia fainii" Qtaro.

  Kentaro Itokawa, Kozue Sato, Yongjin Qiu, Jyunya Yamagishi, Katendi Changula, Naoko Kawai, Kyoko Hayashida, Joseph Ndebe, Bernard Mudenda Hang'ombe, Hirofumi Sawa, Shinji Kasai, Yukihiro Akeda, Hiroki Kawabata

  Microbiology resource announcements 12 (5) e0131822  2023/04/19 

  We report sequences of the complete linear chromosome and five linear plasmids of the relapsing fever spirochete "Candidatus Borrelia fainii" Qtaro. The chromosome sequence of 951,861 bp and the 243,291 bp of plasmid sequences were predicted to contain 852 and 239 protein-coding genes, respectively. The predicted total GC content was 28.4%.
• Morphogenesis of Bullet-Shaped Rabies Virus Particles Regulated by TSG101.

  Yukari Itakura, Koshiro Tabata, Takeshi Saito, Kittiya Intaruck, Nijiho Kawaguchi, Mai Kishimoto, Shiho Torii, Shintaro Kobayashi, Naoto Ito, Michiko Harada, Satoshi Inoue, Ken Maeda, Ayato Takada, William W Hall, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki

  Journal of virology 97 (5) e0043823  2023/04/12 

  Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host endosomal sorting complex required for transport (ESCRT) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection and, specifically, the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a small interfering RNA (siRNA) screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. IMPORTANCE Enveloped viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT complexes. Some enveloped viruses utilize their late (L-) domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped virions, but the underlying molecular mechanisms involved remain elusive. Here, we showed that TSG101, one of the ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via the L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.
• Surveillance and Phylogenetic Characterisation of Avian Influenza Viruses Isolated from Wild Waterfowl in Zambia in 2015, 2020, and 2021

  Annie Kalonda, Ngonda Saasa, Masahiro Kajihara, Naganori Nao, Ladislav Moonga, Joseph Ndebe, Akina Mori-Kajihara, Andrew Nalishuwa Mukubesa, Mulemba Samutela, Samuel Munjita, Yoshihiro Sakoda, Hirofumi Sawa, Ayato Takada, Edgar Simulundu

  Transboundary and Emerging Diseases 2023 1 - 16 1865-1674 2023/03/01 

  In recent years, the southern African region has experienced repeated incursions of highly pathogenic avian influenza viruses (HPAIVs), with wild migratory birds being implicated in the spread. To understand the profile of avian influenza viruses (AIVs) circulating in Zambia, we surveyed wild waterfowl for AIVs and phylogenetically characterised the isolates detected in 2015, 2020, and 2021. A total of 2,851 faecal samples of wild waterfowl were collected from Lochinvar National Park in the Southern Province of Zambia. During the study period, 85 (3.0%) low pathogenicity AIVs belonging to various subtypes were isolated, with H2N9, H8N4, and H10N8 being reported for the first time in avian species in Africa. The majority of the isolates were detected from glossy ibis (order Pelecaniformes) making it the first report of AIV from these birds in Zambia. Phylogenetic analysis of all eight gene segments of the 30 full genomes obtained in this study revealed that all the isolates belonged to the Eurasian lineage with their closest relatives being viruses isolated from wild and/or domestic birds in Bangladesh, Belgium, Egypt, Georgia, Mongolia, the Netherlands, and South Africa. Additionally, the Zambian viruses were grouped into distinct clusters based on the year of isolation. While no notifiable AIVs of the H5 or H7 subtypes were detected in wild birds in Zambia, viral internal protein genes of some viruses were closely related to H7 low pathogenicity AIVs. This study shows that periodically, a considerable diversity of AIV subtypes are introduced into the Zambian ecosystem by wild migratory waterfowl. The findings highlight the importance of continuous surveillance and monitoring of AIVs in wild waterfowl, including birds traditionally not considered to be major AIV reservoirs, for a better understanding of the eco-epidemiology and evolutionary dynamics of AIVs in Africa.
• Structure-activity relationship studies of anti-bunyaviral cap-dependent endonuclease inhibitors.

  Yoshiyuki Taoda, Akihiko Sato, Shinsuke Toba, Yuto Unoh, Makoto Kawai, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa

  Bioorganic & medicinal chemistry letters 83 129175 - 129175 2023/03/01 

  Bunyaviruses, including the Lassa virus (LASV), are known to cause hemorrhagic fever and have a high fatality rate among hospitalized patients, as there are few effective treatments. We focused on the fact that bunyaviruses use cap-dependent endonuclease (CEN) for viral replication, which is similar to influenza viruses. This led us to screen carbamoyl pyridone bicycle (CAB) compounds, which compose a series of baloxavir acid (BXA) derivatives, against lymphocytic choriomeningitis virus (LCMV) and Junin virus (JUNV) among the bunyaviruses. This led to the discovery of 1c, which has potent anti-bunyaviral activities. In SAR studies, we found that a large lipophilic side chain is preferred for the 1-position of the CAB scaffold, similar to the influenza CEN inhibitor, and that a small alkyl group for the 3-position shows high activity. Moreover, the 7‑carboxyl group of the scaffold is essential for anti-bunyaviral activities, and the antiviral activity is reduced by conversion to various carboxylic acid bioisosteres. The SAR results are discussed using a binding model of 9d in the active center of the known LCMV CEN crystal structure. These compounds show promise as broad-spectrum anti-bunyavirus therapeutics, given their relatively favorable metabolic stability and PK profiles.
• Detection of Old and New World Relapsing Fever Borreliae in Ornithodoros Ticks Collected from Warthog Burrows in Zambia.

  Yongjin Qiu, Herman M Chambaro, Kozue Sato, David Squarre, Edgar Simulundu, Masahiro Kajihara, Katendi Changula, Manyando Simbotwe, Hayato Harima, Joseph Ndebe, Ladslav Moonga, Ryo Nakao, Ayato Takada, Bernard Mudenda Hang'ombe, Hirofumi Sawa, Hiroki Kawabata

  Microorganisms 11 (1) 2023/01/12 

  Relapsing fever (RF) is an arthropod-borne disease caused by Borrelia spirochete, which is one of the major public health concerns in endemic regions including Africa. However, information on Borrelia&nbsp;spirochetes is limited in Zambia. Here, we investigate the Borrelia&nbsp;spirochetes harbored by Ornithodoros ticks in Zambian National Parks. We analyzed 182 DNA samples pooled from 886 Ornithodoros ticks. Of these, 43 tested positive, and their sequence revealed that the ticks harbored both Old and New World RF borreliae. This research presents the first evidence of Old-World RF borreliae in Zambia. The New World RF borreliae detected herein differed from the Candidatus Borrelia fainii previously reported in Zambia and were closely related to the pathogenic Borrelia sp. VS4 identified in Tanzania. Additionally, Borrelia theileri was recently reported in Zambia. Hence, at least four different Borrelia species occur in Zambia, and the organisms causing relapsing fever there might be more complex than previously thought. We empirically confirmed that real-time PCR with TaqMan minor groove binder probes accurately and simultaneously detected both Old and New World RF. In this manner, they could facilitate quantitative analyses of both types of RF borreliae. Subsequent investigations should endeavor to isolate the aforementioned Borrelia spp. and perform serosurveys on patients with RF.
• Current status and molecular epidemiology of rabies virus from different hosts and regions in Malawi.

  Henson Kainga, Elisha Chatanga, Marvin Collen Phonera, John Pilate Kothowa, Precious Dzimbiri, Gladson Kamwendo, Malala Mulavu, Cynthia Sipho Khumalo, Katendi Changula, Herman Chambaro, Hayato Harima, Masahiro Kajihara, Kholiwe Mkandawire, Patrick Chikungwa, Julius Chulu, Gilson Njunga, Simbarashe Chitanga, Benjamin Mubemba, Michihito Sasaki, Yasuko Orba, Yongjin Qiu, Junya Yamagishi, Edgar Simulundu, Ayato Takada, Boniface Namangala, Hirofumi Sawa, Walter Muleya

  Archives of virology 168 (2) 61 - 61 2023/01/12 

  Although rabies is endemic in Malawi, there have been no studies in which rabies virus was systematically investigated and characterized in multiple animal hosts in that country. In order to provide molecular epidemiological data on rabies virus in Malawi, 683 suspected rabies case reports from 2008 to 2021 were examined, and 46 (dog = 40, cow = 5, and cat = 1) viable rabies-positive brain samples archived at the Central Veterinary Laboratory (CVL), Lilongwe, Malawi, were analyzed genetically. The results showed an increase in the submission of brain samples from 2008 to 2010, with the highest number of submissions observed in 2020. Of the 683 case reports analyzed for the period under review, 38.1% (260/683) (CI: 34.44 - 41.84) were confirmed by direct fluorescent antibody test. Among the confirmed cases, 65.4% (170/260) (CI: 59.23 - 71.09) were canine rabies. Further, phylogenetic analysis revealed that sequences from different animal hosts clustered together within the Africa 1b lineage, suggesting that the strains circulating in livestock are similar to those in domestic dogs. This finding supports the hypothesis that canine rabies is spilling over to livestock and emphasizes the need for further studies to provide data for effective control of rabies in Malawi.
• Isolation and Characterization of Distinct Rotavirus A in Bat and Rodent Hosts.

  Mai Kishimoto, Masahiro Kajihara, Koshiro Tabata, Yukari Itakura, Shinsuke Toba, Seiya Ozono, Yuko Sato, Tadaki Suzuki, Naoto Ito, Katendi Changula, Yongjin Qiu, Akina Mori-Kajihara, Yoshiki Eto, Hayato Harima, Daniel Mwizabi, Bernard M Hang'ombe, William W Hall, Ayato Takada, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki

  Journal of virology 97 (1) e0145522  2023/01/12 

  Rotavirus A (RVA) causes diarrheal disease in humans and various animals. Recent studies have identified bat and rodent RVAs with evidence of zoonotic transmission and genome reassortment. However, the virological properties of bat and rodent RVAs with currently identified genotypes still need to be better clarified. Here, we performed virus isolation-based screening for RVA in animal specimens and isolated RVAs (representative strains: 16-06 and MpR12) from Egyptian fruit bat and Natal multimammate mouse collected in Zambia. Whole-genome sequencing and phylogenetic analysis revealed that the genotypes of bat RVA 16-06 were identical to that of RVA BATp39 strain from the Kenyan fruit bat, which has not yet been characterized. Moreover, all segments of rodent RVA MpR12 were highly divergent and assigned to novel genotypes, but RVA MpR12 was phylogenetically closer to bat RVAs than to other rodent RVAs, indicating a unique evolutionary history. We further investigated the virological properties of the isolated RVAs. In brief, we found that 16-06 entered cells by binding to sialic acids on the cell surface, while MpR12 entered in a sialic acid-independent manner. Experimental inoculation of suckling mice with 16-06 and MpR12 revealed that these RVAs are causative agents of diarrhea. Moreover, 16-06 and MpR12 demonstrated an ability to infect and replicate in a 3D-reconstructed primary human intestinal epithelium with comparable efficiency to the human RVA. Taken together, our results detail the unique genetic and virological features of bat and rodent RVAs and demonstrate the need for further investigation of their zoonotic potential. IMPORTANCE Recent advances in nucleotide sequence detection methods have enabled the detection of RVA genomes from various animals. These studies have discovered multiple divergent RVAs and have resulted in proposals for the genetic classification of novel genotypes. However, most of these RVAs have been identified via dsRNA viral genomes and not from infectious viruses, and their virological properties, such as cell/host tropisms, transmissibility, and pathogenicity, are unclear and remain to be clarified. Here, we successfully isolated RVAs with novel genome constellations from three bats and one rodent in Zambia. In addition to whole-genome sequencing, the isolated RVAs were characterized by glycan-binding affinity, pathogenicity in mice, and infectivity to the human gut using a 3D culture of primary intestinal epithelium. Our study reveals the first virological properties of bat and rodent RVAs with high genetic diversity and unique evolutional history and provides basic knowledge to begin estimating the potential of zoonotic transmission.
• Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.

  Kyoko Hayashida, Alejandro Garcia, Lavel Chinyama Moonga, Tatsuki Sugi, Kodera Takuya, Mitsuo Kawase, Fumihiro Kodama, Atsushi Nagasaka, Nobuhisa Ishiguro, Ayato Takada, Masahiro Kajihara, Naganori Nao, Masashi Shingai, Hiroshi Kida, Yasuhiko Suzuki, William W Hall, Hirofumi Sawa, Junya Yamagishi

  PloS one 18 (5) e0285861  2023 

  A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.
• Monitoring fusion kinetics of viral and target cell membranes in living cells using a SARS-CoV-2 spike-protein-mediated membrane fusion assay

  Hesham Nasser, Ryo Shimizu, Jumpei Ito, Akatsuki Saito, Kei Sato, Terumasa Ikeda, Keita Matsuno, Naganori Nao, Hirofumi Sawa, Mai Kishimoto, Shinya Tanaka, Masumi Tsuda, Lei Wang, Yoshikata Oda, Marie Kato, Zannatul Ferdous, Hiromi Mouri, Kenji Shishido, Takasuke Fukuhara, Tomokazu Tamura, Rigel Suzuki, Hayato Ito, Daichi Yamasoba, Izumi Kimura, Naoko Misawa, Keiya Uriu, Yusuke Kosugi, Shigeru Fujita, Mai Suganami, Mika Chiba, Ryo Yoshimura, So Nakagawa, Jiaqi Wu, Akifumi Takaori-Kondo, Kotaro Shirakawa, Kayoko Nagata, Yasuhiro Kazuma, Ryosuke Nomura, Yoshihito Horisawa, Yusuke Tashiro, Yugo Kawai, Takashi Irie, Ryoko Kawabata, MST Monira Begum, Otowa Takahashi, Kimiko Ichihara, Takamasa Ueno, Chihiro Motozono, Mako Toyoda, Yuri L. Tanaka, Erika P. Butlertanaka, Maya Shofa, Kazuo Takayama, Rina Hashimoto, Sayaka Deguchi, Takao Hashiguchi, Tateki Suzuki, Kanako Kimura, Jiei Sasaki, Yukari Nakajima, Kaori Tabata

  STAR Protocols 3 (4) 101773 - 101773 2666-1667 2022/12
• Luna Virus and Helminths in Wild Mastomys natalensis in Two Contrasting Habitats in Zambia: Risk Factors and Evidence of Virus Dissemination in Semen.

  Samuel Munalula Munjita, Given Moonga, Andrew Nalishuwa Mukubesa, Joseph Ndebe, Benjamin Mubemba, Manu Vanaerschot, Cristina Tato, John Tembo, Nathan Kapata, Simbarashe Chitanga, Katendi Changula, Mashiro Kajihara, Walter Muleya, Ayato Takada, Elisabeth Fichet-Calvet, Alimuddin Zumla, Hirofumi Sawa, Matthew Bates, Sody Munsaka, Edgar Simulundu

  Pathogens (Basel, Switzerland) 11 (11) 2022/11/14 

  Transmission dynamics and the maintenance of mammarenaviruses in nature are poorly understood. Using metagenomic next-generation sequencing (mNGS) and RT-PCR, we investigated the presence of mammarenaviruses and co-infecting helminths in various tissues of 182 Mastomys natalensis rodents and 68 other small mammals in riverine and non-riverine habitats in Zambia. The Luna virus (LUAV) genome was the only mammarenavirus detected (7.7%; 14/182) from M. natalensis. Only one rodent from the non-riverine habitat was positive, while all six foetuses from one pregnant rodent carried LUAV. LUAV-specific mNGS reads were 24-fold higher in semen than in other tissues from males. Phylogenetically, the viruses were closely related to each other within the LUAV clade. Helminth infections were found in 11.5% (21/182) of M. natalensis. LUAV-helminth co-infections were observed in 50% (7/14) of virus-positive rodents. Juvenility (OR = 9.4; p = 0.018; 95% CI: 1.47-59.84), nematodes (OR = 15.5; p = 0.001; 95% CI: 3.11-76.70), cestodes (OR = 10.8; p = 0.025; 95% CI: 1.35-86.77), and being male (OR = 4.6; p = 0.036; 95% CI: 1.10-18.90) were associated with increased odds of LUAV RNA detection. The role of possible sexual and/or congenital transmission in the epidemiology of LUAV infections in rodents requires further study, along with the implications of possible helminth co-infection.
• Seroprevalence and Associated Risk Factors of Rift Valley Fever in Livestock from Three Ecological Zones of Malawi.

  Henson Kainga, Marvin Collen Phonera, Elisha Chatanga, Simegnew Adugna Kallu, Prudence Mpundu, Mulemba Samutela, Herman Moses Chambaro, Masahiro Kajihara, Doreen Mainza Shempela, Jay Sikalima, Walter Muleya, Misheck Shawa, Julius Chulu, Gilson Njunga, Martin Simuunza, Ayato Takada, Hirofumi Sawa, Edgar Simulundu, Ngonda Saasa

  Pathogens (Basel, Switzerland) 11 (11) 2022/11/14 

  The epidemiology of Rift Valley fever (RVF) is poorly understood in Malawi. Here, a cross-sectional study was conducted (March-June 2020) to investigate the seroprevalence and potential risk factors of RVF virus (RVFV) in cattle, goats, and sheep in three ecological zones of Malawi. A total of 1523 serum samples were tested for anti-RVFV IgG and IgM antibodies by ELISA. Additionally, a questionnaire survey was used to assess potential RVF risk factors. The overall seroprevalence was 17.14% (261/1523; 95% CI = 15.33-19.11) for individual livestock and 33.24% (120/361; 95% CI = 28.18-38.11) for the livestock herd. Seroprevalence was significantly high in sheep (25.68%, 95% CI = 19.31-33.26) compared with cattle (21.35%, 95% CI = 18.74-24.22) and goats (7.72%, 95% CI = 5.72-10.34), (p = 0.047). At the individual livestock level, the risk was elevated in female livestock (OR: 1.74, 95% CI = 1.08-12.82) (p = 0.016), while at the herd level, areas receiving approximately 1001-1500 mm of rainfall (OR: 2.47, 95% CI = 1.14-5.37) (p = 0.022), areas of rainfall amount greater than approximately 1600 mm (OR: 2.239, 95% CI = 1.07-8.82) (p = 0.023), and mixed species herds (OR: 10.410, 95% CI = 3.04-35.59) (p = 0.001), were significant risk factors. The detection of IgM antibodies confirmed active circulation of RVFV in Malawi. Therefore, monitoring of RVF in animals, humans, and vectors using a "One Health" approach, along with community sensitization among the high-risk populations, could help mitigate the threat posed by this zoonotic disease in Malawi.
• Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant.

  Akatsuki Saito, Tomokazu Tamura, Jiri Zahradnik, Sayaka Deguchi, Koshiro Tabata, Yuki Anraku, Izumi Kimura, Jumpei Ito, Daichi Yamasoba, Hesham Nasser, Mako Toyoda, Kayoko Nagata, Keiya Uriu, Yusuke Kosugi, Shigeru Fujita, Maya Shofa, Mst Monira Begum, Ryo Shimizu, Yoshitaka Oda, Rigel Suzuki, Hayato Ito, Naganori Nao, Lei Wang, Masumi Tsuda, Kumiko Yoshimatsu, Jin Kuramochi, Shunsuke Kita, Kaori Sasaki-Tabata, Hideo Fukuhara, Katsumi Maenaka, Yuki Yamamoto, Tetsuharu Nagamoto, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Takamasa Ueno, Gideon Schreiber, Akifumi Takaori-Kondo, Kotaro Shirakawa, Hirofumi Sawa, Takashi Irie, Takao Hashiguchi, Kazuo Takayama, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, Kei Sato

  Cell host & microbe 30 (11) 1540 - 1555 2022/11/09 

  The SARS-CoV-2 Omicron BA.2.75 variant emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically distinct from BA.5, the currently predominant BA.2 descendant. Here, we show that BA.2.75 has a greater effective reproduction number and different immunogenicity profile than BA.5. We determined the sensitivity of BA.2.75 to vaccinee and convalescent sera as well as a panel of clinically available antiviral drugs and antibodies. Antiviral drugs largely retained potency, but antibody sensitivity varied depending on several key BA.2.75-specific substitutions. The BA.2.75 spike exhibited a profoundly higher affinity for its human receptor, ACE2. Additionally, the fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were greater than those of BA.2. Our multilevel investigations suggest that BA.2.75 acquired virological properties independent of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.
• S-217622, a SARS-CoV-2 main protease inhibitor, decreases viral load and ameliorates COVID-19 severity in hamsters.

  Michihito Sasaki, Koshiro Tabata, Mai Kishimoto, Yukari Itakura, Hiroko Kobayashi, Takuma Ariizumi, Kentaro Uemura, Shinsuke Toba, Shinji Kusakabe, Yuki Maruyama, Shun Iida, Noriko Nakajima, Tadaki Suzuki, Shinpei Yoshida, Haruaki Nobori, Takao Sanaki, Teruhisa Kato, Takao Shishido, William W Hall, Yasuko Orba, Akihiko Sato, Hirofumi Sawa

  Science translational medicine 15 (679) eabq4064  2022/11/03 

  In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity, but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622 (ensitrelvir), an inhibitor of SARS-CoV-2 main protease (Mpro, also known as 3C-like protease), decreases viral load and ameliorates disease severity in SARS-CoV-2-infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to sub-micromolar concentrations in cells. Oral administration of S-217622 demonstrated favorable pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern (VOCs), including the highly pathogenic Delta variant and the recently emerged Omicron BA.5 and BA.2.75 variants. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase 3 clinical trial (clinical trial registration no. jRCT2031210350), possesses remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.
• Discovery of Chlorofluoroacetamide-Based Covalent Inhibitors for Severe Acute Respiratory Syndrome Coronavirus 2 3CL Protease.

  Yuya Hirose, Naoya Shindo, Makiko Mori, Satsuki Onitsuka, Hikaru Isogai, Rui Hamada, Tadanari Hiramoto, Jinta Ochi, Daisuke Takahashi, Tadashi Ueda, Jose M M Caaveiro, Yuya Yoshida, Shigehiro Ohdo, Naoya Matsunaga, Shinsuke Toba, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Akihiko Sato, Eiji Kawanishi, Akio Ojida

  Journal of medicinal chemistry 65 (20) 13852 - 13865 2022/10/27 

  The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 3C-like protease (3CLpro) is a promising target for COVID-19 treatment. Here, we report a new class of covalent inhibitors of 3CLpro that possess chlorofluoroacetamide (CFA) as a cysteine-reactive warhead. Based on an aza-peptide scaffold, we synthesized a series of CFA derivatives in enantiopure form and evaluated their biochemical efficiency. The data revealed that 8a (YH-6) with the R configuration at the CFA unit strongly blocks SARS-CoV-2 replication in infected cells, and its potency is comparable to that of nirmatrelvir. X-ray structural analysis showed that YH-6 formed a covalent bond with Cys145 at the catalytic center of 3CLpro. The strong antiviral activity and favorable pharmacokinetic properties of YH-6 suggest its potential as a lead compound for the treatment of COVID-19.
• Quantitative Analysis of Extracellular Vesicle Uptake and Fusion with Recipient Cells.

  Hisaaki Hirose, Yusuke Hirai, Michihito Sasaki, Hirofumi Sawa, Shiroh Futaki

  Bioconjugate chemistry 33 (10) 1852 - 1859 2022/10/19 

  In precision medicine, extracellular vesicles (EVs) are promising intracellular drug delivery vehicles. The development of a quantitative analysis approach will provide valuable information from the perspective of cell biology and system design for drug delivery. Previous studies have reported quantitative methods to analyze the relative uptake or fusion of EVs to recipient cells. However, relatively few methods have enabled the simultaneous evaluation of the "number" of EVs taken up by recipient cells and those that fuse with cellular membranes. In this study, we report a simple quantitative method based on the NanoBiT system to quantify the uptake and fusion of small and large EVs (sEVs and lEVs, respectively). We assessed the abundance of these two subtypes of EVs and determined that lEVs may be more effective vehicles for transporting cargo to recipient cells. The results also indicated that both sEVs and lEVs have very low fusogenic activity, which can be improved in the presence of a fusogenic protein.
• Dihydromaniwamycin E, a Heat-Shock Metabolite from Thermotolerant Streptomyces sp. JA74, Exhibiting Antiviral Activity against Influenza and SARS-CoV-2 Viruses.

  Shun Saito, Kayo Funayama, Wataru Kato, Mayu Okuda, Meiko Kawamoto, Teruhiko Matsubara, Toshinori Sato, Akihiko Sato, Satoko Otsuguro, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Katsumi Maenaka, Kazutoshi Shindo, Masaya Imoto, Midori A Arai

  Journal of natural products 85 (11) 2583 - 2591 2022/10/12 

  Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50 values of 25.7 and 63.2 μM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50 values (for infection of 293TA cells) of 19.7 and 9.7 μM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50 concentrations.
• Identification of cap-dependent endonuclease inhibitors with broad-spectrum activity against bunyaviruses.

  Shinsuke Toba, Akihiko Sato, Makoto Kawai, Yoshiyuki Taoda, Yuto Unoh, Shinji Kusakabe, Haruaki Nobori, Shota Uehara, Kentaro Uemura, Keiichi Taniguchi, Masanori Kobayashi, Takeshi Noshi, Ryu Yoshida, Akira Naito, Takao Shishido, Junki Maruyama, Slobodan Paessler, Michael J Carr, William W Hall, Kumiko Yoshimatsu, Jiro Arikawa, Keita Matsuno, Yoshihiro Sakoda, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Hiroshi Kida

  Proceedings of the National Academy of Sciences of the United States of America 119 (36) e2206104119  2022/09/06 

  Viral hemorrhagic fevers caused by members of the order Bunyavirales comprise endemic and emerging human infections that are significant public health concerns. Despite the disease severity, there are few therapeutic options available, and therefore effective antiviral drugs are urgently needed to reduce disease burdens. Bunyaviruses, like influenza viruses (IFVs), possess a cap-dependent endonuclease (CEN) that mediates the critical cap-snatching step of viral RNA transcription. We screened compounds from our CEN inhibitor (CENi) library and identified specific structural compounds that are 100 to 1,000 times more active in vitro than ribavirin against bunyaviruses, including Lassa virus, lymphocytic choriomeningitis virus (LCMV), and Junin virus. To investigate their inhibitory mechanism of action, drug-resistant viruses were selected in culture. Whole-genome sequencing revealed that amino acid substitutions in the CEN region of drug-resistant viruses were located in similar positions as those of the CEN α3-helix loop of IFVs derived under drug selection. Thus, our studies suggest that CENi compounds inhibit both bunyavirus and IFV replication in a mechanistically similar manner. Structural analysis revealed that the side chain of the carboxyl group at the seventh position of the main structure of the compound was essential for the high antiviral activity against bunyaviruses. In LCMV-infected mice, the compounds significantly decreased blood viral load, suppressed symptoms such as thrombocytopenia and hepatic dysfunction, and improved survival rates. These data suggest a potential broad-spectrum clinical utility of CENis for the treatment of both severe influenza and hemorrhagic diseases caused by bunyaviruses.
• 3種のヤブカ体内において選択されたジカウイルスのゲノム変異評価

  下岡 誠, 三木 要, 江下 優樹, 大場 靖子, 澤 洋文, 村松 康和, 内田 玲麻

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [F1P - 06] 1347-8621 2022/09
• ザンビアのコウモリにおけるネルソンベイオルソレオウイルスの保有調査および分離ウイルスの病原性比較解析

  播磨 勇人, 佐々木 道仁, 大場 靖子, 邱 永晋, Changula Katendi, 梶原 将大, Simulundu Edgar, 谷口 怜, 高田 礼人, 西條 政幸, Hang'ombe Bernard M., 澤 洋文

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 165回 [F1P - 09] 1347-8621 2022/09
• Genomic Surveillance of SARS-CoV-2 in the Southern Province of Zambia: Detection and Characterization of Alpha, Beta, Delta, and Omicron Variants of Concern.

  Ben Katowa, Annie Kalonda, Benjamin Mubemba, Japhet Matoba, Doreen Mainza Shempela, Jay Sikalima, Boniface Kabungo, Katendi Changula, Simbarashe Chitanga, Mpanga Kasonde, Otridah Kapona, Nathan Kapata, Kunda Musonda, Mwaka Monze, John Tembo, Matthew Bates, Alimuddin Zumla, Catherine G Sutcliffe, Masahiro Kajihara, Junya Yamagishi, Ayato Takada, Hirofumi Sawa, Roma Chilengi, Victor Mukonka, Walter Muleya, Edgar Simulundu

  Viruses 14 (9) 2022/08/24 

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) have significantly impacted the global epidemiology of the pandemic. From December 2020 to April 2022, we conducted genomic surveillance of SARS-CoV-2 in the Southern Province of Zambia, a region that shares international borders with Botswana, Namibia, and Zimbabwe and is a major tourist destination. Genetic analysis of 40 SARS-CoV-2 whole genomes revealed the circulation of Alpha (B.1.1.7), Beta (B.1.351), Delta (AY.116), and multiple Omicron subvariants with the BA.1 subvariant being predominant. Whereas Beta, Delta, and Omicron variants were associated with the second, third, and fourth pandemic waves, respectively, the Alpha variant was not associated with any wave in the country. Phylogenetic analysis showed evidence of local transmission and possible multiple introductions of SARS-CoV-2 VOCs in Zambia from different European and African countries. Across the 40 genomes analysed, a total of 292 mutations were observed, including 182 missense mutations, 66 synonymous mutations, 23 deletions, 9 insertions, 1 stop codon, and 11 mutations in the non-coding region. This study stresses the need for the continued monitoring of SARS-CoV-2 circulation in Zambia, particularly in strategically positioned regions such as the Southern Province which could be at increased risk of introduction of novel VOCs.
• Antiviral effect of cetylpyridinium chloride in mouthwash on SARS-CoV-2

  Ryo Takeda, Hirofumi Sawa, Michihito Sasaki, Yasuko Orba, Nako Maishi, Takuya Tsumita, Natsumi Ushijima, Yasuhiro Hida, Hidehiko Sano, Yoshimasa Kitagawa, Kyoko Hida

  Scientific Reports 12 (1) 14050 - 14050 2022/08/18 

  Abstract Cetylpyridinium chloride (CPC), a quaternary ammonium compound, which is present in mouthwash, is effective against bacteria, fungi, and enveloped viruses. This study was conducted to explore the antiviral effect of CPC on SARS-CoV-2. There are few reports on the effect of CPC against wild-type SARS-CoV-2 at low concentrations such as 0.001%–0.005% (10–50 µg/mL). Interestingly, we found that low concentrations of CPC suppressed the infectivity of human isolated SARS-CoV-2 strains (Wuhan, Alpha, Beta, and Gamma) even in saliva. Furthermore, we demonstrated that CPC shows anti-SARS-CoV-2 effects without disrupting the virus envelope, using sucrose density analysis and electron microscopic examination. In conclusion, this study provided experimental evidence that CPC may inhibit SARS-CoV-2 infection even at lower concentrations.
• Isolation and characterization of an orthoreovirus from Indonesian fruit bats.

  Kittiya Intaruck, Yukari Itakura, Mai Kishimoto, Herman M Chambaro, Agus Setiyono, Ekowati Handharyani, Kentaro Uemura, Hayato Harima, Satoshi Taniguchi, Masayuki Saijo, Takashi Kimura, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki

  Virology 575 10 - 19 2022/08/15 

  Nelson Bay orthoreovirus (NBV) is an emerging bat-borne virus and causes respiratory tract infections in humans sporadically. Over the last two decades, several strains genetically related to NBV were isolated from humans and various bat species, predominantly in Southeast Asia (SEA), suggesting a high prevalence of the NBV species in this region. In this study, an orthoreovirus (ORV) belonging to the NBV species was isolated from Indonesian fruit bats' feces, tentatively named Paguyaman orthoreovirus (PgORV). Serological studies revealed that 81.2% (108/133) of Indonesian fruit bats sera had neutralizing antibodies against PgORV. Whole-genome sequencing and phylogenetic analysis of PgORV suggested the occurrence of past reassortments with other NBV strains isolated in SEA, indicating the dispersal and circulation of NBV species among bats in this region. Intranasal PgORV inoculation of laboratory mice caused severe pneumonia. Our study characterized PgORV's unique genetic background and highlighted the potential risk of PgORV-related diseases in Indonesia.
• Serological characterization of lineage II insect-specific flaviviruses compared with pathogenic mosquito-borne flaviviruses.

  Koshiro Tabata, Yukari Itakura, Shinsuke Toba, Kentaro Uemura, Mai Kishimoto, Michihito Sasaki, Jessica J Harrison, Akihiko Sato, William W Hall, Roy A Hall, Hirofumi Sawa, Yasuko Orba

  Biochemical and biophysical research communications 616 115 - 121 2022/08/06 

  The genus Flavivirus includes pathogenic tick- and mosquito-borne flaviviruses as well as non-pathogenic insect-specific flaviviruses (ISFVs). Phylogenetic analysis based on whole amino acid sequences has indicated that lineage II ISFVs have similarities to pathogenic flaviviruses. In this study, we used reactive analysis with immune serum against Psorophora flavivirus (PSFV) as a lineage IIa ISFV, and Barkeji virus (BJV) as a lineage IIb ISFV, to evaluate the antigenic similarity among lineage IIa and IIb ISFVs, and pathogenic mosquito-borne flaviviruses (MBFVs). Binding and antibody-dependent enhancement assays showed that anti-PSFV sera had broad cross-reactivity with MBFV antigens, while anti-BJV sera had low cross-reactivity. Both of the lineage II ISFV antisera were rarely observed to neutralize MBFVs. These results suggest that lineage IIa ISFV PSFV has more antigenic similarity to MBFVs than lineage IIb ISFV BJV.
• A high-affinity aptamer with base-appended base-modified DNA bound to isolated authentic SARS-CoV-2 strains wild-type and B.1.617.2 (delta variant).

  Hirotaka Minagawa, Hirofumi Sawa, Tomoko Fujita, Shintaro Kato, Asumi Inaguma, Miwako Hirose, Yasuko Orba, Michihito Sasaki, Koshiro Tabata, Naoki Nomura, Masashi Shingai, Yasuhiko Suzuki, Katsunori Horii

  Biochemical and biophysical research communications 614 207 - 212 2022/07/23 

  Simple, highly sensitive detection technologies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial for the effective implementation of public health policies. We used the systematic evolution of ligands by exponential enrichment with a modified DNA library, including a base-appended base (uracil with a guanine base at its fifth position), to create an aptamer with a high affinity for the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. The aptamer had a dissociation constant of 1.2 and < 1 nM for the RBD and spike trimer, respectively. Furthermore, enzyme-linked aptamer assays confirmed that the aptamer binds to isolated authentic SARS-CoV-2 wild-type and B.1.617.2 (delta variant). The binding signal was larger that of commercially available anti-SARS-CoV-2 RBD antibody. Thus, this aptamer as a sensing element will enable the highly sensitive detection of SARS-CoV-2.
• Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike.

  Daichi Yamasoba, Izumi Kimura, Hesham Nasser, Yuhei Morioka, Naganori Nao, Jumpei Ito, Keiya Uriu, Masumi Tsuda, Jiri Zahradnik, Kotaro Shirakawa, Rigel Suzuki, Mai Kishimoto, Yusuke Kosugi, Kouji Kobiyama, Teppei Hara, Mako Toyoda, Yuri L Tanaka, Erika P Butlertanaka, Ryo Shimizu, Hayato Ito, Lei Wang, Yoshitaka Oda, Yasuko Orba, Michihito Sasaki, Kayoko Nagata, Kumiko Yoshimatsu, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Jin Kuramochi, Motoaki Seki, Ryoji Fujiki, Atsushi Kaneda, Tadanaga Shimada, Taka-Aki Nakada, Seiichiro Sakao, Takuji Suzuki, Takamasa Ueno, Akifumi Takaori-Kondo, Ken J Ishii, Gideon Schreiber, Hirofumi Sawa, Akatsuki Saito, Takashi Irie, Shinya Tanaka, Keita Matsuno, Takasuke Fukuhara, Terumasa Ikeda, Kei Sato

  Cell 185 (12) 2103 - 2115 2022/06/09 

  Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.
• An unusually long Rift valley fever inter-epizootic period in Zambia: Evidence for enzootic virus circulation and risk for disease outbreak.

  Herman M Chambaro, Kazuyo Hirose, Michihito Sasaki, Brigadier Libanda, Yona Sinkala, Paul Fandamu, Walter Muleya, Fredrick Banda, Joseph Chizimu, David Squarre, Misheck Shawa, Yongjin Qiu, Hayato Harima, Yuki Eshita, Edgar Simulundu, Hirofumi Sawa, Yasuko Orba

  PLoS neglected tropical diseases 16 (6) e0010420  2022/06 

  Rift valley fever (RVF) is a mosquito-borne disease of animals and humans. Although RVF outbreaks are usually reported at 5-15-year intervals in sub-Saharan Africa, Zambia has experienced an unusually long inter-epizootic/-epidemic period of more than three decades. However, serological evidence of RVF virus (RVFV) infection in domestic ruminants during this period underscores the need for comprehensive investigation of the mechanisms of virus perpetuation and disease emergence. Mosquitoes (n = 16,778) captured from eight of the ten provinces of Zambia between April 2014 and May 2019 were pooled (n = 961) and screened for RVFV genome by a pan-phlebo RT-PCR assay. Aedes mosquito pools (n = 85) were further screened by nested RT-PCR assay. Sera from sheep (n = 13), goats (n = 259) and wild ungulates (n = 285) were screened for RVFV antibodies by ELISA while genome detection in pooled sera (n = 276) from domestic (n = 248) and wild ungulates (n = 37) was performed by real-time RT-PCR assay. To examine the association between the long inter-epizootic period and climatic variables, we examined El Niño-Southern Oscillation indices, precipitation anomalies, and normalized difference vegetation index. We then derived RVF risk maps by exploring climatic variables that would favor emergence of primary RVFV vectors. While no RVFV genome could be detected in pooled mosquito and serum samples, seroprevalence was significantly high (OR = 8.13, 95% CI [4.63-14.25]) in wild ungulates (33.7%; 96/285) compared to domestic ruminants (5.6%; 16/272). Retrospective analysis of RVF epizootics in Zambia showed a positive correlation between anomalous precipitation (La Niña) and disease emergence. On risk mapping, whilst northern and eastern parts of the country were at high risk, domestic ruminant population density was low (< 21 animals/km2) in these areas compared to low risk areas (>21 animals/km2). Besides evidence of silent circulation of RVFV and the risk of disease emergence in some areas, wildlife may play a role in the maintenance of RVFV in Zambia.
• Detection of Tick-Borne Bacterial and Protozoan Pathogens in Ticks from the Zambia-Angola Border.

  Yongjin Qiu, Martin Simuunza, Masahiro Kajihara, Joseph Ndebe, Ngonda Saasa, Penjani Kapila, Hayato Furumoto, Alice C C Lau, Ryo Nakao, Ayato Takada, Hirofumi Sawa

  Pathogens (Basel, Switzerland) 11 (5) 2022/05/10 

  Tick-borne diseases (TBDs), including emerging and re-emerging zoonoses, are of public health importance worldwide; however, TBDs tend to be overlooked, especially in countries with fewer resources, such as Zambia and Angola. Here, we investigated Rickettsia, Anaplasmataceae, and Apicomplexan pathogens in 59 and 96 adult ticks collected from dogs and cattle, respectively, in Shangombo, a town at the Zambia-Angola border. We detected Richkettsia africae and Rickettsia aeschilimannii in 15.6% of Amblyomma variegatum and 41.7% of Hyalomma truncatum ticks, respectively. Ehrlichia minasensis was detected in 18.8% of H. truncatum, and Candidatus Midichloria mitochondrii was determined in Hyalomma marginatum. We also detected Babesia caballi and Theileria velifera in A. variegatum ticks with a 4.4% and 6.7% prevalence, respectively. In addition, Hepatozoon canis was detected in 6.5% of Rhipicephalus lunulatus and 4.3% of Rhipicephalus sanguineus. Coinfection of R. aeshilimannii and E. minasensis were observed in 4.2% of H. truncatum. This is the first report of Ca. M. mitochondrii and E. minasensis, and the second report of B. caballi, in the country. Rickettsia africae and R. aeschlimannii are pathogenic to humans, and E. minasensis, B. caballi, T. velifera, and H. canis are pathogenic to animals. Therefore, individuals, clinicians, veterinarians, and pet owners should be aware of the distribution of these pathogens in the area.
• Glu333 in rabies virus glycoprotein is involved in virus attenuation through astrocyte infection and interferon responses.

  Yukari Itakura, Koshiro Tabata, Kohei Morimoto, Naoto Ito, Herman M Chambaro, Ryota Eguchi, Ken-Ichi Otsuguro, William W Hall, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki

  iScience 25 (4) 104122 - 104122 2022/04/15 

  The amino acid residue at position 333 of the rabies virus (RABV) glycoprotein (G333) is a major determinant of RABV pathogenicity. Virulent RABV strains possess Arg333, whereas the attenuated strain HEP-Flury (HEP) possesses Glu333. To investigate the potential attenuation mechanism dependent on a single amino acid at G333, comparative analysis was performed between HEP and HEP333R mutant with Arg333. We examined their respective tropism for astrocytes and the subsequent immune responses in astrocytes. Virus replication and subsequent interferon (IFN) responses in astrocytes infected with HEP were increased compared with HEP333R both in vitro and in vivo. Furthermore, involvement of IFN in the avirulency of HEP was demonstrated in IFN-receptor knockout mice. These results indicate that Glu333 contributes to RABV attenuation by determining the ability of the virus to infect astrocytes and stimulate subsequent IFN responses.
• Autochthonous Leishmania infantum in Dogs, Zambia, 2021.

  David Squarre, Herman M Chambaro, Kyoko Hayashida, Lavel C Moonga, Yongjin Qiu, Yasuyuki Goto, Elizabeth Oparaocha, Chisoni Mumba, Walter Muleya, Patricia Bwalya, Joseph Chizimu, Mwelwa Chembensofu, Edgar Simulundu, Wizaso Mwasinga, Nelly Banda, Racheal Mwenda, Junya Yamagishi, King S Nalubamba, Fredrick Banda, Musso Munyeme, Hirofumi Sawa, Paul Fandamu

  Emerging infectious diseases 28 (4) 888 - 890 2022/04 

  Leishmaniases are neglected tropical diseases of humans and animals. We detected Leishmania infantum in 3 mixed-breed dogs in Zambia that had no travel history outside the country. Our findings suggest presence of and probable emergence of leishmaniasis in Zambia, indicating the need for physicians and veterinarians to consider the disease during diagnosis.
• Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19.

  Yuto Unoh, Shota Uehara, Kenji Nakahara, Haruaki Nobori, Yukiko Yamatsu, Shiho Yamamoto, Yuki Maruyama, Yoshiyuki Taoda, Koji Kasamatsu, Takahiro Suto, Kensuke Kouki, Atsufumi Nakahashi, Sho Kawashima, Takao Sanaki, Shinsuke Toba, Kentaro Uemura, Tohru Mizutare, Shigeru Ando, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Akihiko Sato, Takafumi Sato, Teruhisa Kato, Yuki Tachibana

  Journal of medicinal chemistry 65 (9) 6499 - 6512 2022/03/30 

  The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.
• SARS-COV-2感染マウスを用いた肺の病理学的解析

  水野 天音, 積田 卓也, 武田 遼, 澤 洋文, 佐々木 道仁, 鳥羽 晋輔, 田中 宏典, 任 子驍, 樋田 泰浩, 樋田 京子

  日本病理学会会誌 (一社)日本病理学会 111 (1) 348 - 348 0300-9181 2022/03
• マウス感染モデルを用いたCOVID-19重症化における血管病態の解析

  積田 卓也, 武田 遼, 澤 洋文, 佐々木 道仁, 間石 奈湖, 樋田 泰浩, 松田 彩, 佐藤 彰彦, 鳥羽 晋輔, 田中 宏典, 樋田 京子

  日本内分泌学会雑誌 (一社)日本内分泌学会 97 (5) 1514 - 1514 0029-0661 2022/03
• Application of Acoustic Ejection MS System to High-Throughput Screening for SARS-CoV-2 3CL Protease Inhibitors.

  Tsukasa Hasegawa, Riyo M Imamura, Tateki Suzuki, Takao Hashiguchi, Takao Nomura, Satoko Otsuguro, Katsumi Maenaka, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Akihiko Sato, Takayoshi Okabe, Tetsuo Nagano, Hirotatsu Kojima

  Chemical & pharmaceutical bulletin 70 (3) 199 - 201 2022/03/01 

  MS is a powerful methodology for chemical screening to directly quantify substrates and products of enzymes, but its low throughput has been an issue. Recently, an acoustic liquid-handling apparatus (Echo®) used for rapid nano-dispensing has been coupled to a high-sensitivity mass spectrometer to create the Echo® MS system, and we applied this system to screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease inhibitors. Primary screening of 32033 chemical samples was completed in 12 h. Among the hits showing selective, dose-dependent 3CL-inhibitory activity, 8 compounds showed antiviral activity in cell-based assay.
• Synthesis and Anti-dengue Virus Activity of 5-Ethynylimidazole-4-carboxamide (EICA) Nucleotide Prodrugs.

  Motoki Nakamura, Kentaro Uemura, Noriko Saito-Tarashima, Akihiko Sato, Yasuko Orba, Hirofumi Sawa, Akira Matsuda, Katsumi Maenaka, Noriaki Minakawa

  Chemical & pharmaceutical bulletin 70 (3) 220 - 225 2022/03/01 

  We previously showed that 5-ethynyl-(1-β-D-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) is a potent anti-dengue virus (DENV) compound but is cytotoxic to some cell lines, while its 4-thio derivative, 5-ethynyl-(4-thio-1-β-D-ribofuranosyl)imidazole-4-carboxamide (2; 4'-thioEICAR), has less cytotoxicity but also less anti-DENV activity. Based on the hypothesis that the lower anti-DENV activity of 2 is due to reduced susceptibility to phosphorylation by cellular kinase(s), we investigated whether a monophosphate prodrug of 2 can improve its activity. Here, we first prepared two types of prodrug of 1, which revealed that the S-acyl-2-thioethyl (SATE) prodrug had stronger anti-DENV activity than the aryloxyphosphoramidate (so-called ProTide) prodrug. Based on these findings, we next prepared the SATE prodrug of 4'-thioEICAR 18. As expected, the resulting 18 showed potent anti-DENV activity, which was comparable to that of 1; however, its cytotoxicity was also increased relative to 2. Our findings suggest that prodrugs of 4'-thioribonucleoside derivatives such as EICAR (1) represent an effective approach to developing potent biologically active compounds; however, the balance between antiviral activity and cytotoxicity remains to be addressed.
• Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant.

  Rigel Suzuki, Daichi Yamasoba, Izumi Kimura, Lei Wang, Mai Kishimoto, Jumpei Ito, Yuhei Morioka, Naganori Nao, Hesham Nasser, Keiya Uriu, Yusuke Kosugi, Masumi Tsuda, Yasuko Orba, Michihito Sasaki, Ryo Shimizu, Ryoko Kawabata, Kumiko Yoshimatsu, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Hirofumi Sawa, Terumasa Ikeda, Takashi Irie, Keita Matsuno, Shinya Tanaka, Takasuke Fukuhara, Kei Sato

  Nature 603 (7902) 700 - 705 2022/03 [Refereed]
   

  The emergence of the Omicron variant of SARS-CoV-2 is an urgent global health concern1. In this study, our statistical modelling suggests that Omicron has spread more rapidly than the Delta variant in several countries including South Africa. Cell culture experiments showed Omicron to be less fusogenic than Delta and than an ancestral strain of SARS-CoV-2. Although the spike (S) protein of Delta is efficiently cleaved into two subunits, which facilitates cell-cell fusion2,3, the Omicron S protein was less efficiently cleaved compared to the S proteins of Delta and ancestral SARS-CoV-2. Furthermore, in a hamster model, Omicron showed decreased lung infectivity and was less pathogenic compared to Delta and ancestral SARS-CoV-2. Our multiscale investigations reveal the virological characteristics of Omicron, including rapid growth in the human population, lower fusogenicity and attenuated pathogenicity.
• Current knowledge of vector-borne zoonotic pathogens in Zambia: A clarion call to scaling-up "One Health" research in the wake of emerging and re-emerging infectious diseases.

  Benjamin Mubemba, Monicah M Mburu, Katendi Changula, Walter Muleya, Lavel C Moonga, Herman M Chambaro, Masahiro Kajihara, Yongjin Qiu, Yasuko Orba, Kyoko Hayashida, Catherine G Sutcliffe, Douglas E Norris, Philip E Thuma, Phillimon Ndubani, Simbarashe Chitanga, Hirofumi Sawa, Ayato Takada, Edgar Simulundu

  PLoS neglected tropical diseases 16 (2) e0010193  2022/02 

  BACKGROUND: Although vector-borne zoonotic diseases are a major public health threat globally, they are usually neglected, especially among resource-constrained countries, including those in sub-Saharan Africa. This scoping review examined the current knowledge and identified research gaps of vector-borne zoonotic pathogens in Zambia. METHODS AND FINDINGS: Major scientific databases (Web of Science, PubMed, Scopus, Google Scholar, CABI, Scientific Information Database (SID)) were searched for articles describing vector-borne (mosquitoes, ticks, fleas and tsetse flies) zoonotic pathogens in Zambia. Several mosquito-borne arboviruses have been reported including Yellow fever, Ntaya, Mayaro, Dengue, Zika, West Nile, Chikungunya, Sindbis, and Rift Valley fever viruses. Flea-borne zoonotic pathogens reported include Yersinia pestis and Rickettsia felis. Trypanosoma sp. was the only tsetse fly-borne pathogen identified. Further, tick-borne zoonotic pathogens reported included Crimean-Congo Haemorrhagic fever virus, Rickettsia sp., Anaplasma sp., Ehrlichia sp., Borrelia sp., and Coxiella burnetii. CONCLUSIONS: This study revealed the presence of many vector-borne zoonotic pathogens circulating in vectors and animals in Zambia. Though reports of human clinical cases were limited, several serological studies provided considerable evidence of zoonotic transmission of vector-borne pathogens in humans. However, the disease burden in humans attributable to vector-borne zoonotic infections could not be ascertained from the available reports and this precludes the formulation of national policies that could help in the control and mitigation of the impact of these diseases in Zambia. Therefore, there is an urgent need to scale-up "One Health" research in emerging and re-emerging infectious diseases to enable the country to prepare for future epidemics, including pandemics.
• Seroprevalence and Risk Factors of Crimean-Congo Hemorrhagic Fever in Cattle of Smallholder Farmers in Central Malawi.

  Marvin Collen Phonera, Martin Chitolongo Simuunza, Henson Kainga, Joseph Ndebe, Mwelwa Chembensofu, Elisha Chatanga, Setiala Kanyanda, Katendi Changula, Walter Muleya, Benjamin Mubemba, Simbarashe Chitanga, Masahiro Kajihara, Hirofumi Sawa, Gilson Njunga, Ayato Takada, Edgar Simulundu

  Pathogens (Basel, Switzerland) 10 (12) 2021/12/10 

  Crimean-Congo hemorrhagic fever virus (CCHFV) is endemic in Africa, Asia, and Eastern Europe where it circulates among animals and ticks causing sporadic outbreaks in humans. Although CCHF is endemic in sub-Saharan Africa, epidemiological information is lacking in many countries, including Malawi. To assess the risk of CCHF in Malawi, we conducted an epidemiological study in cattle reared by smallholder livestock farmers in central Malawi. A cross-sectional study was conducted in April 2020 involving seven districts, four from Kasungu and three from Lilongwe Agriculture Development Divisions. A structured questionnaire was administered to farmers to obtain demographic, animal management, and ecological risk factors data. Sera were collected from randomly selected cattle and screened for CCHF virus (CCHFV) specific antibodies using a commercial ELISA kit. Ticks were collected from cattle and classified morphologically to species level. An overall CCHFV seropositivity rate of 46.9% (n = 416; 95% CI: 42.0-51.8%) was observed. The seropositivity was significantly associated with the age of cattle (p < 0.001), sex (p < 0.001), presence of ticks in herds (p = 0.01), district (p = 0.025), and type of grazing lands (p = 0.013). Five species of ticks were identified, including Hyalomma truncatum, a known vector of CCHFV. Ticks of the species Hyalomma truncatum were not detected in two districts with the highest seroprevalence for CCHF and vector competency must be further explored in the study area. To our knowledge, this is the first report of serologic evidence of the presence of CCHV among smallholder cattle in central Malawi. This study emphasizes the need for continued monitoring of CCHFV infection among livestock, ticks, and humans for the development of data-based risk mitigation strategies.
• Influenza A and D Viruses in Non-Human Mammalian Hosts in Africa: A Systematic Review and Meta-Analysis.

  Annie Kalonda, Marvin Phonera, Ngonda Saasa, Masahiro Kajihara, Catherine G Sutcliffe, Hirofumi Sawa, Ayato Takada, Edgar Simulundu

  Viruses 13 (12) 2021/12/02 

  We conducted a systematic review and meta-analysis to investigate the prevalence and current knowledge of influenza A virus (IAV) and influenza D virus (IDV) in non-human mammalian hosts in Africa. PubMed, Google Scholar, Wiley Online Library and World Organisation for Animal Health (OIE-WAHIS) were searched for studies on IAV and IDV from 2000 to 2020. Pooled prevalence and seroprevalences were estimated using the quality effects meta-analysis model. The estimated pooled prevalence and seroprevalence of IAV in pigs in Africa was 1.6% (95% CI: 0-5%) and 14.9% (95% CI: 5-28%), respectively. The seroprevalence of IDV was 87.2% (95% CI: 24-100%) in camels, 9.3% (95% CI: 0-24%) in cattle, 2.2% (95% CI: 0-4%) in small ruminants and 0.0% (95% CI: 0-2%) in pigs. In pigs, H1N1 and H1N1pdm09 IAVs were commonly detected. Notably, the highly pathogenic H5N1 virus was also detected in pigs. Other subtypes detected serologically and/or virologically included H3N8 and H7N7 in equids, H1N1, and H3N8 and H5N1 in dogs and cats. Furthermore, various wildlife animals were exposed to different IAV subtypes. For prudent mitigation of influenza epizootics and possible human infections, influenza surveillance efforts in Africa should not neglect non-human mammalian hosts. The impact of IAV and IDV in non-human mammalian hosts in Africa deserves further investigation.
• Hepatitis E virus infection in pigs: a first report from Zambia.

  Herman M Chambaro, Michihito Sasaki, Walter Muleya, Masahiro Kajihara, Misheck Shawa, Kabemba E Mwape, Hayato Harima, Yongjin Qiu, William W Hall, Paul Fandamu, David Squarre, Edgar Simulundu, Hirofumi Sawa, Yasuko Orba

  Emerging microbes & infections 10 (1) 2169 - 2172 2021/12 

  While evidence suggests presence of HEV infection in humans in Zambia, currently, there is no information on its occurrence in domestic pigs. Here, we investigated the presence of HEV antibodies and genome in domestic pigs in Zambia. Sera (n = 484) from domestic pigs were screened for antibodies against HEV by ELISA while genome detection in fecal (n = 25) and liver (n = 100) samples from slaughter pigs was conducted using nested RT-PCR assay. Overall, seroprevalence was 47.7% (231/484) while zoonotic genotype 3 HEV RNA was detected in 16.0% (20/125) of slaughtered pigs. This is the first report to highlight occurrence of HEV infection in domestic pigs in Zambia. This finding suggests possible contamination of the pork supply chain. Moreover, there is a potential risk of zoonotic transmission of HEV to abattoir workers, pig farmers and handlers.
• SARS-CoV-2 inhibits induction of the MHC class I pathway by targeting the STAT1-IRF1-NLRC5 axis

  Ji-Seung Yoo, Michihito Sasaki, Steven X. Cho, Yusuke Kasuga, Baohui Zhu, Ryota Ouda, Yasuko Orba, Paul de Figueiredo, Hirofumi Sawa, Koichi S. Kobayashi

  Nature Communications 12 (1) 6602 - 6602 2021/12 

  The MHC class I-mediated antigen presentation pathway plays a critical role in antiviral immunity. Here we show that the MHC class I pathway is targeted by SARS-CoV-2. Analysis of the gene expression profile from COVID-19 patients as well as SARS-CoV-2 infected epithelial cell lines reveals that the induction of the MHC class I pathway is inhibited by SARS-CoV-2 infection. We show that NLRC5, an MHC class I transactivator, is suppressed both transcriptionally and functionally by the SARS-CoV-2 ORF6 protein, providing a mechanistic link. SARS-CoV-2 ORF6 hampers type II interferon-mediated STAT1 signaling, resulting in diminished upregulation of NLRC5 and IRF1 gene expression. Moreover, SARS-CoV-2 ORF6 inhibits NLRC5 function via blocking karyopherin complex-dependent nuclear import of NLRC5. Collectively, our study uncovers an immune evasion mechanism of SARS-CoV-2 that targets the function of key MHC class I transcriptional regulators, STAT1-IRF1-NLRC5.
• Evidence of Borrelia theileri in Wild and Domestic Animals in the Kafue Ecosystem of Zambia.

  Yongjin Qiu, David Squarre, Yukiko Nakamura, Alice C C Lau, Lavel Chinyama Moonga, Naoko Kawai, Aiko Ohnuma, Kyoko Hayashida, Ryo Nakao, Junya Yamagishi, Hirofumi Sawa, Boniface Namangala, Hiroki Kawabata

  Microorganisms 9 (11) 2021/11/22 

  Members of the genus Borrelia are arthropod-borne spirochetes that are human and animal pathogens. Vertebrate hosts, including wild animals, are pivotal to the circulation and maintenance of Borrelia spirochetes. However, information on Borrelia spirochetes in vertebrate hosts in Zambia is limited. Thus, we aimed to investigate the presence of Borrelia spirochetes in wild animals and cattle in Zambia. A total of 140 wild animals of four species and 488 cattle DNA samples from /near the Kafue National Park were collected for real-time PCR screening, followed by characterization using three different genes with positive samples. Five impalas and 20 cattle tested positive using real-time PCR, and sequence analysis revealed that the detected Borrelia were identified to be Borrelia theileri, a causative agent of bovine borreliosis. This is the first evidence of Borrelia theileri in African wildlife and cattle in Zambia. Our results suggest that clinical differentiation between bovine borreliosis and other bovine diseases endemic in Zambia is required for better treatment and control measures. As this study only included wild and domestic animals in the Kafue ecosystem, further investigations in other areas and with more wildlife and livestock species are needed to clarify a comprehensive epidemiological status of Borrelia theileri in Zambia.
• Air-liquid interphase culture confers SARS-CoV-2 susceptibility to A549 alveolar epithelial cells.

  Michihito Sasaki, Mai Kishimoto, Yukari Itakura, Koshiro Tabata, Kittiya Intaruck, Kentaro Uemura, Shinsuke Toba, Takao Sanaki, Akihiko Sato, William W Hall, Yasuko Orba, Hirofumi Sawa

  Biochemical and biophysical research communications 577 146 - 151 2021/11/05 [Refereed]
   

  The human lung cell A549 is susceptible to infection with a number of respiratory viruses. However, A549 cells are resistant to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection in conventional submerged culture, and this would appear to be due to low expression levels of the SARS-CoV-2 entry receptor: angiotensin-converting enzyme-2 (ACE2). Here, we examined SARS-CoV-2 susceptibility to A549 cells after adaptation to air-liquid interface (ALI) culture. A549 cells in ALI culture yielded a layer of mucus on their apical surface, exhibited decreased expression levels of the proliferation marker KI-67 and intriguingly became susceptible to SARS-CoV-2 infection. We found that A549 cells increased the endogenous expression levels of ACE2 and TMPRSS2 following adaptation to ALI culture conditions. Camostat, a TMPRSS2 inhibitor, reduced SARS-CoV-2 infection in ALI-cultured A549 cells. These findings indicate that ALI culture switches the phenotype of A549 cells from resistance to susceptibility to SARS-CoV-2 infection through upregulation of ACE2 and TMPRSS2.
• 細胞外小胞の受容細胞への内在化量および膜融合量の定量化技術の開発

  平井 勇祐, 広瀬 久昭, 佐々木 道仁, 澤 洋文, 二木 史朗

  日本生化学会大会プログラム・講演要旨集 (公社)日本生化学会 94回 [P - 440] 2021/11
• 細胞外小胞の受容細胞への内在化量および膜融合量の定量化技術の開発

  平井 勇祐, 広瀬 久昭, 佐々木 道仁, 澤 洋文, 二木 史朗

  日本生化学会大会プログラム・講演要旨集 (公社)日本生化学会 94回 [P - 440] 2021/11
• 食品成分およびその誘導体群によるコロナウイルス感染抑制効果

  村井 勇太, ムトマイナー, クーラス・サジール, 臼杵 靖剛, 湯山 耕平, 中岡 慎治, 佐々木 道仁, 大場 靖子, 佐藤 彰彦, 五十嵐 靖之, 澤 洋文, 門出 健次

  日本生化学会大会プログラム・講演要旨集 (公社)日本生化学会 94回 [P - 943] 2021/11
• Serological and molecular epidemiological study on swine influenza in Zambia.

  Hayato Harima, Kosuke Okuya, Masahiro Kajihara, Hirohito Ogawa, Edgar Simulundu, Eugene Bwalya, Yongjin Qiu, Akina Mori-Kajihara, Musso Munyeme, Yoshihiro Sakoda, Takehiko Saito, Bernard M Hang'ombe, Hirofumi Sawa, Aaron S Mweene, Ayato Takada

  Transboundary and emerging diseases 69 (4) e931-e943  2021/11/01 

  Influenza A viruses (IAVs) cause highly contagious respiratory diseases in humans and animals. In 2009, a swine-origin pandemic H1N1 IAV, designated A(H1N1)pdm09 virus, spread worldwide, and has since frequently been introduced into pig populations. Since novel reassortant IAVs with pandemic potential may emerge in pigs, surveillance for IAV in pigs is therefore necessary not only for the pig industry but also for public health. However, epidemiological information on IAV infection of pigs in Africa remains sparse. In this study, we collected 246 serum and 605 nasal swab samples from pigs in Zambia during the years 2011-2018. Serological analyses revealed that 49% and 32% of the sera collected in 2011 were positive for hemagglutination-inhibition (HI) and neutralizing antibodies against A(H1N1)pdm09 virus, respectively, whereas less than 5.3% of sera collected during the following period (2012-2018) were positive in both serological tests. The positive rate and the neutralization titres to A(H1N1)pdm09 virus were higher than those to classical swine H1N1 and H1N2 IAVs. On the other hand, the positive rate for swine H3N2 IAV was very low in the pig population in Zambia in 2011-2018 (5.3% and 0% in HI and neutralization tests, respectively). From nasal swab samples, we isolated one H3N2 and eight H1N1 IAV strains with an isolation rate of 1.5%. Phylogenetic analyses of all eight gene segments revealed that the isolated IAVs were closely related to human IAV strains belonging to A(H1N1)pdm09 and seasonal H3N2 lineages. Our findings indicate that reverse zoonotic transmission from humans to pigs occurred during the study period in Zambia and highlight the need for continued surveillance to monitor the status of IAVs circulating in swine populations in Africa.
• Abnormal blood coagulation and kidney damage in aged hamsters infected with severe acute respiratory syndrome coronavirus 2

  Marumi Ohno, Michihito Sasaki, Yasuko Orba, Toshiki Sekiya, Md Abdul Masum, Osamu Ichii, Tatsuya Sawamura, Akemi Kakino, Yasuhiko Suzuki, Hiroshi Kida, Hirofumi Sawa, Masashi Shingai

  Viruses 13 (11) 2021/11 

  Systemic symptoms have often been observed in patients with coronavirus disease 2019 (COVID-19) in addition to pneumonia, however, the details are still unclear due to the lack of an appropriate animal model. In this study, we investigated and compared blood coagulation abnormalities and tissue damage between male Syrian hamsters of 9 (young) and over 36 (aged) weeks old after intranasal infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite similar levels of viral replication and inflammatory responses in the lungs of both age groups, aged but not young hamsters showed significant prolongation of prothrombin time and prominent acute kidney damage. Moreover, aged hamsters demonstrated increased intravascular coagulation time-dependently in the lungs, suggesting that consumption of coagulation factors causes prothrombin time prolongation. Furthermore, proximal urinary tract damage and mesangial matrix expansion were observed in the kidneys of the aged hamsters at early and later disease stages, respectively. Given that the severity and mortality of COVID-19 are higher in elderly human patients, the effect of aging on pathogenesis needs to be understood and should be considered for the selection of animal models. We, thus, propose that the aged hamster is a good small animal model for COVID-19 research.
• 5-Hydroxymethyltubercidin exhibits potent antiviral activity against flaviviruses and coronaviruses, including SARS-CoV-2.

  Kentaro Uemura, Haruaki Nobori, Akihiko Sato, Takao Sanaki, Shinsuke Toba, Michihito Sasaki, Akiho Murai, Noriko Saito-Tarashima, Noriaki Minakawa, Yasuko Orba, Hiroaki Kariwa, William W Hall, Hirofumi Sawa, Akira Matsuda, Katsumi Maenaka

  iScience 24 (10) 103120 - 103120 2021/10/22 [Refereed]
   

  Newly emerging or re-emerging viral infections continue to cause significant morbidity and mortality every year worldwide, resulting in serious effects on both health and the global economy. Despite significant drug discovery research against dengue viruses (DENVs) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), no fully effective and specific drugs directed against these viruses have been discovered. Here, we examined the anti-DENV activity of tubercidin derivatives from a compound library from Hokkaido University and demonstrated that 5-hydroxymethyltubercidin (HMTU, HUP1108) possessed both potent anti-flavivirus and anti-coronavirus activities at submicromolar levels without significant cytotoxicity. Furthermore, HMTU inhibited viral RNA replication and specifically inhibited replication at the late stages of the SARS-CoV-2 infection process. Finally, we demonstrated that HMTU 5'-triphosphate inhibited RNA extension catalyzed by the viral RNA-dependent RNA polymerase. Our findings suggest that HMTU has the potential of serving as a lead compound for the development of a broad spectrum of antiviral agents, including SARS-CoV-2.
• Rickettsia lusitaniae in Ornithodoros Porcinus Ticks, Zambia.

  Simbarashe Chitanga, Herman M Chambaro, Lavel C Moonga, Kyoko Hayashida, Junya Yamagishi, Walter Muleya, Katendi Changula, Benjamin Mubemba, Manyando Simbotwe, David Squarre, Paul Fandamu, King S Nalubamba, Yongjin Qiu, Sawa Hirofumi, Edgar Simulundu

  Pathogens (Basel, Switzerland) 10 (10) 2021/10/12 

  Rickettsial pathogens are amongst the emerging and re-emerging vector-borne zoonoses of public health importance. Though traditionally considered to be transmitted by ixodid ticks, the role of argasid ticks as vectors of these pathogens is increasingly being recognized. While bat-feeding (Ornithodoros faini) and chicken-feeding (Argas walkerae) argasid ticks have been shown to harbor Rickettsia pathogens in Zambia, there are currently no reports of Rickettsia infection in southern Africa from warthog-feeding (Phacochoerus africanus) soft ticks, particularly Ornithodoros moubata and Ornithodoros porcinus. Our study sought to expand on the existing knowledge on the role of soft ticks in the epidemiology of Rickettsia species through screening for Rickettsia pathogens in warthog burrow-dwelling soft ticks from two national parks in Zambia. The tick species from which Rickettsia were detected in this study were identified as Ornithodoros porcinus, and an overall minimal Rickettsia infection rate of 19.8% (32/162) was observed. All of the sequenced Rickettsia were identified as Rickettsia lusitaniae based on nucleotide sequence similarity and phylogenetic analysis of the citrate synthase (gltA) and 17kDa common antigen (htrA) genes. Utilizing all of the gltA (n = 10) and htrA (n = 12) nucleotide sequences obtained in this study, BLAST analysis showed 100% nucleotide similarity to Rickettsia lusitaniae. Phylogenetic analysis revealed that all of the Zambian gltA and htrA gene sequences could be grouped with those of Rickettsia lusitaniae obtained in various parts of the world. Our data suggest that Rickettsia lusitaniae has a wider geographic and vector range, enhancing to our understanding of Rickettsia lusitaniae epidemiology in sub-Saharan Africa.
• A SARS-CoV-2 antibody broadly neutralizes SARS-related coronaviruses and variants by coordinated recognition of a virus-vulnerable site.

  Taishi Onodera, Shunsuke Kita, Yu Adachi, Saya Moriyama, Akihiko Sato, Takao Nomura, Shuhei Sakakibara, Takeshi Inoue, Takashi Tadokoro, Yuki Anraku, Kohei Yumoto, Cong Tian, Hideo Fukuhara, Michihito Sasaki, Yasuko Orba, Nozomi Shiwa, Naoko Iwata, Noriyo Nagata, Tateki Suzuki, Jiei Sasaki, Tsuyoshi Sekizuka, Keisuke Tonouchi, Lin Sun, Shuetsu Fukushi, Hiroyuki Satofuka, Yasuhiro Kazuki, Mitsuo Oshimura, Tomohiro Kurosaki, Makoto Kuroda, Yoshiharu Matsuura, Tadaki Suzuki, Hirofumi Sawa, Takao Hashiguchi, Katsumi Maenaka, Yoshimasa Takahashi

  Immunity 54 (10) 2385 - 2398 2021/10/12 [Refereed]
   

  Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.
• Novel Virulent Bacteriophage ΦSG005, Which Infects Streptococcus gordonii, Forms a Distinct Clade among Streptococcus Viruses.

  Jumpei Fujiki, Shin-Ichi Yoshida, Tomohiro Nakamura, Keisuke Nakamura, Yurika Amano, Keita Nishida, Keitaro Nishi, Michihito Sasaki, Tomohito Iwasaki, Hirofumi Sawa, Hitoshi Komatsuzawa, Hiroshi Hijioka, Hidetomo Iwano

  Viruses 13 (10) 2021/09/29 

  Bacteriophages are viruses that specifically infect bacteria and are classified as either virulent phages or temperate phages. Despite virulent phages being promising antimicrobial agents due to their bactericidal effects, the implementation of phage therapy depends on the availability of virulent phages against target bacteria. Notably, virulent phages of Streptococcus gordonii, which resides in the oral cavity and is an opportunistic pathogen that can cause periodontitis and endocarditis have previously never been found. We thus attempted to isolate virulent phages against S. gordonii. In the present study, we report for the first time a virulent bacteriophage against S. gordonii, ΦSG005, discovered from drainage water. ΦSG005 is composed of a short, non-contractile tail and a long head, revealing Podoviridae characteristics via electron microscopic analysis. In turbidity reduction assays, ΦSG005 showed efficient bactericidal effects on S. gordonii. Whole-genome sequencing showed that the virus has a DNA genome of 16,127 bp with 21 coding sequences. We identified no prophage-related elements such as integrase in the ΦSG005 genome, demonstrating that the virus is a virulent phage. Phylogenetic analysis indicated that ΦSG005 forms a distinct clade among the streptococcus viruses and is positioned next to streptococcus virus C1. Molecular characterization revealed the presence of an anti-CRISPR (Acr) IIA5-like protein in the ΦSG005 genome. These findings facilitate our understanding of streptococcus viruses and advance the development of phage therapy against S. gordonii infection.
• LIGHTHOUSE illuminates therapeutics for a variety of diseases including COVID-19

  Hideyuki Shimizu, Manabu Kodama, Masaki Matsumoto, Yasuko Orba, Michihito Sasaki, Akihiko Sato, Hirofumi Sawa, Keiichi I. Nakayama

  bioRxiv 25 (11) 105314 - 105314 2021/09/25 [Not refereed]
   

  Although numerous promising therapeutic targets for human diseases have been discovered, most have not been successfully translated into clinical practice¹. A bottleneck in the application of basic research findings to patients is the enormous cost, time, and effort required for high-throughput screening of potential drugs² for given therapeutic targets. Recent advances in 3D docking simulations have not solved this problem, given that 3D protein structures with sufficient resolution are not always available and that they are computationally expensive to obtain. Here we have developed LIGHTHOUSE, a graph-based deep learning approach for discovery of the hidden principles underlying the association of small-molecule compounds with target proteins, and we present its validation by identifying potential therapeutic compounds for various human diseases. Without any 3D structural information for proteins or chemicals, LIGHTHOUSE estimates protein-compound scores that incorporate known evolutionary relations and available experimental data. It identified novel therapeutics for cancer, lifestyle-related disease, and bacterial infection. Moreover, LIGHTHOUSE predicted ethoxzolamide as a therapeutic for coronavirus disease 2019 (COVID-19), and this agent was indeed effective against alpha, beta, gamma, and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that are rampant worldwide. Given that ethoxzolamide is already approved for several diseases, it could be rapidly deployed for the treatment of patients with COVID-19. We envision that LIGHTHOUSE will bring about a paradigm shift in translational medicine, providing a bridge from bench side to bedside.
• A targeted approach with nanopore sequencing for the universal detection and identification of flaviviruses.

  Patrick Reteng, Linh Nguyen Thuy, Tam Tran Thi Minh, Maria Angélica Monteiro de Mello Mares-Guia, Maria Celeste Torres, Ana Maria Bispo de Filippis, Yasuko Orba, Shintaro Kobayashi, Kyoko Hayashida, Hirofumi Sawa, William W Hall, Lan Anh Nguyen Thi, Junya Yamagishi

  Scientific reports 11 (1) 19031 - 19031 2021/09/24 

  Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.
• A novel nairovirus associated with acute febrile illness in Hokkaido, Japan.

  Fumihiro Kodama, Hiroki Yamaguchi, Eunsil Park, Kango Tatemoto, Mariko Sashika, Ryo Nakao, Yurino Terauchi, Keita Mizuma, Yasuko Orba, Hiroaki Kariwa, Katsuro Hagiwara, Katsunori Okazaki, Akiko Goto, Rika Komagome, Masahiro Miyoshi, Takuya Ito, Kimiaki Yamano, Kentaro Yoshii, Chiaki Funaki, Mariko Ishizuka, Asako Shigeno, Yukari Itakura, Lesley Bell-Sakyi, Shunji Edagawa, Atsushi Nagasaka, Yoshihiro Sakoda, Hirofumi Sawa, Ken Maeda, Masayuki Saijo, Keita Matsuno

  Nature communications 12 (1) 5539 - 5539 2021/09/20 

  The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014-2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan.
• Molecular Survey of Babesia and Anaplasma Infection in Cattle in Bolivia

  Shohei Ogata, Juan Antonio Cristian Pereira, Loza Vega Ariel Jhonny, Herbas Perez Gladys Carolina, Keita Matsuno, Yasuko Orba, Hirofumi Sawa, Fumihiko Kawamori, Nariaki Nonaka, Ryo Nakao

  Veterinary Sciences 8 (9) 188 - 188 2021/09/07 [Refereed]
   

  Latin American countries produce more than a quarter of the world’s beef and are a major global supplier of livestock protein. Tick-borne diseases (TBDs) are a major constraint to the livestock industry worldwide, including in Latin America. The aim of this study was to detect and characterise tick-borne pathogens in cattle from Santa Cruz, Bolivia, where no detailed epidemiological data are available. Blood samples were collected from 104 cattle. Apicomplexan parasites were detected by nested PCR amplification of the 18S ribosomal RNA gene (rDNA), and Anaplasmataceae was screened by the PCR amplification of 16S rDNA, followed by characterisation based on the heat shock protein and citrate synthase gene sequences. Babesia infection was observed in nine cattle (one Babesia bovis and eight Babesia bigemina), while Anaplasmataceae infection was detected in thirty-two cattle. A sequencing analysis confirmed the presence of Anaplasma marginale and Anaplasma platys-like. These results provide the first molecular evidence for the four above-mentioned tick-borne pathogens in cattle in Bolivia. This information improves our understanding of the epidemiology of TBDs and will help in formulating appropriate and improved pathogen control strategies.
• ウエストナイルウイルス感染によるタンパク質の核内輸送への影響

  小林 進太郎, 好井 健太朗, 澤 洋文, 苅和 宏明

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [FO - 24] 1347-8621 2021/09
• Attenuated infection by a Pteropine orthoreovirus isolated from an Egyptian fruit bat in Zambia.

  Hayato Harima, Michihito Sasaki, Yasuko Orba, Kosuke Okuya, Yongjin Qiu, Christida E Wastika, Katendi Changula, Masahiro Kajihara, Edgar Simulundu, Tomoyuki Yamaguchi, Yoshiki Eto, Akina Mori-Kajihara, Akihiko Sato, Satoshi Taniguchi, Ayato Takada, Masayuki Saijo, Bernard M Hang'ombe, Hirofumi Sawa

  PLoS neglected tropical diseases 15 (9) e0009768  2021/09 [Refereed]
   

  BACKGROUND: Pteropine orthoreovirus (PRV) is an emerging bat-borne zoonotic virus that causes severe respiratory illness in humans. Although PRVs have been identified in fruit bats and humans in Australia and Asia, little is known about the prevalence of PRV infection in Africa. Therefore, this study performed an PRV surveillance in fruit bats in Zambia. METHODS: Egyptian fruit bats (Rousettus aegyptiacus, n = 47) and straw-colored fruit bats (Eidolon helvum, n = 33) captured in Zambia in 2017-2018 were screened for PRV infection using RT-PCR and serum neutralization tests. The complete genome sequence of an isolated PRV strain was determined by next generation sequencing and subjected to BLAST and phylogenetic analyses. Replication capacity and pathogenicity of the strain were investigated using Vero E6 cell cultures and BALB/c mice, respectively. RESULTS: An PRV strain, tentatively named Nachunsulwe-57, was isolated from one Egyptian fruit bat. Serological assays demonstrated that 98% of sera (69/70) collected from Egyptian fruit bats (n = 37) and straw-colored fruit bats (n = 33) had neutralizing antibodies against PRV. Genetic analyses revealed that all 10 genome segments of Nachunsulwe-57 were closely related to a bat-derived Kasama strain found in Uganda. Nachunsulwe-57 showed less efficiency in viral growth and lower pathogenicity in mice than another PRV strain, Miyazaki-Bali/2007, isolated from a patient. CONCLUSIONS: A high proportion of Egyptian fruit bats and straw-colored fruit bats were found to be seropositive to PRV in Zambia. Importantly, a new PRV strain (Nachunsulwe-57) was isolated from an Egyptian fruit bat in Zambia, which had relatively weak pathogenicity in mice. Taken together, our findings provide new epidemiological insights about PRV infection in bats and indicate the first isolation of an PRV strain that may have low pathogenicity to humans.
• SARS-CoV-2 Bearing a Mutation at the S1/S2 Cleavage Site Exhibits Attenuated Virulence and Confers Protective Immunity.

  Michihito Sasaki, Shinsuke Toba, Yukari Itakura, Herman M Chambaro, Mai Kishimoto, Koshiro Tabata, Kittiya Intaruck, Kentaro Uemura, Takao Sanaki, Akihiko Sato, William W Hall, Yasuko Orba, Hirofumi Sawa

  mBio 12 (4) e0141521  2021/08/31 [Refereed]
   

  Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by the host furin protease. Proteolytic cleavage activates the spike protein, thereby affecting both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogenesis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited low growth properties. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, thereby indicating the attenuated variant nature of S gene mutants. This transient infection was sufficient for inducing protective neutralizing antibodies that cross-react with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Taken together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of hamsters and highlighted the potential benefits of S gene mutants as potential immunogens. IMPORTANCE SARS-CoV-2 uses its spike protein to enter target cells. The spike protein is cleaved by a host protease, and this event facilitates viral entry and broadens cell tropism. In this study, we employed SARS-CoV-2 mutants lacking the S protein cleavage site and characterized their growth and pathogenicity using hamsters, a laboratory animal model for SARS-CoV-2 infection. These mutants exerted low pathogenicity but induced sufficient levels of neutralizing antibodies in hamsters, which protected hamsters from rechallenge with pathogenic clinical SARS-CoV-2 strains. These virus mutants may be used as protective immunogens against SARS-CoV-2 infection.
• Dual Effect of Organogermanium Compound THGP on RIG-I-Mediated Viral Sensing and Viral Replication during Influenza a Virus Infection

  Sunanda Baidya, Yoko Nishimoto, Seiichi Sato, Yasuhiro Shimada, Nozomi Sakurai, Hirotaka Nonaka, Koki Noguchi, Mizuki Kido, Satoshi Tadano, Kozo Ishikawa, Kai Li, Aoi Okubo, Taisho Yamada, Yasuko Orba, Michihito Sasaki, Hirofumi Sawa, Hiroko Miyamoto, Ayato Takada, Takashi Nakamura, Akinori Takaoka

  Viruses 13 (9) 1674 - 1674 2021/08/24 [Refereed]
   

  The interaction of viral nucleic acid with protein factors is a crucial process for initiating viral polymerase-mediated viral genome replication while activating pattern recognition receptor (PRR)-mediated innate immune responses. It has previously been reported that a hydrolysate of Ge-132, 3-(trihydroxygermyl) propanoic acid (THGP), shows a modulatory effect on microbial infections, inflammation, and immune responses. However, the detailed mechanism by which THGP can modify these processes during viral infections remained unknown. Here, we show that THGP can specifically downregulate type I interferon (IFN) production in response to stimulation with a cytosolic RNA sensor RIG-I ligand 5′-triphosphate RNA (3pRNA) but not double-stranded RNA, DNA, or lipopolysaccharide. Consistently, treatment with THGP resulted in the dose-dependent suppression of type I IFN induction upon infections with influenza virus (IAV) and vesicular stomatitis virus, which are known to be mainly sensed by RIG-I. Mechanistically, THGP directly binds to the 5′-triphosphate moiety of viral RNA and competes with RIG-I-mediated recognition. Furthermore, we found that THGP can directly counteract the replication of IAV but not EMCV (encephalitismyocarditis virus), by inhibiting the interaction of viral polymerase with RNA genome. Finally, IAV RNA levels were significantly reduced in the lung tissues of THGP-treated mice when compared with untreated mice. These results suggest a possible therapeutic implication of THGP and show direct antiviral action, together with the suppressive activity of innate inflammation.
• Mosquito-Borne Viral Pathogens Detected in Zambia: A Systematic Review.

  Rachel Milomba Velu, Geoffrey Kwenda, Liyali Libonda, Caroline Cleopatra Chisenga, Bumbangi Nsoni Flavien, Obvious Nchimunya Chilyabanyama, Michelo Simunyandi, Samuel Bosomprah, Nicholus Chintu Sande, Katendi Changula, Walter Muleya, Monicah Mirai Mburu, Benjamin Mubemba, Simbarashe Chitanga, John Tembo, Matthew Bates, Nathan Kapata, Yasuko Orba, Masahiro Kajihara, Ayato Takada, Hirofumi Sawa, Roma Chilengi, Edgar Simulundu

  Pathogens (Basel, Switzerland) 10 (8) 2021/08/10 [Refereed]
   

  Emerging and re-emerging mosquito-borne viral diseases are a threat to global health. This systematic review aimed to investigate the available evidence of mosquito-borne viral pathogens reported in Zambia. A search of literature was conducted in PubMed and Google Scholar for articles published from 1 January 1930 to 30 June 2020 using a combination of keywords. Eight mosquito-borne viruses belonging to three families, Togaviridae, Flaviviridae and Phenuiviridae were reported. Three viruses (Chikungunya virus, Mayaro virus, Mwinilunga virus) were reported among the togaviruses whilst four (dengue virus, West Nile virus, yellow fever virus, Zika virus) were among the flavivirus and only one virus, Rift Valley fever virus, was reported in the Phenuiviridae family. The majority of these mosquito-borne viruses were reported in Western and North-Western provinces. Aedes and Culex species were the main mosquito-borne viral vectors reported. Farming, fishing, movement of people and rain patterns were among factors associated with mosquito-borne viral infection in Zambia. Better diagnostic methods, such as the use of molecular tools, to detect the viruses in potential vectors, humans, and animals, including the recognition of arboviral risk zones and how the viruses circulate, are important for improved surveillance and design of effective prevention and control measures.
• Complete Genome Sequence of a Veterinary Pseudomonas aeruginosa Isolate, Pa12.

  Keisuke Nakamura, Jumpei Fujiki, Takaaki Furusawa, Tomohiro Nakamura, Satoshi Gondaira, Michihito Sasaki, Masaru Usui, Hidetoshi Higuchi, Hirofumi Sawa, Yutaka Tamura, Hidetomo Iwano

  Microbiology resource announcements 10 (26) e0039821  2021/07 [Refereed]
   

  Pseudomonas aeruginosa causes various opportunistic infections in animals. Here, we report the complete genome sequence of P. aeruginosa strain Pa12, a fluoroquinolone-resistant isolate from a canine skin lesion. To expand the molecular antimicrobial characteristics of the isolate, the whole Pa12 genome was sequenced and assembled via long- and short-read platforms.
• RIG-I triggers a signaling-abortive anti-SARS-CoV-2 defense in human lung cells

  Taisho Yamada, Seiichi Sato, Yuki Sotoyama, Yasuko Orba, Hirofumi Sawa, Hajime Yamauchi, Michihito Sasaki, Akinori Takaoka

  Nature Immunology 22 (7) 820 - 828 1529-2908 2021/07 [Refereed]
   

  Efficient immune responses against viral infection are determined by sufficient activation of nucleic acid sensor-mediated innate immunity1,2. Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains an ongoing global pandemic. It is an urgent challenge to clarify the innate recognition mechanism to control this virus. Here we show that retinoic acid-inducible gene-I (RIG-I) sufficiently restrains SARS-CoV-2 replication in human lung cells in a type I/III interferon (IFN)-independent manner. RIG-I recognizes the 3' untranslated region of the SARS-CoV-2 RNA genome via the helicase domains, but not the C-terminal domain. This new mode of RIG-I recognition does not stimulate its ATPase, thereby aborting the activation of the conventional mitochondrial antiviral-signaling protein-dependent pathways, which is in accordance with lack of cytokine induction. Nevertheless, the interaction of RIG-I with the viral genome directly abrogates viral RNA-dependent RNA polymerase mediation of the first step of replication. Consistently, genetic ablation of RIG-I allows lung cells to produce viral particles that expressed the viral spike protein. By contrast, the anti-SARS-CoV-2 activity was restored by all-trans retinoic acid treatment through upregulation of RIG-I protein expression in primary lung cells derived from patients with chronic obstructive pulmonary disease. Thus, our findings demonstrate the distinctive role of RIG-I as a restraining factor in the early phase of SARS-CoV-2 infection in human lung cells.
• Screening of tick-borne pathogens in argasid ticks in Zambia: Expansion of the geographic distribution of Rickettsia lusitaniae and Rickettsia hoogstraalii and detection of putative novel Anaplasma species.

  Yongjin Qiu, Martin Simuunza, Masahiro Kajihara, Herman Chambaro, Hayato Harima, Yoshiki Eto, Edgar Simulundu, David Squarre, Shiho Torii, Ayato Takada, Bernard Mudenda Hang'ombe, Hirofumi Sawa, Chihiro Sugimoto, Ryo Nakao

  Ticks and tick-borne diseases 12 (4) 101720 - 101720 2021/07 [Refereed]
   

  Ticks (Ixodidae and Argasidae) are important arthropod vectors of various pathogens that cause human and animal infectious diseases. Many previously published studies on tick-borne pathogens focused on those transmitted by ixodid ticks. Although there are increasing reports of viral pathogens associated with argasid ticks, information on bacterial pathogens they transmit is scarce. The aim of this molecular study was to detect and characterize Rickettsia and Anaplasmataceae in three different argasid tick species, Ornithodoros faini, Ornithodoros moubata, and Argas walkerae collected in Zambia. Rickettsia hoogstraalii and Rickettsia lusitaniae were detected in 77 % (77/100) of Ar. walkerae and 10 % (5/50) of O. faini, respectively. All O. moubata pool samples (n = 124) were negative for rickettsial infections. Anaplasmataceae were detected in 63 % (63/100) of Ar. walkerae and in 82.2 % (102/124) of O. moubata pools, but not in O. faini. Phylogenetic analysis based on the concatenated sequences of 16S rRNA and groEL genes revealed that Anaplasma spp. detected in the present study were distinct from previously validated Anaplasma species, indicating that the current knowledge on the diversity and vector range of Anaplasma spp. is incomplete. Our findings highlight new geographical records of R. lusitaniae and R. hoogstraalii and confirm that the wide geographic distribution of these species includes the African continent. The data presented here increase our knowledge on argasid tick-borne bacteria and contribute toward understanding their epidemiology.
• Serological Evidence of Filovirus Infection in Nonhuman Primates in Zambia.

  Katendi Changula, Edgar Simulundu, Boniface Pongombo Lombe, Eri Nakayama, Hiroko Miyamoto, Yuji Takahashi, Hirofumi Sawa, Chuma Simukonda, Bernard M Hang'ombe, Ayato Takada

  Viruses 13 (7) 2021/06/30 [Refereed]
   

  Ebolaviruses and marburgviruses are filoviruses that are known to cause severe hemorrhagic fever in humans and nonhuman primates (NHPs). While some bat species are suspected to be natural reservoirs of these filoviruses, wild NHPs often act as intermediate hosts for viral transmission to humans. Using an enzyme-linked immunosorbent assay, we screened two NHP species, wild baboons and vervet monkeys captured in Zambia, for their serum IgG antibodies specific to the envelope glycoproteins of filoviruses. From 243 samples tested, 39 NHPs (16%) were found to be seropositive either for ebolaviruses or marburgviruses with endpoint antibody titers ranging from 100 to 25,600. Interestingly, antibodies reactive to Reston virus, which is found only in Asia, were detected in both NHP species. There was a significant difference in the seropositivity for the marburgvirus antigen between the two NHP species, with baboons having a higher positive rate. These results suggest that wild NHPs in Zambia might be nonlethally exposed to these filoviruses, and this emphasizes the need for continuous monitoring of filovirus infection in wild animals to better understand the ecology of filoviruses and to assess potential risks of outbreaks in humans in previously nonendemic countries.
• Safety enhancement of a genetically modified live rabies vaccine strain by introducing an attenuating Leu residue at position 333 in the glycoprotein.

  Naoto Ito, Takuya Okamoto, Michihito Sasaki, Shoya Miyamoto, Tatsuki Takahashi, Fumiki Izumi, Maho Inukai, Supasiri Jarusombuti, Kazuma Okada, Kento Nakagawa, Yuji Fujii, Shoko Nishiyama, Tatsunori Masatani, Hirofumi Sawa, Makoto Sugiyama

  Vaccine 39 (28) 3777 - 3784 2021/06/23 [Refereed]
   

  To improve the safety of genetically modified live rabies vaccine strains, most studies have utilized an attenuating Arg-to-Glu mutation at position 333 in the glycoprotein (G333), which is responsible for attenuation of the live vaccine strain SAG2. The Glu residue requires two nucleotide substitutions to revert to pathogenic Arg, thus significantly lowering the probability of pathogenic reversion caused by the Glu-to-Arg mutation at G333. However, only one nucleotide substitution is sufficient to convert the Glu residue to another pathogenic residue, Lys, and thereby to cause pathogenic reversion. This indicates a potential safety problem of SAG2 and the live vaccine candidates attenuated by Glu at G333. In this study, aiming to solve this problem, we examined the utility of a Leu residue, which requires two nucleotide substitutions to be both Arg and Lys, as an attenuating mutation at G333. Using a reverse genetics system of the live vaccine strain ERA, we generated ERA-G333Leu by introducing an Arg-to-Leu mutation at G333. Similar to ERA-G333Glu, which is attenuated by an Arg-to-Glu mutation at G333, ERA-G333Leu did not cause obvious clinical signs in 6-week-old mice after intracerebral inoculation. Importantly, after 10 passages in suckling mouse brains, ERA-G333Glu acquired a pathogenic Lys or Arg at G333 and a high level of lethality in mice, whereas ERA-G333Leu retained the attenuating Leu at G333 and only showed a modest level of virulence probably caused by a mutation at G194. In addition, ERA-G333Leu and ERA-G333Glu induced neutralizing antibody response and protective immunity in mice with similar efficiencies. The results demonstrate that, compared to ERA-G333Glu, ERA-G333Leu is more stably attenuated, also indicating the high utility of a Leu residue as an attenuating mutation at G333 in the development of live rabies vaccine strains with a high level of safety.
• Molecular Detection and Genotyping of Coxiella-Like Endosymbionts in Ticks Collected from Animals and Vegetation in Zambia.

  Toshiya Kobayashi, Elisha Chatanga, Yongjin Qiu, Martin Simuunza, Masahiro Kajihara, Bernard Mudenda Hang'ombe, Yoshiki Eto, Ngonda Saasa, Akina Mori-Kajihara, Edgar Simulundu, Ayato Takada, Hirofumi Sawa, Ken Katakura, Nariaki Nonaka, Ryo Nakao

  Pathogens (Basel, Switzerland) 10 (6) 2021/06/21 [Refereed]
   

  Ticks are obligate ectoparasites as they require to feed on their host blood during some or all stages of their life cycle. In addition to the pathogens that ticks harbor and transmit to vertebrate hosts, they also harbor other seemingly nonpathogenic microorganisms including nutritional mutualistic symbionts. Tick nutritional mutualistic symbionts play important roles in the physiology of the host ticks as they are involved in tick reproduction and growth through the supply of B vitamins as well as in pathogen maintenance and propagation. Coxiella-like endosymbionts (CLEs) are the most widespread endosymbionts exclusively reported in ticks. Although CLEs have been investigated in ticks in other parts of the world, there is no report of their investigation in ticks in Zambia. To investigate the occurrence of CLEs, their maintenance, and association with host ticks in Zambia, 175 ticks belonging to six genera, namely Amblyomma, Argas, Haemaphysalis, Hyalomma, Ornithodoros, and Rhipicephalus, were screened for CLEs, followed by characterization of CLEs by multi-locus sequence typing of the five Coxiella housekeeping genes (dnaK, groEL, rpoB, 16S rRNA, and 23S rRNA). The results showed that 45.7% (n = 80) were positive for CLEs. The comparison of the tick 16S rDNA phylogenetic tree with that of the CLEs concatenated sequences showed that there was a strong correlation between the topology of the trees. The results suggest that most of the CLEs have evolved within tick species, supporting the vertical transmission phenomenon. However, the negative results for CLE in some ticks warrants further investigations of other endosymbionts that the ticks in Zambia may also harbor.
• Immunization Coverage and Antibody Retention against Rabies in Domestic Dogs in Lusaka District, Zambia

  Chiho Kaneko, Michihito Sasaki, Ryosuke Omori, Ryo Nakao, Chikako Kataoka-Nakamura, Ladslav Moonga, Joseph Ndebe, Walter Muleya, Edgar Simulundu, Bernard M. Hang’ombe, George Dautu, Masahiro Kajihara, Akina Mori-Kajihara, Yongjin Qiu, Naoto Ito, Herman M. Chambaro, Chihiro Sugimoto, Hideaki Higashi, Ayato Takada, Hirofumi Sawa, Aaron S. Mweene, Norikazu Isoda

  Pathogens 10 (6) 738 - 738 2021/06/11 [Refereed]
   

  Rabies remains endemic in Zambia. Despite conducting canine vaccinations in Lusaka district, the vaccination coverage and actual seropositivity in the dog population in Lusaka district are rarely evaluated. This study estimated the seropositivity-based immunization coverage in the owned dog population in Lusaka district using the expanded program on immunization cluster survey method. The time-series trend of neutralizing antibodies against rabies in vaccinated dogs was also evaluated. Of 366 dogs in 200 dog-owning households in Lusaka district, blood samples were collected successfully from 251 dogs. In the sampled dogs, 42.2% (106/251) had an antibody titer ≥0.5 IU/mL. When the 115 dogs whose blood was not collected were assumed to be seronegative, the minimum immunization coverage in Lusaka district’s owned dog population was estimated at 29.0% (95% confidence interval: 22.4–35.5). It was also found that a single vaccination with certified vaccines is capable of inducing protective levels of antibodies. In contrast, higher antibody titers were observed in multiple-vaccinated dogs than in single-vaccinated dogs, coupled with the observation of a decline in antibody titer over time. These results suggest the importance of continuous booster immunization to maintain herd immunity and provide useful information to plan mass vaccination against rabies in Zambia.
• Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Zambia.

  Masahiro Kajihara, Martin Simuunza, Ngonda Saasa, George Dautu, Akina Mori-Kajihara, Yongjin Qiu, Ryo Nakao, Yoshiki Eto, Hayato Furumoto, Bernard M Hang'ombe, Yasuko Orba, Hirofumi Sawa, Edgar Simulundu, Shuetsu Fukushi, Shigeru Morikawa, Masayuki Saijo, Jiro Arikawa, Swithine Kabilika, Mwaka Monze, Victor Mukonka, Aaron Mweene, Ayato Takada, Kumiko Yoshimatsu

  PLoS neglected tropical diseases 15 (6) e0009452  2021/06 [Refereed]
   

  Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis with a high case fatality rate in humans. Although the disease is widely found in Africa, Europe, and Asia, the distribution and genetic diversity of CCHF virus (CCHFV) are poorly understood in African countries. To assess the risks of CCHF in Zambia, where CCHF has never been reported, epidemiologic studies in cattle and ticks were conducted. Through an indirect immunofluorescence assay, CCHFV nucleoprotein-specific serum IgG was detected in 8.4% (88/1,047) of cattle. Among 290 Hyalomma ticks, the principal vector of CCHFV, the viral genome was detected in 11 ticks. Phylogenetic analyses of the CCHFV S and M genome segments revealed that one of the detected viruses was a genetic reassortant between African and Asian strains. This study provides compelling evidence for the presence of CCHFV in Zambia and its transmission to vertebrate hosts.
• First Molecular Detection of Coxiella burnetii in Beef Cattle in West Java, Indonesia.

  Elok Puspita Rini, Michihito Sasaki, Dwi Astuti, Vetnizah Juniantito, I Wayan Teguh Wibawan, Hirofumi Sawa, Agus Setiyono

  Japanese journal of infectious diseases 75 (1) 83 - 85 2021/04/30 [Refereed]
   

  Coxiella burnetii (C. burnetii) is a bacterial agent causing Q fever which is widespread all over the world. Livestock such as cattle, goat, and sheep are the main sources of infection for this disease. Infection of C. burnetii causes abortion of livestock, resulting in economic damage. Q fever is zoonotic disease and potential public health hazard. To date, little is known about the infection of C. burnetii in livestock in Indonesia. The objective of this research is to screen the genome of C. burnetii bacteria in beef cattle in West Java, Indonesia. Organ tissue samples were collected from cattle slaughtered in slaughterhouses, West Java. C. burnetii genome was detected from cattle samples in all three samplings area by nested PCR (nPCR) targeting com1 gene of C. burnetii. Sequencing analysis of 16S rRNA gene revealed the amplicons showed 99.9% nucleotide identity to C. burnetii strains Heizberg, 1843, 2574, 701CbB1, and 14160-001. Our results indicate that the infection of C. burnetii occurs in Indonesian beef cattles and highlight the risk of exposure to C. burnetii infection in human.
• Domestic dog demographics and estimates of canine vaccination coverage in a rural area of Zambia for the elimination of rabies

  Chiho Kaneko, Ryosuke Omori, Michihito Sasaki, Chikako Kataoka-Nakamura, Edgar Simulundu, Walter Muleya, Ladslav Moonga, Joseph Ndebe, Bernard M. Hang’ombe, George Dautu, Yongjin Qiu, Ryo Nakao, Masahiro Kajihara, Akina Mori-Kajihara, Herman M. Chambaro, Hideaki Higashi, Chihiro Sugimoto, Hirofumi Sawa, Aaron S. Mweene, Ayato Takada, Norikazu Isoda

  PLOS Neglected Tropical Diseases 15 (4) e0009222 - e0009222 2021/04/28 [Refereed]
   

  An estimated 75% or more of the human rabies cases in Africa occur in rural settings, which underscores the importance of rabies control in these areas. Understanding dog demographics can help design strategies for rabies control and plan and conduct canine mass vaccination campaigns effectively in African countries. A cross-sectional survey was conducted to investigate domestic dog demographics in Kalambabakali, in the rural Mazabuka District of Zambia. The population of ownerless dogs and the total achievable vaccination coverage among the total dog population was estimated using the capture-recapture-based Bayesian model by conducting a canine mass vaccination campaign. This study revealed that 29% of the domestic dog population was under one year old, and 57.7% of those were under three months old and thus were not eligible for the canine rabies vaccination in Zambia. The population growth was estimated at 15% per annum based on the cross-sectional household survey. The population of ownerless dogs was estimated to be small, with an ownerless-to-owned-dog ratio of 0.01–0.06 in the target zones. The achieved overall vaccination coverage from the first mass vaccination was estimated 19.8–51.6%. This low coverage was principally attributed to the owners’ lack of information, unavailability, and dog-handling difficulties. The follow-up mass vaccination campaign achieved an overall coverage of 54.8–76.2%. This paper indicates the potential for controlling canine rabies through mass vaccination in rural Zambia. Rabies education and responsible dog ownership are required to achieve high and sustainable vaccination coverage. Our findings also propose including puppies below three months old in the target population for rabies vaccination and emphasize that securing an annual enforcement of canine mass vaccination that reaches 70% coverage in the dog population is necessary to maintain protective herd immunity.
• First report of Mycobacterium bovis in wild chacma baboons (Papio ursinus) at the human-wildlife interface area in Zambia.

  David Squarre, Joseph Chizimu, Chie Nakajima, John B Muma, Bernard M Hang'ombe, Edgar Simulundu, Wizaso Mwasinga, Jackson Katampi, Paul Fandamu, Victor Mukonka, Yasuhiko Suzuki, Hirofumi Sawa, Hetron M Munang'andu, Griffin Shanungu, Herman M Chambaro, Musso Munyeme

  Transboundary and emerging diseases 69 (3) 1659 - 1662 2021/04/26 [Refereed]
   

  Mycobacterium bovis (M. bovis) causes tuberculosis in mammals and is a major public health threat worldwide. While M. bovis has been reported in humans, domestic and wild ruminants at the human-wildlife-livestock interface area in Zambia, there is paucity of information on the role of primates as reservoir hosts. We screened seven wild chacma baboons (Papio ursinus) for tuberculosis at the human-wildlife interface area in Lochinvar National Park in the Kafue Flats, Zambia. Following necropsy, lung tissue and associated lymph nodes with tuberculous-like lesions collected from four adult male baboons were prepared for Mycobacterium culture. The isolates were initially typed using the Mycobacterium tuberculosis complex-discrimination multiplex PCR assay and further characterized by spoligotyping and 26-loci MIRU-VNTR. Mycobacteria were isolated from all four animals and identified as M. bovis by PCR. On Spoligotyping, all isolates belonged to SB 0120 spoligotype, which is similar to what was previously reported in humans, cattle and Kafue lechwe antelopes in Kafue Flats ecosystem. Furthermore, on MIRU-VNTR typing, the baboon isolates clustered with cattle and Kafue lechwe isolates from the same catchment area. This finding intimates probable cross-species transmission of M. bovis in the Kafue Flats ecosystem. Due to the close interaction of baboons and humans at interface areas in Zambia, our results have potential implications for public health. Equally, this finding raises concerns for conservation.
• Combinatorial approach with mass spectrometry and lectin microarray dissected glycoproteomic features of virion-derived spike protein of SARS-CoV-2

  Takahiro Hiono, Azusa Tomioka, Hiroyuki Kaji, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Atsushi Kuno

  2021/04/12 [Refereed]
   

  The COVID-19 pandemic caused by the novel coronavirus, SARS-CoV-2, has a global impact on public health. Since glycosylation of the viral envelope glycoproteins is known to be deeply associated with their immunogenicity, intensive studies on the glycans of its major glycoprotein, S protein, have been conducted. Nevertheless, the detailed site-specific glycan compositions of virion-associated S protein have not yet been clarified. Here, we conducted intensive glycoproteomic analyses of SARS-CoV-2 S protein using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarray. Using our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, we identified 1,759 site-specific glycan compositions. The most frequent was HexNAc:Hex:Fuc:NeuAc:NeuGc = 6:6:1:0:0, suggesting a tri-antennary N-glycan terminating with LacNAc and having bisecting GlcNAc and a core fucose, which was found in 20 of 22 glycosylated sites. The subsequent lectin microarray analysis emphasized intensive outer arm fucosylation of glycans, which efficiently complemented the glycoproteomic features. The present results illustrate the high-resolution glycoproteomic features of SARS-CoV-2 S protein and significantly contribute to vaccine design, as well as the understanding of viral protein synthesis.
• Host serine proteases TMPRSS2 and TMPRSS11D mediate proteolytic activation and trypsin-independent infection in group A rotaviruses.

  Michihito Sasaki, Yukari Itakura, Mai Kishimoto, Koshiro Tabata, Kentaro Uemura, Naoto Ito, Makoto Sugiyama, Christida E Wastika, Yasuko Orba, Hirofumi Sawa

  Journal of virology 95 (11) 2021/03/24 [Refereed]
   

  Group A rotaviruses (RVAs) are representative enteric virus species and major causes of diarrhea in humans and animals. The RVA virion is a triple-layered particle, and the outermost layer consists of the glycoprotein VP7 and spike protein VP4. To increase the infectivity of RVA, VP4 is proteolytically cleaved into VP5* and VP8* subunits by trypsin; and these subunits form a rigid spike structure on the virion surface. In this study, we investigated the growth of RVAs in cells transduced with type II transmembrane serine proteases (TTSPs), which cleave fusion proteins and promote infection by respiratory viruses, such as influenza viruses, paramyxoviruses, and coronaviruses. We identified TMPRSS2 and TMPRSS11D as host TTSPs that mediate trypsin-independent and multi-cycle infection by human and animal RVA strains. In vitro cleavage assays revealed that recombinant TMPRSS11D cleaved RVA VP4. We also found that TMPRSS2 and TMPRSS11D promote the infectious entry of immature RVA virions, but they could not activate nascent progeny virions in the late phase of infection. This observation differed from the TTSP-mediated activation process of paramyxoviruses, revealing the existence of virus species-specific activation processes in TTSPs. Our study provides new insights into the interaction between RVAs and host factors, and TTSP-transduced cells offer potential advantages for RVA research and development.ImportanceProteolytic cleavage of the viral VP4 protein is essential for virion maturation and infectivity in group A rotaviruses (RVAs). In cell culture, RVAs are propagated in culture medium supplemented with the exogenous protease trypsin, which cleaves VP4 and induces the maturation of progeny RVA virions. In this study, we demonstrated that the host proteases TMPRSS2 and TMPRSS11D mediate the trypsin-independent infection and growth of RVA. Our data revealed that the proteolytic activation of RVAs by TMPRSS2 and TMPRSS11D occurs at the viral entry step. Because TMPRSS2 and TMPRSS11D gene expression induced similar or higher levels of RVA growth as trypsin-supplemented culture, this approach offers potential advantages for RVA research and development.
• MRC5 cells engineered to express ACE2 serve as a model system for the discovery of antivirals targeting SARS-CoV-2.

  Kentaro Uemura, Michihito Sasaki, Takao Sanaki, Shinsuke Toba, Yoshimasa Takahashi, Yasuko Orba, William W Hall, Katsumi Maenaka, Hirofumi Sawa, Akihiko Sato

  Scientific reports 11 (1) 5376 - 5376 2021/03/08 [Refereed]
   

  Although the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in a worldwide pandemic, there are currently no virus-specific drugs that are fully effective against SARS-CoV-2. Only a limited number of human-derived cells are capable of supporting SARS-CoV-2 replication and the infectivity of SARS-CoV-2 in these cells remains poor. In contrast, monkey-derived Vero cells are highly susceptibility to infection with SARS-CoV-2, although they are not suitable for the study of antiviral effects by small molecules due to their limited capacity to metabolize drugs compared to human-derived cells. In this study, our goal was to generate a virus-susceptible human cell line that would be useful for the identification and testing of candidate drugs. Towards this end, we stably transfected human lung-derived MRC5 cells with a lentiviral vector encoding angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2. Our results revealed that SARS-CoV-2 replicates efficiently in MRC5/ACE2 cells. Furthermore, viral RNA replication and progeny virus production were significantly reduced in response to administration of the replication inhibitor, remdesivir, in MRC5/ACE2 cells compared with Vero cells. We conclude that the MRC5/ACE2 cells will be important in developing specific anti-viral therapeutics and will assist in vaccine development to combat SARS-CoV-2 infections.
• Prevalence and genetic diversity of Shibuyunji virus, a novel tick-borne phlebovirus identified in Zambia.

  Edgar Simulundu, Saidon Mbambara, Herman M Chambaro, Karen Sichibalo, Masahiro Kajihara, King S Nalubamba, Hirofumi Sawa, Ayato Takada, Katendi Changula, Simbarashe Chitanga

  Archives of virology 166 (3) 915 - 919 2021/03 [Refereed]
   

  Tick-borne pathogens are an emerging public health threat worldwide. However, information on tick-borne viruses is scanty in sub-Saharan Africa. Here, by RT-PCR, 363 ticks (Amblyomma, Hyalomma and Rhipicephalus) in the Namwala and Livingstone districts of Zambia were screened for tick-borne phleboviruses (TBPVs). TBPVs (L gene) were detected in 19 (5.2%) Rhipicephalus ticks in Namwala. All the detected TBPVs were Shibuyunji viruses. Phylogenetically, they were closely related to American dog tick phlebovirus. This study highlights the possible role of Rhipicephalus ticks as the main host of Shibuyunji virus and suggests that these viruses may be present outside the area where they were initially discovered.
• Mastomys natalensis is a possible natural rodent reservoir for encephalomyocarditis virus.

  Mai Kishimoto, Bernard M Hang'ombe, William W Hall, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki

  The Journal of general virology 102 (3) 2021/03 [Refereed]
   

  Encephalomyocarditis virus (EMCV) infects a wide range of hosts and can cause encephalitis, myocarditis, reproductive disorders and diabetes mellitus in selected mammalian species. As for humans, EMCV infection seems to occur by the contact with animals and can cause febrile illnesses in some infected patients. Here we isolated EMCV strain ZM12/14 from a natal multimammate mouse (Mastomys natalensis: M. natalensis) in Zambia. Pairwise sequence similarity of the ZM12/14 P1 region consisting of antigenic capsid proteins showed the highest similarity of nucleotide (80.7 %) and amino acid (96.2%) sequence with EMCV serotype 1 (EMCV-1). Phylogenetic analysis revealed that ZM12/14 clustered into EMCV-1 at the P1 and P3 regions but segregated from known EMCV strains at the P2 region, suggesting a unique evolutionary history. Reverse transcription PCR (RT-PCR) screening and neutralizing antibody assays for EMCV were performed using collected tissues and serum from various rodents (n=179) captured in different areas in Zambia. We detected the EMCV genome in 19 M. natalensis (19/179=10.6 %) and neutralizing antibody for EMCV in 33 M. natalensis (33/179=18.4 %). However, we did not detect either the genome or neutralizing antibody in other rodent species. High neutralizing antibody litres (≧320) were observed in both RT-PCR-negative and -positive animals. Inoculation of ZM12/14 caused asymptomatic persistent infection in BALB/c mice with high antibody titres and high viral loads in some organs, consistent with the above epidemiological results. This study is the first report of the isolation of EMCV in Zambia, suggesting that M. natalensis may play a role as a natural reservoir of infection.
• Diverse mosquito-specific flaviviruses in the Bolivian Amazon basin.

  Yasuko Orba, Keita Matsuno, Ryo Nakao, Kirill Kryukov, Yumi Saito, Fumihiko Kawamori, Ariel Loza Vega, Tokiko Watanabe, Tadashi Maemura, Michihito Sasaki, William W Hall, Roy A Hall, Juan Antonio Pereira, So Nakagawa, Hirofumi Sawa

  The Journal of general virology 102 (3) 2021/03 

  The genus Flavivirus includes a range of mosquito-specific viruses in addition to well-known medically important arboviruses. Isolation and comprehensive genomic analyses of viruses in mosquitoes collected in Bolivia resulted in the identification of three novel flavivirus species. Psorophora flavivirus (PSFV) was isolated from Psorophora albigenu. The coding sequence of the PSFV polyprotein shares 60 % identity with that of the Aedes-associated lineage II insect-specific flavivirus (ISF), Marisma virus. Isolated PSFV replicates in both Aedes albopictus- and Aedes aegypti-derived cells, but not in mammalian Vero or BHK-21 cell lines. Two other flaviviruses, Ochlerotatus scapularis flavivirus (OSFV) and Mansonia flavivirus (MAFV), which were identified from Ochlerotatus scapularis and Mansonia titillans, respectively, group with the classical lineage I ISFs. The protein coding sequences of these viruses share only 60 and 40 % identity with the most closely related of known lineage I ISFs, including Xishuangbanna aedes flavivirus and Sabethes flavivirus, respectively. Phylogenetic analysis suggests that MAFV is clearly distinct from the groups of the current known Culicinae-associated lineage I ISFs. Interestingly, the predicted amino acid sequence of the MAFV capsid protein is approximately two times longer than that of any of the other known flaviviruses. Our results indicate that flaviviruses with distinct features can be found at the edge of the Bolivian Amazon basin at sites that are also home to dense populations of human-biting mosquitoes.
• An African tick flavivirus forming an independent clade exhibits unique exoribonuclease-resistant RNA structures in the genomic 3'-untranslated region.

  Hayato Harima, Yasuko Orba, Shiho Torii, Yongjin Qiu, Masahiro Kajihara, Yoshiki Eto, Naoya Matsuta, Bernard M Hang'ombe, Yuki Eshita, Kentaro Uemura, Keita Matsuno, Michihito Sasaki, Kentaro Yoshii, Ryo Nakao, William W Hall, Ayato Takada, Takashi Abe, Michael T Wolfinger, Martin Simuunza, Hirofumi Sawa

  Scientific reports 11 (1) 4883 - 4883 2021/03/01 [Refereed]
   

  Tick-borne flaviviruses (TBFVs) infect mammalian hosts through tick bites and can cause various serious illnesses, such as encephalitis and hemorrhagic fevers, both in humans and animals. Despite their importance to public health, there is limited epidemiological information on TBFV infection in Africa. Herein, we report that a novel flavivirus, Mpulungu flavivirus (MPFV), was discovered in a Rhipicephalus muhsamae tick in Zambia. MPFV was found to be genetically related to Ngoye virus detected in ticks in Senegal, and these viruses formed a unique lineage in the genus Flavivirus. Analyses of dinucleotide contents of flaviviruses indicated that MPFV was similar to those of other TBFVs with a typical vertebrate genome signature, suggesting that MPFV may infect vertebrate hosts. Bioinformatic analyses of the secondary structures in the 3'-untranslated regions (UTRs) revealed that MPFV exhibited unique exoribonuclease-resistant RNA (xrRNA) structures. Utilizing biochemical approaches, we clarified that two xrRNA structures of MPFV in the 3'-UTR could prevent exoribonuclease activity. In summary, our findings provide new information regarding the geographical distribution of TBFV and xrRNA structures in the 3'-UTR of flaviviruses.
• TMPRSS11D and TMPRSS13 Activate the SARS-CoV-2 Spike Protein.

  Mai Kishimoto, Kentaro Uemura, Takao Sanaki, Akihiko Sato, William W Hall, Hiroaki Kariwa, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki

  Viruses 13 (3) 2021/02/28 [Refereed]
   

  Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) utilizes host proteases, including a plasma membrane-associated transmembrane protease, serine 2 (TMPRSS2) to cleave and activate the virus spike protein to facilitate cellular entry. Although TMPRSS2 is a well-characterized type II transmembrane serine protease (TTSP), the role of other TTSPs on the replication of SARS-CoV-2 remains to be elucidated. Here, we have screened 12 TTSPs using human angiotensin-converting enzyme 2-expressing HEK293T (293T-ACE2) cells and Vero E6 cells and demonstrated that exogenous expression of TMPRSS11D and TMPRSS13 enhanced cellular uptake and subsequent replication of SARS-CoV-2. In addition, SARS-CoV-1 and SARS-CoV-2 share the same TTSPs in the viral entry process. Our study demonstrates the impact of host TTSPs on infection of SARS-CoV-2, which may have implications for cell and tissue tropism, for pathogenicity, and potentially for vaccine development.
• Detection of B.1.351 SARS-CoV-2 Variant Strain - Zambia, December 2020.

  Mulenga Mwenda, Ngonda Saasa, Nyambe Sinyange, George Busby, Peter J Chipimo, Jason Hendry, Otridah Kapona, Samuel Yingst, Jonas Z Hines, Peter Minchella, Edgar Simulundu, Katendi Changula, King Shimumbo Nalubamba, Hirofumi Sawa, Masahiro Kajihara, Junya Yamagishi, Muzala Kapin'a, Nathan Kapata, Sombo Fwoloshi, Paul Zulu, Lloyd B Mulenga, Simon Agolory, Victor Mukonka, Daniel J Bridges

  MMWR. Morbidity and mortality weekly report 70 (8) 280 - 282 2021/02/26 [Refereed]
   

  The first laboratory-confirmed cases of coronavirus disease 2019 (COVID-19), the illness caused by SARS-CoV-2, in Zambia were detected in March 2020 (1). Beginning in July, the number of confirmed cases began to increase rapidly, first peaking during July-August, and then declining in September and October (Figure). After 3 months of relatively low case counts, COVID-19 cases began rapidly rising throughout the country in mid-December. On December 18, 2020, South Africa published the genome of a SARS-CoV-2 variant strain with several mutations that affect the spike protein (2). The variant included a mutation (N501Y) associated with increased transmissibility.†,§ SARS-CoV-2 lineages with this mutation have rapidly expanded geographically.¶,** The variant strain (PANGO [Phylogenetic Assignment of Named Global Outbreak] lineage B.1.351††) was first detected in the Eastern Cape Province of South Africa from specimens collected in early August, spread within South Africa, and appears to have displaced the majority of other SARS-CoV-2 lineages circulating in that country (2). As of January 10, 2021, eight countries had reported cases with the B.1.351 variant. In Zambia, the average number of daily confirmed COVID-19 cases increased 16-fold, from 44 cases during December 1-10 to 700 during January 1-10, after detection of the B.1.351 variant in specimens collected during December 16-23. Zambia is a southern African country that shares substantial commerce and tourism linkages with South Africa, which might have contributed to the transmission of the B.1.351 variant between the two countries.
• Inactivation of sars-cov-2 by povidone-iodine products: Implications for effective mouth rinsing and gargling

  Hiroaki Kariwa, Hirofumi Sawa, Shintaro Kobayashi

  Japanese Journal of Veterinary Research 69 (3) 183 - 187 0047-1917 2021 

  Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is transmitted mainly via droplets and contact. The implementation of infection control measures is important to reduce the number of COVID-19 cases. Thus, the ability of several povidone-iodine (PVP-I) products to inactivate SARS-CoV-2 was evaluated based on their in vitro inactivation efficacy. PVP-I solutions such as Isodine Gargle® (ethical and consumer products), Isodine Gargle C®, and Isodine Nodo Fresh® for 30 or 60 s decreased the viral infectivity level from 2-4 × 106 TCID50/ml to below the detectable level (> 99.9% reduction). Our results indicate that the use of Isodine® mouthwash and gargle products is an effective infection control measure against SARS-CoV-2.
• First COVID-19 case in Zambia - Comparative phylogenomic analyses of SARS-CoV-2 detected in African countries.

  Edgar Simulundu, Francis Mupeta, Pascalina Chanda-Kapata, Ngonda Saasa, Katendi Changula, Walter Muleya, Simbarashe Chitanga, Miniva Mwanza, Paul Simusika, Herman Chambaro, Benjamin Mubemba, Masahiro Kajihara, Duncan Chanda, Lloyd Mulenga, Sombo Fwoloshi, Aaron Lunda Shibemba, Fred Kapaya, Paul Zulu, Kunda Musonda, Mwaka Monze, Nyambe Sinyange, Mazyanga L Mazaba, Muzala Kapin'a, Peter J Chipimo, Raymond Hamoonga, Davie Simwaba, William Ngosa, Albertina N Morales, Nkomba Kayeyi, John Tembo, Mathew Bates, Yasuko Orba, Hirofumi Sawa, Ayato Takada, King S Nalubamba, Kennedy Malama, Victor Mukonka, Alimuddin Zumla, Nathan Kapata

  International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 102 455 - 459 2021/01 [Refereed]
   

  Since its first discovery in December 2019 in Wuhan, China, COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread rapidly worldwide. While African countries were relatively spared initially, the initial low incidence of COVID-19 cases was not sustained for long due to continuing travel links between China, Europe and Africa. In preparation, Zambia had applied a multisectoral national epidemic disease surveillance and response system resulting in the identification of the first case within 48 h of the individual entering the country by air travel from a trip to France. Contact tracing showed that SARS-CoV-2 infection was contained within the patient's household, with no further spread to attending health care workers or community members. Phylogenomic analysis of the patient's SARS-CoV-2 strain showed that it belonged to lineage B.1.1., sharing the last common ancestor with SARS-CoV-2 strains recovered from South Africa. At the African continental level, our analysis showed that B.1 and B.1.1 lineages appear to be predominant in Africa. Whole genome sequence analysis should be part of all surveillance and case detection activities in order to monitor the origin and evolution of SARS-CoV-2 lineages across Africa.
• SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells.

  Michihito Sasaki, Kentaro Uemura, Akihiko Sato, Shinsuke Toba, Takao Sanaki, Katsumi Maenaka, William W Hall, Yasuko Orba, Hirofumi Sawa

  PLoS pathogens 17 (1) e1009233  2021/01 [Refereed]
   

  The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.
• The occurrence of coxiella burnetiid nfection in local goat in malang, East Java, Indonesia: A first report

  Handayu Untari, Agus Setiyono, Ekowati Handharyani, Masdiana C. Padaga, Dwi Astuti, Michihito Sasaki, Hirofumi Sawa

  Veterinary Practitioner 21 (2) 488 - 493 0972-4036 2020/12/01 

  Q fever is worldwide zoonotic disease caused by Coxiella burnetii with ruminant as the main reservoir for human infection. These bacteria could persist in environment for months due to their capability to form spore like-bacteria. Human could be infected by Coxiella burnetii through inhalation or ingestion. Report of Coxiella burnetii infection in ruminant in East Java, Indonesia is still scanty. Previous research revealed that this bacteria were detected in various type of ruminant in some region in Indonesia but not in Malang region in East Java which known as one of the highest ruminant population. Visceral samples consist of heart, lung, spleen, kidney and liver from 30 cows and 78 goats were taken from abattoirs in Malang East Java. Nested PCR with OMP primers used to screen the existence of Coxiella burnetii and showed that only 10 (8 lungs, 1 liver, and 1 kidney) samples from 78 goat (12.8%) were detected positive. Further examination for all positive samples by nested PCR using 16S rRNA gene noted that sequence from local goat in Malang have close relation with Coxiella burnetii isolate sequence from various host and country. This result indicated that Coxiella burnetii detected in local goat in Malang, East Java, Indonesia have molecular similarity with some established strain before, but it still need further examination.
• Identification of quinolone derivatives as effective anti-Dengue virus agents.

  Haruaki Nobori, Kentaro Uemura, Shinsuke Toba, Takao Sanaki, Takao Shishido, William W Hall, Yasuko Orba, Hirofumi Sawa, Akihiko Sato

  Antiviral research 184 104969 - 104969 2020/12 [Refereed]
   

  Dengue virus (DENV) infection is one of the most important infectious diseases in tropical and subtropical regions around the world. Previously, we performed an initial phenotypic screening of 7000 compounds using DENV type 2 (DENV2)-infected BHK-21 cells to identify small molecules which could inhibit virus replication. In this study, we describe two novel compounds with anti-DENV2 activity, tentatively named Compound-X and Compound-Y. Both compounds possess a quinolone skeleton, and the EC50s of Compound-X and Compound-Y against DENV2 were 3.9 μM and 9.2 μM, respectively. Based on a DENV replicon assay, it was suggested that these compounds have anti-DENV2 activity by inhibition of a step in virus replication. Furthermore, using mutational analysis we obtained compounds-resistant to DENV2 infection and identified a mutation, V130A in the NS5 methyltransferase (MTase) domain. However, these compounds did not inhibit MTase activity. In addition, incorporation of an additional NS1 N246D mutation with the NS5 V130A mutation in DENV2 resulted in recovery of viral replication and a further reduction of the sensitivity to the quinolone compounds by an unknown mechanism. Therefore further investigations are required to clarify the antiviral mechanisms of these quinolone compounds.
• Whole-Genome Sequence of Fluoroquinolone-Resistant Escherichia coli HUE1, Isolated in Hokkaido, Japan.

  Montgomery Munby, Jumpei Fujiki, Kotaro Aoki, Chika Kawaguchi, Keisuke Nakamura, Tomohiro Nakamura, Michihito Sasaki, Toyotaka Sato, Masaru Usui, Hirofumi Sawa, Shin-Ichi Yokota, Yutaka Tamura, Hidetomo Iwano

  Microbiology resource announcements 9 (46) 2020/11/12 [Refereed]
   

  We report the complete genome sequence of Escherichia coli strain HUE1, isolated from the urinary catheter of a female patient, showing fluoroquinolone resistance without quinolone resistance-determining region mutations. To facilitate the exploration of the molecular characteristics of HUE1, the whole genome was sequenced using long- and short-read platforms.
• Susceptibility of Pseudomonas aeruginosa veterinary isolates to Pbunavirus PB1-like phages.

  Jumpei Fujiki, Takaaki Furusawa, Montgomery Munby, Chika Kawaguchi, Yumie Matsuda, Yusei Shiokura, Keisuke Nakamura, Tomohiro Nakamura, Michihito Sasaki, Masaru Usui, Tomohito Iwasaki, Satoshi Gondaira, Hidetoshi Higuchi, Hirofumi Sawa, Yutaka Tamura, Hidetomo Iwano

  Microbiology and immunology 64 (11) 778 - 782 2020/11 [Refereed]
   

  In recent years, antimicrobial-resistant Pseudomonas aeruginosa strains have increased in the veterinary field. Therefore, phage therapy has received significant attention as an approach for overcoming antimicrobial resistance. In this context, we isolated and characterized four Pseudomonas bacteriophages. Phylogenetic analysis showed that the isolated phages are novel Myoviridae Pbunavirus PB1-like phages with ØR12 belonging to a different clade compared with the other three. These phages had distinct lytic activity against 22 P. aeruginosa veterinary isolates. The phage cocktail composed from the PB1-like phages clearly inhibited the occurrence of the phage-resistant variant, suggesting that these phages could be useful in phage therapy.
• The Lethal(2)-Essential-for-Life [L(2)EFL] Gene Family Modulates Dengue Virus Infection in Aedes aegypti.

  Lucky R Runtuwene, Shuichi Kawashima, Victor D Pijoh, Josef S B Tuda, Kyoko Hayashida, Junya Yamagishi, Chihiro Sugimoto, Shoko Nishiyama, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Tomohiko Takasaki, Anthony A James, Takashi Kobayashi, Yuki Eshita

  International journal of molecular sciences 21 (20) 2020/10/12 [Refereed]
   

  Efforts to determine the mosquito genes that affect dengue virus replication have identified a number of candidates that positively or negatively modify amplification in the invertebrate host. We used deep sequencing to compare the differential transcript abundances in Aedes aegypti 14 days post dengue infection to those of uninfected A. aegypti. The gene lethal(2)-essential-for-life [l(2)efl], which encodes a member of the heat shock 20 protein (HSP20) family, was upregulated following dengue virus type 2 (DENV-2) infection in vivo. The transcripts of this gene did not exhibit differential accumulation in mosquitoes exposed to insecticides or pollutants. The induction and overexpression of l(2)efl gene products using poly(I:C) resulted in decreased DENV-2 replication in the cell line. In contrast, the RNAi-mediated suppression of l(2)efl gene products resulted in enhanced DENV-2 replication, but this enhancement occurred only if multiple l(2)efl genes were suppressed. l(2)efl homologs induce the phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the fruit fly Drosophila melanogaster, and we confirmed this finding in the cell line. However, the mechanism by which l(2)efl phosphorylates eIF2α remains unclear. We conclude that l(2)efl encodes a potential anti-dengue protein in the vector mosquito.
• 1940年代に日本国内において分離された狂犬病ウイルス株のゲノム解析

  犬飼 真秀, 高橋 龍樹, 佐々木 道仁, 藤井 祐至, Jarusombuti Supasiri, 西山 祥子, 澤 洋文, 杉山 誠, 伊藤 直人

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 163回 262 - 262 1347-8621 2020/10
• Discoveries of Exoribonuclease-Resistant Structures of Insect-Specific Flaviviruses Isolated in Zambia.

  Christida E Wastika, Hayato Harima, Michihito Sasaki, Bernard M Hang'ombe, Yuki Eshita, Yongjin Qiu, William W Hall, Michael T Wolfinger, Hirofumi Sawa, Yasuko Orba

  Viruses 12 (9) 2020/09/11 [Refereed]
   

  To monitor the arthropod-borne virus transmission in mosquitoes, we have attempted both to detect and isolate viruses from 3304 wild-caught female mosquitoes in the Livingstone (Southern Province) and Mongu (Western Province) regions in Zambia in 2017. A pan-flavivirus RT-PCR assay was performed to identify flavivirus genomes in total RNA extracted from mosquito lysates, followed by virus isolation and full genome sequence analysis using next-generation sequencing and rapid amplification of cDNA ends. We isolated a newly identified Barkedji virus (BJV Zambia) (10,899 nt) and a novel flavivirus, tentatively termed Barkedji-like virus (BJLV) (10,885 nt) from Culex spp. mosquitoes which shared 96% and 75% nucleotide identity with BJV which has been isolated in Israel, respectively. These viruses could replicate in C6/36 cells but not in mammalian and avian cell lines. In parallel, a comparative genomics screening was conducted to study evolutionary traits of the 5'- and 3'-untranslated regions (UTRs) of isolated viruses. Bioinformatic analyses of the secondary structures in the UTRs of both viruses revealed that the 5'-UTRs exhibit canonical stem-loop structures, while the 3'-UTRs contain structural homologs to exoribonuclease-resistant RNAs (xrRNAs), SL-III, dumbbell, and terminal stem-loop (3'SL) structures. The function of predicted xrRNA structures to stop RNA degradation by Xrn1 exoribonuclease was further proved by the in vitro Xrn1 resistance assay.
• Avian Influenza Viruses Detected in Birds in Sub-Saharan Africa: A Systematic Review.

  Annie Kalonda, Ngonda Saasa, Panji Nkhoma, Masahiro Kajihara, Hirofumi Sawa, Ayato Takada, Edgar Simulundu

  Viruses 12 (9) 2020/09/07 

  In the recent past, sub-Saharan Africa has not escaped the devastating effects of avian influenza virus (AIV) in poultry and wild birds. This systematic review describes the prevalence, spatiotemporal distribution, and virus subtypes detected in domestic and wild birds for the past two decades (2000-2019). We collected data from three electronic databases, PubMed, SpringerLink electronic journals and African Journals Online, using the Preferred Reporting Items for Systematic reviews and Meta-Analyses protocol. A total of 1656 articles were reviewed, from which 68 were selected. An overall prevalence of 3.0% AIV in birds was observed. The prevalence varied between regions and ranged from 1.1% to 7.1%. The Kruskal-Wallis and Wilcoxon signed-rank sum test showed no significant difference in the prevalence of AIV across regions, χ2(3) = 5.237, p = 0.1553 and seasons, T = 820, z = -1.244, p = 0.2136. Nineteen hemagglutinin/neuraminidase subtype combinations were detected during the reviewed period, with southern Africa recording more diverse AIV subtypes than other regions. The most detected subtype was H5N1, followed by H9N2, H5N2, H5N8 and H6N2. Whilst these predominant subtypes were mostly detected in domestic poultry, H1N6, H3N6, H4N6, H4N8, H9N1 and H11N9 were exclusively detected in wild birds. Meanwhile, H5N1, H5N2 and H5N8 were detected in both wild and domestic birds suggesting circulation of these subtypes among wild and domestic birds. Our findings provide critical information on the eco-epidemiology of AIVs that can be used to improve surveillance strategies for the prevention and control of avian influenza in sub-Saharan Africa.
• Co-Circulation of Multiple Serotypes of Bluetongue Virus in Zambia.

  Herman M Chambaro, Michihito Sasaki, Edgar Simulundu, Isaac Silwamba, Yona Sinkala, Gabriel Gonzalez, David Squarre, Paul Fandamu, Caesar H Lubaba, Musso Munyeme, Alikhadio Maseko, Choopa Chimvwele, Liywalii Mataa, Lynnfield E Mooya, Andrew N Mukubesa, Hayato Harima, Kenny L Samui, Hetron M Munang'andu, Martin Simuunza, King S Nalubamba, Yongjin Qiu, Michael J Carr, William W Hall, Yuki Eshita, Hirofumi Sawa, Yasuko Orba

  Viruses 12 (9) 2020/08/31 [Refereed]
   

  Bluetongue (BT) is an arthropod-borne viral disease of ruminants with serious trade and socio-economic implications. Although the disease has been reported in a number of countries in sub-Saharan Africa, there is currently no information on circulating serotypes and disease distribution in Zambia. Following surveillance for BT in domestic and wild ruminants in Zambia, BT virus (BTV) nucleic acid and antibodies were detected in eight of the 10 provinces of the country. About 40% (87/215) of pooled blood samples from cattle and goats were positive for BTV nucleic acid, while one hartebeest pool (1/43) was positive among wildlife samples. Sequence analysis of segment 2 revealed presence of serotypes 3, 5, 7, 12 and 15, with five nucleotypes (B, E, F, G and J) being identified. Segment 10 phylogeny showed Zambian BTV sequences clustering with Western topotype strains from South Africa, intimating likely transboundary spread of BTV in Southern Africa. Interestingly, two Zambian viruses and one isolate from Israel formed a novel clade, which we designated as Western topotype 4. The high seroprevalence (96.2%) in cattle from Lusaka and Central provinces and co-circulation of multiple serotypes showed that BT is widespread, underscoring the need for prevention and control strategies.
• Genetic and Phenotypic Characterization of a Rabies Virus Strain Isolated from a Dog in Tokyo, Japan in the 1940s.

  Tatsuki Takahashi, Maho Inukai, Michihito Sasaki, Madlin Potratz, Supasiri Jarusombuti, Yuji Fujii, Shoko Nishiyama, Stefan Finke, Kentaro Yamada, Hiroki Sakai, Hirofumi Sawa, Akira Nishizono, Makoto Sugiyama, Naoto Ito

  Viruses 12 (9) 2020/08/20 

  The rabies virus strain Komatsugawa (Koma), which was isolated from a dog in Tokyo in the 1940s before eradication of rabies in Japan in 1957, is known as the only existent Japanese field strain (street strain). Although this strain potentially provides a useful model to study rabies pathogenesis, little is known about its genetic and phenotypic properties. Notably, this strain underwent serial passages in rodents after isolation, indicating the possibility that it may have lost biological characteristics as a street strain. In this study, to evaluate the utility of the Koma strain for studying rabies pathogenesis, we examined the genetic properties and in vitro and in vivo phenotypes. Genome-wide genetic analyses showed that, consistent with previous findings from partial sequence analyses, the Koma strain is closely related to a Russian street strain within the Arctic-related phylogenetic clade. Phenotypic examinations in vitro revealed that the Koma strain and the representative street strains are less neurotropic than the laboratory strains. Examination by using a mouse model demonstrated that the Koma strain and the street strains are more neuroinvasive than the laboratory strains. These findings indicate that the Koma strain retains phenotypes similar to those of street strains, and is therefore useful for studying rabies pathogenesis.
• Characterization of mammalian orthoreoviruses isolated from faeces of pigs in Zambia.

  Hayato Harima, Michihito Sasaki, Masahiro Kajihara, Gabriel Gonzalez, Edgar Simulundu, Eugene C Bwalya, Yongjin Qiu, Kosuke Okuya, Mao Isono, Yasuko Orba, Ayato Takada, Bernard M Hang'ombe, Aaron S Mweene, Hirofumi Sawa

  The Journal of general virology 101 (10) 1027 - 1036 2020/07/24 [Refereed][Not invited]
   

  Mammalian orthoreovirus (MRV) has been identified in humans, livestock and wild animals; this wide host range allows individual MRV to transmit into multiple species. Although several interspecies transmission and genetic reassortment events of MRVs among humans, livestock and wildlife have been reported, the genetic diversity and geographic distribution of MRVs in Africa are poorly understood. In this study, we report the first isolation and characterization of MRVs circulating in a pig population in Zambia. In our screening, MRV genomes were detected in 19.7 % (29/147) of faecal samples collected from pigs by reverse transcription PCR. Three infectious MRV strains (MRV-85, MRV-96 and MRV-117) were successfully isolated, and their complete genomes were sequenced. Recombination analyses based on the complete genome sequences of the isolated MRVs demonstrated that MRV-96 shared the S3 segment with a different MRV isolated from bats, and that the L1 and M3 segments of MRV-117 originated from bat and human MRVs, respectively. Our results suggest that the isolated MRVs emerged through genetic reassortment events with interspecies transmission. Given the lack of information regarding MRVs in Africa, further surveillance of MRVs circulating among humans, domestic animals and wildlife is required to assess potential risk for humans and animals.
• Isolation of Candidatus Bartonella rousetti and Other Bat-associated Bartonellae from Bats and Their Flies in Zambia.

  Yongjin Qiu, Masahiro Kajihara, Ryo Nakao, Evans Mulenga, Hayato Harima, Bernard Mudenda Hang'ombe, Yoshiki Eto, Katendi Changula, Daniel Mwizabi, Hirofumi Sawa, Hideaki Higashi, Aaron Mweene, Ayato Takada, Martin Simuunza, Chihiro Sugimoto

  Pathogens (Basel, Switzerland) 9 (6) 2020/06/13 [Refereed][Not invited]
   

  Bat-associated bartonellae, including Bartonella mayotimonensis and Candidatus Bartonella rousetti, were recently identified as emerging and potential zoonotic agents, respectively. However, there is no report of bat-associated bartonellae in Zambia. Thus, we aimed to isolate and characterize Bartonella spp. from bats and bat flies captured in Zambia by culturing and PCR. Overall, Bartonella spp. were isolated from six out of 36 bats (16.7%), while Bartonella DNA was detected in nine out of 19 bat flies (47.3%). Subsequent characterization using a sequence of five different genes revealed that three isolates obtained from Egyptian fruit bats (Rousettus aegyptiacus) were Ca. B. rousetti. The isolates obtained from insectivorous bats (Macronycteris vittatus) were divided into two previously unclassified bat-associated bartonellae. A phylogenetic analysis of the six genotypes of Bartonella gltA sequences from nine pathogen-positive bat flies revealed that three genotypes belonged to the same clades as bat-associated bartonellae, including Ca. B. rousetti. The other three genotypes represented arthropod-associated bartonellae, which have previously been isolated only from ectoparasites. We demonstrated that Ca. B. rousetti is maintained between bats (R. aegyptiacus) and bat flies in Zambia. Continuous surveillance of Bartonella spp. in bats and serological surveys in humans in Africa are warranted to evaluate the public health importance of bat-associated bartonellae.
• Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle.

  Shiho Torii, Yasuko Orba, Michihito Sasaki, Koshiro Tabata, Yuji Wada, Michael Carr, Jody Hobson-Peters, Roy A Hall, Ayato Takada, Takasuke Fukuhara, Yoshiharu Matsuura, William W Hall, Hirofumi Sawa

  The Journal of biological chemistry 295 (23) 7941 - 7957 2020/06/05 [Refereed][Not invited]
   

  Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins.
• Evidence for exposure of asymptomatic domestic pigs to African Swine fever virus during an inter-epidemic period in Zambia.

  Herman M Chambaro, Michihito Sasaki, Yona Sinkala, Gabriel Gonzalez, David Squarre, Paul Fandamu, Caesar Lubaba, Liywalii Mataa, Misheck Shawa, Kabemba E Mwape, Sarah Gabriël, Mwelwa Chembensofu, Michael J Carr, William W Hall, Yongjin Qiu, Masahiro Kajihara, Ayato Takada, Yasuko Orba, Edgar Simulundu, Hirofumi Sawa

  Transboundary and emerging diseases 67 (6) 2741 - 2752 2020/05/20 [Refereed][Not invited]
   

  African swine fever (ASF) causes persistent outbreaks in endemic and non-endemic regions in Zambia. However, the epidemiology of the disease is poorly understood, particularly during the inter-epidemic periods. We conducted surveillance for ASF in asymptomatic domestic pigs and soft ticks in selected Zambian provinces. Whilst serum samples (n=1,134) were collected from crossbred pigs from all study sites between 2014 and 2017, whole blood (n=300) was collected from both crossbred and indigenous pigs in Eastern Province (EP) in 2017. Soft ticks were collected from Mosi-oa-Tunya National Park in Southern Province (SP) in 2019. Sera were screened for antibodies against ASF by ELISA while genome detection in whole blood and soft ticks was conducted by PCR. Ticks were identified morphologically and by phylogenetic analysis of the 16S rRNA gene. Seroprevalence was highest in EP (50.9%, 95% CI [47.0 - 54.9]) compared to significantly lower rates in SP (2.9%, 95% CI [1.6 - 5.1]). No antibodies to ASFV were detected in Lusaka Province. In EP, the prevalence of ASFV genome was 11.7% (35/300), significantly higher (OR = 6.2, 95% CI [2.4 - 16.6]) in indigenous pigs compared to crossbred pigs. The pooled prevalence of ASFV genome in ticks was 11.0%, 95% CI [8.5-13.9]. Free-range husbandry system was the only factor that was significantly associated with seropositive (p < 0.0001, OR = 39.3) and PCR positive results (p < 0.001, OR = 5.7). Phylogenetically, based on the p72 gene, ASFV from Ornithodoros moubata ticks detected in this study belonged to genotype I, but they separated into two distinct clusters. Besides confirming ASF endemicity in EP and the presence of ASFV-infected ticks in SP, these results provide evidence for exposure of domestic pigs to ASFV in non-endemic regions during the inter-epidemic period.
• Amino acid 159 of the envelope protein affects viral replication and T-cell infiltration by West Nile virus in intracranial infection.

  Shintaro Kobayashi, Chisato Kaneko, Ryoko Kawakami, Rie Hasebe, Hirofumi Sawa, Kentaro Yoshii, Hiroaki Kariwa

  Scientific reports 10 (1) 7168 - 7168 2020/04/28 [Refereed][Not invited]
   

  West Nile virus (WNV) is an important cause of viral encephalitis in birds and animals, including humans. Amino acid 159 of the envelope (E) protein is reportedly implicated in the different levels of neurovirulence in mice infected with WNV NY99 or Eg101. We investigated the role of amino acid 159 of the E protein in the pathogenesis of WNV infection. We produced recombinant WNV with the structural proteins of the NY99 or Eg101 strain (NY-WT or EgCME-WT) and mutant viruses with substitutions of amino acid 159 of the E protein (NY-E-V159I or EgCME-E-I159V). The NY-WT and NY-E-V159I or EgCME-WT and EgCME-E-I159V titers in culture supernatant were similar. The mortality rate and viral titer in the brains of mice inoculated intraperitoneally with NY-WT or NY-E-V159I were also similar. In contrast, the mortality rate and viral titer in the brains of mice inoculated intracranially with EgCME-E-I159V were significantly higher than those of mice inoculated with EgCME-WT. The numbers of CD3-positive and CD8-positive T cells were greater in brains inoculated with EgCME-E-I159V than in those inoculated with EgCME-WT. Therefore, amino acid 159 of the E protein modulates the pathogenicity of WNV by affecting viral replication and T-cell infiltration in the brain.
• Interferon lambda rs368234815 ΔG/ΔG is associated with higher CD4+:CD8+ T-cell ratio in treated HIV-1 infection.

  Inês T Freitas, Willard Tinago, Hirofumi Sawa, Julie McAndrews, Brenda Doak, Charlotte Prior-Fuller, Gerard Sheehan, John S Lambert, Eavan Muldoon, Aoife G Cotter, William W Hall, Patrick W G Mallon, Michael J Carr

  AIDS research and therapy 17 (1) 13 - 13 2020/04/15 

  BACKGROUND: The objectives of this study were to investigate the relationships between polymorphisms at the interferon lambda (IFNL) locus and CD4+:CD8+ ratio normalisation in people living with HIV (PLWH) on effective antiretroviral therapy (ART); and to examine whether these polymorphisms influence the composition of T lymphocyte compartments in long-term treated HIV-1 infection. METHODS: A cross-sectional study in PLWH enrolled into the Mater Immunology study. We performed IFNL genotyping on stored samples and evaluated the association of IFNL single-nucleotide polymorphisms (rs368234815 and rs12979860) with CD4+:CD8+ ratio normalization (> 1) and expanded CD4+ and CD8+ T-cell subsets; CD45RO+CD62L+ (central-memory), CD45RO+ CD62L-(effector-memory) and CD45RO-CD62L+ (naïve), using logistic and linear regression models, respectively. RESULTS: 190 ambulatory PLWH recruited to the main study, 143 were included in the analysis (38 had no stored DNA and 9 no T-lymphocyte subpopulation). Of 143 included, the median age (IQR) was 45(39-48) years, 64% were male and 66% were of Caucasian ethnicity. Heterosexual-contact (36%), injecting drug-use (33%) and men who have sex with men (24%) were the most presented HIV-transmission risk groups. The majority of subjects (90.2%) were on ART with 79% of the cohort having an undetectable HIV-RNA (< 40 copies/ml) and the time since ART initiation was 7.5 (3.7-10.4) year. rs368234815 and rs12979860 displayed similar allelic frequencies, with minor alleles ΔG and T representing 39% and 42%, respectively, of circulating alleles. rs368234815 ΔG/ΔG minor homozygotes were significantly associated with increased odds for attaining a normalised CD4+:CD8+ ratio compared to rs368234815 T/T major homozygotes in PLWH virologically suppressed on effective ART (OR = 3.11; 95% CI [1.01:9.56]). rs368234815 ΔG/ΔG homozygosity was also significantly associated with lower levels of CD4+ effector memory T-cells (regression coefficient: - 7.1%, p = 0.04) and CD8+ naïve T-cell subsets were significantly higher in HIV-1 mono-infected PLWH with rs368234815 ΔG/ΔG (regression coefficient: + 7.2%, p = 0.04). CONCLUSIONS: In virally-suppressed, long-term ART-treated PLWH, rs368234815 ΔG/ΔG homozygotes were more likely to have attained normalisation of their CD4+:CD8+ ratio, displayed lower CD4+ effector memory and higher naive CD8+ T-cells. Further studies are needed to replicate our findings in other, larger and more diverse cohorts and to determine the impact of IFNL genetic-variation on CD4+:CD8+ ratio normalisation and clinical outcomes in PLWH.
• West Nile Virus in Farmed Crocodiles, Zambia, 2019.

  Edgar Simulundu, Kunda Ndashe, Herman M Chambaro, David Squarre, Paul Michael Reilly, Simbarashe Chitanga, Katendi Changula, Andrew N Mukubesa, Joseph Ndebe, John Tembo, Nathan Kapata, Matthew Bates, Yona Sinkala, Bernard M Hang'ombe, King S Nalubamba, Masahiro Kajihara, Michihito Sasaki, Yasuko Orba, Ayato Takada, Hirofumi Sawa

  Emerging infectious diseases 26 (4) 811 - 814 2020/04 [Refereed]
   

  We detected West Nile virus (WNV) nucleic acid in crocodiles (Crocodylus niloticus) in Zambia. Phylogenetically, the virus belonged to lineage 1a, which is predominant in the Northern Hemisphere. These data provide evidence that WNV is circulating in crocodiles in Africa and increases the risk for animal and human transmission.
• Genetic and Biological Diversity of Porcine Sapeloviruses Prevailing in Zambia.

  Hayato Harima, Masahiro Kajihara, Edgar Simulundu, Eugene Bwalya, Yongjin Qiu, Mao Isono, Kosuke Okuya, Gabriel Gonzalez, Junya Yamagishi, Bernard M Hang'ombe, Hirofumi Sawa, Aaron S Mweene, Ayato Takada

  Viruses 12 (2) 2020/02/05 [Refereed][Not invited]
   

  Porcine sapelovirus (PSV) has been detected worldwide in pig populations. Although PSV causes various symptoms such as encephalomyelitis, diarrhea, and pneumonia in pigs, the economic impact of PSV infection remains to be determined. However, information on the distribution and genetic diversity of PSV is quite limited, particularly in Africa. In this study, we investigated the prevalence of PSV infection in Zambia and characterized the isolated PSVs genetically and biologically. We screened 147 fecal samples collected in 2018 and found that the prevalences of PSV infection in suckling pigs and fattening pigs were high (36.2% and 94.0%, respectively). Phylogenetic analyses revealed that the Zambian PSVs were divided into three different lineages (Lineages 1-3) in the clade consisting of Chinese strains. The Zambian PSVs belonging to Lineages 2 and 3 replicated more efficiently than those belonging to Lineage 1 in Vero E6 and BHK cells. Bioinformatic analyses revealed that genetic recombination events had occurred and the recombination breakpoints were located in the L and 2A genes. Our results indicated that at least two biologically distinct PSVs could be circulating in the Zambian pig population and that genetic recombination played a role in the evolution of PSVs.
• Detection of novel orthoreovirus genomes in shrew (Crocidura hirta) and fruit bat (Rousettus aegyptiacus).

  Hayato Harima, Michihito Sasaki, Masahiro Kajihara, Akina Mori-Kajihara, Bernard M Hang'ombe, Katendi Changula, Yasuko Orba, Hirohito Ogawa, Martin Simuunza, Reiko Yoshida, Aaron Mweene, Ayato Takada, Hirofumi Sawa

  The Journal of veterinary medical science 82 (2) 162 - 167 2020/02/04 [Refereed][Not invited]
   

  Orthoreoviruses have been indentified in several mammals, however, there is no information about orthoreoviruses in shrews. In this study, we screened wild animals in Zambia, including shrews, rodents, and bats for the detection of orthoreoviruses. Two orthoreovirus RNA genomes were detected from a shrew intestinal-contents (1/24) and a bat colon (1/96) sample by reverse-transcription (RT)-PCR targeting the RNA-dependent RNA polymerase gene of orthoreoviruses. Phylogenetic analyses revealed that each of the identified orthoreoviruses formed a distinct branch among members of the Orthoreovirus genus. This is the first report that shrews are susceptible to orthoreovirus infection. Our results suggest the existence of undiscovered orthoreoviruses in shrews and provide important information about the genetic diversity of orthoreoviruses.
• 北海道における新規オルソナイロウイルス(エゾウイルス:Yezo virus)によるマダニ媒介性急性発熱性疾患の発見

  児玉 文宏, 枝川 峻二, 永坂 敦, 松野 啓太, 好井 健太朗, 澤 洋文, 山岸 彩沙, 古澤 弥, 山口 亮, 矢野 公一, 山口 宏樹, 後藤 明子, 駒込 理佳, 三好 正浩, 伊東 拓也, 小山内 佑太, 角 千春, 堀田 明豊, 前田 健, 安藤 秀二, 西條 政幸

  病原微生物検出情報月報 国立感染症研究所 41 (1) 11 - 13 0915-5813 2020/01
• 生体防御 Fitness costによって誘導されるP.aeruginosa変異株のファージ感受性トレードオフ

  藤木 純平, マンビ・モンゴメリ, 中村 暢宏, 権平 智, 佐々木 道仁, 臼井 優, 樋口 豪紀, 澤 洋文, 田村 豊, 岩野 英知

  日本細菌学雑誌 日本細菌学会 75 (1) 57 - 57 0021-4930 2020/01
• West Nile virus capsid protein inhibits autophagy by AMP-activated protein kinase degradation in neurological disease development.

  Shintaro Kobayashi, Kentaro Yoshii, Wallaya Phongphaew, Memi Muto, Minato Hirano, Yasuko Orba, Hirofumi Sawa, Hiroaki Kariwa

  PLoS pathogens 16 (1) e1008238  2020/01 [Refereed][Not invited]
   

  West Nile virus (WNV) belongs to the Flaviviridae family and has emerged as a significant cause of viral encephalitis in birds and animals including humans. WNV replication directly induces neuronal injury, followed by neuronal cell death. We previously showed that accumulation of ubiquitinated protein aggregates was involved in neuronal cell death in the WNV-infected mouse brain. In this study, we attempted to elucidate the mechanisms of the accumulation of protein aggregates in the WNV-infected cells. To identify the viral factor inducing the accumulation of ubiquitinated proteins, intracellular accumulation of ubiquitinated proteins was examined in the cells expressing the viral protein. Expression of capsid (C) protein induced the accumulation, while mutations at residues L51 and A52 in C protein abrogated the accumulation. Wild-type (WT) or mutant WNV in which mutations were introduced into the residues was inoculated into human neuroblastoma cells. The expression levels of LC3-II, an autophagy-related protein, and AMP-activated protein kinase (AMPK), an autophagy inducer, were reduced in the cells infected with WT WNV, while the reduction was not observed in the cells infected with WNV with the mutations in C protein. Similarly, ubiquitination and degradation of AMPK were only observed in the cells infected with WT WNV. In the cells expressing C protein, AMPK was co-precipitated with C protein and mutations in L51 and A52 reduced the interaction. Although the viral replication was not affected, the accumulation of ubiquitinated proteins in brain and neurological symptoms were attenuated in the mouse inoculated with WNV with the mutations in C protein as compared with that with WT WNV. Taken together, ubiquitination and degradation of AMPK by C protein resulted in the inhibition of autophagy and the accumulation of protein aggregates, which contributes to the development of neurological disease.
• Inhibition of dengue virus infection by 1-stearoyl-2-arachidonoyl-phosphatidylinositol in vitro.

  Takao Sanaki, Masato Wakabayashi, Takeshi Yoshioka, Ryu Yoshida, Takao Shishido, William W Hall, Hirofumi Sawa, Akihiko Sato

  FASEB journal : official publication of the Federation of American Societies for Experimental Biology 33 (12) 13866 - 13881 2019/12 

  Dengue fever is an acute febrile infectious disease caused by dengue virus (DENV). Despite the significant public health concerns posed by DENV, there are currently no effective anti-DENV therapeutic agents. To develop such drugs, a better understanding of the detailed mechanisms of DENV infection is needed. Both lipid metabolism and lipid synthesis are activated in DENV-infected cells, so we used lipid screening to identify potential antiviral lipid molecules. We identified 1-stearoyl-2-arachidonoyl-phosphatidylinositol (SAPI), which is the most abundant endogenous phosphatidylinositol (PI) molecular species, as an anti-DENV lipid molecule. SAPI suppressed the cytopathic effects induced by DENV2 infection as well as the replication of all DENV serotypes without inhibiting the entry of DENV2 into host cells. However, no other PI molecular species or PI metabolites, including lysophosphatidylinositols and phosphoinositides, displayed anti-DENV2 activity. Furthermore, SAPI suppressed the production of DENV2 infection-induced cytokines and chemokines, including C-C motif chemokine ligand (CCL)5, CCL20, C-X-C chemokine ligand 8, IL-6, and IFN-β. SAPI also suppressed the TNF-α production induced by LPS stimulation in macrophage cells differentiated from THP-1 cells. Our results demonstrated that SAPI is an endogenous inhibitor of DENV and modulated inflammatory responses in DENV2-infected cells, at least in part via TLR 4.-Sanaki, T., Wakabayashi, M., Yoshioka, T., Yoshida, R., Shishido, T., Hall, W. W., Sawa, H., Sato, A. Inhibition of dengue virus infection by 1-stearoyl-2-arachidonoyl-phosphatidylinositol in vitro.
• Neo-virology: The raison d'etre of viruses

  Tokiko Watanabe, Nobuhiro Suzuki, Keizo Tomonaga, Hirofumi Sawa, Yoshiharu Matsuura, Yasushi Kawaguchi, Hideki Takahashi, Keizo Nagasaki, Yoshihiro Kawaoka

  Virus Research 274 197751 - 197751 0168-1702 2019/12 [Refereed]
   

  Given that approximately 10 virus particles exist on Earth and all of them are parasitic in living organisms, it is not hard to imagine how virus infection might affect the physiology of hosts and their ecosystems. However, traditional virology research tends to focus on viral pathogenicity or the individual pathogenic viruses; hence, the significance of viruses and viral-mediated processes in the global ecosystem has been poorly understood. To identify the previously unrecognized “raison d'etre of viruses” in nature, we established a research community, designated as the ‘Neo-virology’ consortium. In this consortium, we define a virus as a component of the global ecosystem and our aim is to elucidate its key roles in host organisms, that is, the intra-host ecosystem. 31
• Seroprevalence of Rift Valley fever in cattle of smallholder farmers in Kwilu Province in the Democratic Republic of Congo.

  Abdel-Amir Dib Halawi, Ngonda Saasa, Boniface Lombe Pongombo, Masahiro Kajihara, Herman Moses Chambaro, Mutambel Hity, Hirofumi Sawa, Ayato Takada, Aaron S Mweene, Luamba Lua Nsembo, Edgar Simulundu

  Tropical animal health and production 51 (8) 2619 - 2627 2019/11 

  Rift Valley fever (RVF) is a zoonotic mosquito-borne disease caused by RVF virus (RVFV) that causes abortions and high mortalities in livestock and is also associated with acute and fatal disease in humans. In the Democratic Republic of Congo (DRC), information on the epidemiology of RVF is limited, particularly among cattle reared by smallholder farmers. This cross-sectional study was conducted to investigate the seroprevalence of RVF in cattle raised by smallholder farmers in Kwilu Province of DRC, which has not yet reported an RVF epidemic. A total of 677 cattle sera were collected from four territories and tested for anti-RVFV antibodies using immunofluorescent assay and enzyme-linked immunosorbent assay. The overall seroprevalence of anti-RVFV IgG was 6.5% (44/677) (95% CI 4.81-8.7). There was a statistically significant difference in the seroprevalence among the territories (χ2 = 28.79, p < 0.001). Territory seroprevalences were as follows: Idiofa 14.08% (95% CI 9.78-19.76), Bulungu 4.14% (95% CI 1.83-8.68), Gungu 3.21% (95% CI 1.41-6.78), and Masi-Manimba 1.19% (95% CI 0.06-7.37). Seroprevalence differed significantly among age categories (p = 0.0017) and ecosystem (p < 0.001). The seroprevalence of animals aged between 1 and 2 years was 20.0% (95% CI 8.4-39.13) and was higher than group aged <1 year, between 2 and 3 years, and > 3 years. Forest area (18.92% (95% CI 12.35-27.7)) had higher seropositivity than savannah area (4.06% (95% CI 2.65-6.12)). Sex difference was not significant (χ2 = 0.14, p = 0.704). These findings indicate that cattle in Kwilu Province had been exposed to RVFV, which represents a significant risk for both livestock and human health.
• Genetic diversity of rabies virus in different host species and geographic regions of Zambia and Zimbabwe.

  Muleya W, Chambaro HM, Sasaki M, Gwenhure LF, Mwenechanya R, Kajihara M, Saasa N, Mupila Z, Mori-Kajihara A, Qiu Y, Kangwa E, Mweene A, Namangala B, Takada A, Sawa H

  Virus genes 55 (5) 713 - 719 0920-8569 2019/10 [Refereed][Not invited]
   

  Rabies is endemic in Zambia and Zimbabwe. The previously investigated strains of rabies virus in central Zambia belong to the Africa 1b lineage, with similar circulating virus strains found in the various tested hosts and regions. However, prior work assessed only limited regions and host species. Thus, this study aimed to more comprehensively determine the genetic diversity of rabies virus across regions of Zambia and Zimbabwe. RNA (n = 76) was extracted from positive direct fluorescent antibody test brain tissues from dog, cow, goat, cat, pig, human, and jackal collected from Zambia and Zimbabwe. The amplicons of the nucleoprotein and glycoprotein genes were obtained from all examined samples by nested RT-PCR and subsequently sequenced. A phylogenetic analysis of the N gene confirmed that all the endemic strains of rabies virus in Zambia and Zimbabwe belong to the Africa 1b lineage. The obtained viral gene sequences were phylogenetically divided into two clusters. Cluster II comprised only Zambian strains. In contrast, cluster I comprised both Zambia and Zimbabwe strains, with strains from Zimbabwe forming a distinct lineage from Zambian strains, implying viral genetic divergence due to geographical barriers. However, no evidence of clustering based on host or region was observed, implying the circulation of similar virus strains occurs in different hosts and regions of Zambia and Zimbabwe. The clustering of rabies virus strains from jackals with those from domestic animals provides evidence of similar virus strains circulating in both wildlife and domestic animals, and that the jackal might be one of the potential reservoirs of rabies virus infection. In this study, no strains circulating in Zimbabwe were detected in Zambia.
• Upregulated expression of the antioxidant sestrin 2 identified by transcriptomic analysis of Japanese encephalitis virus-infected SH-SY5Y neuroblastoma cells.

  Carr M, Gonzalez G, Martinelli A, Wastika CE, Ito K, Orba Y, Sasaki M, Hall WW, Sawa H

  Virus genes 55 (5) 630 - 642 0920-8569 2019/10 [Refereed][Not invited]
   

  Japanese encephalitis virus (JEV) exerts a profound burden of viral encephalitis. We have investigated the differentially expressed transcripts in the neuronal transcriptome during JEV infection by RNA sequencing (RNA-Seq) of virus-infected SH-SY5Y human neuroblastoma cells. Gene ontology analysis revealed significant enrichment from two main pathways: endoplasmic reticulum (ER)-nucleus signaling (P value: 5.75E-18; false discovery rate [FDR] 3.11E-15) and the ER unfolded protein response (P value: 7.58E-18; FDR 3.11E-15). qPCR validation showed significant upregulation and differential expression (P < 0.01) of ER stress-signaling transcripts (SESN2, TRIB3, DDIT3, DDIT4, XBP1, and ATF4) at 24 h post-infection for both low (LN) and high (HN) neurovirulence JEV strains. Immunoblot analysis following JEV infection of SH-SY5Y cells showed an increase in levels of SESN2 protein following JEV infection. Similarly, Zika virus (MR766) infection of SH-SY5Y showed a titer-dependent increase in ER stress-signaling transcripts; however, this was absent or diminished for DDIT4 and ATF4, respectively, suggestive of differences in the induction of stress-response transcripts between flaviviruses. Interestingly, SLC7A11 and SLC3A2 mRNA were also both deregulated in JEV-infected SH-SY5Y cells and encode the two constituent subunits of the plasma membrane xCT amino acid antiporter that relieves oxidative stress by export of glutamate and import of cystine. Infection of SH-SY5Y and HEK293T cells by the JEV HN strain Sw/Mie/40/2004 lead to significant upregulation of the SLC7A11 mRNA to levels comparable to DDIT3. Our findings suggest upregulation of antioxidants including SESN2 and, also, the xCT antiporter occurs to counteract the oxidative stress elicited by JEV infection.
• Autopsy findings in the early stage of amyotrophic lateral sclerosis with "dropped head" syndrome.

  Tanikawa S, Tanino M, Wang L, Ishikawa M, Miyazaki M, Tsuda M, Orba Y, Sawa H, Matoba K, Nakamura N, Nagashima K, Hall WW, Tanaka S

  Neuropathology : official journal of the Japanese Society of Neuropathology 39 (5) 374 - 377 0919-6544 2019/10 [Refereed][Not invited]
  
• 狂犬病ウイルスの増殖におけるL蛋白質NPYNE配列の重要性

  牧野 真知子, 伊藤 直人, 中川 賢人, 佐々木 道仁, 高橋 龍樹, 岡田 和真, 西山 祥子, 澤 洋文, 杉山 誠

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 162回 432 - 432 1347-8621 2019/08
• Marburgvirus in Egyptian Fruit Bats, Zambia.

  Kajihara M, Hang'ombe BM, Changula K, Harima H, Isono M, Okuya K, Yoshida R, Mori-Kajihara A, Eto Y, Orba Y, Ogawa H, Qiu Y, Sawa H, Simulundu E, Mwizabi D, Munyeme M, Squarre D, Mukonka V, Mweene A, Takada A

  Emerging infectious diseases 25 (8) 1577 - 1580 1080-6040 2019/08 [Refereed][Not invited]
   

  We detected Marburg virus genome in Egyptian fruit bats (Rousettus aegyptiacus) captured in Zambia in September 2018. The virus was closely related phylogenetically to the viruses that previously caused Marburg outbreaks in the Democratic Republic of the Congo. This finding demonstrates that Zambia is at risk for Marburg virus disease.
• Publisher Correction: Discovery and genetic characterization of diverse smacoviruses in Zambian non-human primates.

  Anindita PD, Sasaki M, Gonzalez G, Phongphaew W, Carr M, Hang'ombe BM, Mweene AS, Ito K, Orba Y, Sawa H

  Scientific reports 9 (1) 8502 - 8502 2019/06 [Refereed][Not invited]
   

  A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
• Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing.

  Hayashida K, Orba Y, Sequeira PC, Sugimoto C, Hall WW, Eshita Y, Suzuki Y, Runtuwene L, Brasil P, Calvet G, Rodrigues CDS, Santos CCD, Mares-Guia MAM, Yamagishi J, Filippis AMB, Sawa H

  PLoS neglected tropical diseases 13 (6) e0007480  1935-2727 2019/06 [Refereed][Not invited]
   

  Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas.
• Serological evidence of Zika virus infection in non-human primates in Zambia.

  Wastika CE, Sasaki M, Yoshii K, Anindita PD, Hang'ombe BM, Mweene AS, Kobayashi S, Kariwa H, Carr MJ, Hall WW, Eshita Y, Orba Y, Sawa H

  Archives of virology 164 (8) 2165 - 2170 0304-8608 2019/06 [Refereed][Not invited]
   

  Zika virus (ZIKV) circulation occurs between non-human primates (NHPs) in a sylvatic transmission cycle. To investigate evidence of flavivirus infection in NHPs in Zambia, we performed a plaque reduction neutralization test (PRNT) to quantify neutralizing antibodies. PRNT revealed that sera from NHPs (African green monkeys and baboons) exhibited neutralizing activity against ZIKV (34.4%; 33/96), whereas a PRNT for yellow fever virus using NHP sera showed no neutralization activity. ZIKV genomic RNA was not detected in splenic tissues from NHPs, suggesting that the presence of anti-ZIKV neutralizing antibodies represented resolved infections. Our evidence suggests that ZIKV is maintained in NHP reservoirs in Zambia.
• Human borreliosis caused by a New World relapsing fever Borrelia-like organism in the Old World.

  Qiu Y, Nakao R, Hang’ombe BM, Sato K, Kajihara M, Kanchela S, Changula K, Eto Y, Ndebe J, Sasaki M, Thu MJ, Takada A, Sawa H, Sugimoto C, Kawabata H

  Clinical Infectious Diseases 69 (1) 107 - 112 2019/06 [Refereed][Not invited]
  
• Discovery and genetic characterization of diverse smacoviruses in Zambian non-human primates.

  Anindita PD, Sasaki M, Gonzalez G, Phongphaew W, Carr M, Hang'ombe BM, Mweene AS, Ito K, Orba Y, Sawa H

  Scientific reports 9 (1) 5045 - 5045 2019/04 [Refereed][Not invited]
   

  The Smacoviridae has recently been classified as a family of small circular single-stranded DNA viruses. An increasing number of smacovirus genomes have been identified exclusively in faecal matter of various vertebrate species and from insect body parts. However, the genetic diversity and host range of smacoviruses remains to be fully elucidated. Herein, we report the genetic characterization of eleven circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses detected in the faeces of Zambian non-human primates. Based on pairwise genome-wide and amino acid identities with reference smacovirus species, ten of the identified CRESS DNA viruses are assigned to the genera Porprismacovirus and Huchismacovirus of the family Smacoviridae, which bidirectionally encode two major open reading frames (ORFs): Rep and capsid protein (CP) characteristic of a type IV genome organization. The remaining unclassified CRESS DNA virus was related to smacoviruses but possessed a genome harbouring a unidirectionally oriented CP and Rep, assigned as a type V genome organization. Moreover, phylogenetic and recombination analyses provided evidence for recombination events encompassing the 3'-end of the Rep ORF in the unclassified CRESS DNA virus. Our findings increase the knowledge of the known genetic diversity of smacoviruses and highlight African non-human primates as carrier animals.
• Molecular characterization and phylogenetic analysis of Trypanosoma spp. isolated from striped leaf-nosed bat (Hipposideros vittatus) in Zambia.

  Qiu Y, Simuunza M, Mwizabi D, Changula K, Harima H, Takada A, Kajihara M, Takadate Y, Nakao R, Kawabata H, Sugimoto C, Mweene A, Sawa H, Eto Y, Mudenda Hang’ombe B, Hayashida K, Yoshida R, Mori-Kajihara A, Ndebe J

  International Journal for Parasitology 9 234 - 238 2019/04 [Refereed][Not invited]
   

  Bat trypanosomes consist of more than 30 trypanosome species from over 70 species of bats. Recent studies suggest that bats play a role in disseminating trypanosomes from African continent to the terrestrial mammals both in the Afrotropic-Palearctic Ecozones and Nearctic Ecozone. However, the diversity, distribution, and evolution of bat trypanosomes are still unclear. To better understand their evolution, more genetic data of bat trypanosomes from a variety of locations are required. During a survey of Borrelia spp. of bats inhabiting a cave in Zambia, we observed flagellate parasites from 5 of 43 hemocultures. Sequence and phylogenetic analyses of the glycosomal glyceraldehyde 3-phosphate dehydrogenase gene (gGAPDH; 572 bp) and the 18S ribosomal RNA gene (18S rRNA gene; 1,079-1,091 bp) revealed that all were Trypanosoma spp. belonged to the Trypanosoma cruzi clade. Three and two of them exhibited the similarity with T. conorhini and T. dionisii, respectively. The present study provides the first genetic data on Trypanosoma spp. of bats inhabiting Zambia.
• A novel combination of prion strain co-occurrence in patients with sporadic Creutzfeldt-Jakob disease.

  Kobayashi A, Iwasaki Y, Takao M, Saito Y, Iwaki T, Qi Z, Torimoto R, Shimazaki T, Munesue Y, Isoda N, Sawa H, Aoshima K, Kimura T, Kondo H, Mohri S, Kitamoto T

  The American journal of pathology 189 (6) 1276 - 1283 0002-9440 2019/03 [Refereed][Not invited]
   

  Six subgroups of sporadic Creutzfeldt-Jakob disease have been identified by distinctive clinicopathologic features, genotype at polymorphic codon 129 [methionine (M)/valine (V)] of the PRNP gene, and type of abnormal prion proteins (type 1 or 2). In addition to the pure subgroups, mixed neuropathologic features and the coexistence of two types of abnormal prion proteins in the same patient also have been reported. Here, we found that a portion of the patients previously diagnosed as MM1 had neuropathologic characteristics of the MM2 thalamic form (ie, neuronal loss of the inferior olivary nucleus of the medulla). Furthermore, coexistence of biochemical features of the MM2 thalamic form also was confirmed in the identified cases. In addition, in transmission experiments using prion protein-humanized mice, the brain material from the identified case showed weak infectivity and generated characteristic abnormal prion proteins in the inoculated mice resembling those after inoculation with brain material of MM2 thalamic form. Taken together, these results show that the co-occurrence of MM1 and MM2 thalamic form is a novel entity of sporadic Creutzfeldt-Jakob disease prion strain co-occurrence. The present study raises the possibility that the co-occurrence of the MM2 thalamic form might have been overlooked so far because of the scarcity of abnormal prion protein accumulation and restricted neuropathology.
• Infection of newly identified phleboviruses in ticks and wild animals in Hokkaido, Japan indicating tick-borne life cycles.

  Shiho Torii, Keita Matsuno, Yongjin Qiu, Akina Mori-Kajihara, Masahiro Kajihara, Ryo Nakao, Naganori Nao, Katsunori Okazaki, Mariko Sashika, Takahiro Hiono, Masatoshi Okamatsu, Yoshihiro Sakoda, Hideki Ebihara, Ayato Takada, Hirofumi Sawa

  Ticks and tick-borne diseases 10 (2) 328 - 335 1877-959X 2019/02 [Refereed][Not invited]
   

  Recent discoveries of tick-borne pathogens have raised public health concerns on tick-borne infectious diseases and emphasize the need to assess potential risks of unrecognized tick-borne pathogens. First, to determine the existence of tick-borne phleboviruses (TBPVs), genetic surveillance of phleboviruses in ticks was conducted mainly in Hokkaido, the northernmost island in Japan from 2013 to 2015. Genes of two TBPVs, previously reported as Mukawa virus (MKWV) and a newly identified relative of MKWV, Kuriyama virus (KURV), were detected and the viruses were isolated from Ixodes persulcatus collected in Hokkaido, but not in I. persulcatus collected from other areas of Japan. These viruses were phylogenetically and antigenically similar to each other. Next, to investigate the infection of MKWV in mammals, serum samples from wildlife captured in Hokkaido from 2007 to 2011 were used for serological screening. Neutralizing antibodies against MKWV were detected in both Yezo-deer (Cervus nippon yesoensis) (2/50) and raccoons (Procyon lotor) (16/64). However, no infectious MKWV was recovered from laboratory mice in experimental infections, though viral RNAs were detected in their tissues. Thus, MKWV and KURV may maintain tick-mammalian life cycles in Hokkaido, suggesting their potential as causative agents of tick-borne diseases in mammals.
• "Integrated diagnosis" of pilocytic astrocytoma: Molecular diagnostic procedure for an unusual case.

  Ishida Y, Tsuda M, Sawamura Y, Fujii K, Murai H, Horiuchi N, Orba Y, Sawa H, Hall WW, Nagashima K, Tanaka S

  Pathology international 68 (12) 694 - 699 1320-5463 2018/12 [Refereed][Not invited]
   

  A 24 year-old female presented with a mass lesion in the right temporal lobe. This case was difficult to diagnose using histological and immunological methods and therefore molecular analyses were applied to provide a definitive diagnosis. The tumor was well-demarcated, partially cystic, and irregularly-enhanced on gadolinium-enhanced T1-weighted magnetic resonance images. Pathologically, a large part of the tumor consisted of cells with fine cytoplasmic processes on a myxoid and mucinous background. Cells formed a microcystic structure around the mucinous tissue. Numerous eosinophilic granular bodies, but not Rosenthal fibers, were present. The solid and compact regions of the tumor were composed of fasciculation of dense fibrous glial tissues and occasional multinucleated giant cells. Tumor cells and their fragmented cytoplasmic processes were positively stained with GFAP, while eosinophilic granular bodies were both positive and negative. Xanthomatous changes were not detected and the reticulin fibers were restricted to vascular tissues. The MIB1 index was scored as approximately 10%. In molecular analyses of BRAF, the KIAA1549-BRAF (K16-B9) fusion gene was detected in all tumor regions, whereas BRAF V600E mutation was not detected by either conventional Sanger sequencing or the Eprobe-PCR method. Based on the results of the molecular analyses, this case was diagnosed as pilocytic astrocytoma.
• Ganglioside synthase knock-out reduces prion disease incubation time in mouse models.

  Kobayashi A, Qi Z, Shimazaki T, Munesue Y, Miyamoto T, Isoda N, Sawa H, Aoshima K, Kimura T, Mohri S, Kitamoto T, Yamashita T, Miyoshi I

  The American journal of pathology 189 (3) 677 - 686 0002-9440 2018/12 [Refereed][Not invited]
   

  Localization of the abnormal and normal isoforms of prion proteins to detergent-resistant membrane microdomains, lipid rafts, is important for the conformational conversion. Lipid rafts are enriched in sialic acid-containing glycosphingolipids (namely, gangliosides). Alteration in the ganglioside composition of lipid rafts can affect the localization of lipid raft-associated proteins. To investigate the role of gangliosides in the pathogenesis of prion diseases, we performed intracerebral transmission study of a scrapie prion strain Chandler and a Gerstmann-Sträussler-Scheinker syndrome prion strain Fukuoka-1 using various knockout mouse strains ablated with ganglioside synthase gene (ie, GD2/GM2 synthase, GD3 synthase, or GM3 synthase). After challenge with the Chandler strain, GD2/GM2 synthase knockout mice showed 20% reduction of incubation time, reduced prion protein deposition in the brain with attenuated glial reactions, and reduced localization of prion proteins to lipid rafts. These results raise the possibility that the gangliosides may have an important role in prion disease pathogenesis by affecting the localization of prion proteins to lipid rafts.
• Identification of group A rotaviruses from Zambian fruit bats provides evidence for long-distance dispersal events in Africa.

  Sasaki M, Kajihara M, Changula K, Mori-Kajihara A, Ogawa H, Hang'ombe BM, Mweene AS, Simuunza M, Yoshida R, Carr M, Orba Y, Takada A, Sawa H

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 63 104 - 109 1567-1348 2018/09 [Refereed][Not invited]
   

  Group A rotavirus (RVA) is a major cause of diarrhea in children worldwide. Although RVA infects many animals, little is known about RVA in bats. The present study investigated the genetic diversity of RVA in Zambian bats. We identified RVA from two straw-colored fruit bats (Eidolon helvum) and an Egyptian fruit bat (Rousettus aegyptiacus), and analyzed the genome sequences of these strains. Genome segments of the RVA strains from Zambian E. helvum showed 97%-99% nucleotide sequence identity with those of other RVA strains from E. helvum in Cameroon, which is 2800 km from the sampling locations. These findings suggest that migratory straw-colored fruit bat species, distributed across sub-Saharan Africa, have the potential to disseminate RVA across long distances. By contrast, the RVA strain from Zambian R. aegyptiacus carried highly divergent NSP2 and NSP4 genes, leading us to propose novel genotypes N21 and E27, respectively. Notably, this RVA strain also shared the same genotype for VP6 and NSP3 with the RVA strains from Zambian E. helvum, suggesting interspecies transmission and genetic reassortment may have occurred between these two bat species in the past. Our study has important implications for RVA dispersal in bat populations, and expands our knowledge of the ecology, diversity and evolutionary relationships of RVA.
• チクングニアウイルスの増殖を制御する宿主因子の解析

  鳥居 志保, 和田 雄二, 佐々木 道仁, Hobson-Peters Jody, Hall Roy A., 大場 靖子, 澤 洋文

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 161回 345 - 345 1347-8621 2018/08
• First isolation of West Nile virus in Zambia from mosquitoes.

  Orba Y, Hang'ombe BM, Mweene AS, Wada Y, Anindita PD, Phongphaew W, Qiu Y, Kajihara M, Mori-Kajihara A, Eto Y, Sasaki M, Hall WW, Eshita Y, Sawa H

  Transboundary and emerging diseases 65 (4) 933 - 938 1865-1674 2018/08 [Refereed][Not invited]
  
• Single amino acid mutation in dengue virus NS4B protein has opposing effects on viral proliferation in mammalian and mosquito cells.

  Fujiki J, Nobori H, Sato A, Sasaki M, Carr M, Hall WW, Orba Y, Sawa H

  Japanese journal of infectious diseases 71 (6) 448 - 454 1344-6304 2018/07 [Refereed][Not invited]
   

  Dengue virus (DENV) has a considerable impact on the global health and is known to cause morbidity and mortality every year. By passaging DENV2 in baby hamster kidney (BHK)-21 cells, we isolated a mutant clone of DENV2 that shows rapid cytopathic effects in BHK-21 cells as compared with that showed by the parent strain. To investigate the relationship between amino acid mutations and proliferation activity of the isolated DENV2 clone, we performed full genome sequencing and identified 3 amino acid mutations in the coding region, the envelope T120K, NS4A M85T, and NS4B G124A. Genetically modified recombinant DENV2 (rDENV2) carrying the NS4A M85T and NS4B G124A mutations produced higher titers of progeny virus in BHK-21, Vero, and Huh-7 cells than in the wild-type (WT) rDENV2. rDENV2 with mutations at NS4A M85T and NS4B G124A failed to produce any plaques in C6/36 mosquito cell lines. Furthermore, rDENV2 possessing only the NS4B G124A mutation showed no plaque production in C6/36 cells but had higher viral titers in Vero and Huh-7 cells than the WT rDENV2 had. Our results clearly showed that the DENV2 NS4B G124A mutation has opposing effects on the virus proliferation in mosquito and certain mammalian cell lines.
• Identification of Compound-B, a novel anti-dengue virus agent targeting the non-structural protein 4A.

  Nobori H, Toba S, Yoshida R, Hall WW, Orba Y, Sawa H, Sato A

  Antiviral research 155 60 - 66 0166-3542 2018/07 [Refereed][Not invited]
   

  Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever/dengue shock syndrome. At present, no antiviral drugs are available for treatment DENV infections. In this study, a screening system based on a DENV-infected cell-based assay identified a novel anti-DENV agent with a benzimidazole skeleton, named Compound-B, which demonstrated antiviral activity specific to four DENV serotypes (EC50: 1.32-4.12 μM). Analysis of a single amino acid substitution of Compound-B-resistant DENV2 revealed that mutation C87S in the non-structural protein 4A (NS4A) contributes to resistance to Compound-B.
• The Unique Phylogenetic Position of a Novel Tick-Borne Phlebovirus Ensures an Ixodid Origin of the Genus Phlebovirus.

  Keita Matsuno, Masahiro Kajihara, Ryo Nakao, Naganori Nao, Akina Mori-Kajihara, Mieko Muramatsu, Yongjin Qiu, Shiho Torii, Manabu Igarashi, Nodoka Kasajima, Keita Mizuma, Kentaro Yoshii, Hirofumi Sawa, Chihiro Sugimoto, Ayato Takada, Hideki Ebihara

  mSphere 3 (3) 2018/06/27 [Refereed][Not invited]
   

  The recent emergence of novel tick-borne RNA viruses has complicated the epidemiological landscape of tick-borne infectious diseases, posing a significant challenge to public health systems that seek to counteract tick-borne diseases. The identification of two novel tick-borne phleboviruses (TBPVs), severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV), as causative agents of severe illness in humans has accelerated the investigation and discoveries of novel TBPVs. In the present study, we isolated a novel TBPV designated Mukawa virus (MKWV) from host-questing Ixodes persulcatus females captured in Japan. Genetic characterization revealed that MKWV is a member of the genus Phlebovirus in the family Phenuiviridae Interestingly, MKWV is genetically distinct from other known TBPVs and shares a most recent common ancestor with mosquito/sandfly-borne (insect-borne) phleboviruses. Despite its genetic similarity to insect-borne phleboviruses, the molecular footprints of its viral proteins and its biological characteristics define MKWV as a tick-borne virus that can be transmitted to mammals. A phylogenetic ancestral-state reconstruction for arthropod vectors of phleboviruses including MKWV based on viral L segment sequences indicated that ticks likely harbored ancestral phleboviruses that evolved into both the tick-borne and MKWV/insect-borne phlebovirus lineages. Overall, our findings suggest that most of the phlebovirus evolution has occurred in hard ticks to generate divergent viruses, which may provide a seminal foundation for understanding the mechanisms underlying the evolution and emergence of pathogenic phleboviruses, such as Rift Valley fever virus and SFTSV/HRTV.IMPORTANCE The emergence of novel tick-borne RNA viruses causing severe illness in humans has complicated the epidemiological landscape of tick-borne diseases, requiring further investigation to safeguard public health. In the present study, we discovered a novel tick-borne phlebovirus from Ixodes persulcatus ticks in Japan. While its viral RNA genome sequences were similar to those of mosquito/sandfly-borne viruses, molecular and biological footprints confirmed that this is a tick-borne virus. The unique evolutionary position of the virus allowed us to estimate the ancestral phlebovirus vector, which was likely a hard tick. Our findings may provide a better understanding of the evolution and emergence of phleboviruses associated with emerging infectious diseases, such as severe fever with thrombocytopenia syndrome (SFTS) and Heartland virus disease.
• Ribavirin-related compounds exert in vitro inhibitory effects toward rabies virus.

  Anindita PD, Sasaki M, Okada K, Ito N, Sugiyama M, Saito-Tarashima N, Minakawa N, Shuto S, Otsuguro S, Ichikawa S, Matsuda A, Maenaka K, Orba Y, Sawa H

  Antiviral research 154 1 - 9 0166-3542 2018/06 [Refereed][Not invited]
   

  Rabies remains an invariably fatal neurological disease despite the availability of a preventive vaccination and post-exposure prophylaxis that must be immediately administered to the exposed individual before symptom onset. There is no effective medication for treatment during the symptomatic phase. Ribavirin, a guanine nucleoside analog, is a potent inhibitor of rabies virus (RABV) replication in vitro but lacks clinical efficacy. Therefore, we attempted to identify potential ribavirin analogs with comparable or superior anti-RABV activity. Antiviral activity and cytotoxicity of the compounds were initially examined in human neuroblastoma cells. Among the tested compounds, two exhibited a 5- to 27-fold higher anti-RABV activity than ribavirin. Examination of the anti-RABV mechanisms of action of the compounds using time-of-addition and minigenome assays revealed that they inhibited viral genome replication and transcription. Addition of exogenous guanosine to RABV-infected cells diminished the antiviral activity of the compounds, suggesting that they are involved in guanosine triphosphate (GTP) pool depletion by inhibiting inosine monophosphate dehydrogenase (IMPDH). Taken together, our findings underline the potency of nucleoside analogs as a class of antiviral compounds for the development of novel agents against RABV.
• Tick-borne haemoparasites and Anaplasmataceae in domestic dogs in Zambia.

  Qiu Y, Kaneko C, Kajihara M, Ngonda S, Simulundu E, Muleya W, Thu MJ, Hang'ombe MB, Katakura K, Takada A, Sawa H, Simuunza M, Nakao R

  Ticks and tick-borne diseases 9 (4) 988 - 995 1877-959X 2018/05 [Refereed][Not invited]
   

  Tick-borne diseases (TBDs), including emerging and re-emerging infectious diseases, are important threats to human and animal health worldwide. Indeed, the number of reported human and animal infectious cases of novel TBD agents has increased in recent decades. However, TBDs tend to be neglected, especially in resource-limited countries that often have limited diagnostic capacity. The aim of this molecular survey was to detect and characterise tick-borne pathogens (Babesia, Theileria, and Hepatozoon parasites and Anaplasmataceae bacteria) in domestic dogs in Zambia. In total, 247 canine peripheral blood samples were collected in Lusaka, Mazabuka, Monze, and Shangombo. Conventional PCR to detect the selected pathogens was performed using DNA extracted from canine blood. One hundred eleven samples were positive for protozoa and 5 were positive for Anaplasmataceae. Sequencing of thirty-five randomly selected protozoa-positive samples revealed the presence of Babesia rossi, Babesia vogeli, and Hepatozoon canis 18S rDNA. Based on these sequences, a multiplex PCR system was developed to yield PCR products with different amplicons, the size of which depended on the parasite species; thus, each species could be identified without the need for sequence analysis. Approximately 40% of dogs were positive for H. canis. In particular, the positive rate (75.2%) of H. canis infection was significantly higher in Shangombo than in other sampling sites. Multiplex PCR assay detected B. rossi and B. vogeli infections in five and seven dogs, respectively, indicating that this approach is useful for detecting parasites with low prevalence. Sequencing analysis of gltA and groEL genes of Anaplasmataceae revealed that two and one dogs in Lusaka were infected with Anaplasma platys and Ehrlichia canis, respectively. The data indicated that Zambian dogs were infected with multiple tick-borne pathogens such as H. canis, B. rossi, B. vogeli, A. platys, E. canis and uncharacterized Ehrlichia sp. Since some of these parasites are zoonotic, concerted efforts are needed to raise awareness of, and control, these tick-borne pathogens.
• The Role of Heparan Sulfate Proteoglycans as an Attachment Factor for Rabies Virus Entry and Infection.

  Sasaki M, Anindita PD, Ito N, Sugiyama M, Carr M, Fukuhara H, Ose T, Maenaka K, Takada A, Hall WW, Orba Y, Sawa H

  The Journal of infectious diseases 217 (11) 1740 - 1749 0022-1899 2018/05 [Refereed][Not invited]
   

  Rabies virus (RABV) is the causative agent of fatal neurological disease. Cellular attachment is the initial and essential step for viral infections. Although extensive studies have demonstrated that RABV uses various target cell molecules to mediate infection, no specific molecule has been identified as an attachment factor for RABV infection. Here we demonstrate that cellular heparan sulfate (HS) supports RABV adhesion and subsequent entry into target cells. Enzymatic removal of HS reduced cellular susceptibility to RABV infection, and heparin, a highly sulfated form of HS, blocked viral adhesion and infection. The direct binding between RABV glycoprotein and heparin was demonstrated, and this interaction was shown to require HS N- and 6-O-sulfation. We also revealed that basic amino acids in the ectodomain of RABV glycoprotein serve as major determinants for the RABV-HS interaction. Collectively, our study highlights a previously undescribed role of HS as an attachment factor for RABV infection.
• Discovery of Mwinilunga alphavirus: A novel alphavirus in Culex mosquitoes in Zambia.

  Torii S, Orba Y, Hang'ombe BM, Mweene AS, Wada Y, Anindita PD, Phongphaew W, Qiu Y, Kajihara M, Mori-Kajihara A, Eto Y, Harima H, Sasaki M, Carr M, Hall WW, Eshita Y, Abe T, Sawa H

  Virus research 250 31 - 36 0168-1702 2018/05 [Refereed][Not invited]
   

  Mosquito-borne alphaviruses are disseminated globally and cause febrile illness in humans and animals. Since the prevalence and diversity of alphaviruses has not been previously investigated in Zambia, reverse transcription PCR was employed as a broad-spectrum approach for the detection of alphaviruses in mosquitoes. From 552 mosquito pools, a novel alphavirus, tentatively named Mwinilunga alphavirus (MWAV), was discovered from a single Culex quinquefasciatus mosquito pool. The full genome of MWAV was subsequently determined, and pairwise comparisons demonstrated that MWAV represented a new alphavirus species. Phylogenetic analyses and a linear discriminant analysis based on the dinucleotide ratios in various virus sequences indicated that MWAV is related to a mosquito-specific alphavirus distinct from other known mosquito-borne alphaviruses due to its inability to replicate in vertebrate cell lines. Further analyses of these novel alphaviruses will help to facilitate a greater understanding of the molecular determinants of host range restriction and the evolutionary relationships of alphaviruses.
• Detection of novel gammaherpesviruses from fruit bats in Indonesia.

  Wada Y, Sasaki M, Setiyono A, Handharyani E, Rahmadani I, Taha S, Adiani S, Latief M, Kholilullah ZA, Subangkit M, Kobayashi S, Nakamura I, Kimura T, Orba Y, Sawa H

  Journal of medical microbiology 67 (3) 415 - 422 0022-2615 2018/03 [Refereed][Not invited]
   

  Bats are an important natural reservoir of zoonotic viral pathogens. We previously isolated an alphaherpesvirus in fruit bats in Indonesia, and here establish the presence of viruses belonging to other taxa of the family Herpesviridae. We screened the same fruit bat population with pan-herpesvirus PCR and discovered 68 sequences of novel gammaherpesvirus, designated 'megabat gammaherpesvirus' (MgGHV). A phylogenetic analysis of approximately 3.4 kbp of continuous MgGHV sequences encompassing the glycoprotein B gene and DNA polymerase gene revealed that the MgGHV sequences are distinct from those of other reported gammaherpesviruses. Further analysis suggested the existence of co-infections of herpesviruses in Indonesian fruit bats. Our findings extend our understanding of the infectious cycles of herpesviruses in bats in Indonesia and the phylogenetic diversity of the gammaherpesviruses.
• Shape-dependent adjuvanticity of nanoparticle-conjugated RNA adjuvants for intranasal inactivated influenza vaccines

  Tazaki Taiyu, Tabata Koshiro, Ainai Akira, Ohara Yuki, Kobayashi Shintaro, Ninomiya Takafumi, Orba Yasuko, Mitomo Hideyuki, Nakano Tetsuo, Hasegawa Hideki, Ijiro Kuniharu, Sawa Hirofumi, Suzuki Tadaki, Niikura Kenichi

  RSC ADVANCES 8 (30) 16527 - 16536 2046-2069 2018 [Refereed][Not invited]
   

  Intranasal inactivated influenza vaccines can elicit mucosal immune responses that protect against virus infection. For the development of intranasal inactivated influenza vaccines, effective adjuvants inducing minimal adverse reactions are required. Generally, however, lower toxicity adjuvants have lower adjuvanticity. In this research, we fabricated nanoparticle-based adjuvants to enhance its adjuvanticity. Herein, we focused on low-molecular-weight polyinosinic-polycytidylic acid, referred to as uPIC(40:400), as a weak and less toxic RNA adjuvant. We conjugated uPIC(40:400) with different shaped gold nanoparticles (AuNPs) electrostatically. Conjugation with gold nanorods, but not spherical AuNPs, markedly enhanced the adjuvanticity of uPIC(40:400), leading to the suppression of viral infection in mice. Notably, conjugation with gold nanorods did not increase the inflammatory cytokine production in dendritic cells. These data indicated that gold nanorods can provide a good platform for enhancing the weak adjuvanticity of uPIC(40:400) while maintaining low inflammatory cytokine production toward the development of intranasal inactivated influenza vaccines.
• Development of a quick bioassay for the evaluation of transmission properties of acquired prion diseases.

  Munesue Y, Shimazaki T, Qi Z, Isoda N, Sawa H, Aoshima K, Kimura T, Mohri S, Kitamoto T, Kobayashi A

  Neuroscience letters 668 43 - 47 0304-3940 2018/01 [Refereed][Not invited]
   

  Evaluation of transmission properties is important for the differential diagnosis of a subgroup of acquired Creutzfeldt-Jakob disease (CJD) with methionine homozygosity at polymorphic codon 129 of the PRNP gene, an intermediate type abnormal prion protein (PrP), and kuru plaques, denoted as acquired CJD-MMiK. The present study aimed to develop a quick evaluation system of the transmission properties of acquired CJD-MMiK. In the PrP-humanized mice intraperitoneally inoculated with brain homogenates from an acquired CJD-MMiK patient, accumulation of abnormal PrP was observed in follicular dendritic cells of the spleen at 75 days post-inoculation. The transmission properties of acquired CJD-MMiK were quite different from those of sporadic CJD with the same PRNP codon 129 genotype. Moreover, even at 14 days post-inoculation, the characteristic transmission properties of acquired CJD-MMiK could be detected. These findings suggest that the bioassay using follicular dendritic cells of the spleen, named as a FDC assay, can be an easy, time-saving, and useful method to distinguish acquired CJD-MMiK from sporadic CJD.
• Development of a rapid and quantitative method for the analysis of viral entry and release using a NanoLuc luciferase complementation assay

  Michihito Sasaki, Paulina D. Anindita, Wallaya Phongphaew, Michael Carr, Shintaro Kobayashi, Yasuko Orba, Hirofumi Sawa

  VIRUS RESEARCH 243 69 - 74 0168-1702 2018/01 [Refereed][Not invited]
   

  Subviral particles (SVPs) self-assemble and are released from cells transfected with expression plasmids encoding flavivirus structural proteins. Flavivirus-like particles (VLPs), consisting of flavivirus structural proteins and a subgenomic replicon, can enter cells and cause single-round infections. Neither SVPs or VLPs possess complete viral RNA genomes, therefore are replication-incompetent systems; however, they retain the capacity to fuse and bud from target cells and follow the same maturation process as whole virions. SVPs and VLPs have been previously employed in studies analyzing entry and release steps of viral life cycles. In this study, we have developed quantitative methods for the detection of cellular entry and release of SVPs and VLPs by applying a luciferase complementation assay based on the high affinity interaction between the split NanoLuc luciferase protein, LgBiT and the small peptide, HiBiT. We introduced HiBiT into the structural protein of West Nile virus and generated SVPs and VLPs harboring HiBiT (SVP-HiBiT and VLP-HiBiT, respectively). As SVP-HiBiT emitted strong luminescence upon exposure to LgBiT and its substrate, the nascently budded SVP-HiBiT in the supernatant was readily quantified by luminometry. Similarly, the cellular entry of VLP-HiBiT generated luminescence when VLP-HiBiT was infected into LgBiT-expressing cells. These methods utilizing SVP-HiBiT and VLP-HiBiT will facilitate research into life cycles of flaviviruses, including WNV.
• Characterization of a Novel Bat Adenovirus Isolated from Straw-Colored Fruit Bat (Eidolon helvum).

  Hirohito Ogawa, Masahiro Kajihara, Naganori Nao, Asako Shigeno, Daisuke Fujikura, Bernard M Hang'ombe, Aaron S Mweene, Alisheke Mutemwa, David Squarre, Masao Yamada, Hideaki Higashi, Hirofumi Sawa, Ayato Takada

  Viruses 9 (12) 2017/12/04 [Refereed][Not invited]
   

  Bats are important reservoirs for emerging zoonotic viruses. For extensive surveys of potential pathogens in straw-colored fruit bats (Eidolon helvum) in Zambia, a total of 107 spleen samples of E. helvum in 2006 were inoculated onto Vero E6 cells. The cell culture inoculated with one of the samples (ZFB06-106) exhibited remarkable cytopathic changes. Based on the ultrastructural property in negative staining and cross-reactivity in immunofluorescence assays, the virus was suspected to be an adenovirus, and tentatively named E. helvum adenovirus 06-106 (EhAdV 06-106). Analysis of the full-length genome of 30,134 bp, determined by next-generation sequencing, showed the presence of 28 open reading frames. Phylogenetic analyses confirmed that EhAdV 06-106 represented a novel bat adenovirus species in the genus Mastadenovirus. The virus shared similar characteristics of low G + C contents with recently isolated members of species Bat mastadenoviruses E, F and G, from which EhAdV 06-106 diverged by more than 15% based on the distance matrix analysis of DNA polymerase amino acid sequences. According to the taxonomic criteria, we propose the tentative new species name "Bat mastadenovirus H". Because EhAdV 06-106 exhibited a wide in vitro cell tropism, the virus might have a potential risk as an emerging virus through cross-species transmission.
• 発光タグHiBiTを使用したフラビウイルスの細胞内侵入および出芽機構の解析

  佐々木 道仁, アニンディタ・パウリナ, ポンペオウ・ワンラヤー, カー・マイケル, 小林 進太郎, 大場 靖子, 澤 洋文

  生命科学系学会合同年次大会 生命科学系学会合同年次大会運営事務局 2017年度 [1P - 1118] 2017/12
• Identification of the same polyomavirus species in different African horseshoe bat species is indicative of short-range host-switching events

  Michael Carr, Gabriel Gonzalez, Michihito Sasaki, Serena E. Dool, Kimihito Ito, Akihiro Ishii, Bernard M. Hang'ombe, Aaron S. Mweene, Emma C. Teeling, William W. Hall, Yasuko Orba, Hirofumi Sawa

  JOURNAL OF GENERAL VIROLOGY 98 (11) 2771 - 2785 0022-1317 2017/11 [Refereed][Not invited]
   

  Polyomaviruses (PyVs) are considered to be highly host-specific in different mammalian species, with no well-supported evidence for host-switching events. We examined the species diversity and host specificity of PyVs in horseshoe bats (Rhinolophus spp.), a broadly distributed and highly speciose mammalian genus. We annotated six PyV genomes, comprising four new PyV species, based on pairwise identity within the large T antigen (LTAg) coding region. Phylogenetic comparisons revealed two instances of highly related PyV species, one in each of the Alphapolyomavirus and Betapolyomavirus genera, present in different horseshoe bat host species (Rhinolophus blasii and R. simulator), suggestive of short-range host-switching events. The two pairs of Rhinolophus PyVs in different horseshoe bat host species were 99.9 and 88.8% identical with each other over their respective LTAg coding sequences and thus constitute the same virus species. To corroborate the species identification of the bat hosts, we analysed mitochondrial cytb and a large nuclear intron dataset derived from six independent and neutrally evolving loci for bat taxa of interest. Bayesian estimates of the ages of the most recent common ancestors suggested that the near-identical and more distantly related PyV species diverged approximately 9.1 E4 (5E3-2.8E5) and 9.9E6 (4E6-18E6) years before the present, respectively, in contrast to the divergence times of the bat host species: 12.4E6 (10.4E6-15.4E6). Our findings provide evidence that short-range host-switching of PyVs is possible in horseshoe bats, suggesting that PyV transmission between closely related mammalian species can occur.
• チクングニアウイルスのRNA依存性RNAポリメラーゼを阻害する新規抗ウイルス化合物の探索と解析

  和田 雄治, 大場 靖子, 佐々木 道仁, 小林 進太郎, Carr Michael, 登 治謙, 佐藤 彰彦, Hall William, 澤 洋文

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 160回 391 - 391 1347-8621 2017/08
• Discovery of novel cyclic peptide inhibitors of dengue virus NS2B-NS3 protease with antiviral activity

  Youhei Takagi, Kouhei Matsui, Haruaki Nobori, Haruka Maeda, Akihiko Sato, Takeshi Kurosu, Yasuko Orba, Hirofumi Sawa, Kazunari Hattori, Kenichi Higashino, Yoshito Numata, Yutaka Yoshida

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 27 (15) 3586 - 3590 0960-894X 2017/08 [Refereed][Not invited]
   

  NS2B-NS3 protease is an essential enzyme for the replication of dengue virus (DENV), which continues to be a serious threat to worldwide public health. We designed and synthesized a series of cyclic peptides mimicking the substrates of this enzyme, and assayed their activity against the DENV-2 NS2B-NS3 protease. The introduction of aromatic residues at the appropriate positions and conformational restriction generated the most promising cyclic peptide with an IC50 of 0.95 mu M against NS2B-NS3 protease. Cyclic peptides with proper positioning of additional arginines and aromatic residues exhibited antiviral activity against DENV. Furthermore, replacing the C-terminal amide bond of the polybasic amino acid sequence with an amino methylene moiety stabilized the cyclic peptides against hydrolysis by NS2B-NS3 protease, while maintaining their enzyme inhibitory activity and antiviral activity. (C) 2017 Elsevier Ltd. All rights reserved.
• 2016 International meeting of the Global Virus Network

  Ramesh Akkina, Heinz Ellerbrok, William Hall, Hideki Hasegawa, Yasushi Kawaguchi, Harold Kleanthous, Edward McSweegan, Natalia Mercer, Victor Romanowski, Hirofumi Sawa, Anders Vahlne

  ANTIVIRAL RESEARCH 142 21 - 29 0166-3542 2017/06 [Refereed][Not invited]
   

  The Global Virus Network (GVN) was established in 2011 in order to strengthen research and responses to current viral causes of human disease and to prepare against new viral pandemic threats. There are now 38 GVN Centers of Excellence and 6 Affiliate laboratories in 24 countries. GVN scientists meet annually to learn about each other's current research, address collaborative priorities and plan future programs. The 2016 meeting was held from October 23-25 in Hokkaido, Japan, in partnership with the Japanese Society for Virology, the National Institute of Infectious Diseases of Japan and the Research Center for Zoonosis Control of Hokkaido University. This report highlights the accomplishments of GVN researchers in many priority areas of medical virology, including the current Zika epidemic, infections by human papillomavirus, influenza, Ebola, Lassa, dengue, HIV, hepatitis C, and chikungunya viruses, and the development of improved diagnostics and new vaccines. Published by Elsevier B.V.
• Listeria monocytogenes serotype 4b strains replicate in monocytes/macrophages more than the other serotypes

  Rie Hasebe, Ryo Nakao, Aiko Ohnuma, Takeshi Yamasaki, Hirofumi Sawa, Shinji Takai, Motohiro Horiuchi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 79 (6) 962 - 969 0916-7250 2017/06 [Refereed][Not invited]
   

  We analyzed the pathogenicity of various serotypes of Listeria monocytogenes using a Balb/c mouse intravenous injection model. The survival rates of mice inoculated with strains NS1/2b (serotype 1/2b), NS3b (serotype 3b) and NS 4b (serotype 4b) were 60, 63.6 and 63.6%, respectively. Although the survival rates were similar, the bacterial growth in the liver of NS3b-infected mice was 144.5-fold higher than that in the liver of NS4b-infected mice. Histopathological analyses suggest that the NS4b strain replicated more in monocytes/macrophages, whereas the NS3b strain replicated more in hepatocytes. These results raise a possibility that the serotype 4b strains replicated more in monocytes/macrophages compared to the other serotype strains. To assess this, we isolated CD11b-positive cells from mouse livers infected with EGDe (serotype 1/2a), NS1/2b, NS3b, NS4b and the serotype 4b strains 51414 and F17 and counted the number of live bacteria in these cells. CD11b-positive cells from the NS4b-, 51414- and F17-infected mice possessed 24.4- to 42.7-fold higher numbers of live bacteria than those from mice infected with EGDe and NS3b strains. These results suggest that serotype 4b strains replicated more in monocytes/macrophages than the other serotypes, and this may be involved in the pathogenicity of serotype 4b strains, particularly in the dissemination of L. monocytogenes through the host body.
• Discovery of a novel antiviral agent targeting the nonstructural protein 4 (nsP4) of chikungunya virus

  Yuji Wada, Yasuko Orba, Michihito Sasaki, Shintaro Kobayashi, Michael J. Carr, Haruaki Nobori, Akihiko Sato, William W. Hall, Hirofumi Sawa

  VIROLOGY 505 102 - 112 0042-6822 2017/05 [Refereed][Not invited]
   

  Chikungunya fever (CHIKF) is caused by chikungunya virus (CHIKV) infection which is a re-emerging mosquito-borne zoonosis. At present, there are no approved therapeutics for CHIKF. Herein, we have investigated candidate compounds which can inhibit CHIKV infection. Screening of chemical compound libraries were performed and one candidate, a benzimidazole-related compound designated Compound-A was found to inhibit infection by several CHIKV strains and a Sindbis virus strain at nanomolar concentrations. To investigate the inhibitory mechanism of action, a Compound-A resistant CHIKV (res-CHIKV) was isolated and a key mutation associated with resistance was identified by reverse-genetic recombinant CHIICVs containing amino acid substitutions present in res-CHIKV. These results demonstrated that the target site of Compound-A was the M2295 residue in the nonstructural protein 4 (nsP4), which is located in one of the functional domains of RNA-dependent RNA-polymerase (RdRp). We also confirmed that Compound-A inhibits RdRp function of CHIKV by using CHIKV replicons.
• Discovery of African bat polyomaviruses and infrequent recombination in the large T antigen in the Polyomaviridae

  Michael Carr, Gabriel Gonzalez, Michihito Sasaki, Kimihito Ito, Akihiro Ishii, Bernard M. Hang'ombe, Aaron S. Mweene, Yasuko Orba, Hirofumi Sawa

  JOURNAL OF GENERAL VIROLOGY 98 (4) 726 - 738 0022-1317 2017/04 [Refereed][Not invited]
   

  Bat species represent natural reservoirs for a number of high-consequence human pathogens. The present study investigated the diversity of polyomaviruses (PyVs) in Zambian insectivorous and fruit bat species. We describe the complete genomes from four newly proposed African bat PyV species employing the recently recommended criteria provided by the Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses. A comprehensive phylogenetic and recombination analysis was performed to determine genetic relationships and the distribution of recombination events in PyV from mammalian and avian species. The novel species of PyV from Zambian bats segregated with members of the genera Alphapolyomavirus and Betapolyomavirus, forming monophyletic clades with bat and non-human primate PyVs. Miniopterus schreibersii polyomavirus 1 and 2 segregated in a clade with South American bat PyV species, Old World monkey and chimpanzee PyVs and Human polyomavirus 13 (New Jersey PyV). Interestingly, the newly described Egyptian fruit bat PyV, tentatively named Rousettus aegyptiacus polyomavirus 1, had the highest nucleotide sequence identity to species of PyV from Indonesian fruit bats, and Rhinolophus hildebrandtii polyomavirus 1 was most closely related to New World monkey PyVs. The distribution of recombination events in PyV genomes was non-random: recombination boundaries existed in the intergene region between VP1 and LTAg and also at the 3' end of VP2/3 in the structural genes, whereas infrequent recombination was present within the LTAg gene. These findings indicate that recombination within the LTAg gene has been negatively selected against during polyomaviral evolution and support the recent proposal for taxonomic classification based on LTAg to define novel PyV species.
• Isolation of a simian immunodeficiency virus from a malbrouck (Chlorocebus cynosuros)

  Michael Carr, Akira Kawaguchi, Michihito Sasaki, Gabriel Gonzalez, Kimihito Ito, Yuka Thomas, Bernard M. Hang'ombe, Aaron S. Mweene, Guoyan Zhao, David Wang, Yasuko Orba, Akihiro Ishii, Hirofumi Sawa

  ARCHIVES OF VIROLOGY 162 (2) 543 - 548 0304-8608 2017/02 [Refereed][Not invited]
   

  To investigate the diversity of simian immunodeficiency virus (SIV) among nonhuman primates (NHPs) in Zambia, next-generation sequencing was performed to determine the complete genome sequence of a novel SIV recovered by co-culturing African green monkey (AGM) peripheral blood lymphocytes with human CD4(+) T-cell lines. We report the first described SIV (SIVagmMAL-ZMB) from a malbrouck (Chlorocebus cynosuros). SIVagmMAL-ZMB was detected by real-time PCR analysis of splenic RNA in 3.2% (3/94) of AGMs and was undetectable in baboons (0/105). SIVagmMAL-ZMB possessed < 80% nucleotide sequence identity to known SIV isolates and was located basally to vervet monkey SIV strains in all phylogenies.
• Putative RNA viral sequences detected in an Ixodes scapularis-derived cell line

  Ryo Nakao, Keita Matsuno, Yongjin Qiu, Junki Marilyama, Nao Eguchi, Naganori Nao, Masahiro Kajihara, Kentaro Yoshii, Hirofumi Sawa, Ayato Takada, Chihiro Sugimoto

  TICKS AND TICK-BORNE DISEASES 8 (1) 103 - 111 1877-959X 2017 [Refereed][Not invited]
   

  Ticks harbour various microorganisms, some of which act as pathogens of humans and animals. The recent advancement of genome sequencing technologies revealed that a wide range of previously unrecognised microorganisms exist in ticks. Continuous cell lines established from ticks could play a key role in the isolation of such microorganisms; however, tick cells themselves have been known to harbour symbiotic microorganisms. The present study aimed to characterise putative RNA viral sequences detected in the culture supernatant of one of the most frequently used tick cell lines, ISE6, which was derived from embryos of the blacklegged tick Ixodes scapularis. Viral particles purified from the culture supernatant were used for RNA extraction, followed by Illumina sequencing. The reads were de novo assembled and the resulting contigs were annotated by tBLASTx search. The results suggested that there were at least five putative viral sequences of four phylogenetically distinct lineages in ISE6 cells. The predominant viral sequence found in ISE6 cells, designated L scapularis iflavirus, was a member of the family Iflaviridae, which is an arthropod-infecting virus group. We also identified L and M segments of the family Bunyaviridae, which could not be classified into any of the five known genera, and a potential capsid protein related to Drosophila A virus. In addition to these previously unrecognised viruses, ISE6 was revealed to harbour a putative genome sequence of L scapularis-associated virus-1, which was reported in a recent metagenomic study of L scapularis itself. All the five putative viral sequences were detected by RT-PCR in both ISE6 cells and the culture supernatant. Electron microscopic analysis showed the existence of spherical virions with a varying diameter of 50-70 nm in the culture supernatant of ISE6 cells. Further studies are required to investigate the potential roles of ISE6-associated viruses in ticks. (C) 2016 Elsevier GmbH. All rights reserved.
• Valosin-containing protein (VCP/p97) plays a role in the replication of West Nile virus

  Wallaya Phongphaew, Shintaro Kobayashi, Michihito Sasaki, Michael Carr, William W. Hall, Yasuko Orba, Hirofumi Sawa

  VIRUS RESEARCH 228 114 - 123 0168-1702 2017/01 [Refereed][Not invited]
   

  Valosin-containing protein (VCP) is classified as a member of the type II AAA(+) ATPase protein family. VCP functions in several cellular processes, including protein degradation, membrane fusion, vesicular trafficking and disassembly of stress granules. Moreover, VCP is considered to play a role in the replication of several viruses, albeit through different mechanisms. In the present study, we have investigated the role of VCP in West Nile virus (WNV) infection. Endogenous VCP expression was inhibited using either VCP inhibitors or by siRNA knockdown. It could be shown that the inhibition of endogenous VCP expression significantly inhibited WNV infection. The entry assay revealed that silencing of endogenous VCP caused a significant reduction in the expression levels of WNV-RNA compared to control siRNA-treated cells. This indicates that VCP may play a role in early steps either the binding or entry steps of the WNV life cycle. Using WNV virus like particles and WNV-DNA-based replicon, it could be demonstrated that perturbation of VCP expression decreased levels of newly synthesized WNV genomic RNA. These findings suggest that VCP is involved in early steps and during genome replication of the WNV life cycle. (C) 2016 Elsevier B.V. All rights reserved.
• An optimistic protein assembly from sequence reads salvaged an uncharacterized segment of mouse picobirnavirus

  Gabriel Gonzalez, Michihito Sasaki, Lucy Burkitt-Gray, Tomonori Kamiya, Noriko M. Tsuji, Hirofumi Sawa, Kimihito Ito

  SCIENTIFIC REPORTS 7 40447  2045-2322 2017/01 [Refereed][Not invited]
   

  Advances in Next Generation Sequencing technologies have enabled the generation of millions of sequences from microorganisms. However, distinguishing the sequence of a novel species from sequencing errors remains a technical challenge when the novel species is highly divergent from the closest known species. To solve such a problem, we developed a new method called Optimistic Protein Assembly from Reads (OPAR). This method is based on the assumption that protein sequences could be more conserved than the nucleotide sequences encoding them. By taking advantage of metagenomics, bioinformatics and conventional Sanger sequencing, our method successfully identified all coding regions of the mouse picobirnavirus for the first time. The salvaged sequences indicated that segment 1 of this virus was more divergent from its homologues in other Picobirnaviridae species than segment 2. For this reason, only segment 2 of mouse picobirnavirus has been detected in previous studies.
• Multi-reassortant G3P[3] group A rotavirus in a horseshoe bat in Zambia

  Michihito Sasaki, Yasuko Orba, Satoko Sasaki, Gabriel Gonzalez, Akihiro Ishii, Bernard M. Hang'ombe, Aaron S. Mweene, Kimihito Ito, Hirofumi Sawa

  JOURNAL OF GENERAL VIROLOGY 97 (10) 2488 - 2493 0022-1317 2016/10 [Refereed][Not invited]
   

  Group A rotavirus is a major cause of diarrhoea in humans, especially in young children. Bats also harbour group A rotaviruses, but the genetic backgrounds of bat rotavirus strains are usually distinct from those of human rotavirus strains. We identified a new strain of group A rotavirus in the intestinal contents of a horseshoe bat in Zambia. Whole genome sequencing revealed that the identified virus, named RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3], possessed the genotype constellation G3-P[3]-I3-R2-C2-M3-A9-N2-T3-E2-H3. Several genome segments of LUS12-14 were highly similar to those of group A rotaviruses identified from humans, cows and antelopes, indicating interspecies transmission of rotaviruses between bats and other mammals with possible multiple genomic reassortment events.
• Generation of recombinant rabies viruses encoding NanoLuc luciferase for antiviral activity assays

  Paulina Duhita Anindita, Michihito Sasaki, Haruaki Nobori, Akihiko Sato, Michael Carr, Naoto Ito, Makoto Sugiyama, Yasuko Orba, Hirofumi Sawa

  VIRUS RESEARCH 215 121 - 128 0168-1702 2016/04 [Refereed][Not invited]
   

  Rabies is an invariably fatal disease caused by Rabies virus (RABV), a member of the family Rhabdoviridae, genus Lyssavirus. Once central nervous infection occurs and symptoms develop, the case fatality rate approaches 100% despite availability of post-exposure prophylaxis. Therefore, new antiviral therapies for rabies are urgently required. Antivirals which can inhibit virus replication can be identified through screening of small compounds, however, as RABV infection does not generate easily discernible cytopathic effects in vitro, cell viability assays may not be feasible to observe antiviral activity of small compounds against RABV. In this study, recombinant RABVs (rRABVs) encoding NanoLuc luciferase (NanoLuc) were generated to facilitate the screening of small compound libraries. NanoLuc expression was confirmed in single-step growth cures of virus infection and showed that the rRABVs were capable of viral replication without decrease of luciferase activity through ten serial passages. Furthermore, the rRABVs were able to quantify the antiviral activity of the nucleoside analogue ribavirin against RABV in vitro. These findings confirm the potential of the rRABV encoding NanoLuc system to facilitate screening of small compounds to inhibit RABV infection. (C) 2016 Elsevier B.V. All rights reserved.
• Divergent bufavirus harboured in megabats represents a new lineage of parvoviruses

  Michihito Sasaki, Gabriel Gonzalez, Yuji Wada, Agus Setiyono, Ekowati Handharyani, Ibenu Rahmadani, Siswatiana Taha, Sri Adiani, Munira Latief, Zainal Abidin Kholilullah, Mawar Subangkit, Shintaro Kobayashi, Ichiro Nakamura, Takashi Kimura, Yasuko Orba, Kimihito Ito, Hirofumi Sawa

  SCIENTIFIC REPORTS 6 24257  2045-2322 2016/04 [Refereed][Not invited]
   

  Bufavirus is a recently recognized member of the genus Protoparvovirus in the subfamily Parvovirinae. It has been reported that human bufavirus was detected predominantly in patients with diarrhoea in several countries. However, little is known about bufavirus or its close relatives in nonhuman mammals. In this study, we performed nested-PCR screening and identified bufavirus from 12 megabats of Pteropus spp. in Indonesia. Furthermore, we determined nearly the full genome sequence of a novel megabat-borne bufavirus, tentatively named megabat bufavirus 1. Phylogenetic analyses showed that megabat bufavirus 1 clustered with known protoparvoviruses, including human bufavirus but represented a distinct lineage of bufavirus. Our analyses also inferred phylogenetic relationships among animal-borne bufaviruses recently reported by other studies. Recombination analyses suggested that the most common recent ancestor of megabat bufavirus 1 might have arisen from multiple genetic recombination events. These results characterized megabat bufavirus 1 as the first protoparvovirus discovered from megabats and indicates the high genetic divergence of bufavirus.
• Rab8b Regulates Transport of West Nile Virus Particles from Recycling Endosomes

  Shintaro Kobayashi, Tadaki Suzuki, Akira Kawaguchi, Wallaya Phongphaew, Kentaro Yoshii, Tomohiko Iwano, Akihiro Harada, Hiroaki Kariwa, Yasuko Orba, Hirofumi Sawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 291 (12) 6559 - 6568 0021-9258 2016/03 [Refereed][Not invited]
   

  West Nile virus (WNV) particles assemble at and bud into the endoplasmic reticulum (ER) and are secreted from infected cells through the secretory pathway. However, the host factor related to these steps is not fully understood. Rab proteins, belonging to the Ras superfamily, play essential roles in regulating many aspects of vesicular trafficking. In this study, we sought to determine which Rab proteins are involved in intracellular trafficking of nascent WNV particles. RNAi analysis revealed that Rab8b plays a role in WNV particle release. We found that Rab8 and WNV antigen were colocalized in WNV-infected human neuroblastoma cells, and that WNV infection enhanced Rab8 expression in the cells. In addition, the amount of WNV particles in the supernatant of Rab8b-deficient cells was significantly decreased compared with that of wild-type cells. We also demonstrated that WNV particles accumulated in the recycling endosomes in WNV-infected cells. In summary, these results suggest that Rab8b is involved in trafficking of WNV particles from recycling endosomes to the plasma membrane.
• チクングニアウイルスの増殖を抑制する新規化合物の探索及び作用機序の解明に向けた基礎研究

  和田 雄治, 大場 靖子, 佐々木 道仁, 登 治謙, 佐藤 彰彦, 澤 洋文

  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [1P1143] - [1P1143] 2015/12
• Seroepidemiological Prevalence of Multiple Species of Filoviruses in Fruit Bats (Eidolon helvum) Migrating in Africa

  Hirohito Ogawa, Hiroko Miyamoto, Eri Nakayama, Reiko Yoshida, Ichiro Nakamura, Hirofumi Sawa, Akihiro Ishii, Yuka Thomas, Emiko Nakagawa, Keita Matsuno, Masahiro Kajihara, Junki Maruyama, Naganori Nao, Mieko Muramatsu, Makoto Kuroda, Edgar Simulundu, Katendi Changula, Bernard Hang'ombe, Boniface Namangala, Andrew Nambota, Jackson Katampi, Manabu Igarashi, Kimihito Ito, Heinz Feldmann, Chihiro Sugimoto, Ladslav Moonga, Aaron Mweene, Ayato Takada

  JOURNAL OF INFECTIOUS DISEASES 212 S101 - S108 0022-1899 2015/10 [Refereed][Not invited]
   

  Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.
• 野生動物から検出された新規パルボウイルスの系統解析

  佐々木 道仁, 大場 靖子, Anindita D. Paulina, 石井 秋宏, 伊藤 公人, 澤 洋文

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 158回 340 - 340 1347-8621 2015/08
• Distinct Lineages of Bufavirus in Wild Shrews and Nonhuman Primates

  Michihito Sasaki, Yasuko Orba, Paulina D. Anindita, Akihiro Ishii, Keisuke Ueno, Bernard M. Hang'ombe, Aaron S. Mweeile, Kimihito Ito, Hirofumi Sawa

  EMERGING INFECTIOUS DISEASES 21 (7) 1230 - 1233 1080-6040 2015/07 [Refereed][Not invited]
   

  Viral metagenomic analysis identified a new parvovirus genome in the intestinal contents of wild shrews in Zambia. Related viruses were detected in spleen tissues from wild shrews and nonhuman primates. Phylogenetic analyses showed that these viruses are related to human bufaviruses, highlighting the presence and genetic diversity of bufaviruses in wildlife.
• Tyr724 phosphorylation of ELMO1 by Src is involved in cell spreading and migration via Rac1 activation

  Yoshinori Makino, Masumi Tsuda, Yusuke Ohba, Hiroshi Nishihara, Hirofumi Sawa, Kazuo Nagashima, Shinya Tanaka

  CELL COMMUNICATION AND SIGNALING 13 35  1478-811X 2015/07 [Refereed][Not invited]
   

  Background: The complex of Dock180/ELMO1 that functions as a bipartite guanine nucleotide exchange factor for Rac is essential for diverse physiological and pathological processes of cells such as cell migration, phagocytosis, and invasion of cancer cells. Among the Src-family tyrosine kinases (SFKs), it has been reported that Hck directly phosphorylates ELMO1, regulating phagocytosis by promoting activation of Rac1; however, the involvement of other SFKs in ELMO1 phosphorylation has remained unknown. Here, we identified novel tyrosine (Y) residues of ELMO1 phosphorylated by SFKs, and examined the effects on Rac1 activity, cell adhesion, spreading, and cell motility on extracellular matrix (ECM). Results: In this study, we unveiled that Src and Fyn can induce tyrosine phosphorylation of ELMO1 in in vivo and in vitro phosphorylation assays. Mutational analyses identified both Y720 and Y724 residues of ELMO1 as Src-mediated phosphorylation sites, preferentially on Y724. Single substitution of Y724 to Phe abrogated Rac1 activation triggered by Src. To elucidate the biological function of pY724, we established NIH3T3 cells stably expressing wild-type ELMO1 or its Y724F mutant together with Dock180. Among them, Y724-deficient cells exhibited a depletion of Rac1 activity with diminished phosphorylation of ELMO1 even upon the ECM-stimulation. It is noteworthy that NIH3T3 cells with ELMO1 Y724F were strikingly defective to promote cell spreading on fibronectin-coated dish, concomitantly exhibiting immature assemblies of actin stress fibers and focal adhesions. Eventually, ELMO1 Y724F significantly impaired cell migration. Conclusion: These results define that Src-mediated Y724 phosphorylation in ELMO1 plays a critical role for cell spreading via activation of Rac1, leading to promotion of cell migration. As the overexpression and/or hyperactivation of Src have been shown in a wide variety of human cancers, Src-mediated phosphorylation of Y724 in ELMO1 may regulate cancer cell adhesion to the ECM, invasion into surrounding tissues, and subsequent distant metastasis.
• Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development

  Keisuke Aoshima, Erina Inoue, Hirofumi Sawa, Yuki Okada

  EMBO REPORTS 16 (7) 803 - 812 1469-221X 2015/07 [Refereed][Not invited]
   

  Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K-M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K-M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice.
• Molecular epidemiology of pathogenic Leptospira spp. in the straw-colored fruit bat (Eidolon helvum) migrating to Zambia from the Democratic Republic of Congo

  Hirohito Ogawa, Nobuo Koizumi, Aiko Ohnuma, Alisheke Mutemwa, Bernard M. Hang'ombe, Aaron S. Mweene, Ayato Takada, Chihiro Sugimoto, Yasuhiko Suzuki, Hiroshi Kida, Hirofumi Sawa

  INFECTION GENETICS AND EVOLUTION 32 143 - 147 1567-1348 2015/06 [Refereed][Not invited]
   

  The role played by bats as a potential source of transmission of Leptospira spp. to humans is poorly understood, despite various pathogenic Leptospira spp. being identified in these mammals. Here, we investigated the prevalence and diversity of pathogenic Leptospira spp. that infect the straw-colored fruit bat (Eidolon helvum). We captured this bat species, which is widely distributed in Africa, in Zambia during 2008-2013. We detected the flagellin B gene (flaB) from pathogenic Leptospira spp. in kidney samples from 79 of 529 E. helvum (14.9%) bats. Phylogenetic analysis of 70 flaB fragments amplified from E. helvum samples and previously reported sequences, revealed that 12 of the fragments grouped with Leptospira borgpetersenii and Leptospira kirschneri; however, the remaining 58 flaB fragments appeared not to be associated with any reported species. Additionally, the 16S ribosomal RNA gene (rrs) amplified from 27 randomly chosen flaB-positive samples was compared with previously reported sequences, including bat-derived Leptospira spp. All 27 rrs fragments clustered into a pathogenic group. Eight fragments were located in unique branches, the other 19 fragments were closely related to Leptospira spp. detected in bats. These results show that rrs sequences in bats are genetically related to each other without regional variation, suggesting that Leptospira are evolutionarily well-adapted to bats and have uniquely evolved in the bat population. Our study indicates that pathogenic Leptospira spp. in E. helvum in Zambia have unique genotypes. (C) 2015 The Authors. Published by Elsevier B.V.
• Neurogenic cardiomyopathy in rabbits with experimentally induced rabies.

  Kesdangsakonwut S, Sunden Y, Yamada K, Nishizono A, Sawa H, Umemura T

  Veterinary pathology 52 (3) 573 - 575 0300-9858 2015/05 [Refereed][Not invited]
  
• Detection of coronavirus genomes in Moluccan naked-backed fruit bats in Indonesia

  Paulina Duhita Anindita, Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Yasuko Orba, Shintaro Kobayashi, Ibnu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Ichiro Nakamura, Hirofumi Sawa, Takashi Kimura

  ARCHIVES OF VIROLOGY 160 (4) 1113 - 1118 0304-8608 2015/04 [Refereed][Not invited]
   

  Bats have been shown to serve as natural reservoirs for numerous emerging viruses including severe acute respiratory syndrome coronavirus (SARS-CoV). In the present study, we report the discovery of bat CoV genes in Indonesian Moluccan naked-backed fruit bats (Dobsonia moluccensis). A partial RNA-dependent RNA polymerase gene sequence was detected in feces and tissues samples from the fruit bats, and the region between the RdRp and helicase genes could also be amplified from fecal samples. Phylogenetic analysis suggested that these bat CoVs are related to members of the genus Betacoronavirus.
• Detection of novel polyomaviruses in fruit bats in Indonesia

  Shintaro Kobayashi, Michihito Sasaki, Ryo Nakao, Agus Setiyono, Ekowati Handharyani, Yasuko Orba, Ibnu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Ichiro Nakamura, Takashi Kimura, Hirofumi Sawa

  ARCHIVES OF VIROLOGY 160 (4) 1075 - 1082 0304-8608 2015/04 [Refereed][Not invited]
   

  Bats are an important natural reservoir for a variety of viral pathogens, including polyomaviruses (PyVs). The aims of this study were: (i) to determine which PyVs are present in bats in Indonesia and (ii) to analyze the evolutionary relationships between bat PyVs and other known PyVs. Using broad-spectrum polymerase chain reaction (PCR)-based assays, we screened PyV DNA isolated from spleen samples from 82 wild fruit bats captured in Indonesia. Fragments of the PyV genome were detected in 10 of the 82 spleen samples screened, and eight full-length viral genome sequences were obtained using an inverse PCR method. A phylogenetic analysis of eight whole viral genome sequences showed that BatPyVs form two distinct genetic clusters within the proposed genus Orthopolyomavirus that are genetically different from previously described BatPyVs. Interestingly, one group of BatPyVs is genetically related to the primate PyVs, including human PyV9 and trichodysplasia spinulosa-associated PyV. This study has identified the presence of novel PyVs in fruit bats in Indonesia and provides genetic information about these BatPyVs.
• Orthopoxvirus infection among wildlife in Zambia

  Yasuko Orba, Michihito Sasaki, Hiroki Yamaguchi, Akihiro Ishii, Yuka Thomas, Hirohito Ogawa, Bernard M. Hang'ombe, Aaron S. Mweene, Shigeru Morikawa, Masayuki Saijo, Hirofumi Sawa

  JOURNAL OF GENERAL VIROLOGY 96 (Pt 2) 390 - 394 0022-1317 2015/02 [Refereed][Not invited]
   

  Human monkeypox is a viral zoonosis caused by monkeypox virus, an orthopoxvirus (OPXV). The majority of human monkeypox cases have been reported in moist forested regions in West and Central Africa, particularly in the Democratic Republic of the Congo (DRC). In this study we investigated zoonotic OPXV infection among wild animals in Zambia, which shares a border with DRC, to assess the geographical distribution of OPXV. We screened for OPXV antibodies in sera from non-human primates (NHPs), rodents and shrews by ELISA, and performed real-time PCR to detect OPXV DNA in spleen samples. Serological analysis indicated that 38 of 259 (14.7 %) rodents, 14 of 42 (33.3%) shrews and 4 of 188 (2.1 %) NHPs had antibodies against OPXV. The OPXV DNA could not be detected in spleens from any animals tested. Our results indicated that wild animals living in rural human habitation areas of Zambia have been infected with OPXV.
• Metagenomic analysis of the shrew enteric virome reveals novel viruses related to human stool-associated viruses

  Michihito Sasaki, Yasuko Orba, Keisuke Ueno, Akihiro Ishii, Ladslav Moonga, Bernard M. Hang'ombe, Aaron S. Mweene, Kimihito Ito, Hirofumi Sawa

  JOURNAL OF GENERAL VIROLOGY 96 (Pt 2) 440 - 452 0022-1317 2015/02 [Refereed][Not invited]
   

  Shrews are small insectivorous mammals that are distributed worldwide. Similar to rodents, shrews live on the ground and are commonly found near human residences. In this study, we investigated the enteric virome of wild shrews in the genus Crocidura using a sequence-independent viral metagenomics approach. A large portion of the shrew enteric virome was composed of insect viruses, whilst novel viruses including cyclovirus, picornavirus and picorna-like virus were also identified. Several cycloviruses, including variants of human cycloviruses detected in cerebrospinal fluid and stools, were detected in wild shrews at a high prevalence rate. The identified picornavirus was distantly related to human parechovirus, inferring the presence of a new genus in this family. The identified picorna-like viruses were characterized as different species of calhevirus 1, which was discovered previously in human stools. Complete or nearly complete genome sequences of these novel viruses were determined in this study and then were subjected to further genetic characterization. Our study provides an initial view of the diversity and distinctiveness of the shrew enteric virome and highlights unique novel viruses related to human stool-associated viruses.
• Pathological and molecular diagnosis of the 2013 African swine fever outbreak in Lusaka, Zambia

  John Yabe, Pharaoh Hamambulu, Edgar Simulundu, Hirohito Ogawa, Masahiro Kajihara, Akina Mori-Kajihara, Katendi Changula-Chitanga, Max Mwase, Mutinta Mweemba-Muwowo, Herman Moses Chambaro, Liywalii Mataa, Bernard Hang'ombe, Bonniface Namangala, Paul Fandamu, Hirofumi Sawa, Ayato Takada, Hideaki Higashi, Aaron Simanyengwe Mweene

  TROPICAL ANIMAL HEALTH AND PRODUCTION 47 (2) 459 - 463 0049-4747 2015/02 [Refereed][Not invited]
   

  African swine fever (ASF) is a highly contagious and fatal hemorrhagic viral disease of domestic pigs. The disease is widespread in sub-Saharan Africa and has repeatedly been introduced into other continents. The current study describes the diagnostic investigations of a hemorrhagic disease that was reported in pigs in Lusaka (October 2013), Zambia. Necropsy, histopathology, and molecular diagnosis using polymerase chain reaction and sequence analysis confirmed the disease to be ASF. The sequences obtained showed high similarity to previously isolated ASF viruses. Consistent surveillance and rapid diagnosis of the disease is recommended to prevent future outbreaks and economic losses as there is currently no vaccine against the disease.
• A nairovirus isolated from African bats causes haemorrhagic gastroenteritis and severe hepatic disease in mice

  Akihiro Ishii, Keisuke Ueno, Yasuko Orba, Michihito Sasaki, Ladslav Moonga, Bernard M. Hang'ombe, Aaron S. Mweene, Takashi Umemura, Kimihito Ito, William W. Hall, Hirofumi Sawa

  NATURE COMMUNICATIONS 5 5651  2041-1723 2014/12 [Refereed][Not invited]
   

  Bats can carry important zoonotic pathogens. Here we use a combination of next-generation sequencing and classical virus isolation methods to identify novel nairoviruses from bats captured from a cave in Zambia. This nairovirus infection is highly prevalent among giant leaf-nosed bats, Hipposideros gigas (detected in samples from 16 individuals out of 38). Whole-genome analysis of three viral isolates (11SB17, 11SB19 and 11SB23) reveals a typical bunyavirus tri-segmented genome. The strains form a single phylogenetic clade that is divergent from other known nairoviruses, and are hereafter designated as Leopards Hill virus (LPHV). When i.p. injected into mice, the 11SB17 strain causes only slight body weight loss, whereas 11SB23 produces acute and lethal disease closely resembling that observed with Crimean-Congo Haemorrhagic Fever virus in humans. We believe that our LPHV mouse model will be useful for research on the pathogenesis of nairoviral haemorrhagic disease.
• The zoonotic potential of avian influenza viruses isolated from wild waterfowl in Zambia

  Edgar Simulundu, Naganori Nao, John Yabe, Nilton A. Muto, Thami Sithebe, Hirofumi Sawa, Rashid Manzoor, Masahiro Kajihara, Mieko Muramatsu, Akihiro Ishii, Hirohito Ogawa, Aaron S. Mweene, Ayato Takada

  ARCHIVES OF VIROLOGY 159 (10) 2633 - 2640 0304-8608 2014/10 [Refereed][Not invited]
   

  Whilst remarkable progress in elucidating the mechanisms governing interspecies transmission and pathogenicity of highly pathogenic avian influenza viruses (AIVs) has been made, similar studies focusing on low-pathogenic AIVs isolated from the wild waterfowl reservoir are limited. We previously reported that two AIV strains (subtypes H6N2 and H3N8) isolated from wild waterfowl in Zambia harbored some amino acid residues preferentially associated with human influenza virus proteins (so-called human signatures) and replicated better in the lungs of infected mice and caused more morbidity than a strain lacking such residues. To further substantiate these observations, we infected chickens and mice intranasally with AIV strains of various subtypes (H3N6, H3N8, H4N6, H6N2, H9N1 and H11N9) isolated from wild waterfowl in Zambia. Although some strains induced seroconversion, all of the tested strains replicated poorly and were nonpathogenic for chickens. In contrast, most of the strains having human signatures replicated well in the lungs of mice, and one of these strains caused severe illness in mice and induced lung injury that was characterized by a severe accumulation of polymorphonuclear leukocytes. These results suggest that some strains tested in this study may have the potential to infect mammalian hosts directly without adaptation, which might possibly be associated with the possession of human signature residues. Close monitoring and evaluation of host-associated signatures may help to elucidate the prevalence and emergence of AIVs with potential for causing zoonotic infections.
• Autophagy inhibits viral genome replication and gene expression stages in West Nile virus infection

  Shintaro Kobayashi, Yasuko Orba, Hiroki Yamaguchi, Kenta Takahashi, Michihito Sasaki, Rie Hasebe, Takashi Kimura, Hirofumi Sawa

  VIRUS RESEARCH 191 83 - 91 0168-1702 2014/10 [Refereed][Not invited]
   

  Autophagy is a lysosomal degradation pathway that is implicated in many viral infections. However, its role in West Nile virus (WNV) infection remains controversial. In the present study, we examined the relationship between WNV infection and autophagy in infected cells. We demonstrated that LC3-II expression, a molecular marker for autophagosomal membranes, was enhanced in WNV-infected cells 6 h post-infection. LC3-II expression was further enhanced in WNV-inoculated cells when treated with a lysosomal protease inhibitor. Meanwhile, WNV replication in cells lacking Atg5, an essential factor for autophagy, was increased compared with replication in wild-type cells. In addition, WNV replication was inhibited in cells lacking Atg5 when they were transfected with an ATG5 expression plasmid. These results suggest an antiviral role for autophagy in WNV-infected cells. We also examined which viral replication stages were affected by autophagy by using a Tat-beclin 1 peptide to induce autophagy and pseudo-infectious WNV reporter virus particles (WNV-RVPs) that monitor viral genome replication and gene expression stages via GFP expression. We found that autophagy induction in HeLa cells by Tat-beclin 1 peptide 3 h after WNV inoculation inhibited viral replication, and GFP expression was significantly inhibited in wild-type cells when compared with cells lacking Atg5. Taken together, these results suggest that autophagy is induced by WNV infection, and that this induction inhibits WNV replication at the viral genome replication and gene expression stages. (C) 2014 Elsevier B.V. All rights reserved.
• Detection and characterization of zoonotic pathogens of free-ranging non-human primates from Zambia

  Jesca Nakayima, Kyoko Hayashida, Ryo Nakao, Akihiro Ishii, Hirohito Ogawa, Ichiro Nakamura, Ladslav Moonga, Bernard M. Hang'ombe, Aaron S. Mweene, Yuka Thomas, Yasuko Orba, Hirofumi Sawa, Chihiro Sugimoto

  PARASITES & VECTORS 7 490  1756-3305 2014/10 [Refereed][Not invited]
   

  Background: Wildlife may harbor infectious pathogens that are of zoonotic concern acting as a reservoir of diseases transmissible to humans and domestic animals. This is due to human-wildlife conflicts that have become more frequent and severe over recent decades, competition for the available natural habitats and resources leading to increased human encroachment on previously wild and uninhabited areas. Methods: A total of 88 spleen DNA samples from baboons and vervet monkeys from Zambia were tested for zoonotic pathogens using genus or species-specific PCR. The amplified products were then subjected to sequencing analysis. Results: We detected three different pathogenic agents, including Anaplasma phagocytophilum in 12 samples (13.6%), Rickettsia spp. in 35 samples (39.8%) and Babesia spp. in 2 samples (2.3%). Conclusion: The continuously increasing contacts between humans and primate populations raise concerns about transmission of pathogens between these groups. Therefore, increased medical and public awareness and public health surveillance support will be required to detect and control infections caused by these agents at the interface between humans and wildlife.
• Isolation and Characterization of a Novel Alphaherpesvirus in Fruit Bats

  Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Shintaro Kobayashi, Ibenu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Ichiro Nakamura, Hirofumi Sawa, Takashi Kimura

  JOURNAL OF VIROLOGY 88 (17) 9819 - 9829 0022-538X 2014/09 [Refereed][Not invited]
   

  Bats are known to harbor emerging RNA viruses. Recent studies have used high-throughput sequencing technology to identify various virus species, including DNA viruses that are harbored by bats; however, little is known about the nature of these potentially novel viruses. Here, we report the characterization of a novel herpesvirus isolated from an Indonesian pteropodid bat. The virus, tentatively named fruit bat alphaherpesvirus 1 (FBAHV1), has a double-stranded DNA genome of 149,459 bp. The phylogenetic analyses suggested that FBAHV1 is phylogenetically grouped with simplexviruses within the subfamily Alphaherpesvirinae. Inoculation of FBAHV1 into laboratory mice caused a lethal infection. Virus infection was observed in lung, liver, and brain tissue. Serological and PCR screening revealed that fruit bats infected with FBAHV1 or its related virus are widely distributed in Indonesia. The identification of FBAHV1 makes a considerable contribution to our understanding of simplexviruses associated with bats. IMPORTANCE Bats are known to harbor emerging viruses, such as lyssaviruses, henipaviruses, severe acute respiratory syndrome-like coronaviruses, and filoviruses. Although alphaherpesviruses are disseminated in humans and other animals, there is little information about their distribution in bats. Here, we isolated a previously unknown alphaherpesvirus from an Indonesian fruit bat. Genome sequence analysis suggested that the virus is a member of the genus Simplexvirus within the subfamily Alphaherpesvirinae, which also includes common human viruses, such as herpes simplex virus 1 and herpes simplex virus 2. FBAHV1 is the first batderived alphaherpesvirus whose complete genome has been sequenced.
• Survival of rabid rabbits after intrathecal immunization

  Sawang Kesdangsakonwut, Yuji Sunden, Keisuke Aoshima, Yoshimi Iwaki, Masahiro Okumura, Hirofumi Sawa, Takashi Umemura

  NEUROPATHOLOGY 34 (3) 277 - 283 0919-6544 2014/06 [Refereed][Not invited]
   

  Rabies is a fatal zoonotic disease for which no effective treatment measures are currently available. Rabies virus (RABV) has anti-apoptotic and anti-inflammatory properties that suppress nerve cell damage and inflammation in the CNS. These features imply that the elimination of RABV from the CNS by appropriate treatment could lead to complete recovery from rabies. Ten rabbits showing neuromuscular symptoms of rabies after subcutaneous (SC) immunization using commercially available vaccine containing inactivated whole RABV particles and subsequent fixed RABV (CVS strain) inoculation into hind limb muscles were allocated into three groups. Three rabbits received no further treatment (the SC group), three rabbits received three additional SC immunizations using the same vaccine, and four rabbits received three intrathecal (IT) immunizations, in which the vaccine was inoculated directly into the cerebrospinal fluid (the SC/IT group). An additional three naive rabbits were inoculated intramuscularly with RABV and not vaccinated. The rabbits exhibited neuromuscular symptoms of rabies within 4-8 days post-inoculation (dpi) of RABV. All of the rabbits died within 8-12 dpi with the exception of one rabbit in the SC group and all four rabbits in SC/IT group, which recovered and started to respond to external stimuli at 11-18 dpi and survived until the end of the experimental period. RABV was eliminated from the CNS of the surviving rabbits. We report here a possible, although still incomplete, therapy for rabies using IT immunization. Our protocol may rescue the life of rabid patients and prompt the future development of novel therapies against rabies.
• Role of the C-Terminal Region of Vervet Monkey Polyomavirus 1 VP1 in Virion Formation

  Hiroki Yamaguchi, Shintaro Kobayashi, Junki Maruyama, Michihito Sasaki, Ayato Takada, Takashi Kimura, Hirofumi Sawa, Yasuko Orba

  JOURNAL OF VETERINARY MEDICAL SCIENCE 76 (5) 637 - 644 0916-7250 2014/05 [Refereed][Not invited]
   

  Recently, we detected novel vervet monkey polyomavirus 1 (VmPyV) in a vervet monkey. Among amino acid sequences of major capsid protein VP1s of other polyomaviruses, VmPyV VP1 is the longest with additional amino acid residues in the C-terminal region. To examine the role of VmPyV VP1 in virion formation, we generated virus-like particles (VLPs) of VmPyV VP1, because VLP is a useful tool for the investigation of the morphological characters of polyomavirus virions. After the full-length VmPyV VP1 was subcloned into a mammalian expression plasmid, the plasmid was transfected into human embryonic kidney 293T (HEK293T) cells. Thereafter, VmPyV VLPs were purified from the cell lysates of the transfected cells via sucrose gradient sedimentation. Electron microscopic analyses revealed that VmPyV VP1 forms VLPs with a diameter of approximately 50 nm that are exclusively localized in cell nuclei. Furthermore, we generated VLPs consisting of the deletion mutant VmPyV VP1 (Delta C VP1) lacking the C-terminal 116 amino acid residues and compared its VLP formation efficiency and morphology to those of VLPs from wild-type VmPyV VP1 (WT VP1). WT and Delta C VP1 VLPs were similar in size, but the number of Delta C VP1 VLPs was much lower than that of WT VP1 VLPs in VP1-expressing HEK293T cells. These results suggest that the length of VP1 is unrelated to virion morphology; however, the C-terminal region of VmPyV VP1 affects the efficiency of its VLP formation.
• Molecular Epidemiology of Paramyxoviruses in Frugivorous Eidolon helvum Bats in Zambia

  Walter Muleya, Michihito Sasaki, Yasuko Orba, Akihiro Ishii, Yuka Thomas, Emiko Nakagawa, Hirohito Ogawa, Bernard Hang'ombe, Boniface Namangala, Aaron Mweene, Ayato Takada, Takashi Kimura, Hirofumi Sawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 76 (4) 611 - 614 0916-7250 2014/04 [Refereed][Not invited]
   

  In this study, we describe the detection of novel paramyxoviruses from the Eidolon helvum species of fruit bats. We extracted RNA from 312 spleen samples from bats captured in Zambia over a period of 4 years (2008-2011). Semi-nested RT-PCR detected a total of 25 (8%) positive samples for paramyxoviruses which were then directly sequenced and analyzed using phylogenetic analysis. Among the positive samples, seven novel paramyxoviruses were detected. Five viruses were closely related to the genus Henipavirus, while two viruses were related to the unclassified Bat paramyxoviruses from Ghana and Congo Brazzaville. Our study identified novel Henipavirus-related and unrelated viruses using RT-PCR in fruit bats from Kansaka National Park and indicated the presence of similar Bat paramyxoviruses originating from wide geographic areas, suggesting the ability of bats to harbor and transmit viruses. The presence of these viruses in fruit bats might pose a public health risk.
• Molecular epidemiology of paramyxoviruses in Zambian wild rodents and shrews

  Michihito Sasaki, Walter Muleya, Akihiro Ishii, Yasuko Orba, Bernard M. Hang'ombe, Aaron S. Mweene, Ladslav Moonga, Yuka Thomas, Takashi Kimura, Hirofumi Sawa

  JOURNAL OF GENERAL VIROLOGY 95 (Pt 2) 325 - 330 0022-1317 2014/02 [Refereed][Not invited]
   

  Rodents and shrews are known to harbour various viruses. Paramyxoviruses have been isolated from Asian and Australian rodents, but little is known about them in African rodents. Recently, previously unknown paramyxovirus sequences were found in South African rodents. To date, there have been no reports related to the presence and prevalence of paramyxoviruses in shrews. We found a high prevalence of paramyxoviruses in wild rodents and shrews from Zambia. Seminested reverse transcription-PCR assays were used to detect paramyxovirus RNA in 21% (96/ 462) of specimens analysed. Phylogenetic analysis revealed that these viruses were novel paramyxoviruses and could be classified as morbillivirus- and henipavirus-related viruses, and previously identified rodent paramyxovirus-related viruses. Our findings suggest the circulation of previously unknown paramyxoviruses in African rodents and shrews, and provide new information regarding the geographical distribution and genetic diversity of paramyxoviruses.
• Establishment of tracking system for West Nile virus entry and evidence of microtubule involvement in particle transport

  Yoshinori Makino, Tadaki Suzuki, Rie Hasebe, Takashi Kimura, Akihiko Maeda, Hidehiro Takahashi, Hirofumi Sawa

  JOURNAL OF VIROLOGICAL METHODS 195 250 - 257 0166-0934 2014/01 [Refereed][Not invited]
   

  West Nile virus (WNV) is one of flaviviruses and has emerged recently in the United States as a significant cause of viral encephalitis. Although cellular entry of WNV is important for viral pathogenesis, its mechanisms have not been elucidated fully. To explore the entry mechanisms, a virus-particle tracking system in live cells by using fluorescently labeled subviral particles (SVPs) and time-lapse epifluorescence microscopy was established. This study revealed that, following cellular entry, SVP movements could be divided into two phases: early (slow movement) and late (fast movement) phase. Moreover, fast viral particle movements at the late phase correlated with SVP-microtubule association. (C) 2013 Elsevier B.V. All rights reserved.
• A novel HSP90 inhibitor, 17-DMAG, induces Tax down-regulation and its oral administration to ATL-model mice intervenes against the infiltration property of the ATL-like lymphocytes and provides extended survival period

  Emi Ikebe, Akira Kawaguchi, Kenta Tezuka, Shinya Taguchi, Satoshi Hirose, Takashi Matsumoto, Takahiro Mitsui, Kazuyo Senba, Akira Nishizono, Mitsuo Hori, Hiroo Hasegawa, Yasuaki Yamada, Takaharu Ueno, Yuetsu Tanaka, Hirofumi Sawa, William Hall, Yasuaki Minami, Kuan-Teh Jeang, Masao Ogata, Kazuhiro Morishita, Hideki Hasegawa, Jun-ichi Fujisawa, Hidekatsu Iha

  Retrovirology 11(Suppl 1) P44  2014 [Refereed][Not invited]
  
• Surveillance and diagnosis of plague and anthrax in Tanzania and Zambia

  B. M. Hang’ombe, M. Ziwa, M. Haule, I. Nakamura, K. L. Samui, D. Kaile, A. S. Mweene, B. S. Kilonzo, E. F. Lyamuya, M. Matee, C. Sugimoto, H. Sawa, B. W. Wren

  Onderstepoort Journal of Veterinary Research 81 (2) 2219-0635 2014 [Refereed][Not invited]
   

  Yersinia pestis and Bacillus anthracis are diseases that rarely occur, with devastating consequences. In Africa, despite the diseases being rare, they are reported on a yearly basis in remote areas of the continent due to lack of proper surveillance and detection systems. In Tanzania and Zambia, studies have been ongoing to understand these pathogens in endemic areas.
• [Epidemiological and basic research activity targeting polyomaviruses].

  Sawa H, Kobayashi S, Suzuki T, Orba Y

  Uirusu 64 (1) 25 - 34 0042-6857 2014 [Refereed][Not invited]
  
• Viroporin activity of the JC polyomavirus is regulated by interactions with the adaptor protein complex 3

  Tadaki Suzuki, Yasuko Orba, Yoshinori Makino, Yuki Okada, Yuji Sunden, Hideki Hasegawa, William W. Hall, Hirofumi Sawa

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 (46) 18668 - 18673 0027-8424 2013/11 [Refereed][Not invited]
   

  Viroporins, which are encoded by a wide range of animal viruses, oligomerize in host cell membranes and form hydrophilic pores that can disrupt a number of physiological properties of the cell. Little is known about the relationship between host cell proteins and viroporin activity. The human JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy. The JCV-encoded agnoprotein, which is essential for viral replication, has been shown to act as a viroporin. Here we demonstrate that the JCV agnoprotein specifically interacts with adaptor protein complex 3 through its delta subunit. This interaction interrupts adaptor protein complex 3-mediated vesicular trafficking with suppression of the targeting of the protein to the lysosomal degradation pathway and instead permits the transport of agnoprotein to the cell surface with resulting membrane permeabilization. The findings demonstrate a previously undescribed paradigm in virus-host interactions allowing the host to regulate viroporin activity and suggest that the viroporins of other viruses may also be highly regulated by specific interactions with host cell proteins.
• Cysteine Residues in the Major Capsid Protein, Vp1, of the JC Virus Are Important for Protein Stability and Oligomer Formation

  Shintaro Kobayashi, Tadaki Suzuki, Manabu Igarashi, Yasuko Orba, Noriko Ohtake, Keita Nagakawa, Kenichi Niikura, Takashi Kimura, Harumi Kasamatsu, Hirofumi Sawa

  PLoS ONE 8 (10) e76668  1932-6203 2013/10/09 [Refereed][Not invited]
   

  The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.
• Characterization of Japanese encephalitis virus infection in an immortalized mesencephalic cell line, CSM14.1

  Takashi Kimura, Megumi Okumura, Eunmi Kim, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa

  MICROBIOLOGY AND IMMUNOLOGY 57 (10) 723 - 731 0385-5600 2013/10 [Refereed][Not invited]
   

  Neurons are the major target cell of Japanese encephalitis virus (JEV). Rats intracerebrally inoculated with JEV show an age-dependent pattern of resistance to infection in which resistance is closely associated with neuronal maturation. However, because there is no reliable and convenient cell culture system that mimics the in vivo properties of JEV infection of immature and mature neurons, the mechanisms underlying this association remain poorly understood. The aim of the present study was to examine JEV infection in immortalized CSM14.1 rat neuronal cells, which can be induced to differentiate into neurons by culture under non-permissive conditions. JEV infected undifferentiated CSM14.1 cells more efficiently than differentiated cells, resulting in production of more progeny virus in the former setting than in the latter. An infectious virus recovery assay detected more internalized virions in undifferentiated cells. On the other hand, JEV infection of differentiated cells induced more rapid and stronger expression of interferon- gene, along with smaller amounts of JEV RNA. Taken together, these results show that the initial phase of viral infection and the later IFN response play roles in the viral susceptibility of undifferentiated and differentiated CSM14.1 cells. Because CSM14.1 cells became more resistant to JEV infection as they mature, this culture system can be used as an in vitro model for studying age-dependent resistance of neurons to JEV infection.
• Human Parainfluenza Virus Type 3 in Wild Nonhuman Primates, Zambia

  Michihito Sasaki, Akihiro Ishii, Yasuko Orba, Yuka Thomas, Bernard M. Hang'ombe, Ladslav Moonga, Aaron S. Mweene, Hirohito Ogawa, Ichiro Nakamura, Takashi Kimura, Hirofumi Sawa

  EMERGING INFECTIOUS DISEASES 19 (9) 1500 - 1503 1080-6040 2013/09 [Refereed][Not invited]
   

  Human parainfluenza virus type 3 (HPIV3) genome was detected in 4 baboons in Zambia. Antibody for HPIV3 was detected in 13 baboons and 6 vervet monkeys in 2 distinct areas in Zambia. Our findings suggest that wild nonhuman primates are susceptible to HPIV3 infection.
• De novo sequence analysis of cytochrome P450 1-3 genes expressed in ostrich liver with highest expression of CYP2G19

  Yusuke K. Kawai, Kensuke P. Watanabe, Akihiro Ishii, Aiko Ohnuma, Hirofumi Sawa, Yoshinori Ikenaka, Mayumi Ishizuka

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY D-GENOMICS & PROTEOMICS 8 (3) 201 - 208 1744-117X 2013/09 [Refereed][Not invited]
   

  The cytochrome P450 (CYP) 1-3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1-3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2-10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism. (C) 2013 Elsevier Inc. All rights reserved.
• Oral administration of an HSP90 inhibitor, 17-DMAG, intervenes tumor-cell infiltration into multiple organs and improves survival period for ATL model mice

  E. Ikebe, A. Kawaguchi, K. Tezuka, S. Taguchi, S. Hirose, T. Matsumoto, T. Mitsui, K. Senba, A. Nishizono, M. Hori, H. Hasegawa, Y. Yamada, T. Ueno, Y. Tanaka, H. Sawa, W. Hall, Y. Minami, K. T. Jeang, M. Ogata, K. Morishita, H. Hasegawa, J. Fujisawa, H. Iha

  Blood Cancer Journal 3 (8) e132  2044-5385 2013/08 [Refereed][Not invited]
   

  In the peripheral blood leukocytes (PBLs) from the carriers of the human T-lymphotropic virus type-1 (HTLV-1) or the patients with adult T-cell leukemia (ATL), nuclear factor kappaB (NF-κB)-mediated antiapoptotic signals are constitutively activated primarily by the HTLV-1-encoded oncoprotein Tax. Tax interacts with the I κB kinase regulatory subunit NEMO (NF-κB essential modulator) to activate NF-κB, and this interaction is maintained in part by a molecular chaperone, heat-shock protein 90 (HSP90), and its co-chaperone cell division cycle 37 (CDC37). The antibiotic geldanamycin (GA) inhibits HSP90's ATP binding for its proper interaction with client proteins. Administration of a novel water-soluble and less toxic GA derivative, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), to Tax-expressing ATL-transformed cell lines, C8166 and MT4, induced significant degradation of Tax. 17-DMAG also facilitated growth arrest and cellular apoptosis to C8166 and MT4 and other ATL cell lines, although this treatment has no apparent effects on normal PBLs. 17-DMAG also downregulated Tax-mediated intracellular signals including the activation of NF-κB, activator protein 1 or HTLV-1 long terminal repeat in Tax-transfected HEK293 cells. Oral administration of 17-DMAG to ATL model mice xenografted with lymphomatous transgenic Lck-Tax (Lck proximal promoter-driven Tax transgene) cells or HTLV-1-producing tumor cells dramatically attenuated aggressive infiltration into multiple organs, inhibited de novo viral production and improved survival period. These observations identified 17-DMAG as a promising candidate for the prevention of ATL progression.© 2013 Macmillan Publishers Limited All rights reserved.
• ラット中脳由来神経細胞株CSM14.1における日本脳炎ウイルス感染様式

  木村 享史, 奥村 恵, 金 恩美, 佐々木 道仁, 大場 靖子, 澤 洋文

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 156回 214 - 214 1347-8621 2013/08
• オートファジーによるウエストナイルウイルス増殖抑制機構の解明

  小林 進太郎, 大場 靖子, 山口 宏樹, 佐々木 道仁, 長谷部 理絵, 木村 享史, 澤 洋文

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 156回 267 - 267 1347-8621 2013/08
• インドネシア共和国に生息するオオコウモリから分離した新規ヘルペスウイルスの性状解析

  佐々木 道仁, Agus Setiyono, Ekowati Handharyani, 中村 一郎, 澤 洋文, 木村 享史

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 156回 283 - 283 1347-8621 2013/08
• Cross-Reactivity of Secondary Antibodies against African Rodents and Application for Sero-Surveillance

  Ichiro Nakamura, Bernard Mudenda Hang'ombe, Hirofumi Sawa, Shintaro Kobayashi, Yasuko Orba, Akihiro Ishii, Yuka Thomas, Rie Isozumi, Kumiko Yoshimatsu, Aaron S. Mweene, Ayato Takada, Chihiro Sugimoto, Jiro Arikawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 75 (6) 819 - 825 0916-7250 2013/06 [Refereed][Not invited]
   

  A total of 466 rodents were captured in the Republic of Zambia from 2006 to 2010. Based on morphological observations and phylogenetic analyses of mitochondrial gene sequences, rodents were divided into 10 groups consisting of 39 Rattus rodents, 263 multimammate rats, 18 other Murinae rodents, 95 gerbils, 11 pouched mice, 1 giant-pouched rat, 38 fat mice and 1 dormouse. Rodent antibodies except that from Rattus were examined for their cross-reactivity to commercially available antibody detection reagents. Anti-mouse immunoglobulin G (IgG) was most cross-reactive to heterologous antibodies including multimammate rat, gerbil, pouched mouse and fat mouse. Thus, anti-mouse IgG would be a useful detection tool in serological examination of the Zambian rodent population. Preliminary sero-surveillance for plague, leptospirosis and hantavirus infection was performed by ELISA.
• Identification of a novel polyomavirus from vervet monkeys in Zambia

  Hiroki Yamaguchi, Shintaro Kobayashi, Akihiro Ishii, Hirohito Ogawa, Ichiro Nakamura, Ladslav Moonga, Bernard M. Hang'ombe, Aaron S. Mweene, Yuka Thomas, Takashi Kimura, Hirofumi Sawa, Yasuko Orba

  Journal of General Virology 94 (6) 1357 - 1364 0022-1317 2013/06/01 [Refereed][Not invited]
   

  To examine polyomavirus (PyV) infection in wildlife, we investigated the presence of PyVs in Zambia with permission from the Zambia Wildlife Authority. We analysed 200 DNA samples from the spleens and kidneys (n5100 each) of yellow baboons and vervet monkeys (VMs) (n550 each). We detected seven PyV genome fragments in 200 DNA samples using a nested broadspectrum PCR method, and identified five full-length viral genomes using an inverse PCR method. Phylogenetic analysis of virally encoded proteins revealed that four PyVs were closely related to either African green monkey PyV or simian agent 12. Only one virus detected from a VM spleen was found to be related, with relatively low nucleotide sequence identity (74 %), to the chimpanzee PyV, which shares 48% nucleotide sequence identity with the human Merkel cell PyV identified from Merkel cell carcinoma. The obtained entire genome of this virus was 5157 bp and had large T- and small t-antigens, and VP1 and VP2 ORFs. This virus was tentatively named vervet monkey PyV 1 (VmPyV1) as a novel PyV. Comparison with other PyVs revealed that VmPyV1, like chimpanzee PyV, had a longer VP1 ORF. To examine whether the VmPyV1 genome could produce viral proteins in cultured cells, the whole genome was transfected into HEK293T cells. We detected VP1 protein expression in the transfected HEK293T cells by immunocytochemical and immunoblot analyses. Thus, we identified a novel PyV genome from VM spleen. © 2013 SGM.
• Gold Nanoparticles as a Vaccine Platform: Influence of Size and Shape on Immunological Responses in Vitro and in Vivo

  Kenichi Niikura, Tatsuya Matsunaga, Tadaki Suzuki, Shintaro Kobayashi, Hiroki Yamaguchi, Yasuko Orba, Akira Kawaguchi, Hideki Hasegawa, Kiichi Kajino, Takafumi Ninomiya, Kuniharu Ijiro, Hirofumi Sawa

  ACS NANO 7 (5) 3926 - 3938 1936-0851 2013/05 [Refereed][Not invited]
   

  This paper demonstrates how the shape and size of gold nanoparticles (AuNPs) affect immunological responses in vivo and in vitro for the production of antibodies for West Nile virus (WNV). We prepared spherical (20 and 40 nm in diameter), rod (40 x 10 nm), and cubic (40 x 40 x 40 nm) AuNPs as adjuvants and coated them with WNV envelope (E) protein. We measured anti-WNVE antibodies after inoculation of these WNVE-coated AuNPs (AuNP-Es) into mice. The 40 nm spherical AuNP-Es (Sphere40-Es) induced the highest level of WNVE-specific antibodies, while rod AuNP-Es (Rod-Es) induced only 50% of that of Sphere40-E. To examine the mechanisms of the shape-dependent WNVE antibody production, we next measured the efficiency of cellular uptake of AuNP-Es into RAW264.7 macrophage cells and bone-marrow-derived dendritic cells (BMDCs) and the subsequent cytokine secretion from BMDCs. The uptake of Rod-Es into the cells proceeded more efficiently than those of Sphere-Es or cubic WNVE-coated AuNPs (Cube-Es), suggesting that antibody production was not dependent on the uptake efficiency of the different AuNP-Es. Cytokine production from BMDCs treated with the AuNP-Es revealed that only Rod-E-treated cells produced significant levels of interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18), indicating that Rod-Es activated inflammasome-dependent cytokine secretion. Meanwhile, Sphere40-Es and Cube-Es both significantly induced inflammatory cytokine production, including tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-12, and granulocyte macrophage colony-stimulating factor (GM-CSF). These results suggested that AuNPs are effective vaccine adjuvants and enhance the immune response via different cytokine pathways depending on their sizes and shapes.
• Virus-like particles with removable cyclodextrins enable glutathione-triggered drug release in cells

  Kenichi Niikura, Naotoshi Sugimura, Yusuke Musashi, Shintaro Mikuni, Yasutaka Matsuo, Shintaro Kobayashi, Keita Nagakawa, Shuko Takahara, Chie Takeuchi, Hirofumi Sawa, Masataka Kinjo, Kuniharu Ijiro

  Molecular BioSystems 9 (3) 501 - 507 1742-206X 2013/03 [Refereed][Not invited]
   

  The efficient delivery of hydrophobic drugs into target cells without the use of organic solvents or chemical linkage to delivery carriers is an important theme in the biomedical and pharmaceutical field. In this study, we synthesized virus-like particles (VLPs) coupled with cyclodextrins (CDs) as hydrophobic pockets through disulfide bonds inside the VLPs, where hydrophobic drugs can be incorporated. We report here the intracellular delivery of hydrophobic dyes or drugs encapsulated in VLPs through CDs with high efficiency and their subsequent release in cells in response to glutathione. As a model anticancer drug, paclitaxel (PTX)-CD complexes were encapsulated inside VLPs and the cytotoxic drug activity of PTX loaded VLPs against NIH3T3 cells was evaluated by CCK-8 assay. PTX-loaded VLPs exhibited a dose-dependent cytotoxic effect with a 20-fold smaller IC50 than that of free PTX dissolved in DMSO. These results indicate that VLPs with removable CDs afford highly promising carriers of hydrophobic drugs without chemical modification of drugs. © 2013 The Royal Society of Chemistry.
• Relationship between Methyl CpG Binding Protein 2 and JC Viral Proteins

  Kenta Takahashi, Yasuko Orba, Taichi Kimura, Lei Wang, Shinji Kohsaka, Masumi Tsuda, Mishie Tanino, Hiroshi Nishihara, Kazuo Nagashima, Hirofumi Sawa, Shinya Tanaka

  JAPANESE JOURNAL OF INFECTIOUS DISEASES 66 (2) 126 - 132 1344-6304 2013/03 [Refereed][Not invited]
   

  JC virus (JCV) is a causative agent of progressive multifocal leukoencephalopathy (PML). Methyl CpG binding protein 2 (MeCP2) is a transcriptional control nuclear protein that is abundantly expressed in neurons. We previously observed that the MeCP2 protein is expressed in JCV large T antigen (TAg)-expressing glial cells in PML brains. To investigate the relationship between MeCP2 and JCV TAg, we examined the promoter activity and mRNA and protein expression levels of MeCP2 in JCV TAg-expressing cells. We found that JCV TAg enhances the promoter activity of MeCP2, but does not enhance the mRNA and protein levels of MeCP2. These results suggest that post-transcriptional mechanisms may play a role in MeCP2 expression.
• Population genetic analysis and sub-structuring of Theileria parva in the northern and eastern parts of Zambia

  Walter Muleya, Boniface Namangala, Martin Simuunza, Ryo Nakao, Noboru Inoue, Takashi Kimura, Kimihito Ito, Chihiro Sugimoto, Hirofumi Sawa

  PARASITES & VECTORS 5 255  1756-3305 2012/11 [Refereed][Not invited]
   

  Background: Theileriosis, caused by Theileria parva, is an economically important disease in Africa. It is a major constraint to the development of the livestock industry in some parts of eastern, central and southern Africa. In Zambia, theileriosis causes losses of up to 10,000 cattle annually. Methods: Cattle blood samples were collected for genetic analysis of Theileria parva from Isoka and Petauke districts in Zambia. Microsatellite analysis was then performed on all Theileria parva positive samples for PCR using a panel of 9 microsatellite markers. Microsatellite data was analyzed using microsatellite toolkit, GenAlEx ver. 6, Fstat ver. 2.9.3.2, and LIAN computer softwares. Results: The combined percentage of positive samples in both districts determined by PCR using the p104 gene primers was 54.9% (95% CI: 46.7 - 63.1%, 78/142), while in each district, it was 44.8% (95% CI: 34.8 - 54.8%) and 76.1% (95% CI = 63.9 - 88.4%) for Isoka and Petauke districts, respectively. We analyzed the population genetic structure of Theileria parva from a total of 61 samples (33 from Isoka and 28 from Petauke) using a panel of 9 microsatellite markers encompassing the 4 chromosomes of Theileria parva. Wright's F index (F-ST = 0.178) showed significant differentiation between the Isoka and Petauke populations. Linkage disequilibrium was observed when populations from both districts were treated as a single population. When analyzed separately, linkage disequilibrium was observed in Kanyelele and Kalembe areas in Isoka district, Isoka district overall and in Petauke district. Petauke district had a higher multiplicity of infection than Isoka district. Conclusion: Population genetic analyses of Theileria parva from Isoka and Petauke districts showed a low level of genotype exchange between the districts, but a high level of genetic diversity within each district population, implying genetic and geographic sub-structuring between the districts. The sub-structuring observed, along with the lack of panmixia in the populations, could have been due to low transmission levels at the time of sampling. However, the Isoka population was less diverse than the Petauke population.
• Molecular surveillance and phylogenetic analysis of Old World arenaviruses in Zambia

  Akihiro Ishii, Yuka Thomas, Ladslav Moonga, Ichiro Nakamura, Aiko Ohnuma, Bernard M. Hang'ombe, Ayato Takada, Aaron S. Mweene, Hirofumi Sawa

  JOURNAL OF GENERAL VIROLOGY 93 (10) 2247 - 2251 0022-1317 2012/10 [Refereed][Not invited]
   

  In order to survey arenaviruses in the Republic of Zambia, we captured 335 rodents from three cities between 2010 and 2011. Eighteen Luna virus (LUNV) and one lymphocytic choriomeningitis virus (LCMV)-related virus RNAs were detected by one-step RT-PCR from Mastomys natalensis and Mus minutoides, respectively. Four LUNV strains and one LCMV-related virus were isolated, and the whole genome nucleotide sequence was determined by pyrosequencing. Phylogenetic analyses revealed that the LUNV clade consists of two branches that are distinguished by geographical location and that the LCMV-related virus belongs to the LCMV clade, but diverges from the typical LCMVs. Comparison of nucleoprotein amino acid sequences indicated that the LCMV-related virus could be designated a novel arenavirus, which was tentatively named as the Lunk virus. Amino acid sequences of the GP, NP, Z and L proteins showed poor similarity among the three Zambian arenavirus strains, i.e. Luna, Lunk and Lujo virus.
• Molecular detection of a novel paramyxovirus in fruit bats from Indonesia

  Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Ibenu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Hirofumi Sawa, Ichiro Nakamura, Takashi Kimura

  VIROLOGY JOURNAL 9 240  1743-422X 2012/10 [Refereed][Not invited]
   

  Background: Fruit bats are known to harbor zoonotic paramyxoviruses including Nipah, Hendra, and Menangle viruses. The aim of this study was to detect the presence of paramyxovirus RNA in fruit bats from Indonesia. Methods: RNA samples were obtained from the spleens of 110 fruit bats collected from four locations in Indonesia. All samples were screened by semi-nested broad spectrum reverse transcription PCR targeting the paramyxovirus polymerase (L) genes. Results: Semi-nested reverse transcription PCR detected five previously unidentified paramyxoviruses from six fruit bats. Phylogenetic analysis showed that these virus sequences were related to henipavirus or rubulavirus. Conclusions: This study indicates the presence of novel paramyxoviruses among fruit bat populations in Indonesia.
• Role of JC virus agnoprotein in virion formation

  Tadaki Suzuki, Shingo Semba, Yuji Sunden, Yasuko Orba, Shintaro Kobayashi, Kazuo Nagashima, Takashi Kimura, Hideki Hasegawa, Hirofumi Sawa

  MICROBIOLOGY AND IMMUNOLOGY 56 (9) 639 - 646 0385-5600 2012/09 [Refereed][Not invited]
   

  JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T' antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein-negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus-like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S-transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1-pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.
• Pathological Examination of Lung Tissues in Influenza A Virus-Infected Mice

  Nilton Akio Muto, Yuji Sunden, Tomoe Hattori, Daisuke Fujikura, Yosuke Nakayama, Tadaaki Miyazaki, Mitsuo Maruyama, Takashi Kimura, Hirofumi Sawa

  JAPANESE JOURNAL OF INFECTIOUS DISEASES 65 (5) 383 - 391 1344-6304 2012/09 [Refereed][Not invited]
   

  This study examined pathological changes in the lung tissues of young and aged mice infected with influenza virus. Young mice inoculated with influenza virus showed body weight loss at 4 days post-infection (dpi), meanwhile body weight decrease started from 9 dpi in the aged mice. We histopathologically examined the lungs of these mice. Immunohistochemical examination revealed that viral antigen-positive bronchiolar and alveolar epithelial cell numbers at 3 dpi were significantly higher in young mice than in the aged ones. Further, viral antigen-positive cells were observed at 9 dpi in the aged mice, but not in the young ones. Diffuse and severe bronchointerstitial pneumonia characterized by the accumulation of polymorphonuclear leukocytes (PMNs) was observed in young mice at 6 dpi. Histopathological changes in the aged mice were milder than those in the young mice. Moreover, T cell and macrophage accumulation in the lungs was significantly higher in the young mice than in the aged mice at 9 dpi. These results suggest that there may be a correlation between the relatively low level of infiltration of PMNs, macrophages, and T lymphocytes and the delayed body weight loss and longer lasting infections observed in the lungs of the aged mice. These findings provide detailed insights into the age-specific course of infection in young and aged populations with associated differences in lung pathology.
• Accumulation of ubiquitinated proteins is related to West Nile virus-induced neuronal apoptosis

  Shintaro Kobayashi, Yasuko Orba, Hiroki Yamaguchi, Takashi Kimura, Hirofumi Sawa

  NEUROPATHOLOGY 32 (4) 398 - 405 0919-6544 2012/08 [Refereed][Not invited]
   

  West Nile virus (WNV) belongs to the Flaviviridae family of viruses and has emerged as a significant cause of viral encephalitis in humans, animals and birds. It has been reported that WNV replication directly induces neuronal injury, followed by neuronal cell death proven as apoptosis. Therefore, it is important to understand the mechanism of neuronal apoptosis caused by this virus to develop strategies to control its pathogenicity. Accumulation of ubiquitinated abnormal proteins has been reported to be associated with neuronal apoptosis in some pathological conditions. A lot of cellular stresses prevent cellular protein quality control mechanisms, resulting in the accumulation of ubiquitinated abnormal proteins. To obtain a better understanding of the mechanisms of WNV-induced neuronal apoptosis, we evaluated the accumulation of ubiquitinated proteins in the WNV-infected neuronal cells. We have observed that WNV infection caused massive neuronal injury in the brains of mice. Viral antigen was detected in the neuronal cytoplasm of the cells exhibiting neuronal apoptosis. Notably, ubiquitinated proteins were detected in WNV-infected neuronal cells. In addition, accumulation of ubiquitinated proteins was markedly enhanced in mouse neuroblastoma, Neuro-2a cells after WNV infection. Our histopathological and in vitro studies suggest that accumulation of ubiquitinated proteins in neuronal cells might be associated with neuronal apoptosis caused by WNV infection.
• Inhibitory effects of an M2-specific monoclonal antibody on different strains of influenza A virus

  Nilton Akio Muto, Reiko Yoshida, Tadaki Suzuki, Shintaro Kobayashi, Hiroichi Ozaki, Daisuke Fujikura, Rashid Manzoor, Mieko Muramatsu, Ayato Takada, Takashi Kimura, Hirofumi Sawa

  JAPANESE JOURNAL OF VETERINARY RESEARCH 60 (2-3) 71 - 83 0047-1917 2012/08 [Refereed][Not invited]
   

  New approaches to the treatment of influenza have been designed based on the highly conserved antigenicity of the M2 envelope protein among influenza A virus strains. The present study examined the anti-viral activities of an anti-M2 ectodomain (M2e) monoclonal antibody (clone rM2ss23), which binds to the M2 proteins of the influenza A virus strains A/Aichi/2/68 (H3N2) (Aichi) and A/PR/8/34 (H1N1) (PR8). The results showed that rM2ss23 bound to both Aichi and PR8 M2 proteins expressed on the cell surface. While the antibody did not prevent virus entry into cells, it significantly inhibited plaque formation by the Aichi strain in a dose-dependent manner when infected cells were cultured in the presence of the antibody. By contrast, the growth of PR8 (H1N1) was not affected by the antibody. A reverse genetics approach revealed that the inhibitory effect of rM2ss23 on the Aichi virus was abolished by replacing the genes encoding the HA and/or M proteins with those of the PR8 strain. These results suggest that rM2ss23 prevents virus release from infected cells and further suggest that the mechanisms underlying the virus budding mediates by HA and M2 proteins might differ between the Aichi and PR8 strains.
• Human-animal anthrax outbreak in the Luangwa valley of Zambia in 2011

  Mudenda B. Hang'ombe, James C. L. Mwansa, Sergio Muwowo, Phillip Mulenga, Muzala Kapina, Eric Musenga, David Squarre, Liywali Mataa, Suzuki Y. Thomas, Hirohito Ogawa, Hirofumi Sawa, Hideaki Higashi

  TROPICAL DOCTOR 42 (3) 136 - 139 0049-4755 2012/07 [Refereed][Not invited]
   

  There has been a reduction of incidences of anthrax in the developed countries but it is still a public health problem in the developing countries where communities live in interface areas with wildlife. An outbreak of anthrax in Hippopotamus amphibious was observed in Zambia. Following the death of hippopotamuses, suspected human cases were reported. The objective of this study was to isolate and confirm Bacillus anthracis and to determine the antimicrobial susceptibility for the management of the disease. Of the specimens collected, 29.4% (95% confidence interval [CI], 11.4-56.0) were from humans, 42.1% (95% CI, 21.1-66.0) were from hippopotamuses and 20.0% (95% CI, 6.61-44.3) from the soil were found to be positive were for B. anthracis. An antimicrobial susceptibility test revealed that all the isolates were found to be sensitive to the recommended antibiotics. The disease control was achieved by case management and by explaining to the communities that they should avoid contact with animals that die from unknown causes.
• Alpha-Defensin Overexpression in Patients With Bell's Palsy and Ramsay Hunt Syndrome

  Yasushi Furuta, Nobuhiko Oridate, Norihito Takeichi, Satoshi Fukuda, Hirofumi Sawa

  ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY 121 (6) 419 - 425 0003-4894 2012/06 [Refereed][Not invited]
   

  Objectives: To investigate the biological factors related to the onset of Bell's palsy, we sought to identify differentially expressed genes in peripheral blood mononuclear cells (PBMCs) and plasma of patients with Bell's palsy and Ramsay Hunt syndrome (RHS). Methods: We carried out DNA microarray analyses using PBMCs taken from patients with Bell's palsy at their initial visit and 2 to 4 weeks later. To validate these analyses, we measured the relative messenger RNA levels of alpha-defensin in paired PBMCs by reverse transcription-polymerase chain reaction. The plasma concentrations of alpha-defensin in patients and healthy volunteers were quantified by enzyme-linked immunosorbent assay. Results: The DNA microarray analysis identified alpha-defensin as a candidate gene related to the onset of Bell's palsy. Reverse transcription polymerase chain reaction analysis showed that the relative alpha-defensin messenger RNA levels in PBMCs from the later visit were increased at least twofold in 9 of 13 patients (69%) with Bell's palsy and in 4 of 6 patients (67%) with RHS. The plasma alpha-defensin concentrations in the patients with RHS were significantly higher than those in healthy volunteers (p = 0.0013) and in the patients with Bell's palsy (p = 0.0306). Elevations of plasma alpha-defensin were observed in 5 of the 9 patients with Bell's palsy who demonstrated alpha-defensin overexpression in PBMCs. Conclusions: alpha-Defensin may be one of the biological factors related to the onset of Bell's palsy and RHS.
• The sphingosine-1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice

  Shigetomo Fukuhara, Szandor Simmons, Shunsuke Kawamura, Asuka Inoue, Yasuko Orba, Takeshi Tokudome, Yuji Sunden, Yuji Arai, Kazumasa Moriwaki, Junji Ishida, Akiyoshi Uemura, Hiroshi Kiyonari, Takaya Abe, Akiyoshi Fukamizu, Masanori Hirashima, Hirofumi Sawa, Junken Aoki, Masaru Ishii, Naoki Mochizuki

  JOURNAL OF CLINICAL INVESTIGATION 122 (4) 1416 - 1426 0021-9738 2012/04 [Refereed][Not invited]
   

  The bioactive lysophospholipid mediator sphingosine-l-phosphate (SIP) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how SIP is released. Here, we show that in mice, the SIP transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in SW release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.
• JC Virus Encephalopathy Is Associated with a Novel Agnoprotein-Deletion JCV Variant

  Xin Dang, Christian Wuethrich, Jennifer Gordon, Hirofumi Sawa, Igor J. Koralnik

  PLOS ONE 7 (4) e35793  1932-6203 2012/04 [Refereed][Not invited]
   

  JC virus encephalopathy (JCVE) is a newly described gray matter disease of the brain caused by productive infection of cortical pyramidal neurons. We characterized the full length sequence of JCV isolated from the brain of a JCVE patient, analyzed its distribution in various compartments by PCR, and determined viral gene expression in the brain by immunohistochemistry(IHC). We identified a novel JCV variant, JCV(CPN1), with a unique 143 bp deletion in the Agno gene encoding a truncated 10 amino acid peptide, and harboring an archetype-like regulatory region. This variant lacked one of three nuclear protein binding regions in the Agno gene. It was predominant in the brain, where it coexisted with an Agno-intact wild-type strain. Double immunostaining with anti-Agno and anti-VP1 antibodies demonstrated that the truncated JCVCPN1 Agno peptide was present in the majority of cortical cells productively infected with JCV. We then screened 68 DNA samples from 8 brain, 30 CSF and 30 PBMC samples of PML patients, HIV+and HIV-control subjects. Another JCV(CPN) strain with a different pattern of Agno-deletion was found in the CSF of an HIV+/PML patient, where it also coexisted with wildtype, Agno-intact JCV. These findings suggest that the novel tropism for cortical pyramidal neurons of JCV(CPN1,) may be associated with the Agno deletion. Productive and lytic infection of these cells, resulting in fulminant JCV encephalopathy and death may have been facilitated by the co-infection with a wild-type strain of JCV.
• A case of clear cell variant of solid-pseudopapillary tumor of the pancreas in an adult male patient.

  Tanino M, Kohsaka S, Kimura T, Tabu K, Nishihara H, Sawa H, Kawami H, Kamada H, Shimizu M, Tanaka S

  Annals of diagnostic pathology 16 (2) 134 - 140 1092-9134 2012/04 [Refereed][Not invited]
  
• Molecular epidemiology and a loop-mediated isothermal amplification method for diagnosis of infection with rabies virus in Zambia

  Walter Muleya, Boniface Namangala, Aaron Mweene, Luke Zulu, Paul Fandamu, Douglas Banda, Takashi Kimura, Hirofumi Sawa, Akihiro Ishii

  VIRUS RESEARCH 163 (1) 160 - 168 0168-1702 2012/01 [Refereed][Not invited]
   

  The National Livestock Epidemiology and Information Center (NALEIC) in Zambia reported over 132 cases of canine rabies diagnosed by the direct fluorescent antibody test (DFAT) from 2004 to 2009. In this study, the lineage of rabies virus (RABV) in Zambia was determined by phylogenetic analysis of the nucleoprotein (N) and glycoprotein (G) gene sequences. Total RNA was extracted from 87-DFAT brain specimens out of which only 35 (40%) were positive on nested reverse transcription polymerase chain reaction (RT-PCR) for each gene, and 26 being positive for both genes. Positive specimens for the N (n =33) and G (n = 35) genes were used for phylogenetic analysis. Phylogenetic analysis of the N gene showed two phylogenetic clusters in Zambia belonging to the Africa 1b lineage present in eastern and southern Africa. While one cluster exclusively comprised Zambian strains, the other was more heterogeneous regarding the RABV origins and included strains from Tanzania, Mozambique and Zambia. Phylogenetic analysis of the G gene revealed similar RABV strains in different hosts and regions of Zambia. We designed primers for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay from the consensus sequence of the N gene in an attempt to improve the molecular diagnosis of RABV in Zambia. The specificity and reproducibility of the RI-LAMP assay was confirmed with actual clinical specimens. Therefore, the RI-LAMP assay presented in this study may prove to be useful for routine diagnosis of rabies in Zambia. (C) 2011 Elsevier B.V. All rights reserved.
• Evidence of Yersinia pestis DNA from fleas in an endemic plague area of Zambia

  Bernard M Hang'Ombe, Ichiro Nakamura, Kenny L Samui, Davy Kaile, Aaron S Mweene, Bukheti S Kilonzo, Hirofumi Sawa, Chihiro Sugimoto, Brendan W Wren

  BMC Research Notes 5 72  1756-0500 2012 [Refereed][Not invited]
   

  Background: Yersinia pestis is a bacterium that causes plague which infects a variety of mammals throughout the world. The disease is usually transmitted among wild rodents through a flea vector. The sources and routes of transmission of plague are poorly researched in Africa, yet remains a concern in several sub-Saharan countries. In Zambia, the disease has been reported on annual basis with up to 20 cases per year, without investigating animal reservoirs or vectors that may be responsible in the maintenance and propagation of the bacterium. In this study, we undertook plague surveillance by using PCR amplification of the plasminogen activator gene in fleas. Findings. Xenopsylla species of fleas were collected from 83 rodents trapped in a plague endemic area of Zambia. Of these rodents 5 had fleas positive (6.02%) for Y. pestis plasminogen activator gene. All the Y. pestis positive rodents were gerbils. Conclusions: We conclude that fleas may be responsible in the transmission of Y. pestis and that PCR may provide means of plague surveillance in the endemic areas of Zambia. © 2012 Hang'ombe et al BioMed Central Ltd.
• Virus Capsid Coating of Gold Nanoparticles via Cysteine-Au Interactions and their Effective Cellular Uptakes.

  Nagakawa K, Niikura K, Suzuki T, Matsuo Y, Igarashi M, Sawa H, Ijiro K

  Chemistry Letters 41 (1) 113 - 115 0366-7022 2012 [Refereed][Not invited]
   

  This manuscript describes the synthesis of virus capsid protein-coated Au nanoparticle (VP-AuNP) without the use of the inherent self-assembly of virus proteins into virus particles. Covalent binding between Au and cysteines in the virus proteins keep the cell-surface binding sites on the external surface. Based on this method, various sizes of VP-AuNP can be created in a similar manner to native virus particles. We clarified the optimum size of the VP-AuNP for internalization into cells.
• 難治性神経感染症update 進行性多巣性白質脳症

  水澤 英洋, 岸田 修二, 西條 政幸, 雪下 基弘, 宍戸 由紀子[原], 澤 洋文, 長嶋 和郎, 奴久妻 聡一, 山田 正仁

  臨床神経学 (一社)日本神経学会 51 (11) 1051 - 1057 0009-918X 2011/11 [Not refereed][Not invited]
  
• Single Amino Acid Residue in the A2 Domain of Major Histocompatibility Complex Class I Is Involved in the Efficiency of Equine Herpesvirus-1 Entry

  Michihito Sasaki, Eunmi Kim, Manabu Igarashi, Kimihito Ito, Rie Hasebe, Hideto Fukushi, Hirofumi Sawa, Takashi Kimura

  JOURNAL OF BIOLOGICAL CHEMISTRY 286 (45) 39370 - 39378 0021-9258 2011/11 [Refereed][Not invited]
   

  Equine herpesvirus-1 (EHV-1), an alpha-herpesvirus of the family Herpesviridae, causes respiratory disease, abortion, and encephalomyelitis in horses. EHV-1 utilizes equine MHC class I molecules as entry receptors. However, hamster MHC class I molecules on EHV-1-susceptible CHO-K1 cells play no role in EHV-1 entry. To identify the MHC class I molecule region that is responsible for EHV-1 entry, domain exchange and site-directed mutagenesis experiments were performed, in which parts of the extracellular region of hamster MHC class I (clone C5) were replaced with corresponding sequences from equine MHC class I (clone A68). Substitution of alanine for glutamine at position 173 (Q173A) within the alpha 2 domain of the MHC class I molecule enabled hamster MHC class I C5 to mediate EHV-1 entry into cells. Conversely, substitution of glutamine for alanine at position 173 (A173Q) in equine MHC class I A68 resulted in loss of EHV-1 receptor function. Equine MHC class I clone 3.4, which possesses threonine at position 173, was unable to act as an EHV-1 receptor. Substitution of alanine for threonine at position 173 (T173A) enabled MHC class I 3.4 to mediate EHV-1 entry into cells. These results suggest that the amino acid residue at position 173 of the MHC class I molecule is involved in the efficiency of EHV-1 entry.
• A novel function of the N-terminal domain of PA in assembly of influenza A virus RNA polymerase

  Tadaki Suzuki, Akira Ainai, Noriyo Nagata, Tetsutaro Sata, Hirofumi Sawa, Hideki Hasegawa

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 414 (4) 719 - 726 0006-291X 2011/11 [Refereed][Not invited]
   

  Transcription and replication of the negative-sense single-stranded influenza A virus genomic viral RNA are catalyzed by the viral RNA polymerase, which is a trimeric complex encoded by the three largest segments of the influenza virus genome: PB1, PB2, and PA. Numerous studies of the trimeric polymerase complex assembly have substantially contributed to current understanding of influenza virus replication. However, the dynamics of spatial and temporal macromolecular interactions involving virus and host proteins during the formation of the trimeric polymerase complex (PA-PB1-PB2) are still not completely understood. In this study, bimolecular fluorescence complementation (BiFC) and Raster image correlation spectroscopy (RICS) were applied to monitor the interactions between PB1, PB2, and PA. The BiFC probes of PA-PB1 and PB1-PB2 could monitor the trimeric polymerase complex as well as the binary complex. Furthermore, the C-terminal domain of PA (PAC) promoted interaction between PB1 and PB2 in the cytoplasm and that the N-terminal domain of PA (PAN) inhibited the aberrant trimeric complex formation and assembly of higher-order oligomers induced by PAC in the cytoplasm. Taken together, these results revealed a novel function of PAN in the formation of the trimeric polymerase complexes of influenza A virus. (C) 2011 Elsevier Inc. All rights reserved.
• Novel Arenavirus, Zambia

  Akihiro Ishii, Yuka Thomas, Ladslav Moonga, Ichiro Nakamura, Aiko Ohnuma, Bernard Hang'ombe, Ayato Takada, Aaron Mweene, Hirofumi Sawa

  EMERGING INFECTIOUS DISEASES 17 (10) 1921 - 1924 1080-6040 2011/10 [Refereed][Not invited]
   

  To investigate arenavirus in Zambia, we characterized virus from the kidneys of 5 arenavirus RNA-positive rodents (Mastomys natalensis) among 263 captured. Full-genome sequences of the viruses suggested that they were new strains similar to Lassa virus-related arenaviruses. Analyzing samples from additional rodents and other species can elucidate epizootiologic aspects of arenaviruses.
• Novel Arenavirus, Zambia

  Akihiro Ishii, Yuka Thomas, Ladslav Moonga, Ichiro Nakamura, Aiko Ohnuma, Bernard Hang'ombe, Ayato Takada, Aaron Mweene, Hirofumi Sawa

  EMERGING INFECTIOUS DISEASES 17 (10) 1921 - 1924 1080-6040 2011/10 [Refereed][Not invited]
   

  To investigate arenavirus in Zambia, we characterized virus from the kidneys of 5 arenavirus RNA-positive rodents (Mastomys natalensis) among 263 captured. Full-genome sequences of the viruses suggested that they were new strains similar to Lassa virus-related arenaviruses. Analyzing samples from additional rodents and other species can elucidate epizootiologic aspects of arenaviruses.
• ウマMHCクラスI遺伝子導入マウスのウマヘルペスウイルス1型に対する感受性

  木村 享史, 佐々木 道仁, 金 亨振, 福士 秀人, 澤 洋文

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 152回 184 - 184 1347-8621 2011/08
• Detection of KI polyomavirus and WU polyomavirus DNA by real-time polymerase chain reaction in nasopharyngeal swabs and in normal lung and lung adenocarcinoma tissues

  Shinobu Teramoto, Miki Kaiho, Yasuo Takano, Rika Endo, Hideaki Kikuta, Hirofumi Sawa, Tadashi Ariga, Nobuhisa Ishiguro

  MICROBIOLOGY AND IMMUNOLOGY 55 (7) 525 - 530 0385-5600 2011/07 [Not refereed][Not invited]
   

  Polyomaviruses KI (KIPyV) and WU (WUPyV) were detected from 7 (3.0%) and 38 (16.4%) of 232 children with respiratory tract infections by real-time PCR. The rates of infection by KIPyV and WUPyV alone were 3 of 7 (42.9%) and 20 of 38 (52.6%), respectively. In the other samples, various viruses (human respiratory syncytial virus, human metapneumovirus, human rhinovirus, parainfluenza virus 1 and human bocavirus) were detected simultaneously. One case was positive for KIPyV, WUPyV and hMPV. There was no obvious difference in clinical symptoms between KIPyV-positive and WUPyV-positive patients with or without coinfection. KIPyV was detected in one of 30 specimens of lung tissue (3.3%). Neither of the viruses was detected in 30 samples of lung adenocarcinoma tissue.
• Detection of KI polyomavirus and WU polyomavirus DNA by real-time polymerase chain reaction in nasopharyngeal swabs and in normal lung and lung adenocarcinoma tissues

  Shinobu Teramoto, Miki Kaiho, Yasuo Takano, Rika Endo, Hideaki Kikuta, Hirofumi Sawa, Tadashi Ariga, Nobuhisa Ishiguro

  MICROBIOLOGY AND IMMUNOLOGY 55 (7) 525 - 530 0385-5600 2011/07 [Refereed][Not invited]
   

  Polyomaviruses KI (KIPyV) and WU (WUPyV) were detected from 7 (3.0%) and 38 (16.4%) of 232 children with respiratory tract infections by real-time PCR. The rates of infection by KIPyV and WUPyV alone were 3 of 7 (42.9%) and 20 of 38 (52.6%), respectively. In the other samples, various viruses (human respiratory syncytial virus, human metapneumovirus, human rhinovirus, parainfluenza virus 1 and human bocavirus) were detected simultaneously. One case was positive for KIPyV, WUPyV and hMPV. There was no obvious difference in clinical symptoms between KIPyV-positive and WUPyV-positive patients with or without coinfection. KIPyV was detected in one of 30 specimens of lung tissue (3.3%). Neither of the viruses was detected in 30 samples of lung adenocarcinoma tissue.
• Characterization of influenza A viruses isolated from wild waterfowl in Zambia

  Edgar Simulundu, Akihiro Ishii, Manabu Igarashi, Aaron S. Mweene, Yuka Suzuki, Bernard M. Hang'ombe, Boniface Namangala, Ladslav Moonga, Rashid Manzoor, Kimihito Ito, Ichiro Nakamura, Hirofumi Sawa, Chihiro Sugimoto, Hiroshi Kida, Chuma Simukonda, Wilbroad Chansa, Jack Chulu, Ayato Takada

  JOURNAL OF GENERAL VIROLOGY 92 (6) 1416 - 1427 0022-1317 2011/06 [Refereed][Not invited]
   

  Although the quest to clarify the role of wild birds in the spread of the highly pathogenic H5N1 avian influenza virus (AIV) has yielded considerable data on AIVs in wild birds worldwide, information regarding the ecology and epidemiology of AIVs in African wild birds is still very limited. During AIV surveillance in Zambia (2008-2009), 12 viruses of distinct subtypes (H3N8, H4N6, H6N2, H9N1 and H11N9) were isolated from wild waterfowl. Phylogenetic analyses demonstrated that all the isolates were of the Eurasian lineage. Whilst some genes were closely related to those of AIVs isolated from wild and domestic birds in South Africa, intimating possible AIV exchange between wild birds and poultry in southern Africa, some gene segments were closely related to those of AIVs isolated in Europe and Asia, thus confirming the inter-regional AIV gene flow among these continents. Analysis of the deduced amino acid sequences of internal proteins revealed that several isolates harboured particular residues predominantly observed in human influenza viruses. Interestingly, the isolates with human-associated residues exhibited higher levels of virus replication in the lungs of infected mice and caused more morbidity as measured by weight loss than an isolate lacking such residues. This study stresses the need for continued monitoring of AIVs in wild and domestic birds in southern Africa to gain a better understanding of the emergence of strains with the potential to infect mammals.
• Paradoxical effects of chondroitin sulfate-E on Japanese encephalitis viral infection

  Eunmi Kim, Megumi Okumura, Hirofumi Sawa, Tadaaki Miyazaki, Daisuke Fujikura, Shuhei Yamada, Kazuyuki Sugahara, Michihito Sasaki, Takashi Kimura

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 409 (4) 717 - 722 0006-291X 2011/06 [Refereed][Not invited]
   

  Glycosaminoglycans (GAGs) have diverse functions in the body and are involved in viral infection. The purpose of this study was to evaluate the possible roles of the E-disaccharide units GlcA beta 1-3Gal-NAc(4,6-O-disulfate) of chondroitin sulfate (CS), a GAG involved in neuritogenesis and neuronal migration, in Japanese encephalitis virus (JEV) infection. Soluble CS-E (sCS-E) derived from squid cartilage inhibited JEV infection in African green monkey kidney-derived Vero cells and baby hamster kidney-derived BHK cells by interfering with viral attachment. In contrast, sCS-E enhanced viral infection in the mouse neuroblastoma cell line Neuro-2a, despite the fact that viral attachment to Neuro-2a cells was inhibited by sCS-E. This enhancement effect in Neuro-2a cells seemed to be related to increased viral RNA replication and was also observed in a rat infection model in which intracerebral coadministration of sCS-E with JEV in 17-day-old rats resulted in higher brain viral loads than in rats infected without sCS-E administration. These results show the paradoxical effects of sCS-E on JEV infection in different cell types and indicate that potential use of sCS-E as an antiviral agent against JEV infection should be approached with caution considering its effects in the neuron, the major target of JEV. (C) 2011 Elsevier Inc. All rights reserved.
• Detection and characterization of a novel polyomavirus in wild rodents

  Yasuko Orba, Shintaro Kobayashi, Ichiro Nakamura, Akihiro Ishii, Bernard M. Hang'ombe, Aaron S. Mweene, Yuka Thomas, Takashi Kimura, Hirofumi Sawa

  JOURNAL OF GENERAL VIROLOGY 92 (4) 789 - 795 0022-1317 2011/04 [Refereed][Not invited]
   

  To investigate polyomavirus infection in wild rodents, we analysed DNA samples from the spleens of 100 wild rodents from Zambia using a broad-spectrum PCR-based assay. A previously unknown polyomavirus genome was identified in a sample from a multimammate mouse (Mastomys species) and the entire viral genome of 4899 bp was :subsequently sequenced. This viral genome contained potential ORFs for the capsid proteins, VP1, VP2 and VP3, and early proteins, small t antigen and large T antigen. Phylogenetic analysis showed that it was a novel member of the family Polyomaviridae, and thus the virus was tentatively named mastomys polyomavirus. After transfection of the viral genome into several mammalian cell lines, transient expression of the VP1 and large T antigen proteins was confirmed by immunoblotting and immunocytochemical analyses. Comparison of large T antigen function in mastomys polyomavirus with that in rhesus monkey polyomavirus SV40 and human polyomavirus JC virus revealed that the large T antigen from mastomys polyomavirus interacted with the tumour suppressor protein pRb, but not with p53.
• Equine major histocompatibility complex class I molecules act as entry receptors that bind to equine herpesvirus-1 glycoprotein D

  Michihito Sasaki, Rie Hasebe, Yoshinori Makino, Tadaki Suzuki, Hideto Fukushi, Minoru Okamoto, Kazuya Matsuda, Hiroyuki Taniyama, Hirofumi Sawa, Takashi Kimura

  GENES TO CELLS 16 (4) 343 - 357 1356-9597 2011/04 [Refereed][Not invited]
   

  The endotheliotropism of equine herpesvirus-1 (EHV-1) leads to encephalomyelitis secondary to vasculitis and thrombosis in the infected horse central nervous system (CNS). To identify the host factors involved in EHV-1 infection of CNS endothelial cells, we performed functional cloning using an equine brain microvascular endothelial cell cDNA library. Exogenous expression of equine major histocompatibility complex (MHC) class I heavy chain genes conferred susceptibility to EHV-1 infection in mouse NIH3T3 cells, which are not naturally susceptible to EHV-1 infection. Equine MHC class I molecules bound to EHV-1 glycoprotein D (gD), and both anti-gD antibodies and a soluble form of gD blocked viral entry into NIH3T3 cells stably expressing the equine MHC class I heavy chain gene (3T3-A68 cells). Treatment with an anti-equine MHC class I monoclonal antibody blocked EHV-1 entry into 3T3-A68 cells, equine dermis (E. Derm) cells and equine brain microvascular endothelial cells. In addition, inhibition of cell surface expression of MHC class I molecules in E. Derm cells drastically reduced their susceptibility to EHV-1 infection. These results suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV-1 entry into equine cells.
• High expression of MeCP2 in JC virus-infected cells of progressive multifocal leukoencephalopathy brains

  Saya Shirai, Kenta Takahashi, Shinji Kohsaka, Tetsu Tsukamoto, Hiroshi Isogai, Shinichi Kudo, Hirofumi Sawa, Kazuo Nagashima, Shinya Tanaka

  NEUROPATHOLOGY 31 (1) 38 - 41 0919-6544 2011/02 [Refereed][Not invited]
   

  Mutations of the methyl CpG binding protein 2 (MeCP2) gene are a major cause of Rett syndrome. To investigate whether the expression of this gene was related to JC virus (JCV) infection, we examined brains of four progressive multifocal leukoencephalopathy (PML) patients. JCV infection was confirmed by immunohistochemical labeling with antibodies against JCV VP1, agnoprotein and large T antigen. MeCP2 expression was examined by immunohistochemistry using a specific polyclonal antibody against MeCP2. In normal brains and uninfected cortices of PML brains, MeCP2 expression was observed in the nuclei of neurons, but not observed in glial and endothelial cell nuclei. However, in PML brains intense immunolabeling was observed in abnormally enlarged glial nuclei of JCV-infected cells. Double immunolabeling using antibodies against large T antigen (visualized as blue) and MeCP2 (visualised as red) revealed dark red JCV-infected nuclei, which confirmed that the JCV infected nuclei expressed MeCP2. We conclude that MeCP2 is highly expressed in the JCV-infected nuclei of PML brain and these results may provide a new insight into the mechanism which regulates the MeCP2 expression in glial cells by the infection of JCV.
• ウマヘルペスウイルス1型の新たな細胞内侵入経路(Novel entry pathway of equine herpesvirus-1)

  佐々木 道仁, 長谷部 理絵, 澤 洋文, 福士 秀人, 谷山 弘行

  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 (公社)日本生化学会 83回・33回 1P - 1055 2010/12
• Chondroitin sulfate-E induced enhancement of Japanese encephalitis virus infection

  Kim Eunmi, Okumura Megumi, Sasaki Michihito, Fujikura Daisuke, Miyazaki Tadaaki, Sawa Hirofumi, Kimura Takashi

  JOURNAL OF NEUROVIROLOGY 16 44  1355-0284 2010/10 [Refereed][Not invited]
  
• Flavivirus Encephalitis: Pathological Aspects of Mouse and Other Animal Models

  T. Kimura, M. Sasaki, M. Okumura, E. Kim, H. Sawa

  VETERINARY PATHOLOGY 47 (5) 806 - 818 0300-9858 2010/09 [Not refereed][Not invited]
   

  Encephalitic flaviviruses are important arthropod-borne pathogens of humans and other animals. In particular, the recent emergence of the West Nile virus (WNV) and Japanese encephalitis virus (JEV) in new geographic areas has caused a considerable public health alert and international concern. Among the experimental in vivo models of WNV and JEV infection, mice and other laboratory rodents are the most thoroughly studied and well-characterized systems, having provided data that are important for understanding the infectious process in humans. Macaca monkeys have also been used as a model for WNV and JEV infection, mainly for the evaluation of vaccine efficacy, although a limited number of published studies have addressed pathomorphology. These animal models demonstrate the development of encephalitis with many similarities to the human disease; however, the histological events that occur during infection, especially in peripheral tissues, have not been fully characterized.
• Functional analysis of an alpha-helical antimicrobial peptide derived from a novel mouse defensin-like gene

  Akira Kawaguchi, Tadaki Suzuki, Takashi Kimura, Naoki Sakai, Tokiyoshi Ayabe, Hirofumi Sawa, Hideki Hasegawa

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 398 (4) 778 - 784 0006-291X 2010/08 [Refereed][Not invited]
   

  Gene-encoded antimicrobial peptides (AMPS) are an essential component of the innate immune system in many species. Analysis of beta-defensin gene expression in mouse tissue using primers that were specific for conserved sequences located outside of the beta-defensin translated region identified a novel small gene. The novel gene had an open reading frame of 114 bp and encoded a predicted protein of 37 amino acid residues. A search of the genome database revealed that the gene locus and the sequence of exon 1 of this novel gene were similar to subgroup 1 mouse beta-defensins. A small peptide, K17 (FSPQMLQDIIEKKTKIL), derived from the amino acid sequence of this novel gene was synthesized. Circular dichroism (CD) spectroscopic analysis of chemically synthesized peptide demonstrated that the peptide exhibited random coil conformation in aqueous solution, but the peptide adopted helical conformation in the presence of trifluoroethanol or sodium dodecyl sulfate, a membrane-mimicking environment. The peptide exhibited bactericidal activity against Salmonella enterica serovar Typhimurium (Gram negative) and Staphylococcus aureus (Gram positive); it was not cytotoxic in cultures of mammalian cells or hemolytic in cultures of erythrocytes. These results suggested that K17 may be a candidate therapeutic for the treatment of bacterial infection. (C) 2010 Elsevier Inc. All rights reserved.
• Functional analysis of an alpha-helical antimicrobial peptide derived from a novel mouse defensin-like gene

  Akira Kawaguchi, Tadaki Suzuki, Takashi Kimura, Naoki Sakai, Tokiyoshi Ayabe, Hirofumi Sawa, Hideki Hasegawa

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 398 (4) 778 - 784 0006-291X 2010/08 [Not refereed][Not invited]
   

  Gene-encoded antimicrobial peptides (AMPS) are an essential component of the innate immune system in many species. Analysis of beta-defensin gene expression in mouse tissue using primers that were specific for conserved sequences located outside of the beta-defensin translated region identified a novel small gene. The novel gene had an open reading frame of 114 bp and encoded a predicted protein of 37 amino acid residues. A search of the genome database revealed that the gene locus and the sequence of exon 1 of this novel gene were similar to subgroup 1 mouse beta-defensins. A small peptide, K17 (FSPQMLQDIIEKKTKIL), derived from the amino acid sequence of this novel gene was synthesized. Circular dichroism (CD) spectroscopic analysis of chemically synthesized peptide demonstrated that the peptide exhibited random coil conformation in aqueous solution, but the peptide adopted helical conformation in the presence of trifluoroethanol or sodium dodecyl sulfate, a membrane-mimicking environment. The peptide exhibited bactericidal activity against Salmonella enterica serovar Typhimurium (Gram negative) and Staphylococcus aureus (Gram positive); it was not cytotoxic in cultures of mammalian cells or hemolytic in cultures of erythrocytes. These results suggested that K17 may be a candidate therapeutic for the treatment of bacterial infection. (C) 2010 Elsevier Inc. All rights reserved.
• Natalizumab Has No Direct Biological Effect on JC Virus Infectivity in Permissive Human Neural Cell Lines

  Tadaki Suzuki, Satoko Yamanouchi, Yuji Sunden, Yasuko Orba, Takashi Kimura, Hirofumi Sawa

  JOURNAL OF MEDICAL VIROLOGY 82 (7) 1229 - 1235 0146-6615 2010/07 [Refereed][Not invited]
   

  The human polyomavirus JC virus (JCV) infects 70-80% of humans and establishes latent infection in the kidney. In immunosuppressed patients, JCV reactivates and causes a fatal and progressive neurological disease known as progressive multifocal leukoencephalopathy (PML). Over the past three decades, PML has become an important neurological complication in acquired immunodeficiency syndrome (AIDS) patients. Recently, it was reported that patients treated with therapeutics that target the integrin receptor very late antigen (VLA)-4 are at increased risk of developing PML. However, the relationship between Natalizumab and this unexpected onset of PML has yet to be elucidated. Here, we investigated the effect of Natalizumab on the growth of JCV in the permissive human neural cell line IMR-32 following viral inoculation. Natalizumab had no effect either on the expression levels of viral proteins as determined by immunoblot analysis using specific antibodies or on the hemagglutination activity of cellular lysates from infected cells. These results suggest that there is no direct effect of Natalizumab on JCV infectivity in IMR-32 cells in vitro. J. Med. Virol. 82:1229-1235, 2010. (C) 2010 Wiley-Liss, Inc.
• Transcellular transport of West Nile virus-like particles across human endothelial cells depends on residues 156 and 159 of envelope protein

  Rie Hasebe, Tadaki Suzuki, Yoshinori Makino, Manabu Igarashi, Satoko Yamanouchi, Akihiko Maeda, Motohiro Horiuchi, Hirofumi Sawa, Takashi Kimura

  BMC MICROBIOLOGY 10 165  1471-2180 2010/06 [Refereed][Not invited]
   

  Background: West Nile virus (WNV) causes viremia after invasion to the hosts by mosquito bite. Endothelial cells could play an important role in WNV spread from the blood stream into the central nervous system and peripheral tissues. Here, we analyzed the capacity of virus-like particles (VLPs) of the highly virulent NY99 6-LP strain (6-LP VLPs) and the low virulence Eg101 strain (Eg VLPs) to cross cultured human endothelial cells. Results: 6-LP VLPs were transported from the apical to basolateral side of endothelial cells, whereas Eg VLPs were hardly transported. The localization of tight junction marker ZO-1 and the integrity of tight junctions were not impaired during the transport of 6-LP VLPs. The transport of 6-LP VLPs was inhibited by treatment with filipin, which prevents the formation of cholesterol-dependent membrane rafts, suggesting the involvement of raft-associated membrane transport. To determine the amino acid residues responsible for the transport of VLPs, we produced mutant VLPs, in which residues of E protein were exchanged between the 6-LP and Eg strains. Double amino acid substitution of the residues 156 and 159 greatly impaired the transport of VLPs. Conclusion: Our results suggest that a transcellular pathway is associated with 6-LP VLPs transport. We also showed that the combination of the residues 156 and 159 plays an important role in the transport of VLPs across endothelial cells.
• Cytokine and Growth Factor Expression by HTLV-1 Lck-tax Transgenic Cells in SCID Mice

  Karen M. Watters, Jonathan Dean, Hideki Hasegawa, Hirofumi Sawa, William Hall, Noreen Sheehy

  AIDS RESEARCH AND HUMAN RETROVIRUSES 26 (5) 593 - 603 0889-2229 2010/05 [Refereed][Not invited]
   

  The Tax protein encoded by HTLV-1 plays a key role in the development of ATL in infected individuals. Our previous studies showed that tax transgenic mice develop disease that is almost identical to human ATL, with widespread organ invasion by lymphomatous cells and the development of leukemia. The same pathology develops rapidly in SCID mice engrafted with cells from the transgenic animals. In the present study, we used this SCID model to analyze the expression levels of several cytokines, growth factors, and adhesion molecules to determine their possible involvement in the development of disease. We showed that Tax expression was undetectable at the protein level in the tax-transformed cells used to inoculate the SCID mice and that these cells displayed constitutive NF-kappa B and Akt activity. We demonstrated significant differences in the levels of circulating PDGF-BB, TNF-alpha, sICAM-1, and sVCAM-1 in inoculated animals. Cell-surface staining of the tax transgenic cells showed that they do not express receptors for any of the upregulated growth factors. Significant differences were not found in the secreted levels of bFGF, MMP9, VEGF, or E-selectin, whereas IL-2, IL-15, IL-6, IL-1 beta, and IFN-gamma expression was undetectable. Even though the number of factors analyzed is limited, our study identified TNF-alpha, PDGF-BB, and the adhesion molecules sICAM-1 and sVCAM-1 as factors that may contribute to the high levels of organ infiltration by leukemic cells in this tax transgenic SCID model.
• Low pH-Triggered Model Drug Molecule Release from Virus-Like Particles

  Noriko Ohtake, Kenichi Niikura, Tadaki Suzuki, Keita Nagakawa, Shintaro Mikuni, Yasutaka Matsuo, Masataka Kinjo, Hirofumi Sawa, Kuniharu Ijiro

  CHEMBIOCHEM 11 (7) 959 - 962 1439-4227 2010/05 [Refereed][Not invited]
  
• The Human Polyoma JC Virus Agnoprotein Acts as a Viroporin

  Tadaki Suzuki, Yasuko Orba, Yuki Okada, Yuji Sunden, Takashi Kimura, Shinya Tanaka, Kazuo Nagashima, William W. Hall, Hirofumi Sawa

  PLOS PATHOGENS 6 (3) e1000801  1553-7366 2010/03 [Refereed][Not invited]
   

  Virus infections can result in a range of cellular injuries and commonly this involves both the plasma and intracellular membranes, resulting in enhanced permeability. Viroporins are a group of proteins that interact with plasma membranes modifying permeability and can promote the release of viral particles. While these proteins are not essential for virus replication, their activity certainly promotes virus growth. Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease resulting from lytic infection of oligodendrocytes by the polyomavirus JC virus (JCV). The genome of JCV encodes six major proteins including a small auxiliary protein known as agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to viral propagation at various stages in the replication cycle, including transcription, translation, processing of late viral proteins, assembly of virions, and viral propagation. Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV. Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins. Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca(2+); (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity. The viroporin-like properties of agnoprotein result in increased membrane permeability and alterations in intracellular Ca(2+) homeostasis leading to membrane dysfunction and enhancement of virus release.
• Induction of Cross-Protective Immunity Against Influenza A Virus H5N1 by an Intranasal Vaccine With Extracts of Mushroom Mycelia

  Takeshi Ichinohe, Akira Ainai, Tomoyuki Nakamura, Yukihito Akiyama, Jun-ichi Maeyama, Takato Odagiri, Masato Tashiro, Hidehiro Takahashi, Hirofumi Sawa, Shin-ichi Tamura, Joe Chiba, Takeshi Kurata, Tetsutaro Sata, Hideki Hasegawa

  JOURNAL OF MEDICAL VIROLOGY 82 (1) 128 - 137 0146-6615 2010/01 [Refereed][Not invited]
   

  The identification of a safe and effective adjuvant that is able to enhance mucosal immune responses is necessary for the development of an efficient inactivated intranasal influenza vaccine, The present study demonstrated the effectiveness of extracts of mycelia derived from edible mushrooms as adjuvants for intranasal influenza vaccine. The adjuvant effect of extracts of mycelia was examined by intranasal co-administration of the extracts and inactivated A/PR8 (H1N1) influenza virus hemagglutinin (HA) vaccine in BALB/c mice. The inactivated vaccine in combination with mycelial extracts induced a high anti-A/PR8 HA-specific IgA and IgG response in nasal washings and serum, respectively. Virus-specific cytotoxic T-lymphocyte responses were also induced by administration of the vaccine with extract of mycelia, resulting in protection against lethal lung infection with influenza virus A/PR8. In addition, intranasal administration of NIBRG14 vaccine derived from the influenza A/Vietnam/1194/2004 (H5N1) virus strain administered in conjunction with mycelial extracts from Phellinus linteus conferred cross-protection against heterologous influenza A/Indonesia/6/2005 virus challenge in the nasal infection model. In addition, mycelial extracts induced proinflammatory cytokines and CD40 expression in bone marrow-derived dendritic cells. These results suggest that mycelial extract-adjuvanted vaccines can confer cross-protection against variant H5N1 influenza viruses. The use of extracts of mycelia derived from edible mushrooms is proposed as a new safe and effective mucosal adjuvant for use for nasal vaccination against influenza virus infection. J. Med. Virol. 82:128-137, 2010. (C) 2009 Wiley-Liss, Inc.
• Large T Antigen Promotes JC Virus Replication in G(2)-arrested Cells by Inducing ATM- and ATR-mediated G(2) Checkpoint Signaling

  Yasuko Orba, Tadaki Suzuki, Yoshinori Makino, Kanako Kubota, Shinya Tanaka, Takashi Kimura, Hirofumi Sawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 285 (2) 1544 - 1554 0021-9258 2010/01 [Refereed][Not invited]
   

  Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.
• Effects of the number of amino acid residues in the signal segment upstream or downstream of the NS2B-3 cleavage site on production and secretion of prM/M-E virus-like particles of West Nile virus

  Hidehiro Takahashi, Naohiro Ohtaki, Masae Maeda-Sato, Michiko Tanaka, Keiko Tanaka, Hirofumi Sawa, Toyokazu Ishikawa, Akihisa Takamizawa, Tomohiko Takasaki, Hideki Hasegawa, Tetsutaro Sata, William W. Hall, Takeshi Kurata, Asato Kojima

  MICROBES AND INFECTION 11 (13) 1019 - 1028 1286-4579 2009/11 [Refereed][Not invited]
   

  Expression of genes for precursor M (prM) and envelope (E) proteins of West Nile virus (WNV) leads to the production of small. capsidless, and non-infectious virus-like particles (VLPs) possessing the E antigen which is responsible for viral entry and immune protection It has been reported that processing of the secretion signal affects viral release We examined the secretion efficiency of VLPs into the culture medium front RK13 or 293 T cells transfected with expression vectors for prM and E proteins of WNV which were constructed to comprise different lengths of signal peptides upstream of the prM-E domain The number of amino acid residues present in the segment markedly affected the production, processing, and secretion of VLPs Secreted VLPs possessed both the processed M protein and the glycosylated E protein In addition, immunization with VLPs induced neutralizing, antibodies in C3H/HeN mice. These results indicate that the number of amino acid residues comprising the N-terminus of the signal segment controls the efficiency of assembly, maturation, and release of VLPs; in the absence of viral protease, which in turn indicates the potential of VLPs as a candidate for an effective WNV subunit vaccine. (C) 2009 Elsevier Masson SAS. All rights reserved
• Covalent bonded Gag multimers in human immunodeficiency virus type-1 particles

  Yoshinori Kitagawa, Masae Maeda-Sato, Keiko Tanaka, Minoru Tobiume, Hirofumi Sawa, Hideki Hasegawa, Asato Kojima, William W. Hall, Takeshi Kurata, Tetsutaro Sata, Hidehiro Takahashi

  MICROBIOLOGY AND IMMUNOLOGY 53 (11) 609 - 620 0385-5600 2009/11 [Refereed][Not invited]
   

  The oligomerization of HIV-1 Gag and Gag-Pol proteins, which are assembled at the plasma membrane, leads to viral budding. The budding generally places the viral components under non-reducing conditions. Here the effects of non-reducing conditions on Gag structures and viral RNA protection were examined. Using different reducing conditions and SDS-PAGE, it was shown that oligomerized Gag possesses intermolecular covalent bonds under non-reducing conditions. In addition, it was demonstrated that the mature viral core contains a large amount of covalent bonded Gag multimers, as does the immature core. Viral genomic RNA becomes sensitive to ribonuclease in reducing conditions. These results suggest that, under non-reducing conditions, covalent bonded Gag multimers are formed within the viral particles and play a role in protection of the viral genome.
• Inhibition of the SDF-1 alpha-CXCR4 axis by the CXCR4 antagonist AMD3100 suppresses the migration of cultured cells from ATL patients and murine lymphoblastoid cells from HTLV-I Tax transgenic mice

  Akira Kawaguchi, Yasuko Orba, Takashi Kimura, Hidekatsu Iha, Masao Ogata, Takahiro Tsuji, Akira Ainai, Tetsutaro Sata, Takashi Okamoto, William W. Hall, Hirofumi Sawa, Hideki Hasegawa

  BLOOD 114 (14) 2961 - 2968 0006-4971 2009/10 [Refereed][Not invited]
   

  Adult T-cell leukemia (ATL) is a T-cell malignancy caused by human T lymphotropic virus type I, and presents as an aggressive leukemia with characteristic widespread leukemic cell infiltration into visceral organs and skin. The molecular mechanisms associated with leukemic cell infiltration are poorly understood. We have used mouse models of ATL to investigate the role of chemokines in this process. Transfer of splenic lymphomatous cells from transgenic to SCID mice reproduces a leukemia and lymphoma that is histologically identical to human disease. It could be shown that lymphomatous cells exhibit specific chemotactic activity in response to stromal cell-derived factor-1 alpha (SDF-1 alpha). Lymphomatous cells exhibited surface expression of CXCR4, the specific receptor of SDF-1 alpha. AMD3100, a CXCR4 antagonist, was found to inhibit both SDF-1 alpha-induced migration and phosphorylation of extracellular signal-related kinase 1/2. Investigation of cultured cells from human ATL patients revealed identical findings. Using the SCID mouse model, it could be demonstrated that AMD3100 inhibited infiltration of lymphomatous cells into liver and lung tissues in vivo. These results demonstrate the involvement of the SDF-1 alpha/CXCR4 interaction as one mechanism of leukemic cell migration and this may provide a novel target as part of combination therapy for ATL. (Blood. 2009; 114: 2961-2968)
• Infectious entry of equine herpesvirus-1 into host cells through different endocytic pathways

  Rie Hasebe, Michihito Sasaki, Hirofumi Sawa, Ryuichi Wada, Takashi Umemura, Takashi Kimura

  VIROLOGY 393 (2) 198 - 209 0042-6822 2009/10 [Refereed][Not invited]
   

  We investigated the mechanism by which equine herpesvirus-1 (EHV-1) enters primary Cultured equine brain microvascular endothelial cells (EBMECs) and equine dermis (E. Derm) cells. EHV-1 colocalized with caveolin in EBMECs and the infection was greatly reduced by the expression of a dominant negative form of equine caveolin-1 (ecavY14F), suggesting that EHV-1 enters EBMECs via caveolar endocytosis. EHV-1 entry into E. Derm cells was significantly reduced by ATP depletion and treatments with lysosomotropic agents. Enveloped virions were detected from E. Derm cells by infectious virus recovery assay after vital internalization, suggesting that EHV-1 enters E. Derm cells via energy- and pH-dependent endocytosis. These results suggest that EHV-1 utilizes multiple endocytic pathways in different cell types to establish productive infection. (C) 2009 Elsevier Inc. All rights reserved.
• Gold Nanoparticle Arrangement on Viral Particles through Carbohydrate Recognition: A Non-Cross-Linking Approach to Optical Virus Detection

  Kenichi Niikura, Keita Nagakawa, Noriko Ohtake, Tadaki Suzuki, Yasutaka Matsuo, Hirofumi Sawa, Kuniharu Ijiro

  BIOCONJUGATE CHEMISTRY 20 (10) 1848 - 1852 1043-1802 2009/10 [Refereed][Not invited]
   

  We propose a new approach to optical virus detection, based on the spatial assembly of gold nanoparticles on the surface of viruses. Since JC virus-like particles (VLPs) comprise a repeating viral capsid protein that binds to sialic acid, the conjugation of sialic acid-linked Au particles with VLPs enables the spatial arrangement of Au particles on the VLP surface. This structure produced a red shift in the absorption spectrum due to plasmon coupling between adjacent Au particles, leading to the, construction of an optical virus detection system. Our system depends not on the simple cross-linking of VLPs and Au particles, but on an ordered Au structure covering the entire surface of the VLPs and can be applied to virus detection systems using the inherent ligand recognition of animal viruses.
• Characterization of H3N6 avian influenza virus isolated from a wild white pelican in Zambia

  Edgar Simulundu, Aaron S. Mweene, Daisuke Tomabechi, Bernard M. Hang'ombe, Akihiro Ishii, Yuka Suzuki, Ichiro Nakamura, Hirofumi Sawa, Chihiro Sugimoto, Kimihito Ito, Hiroshi Kida, Lewis Saiwana, Ayato Takada

  ARCHIVES OF VIROLOGY 154 (9) 1517 - 1522 0304-8608 2009/09 [Refereed][Not invited]
   

  We characterized an influenza virus isolated from a great white pelican in Zambia. Phylogenetic analysis showed that all of its gene segments belonged to the Eurasian lineage and that they appear to have evolved in distinct geographical regions in Europe, Asia, and Africa, suggesting reassortment of virus genes maintained in wild aquatic birds whose flyways overlap across these continents. It is notable that this virus might possess some genes of the same origin as those of highly pathogenic H7 and H5 viruses isolated in Eurasia. The present study underscores the need for continued monitoring of avian influenza viruses in Eurasia and Africa.
• Adaptor Protein Crk Induces Src-Dependent Activation of p38 MAPK in Regulation of Synovial Sarcoma Cell Proliferation

  Takuya Watanabe, Masumi Tsuda, Shinya Tanaka, Yusuke Ohba, Hideaki Kawaguchi, Tokifumi Majima, Hirofumi Sawa, Akio Minami

  MOLECULAR CANCER RESEARCH 7 (9) 1582 - 1592 1541-7786 2009/09 [Refereed][Not invited]
   

  The adaptor protein Crk mediates intracellular signaling related to cell motility and proliferation and is implicated in human tumorigenesis. The role of Crk in the growth of human sarcoma has remained unclear, however. The present study shows that Crk-induced activation of Src and subsequent signaling by p38 mitogen-activated protein kinase (MAPK) contribute to the enhanced proliferation of human synovial sarcoma cells. Depletion of Crk by RNA interference markedly inhibited proliferation of the synovial sarcoma cell lines HS-SYII, SYO-1, and Fuji as well as prevented anchorage-independent growth. Conversely, reconstitution with CrkII by authentic small interfering RNA-resistant Crk gene restored proliferation in Crk-silenced SYO-1 cells. Crk-depleted synovial sarcoma cells manifested enhanced transcriptional activity and expression of the p16(INK4A) gene, resulting in their accumulation in G, phase of the cell cycle. In response to hepatocyte growth factor stimulation, Crk prominently induced the tyrosine phosphorylation of Grb2-associated binder 1 through activation of Src and focal adhesion kinase, and the Src family kinase inhibitor PP2 almost completely inhibited the proliferation of SYO-1 cells. Crk also induced the phosphorylation of p38 MAPK, and SB203580, a p38 MAPK-specific inhibitor, increased expression of p16(INK4A) gene in SYO-1 cells. Furthermore, SB203580 or depletion of p38 MAPK by small interfering RNA suppressed both the phosphorylation of Akt triggered by hepatocyte growth factor and the proliferation of SYO-1 cells. These results suggest that Crk promotes proliferation of human synovial sarcoma cells through activation of Src and its downstream signaling by a novel p38 MAPK-Akt pathway, with these signaling molecules providing potent new targets for molecular therapeutics. (Mol Cancer Res 2009;7(9):1582-92)
• Human synovial sarcoma proto-oncogene Syt is essential for early embryonic development through the regulation of cell migration

  Taichi Kimura, Mieko Sakai, Kouichi Tabu, Lei Wang, Ryosuke Tsunematsu, Masumi Tsuda, Hirofumi Sawa, Kazuo Nagashima, Hiroshi Nishihara, Shigetsugu Hatakeyama, Keiko Nakayama, Marc Ladanyi, Shinya Tanaka, Keiichi I. Nakayama

  LABORATORY INVESTIGATION 89 (6) 645 - 656 0023-6837 2009/06 [Refereed][Not invited]
   

  SYT-SSX protein, resulted from chromosomal translocation, causes synovial sarcoma, which is a malignant tumor accounting for 10% of soft tissue sarcoma. However, biological functions of SYT (synovial sarcoma translocation), also known as SS18, are largely unclear, whereas it has been proven that Syt-null mice die at early stages of embryonic development. Here, we generated Syt-deficient mice and confirmed the reported phenotypes, including growth retardation, open neural tube and haplo-insufficient lethality, and therefore, there is no doubt that Syt is essential for embryonic development. However, placental defects, described in the earlier report, were rarely seen in our mice and we frequently observed cardiac defect in Syt-deficient mice. As the mechanisms responsible for embryonic lethality seem to be complicate, we performed additional experiments. By using primary cultured embryonic fibroblasts, we showed that Syt(-/-) MEFs deregulate actin organization and suppressed cell migration. These observations suggest that Syt may contribute to the signaling pathway important for various cellular functions in vivo and in vitro, and we propose that Syt-deficient MEFs would be a powerful means to understand the biological roles of SYT in vitro. Laboratory Investigation (2009) 89, 645-656; doi:10.1038/labinvest.2009.25; published online 30 March 2009
• A case of central nervous system lymphomatoid granulomatosis; characteristics of PET imaging and pathological findings.

  Nishihara H, Nakasato M, Sawa H, Murakami H, Yamamoto D, Moriyama K, Kato N, Hashimoto I, Kamada H, Tanaka S

  Journal of neuro-oncology 2 93 275 - 278 0167-594X 2009/06 [Refereed][Not invited]
  
• Monoclonal Antibody and siRNAs for Topoisomerase I Suppress Telomerase Activity

  Hidehiro Takahashi, Yoshinori Kitagawa, Masae Maeda-Satoh, Hideki Hasegawa, Hirofumi Sawa, Tetsutaro Sata

  HYBRIDOMA 28 (1) 63 - 65 1554-0014 2009/02 [Refereed][Not invited]
   

  Telomerase, a ribonucleoprotein enzyme, is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric repeats at the eukaryotic chromosomal ends. We previously reported that topoisomerase I dissociates HIV-1 reverse transcriptase from genomic RNAs, and binding of topoisomerase I to RNA template regulates cDNA synthesis. We also found that a monoclonal antibody (MAb) against topoisomerase I, designated as MAb 1, suppresses the reverse transcription efficiency using a detergent-disrupted HIV-1 virion. In this study, we describe how MAb 1 suppresses telomerase activity in cellular lysates. In addition, siRNAs of topoisomerase I has attenuated telomerase activity in culture cells. These results suggest that topoisomerase I is involved in telomerase activity, as well as HIV-1 reverse transcription.
• Monoclonal Antibody and siRNAs for Topoisomerase I Suppress Telomerase Activity

  Hidehiro Takahashi, Yoshinori Kitagawa, Masae Maeda-Satoh, Hideki Hasegawa, Hirofumi Sawa, Tetsutaro Sata

  HYBRIDOMA 28 (1) 63 - 65 1554-0014 2009/02 [Not refereed][Not invited]
   

  Telomerase, a ribonucleoprotein enzyme, is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric repeats at the eukaryotic chromosomal ends. We previously reported that topoisomerase I dissociates HIV-1 reverse transcriptase from genomic RNAs, and binding of topoisomerase I to RNA template regulates cDNA synthesis. We also found that a monoclonal antibody (MAb) against topoisomerase I, designated as MAb 1, suppresses the reverse transcription efficiency using a detergent-disrupted HIV-1 virion. In this study, we describe how MAb 1 suppresses telomerase activity in cellular lysates. In addition, siRNAs of topoisomerase I has attenuated telomerase activity in culture cells. These results suggest that topoisomerase I is involved in telomerase activity, as well as HIV-1 reverse transcription.
• An siRNA against JC virus (JCV) agnoprotein inhibits JCV infection in JCV-producing cells inoculated in nude mice

  Tomoko Matoba, Yasuko Orba, Tadaki Suzuki, Yoshinori Makino, Hideo Shichinohe, Satoshi Kuroda, Takahiro Ochiya, Hiroshi Itoh, Shinya Tanaka, Kazuo Nagashima, Hirofumi Sawa

  NEUROPATHOLOGY 28 (3) 286 - 294 0919-6544 2008/06 [Refereed][Not invited]
   

  JC virus (JCV) is the etiological agent of the demyelinating disease progressive multifocal leukoencephalopathy (PML). Because JCV has a very narrow host range, it has been difficult to develop an animal model of JCV infection; as a result, no effective therapy for PML has been established. In this study, we have tried to create an animal model that replaces an in vivo JCV infection. As a result, we have obtained a stable persistence of JCV-infected human cells in the mouse brain by inoculating the virus-infected cells into the nude mice brains. In this model, the JCV-infected cells were well preserved in the nude mouse brains for 2 weeks. We then treated JCV-injected brains with an siRNA against the JCV agnoprotein that is known to be an effective inhibitor of JCV infection in vitro. A highly purified type I collagen, atelocollagen, was used as a carrier for the siRNA. The siRNA inhibited the expression of JCV protein in inoculated JCV-infected cells in the mouse brain, compared to the medium containing only atelocollagen used as a placebo. Thus, the combination of siRNA and atelocollagen might be a candidate therapeutic agent for the treatment of JCV infection.
• Proteomics- and transcriptomics-based screening of differentially expressed proteins and genes in brain of Wig rat: A model for attention deficit hyperactivity disorder (ADHD) research

  Misato Hirano, Randeep Rakwal, Junko Shibato, Hirofumi Sawa, Kazuo Nagashima, Yoko Ogawa, Yasukazu Yoshida, Hitoshi Wahashi, Etsuo Niki, Yoshinori Masuo

  JOURNAL OF PROTEOME RESEARCH 7 (6) 2471 - 2489 1535-3893 2008/06 [Refereed][Not invited]
   

  Two global omics approaches were applied to develop an inventory of differentially expressed proteins and genes in Wig rat, a promising animal model of attention-deficit hyperactivity disorder (ADHD). The frontal cortex, striatum, and midbrain of Wig rat at 4 weeks of age were dissected for proteomics and transcriptomics analyses. Two-dimensional gel electrophoresis detected 13, 1, and 16 differentially expressed silver nitrate-stained spots in the frontal cortex, striatum, and midbrain, respectively. Peptide mass fingerprinting/tandem mass spectrometry identified 19 nonredundant proteins, belonging to 7 functional categories, namely, signal transduction, energy metabolism, cellular transport, protein with binding function, protein synthesis, cytoskeleton, and cell rescue. Interestingly, 10 proteins that were indentified in the present study were also previously reported in studies involving neurodegenerative diseases and psychiatric disorders, such as Alzheimer's disease (AD), Parkinson's disease, and Schizophrenia. Moreover, some of the proteins identified in the midbrain were involved in synaptic vesicular transport, suggesting abnormality in neurotransmitter release in this region. On the other hand, transcriptomics analysis of combined frontal cortex, striatum, and midbrain by rat whole genome 44K DNA oligo microarray revealed highly up-regulated (28) and down-regulated (33) genes. Functional categorization of these genes showed cellular transport, metabolism, protein fate, signal transduction, and transcription as the major categories, with 26% genes of unknown function. Some of the identified genes were related to AD, fragile X syndrome, and ADHD. This is a first comprehensive study providing insight into molecular components in Wig rat brain, and will help to elucidate the roles of identified proteins and genes in Wig rat brain, hopefully leading to uncovering the pathogenesis of ADHD.
• Progressive multifocal leukoencephalopathy and CD4+T-lymphocytopenia in a patient with Sjogren syndrome

  Yuichi Hayashi, Akio Kimura, Shimon Kato, Akihiro Koumura, Takeo Sakurai, Yuji Tanaka, Isao Hozumi, Yuji Sunden, Yasuko Orba, Hirofumi Sawa, Hitoshi Takahashi, Takashi Inuzuka

  JOURNAL OF THE NEUROLOGICAL SCIENCES 1-2 268 (1-2) 195 - 198 0022-510X 2008/05 [Refereed][Not invited]
   

  We report progressive multifocal leukoencephalopathy (PML) and CD4+ T-lymphocytopenia in a 71-year-old man with Sjogren syndrome (SjS). The patient was admitted to our hospital because of progressive dementia and gait disturbance. T2-weighted MR images showed high-intensity lesions in his left frontal white matter thalamus, cerebellum and brainstem. A pathological diagnosis of PML was made by brain biopsy. SjS is frequently accompanied with immunological complications; however, there are few reports on PML in patients with SjS. Recently, isolated CD4+ T-lymphocytopenia is reported to be one of the based immunological conditions associated with the development of PML. In the present case, CD4+ T-lymphocytopenia was also observed on admission, which is also associated with SjS. (C) 2007 Elsevier B.V. All rights reserved.
• Forkhead transcription factor FoxO1 in adipose tissue regulates energy storage and expenditure

  Jun Nakae, Yongheng Cao, Miyo Oki, Yasuko Orba, Hirofumi Sawa, Hiroshi Kiyonari, Kristy Iskandar, Koji Suga, Marc Lombes, Yoshitake Hayashi

  DIABETES 57 (3) 563 - 576 0012-1797 2008/03 [Refereed][Not invited]
   

  OBJECTIVE-Adipose tissue serves as an integrator of various physiological pathways, energy balance, and glucose homeostasis. Forkhead box-containing protein O subfamily (FoxO) 1 mediates insulin action at the transcriptional level. However, physiological roles of FoxO1 in adipose tissue remain unclear. RESEARCH DESIGN AND METHODS-In the present study, we generated adipose tissue-specific FoxO1. transgenic mice (adipocyte protein 2 [aP(2)]-FLAG-Delta 256) using an aP(2) promoter/enhancer and a mutant FoxO1 (FLAG Delta 256) in which the carboxyl terminal transactivation domain was deleted. Using these mice, we analyzed the effects of the overexpression of FLAG Delta 256 on glucose metabolism and energy homeostasis. RESULTS-The aP(2)-FLAG-Delta 256 mice showed improved glucose tolerance and insulin sensitivity accompanied with smaller-sized adipocytes and increased adiponectin (adipoq) and Glut 4 (Slc2a4) and decreased tumor necrosis factor alpha (Tnf) and chemokine (C-C motif) receptor 2 (Ccr2) gene expression levels in white adipose tissue (WAT) under a high-fat diet. Furthermore, the aP(2)-FLAG-Delta 256 mice had increased oxygen consumption accompanied with increased expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1 alpha protein and uncoupling protein (UCP)-1 (Ucp1), UCP-2 (Ucp2), and beta 3-AR (Adrb3) in brown adipose tissue (BAT). Overexpression of FLAG Delta 256 in T37i cells, which are derived from the hibernoma of SV40 large T antigen transgenic mice, increased expression of PGC-1 alpha protein and Ucp1. Furthermore, knockdown of endogenous FoxO1 in T37i cells increased Pgc1 alpha (Ppargc1a), Pgc1 beta (Ppargc1b), Ucp1, and Adrb3 gene expression. CONCLUSIONS-These data suggest that FoxO1 modulates energy homeostasis in WAT and BAT through regulation of adipocyte size and adipose tissue-specific gene expression in response to excessive calorie intake.
• Forkhead transcription factor FoxO1 in adipose tissue regulates energy storage and expenditure

  Jun Nakae, Yongheng Cao, Miyo Oki, Yasuko Orba, Hirofumi Sawa, Hiroshi Kiyonari, Kristy Iskandar, Koji Suga, Marc Lombes, Yoshitake Hayashi

  DIABETES 57 (3) 563 - 576 0012-1797 2008/03 [Not refereed][Not invited]
   

  OBJECTIVE-Adipose tissue serves as an integrator of various physiological pathways, energy balance, and glucose homeostasis. Forkhead box-containing protein O subfamily (FoxO) 1 mediates insulin action at the transcriptional level. However, physiological roles of FoxO1 in adipose tissue remain unclear. RESEARCH DESIGN AND METHODS-In the present study, we generated adipose tissue-specific FoxO1. transgenic mice (adipocyte protein 2 [aP(2)]-FLAG-Delta 256) using an aP(2) promoter/enhancer and a mutant FoxO1 (FLAG Delta 256) in which the carboxyl terminal transactivation domain was deleted. Using these mice, we analyzed the effects of the overexpression of FLAG Delta 256 on glucose metabolism and energy homeostasis. RESULTS-The aP(2)-FLAG-Delta 256 mice showed improved glucose tolerance and insulin sensitivity accompanied with smaller-sized adipocytes and increased adiponectin (adipoq) and Glut 4 (Slc2a4) and decreased tumor necrosis factor alpha (Tnf) and chemokine (C-C motif) receptor 2 (Ccr2) gene expression levels in white adipose tissue (WAT) under a high-fat diet. Furthermore, the aP(2)-FLAG-Delta 256 mice had increased oxygen consumption accompanied with increased expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1 alpha protein and uncoupling protein (UCP)-1 (Ucp1), UCP-2 (Ucp2), and beta 3-AR (Adrb3) in brown adipose tissue (BAT). Overexpression of FLAG Delta 256 in T37i cells, which are derived from the hibernoma of SV40 large T antigen transgenic mice, increased expression of PGC-1 alpha protein and Ucp1. Furthermore, knockdown of endogenous FoxO1 in T37i cells increased Pgc1 alpha (Ppargc1a), Pgc1 beta (Ppargc1b), Ucp1, and Adrb3 gene expression. CONCLUSIONS-These data suggest that FoxO1 modulates energy homeostasis in WAT and BAT through regulation of adipocyte size and adipose tissue-specific gene expression in response to excessive calorie intake.
• Enhanced cellular uptake of virus-like particles through immobilization on a sialic acid-displaying solid surface

  Noriko Ohtake, Kenichi Niikura, Tadaki Suzuki, Keita Nagakawa, Hirofumi Sawa, Kuniharu Ijiro

  BIOCONJUGATE CHEMISTRY 19 (2) 507 - 515 1043-1802 2008/02 [Refereed][Not invited]
   

  Herein, we present the efficient cellular uptake of immobilized virus-like particles (VLPs) made of recombinant JC virus capsid proteins. VLPs expressed in Escherichia coli were labeled with fluorescein isothiocyanate (FITC). We compared two approaches for the cellular uptake of the FITC-VLPs. In the first approach, FITC-VLPs were immobilized on a polystyrene substrate, and then NIH3T3 cells were cultured on the same substrate. In the second approach, cells were cultured on a polystyrene substrate, and FITC-VLPs were then added to the cell culture medium. Flow cytometric analysis and confocal laser microscopic observation revealed that immobilized VLPs were incorporated into the cells with higher efficiency than were the diffusive VLPs suspended in solution. The cellular uptake of VLPs on the substrate was increased in a VLP density-dependent manner. As a control, disassembling VLPs to form VP1 pentamers abolished incorporation into the cells. Displaying sialic acid on the substrate enhanced VLP density through the specific affinities between the VLPs and sialic acid, resulting in efficient incorporation into the seeded cells. These techniques can be applied to the development of novel drug delivery systems and cell microarrays not only of nucleic acids but also of small molecules and proteins through their encapsulation in VLPs.
• Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in mouse

  Naomi Ohnishi, Hitomi Yuasa, Shinya Tanaka, Hirofumi Sawa, Motohiro Miura, Atsushi Matsui, Hideaki Higashi, Manabu Musashi, Kazuya Lwabuchi, Misao Suzuki, Gen Yamada, Takeshi Azuma, Masanori Hatakeyama

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (3) 1003 - 1008 0027-8424 2008/01 [Refereed][Not invited]
   

  Infection with cagA-positive Helicobacter pylori is associated with gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma of B cell origin. The cagA-encoded CagA protein is delivered into gastric epithelial cells via the bacterial type IV secretion system and, upon tyrosine phosphorylation by Src family kinases, specifically binds to and aberrantly activates SHIP-2 tyrosine phosphatase, a bona fide oncoprotein in human malignancies. CagA also elicits junctional and polarity defects in epithelial cells by interacting with and inhibiting partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) independently of CagA tyrosine phosphorylation. Despite these CagA activities that contribute to neoplastic transformation, a causal link between CagA and in vivo oncogenesis remains unknown. Here, we generated transgenic mice expressing wild-type or phosphorylation-resistant CagA throughout the body or predominantly in the stomach. Wild-type CagA transgenic mice showed gastric epithelial hyperplasia and some of the mice developed gastric polyps and adenocarcinomas of the stomach and small intestine. Systemic expression of wild-type CagA further induced leukocytosis with IL-3/GM-CSF hypersensitivity and some mice developed myeloid leukemias and B cell lymphomas, the hematological malignancies also caused by gain-of-function SHIP-2 mutations. Such pathological abnormalities were not observed in transgenic mice expressing phosphorylation-resistant CagA. These results provide first direct evidence for the role of CagA as a bacterium-derived oncoprotein (bacterial oncoprotein) that acts in mammals and further indicate the importance of CagA tyrosine phosphorylation, which enables CagA to deregulate SHIP-2, in the development of H. pylori-associated neoplasms.
• Pharmacological cdk inhibitor R-Roscovitine suppresses JC virus proliferation

  Yasuko Orba, Yuji Sunden, Tadaki Suzuki, Kazuo Nagashima, Takashi Kimura, Shinya Tanaka, Hirofumi Sawa

  VIROLOGY 370 (1) 173 - 183 0042-6822 2008/01 [Refereed][Not invited]
   

  The human Polyomavirus JC virus (JCV) utilizes cellular proteins for viral replication and transcription in the host cell nucleus. These cellular proteins represent potential targets for antiviral drugs against the JCV. In this study, we examined the antiviral effects of the pharmacological cyclin-dependent kinase (cdk) inhibitor R-Roscovitine, which has been shown to have antiviral activity against other viruses. We found that Roscovitine significantly inhibited the viral production and cytopathic effects of the JCV in a JCV-infected cell line. Roscovitine attenuated the transcriptional activity of JCV late genes, but not early genes, and also prevented viral replication via inhibiting phosphorylation of the viral early protein, large T antigen. These data suggest that the JCV requires cdks to transcribe late genes and to replicate its own DNA. That Roscovitine exhibited antiviral activity in JCV-infected cells suggests that Roscovitine might have therapeutic utility in the treatment of progressive multifocal leukoencephalopathy (PML). (c) 2007 Elsevier Inc. All rights reserved.
• Cross-protection against H5N1 influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine

  Takeshi Ichinohe, Shin-ichi Tamura, Akira Kawaguchi, Ai Ninomiya, Masaki Imai, Shigeyuki Itamura, Takato Odagiri, Masato Tashiro, Hidehiro Takahashi, Hirofumi Sawa, William M. Mitchell, David R. Strayer, William A. Carter, Joe Chiba, Takeshi Kurata, Tetsutaro Sata, Hideki Hasegawa

  JOURNAL OF INFECTIOUS DISEASES 196 (9) 1313 - 1320 0022-1899 2007/11 [Refereed][Not invited]
   

  Background. Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. Methods. BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005-2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I): poly(C 12 U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. Results. Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. Conclusions. Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor -3 agonist, poly(I): poly(C-12 U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.
• Cross-protection against H5N1 influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine

  Takeshi Ichinohe, Shin-ichi Tamura, Akira Kawaguchi, Ai Ninomiya, Masaki Imai, Shigeyuki Itamura, Takato Odagiri, Masato Tashiro, Hidehiro Takahashi, Hirofumi Sawa, William M. Mitchell, David R. Strayer, William A. Carter, Joe Chiba, Takeshi Kurata, Tetsutaro Sata, Hideki Hasegawa

  JOURNAL OF INFECTIOUS DISEASES 196 (9) 1313 - 1320 0022-1899 2007/11 [Not refereed][Not invited]
   

  Background. Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. Methods. BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005-2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I): poly(C 12 U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. Results. Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. Conclusions. Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor -3 agonist, poly(I): poly(C-12 U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.
• Oligodendrocyte lineage transcription factor 2 inhibits the motility of a human glial tumor cell line by activating RhoA

  Kouichi Tabu, Yusuke Ohba, Tadaki Suzuki, Yoshinori Makino, Taichi Kimura, Akiko Ohnishi, Mieko Sakai, Takuya Watanabe, Shinya Tanaka, Hirofumi Sawa

  MOLECULAR CANCER RESEARCH 5 (10) 1099 - 1109 1541-7786 2007/10 [Refereed][Not invited]
   

  The basic helix-loop-helix transcription factor, oligodendrocyte lineage transcription factor 2 (OLIG2), is specifically expressed in the developing and mature central nervous system and plays an important role in oligodendrogenesis from neural progenitors. It is also expressed in various types of glial tumors, but rarely in glioblastoma. Although we previously showed that OLIG2 expression inhibits glioma cell growth, its role in tumorigenesis remains incompletely understood. Here, we investigated the effect of OLIG2 expression on the migration of the human glioblastoma cell line U12-1. In these cells, OLIG2 expression is controlled by the Tet-off system. Induction of OLIG2 expression inhibited both the migration and invasiveness of U12-1 cells. OLIG2 expression also increased the activity of the GTPase RhoA as well as inducing the cells to form stress fibers and focal adhesions. Experiments using short interfering RNA against p27(KiP1) revealed that up-regulation of the p27(Kip1) protein was not essential for RhoA activation, rather it contributed independently to the decreased motility of OLIG2-expressing U12-1 cells. Alternatively, semiquantitative reverse transcription-PCR analysis revealed that mRNA expression of RhoGAP8, which regulates cell migration, was decreased by OLIG2 expression. Furthermore, expression of C3 transferase, which inhibits Rho via ADP ribosylation, attenuated the OLIG2-induced inhibition of cell motility. Imaging by fluorescence resonance energy transfer revealed that in U12-1 cells lacking OLIG2, the active form of RhoA was localized to protrusions of the cell membrane. In contrast, in OLIG2-expressing cells, it lined almost the entire plasma membrane. Thus, OLIG2 suppresses the motile phenotype of glioblastoma cells by activating RhoA.
• A case of highly active anti-retroviral therapy-induced immune reconstitution inflammatory syndrome in AIDS-related progressive multifocal leukoencephalopathy

  Eiji Imamura, Hiroshi Yamashita, Toshiyuki Fukuhara, Hirofumi Sawa, Kazuo Nagashima, Masao Kuwabara, Hiroshi Tokinobu

  Clinical Neurology 47 (10) 650 - 656 0009-918X 2007/10 [Refereed][Not invited]
   

  A 44-year-old man presented with difficulties in gripping a ball with the left hand upon playing tennis. He developed muscle weakness involving the left limbs, as well as memory decline, with subsequent gradual worsening of such symptoms. Cranial MRI showed multiple high intensity lesions in the white matter on T2-weighted imaging and FLAIR imaging. Serologic testing was positive for HIV infection and laboratory studies revealed a CD4 +T cell count of 103 cells/μl and a plasma HIV-1 RNA load of 240,000 copies/ml. Accordingly, the diagnosis of AIDS was made. Although the initial result of DNA amplification by PCR for detection of the JC virus with CSF was negative, PML was suspected because of the presence of multifocal white matter lesions. Four weeks after the commencement of highly active anti-retroviral therapy (HAART), the HIV-1 RNA load was decreased from 680,000 to 480 copies/ml, although the CD4 +T cell count was unchanged. JC virus DNA became positive at the second examination. Five weeks after the commencement of HAART, general fever, disturbance of consciousness, and brain edema suddenly developed, and the patient died within two days. Histological examination of the autopsied brain revealed demyelination and reactive gliosis within the white matter and JC virus antigen was detected in oligodendrocytes, consistent with a diagnosis of PML. The most striking feature was an intense perivascular infiltration by CD8 +T cells with proteinaceous fluid exudation, suggesting an occurrence of HAART-induced immune reconstitution inflammatory syndrome. Immune reconstitution inflammatory syndrome should be considered as a fatal complication of HAART in patients with AIDS-related PML.
• Oligodendrocyte lineage transcription factor 2 inhibits the motility of a human glial tumor cell line by activating RhoA

  Kouichi Tabu, Yusuke Ohba, Tadaki Suzuki, Yoshinori Makino, Taichi Kimura, Akiko Ohnishi, Mieko Sakai, Takuya Watanabe, Shinya Tanaka, Hirofumi Sawa

  MOLECULAR CANCER RESEARCH 5 (10) 1099 - 1109 1541-7786 2007/10 [Not refereed][Not invited]
   

  The basic helix-loop-helix transcription factor, oligodendrocyte lineage transcription factor 2 (OLIG2), is specifically expressed in the developing and mature central nervous system and plays an important role in oligodendrogenesis from neural progenitors. It is also expressed in various types of glial tumors, but rarely in glioblastoma. Although we previously showed that OLIG2 expression inhibits glioma cell growth, its role in tumorigenesis remains incompletely understood. Here, we investigated the effect of OLIG2 expression on the migration of the human glioblastoma cell line U12-1. In these cells, OLIG2 expression is controlled by the Tet-off system. Induction of OLIG2 expression inhibited both the migration and invasiveness of U12-1 cells. OLIG2 expression also increased the activity of the GTPase RhoA as well as inducing the cells to form stress fibers and focal adhesions. Experiments using short interfering RNA against p27(KiP1) revealed that up-regulation of the p27(Kip1) protein was not essential for RhoA activation, rather it contributed independently to the decreased motility of OLIG2-expressing U12-1 cells. Alternatively, semiquantitative reverse transcription-PCR analysis revealed that mRNA expression of RhoGAP8, which regulates cell migration, was decreased by OLIG2 expression. Furthermore, expression of C3 transferase, which inhibits Rho via ADP ribosylation, attenuated the OLIG2-induced inhibition of cell motility. Imaging by fluorescence resonance energy transfer revealed that in U12-1 cells lacking OLIG2, the active form of RhoA was localized to protrusions of the cell membrane. In contrast, in OLIG2-expressing cells, it lined almost the entire plasma membrane. Thus, OLIG2 suppresses the motile phenotype of glioblastoma cells by activating RhoA.
• Axonal guidance protein FEZ1 associates with tubulin and kinesin motor protein to transport mitochondria in neurites of NGF-stimulated PC12 cells

  Toshitsugu Ftijita, Andres D. Maturana, Junko Ikuta, Juri Hamada, Sebastien Walchli, Tadaki Suzuki, Hirofumi Sawa, Marie W. Wooten, Toshihide Okajima, Kenji Taternatsu, Katsuyuki Tanizawa, Shun'ichi Kuroda

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 361 (3) 605 - 610 0006-291X 2007/09 [Refereed][Not invited]
   

  Fasciculation and elongation protein zeta-1 (FEZ1) promotes efficiently the neurite elongation of rat phaeochromocytoma PC12 cells. We here characterized FEZ1 in PC12 cells. Nerve growth factor (NGF) stimulation induces significant expression of endogenous FEZ1 in PC12 cells. Upon NGF stimulation FEZ1 localizes in both cytoplasm and neuritis, co-localizing with mitochondria. Silencing of FEZ1 by RNA interference efficiently reduces NGF-induced neurite elongation and the anterograde motility of mitochondria in PC12 cells. immunoprecipitation and pulldown assay shows that FEZ1 interacts with kinesin superfamily protein 5 (KIF5) and tubulin. Thus, our results suggest that the FEZ1/kinesin complex functions for the transport of mitochondria along microtubules toward the extending neurites in differentiating PC12 cells. (c) 2007 Elsevier Inc. All rights reserved.
• Intranasal immunization with H5N1 vaccine plus Poly I : Poly C12U, a Toll-like receptor agonist, protects mice against homologous and heterologous virus challenge

  Takeshi Ichinohe, Akira Kawaguchi, Shin-ichi Tamura, Hidehiro Takahashi, Hirofumi Sawa, Ai Ninomiya, Masaki Imai, Shigeyuki Itamura, Takato Odagiri, Masato Tashiro, Joe Chiba, Tetsutaro Sata, Takeshi Kurata, Hideki Hasegawa

  MICROBES AND INFECTION 9 (11) 1333 - 1340 1286-4579 2007/09 [Refereed][Not invited]
   

  The avian H5N1 influenza virus has the potential to cause a new pandemic. Since it is difficult to predict which strain of influenza will cause a pandemic, it is advantageous to produce vaccines that confer cross-protective immunity. Mucosal vaccine administration was reported to induce cross-protective immunity by inducing secretion of IgA at the mucosal surface. Adjuvants can also enhance the development of fully protective mucosal immunity. Here we show that a new mucosal adjuvant, polyI:polyC(12)U (Ampligen (R)), a Toll-like receptor 3 agonist proven to be safe in a Phase III human trial, is an effective adjuvant for H5N1 influenza vaccination. Intranasal administration of a candidate influenza vaccine with Ampligen resulted in secretion of IgA, and protected mice that were subsequently challenged with homologous A/Vietnam/1194/2004 and heterologous A/HK/483/97 and A/Indonesia/6/2005 virus. (C) 2007 Elsevier Masson SAS. All rights reserved.
• [Etiological agent and pathogenicity mechanism of PML].

  Suzuki T, Nagashima K, Sawa H

  Nihon rinsho. Japanese journal of clinical medicine 8 65 1495 - 1500 0047-1852 2007/08 [Refereed][Not invited]
  
• Gab family proteins are essential for postnatal maintenance of cardiac function via neuregulin-1/ErbB signaling

  Yoshikazu Nakaoka, Keigo Nishida, Masahiro Narimatsu, Atsunori Kamiya, Takashi Minami, Hirofumi Sawa, Katsuya Okawa, Yasushi Fujio, Tatsuya Koyama, Makiko Maeda, Manami Sone, Satoru Yamasaki, Yuji Arai, Gou Young Koh, Tatsuhiko Kodama, Hisao Hirota, Kinya Otsu, Toshio Hirano, Naoki Mochizuki

  JOURNAL OF CLINICAL INVESTIGATION 117 (7) 1771 - 1781 0021-9738 2007/07 [Refereed][Not invited]
   

  Grb2-associated binder (Gab) family of scaffolding adaptor proteins coordinate signaling cascades downstream of growth factor and cytokine receptors. In the heart, among EGF family members, neuregulin-1 beta (NRG-1 beta, a paracrine factor produced from endothelium) induced remarkable tyrosine phosphorylation of Gab 1 and Gab2 via erythroblastic leukemia viral oncogene (ErbB) receptors. We examined the role of Gab family proteins in NRG-1 beta/ErbB-mediated signal in the heart by creating cardiomyocyte-specific Gab1/Gab2 double knockout mice (DKO mice). Although DKO mice were viable, they exhibited marked ventricular dilatation and reduced contractility with aging. DKO mice showed high mortality after birth because of heart failure. In addition, we noticed remarkable endocardial fibroelastosis and increase of abnormally dilated vessels in the ventricles of DKO mice. NRG-1 beta induced activation of both ERK and AKT in the hearts of control mice but not in those of DKO mice. Using DNA microarray analysis, we found that stimulation with NRG-1 beta upregulated expression of an endothelium-stabilizing factor, angiopoietin 1, in the hearts of control mice but not in those of DKO mice, which accounted for the pathological abnormalities in the DKO hearts. Taken together, our observations indicated that in the NRG-1 beta/ErbB signaling, Gab1 and Gab2 of the myocardium are essential for both maintenance of myocardial function and stabilization of cardiac capillary and endocardial endothelium in the postnatal heart.
• Prophylactic effects of chitin microparticles on highly pathogenic H5N1 influenza virus

  Takeshi Ichinohe, Noriyo Nagata, Peter Strong, Shin-ichi Tamura, Hidehiro Takahashi, Ai Ninomiya, Masaki Imai, Takato Odagiri, Masato Tashiro, Hirofumi Sawa, Joe Chiba, Takeshi Kurata, Tetsutaro Sata, Hideki Hasegawa

  JOURNAL OF MEDICAL VIROLOGY 79 (6) 811 - 819 0146-6615 2007/06 [Refereed][Not invited]
   

  Highly pathogenic avian influenza virus (H5N1) is an emerging pathogen with the potential to cause great harm to humans, and there is concern about the potential for a new influenza pandemic. This virus is resistant to the antiviral effects of interferons and tumor necrosis factor-of.. However, the mechanism of interferon-independent protective innate immunity is not well understood. The prophylactic effects of chitin microparticles as a stimulator of innate mucosal immunity against a recently obtained strain of H5N1 influenza virus infection were examined in mice. Clinical parameters and the survival rate of mice treated by intranasal application of chitin microparticles were significantly improved compared to non-treated mice after a lethal influenza virus challenge. Flow cytometric analysis revealed that the number of natural killer cells that expressed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and that had migrated into the cervical lymph node was markedly increased (26-fold) after intranasal treatment with chitin microparticles. In addition, the level of IL-6 and interferon-gamma-inducible protein-10 (IP-10) in the nasal mucosa after H5N1 influenza virus challenge was decreased by prophylactic treatment with chitin microparticles. These results suggest that prophylactic intranasal administration of chitin microparticles enhanced the local accumulation of natural killer cells and suppressed hyper-induction of cytokines, resulting in an innate immune response to prevent pathogenesis of H5N1 influenza virus. J. Med. Virol. 79:811-819, 2007. (C) 2007 Wiley-Liss, Inc.
• Behavioural characteristics and gene expression in the hyperactive wiggling (Wig) rat

  Yoshinori Masuo, Masami Ishido, Masatoshi Morita, Hirofumi Sawa, Kazuo Nagashima, Etsuo Niki

  EUROPEAN JOURNAL OF NEUROSCIENCE 25 (12) 3659 - 3666 0953-816X 2007/06 [Refereed][Not invited]
   

  Recently, congenic wiggling (Wig) rats were described as a good model for attention-deficit hyperactivity disorder; 12- to 14-week-old animals demonstrated hyperactivity, impulsive behaviour and an impaired working memory. Here, we show that 4- to 5-week-old Wig rats displayed significantly greater spontaneous motor activity than control rats during a period of darkness. Subcutaneous injection of 4 mg/kg methamphetamine exacerbated hyperactivity, the reverse of its effect in rats with neonatally induced 6-hydroxydopamine lesions. Immunohistochemistry showed low levels of tyrosine hydroxylase in the ventral midbrain, similar to 6-hydroxydopamine-treated rats. In cDNA macroarrays, 4-week-old Wig rats showed increased expression of the adenosine A2a receptor in the dorsal striatum, macrophage migration inhibitory factor in the frontal cortex, ventral striatum and midbrain, and calbindin 2 in the dorsal and ventral midbrain. Expression of the gamma-aminobutyric acid (GABA) transporter and sterol carrier protein 2 genes was reduced in all regions. Dopamine transporter gene expression was increased in the dorsal midbrain but decreased in the ventral midbrain, a pattern distinct from that induced by 6-hydroxydopamine. Although abnormal development of dopaminergic neurons may underlie motor hyperactivity, other mechanisms may control responsiveness to methamphetamine. Wig rats may provide a model of attention-deficit hyperactivity disorder in which treatment with psychostimulants accelerate the hyperactivity.
• The nuclear import of the human T lymphotropic virus type I (HTLV-1) tax protein is carrier- and energy-independent

  Takahiro Tsuji, Noreen Sheehy, Virginie W. Gautier, Hitoshi Hayakawa, Hirofumi Sawa, William W. Hall

  JOURNAL OF BIOLOGICAL CHEMISTRY 282 (18) 13875 - 13883 0021-9258 2007/05 [Refereed][Not invited]
   

  HTLV-1 is the etiologic agent of the adult T cell leukemia-lymphoma (ATLL). The viral regulatory protein Tax plays a central role in leukemogenesis as a transcriptional transactivator of both viral and cellular gene expression, and this requires Tax activity in both the cytoplasm and the nucleus. In the present study, we have investigated the mechanisms involved in the nuclear localization of Tax. Employing a GFP fusion expression system and a range of Tax mutants, we could confirm that the N-terminal 60 amino acids, and specifically residues within the zinc finger motif in this region, are important for nuclear localization. Using an in vitro nuclear import assay, it could be demonstrated that the transportation of Tax to the nucleus required neither energy nor carrier proteins. Specific and direct binding between Tax and p62, a nucleoporin with which the importin beta family of proteins have been known to interact was also observed. The nuclear import activity of wild type Tax and its mutants and their binding affinity for p62 were also clearly correlated, suggesting that the entry of Tax into the nucleus involves a direct interaction with nucleoporins within the nuclear pore complex (NPC). The nuclear export of Tax was also shown to be carrier independent. It could be also demonstrated that Tax it self may have a carrier function and that the NF-kappa B subunit p65 could be imported into the nucleus by Tax. These studies suggest that Tax could alter the nucleocytoplasmic distribution of cellular proteins, and this could contribute to the deregulation of cellular processes observed in HTLV-1 infection.
• R-ras regulates exocytosis by Rgl2/Rlf-mediated activation of RalA on endosomes

  Akiyuki Takaya, Takahiro Kamio, Michitaka Masuda, Naoki Mochizuki, Hirofumi Sawa, Mami Sato, Kazuo Nagashima, Akiko Mizutani, Akira Matsuno, Etsuko Kiyokawa, Michiyuki Matsuda

  MOLECULAR BIOLOGY OF THE CELL 18 (5) 1850 - 1860 1059-1524 2007/05 [Refereed][Not invited]
   

  R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/RIf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis.
• [Recent research on the JC virus].

  Sawa H, Suzuki T, Orba Y, Sunden Y, Nagashima K

  Brain and nerve = Shinkei kenkyu no shinpo 2 59 101 - 108 1881-6096 2007/02 [Refereed][Not invited]
  
• Lamprey TLRs with properties distinct from those of the variable lymphocyte receptors

  Akihiro Ishii, Aya Matsuo, Hirofumi Sawa, Tadayuki Tsujita, Kyoko Shida, Misako Matsumoto, Tsukasa Seya

  JOURNAL OF IMMUNOLOGY 178 (1) 397 - 406 0022-1767 2007/01 [Refereed][Not invited]
   

  Fish express mammalian-type (M-type) TLRs consisting of leucine-rich repeats (LRRs) and Toll-IL-1R (TIR) homology domain for immunity, whereas invertebrates in deuterostomes appear to have no orthologs of M-type TLRs. Lampetra japonica (lamprey) belongs to the lowest class of vertebrates with little information about its TLRs. We have identified two cDNA sequences of putative TLRs in the lamprey (laTLRs) that contain LRRs and TIR domains. The two laTLRs were 56% homologous to each other, and their TIRs were similar to those of members of the human TLR2 subfamily, most likely orthologs of fish TLR14. We named them laTLR14a and laTLR14b. We raised a rabbit polyclonal Ab against laTLR14b and identified a 85-kDa protein in a human HEK293 transfectant by immunoblotting using the All. FACS, histochemical, and confocal analyses showed that laTLR14b is expressed intracellularly in lamprey gill cells and that the overexpressed protein resides in the endoplasmic reticulum of human and fish (medaka) cell lines. Because natural agonists of TLR14 remained unidentified, we made a chimera construct of extracellular CD4 and the cytoplasmic domain of laTLR14. The chimera molecule of laTLR14b, when expressed in HEK293 cells, elicited activation of NF-kappa B and, consequently, weak activation of the IFN-beta promoter. laTLR14b mRNA was observed in various organs and leukocytes. This lamprey species expressed a variable lymphocyte receptor structurally independent of laTLR14 in leukocytes. Thus, the jawless vertebrate lamprey possesses two LRR-based recognition systems, the variable lymphocyte receptor and TLR, and the M-type TLRs are conserved across humans, fish, and lampreys.
• Identification of DDX1 as a JC virus transcriptional control region-binding protein

  Yuji Sunden, Shingo Semba, Tadaki Suzuki, Yuki Okada, Yasuko Orba, Kazuo Nagashima, Takashi Umemura, Hirofumi Sawa

  MICROBIOLOGY AND IMMUNOLOGY 51 (3) 327 - 337 0385-5600 2007 [Refereed][Not invited]
   

  To investigate the mechanism behind JC virus (JCV) cell specificity we performed electrophoretic mobility shift assays (EMSA) using probes derived from the JCV transcriptional control region (JCV-TCR). Using nuclear extracts from the JCV-susceptible neuroblastoma cell line IMR-32, EMSA revealed a 670 kDa JCV-TCR-binding protein complex designated as #3-bp. This complex could not be detected in nuclear extracts from non-susceptible cell lines. Using column chromatographic purification and microsequencing, we identified cleavage stimulation factor (CstF) as a component of #3-bp. However, as CstF is present in many cell types, we speculated that the IMR-32-specific component(s) of #3bp bind CAR We performed a yeast two-hybrid assay using CstF-77 as the bait against a HeLa cDNA-subtracted IMR-32 cDNA library. This analysis detected binding between CstF-77 and the RNA helicase DDX1. Subsequently, biotinylated DNA affinity precipitation and chromatin immunoprecipitation assays also confirmed that DDX1 binds specifically to JCV-TCR. Our findings indicate that an association between DDX1 and the JCV-TCR may play a significant role in JCV infection in IMR-32 cells.
• DDX1 promotes proliferation of the JC virus through transactivation of its promoter

  Yuji Sunden, Shingo Semba, Tadaki Suzuki, Yuki Okada, Yasuko Orba, Kazuo Nagashima, Takashi Umemura, Hirofumi Sawa

  MICROBIOLOGY AND IMMUNOLOGY 51 (3) 339 - 347 0385-5600 2007 [Refereed][Not invited]
   

  Recently, we demonstrated that the DEAD box protein 1 (DDX1), an RNA helicase, and the cleavage stimulation factor (CstF) form a complex that binds to the JC virus transcriptional control region (JCV-TCR). Here, we examined the function of DDX1, which is expressed at much higher levels in the JCV-susceptible cell line IMR-32 than in non-susceptible cell lines. DDX1 had no effect on the replication efficiency of JCV, but overexpression of DDX1 significantly increased transactivation of the JCV promoter. Furthermore, DDX1 enhanced the expression of JCV proteins in JCV infected cells, and knockdown of DDX1 using small interfering (si) RNA suppressed the expression of JCV proteins. Our results clearly demonstrate that DDX1 regulates proliferation of JCV in vitro through transcriptional activation.
• 高活性レトロウイルス療法により免疫再構築症候群をきたしたAIDSにともなう進行性多巣性白質脳症の1剖検例

  臨床神経学 65 1495 - 1500 2007 [Not refereed][Not invited]
  
• 進行性多巣性白質脳症 （PML）：原因ウイルスと発病機構。

  日本臨床 65 1495 - 1500 2007 [Not refereed][Not invited]
  
• Intranasal administration of adjuvant-combined recombinant influenza virus HA vaccine protects mice from the lethal H5N1 virus infection

  Yasuko Asahi-Ozaki, Shigeyuki Itamura, Takeshi Ichinohe, Peter Strong, Shin-ichi Tamura, Hidehiro Takahashi, Hirofumi Sawa, Masami Moriyama, Masato Tashiro, Tetsutaro Sata, Takeshi Kurata, Hideki Hasegawa

  MICROBES AND INFECTION 8 (12-13) 2706 - 2714 1286-4579 2006/10 [Refereed][Not invited]
   

  Attenuated recombinant H5N1 influenza virus was constructed to develop a safe H5N1 influenza vaccine. The immunogenicity and protective effect of the vaccine prepared from haemagglutinin-modified recombinant H5N1 influenza virus was evaluated in mice intranasally co-administered with cholera toxin B subunit containing a trace amount of holotoxin (CTB*), synthetic double-stranded RNA, poly (I:C) or chitin microparticles (CMP) as adjuvants. Intranasal administration of recombinant H5 HA split vaccine with CTB* or poly(I:C) and/or CMP elicited an immunological response with both anti-H5 HA IgA in the nasal wash and anti-H5 HA IgG antibody in the serum, and showed a protective against lethal H5N1 A/Hong Kong/483/97 (HK483) infection. We also demonstrated that intranasal co-administration of antigen with both poly (I:C) and CMP enhanced the expression of Toll-like receptor (TLR) 3, TLR7 in the spleen. These results indicate that poly (I:C) and CMP are highly effective as mucosal adjuvants for use with the nasal H5N1 vaccine. (c) 2006 Elsevier Masson SAS. All rights reserved.
• Tristetraprolin inhibits HIV-1 production by binding to genomic RNA

  Masae Maeda, Hirofumi Sawa, Minoru Tobiume, Kenzo Tokunaga, Hideki Hasegawa, Takeshi Ichinohe, Tetsutaro Sata, Masami Moriyama, William W. Hall, Takeshi Kurata, Hidehiro Takahashi

  MICROBES AND INFECTION 8 (11) 2647 - 2656 1286-4579 2006/09 [Refereed][Not invited]
   

  HIV-1 genome has an AU-rich sequence and requires rapid nuclear export by Rev activity to prevent multiple splicing. HIV-1 infection occurs in activated CD4(+) T cells where the decay of mRNAs of cytokines and chemokines is regulated by the binding of AU-rich elements to the mRNA-destabilizing protein tristetraprolin. We here investigated the influence of tristetraprolin on the replication of HIV-1. Treatment of siRNA against tristetraprolin in a latently HIV-1 infected cell line increases HIV-1 production following stimulation. A chloramphenicol acetyltransferase and luciferase assay revealed that exogenous tristetraprolin reduced HIV-1 virion production and in contrast increased the multiply spliced products. Furthermore, quantitative RT-PCR analysis showed tristetraprolin increases the ratio of multiple-spliced RNAs to un-, single-spliced RNA. Moreover. an electrophoretic mobility shift assay showed that tristetraprolin binds to synthesized HIV-1 RNA with AU-rich sequence but not to RNA with less AU sequence. These results suggest that tristetraprolin is a regulator of HIV-1 replication and enhances splicing by direct binding to AU-rich sequence of HIV-1 RNAs. (c) 2006 Elsevier Masson SAS. All rights reserved.
• Protection against influenza virus infection by intranasal vaccine with surf clam microparticles (SMP) as an adjuvant

  T Ichinohe, Watanabe, I, E Tao, S Ito, A Kawaguchi, S Tamura, H Takahashi, H Sawa, M Moriyama, J Chiba, K Komase, Y Suzuki, T Kurata, T Sata, H Hasegawa

  JOURNAL OF MEDICAL VIROLOGY 78 (7) 954 - 963 0146-6615 2006/07 [Refereed][Not invited]
   

  A safe and effective adjuvant is necessary to enhance mucosal immune responses for the development of an inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of surf clam microparticles (SMP) derived from natural surf clams as an adjuvant for an intranasal influenza vaccine. The adjuvant effect of SMP was examined when co-administered intranasally with inactivated A/PR8 (H1N1) influenza virus hemagglutinin vaccine in BALB/c mice. Administration of the vaccine with SMP induced a high anti-PR8 haemagglutinin (HA)specific immunoglobulin A (IgA) response in the nasal wash and immunoglobulin G (IgG) response in the serum, resulting in protection against both nasal-restricted infection and lethal lung infection by A/PR8 virus. In addition, administration of SMP with A/Yamagata (H1N1), A/Beijing (H1N1), or A/Guizhou (H3N2) vaccine conferred complete protection against A/PR8 virus challenge in the nasal infection model, suggesting that SMP adjuvanted vaccine can confer cross-protection against variant influenza viruses. The use of SMP is suggested as a new safe and effective mucosal adjuvant for nasal vaccination against influenza virus infection.
• Adaptor molecule Crk is required for sustained phosphorylation of Grb2-associated binder 1 and hepatocyte growth factor-induced cell motility of human synovial sarcoma cell lines

  Takuya Watanabe, Masumi Tsuda, Yoshinori Makino, Shin Ichihara, Hirofumi Sawa, Akio Minami, Naoki Mochizuki, Kazuo Nagashima, Shinya Tanaka

  MOLECULAR CANCER RESEARCH 4 (7) 499 - 510 1541-7786 2006/07 [Refereed][Not invited]
   

  Activation of the c-Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes mitogenic, motogenic, and morphogenic cellular responses. Aberrant HGF/c-Met signaling has been strongly implicated in tumor cell invasion and metastasis. Both HGF and its receptor c-Met have been shown to be overexpressed in human synovial sarcoma, which often metastasizes to the lung; however, little is known about HGF-mediated biological effects in this sarcoma. Here, we provide evidence that Crk adaptor protein is required for the sustained phosphorylation of c-Met-docking protein Grb2-associated binder 1 (Gab1) in response to HGF, leading to the enhanced cell motility of human synovial sarcoma cell lines SY0-1, HS-SY-II, and Fuji. HGF stimulation induced the sustained phosphorylation on Y307 of Gab1 where Crk was recruited. Crk knockdown by RNA interference disturbed this HGF-induced tyrosine phosphorylation of Gab1. By mutational analysis, we identified that Src homology 2 domain of Crk is indispensable for the induction of the phosphorylation on multiple Tyr-X-X-Pro motifs containing Y307 in Gab1. HGF remarkably stimulated cell motility and scattering of synovial sarcoma cell lines, consistent with the prominent activation of Rac1, extreme filopodia formation, and membrane ruffling. Importantly, the elimination of Crk in these cells induced the disorganization of actin cytoskeleton and complete abolishment of HGF-mediated Rac1 activation and cell motility. Time-lapse microscopic analysis revealed the significant attenuation in scattering of Crk knockdown cells following HGF treatment. Furthermore, the depletion of Crk remarkably inhibited the tumor formation and its invasive growth in vivo. These results suggest that the sustained phosphorylation of Gab1 through Crk in response to HGF contributes to the prominent activation of Rac1 leading to enhanced cell motility, scattering, and cell invasion, which may support the crucial role of Crk in the aggressiveness of human synovial sarcoma.
• Involvement of adaptor protein Crk in malignant feature of human ovarian cancer cell line MCAS

  H Linghu, M Tsuda, Y Makino, M Sakai, T Watanabe, S Ichihara, H Sawa, K Nagashima, N Mochizuki, S Tanaka

  ONCOGENE 25 (25) 3547 - 3556 0950-9232 2006/06 [Not refereed][Not invited]
   

  Signaling adaptor protein Crk regulates cell motility and growth through its targets Dock180 and C3G, those are the guanine-nucleotide exchange factors (GEFs) for small GTPases Rac and Rap, respectively. Recently, overexpression of Crk has been reported in various human cancers. To de. ne the role for Crk in human cancer cells, Crk expression was targeted in the human ovarian cancer cell line MCAS through RNA interference, resulting in the establishment of three Crk knockdown cell lines. These cell lines exhibited disorganized actin fibers, reduced number of focal adhesions, and abolishment of lamellipodia formation. Decreased Rac activity was demonstrated by pull-down assay and FRET-based time-lapse microscopy, in association with suppression of both motility and invasion by phagokinetic track assay and transwell assay in these cells. Furthermore, Crk knockdown cells exhibited slow growth rates in culture and suppressed anchorage-dependent growth in soft agar. Tumor forming potential in nude mice was attenuated, and intraperitoneal dissemination was not observed when Crk knockdown cells were injected into the peritoneal cavity. These results suggest that the Crk is a key component of focal adhesion and involved in cell growth, invasion, and dissemination of human ovarian cancer cell line MCAS.
• Characterization and application of polyclonal antibodies that specifically recognize JC virus large T antigen

  Y Sunden, T Suzuki, Y Orba, T Umemura, M Asamoto, K Nagashima, S Tanaka, H Sawa

  ACTA NEUROPATHOLOGICA 111 (4) 379 - 387 0001-6322 2006/04 [Refereed][Not invited]
   

  Polyomavirus JC virus (JCV) is the causative agent of progressive multifocal leukoencephalopathy, a fatal demyelinating disease of the central nervous system. Similar to other polyomaviruses, such as simian vacuolating virus 40 (SV40) and BK virus (BKV), JCV is also associated with human tumours. The Polyomavirus early protein large T antigen (TAg) plays a crucial role in tumour pathogenesis. An antibody to SV40 TAg (PAb416), which cross-reacts with TAgs of both JCV and BKV, has been used widely for the detection of JCV and BKV TAgs. As a consequence, it is difficult to discriminate between the TAgs of SV40, BKV and JCV by immunohistochemical analyses. In the present study, we generated JCV TAg-specific polyclonal antibodies (JCT629 and JCT652) by immunization of New Zealand white rabbits with synthetic peptides reproducing the JCV TAg carboxyl-terminal region as immunogens. Immunoblotting analyses indicated that the new antibodies bind specifically to JCV TAg, and not to those of SV40 or BKV. We also demonstrated that these antibodies can be used for immunoprecipitation, immunocytochemical analyses and immunohistochemical staining of routinely processed specimens. In conclusion, the newly generated JCV-specific TAg antibodies may be useful both in the investigation of the pathophysiological function of JCV TAg and in discriminating between Polyomavirus-related clinical samples.
• Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I

  H Hasegawa, H Sawa, MJ Lewis, Y Orba, N Sheehy, Y Yamamoto, T Ichinohe, Y Tsunetsugu-Yokota, H Katano, H Takahashi, J Matsuda, T Sata, T Kurata, K Nagashima, WW Hall

  NATURE MEDICINE 12 (4) 466 - 472 1078-8956 2006/04 [Refereed][Not invited]
   

  Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4(-) and CD8(-), but CD44(+), CD25(+) and cytoplasmic CD3(+). This phenotype is indicative of a thymus-derived pre-T-cell phenotype, and disease development was associated with the constitutive activation of NF-kappa B. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics.
• A novel function of OLIG2 to suppress human glial tumor cell growth via p27(Kip1) transactivation

  K Tabu, A Ohnishi, Y Sunden, T Suzuki, M Tsuda, S Tanaka, T Sakai, K Nagashima, H Sawa

  JOURNAL OF CELL SCIENCE 119 (7) 1433 - 1441 0021-9533 2006/04 [Refereed][Not invited]
   

  The basic helix-loop-helix transcription factor OLIG2 is specifically expressed in cells of the oligodendrocyte lineage. It is also expressed in various tumors originating from glial cells; however, the expression of OLIG2 is rare or weak in glioblastomas, the most malignant gliomas. The role of OLIG2 in glioma remains unclear. To investigate the function of OLIG2 in glial tumor cells, we have established a glioblastoma cell line, U12-1, in which the expression of OLIG2 is induced by the Tet-off system. Induction of OLIG2 resulted in suppression of both the proliferation and anchorage-independent growth of U12-1. It also resulted in an increase in the expression of p27(Kip1). A luciferase assay revealed that the CTF site of the p27(Kip1) gene promoter was essential for OLIG2-dependent activation of p27(Kip1) gene transcription. Electrophoretic mobility shift assays confirmed that a nuclear extract of OLIG2-expressing U12-1 cells contained a protein complex that binds to the CTF site of the p27(Kip1) gene promoter. Furthermore, siRNA against p27(Kip1) rescued the OLIG2-mediated growth and DNA synthesis inhibition of U12-1 cells. These results indicate that OLIG2 suppresses the proliferation of U12-1 and that this effect is mediated by transactivation of the p27(Kip1) gene, and low expression of OLIG2 may be related to the malignant behavior of human glioblastoma.
• Elmo1 inhibits ubiquitylation of Dock180

  Y Makino, M Tsuda, S Ichihara, T Watanabe, M Sakai, H Sawa, K Nagashima, S Hatakeyama, S Tanaka

  JOURNAL OF CELL SCIENCE 119 (5) 923 - 932 0021-9533 2006/03 [Refereed][Not invited]
   

  Dock180, a member of the CDM family of proteins, plays roles in biological processes such as phagocytosis and motility through its association with the signalling adaptor protein Crk. Recently, the complex formation between Dock180 and Elmo1 was reported to function as a bipartite guanine nucleotide exchange factor for Rac. In this study, we demonstrated that the amount of Dock180 increased when Elmo1 was co-expressed. Dock180 was found to be ubiquitylated and Dock180 protein levels could be augmented by treatment with proteasome inhibitor. The ubiquitylation of Dock180 was enhanced by epidermal growth factor (EGF), Crk and adhesion-dependent signals. Furthermore, Elmo1 inhibited labiquitylation of Dock180, resulting in the increase in Dock180 levels. The Elmo1 mutant Delta 531, which encompasses amino acids required for Dock180 binding, preserved the inhibitory effects on ubiquitylation of Dock180. Upon EGF stimulation, both Dock180 and ubiquitin were demonstrated to translocate to the cell periphery by immunofluorescence, and we found ubiquitylation of Dock180 and its inhibition by Elmo1 to occur in cellular membrane fractions by in vivo ubiquitylation assay. These data suggest that Dock180 is ubiquitylated on the plasma membrane, and also that Elmo1 functions as an inhibitor of ubiquitylation of Dock180. Therefore, an ubiquitin-proteasome-dependent protein degradation mechanism might contribute to the local activation of Rac on the plasma membrane.
• Dicer and positive charge of proteins decrease the stability of RNA containing the AU-rich element of GM-CSF

  H Takahashi, M Maeda, H Sawa, H Hasegawa, M Moriyama, T Sata, WW Hall, T Kurata

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 340 (3) 807 - 814 0006-291X 2006/02 [Refereed][Not invited]
   

  AU-rich elements (AREs) in the 3'-untranslated region of mRNAs promote rapid decay of the mRNAs for certain cytokines, including that encoding granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that,in RNA molecule based on the ARE of GM-CSF mRNA is cleaved between U and A residues in the presence of bovine serum albumin Of Which cleavage effect is attenuated by acetylation. Furthermore, the expression of RNA molecule containing the ARE of GM-CSF mRNA in human cell lines was increased by inhibition of histone deacetylase activity and attenuation of Dicer expression. These findings suggest that degradation of mRNAs containing an ARE might be regulated by positive charge of polypeptides and Dicer. (c) 2005 Elsevier Inc. All rights reserved.
• Induction of p21(WAF1/CIP1) by human synovial sarcoma-associated chimeric oncoprotein SYT-SSX1

  M Tsuda, T Watanabe, T Seki, T Kimura, H Sawa, A Minami, T Akagi, K Isobe, K Nagashima, S Tanaka

  ONCOGENE 24 (54) 7984 - 7990 0950-9232 2005/12 [Refereed][Not invited]
   

  Oncogenic protein provokes cell cycle arrest termed premature senescence. In this process Ras has been known to induce cyclin-dependent kinase inhibitor (CKI) p16(INK4A) in primary fibroblasts. Here, we present a novel finding that human chimeric oncoprotein SYT-SSX1 induces CKI p21(WAF1/CIP1) (p21) for suppression of cell growth. In human synovial sarcoma cell lines, the expression levels of p21 were high and the transcriptional activity of the p21 gene promoter was significantly elevated. The transient expression of SYT- SSX1-induced activation of the p21 gene promoter in human diploid fibroblasts. The N-terminus deletion form of SYT-SSX1, which failed to bind to hBRM one of the chromatin remodeling factors, preserved the p21 induction ability. This effect of SYT-SSX1 was similar in extent in both wild-type and p53-deficient HCT116 cell lines. Furthermore, the introduction of mutation in Sp1/Sp3 binding sites of the p21 gene promoter abolished the SYT-SSX1-induced transcriptional activity of its promoter. In SW13 cells, the stable expression of SYT-SSX1 suppressed cell growth in culture. These results suggest that SYT-SSX1 is able to induce p21 in a manner independent on hBRM and p53 but dependent on Sp1/Sp3.
• Strategic Approach to Information Security and Assurance in Health Research

  Shunichi Akazawa, Manabu Igarashi, Hirofumi Sawa, Hiko Tamashiro

  Environmental Health and Preventive Medicine 10 (5) 282 - 285 1342-078X 2005/09 [Refereed][Not invited]
   

  Information security and assurance are an increasingly critical issue in health research. Whether health research be in genetics, new drugs, disease outbreaks, biochemistry, or effects of radiation, it deals with information that is highly sensitive and which could be targeted by rogue individuals or groups, corporations, national intelligence agencies, or terrorists, looking for financial, social, or political gains. The advents of the Internet and advances in recent information technologies have also dramatically increased opportunities for attackers to exploit sensitive and valuable information. Government agencies have deployed legislative measures to protect the privacy of health information and developed information security guidelines for epidemiological studies. However, risks are grossly underestimated and little effort has been made to strategically and comprehensively protect health research information by institutions, governments and international communities. There is a need to enforce a set of proactive measures to protect health research information locally and globally. Such measures should be deployed at all levels but will be successful only if research communities collaborate actively, governments enforce appropriate legislative measures at national level, and the international community develops quality standards, concluding treaties if necessary, at the global level. Proactive measures for the best information security and assurance would be achieved through rigorous management process with a cycle of "plan, do, check, and act". Each health research entity, such as hospitals, universities, institutions, or laboratories, should implement this cycle and establish an authoritative security and assurance organization, program and plan coordinated by a designated Chief Security Officer who will ensure implementation of the above process, putting appropriate security controls in place, with key focus areas such as policies and best practices, enforcement and certification, risk assessment and audit, monitoring and incident response, awareness and training, and modern protection method and architecture. Governments should enforce a comprehensive scheme, and international health research communities should adopt standardized innovative methods and approaches.
• The agnoprotein of polyomaviruses: A multifunctional auxiliary protein

  K Khalili, MK White, H Sawa, K Nagashima, M Safak

  JOURNAL OF CELLULAR PHYSIOLOGY 204 (1) 1 - 7 0021-9541 2005/07 [Refereed][Not invited]
   

  The late region of the three primate polyomaviruses (JCV, BKV, and SV40) encodes a small, highly basic protein known as agnoprotein. While much attention during the last two decades has focused on the transforming proteins encoded by the early region (small and large T-antigens), it has become increasingly evident that agnoprotein has a critical role in the regulation of viral gene expression and replication, and in the modulation of certain important host cell functions including cell cycle progression and DNA repair. The importance of agnoprotein is underscored by its expression during lytic infection of glial cells by JCV that occurs in progressive multifocal leukoencephalopathy (PML), and also in some JCV-associated human neural tumors particularly medulloblastoma. In this review, we will discuss the structure and function of agnoprotein in the viral life cycle during the course of lytic infection and the consequences of agnoprotein expression for the host cell.
• Identification of FEZ1 as a protein that interacts with JC virus agnoprotein and microtubules - Role of agnoprotein-induced dissociation of FEZ1 from microtubules in viral propagation

  T Suzuki, Y Okada, S Semba, Y Orba, S Yamanouchi, S Endo, S Tanaka, T Fujita, S Kuroda, K Nagashima, H Sawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 280 (26) 24948 - 24956 0021-9258 2005/07 [Refereed][Not invited]
   

  The human polyomavirus JC virus (JCV) is the causative agent of a fatal demyelinating disease, progressive multifocal leukoencephalopathy, and encodes six major proteins, including agnoprotein. Agnoprotein colocalizes with microtubules in JCV-infected cells, but its function is not fully understood. We have now identified fasciculation and elongation protein zeta 1 (FEZ1) as a protein that interacted with JCV agnoprotein in a yeast two-hybrid screen of a human brain cDNA library. An in vitro binding assay showed that agnoprotein interacted directly with FEZ1 and microtubules. A microtubule co-sedimentation assay revealed that FEZ1 also associates with microtubules and that agnoprotein induces the dissociation of FEZ1 from microtubules. Agnoprotein inhibited the promotion by FEZ1 of neurite outgrowth in PC12 cells. Conversely, overexpression of FEZ1 suppressed JCV protein expression and intracellular trafficking in JCV-infected cells. These results suggest that FEZ1 promotes neurite extension through its interaction with microtubules, and that agnoprotein facilitates JCV propagation by inducing the dissociation of FEZ1 from microtubules.
• Identification of FEZ1 as a protein that interacts with JC virus agnoprotein and microtubules - Role of agnoprotein-induced dissociation of FEZ1 from microtubules in viral propagation

  T Suzuki, Y Okada, S Semba, Y Orba, S Yamanouchi, S Endo, S Tanaka, T Fujita, S Kuroda, K Nagashima, H Sawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 280 (26) 24948 - 24956 0021-9258 2005/07 [Not refereed][Not invited]
   

  The human polyomavirus JC virus (JCV) is the causative agent of a fatal demyelinating disease, progressive multifocal leukoencephalopathy, and encodes six major proteins, including agnoprotein. Agnoprotein colocalizes with microtubules in JCV-infected cells, but its function is not fully understood. We have now identified fasciculation and elongation protein zeta 1 (FEZ1) as a protein that interacted with JCV agnoprotein in a yeast two-hybrid screen of a human brain cDNA library. An in vitro binding assay showed that agnoprotein interacted directly with FEZ1 and microtubules. A microtubule co-sedimentation assay revealed that FEZ1 also associates with microtubules and that agnoprotein induces the dissociation of FEZ1 from microtubules. Agnoprotein inhibited the promotion by FEZ1 of neurite outgrowth in PC12 cells. Conversely, overexpression of FEZ1 suppressed JCV protein expression and intracellular trafficking in JCV-infected cells. These results suggest that FEZ1 promotes neurite extension through its interaction with microtubules, and that agnoprotein facilitates JCV propagation by inducing the dissociation of FEZ1 from microtubules.
• Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles

  Y Okada, T Suzuki, Y Sunden, Y Orba, S Kose, N Imamoto, H Takahashi, S Tanaka, WW Hall, K Nagashima, H Sawa

  EMBO REPORTS 6 (5) 452 - 457 1469-221X 2005/05 [Refereed][Not invited]
   

  The nuclear envelope is one of the chief obstacles to the translocation of macromolecules that are larger than the diameter of nuclear pores. Heterochromatin protein 1 (HP1) bound to the lamin B receptor (LBR) is thought to contribute to reassembly of the nuclear envelope after cell division. Human polyomavirus agnoprotein ( Agno) has been shown to bind to HP1 alpha and to induce its dissociation from LBR, resulting in destabilization of the nuclear envelope. Fluorescence recovery after photobleaching showed that Agno increased the lateral mobility of LBR in the inner nuclear membrane. Biochemical and immunofluorescence analyses showed that Agno is targeted to the nuclear envelope and facilitates the nuclear egress of polyomavirus-like particles. These results indicate that dissociation of HP1a from LBR and consequent perturbation of the nuclear envelope induced by polyomavirus Agno promote the translocation of virions out of the nucleus.
• Clinicopathological and virological analyses of familial human T-lymphotropic virus type I-associated polyneuropathy

  H Sawa, T Nagashima, K Nagashima, T Shinohara, T Chuma, Y Mano, N Tachi, WW Hall

  JOURNAL OF NEUROVIROLOGY 11 (2) 199 - 207 1355-0284 2005/04 [Refereed][Not invited]
   

  Human T-lymphotropic virus type I (HTLV-I) is known to be the causative agent of the chronic myelopathy, HTLV- I-associated myelopathy ( HAM), and on rare occasions infection is also associated with the development of polyneuropathy. Here the authors present an HTLV-I-positive family of whom four members developed a chronic demyelinating polyneuropathy without HAM. Four female patients in a family from Hokkaido in Japan developed distal dominant paresthesia and muscle weakness in the second and third decades of their life. Neurological findings at ages ranging from 50 to 65 years included mild painful sensorimotor disturbances with atrophy of the distal parts of the extremities but without pyramidal signs or hyperactive tendon reflexes. Magnetic resonance imaging (MRI) findings of brain and spinal cord were unremarkable. Serum HTLV- I antibody levels were elevated at 1: 8,192 to 1: 32,768, whereas those in cerebrospinal fluid were low at 1: 4 to 1: 8. Electrophysiological studies revealed polyphasic compound muscle action potentials with denervation potentials on nerve conduction studies and neurogenic patterns by electromyography, which were consistent with signs of chronic motor dominant demyelinating polyneuropathy. Sural nerve biopsy showed decreased myelinated fibers, occurrence of globule formation, myelin ovoid and remyelinated fibers, and an infiltration of CD68-positive macrophages with occasional CD4-positive T cells in the nerve fascicles. The polyneuropathy was responsive to steroid therapy. Analyses of serological human leukocyte antigen (HLA) types indicated that none of the patients possessed a high-risk HLA type known to be associated with adult T-cell leukemia (ATL), whereas they did have high responsive alleles to HTLV- I env similar to that observed in HAM. Nucleotide sequence analysis of the HTLV- I tax region demonstrated the B subgroup in all patients. This study suggests that HTLV- I infection can result in the development of a familial form of polyneuropathy that is associated with distinct HLA class I alleles, which might possibly involve a distinct virus subtype.
• Synthetic double-stranded RNA poly(I : C) combined with mucosal vaccine protects against influenza virus infection

  T Ichinohe, Watanabe, I, S Ito, H Fujii, M Moriyama, S Tamura, H Takahashi, H Sawa, J Chiba, T Kurata, T Sata, H Hasegawa

  JOURNAL OF VIROLOGY 79 (5) 2910 - 2919 0022-538X 2005/03 [Refereed][Not invited]
   

  The mucosal adjuvant effect of synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] against influenza virus was examined under intranasal coadministration with inactivated hemagglutinin (RA) vaccine in BALB/c mice and was shown to have a protective effect against both nasal-restricted infection and lethal lung infection. Intranasal administration of vaccine from PR8 (H1N1) with poly(I:C) induced a high anti-HA immunoglobulin A (IgA) response in the nasal wash and IgG antibody response in the serum, while vaccination without poly(I:C) induced little response. Intracerebral injection confirmed the safety of poly(I:C). In addition, we demonstrated that administration of poly(I:C) with either A/Beijing (H1N1) or A/Yamagata (H1N1) vaccine conferred complete protection against PR8 challenge in this mouse nasal infection model, suggesting that poly(I:C) possessed cross-protection ability against variant viruses. To investigate the mechanism of the protective effect of poly(I:C), mRNA levels of Toll-like receptors and cytokines were examined in the nasal-associated lymphoid tissue after vaccination or virus challenge. Intranasal administration of HA vaccine with poly(I:C) up-regulated expression of Toll-like receptor 3 and alpha/beta interferons as well as Th1- and Th2-related cytokines. We propose that poly(I:C) is a new effective intranasal adjuvant for influenza virus vaccine.
• Establishment of an immunoscreening system using recombinant VP1 protein for the isolation of a monoclonal antibody that blocks JC virus infection

  C Henmi, H Sawa, H Iwata, Y Orba, S Tanaka, K Nagashima

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 327 (1) 242 - 251 0006-291X 2005/02 [Refereed][Not invited]
   

  Polyomavirus JC (JCV) infection causes the fatal human demyelinating disease, progressive multifocal leukoencephalopathy. Although the initial interaction of JCV with host cells occurs through direct binding of the major viral capsid protein (VP1) with cell-surface molecules possessing sialic acid, these molecules have not yet been identified. In order to isolate monoclonal antibodies which inhibit attachment of JCV, we established an immunoscreening system using virus-like particles consisting of the VP1. Using this system, among monoclonal antibodies against the cell membrane fraction from JCV-permissive human neuroblastoma IMR-32 cells, we isolated a monoclonal antibody designated as 24132 that specifically inhibited attachment and infection of JCV to IMR-32 cells. The antibody 24132 recognized a single molecule of around 60 kDa in molecular weight in the IMR-32 membrane fraction. Immunohistochemical staining with 24132 demonstrated immunoreactivity in the cell membrane of JCV-permissive cell lines and glial cells of the human brain. These results suggested that the molecule recognized by 24132 plays a role in JCV infection, and that it might participate as a receptor or a co-receptor in JCV attachment and entry into the cells. (C) 2004 Elsevier Inc. All rights reserved.
• The JC virus-like particle overlay assay.

  Sawa H, Komagome R

  Methods in molecular biology (Clifton, N.J.) 292 175 - 186 1064-3745 2005 [Refereed][Not invited]
  
• Protection against influenza virus infection by intranasal administration of hemagglutinin vaccine with chitin microparticles as an adjuvant

  H Hasegawa, T Ichinohe, P Strong, Watanabe, I, S Ito, S Tamura, H Takahashi, H Sawa, J Chiba, T Kurata, T Sata

  JOURNAL OF MEDICAL VIROLOGY 75 (1) 130 - 136 0146-6615 2005/01 [Refereed][Not invited]
   

  Chitin in the form of microparticles (chitin microparticles, CMP) has been demonstrated to be a potent stimulator of macrophages, promoting T-helper-1 (Th1) activation and cytokine response. In order to examine the mucosal adjuvant effect of CMP co-administered with influenza hemagglutinin (HA) vaccine against influenza infection, CMP were intranasally co-administered with influenza HA vaccine prepared from PR8 (H1N1) virus. Inoculation of the vaccine with CMP induced primary and secondary anti-HA IgA responses in the nasal wash and anti-HA IgG responses in the serum, which were significantly higher than those of nasal vaccination without CMP, and provided a complete protection against a homologous influenza virus challenge in the nasal infection influenza model. In addition, CMP-based immunization using A/Yamagata (H1N1) and A/Guizhou (H3N2) induced PR8 HA-reactive IgA in the nasal washes and specific-IgG in the serum. The immunization with A/Yamagata and CMP resulted in complete protection against a PR8 (H1N1) challenge in A/Yamagata (H1N1)-vaccinated mice, while that with A/Guizhou (H3H2) and CMP exhibited a 100-fold reduction of nasal virus titer, demonstrating the cross-protective effect of CMP and influenza vaccine. It is suggested that CMP provide a safe and effective adjuvant for nasal vaccination with inactivated influenza vaccine. (C) 2005 Wiley-Liss, Inc.
• Sawa H, Nagashima T, Nagashima K, Shinohara T, Chuma T, Mano Y, Tachi N, Hall WW. Clinicopathological and virological analyses of familial HTLV-I associated polyneuropathy. J Neurovirol 11: 199-207, 2005*

  2005 [Not refereed][Not invited]
  
• Hasegawa H, Ichinohe T, Strong P, Watanabe I, Ito S, Tamura S-i, Takahashi H, Sawa H, Chiba J, Kurata T, Sata T: Protection against influenza virus infection by intranasal administration of HA vaccine with chitin microparticles as an adjuvant. J Med V・・・

  2005 [Not refereed][Not invited]
   

  Hasegawa H, Ichinohe T, Strong P, Watanabe I, Ito S, Tamura S-i, Takahashi H, Sawa H, Chiba J, Kurata T, Sata T: Protection against influenza virus infection by intranasal administration of HA vaccine with chitin microparticles as an adjuvant. J Med Virol 75: 130-136, 2005
• Henmi C, Sawa H*, Iwata H, Orba Y, Tanaka S, Nagashima K: Isolation of a monoclonal antibody recognizing a cell-surface molecule as a receptor for JC virus. Biochem Biophys Res Commun 327: 242-251, 2005 (* corresponding author)*

  2005 [Not refereed][Not invited]
  
• Ichinohe T, Watanabe I, Ito S, Moriyama M, Tamura S, Takahashi H, Sawa H, Chiba J, Kurata T, Sata T, Hasegawa H: Synthetic double-stranded RNA [poly (I:C)] combined with mucosal vaccine protects against influenza virus infection. J Virol 79: 2910-2919・・・

  2005 [Not refereed][Not invited]
   

  Ichinohe T, Watanabe I, Ito S, Moriyama M, Tamura S, Takahashi H, Sawa H, Chiba J, Kurata T, Sata T, Hasegawa H: Synthetic double-stranded RNA [poly (I:C)] combined with mucosal vaccine protects against influenza virus infection. J Virol 79: 2910-2919, 2005*
• Khalili K, White MK, Sawa H, Nagashima K, Safak M: The agnoprotein of polyomaviruses: A multifunctional auxiliary protein. J Cell Physiol 204:1-7, 2005*

  2005 [Not refereed][Not invited]
  
• Okada Y, Suzuki T, Sunden Y, Orba Y, Kose S, Imamoto N, Takahashi H, Tanaka S, Hall WW, Nagashima K, Sawa H*: Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral par・・・

  2005 [Not refereed][Not invited]
   

  Okada Y, Suzuki T, Sunden Y, Orba Y, Kose S, Imamoto N, Takahashi H, Tanaka S, Hall WW, Nagashima K, Sawa H*: Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles. EMBO Rep 6: 452-457, 2005 (* corresponding author)*
• Akazawa S, Igarashi M, Sawa H, Tamashiro H: Strategic approach to information security and assurance in health research. Environ Health Prev Med 10: 282-285, 2005

  2005 [Not refereed][Not invited]
  
• Tsuda M, Watanabe T, Seki T, Kimura T, Sawa H, Minami A, Akagi T, Isobe KI, Nagashima K, Tanaka S: Induction of p21(WAF1/CIP1) by human synovial sarcoma-associated chimeric oncoprotein SYT-SSX1. Oncogene. 24: 7984-7990, 2005*

  2005 [Not refereed][Not invited]
  
• Investigation of Simian virus 40 large T antigen in 18 autopsied malignant mesothelioma patients in Japan

  M Jin, H Sawa, T Suzuki, K Shimizu, Y Makino, S Tanaka, T Nojima, Y Fujioka, M Asamoto, N Suko, M Fujita, K Nagashima

  JOURNAL OF MEDICAL VIROLOGY 74 (4) 668 - 676 0146-6615 2004/12 [Refereed][Not invited]
   

  It has been reported that Simian virus 40 (SV40) is linked to human beings by inoculation of contaminated poliovaccines and may have a role in the etiology of malignant mesothelioma. However, there have been no reports describing the relationship between SV40 and malignant mesothelioma in Japan. A study was undertaken to investigate whether SV40 was related to patients of malignant mesothelioma in Japan by the polymerase chain reaction (PCR) assay, DNA sequence analysis, and immunohistochemical methods. Paraffin-embedded samples of the 18 autopsied patients with pleural malignant mesothelioma were collected from five hospitals in Japan. After isolation of DNA from paraffin blocks, PCR analyses followed by sequencing were performed using three different sets of primers for detection of SV40 large T antigen (TAg) gene. All 18 malignant mesothelioma samples were also immunohistochemically evaluated for expression of SV40 TAg protein with two different anti-SV40 TAg antibodies. SV40 TAg genome was detected in eight malignant mesothelioma cases. Only one of three primer pairs successfully amplified SV40 genome in the samples, whereas all pairs yielded a PCR product in the controls, suggesting a low content of virus DNA. No immunopositive staining for SV40 TAg was found in any of the samples. This study shows that SV40 genome was present in a subset of Japanese malignant mesothelioma patients who were unlikely to have received a contaminated polio vaccine based on their age.
• Dysferlinopathy associated with rigid spine syndrome

  T Nagashima, T Chuma, Y Mano, Y Goto, YK Hayashi, N Minami, Nishino, I, Nonaka, I, T Takahashi, H Sawa, M Aoki, K Nagashima

  NEUROPATHOLOGY 24 (4) 341 - 346 0919-6544 2004/12 [Refereed][Not invited]
   

  Dysferlinopathy and rigid spine syndrome occurring in a 50-year-old man is reported. The patient noticed stiffness of knee and ankle joints, which gradually extended to neck, wrist and elbow joints leading to difficulty in anterior flexion. Muscular weakness and wasting of the lower extremities had developed since age 40, accompanied by a limitation of anterior bending of the spine. Elevated serum CK was noticed. Muscle CT revealed atrophy with moderate fatty replacement of muscles in the neck, shoulder and pelvic girdle, and marked replacement in the para-vertebral muscles, posterior compartment of hamstrings and calf muscles. Electromyography showed a typical myogenic pattern, and muscle biopsy disclosed dystrophic changes, compatible with limb-girdle muscular dystrophy 2B. Loss of dysferlin expression was verified by immunohistochemistry, which was confirmed by a mini-multiplex Western blotting system. Gene analyses of the dysferlin gene disclosed compound heterozygotes for frameshift (G3016 + 1A) and a missense mutation (G3370T). This study might propose some clues to resolve the combination of musular dystrophies and rigid spine syndrome.
• Crk associates with ERM proteins and promotes cell motility toward hyaluronic acid

  M Tsuda, Y Makino, T Iwahara, H Nishihara, H Sawa, K Nagashima, H Hanafusa, S Tanaka

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (45) 46843 - 46850 0021-9258 2004/11 [Refereed][Not invited]
   

  Cell migration is a well organized process regulated by the extracellular matrix-mediated cytoskeletal reorganization. The signaling adaptor protein Crk has been shown to regulate cell motility, but its precise role is still under investigation. Herein, we report that Crk associates with ERM family proteins ( including ezrin, radixin, and moesin), activates RhoA, and promotes cell motility toward hyaluronic acid. The binding of Crk with ERMs was demonstrated both by transient and stable protein expression systems in 293T cells and 3Y1 cells, and it was shown that v-Crk translocated the phosphorylated form of ERMs to microvilli in 3Y1 cells by immunofluorescence and immunoelectron microscopy. This v-Crk-dependent formation of microvilli was suppressed by inhibitors of Rho-associated kinase, and the activity of RhoA was elevated by coexpression of c-Crk-II and ERMs in 3Y1 cells. In concert with the activation of RhoA by Crk, Crk was found to associate with Rho-GDI, which has been shown to bind to ERMs. Furthermore, upon hyaluronic acid treatment, coexpression of c-Crk-II and ERMs enhanced cell motility, whereas the sole expression of c-Crk-II or either of the ERMs decreased the motility of 3Y1 cells. These results suggest that Crk may be involved in regulation of cell motility by a hyaluronic acid-dependent mechanism through an association with ERMs.
• Variable patterns of varicella-zoster virus reactivation in Ramsay Hunt syndrome

  H Aizawa, F Ohtani, Y Furuta, H Sawa, S Fukuda

  JOURNAL OF MEDICAL VIROLOGY 74 (2) 355 - 360 0146-6615 2004/10 [Refereed][Not invited]
   

  The mechanism by which reactivation of varicella-zoster virus (VZV) causes facial paralysis in Ramsay Hunt syndrome remains unclear. The relationship between VZV load and the onset of facial paralysis was analyzed in 42 patients with Ramsay Hunt syndrome. The patients were divided into three groups according to the times of appearance of zoster and of facial paralysis; group I (zoster preceding, n = 13), group II (simultaneous, n = 22), group III (paralysis preceding, n = 7). A real-time quantitative PCR assay was used to measure VZV DNA copy number in saliva, and paired sera were assayed for anti-VZV IgG and IgM antibodies. In group I, the VZV DNA-positive rate was low and virus load decreased gradually after the initial hospital visit around the time of onset of paralysis. The level of anti-VZV antibodies had in most cases already increased at that time. In group III, viral load tended to increase after the onset of paralysis and peaked around the time of appearance of zoster. The level of anti-VZV antibodies was low at the onset of paralysis but showed a significant increase when paired sera were tested. In group II, virus load and changes in level of anti-VZV antibodies either resembled group I or group III behavior. These results indicate that facial paralysis in Ramsay Hunt syndrome can occur at various times between the early and the regression phase of VZV reactivation, suggesting that there are variable patterns of development of facial nerve dysfunction caused by VZV reactivation and the progression of neuritis.
• A novel dynamin-associating molecule, formin-binding protein 17, induces tubular membrane invaginations and participates in endocytosis

  Y Kamioka, S Fukuhara, H Sawa, J Nagashima, M Masuda, M Matsuda, N Mochizuki

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (38) 40091 - 40099 0021-9258 2004/09 [Refereed][Not invited]
   

  Dynamin associates with a variety of SH3 domain-containing molecules via a C-terminal proline-rich motif and takes part, with them, in endocytic processes. Here, we have investigated a new dynamin-associating molecule, formin-binding protein 17 (FBP17), involved in deforming the plasma membrane and in endocytosis. FBP17 formed tubular invaginations originating from the plasma membrane. Its N-terminal Fer/CIP4 homology domain, a coiled-coil domain, and a proline-rich motif were required for tubular invagination and self-assembly, by which tubular invagination might be induced. Using anti-FBP17 antibody, we detected positive immunoreactions in the testis that were restricted to the germ cells. We also detected FBP17 in the brain by immunoblotting and in situ hybridization. When COS cells expressing enhanced green fluorescent protein-tagged FBP17 were incubated with fluorescently labeled transferrin, epidermal growth factor, and cholera toxin, these molecules co-localized with FBP17-induced tubular invaginations, suggesting that FBP17 is involved in dynamin-mediated endocytosis in both a clathrin-dependent and -independent manner. These observations therefore indicate that FBP17 interacts with dynamin and regulates endocytosis by forming vesicotubular structures.
• Varicella-zoster virus DNA level and facial paralysis in Ramsay Hunt syndrome

  Y Furuta, H Aizawa, F Ohtani, H Sawa, S Fukuda

  ANNALS OF OTOLOGY RHINOLOGY AND LARYNGOLOGY 113 (9) 700 - 705 0003-4894 2004/09 [Refereed][Not invited]
   

  We have investigated whether the copy number of varicella-zoster virus (VZV) in saliva correlates with the clinical symptoms in patients with Ramsay Hunt syndrome. A real-time quantitative polymerase chain reaction assay was used to examine the VZV DNA copy number in saliva samples from 37 patients. We detected VZV DNA in 6 of the 7 patients with oropharyngeal zoster lesions (86%) and in 17 of the 30 patients who had zoster lesions only on the skin (57%). Patients with oropharyngeal zoster lesions had a high VZV load in their saliva, and the difference between the copy number in patients with oropharyngeal zoster lesions and those without was around 10,000 copies per 50 muL. In addition, patients with oropharyngeal zoster lesions showed worse recovery of facial function than those without. It seems that the VZV DNA level in saliva reflects the kinetics of viral reactivation in the facial nerve, as well as in the oropharyngeal epithelium, in patients with Ramsay Hunt syndrome.
• Clinico-pathological features of pilomyxoid astrocytoma of the optic pathway

  K Chikai, A Ohnishi, T Kato, J Ikeda, Y Sawamura, Y Iwasaki, T Itoh, H Sawa, K Nagashima

  ACTA NEUROPATHOLOGICA 108 (2) 109 - 114 0001-6322 2004/08 [Refereed][Not invited]
   

  Five cases of pilomyxoid astrocytoma (PmA) characterized by a monophasic pattern with a myxoid background were selected for a clinicopathological study from 23 cases previously diagnosed as pilocytic astrocytoma (PA). All PmA patients were either infants or young children (mean age 2.1 years), and all tumors were located in the optic chiasm/hypothalamus region. All cases received chemotherapy, which reduced tumor size, and the location of the tumor became confined to the optic chiasm. In two cases, tumor recurrence occurred 3 and 7 years after chemotherapy. Histology of the recurrent tumors showed the biphasic pattern of classical PA. Hence, we conclude that PmA might be an infantile form of PA and speculate that a subset of PmA in the optic pathway/hypothalamus originates from the optic chiasm, possibly derived from radial glia existing in the embryonic optic chiasm.
• Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen

  T Minamitani, T Ikuta, Y Saito, G Takebe, M Sato, H Sawa, T Nishimura, F Nakamura, K Takahashi, H Ariga, K Matsumoto

  EXPERIMENTAL CELL RESEARCH 298 (1) 305 - 315 0014-4827 2004/08 [Refereed][Not invited]
   

  Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX-/-) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro. (C) 2004 Elsevier Inc. All rights reserved.
• Triglyceride accumulation and altered composition of triglyceride-associated fatty acids in the skin of tenascin-X-deficient mice

  K Matsumoto, T Sato, S Oka, Y Orba, H Sawa, K Kabayama, J Inokuchi, H Ariga

  GENES TO CELLS 9 (8) 737 - 748 1356-9597 2004/08 [Refereed][Not invited]
   

  Tenascin-X (TNX) is a member of the tenascin family of glycoproteins of the extracellular matrix. Here, we observed abnormalities in the skin of TNX-deficient mice in comparison with that of wild-type mice. Histological analysis with Oil Red O staining demonstrated that there was considerable accumulation of lipid in the skin of TNX-deficient (TNX-/-) mice. By thin-layer chromatography of total lipids, it was found that the level of triglyceride was significantly increased in TNX-/- mice. The mRNA levels of most of the lipogenic enzyme genes examined were remarkably increased in TNX-/- mice. By gas chromatography-mass spectrometry analysis of triglyceride-associated fatty acids in the skin, saturated fatty acid palmitoic acid was decreased, whereas unsaturated fatty acids palmitoleic acid and oleic acid were increased in TNX-/- mice compared with those in wild-type mice. Conversely, fibroblast cell lines transfected with TNX showed a significant decrease in the amount of triglyceride. An increase in the saturated fatty acid stearic acid and decreases in the unsaturated fatty acids palmitoleic acid, oleic acid and linoleic acid, compared to those in mock-transfected cells were also caused by over-expression of TNX. These results indicate that TNX is involved in the regulation of triglyceride synthesis and the regulation of composition of triglyceride-associated fatty acids.
• Triglyceride accumulation and altered composition of triglyceride-associated fatty acids in the skin of tenascin-X-deficient mice

  Ken-Ichi Matsumoto, Takashige Sato, Seiko Oka, Yasuko Orba, Hirofumi Sawa, Kazuya Kabayama, Jin-Ichi Inokuchi, Hiroyoshi Ariga

  Genes to Cells 9 (8) 737 - 748 1356-9597 2004/08 [Not refereed][Not invited]
   

  Tenascin-X (TNX) is a member of the tenascin family of glycoproteins of the extracellular matrix. Here, we observed abnormalities in the skin of TNX-deficient mice in comparison with that of wild-type mice. Histological analysis with Oil Red O staining demonstrated that there was considerable accumulation of lipid in the skin of TNX-deficient (TNX-/-) mice. By thin-layer chromatography of total lipids, it was found that the level of triglyceride was significantly increased in TNX-/- mice. The mRNA levels of most of the lipogenic enzyme genes examined were remarkably increased in TNX-/- mice. By gas chromatography-mass spectrometry analysis of triglyceride-associated fatty acids in the skin, saturated fatty acid palmitoic acid was decreased, whereas unsaturated fatty acids palmitoleic acid and oleic acid were increased in TNX-/- mice compared with those in wild-type mice. Conversely, fibroblast cell lines transfected with TNX showed a significant decrease in the amount of triglyceride. An increase in the saturated fatty acid stearic acid and decreases in the unsaturated fatty acids palmitoleic acid, oleic acid and linoleic acid, compared to those in mock-transfected cells were also caused by over-expression of TNX. These results indicate that TNX is involved in the regulation of triglyceride synthesis and the regulation of composition of triglyceride-associated fatty acids. © Blackwell Publishing Limited.
• Inhibition of virus production in JC virus-infected cells by postinfection RNA interference

  Y Orba, H Sawa, H Iwata, S Tanaka, K Nagashima

  JOURNAL OF VIROLOGY 78 (13) 7270 - 7273 0022-538X 2004/07 [Refereed][Not invited]
   

  RNA interference has been applied for the prevention of virus infections in mammalian cells but has not succeeded in eliminating infections from already infected cells. We now show that the transfection of JC virus-infected SVG-A human glial cells with small interfering RNAs that target late viral proteins, including agnoprotein and VP1, results in a marked inhibition both of viral protein expression and of virus production. RNA interference directed against JC virus genes may thus provide a basis for the development of new strategies to control infections with this polyomavirus.
• Induction of matrix metalloproteinase-2 by tenascin-X deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathway

  K Matsumoto, T Minamitani, Y Orba, M Sato, H Sawa, H Ariga

  EXPERIMENTAL CELL RESEARCH 297 (2) 404 - 414 0014-4827 2004/07 [Refereed][Not invited]
   

  The results of our previous study showed that tumor invasion and metastasis are promoted in extracellular matrix (ECM) tenascin-X-deficient (TNX-/-) mice via increased expression of matrix metalloproteinases (MMPs). However, little is known about the relationship between TNX deficiency and activation of MMP genes. In this study, we investigated the molecular mechanism by which TNX deficiency activates the MMP-2 gene. We examined the intracellular signaling pathways that regulate gene expression of the proteinase in isolated fibroblasts. Results of gelatin zymography showed that MMP-2 was induced to a greater extent in TNX-/- fibroblasts embedded in type I collagen than in wild-type fibroblasts. RT-PCR analysis revealed that the increased level of MMP-2 expression was caused at the transcription level. Conversely, stable overexpression of TNX in a fibroblast cell line reduced MMP-2 expression and suppressed MMP-2 promoter activity. In addition, treatment of TNX-/- fibroblasts with SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and genistein, a tyrosine kinase inhibitor, suppressed the increased level of proMMP-2 and increased MMP-2 promoter activity in TNX-/- fibroblasts. Furthermore, increased activation of JNK and tyrosine phosphorylation of certain proteins were observed in TNX-/- fibroblasts. These findings suggest that induction of MMP-2 by TNX deficiency is mediated, at least in part, through the JNK and protein tyrosine kinase phosphorylation pathway. (C) 2004 Elsevier Inc. All rights reserved.
• Nuclear entry mechanism of the human polyomavirus JC virus-like particle - Role of importins and the nuclear pore complex

  QM Qu, H Sawa, T Suzuki, S Semba, C Henmi, Y Okada, M Tsuda, S Tanaka, WJ Atwood, K Nagashima

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (26) 27735 - 27742 0021-9258 2004/06 [Refereed][Not invited]
   

  JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. Although transport of virions to the nucleus is an important step in JCV infection, the mechanism of this process has remained unclear. The outer shell of the JCV virion comprises the major capsid protein VP1, which possesses a putative nuclear localization signal (NLS), and virus-like particles (VLPs) consisting of recombinant VP1 exhibit a virion-like structure and physiological functions ( cellular attachment and intracytoplasmic trafficking) similar to those of JCV virions. We have now investigated the mechanism of nuclear transport of JCV with the use of VLPs. Wild-type VLPs (wtVLPs) entered the nucleus of most HeLa or SVG cells. The virion structure of VLPs was preserved during transport to the nucleus as revealed by confocal microscopy of cells inoculated with fluorescein isothiocyanate-labeled wtVLPs containing packaged Cy3. The nuclear transport of wtVLPs in digitonin-permeabilized cells was dependent on the addition of importins alpha and beta and was prevented by wheat germ agglutinin or by antibodies to the nuclear pore complex. The nuclear entry of VLPs composed of VP1 with a mutated NLS was greatly inhibited, compared with that of wtVLPs, in both intact and permeabilized cells. Unlike wtVLPs, the mutant VLPs did not bind to importins alpha or beta. Limited proteolysis analysis revealed that the NLS of VP1 was exposed on the surface of wtVLPs. These results suggest that JCV VLPs bind to cellular importins via the NLS of VP1 and are transported into the nucleus through the nuclear pore complex.
• Topoisomerase I dissociates human immunodeficiency virus type 1 reverse transcriptase from genomic RNAs

  H Takahashi, H Sawa, H Hasegawa, K Nagashima, T Sata, T Kurata

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 313 (4) 1073 - 1078 0006-291X 2004/01 [Refereed][Not invited]
   

  Both HIV-1 reverse transcriptase (RT) and topoisomerase I bind to structural RNAs and they cooperate to synthesize cDNA during the replication of HIV-1. In this study, we find that human topoisomerase I exclusively dissociated HIV-1 reverse transcriptase, which strongly binds to structural RNAs. Meanwhile, topoisomerase I did not dissociate either HIV-1 nucleocapsid proteins or murine leukemia virus RT which was bound to RNA. We propose that human topoisomerase I may regulate the binding of RT to RNAs and play a pivotal role in HIV-1 replication. (C) 2003 Elsevier Inc. All rights reserved.
• Nucleolin and the packaging signal, psi, promote the budding of human immunodeficiency virus type-1 (HIV-1)

  T Ueno, K Tokunaga, H Sawa, M Maeda, J Chiba, A Kojima, H Hasegawa, Y Shoya, T Sata, T Kurata, H Takahashi

  MICROBIOLOGY AND IMMUNOLOGY 48 (2) 111 - 118 0385-5600 2004 [Refereed][Not invited]
   

  Gag proteins of human immunodeficiency virus type 1 (HIV-1) play a pivotal role in the budding of the virion, in which the zinc finger motifs of the gag proteins recognize the packaging signal of genomic RNA. Nucleolin, an RNA-binding protein, is identified as a cellular protein that binds to murine leukemia virus (MuLV) gag proteins and regulates the viral budding, suggesting that HIV-1 gag proteins, the packaging signal, psi and nucleolin affect the budding of HIV-1. Here we report that nucleolin enhances the release of HIV-1 virions which contain psi. Furthermore, nucleolin and gag proteins form a complex incorporated into virions, and nucleolin promotes the infectivity of HIV-1. Our results suggest that an empty particle which contains neither nucleolin nor the genomic RNA is eliminated during the budding process, and this mechanism is beneficial for escape from the host immune response against HIV-1.
• Qu Q, Sawa H*, Suzuki T, Semba S, Henmi C, Okada Y, Tsuda M, Tanaka S, Nagashima K: Nuclear entry mechanism of the human polyomavirus JC virus like particle: role of importins and the nuclear pore complex. J Biol Chem 279: 27735-27742, 2004 (* corresp・・・

  2004 [Not refereed][Not invited]
   

  Qu Q, Sawa H*, Suzuki T, Semba S, Henmi C, Okada Y, Tsuda M, Tanaka S, Nagashima K: Nuclear entry mechanism of the human polyomavirus JC virus like particle: role of importins and the nuclear pore complex. J Biol Chem 279: 27735-27742, 2004 (* corresponding author)*
• Minamitani T, Ikuta T, Saito Y, Takebe G, Sato M, Sawa H, Nishimura T, Nakamura F, Takahashi K, Ariga H, Matsumoto K: Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen. Exp Cell Res 298: 305-15, 2004*

  2004 [Not refereed][Not invited]
  
• Chikai K, Ohnishi A, kato T, Ikeda J, Sawamura Y, Iwasaki Y, Itoh T, Sawa H, Nagashima K: Clinico-pathological features of piloymyxoid astrocytoma of the optic pathway. Acta Neuropathol 108: 109-114, 2004*

  2004 [Not refereed][Not invited]
  
• Kamioka Y, Fukuhara S, Sawa H, Nagashima K, Masuda M, Matsuda M, Mochizuki N: A novel dynamin-associating molecule, formin-binding Protein 17, induces tubular membrane invaginations and participates in endocytosis. J Biol Chem 279: 40091-40099, 2004*

  2004 [Not refereed][Not invited]
  
• Matsumoto K, Minamitani T, Orba Y, Sato M, Sawa H, Ariga H: Induction of matrix metalloproteinase-2 by tenascin-X deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathway. Exp Cell Res 297: 404-414・・・

  2004 [Not refereed][Not invited]
   

  Matsumoto K, Minamitani T, Orba Y, Sato M, Sawa H, Ariga H: Induction of matrix metalloproteinase-2 by tenascin-X deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathway. Exp Cell Res 297: 404-414, 2004*
• Tsuda M, Makino Y, Iwahara T, Nishihara H, Sawa H, Nagashima K, Hanafusa H, Tanaka S: Crk associates with ERM proteins and promotes cell motility toward hyaluronic acid. J Biol Chem 279: 46843-46850, 2004*

  2004 [Not refereed][Not invited]
  
• Orba Y, Sawa H*, Iwata H, Tanaka S, Nagashima K: Inhibition of virus production in JC virus-infected cells by postinfection RNA interference. J Virol 78: 7270-7273, 2004 (* corresponding author)*

  2004 [Not refereed][Not invited]
  
• Furuta Y, Aizawa H, Ohtani F, Sawa H, Fukuda S: Varicella-zoster virus DNA level and facial paralysis in Ramsay Hunt syndrome. Ann Otol Rhinol Laryngol 113: 700-705, 2004.*

  2004 [Not refereed][Not invited]
  
• Jin M, Sawa H*, Suzuki T, Shimizu K, Makino Y, Tanaka S, Nojima T, Fujioka Y, Asamoto M, Suko N, Nagashima K: Investigation of simian virus 40 large T antigen in 18 autopsied malignant mesothelioma patients in Japan. J Med Virol 74: 668-676, 2004 (* ・・・

  2004 [Not refereed][Not invited]
   

  Jin M, Sawa H*, Suzuki T, Shimizu K, Makino Y, Tanaka S, Nojima T, Fujioka Y, Asamoto M, Suko N, Nagashima K: Investigation of simian virus 40 large T antigen in 18 autopsied malignant mesothelioma patients in Japan. J Med Virol 74: 668-676, 2004
  (* corresponding author)*
• Aizawa H, Ohtani F, Furuta Y, Sawa H, Fukuda S: Variable patterns of varicella-zoster virus reactivation in Ramsay Hunt syndrome. J Med Virol 74: 355-360, 2004.*

  2004 [Not refereed][Not invited]
  
• Ueno T, Tokunaga K, Sawa H, Maeda M, Chiba J, Kojima A, Hasegawa H, Shoya Y, Sata T, Kutrata T, Takahashi H: Nucleolin and the packaging signal,  promote the budding of human immunodeficiency virus type-1 (HIV-1). Microbiol Immunol 48: 111-118, 2004*

  2004 [Not refereed][Not invited]
  
• Takahashi H, Sawa H, Hasegawa H, Nagashima K, Sata T, Kurata T: Topoisomerase I dissociates human immunodeficiency virus type 1 reverse transcriptase from genomic RNAs. Biochem Biophys Res Commun. 313: 1073-1078, 2004.*

  2004 [Not refereed][Not invited]
  
• Nagashima T, Chuma T, Mano Y, Goto Y-i, Hayashi KY, Minami N, Nishino I, Nonaka I, Takahashi T, Sawa H, Aoki M, Nagashima K: Dysferlinopathy associated with rigid spine syndrome. Neuropathology 24: 341-346, 2004.*

  2004 [Not refereed][Not invited]
  
• Chikai K, Ohnishi A, kato T, Ikeda J, Sawamura Y, Iwasaki Y, Itoh T, Sawa H, Nagashima K: Clinico-pathological features of piloymyxoid astrocytoma of the optic pathway. Acta Neuropathol 108: 109-114, 2004*

  2004 [Not refereed][Not invited]
  
• Progressive multifocal leukoencephalopathy in a patient with X-linked agammaglobulinemia

  T Teramoto, H Kaneko, M Funato, H Sawa, K Nagashima, Y Hirose, N Kondo

  SCANDINAVIAN JOURNAL OF INFECTIOUS DISEASES 35 (11-12) 909 - 910 0036-5548 2003/12 [Refereed][Not invited]
   

  To our knowledge, this is the first case report describing progressive multifocal leukoencephalopathy (PML) associated with X-linked agammaglobulinemia. The JC virus was confirmed at autopsy and PML was diagnosed. Humoral immunodeficiency with normal cellular immunity could be infected with JCV.
• Protection against influenza virus infection by intranasal administration of C3d-fused hemagglutinin

  Watanabe, I, TM Ross, S Tamura, T Ichinohe, S Ito, H Takahashi, H Sawa, J Chiba, T Kurata, T Sata, H Hasegawa

  VACCINE 21 (31) 4532 - 4538 0264-410X 2003/11 [Refereed][Not invited]
   

  For the induction of mucosal immune responses by intranasal vaccination, cholera toxin B subunits (CTB) and Escherichia coli heat-labile toxin (I-T) are often administered as mucosal adjuvants in order to enhance immune responses to mucosally co-administered bystander antigens. However, these toxin also are the causative agents of diarrhea. There is a demand for the establishment of an effective and safer adjuvant or vaccine that elicits mucosal immunity, but does not require the use of CTB or LT adjuvants. In order to induce protective mucosal immune responses in the nasal area against influenza virus infection, we have examined the recombinant protein composed of the complement component, CM, which is fused to the secreted form of hemagglutinin (sHA-mC3d(3)) in the influenza-BALB/c mouse model. The fusion protein sHA-mC3d(3), the secretory form of hemagglutinin, and the transmembrane form of HA (tmHA) from the influenza virus were intranasally administered to the mice with or without CTB containing a trace amount of holotoxin (CTB*) as an adjuvant. After intranasal administration of these proteins with CTB*, all mice produced nasal IgA and serum IgG antibodies (Abs) against the viral HA. In addition, viral infection was completely inhibited in these mice. In contrast, in the absence of the adjuvant, only sHA-mC3d(3)-induced locally secreted IgA and serum IgG Abs and provided complete protection against the influenza virus challenge. Thus, C3d fused to the influenza HA antigen is an effective and safe tool for mucosal vaccination. (C) 2003 Elsevier Ltd. All rights reserved.
• Induction of chromosomal instability in colonic cells by the human polyomavirus JC virus

  L Ricciardiello, M Baglioni, C Giovannini, M Pariali, G Cenacchi, A Ripalti, MP Landini, H Sawa, K Nagashima, RJ Frisque, A Goel, CR Boland, M Tognon, E Roda, F Bazzoli

  CANCER RESEARCH 63 (21) 7256 - 7262 0008-5472 2003/11 [Refereed][Not invited]
   

  Most colorectal cancers display chromosomal instability, which is characterized by gross chromosomal rearrangements, loss of heterozygosity and aneuploidy. We have previously demonstrated a link between JC virus strains Mad-1 and Delta98 and colorectal cancer. Others have also associated the virus to the induction of colon cancer and aneuploid brain tumors by producing a highly tumorigenic protein named T antigen (TAg), which binds to beta-catenin and inactivates key proteins such as p53. The aim is to demonstrate that JC virus is capable of inducing chromosomal instability in colonic cells. We used the human colon cancer cell line RKO as a model. The cell line has wild-type p53, wild-type beta-catenin and APC and is diploid. Neuroblastoma JCI cells, which are infected with the virus, VA13 fibroblasts, which are transformed by the SV40 TAg, were used as positive controls. HCT116, which has mutated P-catenin, and SW480, which is a model of CIN, were also used as controls. The genomes of the Mad-1 and Delta98 strains were transfected into cells. As negative controls we used pUC or no plasmids. Cells were collected at 0, 7, 14, and 21 days after transfection. PCR was used for the detection of TAg and the regulatory region DNA sequences at different time frames and Southern blot of whole genomic extracts for viral DNA integration into the host genome. Immuntifluorescence and Western blot were performed for TAg, viral capsid proteins, and nuclear beta-catenin expressions, whereas coimmunoprecipitation was used to detect protein interactions. Karyotype analysis and electron microscopy were performed to seek chromosomal instability and cell abnormalities, respectively. Retention of viral sequences was observed for Mad-1- and Delta98-transfected RKO cells at all time frames with PCR only, whereas Southern blot analysis showed nonintegrated sequences at T7 alone. TAg and capsid protein expressions, as well as increased p53 and nuclear beta-catenin, were observed between TO and T7 for Mad-l and Delta98 alone. Also, interaction between TAg and both p53 and beta-catenin was also observed between TO and T7. Chromosomal instability, characterized by chromosomal breakage, dicentric chromosomes, and increasing ploidy, was observed at all time frames for Mad-l and Delta98, as well as cell abnormalities. In conclusion, we demonstrate that JC virus Mad-1 and Delta98 are able to induce chromosomal instability in colonic cells with a hit and run mechanism that involves an early interaction with beta-catenin and p53.
• Protection against influenza virus infection by intranasal administration of C3d-fused hemagglutinin

  Watanabe, I, TM Ross, S Tamura, T Ichinohe, S Ito, H Takahashi, H Sawa, J Chiba, T Kurata, T Sata, H Hasegawa

  VACCINE 21 (31) 4532 - 4538 0264-410X 2003/11 [Not refereed][Not invited]
   

  For the induction of mucosal immune responses by intranasal vaccination, cholera toxin B subunits (CTB) and Escherichia coli heat-labile toxin (I-T) are often administered as mucosal adjuvants in order to enhance immune responses to mucosally co-administered bystander antigens. However, these toxin also are the causative agents of diarrhea. There is a demand for the establishment of an effective and safer adjuvant or vaccine that elicits mucosal immunity, but does not require the use of CTB or LT adjuvants. In order to induce protective mucosal immune responses in the nasal area against influenza virus infection, we have examined the recombinant protein composed of the complement component, CM, which is fused to the secreted form of hemagglutinin (sHA-mC3d(3)) in the influenza-BALB/c mouse model. The fusion protein sHA-mC3d(3), the secretory form of hemagglutinin, and the transmembrane form of HA (tmHA) from the influenza virus were intranasally administered to the mice with or without CTB containing a trace amount of holotoxin (CTB*) as an adjuvant. After intranasal administration of these proteins with CTB*, all mice produced nasal IgA and serum IgG antibodies (Abs) against the viral HA. In addition, viral infection was completely inhibited in these mice. In contrast, in the absence of the adjuvant, only sHA-mC3d(3)-induced locally secreted IgA and serum IgG Abs and provided complete protection against the influenza virus challenge. Thus, C3d fused to the influenza HA antigen is an effective and safe tool for mucosal vaccination. (C) 2003 Elsevier Ltd. All rights reserved.
• Expression of the oligodendroglial lineage-associated markers Olig1 and Olig2 in different types of human gliomas

  A Ohnishi, H Sawa, M Tsuda, Y Sawamura, T Itoh, Y Iwasaki, K Nagashima

  JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY 62 (10) 1052 - 1059 0022-3069 2003/10 [Refereed][Not invited]
   

  Because a specific group of oligodendrogliomas is susceptible to adjuvant therapy, it is important to elucidate the biological characteristics of these tumors. In situ hybridization analyses have revealed that Olig genes are expressed in oligodendroglial lineage cells and are highly expressed in oligodendrogliomas. To clarify whether OLIG is a tumor-specific marker for oligodendrogliomas, we have investigated the expression of Olig transcripts by semiquantitative RT-PCR assay and OLIG2 protein with a new antibody in a variety of glial tumors. The semiquantitative RT-PCR revealed that high levels of expression of Olig1 and Olig2 mRNAs were present in anaplastic oligodendrogliomas and anaplastic astrocytomas, while expression of these mRNAs in grade IV glioblastomas was lower than in grade II and grade III gliomas (p < 0.01). Immunohistochemical analyses demonstrated that the mean immunopositive proportion of OLIG2 was 82% in anaplastic oligodendrogliomas but only 34% in anaplastic astrocytomas. Therefore, although OLIG2 expression was detected in a range of gliomas not specific for oligodendrogliomas, the expression level in anaplastic oligodendrogliomas was more uniform and intense than that in other glial tumors. In conclusion, combining Olig mRNA expression and immunohistochemistry of OLIG2 enables oligodendrogliomas to be distinguished from glioblastomas and other astrocytic glial tumors.
• Anti-Hu paraneoplastic syndrome presenting with brainstem-cerebellar symptoms and Lambert-Eaton myasthenic syndrome

  T Nagashima, Y Mizutani, H Kawahara, S Maguchi, Y Terayama, T Shinohara, Y Orba, T Chuma, Y Mano, T Itoh, H Sawa, K Sakai, M Motomura, K Nagashima

  NEUROPATHOLOGY 23 (3) 230 - 238 0919-6544 2003/09 [Refereed][Not invited]
   

  Paraneoplastic syndrome (PNS) with two distinct neurological features was reported in a 50-year-old man who presented initially with vertigo, ataxia, dysarthria, tremor, confusion, urinary retention and hypotension. Pulmonary X-ray findings, class IIIb sputum cytology, and positive anti-Hu antibody established the diagnosis of PNS associated with small-cell lung cancer (SCLC). Two cycles of combined chemotherapy resulted in shrinkage of the lung tumor together with complete recovery of neurological symptoms and disappearance of anti-Hu antibody. Relapse of SCLC 4 months later with re-appearance of anti-Hu antibody required additional chemotherapy and irradiation. Eight months later, when multiple liver metastasis of SCLC was noticed, muscular weakness with positive waxing phenomenon compatible with Lambert-Eaton myasthenic syndrome (LEMS) developed. Postmortem examinations revealed residual SCLC in the primary lung, and massive liver metastasis with generalized lymph node involvement, but no tumors in the CNS. In the cerebellum, there was a slight loss of Purkinje cells with torpedo formation but without apparent lymphocytic infiltration. The present PNS was unique in that the relapse of SCLC was accompanied by the appearance of anti-Hu antibody, and that initial signs of brainstem-cerebellar symptoms, encephalopathy and autonomic failure were replaced by LEMS coinciding with the tumor recurrence.
• Application of laser capture microdissection to cytologic specimens for the detection of immunoglobulin heavy chain gene rearrangement in patients with malignant lymphoma

  Y Orba, S Tanaka, H Nishihara, N Kawamura, T Itoh, M Shimizu, H Sawa, K Nagashima

  CANCER CYTOPATHOLOGY 99 (4) 198 - 204 0008-543X 2003/08 [Refereed][Not invited]
   

  BACKGROUND. The demonstration of the monoclonality of immunoglobulin heavy chain (IgH) gene rearrangement is an indispensable method for the diagnosis of B-cell lymphoma as well as histocytochemical analysis. For the detection of IgH gene rearrangement, the extraction of DNA from a homogenous cell population is necessary. Recently, the laser capture microdissection (LCM) technique was shown to isolate specific cells from histopathologic specimens for molecular analysis. However, to the authors' knowledge the applicability of LCM to cytologic specimens has not yet been well established. METHODS. Using LCM, a homogenous population of B-cell lymphoma cells as both histologic sections and cytologic specimens was captured, and genomic DNA was extracted from the captured cells. IgH gene rearrangement was analyzed by the polymerase chain reaction (PCR)-based single-strand conformational polymorphism (SSCP) method. RESULTS. Genomic DNAs were extracted successfully from ethanol-fixed cytologic specimens, but cells were not captured from air-dried specimens. Using PCR-SSCP analysis, the monoclonality of the IgH gene rearrangement was detected in five cases of tissue sections among nine analyzed cases of malignant lymphoma diagnosed immunohistochemically. However, analysis of the cytologic specimens with LCM demonstrated the monoclonality of the IgH gene rearrangement in seven cases of lymphoma. CONCLUSIONS. The results of the current study suggest that the novel application of LCM to cytologic specimens occasionally exhibits high sensitivity for the detection of IgH gene rearrangement monoclonality compared with the use of histologic sections.
• EphA4-mediated rho activation via Vsm-RhoGEF expressed specifically in vascular smooth muscle cells

  H Ogita, S Kunimoto, Y Kamioka, H Sawa, M Masuda, N Mochizuki

  CIRCULATION RESEARCH 93 (1) 23 - 31 0009-7330 2003/07 [Refereed][Not invited]
   

  Rho-kinase, an effector of Rho GTPase, increases the contractility of vascular smooth muscle by phosphorylating myosin light chain ( MLC) and by inactivating MLC phosphatase. A wide variety of extracellular stimuli activate RhoA via G protein-coupled receptors. In the present study, we demonstrate a novel cell-cell interaction - mediated Rho activation signaling pathway in vascular smooth muscle cells (VSMCs). Among many receptor tyrosine kinases, the Eph family receptors are unique in that they require cell-cell interaction to engage their ligands, ephrin. We found that a novel VSMC-specific guanine nucleotide exchange factor (GEF) for Rho ( Vsm-RhoGEF/KIAA0915) was expressed specifically in VSMCs of several organs including the heart, aorta, liver, kidney, and spleen, as examined by the immunohistochemical analysis using a specific antibody against Vsm-RhoGEF. Based on the association of Vsm-RhoGEF with EphA4 in quiescent cells, we tested whether EphA4 and Vsm-RhoGEF were expressed in the same tissue and further studied the molecular mechanism of Vsm-RhoGEF regulation by EphA4. Immunohistochemical analysis showed that EphA4 and Vsm-RhoGEF expression overlapped in VSMCs. Additionally, tyrosine phosphorylation of Vsm-RhoGEF induced by EphA4 upon ephrin-A1 stimulation enhanced the Vsm-RhoGEF activity for RhoA. The requirement of Vsm-RhoGEF for ephrin-A1 - induced assembly of actin stress fibers in VSMCs was shown by the overexpression of a dominant-negative form of VSM-RhoGEF and by the depletion of Vsm-RhoGEF using RNA interference. These results suggested that ephrin-A1 - triggered EphA4-Vsm-RhoGEF-RhoA pathway is involved in the cell-cell interaction - mediated RhoA activation that regulates vascular smooth muscle contractility.
• Human topoisomerase I promotes HIV-1 proviral DNA synthesis: Implications for the species specificity and cellular tropism of HIV-1 infection

  Y Shoya, K Tokunaga, H Sawa, M Maeda, T Ueno, T Yoshikawa, H Hasegawa, T Sata, T Kurata, WW Hall, BR Cullen, H Takahashi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 (14) 8442 - 8447 0027-8424 2003/07 [Refereed][Not invited]
   

  Although HIV type 1 (HIV-1) cannot efficiently replicate in simian cells, the mechanism(s) involved in the restriction of virus tropism remain unclear. To investigate this, we have focused on the identification of human cellular factors that can influence the infectivity of HIV-1 derived from African green monkey producer cells. Whereas the infectivity of HIV-1 derived from such cells was only 10-15% of that of human cell-derived virus, expression of human topoisomerase I in the African green monkey cells resulted in a 5-fold increase of the infectivity of progeny HIV-1 virions. Replacement of glutamate-236 and asparagine-237 of human topoisomerase I with the corresponding residues (aspartate and serine, respectively) of the African green monkey enzyme abolished this enhancement of HIV-1 infectivity. This positive effect of human topoisomerase I expression in the African green monkey producer cells seemed to result from the promotion of HIV-1 cDNA synthesis. Thus, human topoisomerase I plays an important role in HIV-1 replication and infectivity, and differences in the species specificity of HIV-1 infection can at least in part be attributed to differences in topoisomerase I activities.
• EphA4-mediated rho activation via Vsm-RhoGEF expressed specifically in vascular smooth muscle cells

  H Ogita, S Kunimoto, Y Kamioka, H Sawa, M Masuda, N Mochizuki

  CIRCULATION RESEARCH 93 (1) 23 - 31 0009-7330 2003/07 [Not refereed][Not invited]
   

  Rho-kinase, an effector of Rho GTPase, increases the contractility of vascular smooth muscle by phosphorylating myosin light chain ( MLC) and by inactivating MLC phosphatase. A wide variety of extracellular stimuli activate RhoA via G protein-coupled receptors. In the present study, we demonstrate a novel cell-cell interaction - mediated Rho activation signaling pathway in vascular smooth muscle cells (VSMCs). Among many receptor tyrosine kinases, the Eph family receptors are unique in that they require cell-cell interaction to engage their ligands, ephrin. We found that a novel VSMC-specific guanine nucleotide exchange factor (GEF) for Rho ( Vsm-RhoGEF/KIAA0915) was expressed specifically in VSMCs of several organs including the heart, aorta, liver, kidney, and spleen, as examined by the immunohistochemical analysis using a specific antibody against Vsm-RhoGEF. Based on the association of Vsm-RhoGEF with EphA4 in quiescent cells, we tested whether EphA4 and Vsm-RhoGEF were expressed in the same tissue and further studied the molecular mechanism of Vsm-RhoGEF regulation by EphA4. Immunohistochemical analysis showed that EphA4 and Vsm-RhoGEF expression overlapped in VSMCs. Additionally, tyrosine phosphorylation of Vsm-RhoGEF induced by EphA4 upon ephrin-A1 stimulation enhanced the Vsm-RhoGEF activity for RhoA. The requirement of Vsm-RhoGEF for ephrin-A1 - induced assembly of actin stress fibers in VSMCs was shown by the overexpression of a dominant-negative form of VSM-RhoGEF and by the depletion of Vsm-RhoGEF using RNA interference. These results suggested that ephrin-A1 - triggered EphA4-Vsm-RhoGEF-RhoA pathway is involved in the cell-cell interaction - mediated RhoA activation that regulates vascular smooth muscle contractility.
• Human topoisomerase I promotes HIV-1 proviral DNA synthesis: Implications for the species specificity and cellular tropism of HIV-1 infection

  Y Shoya, K Tokunaga, H Sawa, M Maeda, T Ueno, T Yoshikawa, H Hasegawa, T Sata, T Kurata, WW Hall, BR Cullen, H Takahashi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 (14) 8442 - 8447 0027-8424 2003/07 [Not refereed][Not invited]
   

  Although HIV type 1 (HIV-1) cannot efficiently replicate in simian cells, the mechanism(s) involved in the restriction of virus tropism remain unclear. To investigate this, we have focused on the identification of human cellular factors that can influence the infectivity of HIV-1 derived from African green monkey producer cells. Whereas the infectivity of HIV-1 derived from such cells was only 10-15% of that of human cell-derived virus, expression of human topoisomerase I in the African green monkey cells resulted in a 5-fold increase of the infectivity of progeny HIV-1 virions. Replacement of glutamate-236 and asparagine-237 of human topoisomerase I with the corresponding residues (aspartate and serine, respectively) of the African green monkey enzyme abolished this enhancement of HIV-1 infectivity. This positive effect of human topoisomerase I expression in the African green monkey producer cells seemed to result from the promotion of HIV-1 cDNA synthesis. Thus, human topoisomerase I plays an important role in HIV-1 replication and infectivity, and differences in the species specificity of HIV-1 infection can at least in part be attributed to differences in topoisomerase I activities.
• Desmoplastic malignant mesothelioma of the pleura: Autopsy reveals asbestos exposure

  R Ishikawa, H Kikuchi, ML Jin, M Fujita, T Itoh, H Sawa, K Nagashima

  PATHOLOGY INTERNATIONAL 53 (6) 401 - 406 1320-5463 2003/06 [Refereed][Not invited]
   

  Desmoplastic mesothelioma is a rare subtype of diffuse malignant mesothelioma, and is often difficult to distinguish from reactive pleural fibrosis because of associated extensive collagen fibrosis. An 82-year-old woman with a severe cough was revealed to have pleural effusion and diffuse pleural thickening on the right side. Antibiotics were ineffective, and a compression fracture of the ninth and tenth thoracic vertebral bodies was recognized on X-ray. Autopsy revealed a diffuse pleural thickening with hyalinized collagen tissue in the central part of the pleura. However, the peripheral part of the fibrous tissue was composed of spindle and polygonal cell proliferation that were immunohistochemically positive for antibodies against cytokeratin and vimentin. In addition, the ninth and tenth thoracic spines were infiltrated by similar cells. The condition was diagnosed as desmoplastic mesothelioma with bone metastases. Asbestos bodies were detected in the thickened pleura and fibrosed alveolar septa, and it was suggested retrospectively that the patient had been exposed to asbestos. Thus, autopsy analyses of fibrous pleurisy are necessary to detect a desmoplastic variant of mesothelioma of the pleura and its association with asbestos exposure.
• Differentially expressed genes associated with cis-diamminedichloroplatinum (II) resistance in head and neck cancer using differential display and cDNA microarray

  E Higuchi, N Oridata, Y Furuta, S Suzuki, H Hatakeyama, H Sawa, K Sunayashiki-Kusuzaki, K Yamazaki, Y Inuyama, S Fukuda

  HEAD AND NECK-JOURNAL FOR THE SCIENCES AND SPECIALTIES OF THE HEAD AND NECK 25 (3) 187 - 193 1043-3074 2003/03 [Refereed][Not invited]
   

  Background. The mechanism by which cancer cells become resistant to cis-Diamminedichloroplatinum (II) (cDDP) is not completely understood. To investigate the molecular markers involved in the cDDP resistance, we compared the gene expression profiles between a head and neck squamous cell carcinoma (HNSCC) line sensitive to cDDP and its cDDP-resistant variant. Methods. Both a fluorescent differential display and a cDNA microarray analysis were applied to distinguish the gene profiles between KB, a human HNSCC line, and its cDDP-resistant variant (KB/cDDP). These results were confirmed by Northern blot analysis. Results. One up-regulated gene, glycoprotein hormone alpha-subunit, and two down-regulated genes coding membrane proteins, human folate receptor and tumor-associated antigen L6, were identified in KB/cDDP cells. Conclusions. Our findings suggest that development of the cDDP-resistant phenotype is accompanied by alternations of gene expression including a glycoprotein hormone and membrane proteins. These gene products could be new molecular markers for resistance to cDDP. (C) 2003 Wiley Periodicals, Inc.
• Ishikawa R, Kikuchi H, Jin M, Fujita M, Itoh T, Sawa H, Nagashima K: Desmoplastic malignant mesothelioma of the pleura: Autopsy reveals asbestos exposure. Pathol Int 53: 401-406, 2003*

  2003 [Not refereed][Not invited]
  
• Orba Y, Nishihara H, Sawa H, Itoo T, Shimizu M, Tanaka S, Nagashima K: Application of laser capture microdissection on cytological specimens for detection of immunoglobulin heavy chain gene rearrangement of malignant lymphoma. Cancer (Cancer Cytopatho・・・

  2003 [Not refereed][Not invited]
   

  Orba Y, Nishihara H, Sawa H, Itoo T, Shimizu M, Tanaka S, Nagashima K: Application of laser capture microdissection on cytological specimens for detection of immunoglobulin heavy chain gene rearrangement of malignant lymphoma. Cancer (Cancer Cytopathol) 99: 198-204, 2003*
• Nagashima T, Mizutani Y, Kawahara H, Maguchi S, Terayama Y, Shinohara T, Orba Y, Chuma T, Mano Y, Itoh T, Sawa H, Sakai K, Motomura M, Nagashima K. Anti-Hu paraneoplastic syndrome presenting with brainstem-cerebellar symptoms and Lambert-Eaton myasthen・・・

  2003 [Not refereed][Not invited]
   

  Nagashima T, Mizutani Y, Kawahara H, Maguchi S, Terayama Y, Shinohara T, Orba Y, Chuma T, Mano Y, Itoh T, Sawa H, Sakai K, Motomura M, Nagashima K. Anti-Hu paraneoplastic syndrome presenting with brainstem-cerebellar symptoms and Lambert-Eaton myasthenic syndrome. Neuropathology 23: 230-238, 2003*
• Endo S, Okada Y, Orba Y, Nishihara H, Tanaka S, Nagashima K, Sawa H*: JC virus (JCV) agnoprotein colocalizes with tubulin. J Neurovirol 9 (Suppl 1): 10-14, 2003 (*corresponding author)*

  2003 [Not refereed][Not invited]
  
• Higuchi E, Oridate N, Furuta Y, Suzuki S, Hatakeyama H, Sawa H, Sunayashiki-Kusazaki K, Yamazaki-k, Inuyama-Y, Fukuda S: Differentially expressed genes associated with cis-diammeinedichloroplatinum (II) resistance in head and neck cancer using differen・・・

  2003 [Not refereed][Not invited]
   

  Higuchi E, Oridate N, Furuta Y, Suzuki S, Hatakeyama H, Sawa H, Sunayashiki-Kusazaki K, Yamazaki-k, Inuyama-Y, Fukuda S: Differentially expressed genes associated with cis-diammeinedichloroplatinum (II) resistance in head and neck cancer using differential display and cDNA microarray. Head Neck 25:187-93, 2003.*
• Ricciardiello L, Baglioni M, Giovannini C, Pariali M, Cenacchi G, Ripalti A, Landini MP, Sawa H, Nagashima K, Frisque RJ, Goel A, Boland CR, Tognon M, Roda E, Bazzoli F: Induction of Chromosomal Instability in Colonic Cells by the Human Polyomavirus JC・・・

  2003 [Not refereed][Not invited]
   

  Ricciardiello L, Baglioni M, Giovannini C, Pariali M, Cenacchi G, Ripalti A, Landini MP, Sawa H, Nagashima K, Frisque RJ, Goel A, Boland CR, Tognon M, Roda E, Bazzoli F: Induction of Chromosomal Instability in Colonic Cells by the Human Polyomavirus JC Virus. Cancer Res 63:7256-62, 2003*
• Ohnishi A, Sawa H, Tsuda M, Sawamura Y, Marukawa K, Iwasaki Y, Nagashima K: Expression of the oligodendroglial lineage-associated markers Olig1 and Olig2 in different types of human gliomas. J Neuropathol Exp Neurol 62: 1052-1059, 2003*

  2003 [Not refereed][Not invited]
  
• Yamamoto S, Furukawa H, Kitamoto T, Takamaru Y, Morita N, Yasuda M, Okada Y, Sawa H, Nagashima K: An atypical form of sporadic panencephalopathic Cleutzfeldt-Jakob disease in Japan. Neuropathol Appl Neurobiol 29: 77-80, 2003*

  2003 [Not refereed][Not invited]
  
• Teramoto T, Kaneko H, Futano M, Sawa H, Nagashima K, Hirose Y, Kondo N: Progressive multifocal leukoencephalopathy in a patient with X-linked agammaglobulinemia. Scand J Infect Dis 35: 910-911, 2003*

  2003 [Not refereed][Not invited]
  
• Mechanism of white matter damage caused by virus infection

  S Semba, H Sawa, K Nagashima

  ADVANCES IN BRAIN RESEARCH: CEREBROVASCULAR DISORDERS AND NEURODEGENERATION 1251 139 - 147 0531-5131 2003 [Refereed][Not invited]
   

  White matter can be damaged by numerous causes resulting in various neurological deficits. The investigation of white matter damage associated with viruses can be simplified by studying JC virus infection of the central nervous system, which is known as progressive multifocal leukoencephalopathy (PML). JC virus capsid protein VP1 recognizes sialylated molecules of cell membrane and enters into various cells including neural and non-neural cells of humans, monkeys and rodents. The incorporated virus particle is transported along a clathrin pathway to the nucleus where the coat is removed. One of the cell-specific transcriptional factors, p75, may promote viral transcription, and the viral proteins are then transcribed. These findings may clarify the mechanism of viral neurotropism and provide basic knowledge for therapy of neurological disease. (C) 2003 Elsevier Science B.V. All rights reserved.
• JC virus agnoprotein colocalizes with tubulin

  S Endo, Y Okada, Y Orba, H Nishibara, S Tanaka, K Nagashima, H Sawa

  JOURNAL OF NEUROVIROLOGY 9 10 - 14 1355-0284 2003 [Refereed][Not invited]
   

  The human polyomavirus JC (JCV) encodes an agnoprotein that consists of 71 amino acid residues, with a molecular weight of approximately 8 kDa, from the late protein coding region. The agnoprotein of JCV shares 50% to 60% homology with those of simian virus 40 (SV40) and BK virus (BKV), and the carboxyl-terminal region of JCV agnoprotein is relatively unique. By using specific antibody to the carboxyl-terminal region of JCV agnoprotein, the authors have demonstrated that JCV agnoprotein expressed in the JCV-infected cells, where it localized predominantly in the perinuclear region of the cytoplasm, and colocalizes with the cellular cytoskeletal protein, tubulin. The results suggest that JCV agnoprotein may play a role in the stability of microtubules and the preservation of JCV infected cells via an interaction with tubulin.
• DOCK2 associates with CrkL and regulates Rac1 in human leukemia cell lines

  H Nishihara, M Maeda, A Oda, M Tsuda, H Sawa, K Nagashima, S Tanaka

  BLOOD 100 (12) 3968 - 3974 0006-4971 2002/12 [Refereed][Not invited]
   

  The CDM (ced-5 of Caenorhabditis elegans, DOCK180 [downstream of Crk with molecular weight of 180 kDa] of humans, and myoblast city of Drosophila melanogaster) family of proteins has been shown to play a pivotal role in the integrin-mediated signaling pathway under the regulation of an adaptor molecule c-CT10- related kinase II (c-Crk-II) in adherent cells. Recently, hematopoietic cell-specific CDM protein DOCK2 has been shown to be indispensable for lymphocyte migration. However, the regulatory mechanism for DOCK2 is still unknown because DOCK2 lacks a c-Crk-II binding consensus motif. In this study, we demonstrated that DOCK2 bound to CrkL, which is present exclusively in hematopoietic cells both in vivo and in vitro, and we also found that 2 separate regions of DOCK2 contributed to its binding to Src homology 3 (SH3) domain of CrkL. Colocalization of DOCK2 with Crk-like (CrkL) and F-actin was shown by immunocytochemical analysis with the use of Jurkat cells. We also found that CrkL-induced activation of small guanine triphosphatase (GTPase) Rac1 was significantly inhibited by the DOCK2-dCS mutant in 293T cells. Furthermore, the association of DOCK2 and Vav, the guanine-nucleotide exchanging factor (GEF) for Rac1, was demonstrated in Jurkat cells. Finally, the stable expression of DOCK2-dCS mutant in Jurkat cells was shown to reduce cell attachment. These data suggest the presence of a novel protein complex of CrkL, DOCK2, and Vav to regulate Rac1 in leukemia cell lines. (C) 2002 by The American Society of Hematology.
• Oligosaccharides as receptors for JC virus

  R Komagome, H Sawa, T Suzuki, Y Suzuki, S Tanaka, WJ Atwood, K Nagashima

  JOURNAL OF VIROLOGY 76 (24) 12992 - 13000 0022-538X 2002/12 [Refereed][Not invited]
   

  JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and in humans causes a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy. Its hemagglutination activity and entry into host cells have been reported to depend on an N-linked glycoprotein containing sialic acid. In order to identify the receptors of JCV, we generated virus-like particles (VLP) consisting of major viral capsid protein VP1. We then developed an indirect VLP overlay assay to detect VLP binding to glycoproteins and a panel of glycolipids. We found that VLP bound to sialoglycoproteins, including alpha1-acid glycoprotein, fetuin, and transferrin receptor, and that this binding depended on alpha2-3-linked sialic acids and N-linked sugar chains. Neoglycoproteins were synthesized by using ovalbumin and conjugation with oligosaccharides containing the terminal alpha2-3- or alpha2-6-linked sialic acid or the branched alpha2-6-linked sialic acid. We show that the neoglycoprotein containing the terminal alpha2-6-linked sialic acid had the highest affinity for VLP, inhibited the hemagglutination activity of VLP and JCV, and inhibited the attachment of VLP to cells. We also demonstrate that VLP bound to specific glycolipids, such as lactosylceramide, and gangliosides, including GM3, GD2, GD3, GD1b, GT1b, and GQ1b, and that VLP bound weakly to GD1a but did not bind to GM1a, GM2, or galactocerebroside. Furthermore, the neoglycoprotein containing the terminal alpha2-6-linked sialic acid and the ganglioside GT1b inhibited JCV infection in the susceptible cell line IMR-32. These results suggest that the oligosaccharides of glycoproteins and glycolipids work as JCV receptors and may be feasible as anti-JCV agents.
• Processing of the HTLV-II envelope precursor glycoprotein gp63 by furin is essential for cell fusion activity

  H Hasegawa, M Tatsumi, K Ogawa-Goto, H Takahashi, A Kojima, T Iwasaki, T Kurata, T Sata, T Takeuchi, N Sheehy, H Sawa, K Nagashima, WW Hall

  AIDS RESEARCH AND HUMAN RETROVIRUSES 18 (17) 1253 - 1260 0889-2229 2002/11 [Refereed][Not invited]
   

  To investigate the relationship between the fusogenic properties of HTLV-II and the processing of the envelope precursor glycoprotein gp63, recombinant cowpox virus expressing this protein was used to infect a range of cell lines derived from different species. Syncytium formation and gp63 processing were observed in all cells with the exception of LoVo cells, which are known to have a dysfunctional form of the endoprotease, furin. Furin has been shown to be necessary for the processing of a number of viral envelope glycoproteins, and gp63 contains a consensus sequence (305)Arg-Arg-Arg-Arg, which is a furin substrate motif. Pulse-chase studies demonstrated gp63 processing in Vero but not in LoVo cells. In addition it could be shown that expression of recombinant furin restored the processing of gp63 to gp46 in LoVo cells, and this resulted in syncytium formation. Our findings suggest that furin plays a pivotal role in cleavage of the HTLV-II envelope gp63, which in turn is a prerequisite for the fusogenic properties of the virus.
• Distribution of extracellular matrix tenascin-X in sciatic nerves

  K Matsumoto, H Sawa, M Sato, Y Orba, K Nagashima, H Ariga

  ACTA NEUROPATHOLOGICA 104 (5) 448 - 454 0001-6322 2002/11 [Refereed][Not invited]
   

  Tenascin-X (TNX) is an extracellular matrix protein that is highly expressed in the peripheral nervous system as well as muscular tissues, especially the heart and skeletal muscle. However, the expression manner and the physiological role of TNX in the peripheral nervous system have not been fully investigated. In this study, we elucidated its distribution in adult mouse sciatic nerves by immunohistochemical staining. TNX was found to be localized in the perineurium and the endoneurium of sciatic nerve fibers. To examine the physiological role of TNX, we investigated sciatic nerves of TNX-deficient mice that are viable and fertile and have no obvious deficits in general performance. The thickness of myelin sheaths and the size of the individual axons in these mice appeared normal. The ultrastructure of the sciatic nerves of TNX-deficient mice were similar to those of wild-type mice. Thus, the lack of a discernible phenotype in the sciatic nerves of TNX-deficient mice suggests that TNX has either a redundant or a very subtle function in the macromolecular organization in the peripheral nerve.
• Chk2-deficient mice exhibit radioresistance and defective p53-mediated transcription

  H Takai, K Naka, Y Okada, M Watanabe, N Harada, S Saito, CW Anderson, E Appella, M Nakanishi, H Suzuki, K Nagashima, H Sawa, K Ikeda, N Motoyama

  EMBO JOURNAL 21 (19) 5195 - 5205 0261-4189 2002/10 [Refereed][Not invited]
   

  The mammalian Chk2 kinase is thought to mediate ATM-dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2-deficient mice. Although Chk2(-/-) mice appeared normal, they were resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain were also resistant to IR-induced apoptosis. The IR-induced G(1)/S cell cycle checkpoint, but not the G(2)/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2(-/-) mice. IR-induced stabilization of p53 in Chk2(-/-) cells was 50-70% of that in wild-type cells. Caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ ATR-dependent but Chk2-independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53-dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2(-/-) cells. Our results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability.
• Drug-induced Long-QT syndrome associated with a subclinical SCN5A mutation

  N Makita, M Horie, T Nakamura, T Ai, K Sasaki, H Yokoi, M Sakurai, Sakuma, I, H Otani, H Sawa, A Kitabatake

  CIRCULATION 106 (10) 1269 - 1274 0009-7322 2002/09 [Refereed][Not invited]
   

  Background-Subclinical mutations in genes associated with the congenital long-QT syndromes (LQTS) have been suggested as a risk factor for drug-induced LQTS and accompanying life-threatening arrhythmias. Recent studies have identified genetic variants of the cardiac K+ channel genes predisposing affected individuals to acquired LQTS. We have identified a novel Na+ channel mutation in an individual who exhibited drug-induced LQTS. Methods and Results-An elderly Japanese woman with documented QT prolongation and torsade de pointes during treatment with the prokinetic drug cisapride underwent mutational analysis of LQTS-related genes. A novel missense mutation (L1825P) was identified within the C-terminus region of the cardiac Na+ channel (SCN5A). The L1825P channel heterologously expressed in tsA-201 cells showed Na+ cur-rent with slow decay and a prominent tetrodotoxin-sensitive noninactivating component, similar to the gain-of-function phenotype most commonly observed for SCN5A-associated congenital LQTS (LQT3). In addition, L1825P exhibited loss of function Na+ channel features characteristic of Brugada syndrome. Peak Na+ current density observed in cells expressing L1825P was significantly diminished, and the voltage dependence of activation and inactivation was shifted toward more positive and negative potentials, respectively. Conclusions-This study demonstrates that subclinical mutations in the LQTS-related gene SCN5A may predispose certain individuals to drug-induced cardiac arrhythmias.
• Binding and dissociation of human topoisomerase I with hairpin-loop RNAs: implications for the regulation of HIV-1 replication

  H Takahashi, H Sawa, H Hasegawa, T Sata, WW Hall, T Kurata

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 297 (3) 593 - 599 0006-291X 2002/09 [Refereed][Not invited]
   

  Cellular topoisomerase I has been reported to be present in retroviral particles and to enhance viral cDNA synthesis; however, the mechanisms involved remain unknown. In the present study, it has been demonstrated that human topoisomerase I combines with a stem-loop RNA and that the bound topoisomerase I can be dissociated from RNA substrates in the presence of ATP. In addition, in vitro cleaved synthetic RNA bound by topoisomerase I is subsequently religated when the topoisomerase I is dissociated by ATP. A mechanism is proposed in which human topoisomerase I is carried into virions and regulates the repair of genomic RNA by its ligation activity. (C) 2002 Elsevier Science (USA). All rights reserved.
• Expression of JC virus agnoprotein in progressive multifocal leukoencephalopathy brain

  Y Okada, H Sawa, S Endo, Y Orba, T Umemura, H Nishihara, AC Stan, S Tanaka, H Takahashi, K Nagashima

  ACTA NEUROPATHOLOGICA 104 (2) 130 - 136 0001-6322 2002/08 [Refereed][Not invited]
   

  To examine the function of JC virus (JCV) agnoprotein, we examined the brains of cases of progressive multifocal leukoencephalopathy (PML), which is caused by JCV infection, using a newly generated antibody. The antibody reacted with 8 kDa protein specific for JCV agnoprotein by Western blotting. In vitro analyses showed that JCV capsid protein VP1 and large T antigen (T-Ag) were localized in the nuclei, but that agnoprotein was mainly detected in the cytoplasm of JCV-infected cells with an occasional nuclear staining. In the PML brain, an immunoreactive signal for agnoprotein was distributed in the perinuclear areas and cytoplasmic processes with occasional punctate staining in demyelinating lesions as well as adjacent myelinated areas. Agnoprotein presented mostly in the infected oligodendrocytes and partly in the astrocytes. Using double immunostaining, agnoprotein was seen to be expressed in the cytoplasmic processes of the cells, the nuclei of which were labeled with VP1 and T-Ag, where virus particles existed. Thus, JCV agnoprotein was mostly expressed in the infected oligodendrocytes and mainly localized in the cytoplasmic processes apart from virus particles in the demyelinated lesions.
• DOCK2 mediates T cell receptor-induced activation of Rac2 and IL-2 transcription

  H Nishihara, M Maeda, M Tsuda, Y Makino, H Sawa, K Nagashima, S Tanaka

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 296 (3) 716 - 720 0006-291X 2002/08 [Refereed][Not invited]
   

  DOCK2, a CDM family protein exclusively found in hematopoietic cells, has been shown to play a role in lymphocyte migration by the regulation of actin cytoskeleton. Although DOCK2 has been shown to induce the activation of Rac1, the regulatory mechanism of Rac2, which is a hematopoietic cell-specific small GTPase, is still unknown. In this study, we examined the role of DOCK2 in the activation of Rac2 in hematopoietic cells. DOCK2 was found to associate with the zeta subunit of the CD3 complex of T cell receptors in Jurkat cells and to activate forced expressed Rac2 in 293T cells. In addition, the stable expression of DOCK2 in Jurkat cells exhibited the elevated activity of endogenous Rac2. Furthermore, the transcriptional activity of interleukin-2 (IL-2) was enhanced in DOCK2-expressing Jurkat cells and the dominant negative form of Rac2 suppressed its elevated IL-2 promoter activity. These results suggest that DOCK2 mediates TCR-dependent activation of Rac2, leading to the regulation of IL-2 promoter activity in T cells. (C) 2002 Elsevier Science (USA). All rights reserved.
• Molecular and immunohistochemical analysis of signaling adaptor protein Crk in human cancers

  H Nishihara, S Tanaka, M Tsuda, S Oikawa, M Maeda, M Shimizu, H Shinomiya, A Tanigami, H Sawa, K Nagashima

  CANCER LETTERS 180 (1) 55 - 61 0304-3835 2002/06 [Refereed][Not invited]
   

  Crk is a signaling adaptor protein which is mostly composed of SH2 and SH3 domains. and has been shown to play a pivotal role in cell proliferation. differentiation, and migration. Because Crk was originally isolated as an avian sarcoma virus CT10 encoding oncoprotein v-Crk. we examined a potential role for c-Crk in the carcinogenesis of human cancers. First, to analyze gene mutations of c-Crk, we isolated a human bacterial artificial chromosome clone containing Crk genome and exon/intron structures, However, polymerase chain reaction-single strand conformation polymorphism methods failed to show any genomic mutations in the Crk exon which could be related to carcinogenesis. Second, immunohistochemical analysis of c-Crk-II demonstrated that the levels of c-Crk-II were significantly elevated in most of the tumors, particularly in the colon and lung cancers. Furthermore, immunoblot analysis using human lung cancer cell lines revealed that the expression levels of c-Crk-II were correlated to growth rates of L cells. The elevated expression levels of c-Crk-II might be related to the development of human cancers. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.
• Topoisomerase I and ATP activate cDNA synthesis of human immunodeficiency virus type 1

  H Takahashi, H Sawa, H Hasegawa, Y Shoya, T Sata, WW Hall, K Nagashima, T Kurata

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 294 (2) 509 - 517 0006-291X 2002/06 [Refereed][Not invited]
   

  Replication of human immunodeficiency virus type 1 (HIV-1) is regulated at reverse transcription. Cellular topoisomerase I has been reported to be carried into HIV-1 virions and enhance cDNA synthesis in vitro, suggesting that topoisomerase I expressed in virus producer cells regulates reverse transcription. Here, by employing both indicator cell assay and endogenous reverse transcription (ERT) assay, we show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV-1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I-RNA complex and enhance cDNA synthesis in vitro. These findings suggest that cellular topoisomerase I and ATP play a pivotal role in the synthesis of cDNA of HIV-1. (C) 2002 Elsevier Science (USA). All rights reserved.
• Reconstitution of cleavage of human immunodeficiency virus type-1 (HIV-1) RNAs

  H Takahashi, H Sawa, H Hasegawa, T Sata, WW Hall, K Nagashima, T Kurata

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 293 (3) 1084 - 1091 0006-291X 2002/05 [Refereed][Not invited]
   

  A human immunodeficiency virus type 1 (HIV-1) particle contains approximately 1200 molecules of gag proteins and two copies of a 9.2-kb genomic RNA which has been reported to be dimerized and rapidly cleaved and to form a complex with a nucleocapsid protein, p7 (NCp7), during viral budding. These suggest that the cleavage can be reconstituted with gag proteins in vitro. Here we show that the p15(gag) coding region of viral RNA is fragmented in viral particles and that in vitro-synthesized RNA transcripts of HIV-1 undergo cleavage which is activated by NCp7 and other factors. Single-stranded oligoribonucleotides were cleaved between C and A or U and A, leaving 2',3'-cyclic phosphate and 5'-hydroxyl termini. These findings might explain the rapid degradation of genomic RNAs in HIV-1 particles. (C) 2002 Elsevier Science (USA). All rights reserved.
• Hydrocephalus, situs inversus, chronic sinusitis, and male infertility in DNA polymerase lambda-deficient mice: Possible implication for the pathogenesis of immotile cilia syndrome

  Y Kobayashi, M Watanabe, Y Okada, H Sawa, H Takai, M Nakanishi, Y Kawase, H Suzuki, K Nagashima, K Ikeda, N Motoyama

  MOLECULAR AND CELLULAR BIOLOGY 22 (8) 2769 - 2776 0270-7306 2002/04 [Refereed][Not invited]
   

  A growing number of DNA polymerases have been identified, although their physiological function and relation to human disease remain mostly unknown. DNA polymerase lambda (Pol lambda; also known as Pol beta2) has recently been identified as a member of the X fancily of DNA polymerases and shares 32% amino acid sequence identity with DNA Pot R within the polymerase domain. With the use of homologous recombination, we generated Pol lambda(-/-) mice. Pol lambda(-/-) mice develop hydrocephalus with marked dilation of the lateral ventricles and exhibit a high rate of mortality after birth, although embryonic development appears normal. Pol lambda(-/-) mice also show situs inversus totalis and chronic suppurative sinusitis. The surviving male, but not female, Pol lambda(-/-) mice are sterile as a result of spermatozoal immobility. Microinjection of sperm from male Pol lambda(-/-) mice into oocytes gives rise to normal offspring, suggesting that the meiotic process is not impaired. Ultrastructural analysis reveals that inner dynein arms of cilia from both the ependymal cell layer and respiratory epithelium are defective, which may underlie the pathogenesis of hydrocephalus, situs inversus totalis, chronic sinusitis, and male infertility. Sensitivity of Pol lambda(-/-) cells to various kinds of DNA damage is indistinguishable from that of Pol lambda(+/+) cells. Collectively, Pol lambda(-/-) mice may provide a useful model for clarifying the pathogenesis of immotile cilia syndrome.
• Advanced glycation end products induce angiogenesis in vivo

  T Okamoto, S Tanaka, AC Stan, T Koike, M Kase, Z Makita, H Sawa, K Nagashima

  MICROVASCULAR RESEARCH 63 (2) 186 - 195 0026-2862 2002/03 [Refereed][Not invited]
   

  Advanced glycation end products (AGEs) have been thought to participate in diabetic microangiopathy. However, the effects of AGEs on angiogenesis have so far been mainly examined either in vitro or by using cultured cells. In the present study, we have analyzed whether AGES induce angiogenesis in vivo by using the chorioallantoic membrane (CAM) assay. The CAM assay was carried out in embryonated hen eggs to determine the effects of AGEs. Following generation of AGEs based on bovine serum albumin (BSA), either AGE-BSA or nonglycated BSA was administered to the CAM and their effects on angiogenesis were assessed, together with an inhibitory effect of an anti-AGE antibody against AGE-BSA-induced angiogenesis. The histological features of AGE-induced vascular lumens were examined by immunohistochemical analysis for Factor VIII and smooth muscle a-actin. AGE-BSA induced angiogenesis in CAM in a dose- and time-dependent manner. AGE-induced angiogenesis on CAM was neutralized by the anti-AGE antibody. Inummohistochemical analysis demonstrated that AGE-induced vascular lumens were devoid of pericytes. Our data demonstrated that AGES are an angiogenetic factor and that our system of AGE-induced abnormal vessels in CAMs is useful in further investigations of the mechanism of diabetic retinal angiogenesis and can also be used to provide a therapeutic model for diabetic angiopathy. (C) 2002 Elsevier Science (USA).
• Signaling adaptor protein v-Crk activates Rho and regulates cell motility in 3Y1 rat fibroblast cell line

  M Tsuda, S Tanaka, H Sawa, H Hanafusa, K Nagashima

  CELL GROWTH & DIFFERENTIATION 13 (3) 131 - 139 1044-9523 2002/03 [Refereed][Not invited]
   

  The adaptor protein Crk has been reported to associate with focal adhesions and is thought to be involved in integrin-mediated signaling pathway. However, the precise mechanism of Crk-dependent regulation of cytoskeleton still remains under investigation. In this study, we have established a v-Crk-inducible cell line in rat fibroblasts 3Y1 cells and found that v-Crk activated Rho and induced actin stress fiber formation. In addition to the induction of tyrosine-phosphorylation of p130(Cas) and paxillin, we demonstrated that v-Crk induced threonine-phosphorylated bands sized at 72/78 kDa found specifically in 3Y1 cells. Both of the inhibitors of Rho and Rho-associated kinase, C3 and Y27632, respectively, inhibited these v-Crk-induced biochemical effects. Although v-Crk-induced cells exhibited a decrease of cell motility, integrin stimulation recovered the suppression of motility. Furthermore, v-Crk enhanced motility in chemotactic assay toward fibronectin with additional activation of Rho and the increase of levels of CD44 cleavage. These results suggest that v-Crk activated Rho and induced actin stress fiber formation and CD44 cleavage leading to the regulation of cell motility.
• Molecular neuropathology of JC virus

  Yasuko Orba, Hirofumi Sawa, Kazuo Nagashima

  Brain and Nerve 54 (2) 101 - 109 0006-8969 2002 [Refereed][Not invited]
  
• Tsuda, M., Tanaka, S., Sawa, H., Hanafusa, H., Nagashima: Signalling adaptor protein v-Crk activates Rho and regulates cell motility in 3Y1 rat fibroblast cell line. Cell Growth Diff 13: 131-139, 2002*

  2002 [Not refereed][Not invited]
  
• Takahashi H, Sawa H, Hasegawa H, Sata T, Hall WW, Kurata T: Binding and dissociation of human topoisomerase I with hair-pin loop RNAs: implications for the regulation of HIV-1 replication. Biochem Biophys Res Commun 297: 593-599, 2002*

  2002 [Not refereed][Not invited]
  
• Nishihara H, Tanaka S, Tsuda M., Oikawa S, Maeda M, Shimizu M, Shimoyama H, Tanigami A, Sawa H, Nagashima K: Molecular and immunohistochemical analysis of adaptor protein Crk in human cancers. Cancer Lett 180, 55-61, 2002*

  2002 [Not refereed][Not invited]
  
• Takahashi H, Sawa H, Hasegawa H, Sata T, Hall WW, Nagashima K, Kurata T: Reconstitution of cleavage of human immunodeficiency virus type-1 (HIV-1) RNAs. Biochem Biophys Res Commun 293: 1084-1091, 2002*

  2002 [Not refereed][Not invited]
  
• Matsumoto K, Sawa H, Sato M, Orba Y, Nagashima K, Ariga H: Distribution of extracellular matrix tenascin-X in sciatic nerves. Acta Neuropathol 104: 448-454, 2002*

  2002 [Not refereed][Not invited]
  
• Okada Y, Sawa H, Endo S, Orba Y, Umemura T, Nishihara H, Stan AC, Tanaka S, Nagashima K. Expression of JC virus (JCV) agnoprotein in progressive multifocal leukoencephalopathy (PML) brain. Acta Neuropathol 104: 130-136, 2002*

  2002 [Not refereed][Not invited]
  
• Makita N, Horie M, Nakamura T, Ai T, Otani H, Sawa H, Kitabatake A: Drug-induced long-QT syndrome associated with a subclinical SCN5A mutation. Circulation 106: 1269-1274, 2002*

  2002 [Not refereed][Not invited]
  
• Hasegawa H, Tatsumi M, Ogawa-Goto K, Takahashi H, Iwasaki T, Kurata T, Sata T, Takeuchi T, Sheehy N, Sawa H, Nagashima K, Hall WW: Processing of the HTLV-II envelope precursor glycoprotein, gp63 by furin is essential for cell fusion activity. AIDS Res・・・

  2002 [Not refereed][Not invited]
   

  Hasegawa H, Tatsumi M, Ogawa-Goto K, Takahashi H, Iwasaki T, Kurata T, Sata T, Takeuchi T, Sheehy N, Sawa H, Nagashima K, Hall WW: Processing of the HTLV-II envelope precursor glycoprotein, gp63 by furin is essential for cell fusion activity. AIDS Res Hum Retroviruses 18: 1253-1260, 2002*
• Nishihara H, Maeda M, Oda A, Tsuda M, Sawa H, Tanaka S, and Nagashima K: DOCK2 associates with CrkL and regulates Rac1 in hematopoietic cells. Blood 100: 3968-3974, 2002*

  2002 [Not refereed][Not invited]
  
• Yoshida H, Okada Y, Kinoshita N, Hara H, Sasaki M, Sawa H, Nagashima K, Mak TW, Motoyama N: Differential requirement for Apaf1 and Bcl-XL in the regulation of programmed cell death during development. Cell Death Differ 9: 1273-1276, 2002*

  2002 [Not refereed][Not invited]
  
• Nishihara H, Maeda M, Tsuda M, Makino Y, Sawa H, Nagashima K, Tanaka S: DOCK2 mediates T cell receptor-induced activation of Rac2 and IL-2 transcription. Biochem Biophys Res Commun 296: 716-720, 2002*

  2002 [Not refereed][Not invited]
  
• Takai H, Naka K, Okada Y, Watanabe M, Harada N, Saito S, Anderson CW, Appella E, Nakanishi M, Suzuki H, Nagashima K, Sawa H, Ikeda K, Motoyama N: Chk2-deficient mice exhibit increased resistance to ionizing radiation and defective p53-mediated transcri・・・

  2002 [Not refereed][Not invited]
   

  Takai H, Naka K, Okada Y, Watanabe M, Harada N, Saito S, Anderson CW, Appella E, Nakanishi M, Suzuki H, Nagashima K, Sawa H, Ikeda K, Motoyama N: Chk2-deficient mice exhibit increased resistance to ionizing radiation and defective p53-mediated transcription. EMBO J 21: 5195-5205, 2002*
• Komagome R, Sawa H*, Suzuki T, Suzuki Y, Tanaka S, Atwood WJ, Nagashima K: Oligosaccharides as receptors for JC virus. J Virol 76: 12992-13000, 2002 (* corresponding author)*

  2002 [Not refereed][Not invited]
  
• Takahashi H, Sawa H, Hasegawa H, Shoya Y, Sata T, Hall WW, Nagashima K, Kurata T: Topoisomerase I and ATP activate cDNA synthesis of human immunodeficiency virus type-1 (HIV-1). Biochem Biophys Res Commun 294: 509-517, 2002

  2002 [Not refereed][Not invited]
  
• Hasegawa H, Tatsumi M, Ogawa-Goto K, Takahashi H, Iwasaki T, Kurata T, Sata T, Takeuchi T, Sheehy N, Sawa H, Nagashima K, Hall WW: "Processing of the HTLV-II envelope precursor glycoprotein, gp63 by furin is essential for cell fusion activity." AIDS R・・・

  2002 [Not refereed][Not invited]
   

  Hasegawa H, Tatsumi M, Ogawa-Goto K, Takahashi H, Iwasaki T, Kurata T, Sata T, Takeuchi T, Sheehy N, Sawa H, Nagashima K, Hall WW: "Processing of the HTLV-II envelope precursor glycoprotein, gp63 by furin is essential for cell fusion activity." AIDS Res Hum Retroviruses 18: 1253-1260, 2002*
• Nishihara H, Tanaka S, Tsuda M., Oikawa S, Maeda M, Shimizu M, Shimoyama H, Tanigami A, Sawa H, Nagashima K: "Molecular and immunohistochemical analysis of adaptor protein Crk in human cancers." Cancer Lett 180, 55-61, 2002*

  2002 [Not refereed][Not invited]
  
• Matsumoto K, Sawa H, Sato M, Orba Y, Nagashima K, Ariga H: "Distribution of extracellular matrix tenascin-X in sciatic nerves." Acta Neuropathol 104: 448-454, 2002*

  2002 [Not refereed][Not invited]
  
• Tsuda, M., Tanaka, S., Sawa, H., Hanafusa, H., and Nagashima: "Signalling adaptor protein v-Crk activates Rho and regulates cell motility in 3Y1 rat fibroblast cell line." Cell Growth Diff 13: 131-139, 2002*

  2002 [Not refereed][Not invited]
  
• Takahashi H, Sawa H, Hasegawa H, Sata T, Hall WW, Nagashima K, Kurata T: "Reconstitution of cleavage of human immunodeficiency virus type-1 (HIV-1) RNAs." Biochem Biophys Res Commun 293: 1084-1091, 2002*

  2002 [Not refereed][Not invited]
  
• Yoshida H, Okada Y, Kinoshita N, Hara H, Sasaki M, Sawa H, Nagashima K, Mak TW, Motoyama N: "Differential requirement for Apaf1 and Bcl-XL in the regulation of programmed cell death during development." Cell Death Differ 9: 1273-1276, 2002*

  2002 [Not refereed][Not invited]
  
• Takahashi H, Sawa H, Hasegawa H, Sata T, Hall WW, Kurata T: "Binding and dissociation of human topoisomerase I with hair-pin loop RNAs: implications for the regulation of HIV-1 replication." Biochem Biophys Res Commun 297: 593-599, 2002*

  2002 [Not refereed][Not invited]
  
• Komagome R, Sawa H*, Suzuki T, Suzuki Y, Tanaka S, Atwood WJ, Nagashima K: "Oligosaccharides as receptors for JC virus." J Virol 76: 12992-13000, 2002 (* corresponding author)*

  2002 [Not refereed][Not invited]
  
• Nishihara H, Maeda M, Oda A, Tsuda M, Sawa H, Tanaka S, and Nagashima K: "DOCK2 associates with CrkL and regulates Rac1 in hematopoietic cells." Blood 100: 3968-3974, 2002*

  2002 [Not refereed][Not invited]
  
• Takai H, Naka K, Okada Y, Watanabe M, Harada N, Saito S, Anderson CW, Appella E, Nakanishi M, Suzuki H, Nagashima K, Sawa H, Ikeda K, Motoyama N: "Chk2-deficient mice exhibit increased resistance to ionizing radiation and defective p53-mediated transcr・・・

  2002 [Not refereed][Not invited]
   

  Takai H, Naka K, Okada Y, Watanabe M, Harada N, Saito S, Anderson CW, Appella E, Nakanishi M, Suzuki H, Nagashima K, Sawa H, Ikeda K, Motoyama N: "Chk2-deficient mice exhibit increased resistance to ionizing radiation and defective p53-mediated transcription." EMBO J 21: 5195-5205, 2002*
• Nishihara H, Maeda M, Tsuda M, Makino Y, Sawa H, Nagashima K, Tanaka S: "DOCK2 mediates T cell receptor-induced activation of Rac2 and IL-2 transcription." Biochem Biophys Res Commun 296: 716-720, 2002*

  2002 [Not refereed][Not invited]
  
• Okada Y, Sawa H, Endo S, Orba Y, Umemura T, Nishihara H, Stan AC, Tanaka S, Nagashima K. "Expression of JC virus (JCV) agnoprotein in progressive multifocal leukoencephalopathy (PML) brain." Acta Neuropathol 104: 130-136, 2002*

  2002 [Not refereed][Not invited]
  
• Coronary reperfusion following ischemia - Different expression of bcl-2 and bax proteins, and cardiomyocyte apoptosis

  ZL Xie, T Koyama, J Suzuki, Y Fujii, H Togashi, H Sawa, K Nagashima

  JAPANESE HEART JOURNAL 42 (6) 759 - 770 0021-4868 2001/11 [Refereed][Not invited]
   

  The aim of this work was to examine factors that could be involved in the occurrence of apoptosis in rat hearts subjected to coronary occlusion followed by reperfusion. To this end, we studied the expression of the pro- and anti-apoptotic factors, bax and bcl-2. respectively, in reperfused ischemic hearts and in hearts injected with bFGF or saline. In anesthetized rats the left coronary artery was occluded for 45 min, the anesthesia withdrawn and the occlusion removed to allow reperfusion; in sham-operated rats the occlusion was omitted. After 4 hours the rats were decapitated and the heart excised. Sections from the left ventricle were stained with anti-bcl-2-antibody and anti-bax-antibody using the TUNEL method which detects apoptosis. Fragmentation of DNA isolated from reperfused ventricles was examined by agarose electrophoresis. In reperfused hearts no bcl-2 staining was observed in the discrete area in which many cardiomyocyte nuclei were stained by the TUNEL method; outside this area staining for bcl-2 was more marked than in sham-operated rats. Sections from reperfused hearts were stained for bax protein over a wide area including the apoptotic region; sham-operated hearts showed little reaction. Staining for bcl-2 was demonstrable in some nuclei in hearts from saline-injected rats; the numbers were unaffected by i.v. bFGF. Ischemia/reperfusion increases the overall expression of both bcl-2 and bax proteins, but bcl-2 is lost from the reperfused area as indicated by TUNEL staining. Accordingly, the ratio of bcl-2 to bax was reduced in the reperfused area, indicating a pro-apoptotic trend. The marked increase in bcl-2 outside the reperfused area could be a mechanism with which to salvage surviving cardiomyocytes.
• Developmental and functional analyses of CD8+ NK1.1+ T cells in class-I-restricted TCR transgenic mice

  Ryuichiro Ohwatari, Kazuya Iwabuchi, Chikako Iwabuchi, Taiki Morohashi, Hirofumi Sawa, Kyoji Hioki, Kimio Kobayashi, Satoshi Fukuda, Yukio Inuyama, Kazunori Onoé

  Cellular Immunology 213 (1) 24 - 33 0008-8749 2001/10/10 [Refereed][Not invited]
   

  Using a class-I-restricted T cell receptor (TCR) transgenic mice (Tgm), 2C (Vα3.1/Vβ 8.2, specific for Ld + LSPFPFDL), the development and cytokine production of tg-TCR+ NKT cells were analyzed. We found that CD8+ or double negative (DN) NKT cells constituted a major population of NKT cells in the H-2b/b 2C Tgm (positive selecting background) or the H-2b/d 2C Tgm (negative selecting background), respectively. Virtually no NKT cells were generated in the H-2k/k 2C Tgm (neutral selecting background). CD8+ NKT cells in the H-2b/b 2C Tgm expressed CD8αβ heterodimers, whereas those in the H-2b/d 2C Tgm expressed CD8αα homodimers. These findings suggest that development of a subpopulation of NKT cells is influenced by the H-2 molecules. Upon stimulation with anti-CD3 mAb, tg-TCR+ NKT cells generated in the H-2b/b and H-2b/d backgrounds produced IFN-γ but not IL-4. © 2001 Elsevier Science.
• Adult T-cell lymphoma involving the leptomeninges associated with a spinal cord schwannoma

  T Nagashima, M Mori, M Fujimoto, M Nunomura, Y Sakurai, Y Okada, T Itoh, H Sawa, AC Stan, K Nagashima

  NEUROPATHOLOGY 21 (3) 229 - 235 0919-6544 2001/09 [Refereed][Not invited]
   

  Adult T-cell lymphoma (ATL-L) developing initially in the meninges is rare. An autopsy case of ATL-L with an acute onset of meningitis and generalized lymphadenopathy in association with a cervical cord schwannoma is reported here. A 78-year-old woman with sensori-motor weakness of both arms over a 1-year period, developed febrile episodes and drowsiness with neck stiffness. Lumbar puncture revealed an increased protein content (161 mg/dL) and increased cell count (463/3) consisting of 99% of lymphocytes which contained atypical lymphocytes with multilobulated nuclei ('flower cells'), which are characteristic of ATL-L. Viral titers were positive only for HTLV-I antibodies (serum 3 640: CSF 3 16). Biopsy of an enlarged retroperitoneal lymph node revealed malignant lymphoma of the T-cell type. Brain MRI was negative, whereas an intradural extramedullary mass was found at the C4 level. With a diagnosis of ATL-L stage IV, chemotherapy was commenced, which was effective in reducing the generalized lymphadenopathy as well as the cervical mass and restoring the CSF findings to normality. The cervical cord mass was verified to be a solitary schwannoma, and ATL-L involvement was found not only in the leptomeninges, but also within the cervical cord schwannoma.
• Distribution and function of JCV agnoprotein

  Y Okada, S Endo, H Takahashi, H Sawa, T Umemura, K Nagashima

  JOURNAL OF NEUROVIROLOGY 7 (4) 302 - 306 1355-0284 2001/08 [Refereed][Not invited]
   

  JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), encodes six major proteins including agnoprotein, the function of which is unknown. To explore its function, we initially studied the expression and localization of agnoprotein in both cultured cells and PML brain using immunohistochemical methods. Employing a specific polyclonal antibody, agnoprotein was found mostly in the cytoplasm of persistently infected JCI cells and in the finely elaborated cytoplasmic processes of oligodendroglial cells in PML brain. The immunohistochemistry indicated that the cytoplasm of oligodendroglial cells was relatively well-preserved in the demyelinated foci. Agnoprotein coprecipitated with tubulin in immunoprecipitation assays and the colocalization of agnoprotein with cytoplasmic tubulin was verified by double immunostaining with confocal microscopy. Transfection of an agnogene deleted JCV Mad1 strain (Mad1(Delta agno)) into the susceptible cell line failed to produce not only agnoprotein but also VP1 and large T mRNAs, whereas the wild-type JCV Mad1 resulted in the expression of both large T and VP1 mRNAs. The cytoplasmic agnoprotein was phosphorylated and when coexpressed with GST-EGFP, was also localized in the cytoplasm. Inhibition of protein kinase A by its inhibitor H-89, however, reversed the cytoplasmic localization of agnoprotein to the nuclear compartment. Our results suggest that JCV agnoprotein may "shuttle" between the nucleus and cytoplasm in a phosphorylation-dependent manner during viral replication.
• Quantitation of varicella-zoster virus DNA in patients with Ramsay Hunt syndrome and zoster sine herpete

  Y Furuta, F Ohtani, H Sawa, S Fukuda, Y Inuyama

  JOURNAL OF CLINICAL MICROBIOLOGY 39 (8) 2856 - 2859 0095-1137 2001/08 [Refereed][Not invited]
   

  Varicella-zoster virus (VZV) reactivation causes facial nerve palsy in Ramsay Hunt syndrome (RHS) and zoster sine herpete (ZSH) with and without zoster rash, respectively. In the present study, we analyzed the VZV DNA copy number in saliva samples from 25 patients with RHS and 31 patients with ZSH using a TaqMan PCR assay to determine differences in the viral load between the two diseases. VZV copy number in saliva peaked near the day of the appearance of zoster in patients with RHS. Consequently, VZV DNA was less frequently detected in patients with RHS who exhibited facial palsy several days after the appearance of zoster. These findings suggest that the VZV load in saliva samples reflects the kinetics of viral reactivation in patients with RHS. In addition, VZV DNA was equally detected in saliva from patients with RHS and ZSH, and there was no significant difference in the highest viral copy number between patients with RHS and those with ZSH. The VZV load does not appear to reflect a major difference between RHS and ZSH.
• Angiotensin-converting enzyme inhibition attenuates hypofibrinolysis and reduces cardiac perivascular fibrosis in genetically obese diabetic

  AKMT Zaman, S Fujii, H Sawa, D Goto, N Ishimori, K Watano, T Kaneko, T Furumoto, T Sugawara, Sakuma, I, A Kitabatake, BE Sobel

  CIRCULATION 103 (25) 3123 - 3128 0009-7322 2001/06 [Refereed][Not invited]
   

  Background-Obesity and insulin resistance are associated with accelerated macrovascular and microvascular coronary disease, cardiomyopathic phenomena, and increased concentrations and activity in blood of plasminogen activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of fibrinolysis. Methods and Results-To determine whether hypofibrinolysis in blood and tissues and its potential sequelae could be attenuated pharmacologically, we studied genetically modified obese mice. By 10 weeks of age, obese mice exhibited increases in left ventricular weight and glucose and immunoreactive insulin in blood. PAI-1 activity in blood measured spectrophotometrically was significantly elevated as well. The difference compared with values in lean controls widened by 20 weeks of age. Perivascular fibrosis in coronary arterioles and small coronary arteries was evident in obese mice 10 and 20 weeks of age, paralleling increases in PAI-1 and tissue factor expression evident by immunohistochemical image analysis, in situ hybridization, and reverse transcription-polymerase chain reaction. Inhibition of ACE activity initiated in obese mice 10 weeks of age and continued for 20 weeks arrested the increase in PAI-1 activity in blood and in cardiac PAI-I and tissue factor mRNA as well as coronary perivascular fibrosis. Conclusions-Thus, inhibition of proteo(fibrino)lysis and augmented tissue factor expression in the heart precede and may contribute to the coronary perivascular fibrosis seen with obesity and insulin resistance. Furthermore, inhibition of ACE activity can attenuate all 3 phenomena.
• Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2α

  Makoto Nagai, Shinya Tanaka, Masumi Tsuda, Shuichi Endo, Hiroyuki Kato, Hiroshi Sonobe, Akio Minami, Hiroaki Hiraga, Hiroshi Nishihara, Hirofumi Sawa, Kazuo Nagashima

  Proceedings of the National Academy of Sciences of the United States of America 98 (7) 3843 - 3848 0027-8424 2001/03/27 [Not refereed][Not invited]
   

  Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X 18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2α, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1-181) of SYT-SSX1 and 50 amino acids (aa 156-205) of hBRM/hSNF2α and we found that the overexpression of this binding region of hBRM/hSNF2α significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription-PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2α in human cancer.
• Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2 alpha

  M Nagai, S Tanaka, M Tsuda, S Endo, H Kato, H Sonobe, A Minami, H Hiraga, H Nishihara, H Sawa, K Nagashima

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 98 (7) 3843 - 3848 0027-8424 2001/03 [Refereed][Not invited]
   

  Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2 alpha, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1-181) of SYT-SSX1 and 50 amino acids (aa 156-205) of hBRM/hSNF2 alpha and we found that the overexpression of this binding region of hBRM/hSNF2 alpha significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription-PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2 alpha in human cancer.
• Okada Y, Endo S, Takahashi H, Sawa H, Umemura T, Nagashima K: "Distribution and function of JCV agnoprotein." J Neurovirol 7: 302-306, 2001

  2001 [Not refereed][Not invited]
  
• Suzuki S, Sawa H*, Komagome R, Orba Y, Yamada M, Okada Y, Ishida Y, Nishihara H, Tanaka S, Nagashima K: "Broad distribution of the JC virus receptor contrasts with a marked cellular restriction of virus replication." Virology 286: 100-112, 2001 (* cor・・・

  2001 [Not refereed][Not invited]
   

  Suzuki S, Sawa H*, Komagome R, Orba Y, Yamada M, Okada Y, Ishida Y, Nishihara H, Tanaka S, Nagashima K: "Broad distribution of the JC virus receptor contrasts with a marked cellular restriction of virus replication." Virology 286: 100-112, 2001 (* corresponding author)*
• Shirane M, Sawa H, Kobayashi Y, Nakano T, Shinkai Y, Nagashima K, and Negishi I. "Deficiency of phospholipase C-1 impares renal development and hematopoiesis." Development 128: 5173-5180, 2001*

  2001 [Not refereed][Not invited]
  
• Okamoto T, Tanaka S, Stan AC., Koike T, Kase, Makita Z, Sawa H, Ngashima K: "Advanced glycation end products induce angiogenesis in vivo." Microvascular Research 63: 186-195, 2002*

  2001 [Not refereed][Not invited]
  
• Tanaka S, Katano H, Tsukamoto K, Jin M, Oikawa S, Nishihara H, Sawa H, Sawada K, Shimizu M, Sata T, Fujioka Y, Nagashima K. "HHV8-negative primary effusion lymphoma of the peritoneal cavity presenting with a distinct immunohistochemical phenotype." Pa・・・

  2001 [Not refereed][Not invited]
   

  Tanaka S, Katano H, Tsukamoto K, Jin M, Oikawa S, Nishihara H, Sawa H, Sawada K, Shimizu M, Sata T, Fujioka Y, Nagashima K. "HHV8-negative primary effusion lymphoma of the peritoneal cavity presenting with a distinct immunohistochemical phenotype." Pathol Int 51: 293-300, 2001*
• Ohwatari R, Iwabuchi K, Iwabuchi C, Morohashi T, Sawa H, Hioki K, Kobayash i K, Fukuda S, Inuyama Y, and Onoe K. "Developmental and functional analyses of CD8+ NK1.1+ T cells in the class I restricted TCR transgenic mice." Cell Immunol. 213: 24-33, 2001*

  2001 [Not refereed][Not invited]
  
• Nagashima T, Mori M, Fujimoto M, Nunomura M, Sakurai Y, Okada Y, Itoh T, Sawa H, Stan AC, Nagashima K: "Adult T-cell lymphoma involving the leptomeninges associated with a spinal cord schwannoma." Neuropathology 21, 229-235, 2001

  2001 [Not refereed][Not invited]
  
• Furuta Y, Ohtani F, Sawa H, Fukuda S, Inuyama Y: "Quantitation of varicella-zoster virus DNA in patients with Ramsay Hunt syndrome and Zoster Sine Herpete." J Clin Microbiol 39: 2856-2859, 2001.*

  2001 [Not refereed][Not invited]
  
• Kamimura E, UenoY, Tanaka S, Sawa H, Yoshioka M, Ueno K, Ishikawa R, Inoue T, Li X, Koyama T, Nagashima K: "New rat model for attention deficit hyperactive disorder (ADHD)." Comp Med 51: 245-251, 2001*

  2001 [Not refereed][Not invited]
  
• Ohnishi J, Ohnishi E, Hirano W, Nakane D, Matsui H, Kimura A, Sawa H, Nakayama K, Shibuya H, Nagashima K, Takahashi T: Cloning and characterization of a rat ortholog of MMP-23 (matrix metalloproteinase-23), a unique type of membrane-anchored matrix met・・・

  2001 [Not refereed][Not invited]
   

  Ohnishi J, Ohnishi E, Hirano W, Nakane D, Matsui H, Kimura A, Sawa H, Nakayama K, Shibuya H, Nagashima K, Takahashi T: Cloning and characterization of a rat ortholog of MMP-23 (matrix metalloproteinase-23), a unique type of membrane-anchored matrix metalloproteinase and conditioned switching of its expression during the ovarian follicular development. Mol Endocrinol 15, 747-764, 2001*
• Hayashi H, Endo S, Suzuki S, Tanaka S, Sawa H, Ozaki Y, Sawamura Y, Nagashima K: "JC virus large T protein transforms rodent cells but is not involved in human medulloblastoma." Neuropathology 21: 129-137, 2001*

  2001 [Not refereed][Not invited]
  
• Xie Z, Koyama T, Suzuki J, Fujii Y, Togashi H, Sawa H, Nagashima K: Coronary reperfusion following ischemia. Different expression of Bcl-2 and Bax proteins, and cardiomyocyte apoptosis. Jpn Heart J 42: 759-770, 2001*

  2001 [Not refereed][Not invited]
  
• Impression of the 3rd International Symposium of Neurovirology (ISNV) Meeting 2000

  Sawa Hirofumi

  Uirusu 日本ウイルス学会 51 (1) 113 - 118 0042-6857 2001
• Cloning and characterization of a rat ortholog of MMP-23 (matrix metalloproteinase-23), a unique type of membrane-anchored matrix metalloproteinase and conditioned switching of its expression during the ovarian follicular development

  J. Ohnishi, E. Ohnishi, M. Jin, W. Hirano, D. Nakane, H. Matsui, A. Kimura, H. Sawa, K. Nakayama, H. Shibuya, K. Nagashima, T. Takahashi

  Molecular Endocrinology 15 (5) 747 - 764 0888-8809 2001 [Refereed][Not invited]
   

  In our attempt to study the role of matrix metalloproteinases (MMPs) in the process of mammalian ovulation, we isolated a rat ortholog of the recently reported human MMP-23 from gonadotropin-primed immature rat ovaries. Transient expression of epitope-tagged rat and human MMP-23 in COS-1 cells revealed that they were synthesized as a membrane-anchored glycoprotein with type II topology. Indirect immunofluorescent analysis showed that subcellular localization of MMP-23 was predominantly in the perinuclear regions. The transfected human MMP-23 protein was processed endogenously to the soluble form in COS-1 cells. However, cotransfection of MMP-23 with the mouse furin cDNA did not enhance this processing, indicating that furin may not be involved in this event. Notably, in situ hybridization analysis revealed a dramatic switching of MMP-23 mRNA localization from granulosa cells to theca-externa/fibroblasts and ovarian surface epithelium during the follicular development. In serum-free primary culture of rat granulosa cells, a drastic diminution of MMP-23 mRNA expression was observed in response to FSH action between 24 h and 48 h of culture. The observed effect of FSH on MMP-23 expression was mimicked by treatment of granulosa cells with forskolin or 8-bromo (Br)-cAMP. In contrast, MMP-23 mRNA levels increased in theca-interstitial cells regardless of the presence of LH in the culture. However, treatment of theca-interstitial cells with forskolin or 8-Br-cAMP markedly reduced the expression of MMP-23 with a concomitant increase in progesterone production. These results indicate that the MMP-23 gene is spatially and temporally regulated in a cell type-specific manner in ovary via the cAMP signaling pathway.
• Deficiency of phospholipase C-γ1 impairs renal development and hematopoiesis

  Masatoshi Shirane, Hirofumi Sawa, Yoshiyasu Kobayashi, Toru Nakano, Kenji Kitajima, Yoichi Shinkai, Kazuo Nagashima, Izumi Negishi

  Development 128 (24) 5173 - 5180 0950-1991 2001 [Refereed][Not invited]
   

  Phospholipase C-γ1 (PLC-γ1) is involved in a variety of intracellular signaling via many growth factor receptors and T-cell receptor. To explore the role of PLC-γ1 in vivo, we generated the PLC-γ1-deficient (plc-γ1-/-) mice, which died of growth retardation at embryonic day 8.5-9.5 in utero. Therefore, we examined plc-γ1-/- chimeric mice generated with plc-γ1-/- embryonic stem (ES) cells for further study. Pathologically, plc-γ1-/- chimeras showed multicystic kidney due to severe renal dysplasia and renal tube dilation. Flow cytometric analysis and glucose phosphate isomerase assay revealed very few hematopoietic cells derived from the plc-γ1-/- ES cells in the mutant chimeras. However, differentiation of plc-γ1-/- ES cells into erythrocytes and monocytes/macrophages in vitro was observed to a lesser extent compared with control wild-type ES cells. These data suggest that PLC-γ1 plays an essential role in the renal development and hematopoiesis in vivo.
• New rat model for attention deficit hyperactive disorder (ADHD)

  E. Kamimura, Y. Ueno, S. Tanaka, H. Sawa, M. Yoshioka, K. I. Ueno, T. Inoue, X. Li, T. Koyama, R. Ishikawa, K. Nagashima

  Comparative Medicine 51 (3) 245 - 251 0023-6764 2001 [Refereed][Not invited]
   

  Purpose: In a strain of the Long-Evans Cinnamon (LEC) rats, we found spontaneously hyperactive animals designated as "wiggling," and established a congenic wiggling (Wig) rat by transferring the gene from the LEC to the Wistar King-Aptekman/Hokkaido (WKAH) strain. We evaluated the feasibility of the Wig rat for an animal model of human attention deficit hyperactive disorder (ADHD). Methods: Mode of inheritance was examined by use of linkage analyses. Motor activity, behavior, and working memory were assessed by use of electric digital counters, open field test, and Y-maze and water-maze tests. Results: The abnormal behavior, including hyperactivity, was transmitted in autosomal recessive mode. Diurnal and nocturnal motor activity of 12- to 14-week-old Wig rats was markedly higher than that of controls, and this hyperactivity was more prominent during nighttime than daytime. Ambulation in the open-field test was significantly increased in Wig rats, but rearing was decreased in Wig rats, compared with controls. Results of the Y-maze tests indicated that spontaneous alternation behavior was significantly impaired in Wig rats, although there was no difference in the total arm entries. The water-maze test could not be performed because, when exposed to water, Wig rats panicked and almost drowned. Conclusions: Wig rats are hyperactive and have impaired working memory and impulsive behavior, as assessed by the motor activity and open-field tests and the Y-maze test, and these abnormalities are transmitted by a single gene with Mendelian pattern. Wig rats represent an excellent animal model of human ADHD.
• Infection with JC virus and possible dysplastic ganglion-like transformation of the cerebral cortical neurons in a case of progressive multifocal leukoencephalopathy

  M Shintaku, R Matsumoto, H Sawa, K Nagashima

  JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY 59 (10) 921 - 929 0022-3069 2000/10 [Not refereed][Not invited]
   

  Infection of the cerebral cortical neurons with JC virus (JCV) with possible dysplastic ganglion-like alteration of the infected neurons found in a case of progressive multifocal leukoencephalopathy (PML) is described. The patient was a 21-year-old man with common variable immunodeficiency who died of PML after a 9-month clinical course. Ar autopsy, the white matter of the cerebrum, brainstem, cerebellum, and spinal cord exhibited extensive demyelination and necrosis. Numerous inclusion-bearing oligodendrocytes and bizarre astrocytes were found. In the occipital and temporal cortex, thick bandlike aggregates of dysplastic ganglion-like cells (DGLCs) were found. These DGLCs showed immunohistochemical properties of neurons, and nuclei of some DGLCs were immunoreactive for large T antigen of SV40/JCV and p53, but not for capsid protein JCV VP1. In situ hybridization for mRNA of JCV large T antigen revealed positive signals in the nuclei of some DGLCs. These results indicate that JCV infected neurons and it is suggested that binding of the large T antigen with cellular proteins could have resulted in the dysplastic, ganglion cell-like change of the infected neurons, although the possibility that the aggregates of DGLCs represent a pre-existent malformative lesion of the cortex cannot be excluded completely.
• CalDAG-GEFIII activation of Ras, R-Ras, and Rap1

  S. Yamashita, N. Mochizuki, Y. Ohba, M. Tobiume, Y. Okada, H. Sawa, K. Nagashima, M. Matsuda

  Journal of Biological Chemistry 275 (33) 25488 - 25493 0021-9258 2000/08/18 [Not refereed][Not invited]
   

  We characterized a novel guanine nucleotide exchange factor (GEF) for Ras family G proteins that is highly homologous to CalDAG-GEFI, a GEF for Rap1 and R-Ras, and to RasGRP/CalDAG-GEFH, a GEF for Ras and R-Ras. This novel GEF, referred to as CalDAGGEFIII, increased the GTP/GDP ratio of Ha-Ras, R-Ras, and Rap1 in 293T cells. CalDAG-GEFIII promoted the guanine nucleotide exchange of Ha-Ras, R-Ras, and Rap1 in vitro also, indicating that CalDAG-GEFIII exhibited the widest substrate specificity among the known GEFs for Ras family G proteins. Expression of CalDAGGEFIII was detected in the glial cells of the brain and the glomerular mesangial cells of the kidney by in situ hybridization. CalDAG-GEFIII activated ERK/MAPK most efficiently, followed by CalDAG-GEFII and CalDAG-GEFI in 293T cells. JNK activation was most prominent in cells expressing CalDAG-GEFII, followed by CalDAG-GEFIII and CalDAG-GEFI. Expression of CalDAG-GEFIII induced neuronal differentiation of PC12 cells and anchorage-independent growth of Rat1A cells less efficiently than did CalDAG-GEFII. Thus, coactivation of Rap1 by CalDAG-GEFIII apparently attenuated Ras-MAPK-dependent neuronal differentiation and cellular transformation. Altogether, CalDAG-GE-FIII activated a broad range of Ras family G proteins and exhibited a biological activity different from that of either CalDAG-GEFI or CalDAG-GEFII.
• CalDAG-GEFIII activation of Ras, R-Ras, and Rap1

  S Yamashita, N Mochizuki, Y Ohba, M Tobiume, Y Okada, H Sawa, K Nagashima, M Matsuda

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 (33) 25488 - 25493 0021-9258 2000/08 [Refereed][Not invited]
   

  We characterized a novel guanine nucleotide exchange factor (GEF) for Ras family G proteins that is highly homologous to CalDAG-GEFI, a GEF for Rap1 and R-Ras, and to RasGRP/CalDAG-GEFII, a GEF for Res and R-Ras. This novel GEF, referred to as CalDAG-GEFIII, increased the GTP/GDP ratio of Ha-Ras, R-Ras, and Rap1 in 293T cells. CalDAG-GEFIII promoted the guanine nucleotide exchange of Ha-Ras, R-Ras, and Rap1 in vitro also, indicating that CalDAG-GEFIII exhibited the widest substrate specificity among the known GEFs for Res family G proteins. Expression of CalDAG-GEFIII was detected in the glial cells of the brain and the glomerular mesangial cells of the kidney by in. situ hybridization. CalDAG-GEFIII activated ERW MAPK most efficiently, followed by CalDAG-GEFII and CalDAG-GEFI in 293T cells. JNK activation was most prominent in cells expressing CalDAG-GEFII, followed by CalDAG-GEFIII and CaLDAG-GEFI. Expression of CalDAG-GEFIII induced neuronal differentiation of PC12 cells and anchorage-independent growth of Rat1A cells less efficiently than did CalDAG-GEFII. Thus, coactivation of Rap1 by CalDAG-GEFIII apparently attenuated Ras-MAPK-dependent neuronal differentiation and cellular transformation. Altogether, CalDAG-GEFIII activated a broad range of Res family Gr proteins and exhibited a biological activity different from that of either CalDAG-GEFI or CalDAG-GEFII.
• Transcriptional activation of JC virus by human T-lymphotropic virus type I tax protein in human neuronal cell lines

  Y Okada, H Sawa, S Tanaka, A Takada, S Suzuki, H Hasegawa, T Umemura, J Fujisawa, Y Tanaka, WW Hall, K Nagashima

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 (22) 17016 - 17023 0021-9258 2000/06 [Refereed][Not invited]
   

  Polyomavirus JC (JCV) causes the human demyelinating disease, progressive multifocal leukoencephalopathy (PML). The recent demonstration of cases of PML in association with human T-lymphotropic virus type I (HTLV-I) infection prompted us to examine whether the HTLV-I-encoded regulatory protein Tax activates JCV transcription. By employing a dual luciferase assay, we initially found that the expression of Tax activated the transcriptional potential of both early and late promoters of JCV in human neuronal but not in non-neuronal cells. We subsequently analyzed the mechanism of Tax-induced activation of the JCV promoter in neuronal cells with the following results: 1) the JCV promoter that lacks the NF-kappa B-binding motif could not be activated by Tax; 2) the overexpression of I kappa B alpha abolished Tax-induced transcriptional activation of the JCV promoter; 3) a Tax mutant M22) lacking the potential for activation via the NF-kappa B pathway did not activate the JCV promoter. Furthermore, Tax enhances the gene expression of JCV T antigen and VP1. We examined mechanisms of the cell-specific activation of the JCV promoter by Tax. Electrophoretic mobility shift assay demonstrated the presence of Tax-bound protein(s) that were specifically present in non-neuronal cells. This study is the first demonstration of the activation of JCV promoter by HTLV-I Tax in an NF-kappa B-dependent manner.
• Sato-Matsumura KC, Matsumura T, Nakamura H, Sawa H, Nagashima K, Koizumi H: Membranous expression of annexin I is enhanced by calcium and TPA in cultured human keratinocytes. Arch Dermatol Res 292, 496-499, 2000*

  2000 [Not refereed][Not invited]
  
• Hiroi Y, Chen R, Sawa H, Hosoda T, Kudoh S, Kobayashi Y, Aburatani H, Nagashima K, Nagai R, Yazaki Y, Medof ME, Komuro I: Cloning of murine glycosyl phosphatidylinositol anchor attachment protein, GPAA1. Am J Physiol Cell Physiol 279, C205-C212, 2000*

  2000 [Not refereed][Not invited]
  
• Xie Z, Koyama T, Abe K, Fujii Y, Sawa H, Nagashima K: Upregulation of p53 protein in rat heart subjected to a transient occlusion of the coronary artery followed by reperfusion. Jpn J Physiol 50: 159-162, 2000*

  2000 [Not refereed][Not invited]
  
• Membrane expression of annexin I is enhanced by calcium and TPA in cultured human keratinocytes

  K. C. Sato-Matsumura, T. Matsumura, H. Nakamura, H. Sawa, K. Nagashima, H. Koizumi

  Archives of Dermatological Research 292 (10) 496 - 499 0340-3696 2000 [Refereed][Not invited]
   

  Annexin I plays an important role in the process of keratinization as a component of the cornified envelope. To elucidate the function of annexin I in keratinization, we investigated the effects of calcium, epinephrine, hydrocortisone, and 12-O-tetradecanoyl phorbol 13-acetate (TPA) on the expression and localization of annexin I in cultured human keratinocytes. Normal human keratinocytes were cultured in serumfree culture medium (0.15 mM calcium) until 70 % confluence. After incubation with a higher concentration of calcium (1.8 m/M), TPA (100 nM), epinephrine (50 μM), or hydrocortisone (10 μM) for 24 h, the expression of annexin I protein and mRNA was examined using immunofluorescence, Western blot, and Northern blot techniques. Immunofluorescence microscopy showed increased membrane staining of annexin I by calcium, which was inhibited by the addition of epinephrine or hydrocortisone. Western blotting confirmed elevated annexin I on the cell membrane. It was increased in the cell membrane fraction, but not in the cytosol fraction. Interestingly, the mRNA level of annexin I was slightly reduced after incubation with calcium, whereas TPA upregulated both membrane expression and the mRNA level. Secretion of annexin I was increased by TPA but inhibited by calcium. Because calcium and TPA are known to promote keratinization, our data suggest that annexin I expression on the cell membrane is involved in the process of keratinization.
• Upregulation of p53 protein in rat heart subjected to a transient occlusion of the coronary artery followed by reperfusion

  Zhonglin Xie, Tomiyasu Koyama, Kazuhiro Abe, Yukiko Fujii, Hirofumi Sawa, Kazuo Nagashima

  Japanese Journal of Physiology 50 (1) 159 - 162 0021-521X 2000 [Refereed][Not invited]
   

  The accumulation of p53 protein in cardiomyocyte nuclei was immunohistochemically demonstrated by the pronounced staining of p53 antibody in 4 h-reperfused ventricular tissue after a 45-min coronary occlusion. The occurrence of apoptosis in the reperfused ventricular tissue was evidenced by positive TUNEL staining and DNA laddering on agarose gels.
• Cloning of murine glycosyl phosphatidylinositol anchor attachment protein, GPAA1

  Y. Hiroi, R. Chen, H. Sawa, T. Hosoda, S. Kudoh, Y. Kobayashi, H. Aburatani, K. Nagashima, R. Nagai, Y. Yazaki, M. E. Medof, I. Komuro

  American Journal of Physiology - Cell Physiology 279 (1) C205 - C212 0363-6143 2000 [Refereed][Not invited]
   

  Glycosyl phosphatidylinositols (GPIs) are used to anchor many proteins to the cell surface membrane and are utilized in all eukaryotic cells. GPI anchoring units are attached to proteins via a transamidase reaction mediated by a GPI transamidase complex. We isolated one of the components of this complex, mGPAA1 (murine GPI anchor attachment), by the signal sequence trap method, mGPAA1 cDNA is about 2 kb in length and encodes a putative 621 amino acid protein. The mGPAA1 gene has 12 small exons and 11 small introns, mGPAA1 mRNA is ubiquitously expressed in mammalian cells, and in situ hybridization analysis revealed that it is abundant in the choroid plexus, skeletal muscle, osteoblasts of rib, and occipital bone in mouse embryos. Its expression levels and transamidation efficiency decreased with differentiation of embryonic stem cells. The 3T3 cell lines expressing antisense mGPAA1 failed to express GPI-anchored proteins on the cell surface membrane.
• Melanotic peritoneal sarcomatosis originating from clear cell sarcoma

  Y Ohba, H Suzuki, H Hiraga, T Ito, H Sawa, M Nagai, S Satoh, H Iwaki, K Nagashima

  PATHOLOGY INTERNATIONAL 49 (7) 653 - 657 1320-5463 1999/07 [Refereed][Not invited]
   

  Peritoneal sarcomatosis was found in a 53-year-old male who had a history of resection of clear cell sarcoma (CCS) of the right wrist 7 years previously. Both the previous wrist tumor and the peritoneal disseminants consisted of small, spindle-shaped cells occasionally containing melanophages. Histologic features, histochemical demonstration of argentaffin granules, immunohistochemical reaction with HMB 45, and the demonstration of a chimeric transcript of EWS-ATF-1 established the diagnosis of CCS in the peritoneal tumors. As far as we are aware, this is the first case of a peritoneal sarcomatosis associated with CCS.
• Pertussis toxin-sensitive signal controls the trafficking of thymocytes across the corticomedullary junction in the thymus

  Gen Suzuki, Hirofumi Sawa, Yoshiyasu Kobayashi, Yukiko Nakata, Ken-Ichi Nakagawa, Akiko Uzawa, Hisako Sakiyama, Shizuko Kakinuma, Kazuya Iwabuchi, Kazuo Nagashima

  Journal of Immunology 162 (10) 5981 - 5985 0022-1767 1999/05/15 [Refereed][Not invited]
   

  We investigated a role of chemokines in thymocyte trafficking. Genes encoding stromal cell-derived factor-1 and its receptor CXCR4 were detected in the cortex by in situ hybridization. Early immigrant cells did not express CXCR4, whereas their descendant CD44+CD25+CD4-CD8- cells did. CXCR4 expression was down-modulated when CD4+CD8+ double-positive cells became CD4+CD8- or CD4-CD8+ single-positive (SP) cells. Positively selected CD69+ CD3(intermediate) cells gained CCR4, of which ligand, thymus activation-regulated chemokine, was expressed in the medulla. At the next developmental stage, CD69-CD3(high) cells lost CCR4 but gained CCR7. These results suggest that thymocytes use different chemokines along with their development. Blockade of chemokine receptor-mediated signaling by pertussis toxin perturbed the normal distribution of SP cells and resulted in the accumulation of SP cells in the cortex. Thus, a pertussis toxin-sensitive event controls the trafficking of SP cells across the corticomedullary junction.
• JCウイルスcapsid proteinの機能的領域に対する抗体を用いた免疫組織学的検索と感染抑制実験

  西原 広史, 小林 希, 佐藤 真実, 宍戸 由紀子, 岡田 由紀, 田中 伸哉, 澤 洋文, 古林 与志安, 長嶋 和郎

  日本病理学会会誌 (一社)日本病理学会 88 (1) 333 - 333 0300-9181 1999/03 [Refereed][Not invited]
  
• Pertussis toxin-sensitive signal controls the trafficking of′ thymocytes across the corticomedullary junction in the′ thymus.

  Suzuki G., Sawa H., Kobayashi Y., Nakata Y., Nakagawa ′ K., Uzawa A., Sakiyama H., Kakinuma S., Iwabuchi K., ′ Nagashima K.

  Collected papers from Institute for Genetic Medicine Hokkaido University 北海道大学 1 276 - 280 1346-3837 1999 [Not refereed][Not invited]
   

  Suzuki G, Sawa H, Kobayashi Y, Nakata Y, Nakagawa Ki, Uzawa A, Sakiyama H, Kakinuma S, Iwabuchi K, Nagashima K: Pertussis toxin-sensitive signal controls the trafficking of thymocytes across corticomedullary junction in the thymus. J Immunol 162: 5981-5985, 1999*
• Ohba Y, Suzuki H, Hiraga H, Ito T, Sawa H, Nagai M, Satoh S, Iwaki H, Nagashima K. Melanotic peritoneal sarcomatosis originating from clear cell sarcoma. Pathol Int 49: 653-657, 1999*

  1999 [Not refereed][Not invited]
  
• Calbindin D immunoreactivity and chronic lesions of rat cerebella in methylmercury chloride intoxication

  Y Kobayashi, H Sawa, M Watanabe, H Furuoka, T Matsui, K Nagashima

  NEUROPATHOLOGY 18 (4) 402 - 407 0919-6544 1998/12 [Refereed][Not invited]
   

  This report deals with the immunoreactivity for calbindin D, an intracellular calcium-binding protein, and chronic lesions of rat cerebella in methylmercury chloride (MMC) intoxication. The rats administered MMC showed clinical signs of spasticity on walking as well as hind limb paralysis at around day 19; these developed to moribund by day 26, Pathologic examination of the moribund animals revealed selective apoptotic neuronal deaths of the granule cells. Even in these affected areas, calbindin D, which indirectly reflects Purkinje cell function, was well demonstrated in Purkinje cell dendrites and cytoplasm as well as in unaffected areas. These results suggest that the Purkinje cells of the MMC administered rats were functionally preserved in spite of granule cell deaths. Pathologic examination of rats recovered from acute MMC intoxication and maintained for 3 months without MMC administration revealed severe loss of granule cells with calcium salt deposition. Purkinje cells were again well preserved. Furthermore, a large number of the remaining granule cells were stained positively using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end-labeling (TUNEL) method, which showed an ongoing apoptotic process. These results indicate that the involvement of the cerebellar granule cell was in progression for at least 3 months after MMC intoxication with calcium salt deposition.
• CXCR4の発現と胸腺細胞分化

  中川 憲一, 中田 有紀子, 鵜沢 玲子, 澤 洋文, 古林 与志安, 長嶋 和郎, 岩渕 和也, 鈴木 元

  日本臨床免疫学会会誌 日本臨床免疫学会 (26回抄録集) 266 - 266 0911-4300 1998/10 [Refereed][Not invited]
  
• Augmentation of arterial endothelial cell expression of the plasminogen activator inhibitor type-1 (PAI-1) gene by proinsulin and insulin in vivo

  TK Nordt, H Sawa, S Fujii, C Bode, BE Sobel

  JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 30 (8) 1535 - 1543 0022-2828 1998/08 [Not refereed][Not invited]
   

  Diabetes mellitus is associated with an increased incidence and greater severity of primary atherosclerosis as well as restenosis after angioplasty for reasons not yet clear. We have shown previously that insulin and proinsulin-typically elevated in blood in patients with type II diabetes-increase plasma activity of plasminogen activator inhibitor type 1 (PAI-1) in vivo. Others have demonstrated that increased PAI-1 activity is associated with coronary heart disease. Accordingly the present study was performed to identify sites of increased expression of the PAI-1 gene within the Vessel wall. Equimolar concentrations of insulin, proinsulin, or vehicle alone as a control, were administered intravenously over Ih to conscious rabbits that were kept euglycemic throughout by the use of glucose clamping. Within 3 h plasma PAI-1 activity increased from 1.15 +/- 1.34 to 11.33 +/- 4.30 AU/ml with insulin (mean +/- S.D., P = 0.015) and from 2.83 +/- 0.74 to 15.43 +/- 4.70 AU/ml with proinsulin (P = 0.035). This was found to be in contrast to the controls where the increase in plasma PAI-1 activity was of lesser degree (2.43 +/- 1.86 to 6.80 +/- 1.10 AU/ml, P = N.S., n = 4 each). As judged from the results of in situ hybridization; the site of prominent aortic expression of the PAI-1 gene was the endothelium, Furthermore, expression increased further in this site after administration of insulin or proinsulin. As judged from results of immunohistochemistry, PAI-I protein in the aorta was also prominent in endothelium. These results suggest that "hyper(pro)insulinemia", increases PAI-1 not only in blood but also in arterial endothelium. Thus, attenuation of vasculopathy and especially of restenosis after angioplasty in type II diabetes may be possible with somatic gene therapy targeting PAI-I expression in endothelial cells. (C) 1998 Academic Press
• Augmentation of arterial endothelial cell expression of the plasminogen activator inhibitor type-1 (PAI-1) gene by proinsulin and insulin in vivo

  TK Nordt, H Sawa, S Fujii, C Bode, BE Sobel

  JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 30 (8) 1535 - 1543 0022-2828 1998/08 [Not refereed][Not invited]
   

  Diabetes mellitus is associated with an increased incidence and greater severity of primary atherosclerosis as well as restenosis after angioplasty for reasons not yet clear. We have shown previously that insulin and proinsulin-typically elevated in blood in patients with type II diabetes-increase plasma activity of plasminogen activator inhibitor type 1 (PAI-1) in vivo. Others have demonstrated that increased PAI-1 activity is associated with coronary heart disease. Accordingly the present study was performed to identify sites of increased expression of the PAI-1 gene within the Vessel wall. Equimolar concentrations of insulin, proinsulin, or vehicle alone as a control, were administered intravenously over Ih to conscious rabbits that were kept euglycemic throughout by the use of glucose clamping. Within 3 h plasma PAI-1 activity increased from 1.15 +/- 1.34 to 11.33 +/- 4.30 AU/ml with insulin (mean +/- S.D., P = 0.015) and from 2.83 +/- 0.74 to 15.43 +/- 4.70 AU/ml with proinsulin (P = 0.035). This was found to be in contrast to the controls where the increase in plasma PAI-1 activity was of lesser degree (2.43 +/- 1.86 to 6.80 +/- 1.10 AU/ml, P = N.S., n = 4 each). As judged from the results of in situ hybridization; the site of prominent aortic expression of the PAI-1 gene was the endothelium, Furthermore, expression increased further in this site after administration of insulin or proinsulin. As judged from results of immunohistochemistry, PAI-I protein in the aorta was also prominent in endothelium. These results suggest that "hyper(pro)insulinemia", increases PAI-1 not only in blood but also in arterial endothelium. Thus, attenuation of vasculopathy and especially of restenosis after angioplasty in type II diabetes may be possible with somatic gene therapy targeting PAI-I expression in endothelial cells. (C) 1998 Academic Press
• Diagnosis of synovial sarcoma with the reverse transcriptase-polymerase chain reaction: Analyses of 84 soft tissue and bone tumors

  H Hiraga, T Nojima, S Abe, H Sawa, K Yamashiro, S Yamawaki, K Kaneda, K Nagashima

  DIAGNOSTIC MOLECULAR PATHOLOGY 7 (2) 102 - 110 1052-9551 1998/04 [Refereed][Not invited]
   

  The chimeric transcript SYT-SSX is generated as a result of reciprocal translocation t(X;18), which is the primary cytogenetic abnormality found in, and appears to be specific for, synovial sarcoma. We performed a reverse transcriptase-polymerase chain reaction (RT-PCR) for SYT-SSX transcripts in a series of 84 tumors (61 soft tissue tumors and 23 bone tumors), including a variety of histologic types, to assess its usefulness in molecular diagnosis. Ten synovial sarcomas, three tumors initially unclassified, and one malignant peripheral nerve sheath tumor contained the chimeric transcripts. A review of the original slides and additional examination showed that a diagnosis of synovial sarcoma was appropriate for these cases. Additionally, in situ hybridization with an SSX1 probe indicated that the chimeric transcripts exist not only in the cells of special components but also in cells showing a variety of histologic patterns. Therefore, RT-PCR can be considered a useful molecular biological technique that can provide objective evidence for diagnosis of synovial sarcoma. Northern blot analysis with an SSX1 probe also detected chimeric SYT-SSX transcripts in the synovial sarcoma cases. The additional smaller bands, however, were also detected in six peripheral primitive neuroectodermal tumors (pPNETs) and one embryonal rhabdomyosarcoma. In five of these pPNETs, other bands ranging in size from 2.0 to 2.2 kb were also found, and it seems possible that these bands might represent novel karyotypic aberrations and/or splicing variants of SSX.
• Hiraga H, Nojima T, Abe S, Sawa H, Yamashiro K, Yamawaki S, Kaneda K, Nagashima K: Diagnosis of synovial sarcoma with the reverse transcriptase-polymerase chain reaction: analyses of 84 soft tissue and bone tumors. Diagn Mol Pathol 7: 102-110, (1998)*

  1998 [Not refereed][Not invited]
  
• Sasaki H, Kojima H, Yabe I, Tashiro K, Hamada T, Sawa H, Hiraga H, Nagashima K: Neuropathological and molecular studies of spinocerebellar ataxia type 6 (SCA6). Acta Neuropathology (Berl) 95: 199-204, 1998*

  1998 [Not refereed][Not invited]
  
• Kobayashi Y, Sawa H, Akagi H, Itakura C, Fujioka Y, Nagashima K: Distributional pattern of apoptotic cells in rat cerebellar vermis experimentally induced by methylmercury intoxication. Neuropathology 18: 33-37, 1998*

  1998 [Not refereed][Not invited]
  
• Kobayashi Y, Sawa H, Watanabe M, Furuoka H, Matsui T, Nagashima K: Calbindin D immunoreactivity and chronic lesions of rat cerebella in methylmercury chrolide intoxication. Neuropathology 18: 402-407, 1998*

  1998 [Not refereed][Not invited]
  
• Neuropathological and molecular studies of spinocerebellar ataxia type 6 (SCA6)

  Hidenao Sasaki, Hideaki Kojima, Ichiro Yabe, Kunio Tashiro, Takeshi Hamada, Hirofumi Sawa, Hiroaki Hiraga, Kazuo Nagashima

  Acta Neuropathologica 95 (2) 199 - 204 0001-6322 1998 [Refereed][Not invited]
   

  SCA6 is an autosomal dominant spinocerebellar ataxia (SCA) caused by a small CAG repeat expansion of the gene encoding an α-1A-voltage-dependent Ca channel gene subunit on chromosome 19p13. A Japanese woman with SCA6, with a 7-year history of progressive pure cerebellar ataxia, died of malignant lymphoma. Systematic neuropathological examination showed that neuronal degeneration was confined to the cerebellar Purkinje cells and, to a lesser degree, the granular cells, without any involvement of other central nervous system structures. Such pathological selectivity correlates with the localized expression of the responsible gene, and coincides with the neurological manifestation. These findings might contribute to establishing the phenotype of the SCA6 via comparison with other dominant ataxias.
• Nordt TK, Sawa H, Fujii S, Sobel BE: Hyperinsulinemia increases plasma activity of PAI-1 in vivo independently of an acute phase reaction. Fibrinolysis Proteolysis 11 : 51-54, 1997

  1997 [Not refereed][Not invited]
  
• Takahashi HR, Sawa H, Takada A, Kitabatake A, Nagashima K: Expression of beta-amyloid precursor protein mRNAs in the vascular smooth muscle cell of human brain. Neuropathology 17: 11-14, 1997*

  1997 [Not refereed][Not invited]
  
• Localization of annexin I (lipocortin I, p35) mRNA in normal and diseased human skin by in situ hybridization

  KC SatoMatsumura, H Koizumi, T Matsumura, A Ohkawara, T Takasu, Y Furuta, H Sawa, K Nagashima

  ARCHIVES OF DERMATOLOGICAL RESEARCH 288 (10) 565 - 569 0340-3696 1996/09 [Refereed][Not invited]
   

  Annexin I is a calcium- and phospholipid-binding protein that is involved in the regulation of cellular differentiation. The aim of the present study was to determine the localization of annexin I mRNA expression in normal and diseased human skin, In situ hybridization with a specific digoxigenin-labelled RNA probe was used throughout, We detected no annexin I mRNA signals in basal and suprabasal cells of normal epidermis, but positive signals were evident in the sudoriferous ducts, Annexin I mRNA expression was detected in the keratinizing squamous cells in keratotic type seborrhoeic keratosis and in keratinocytes at the periphery of the horn pearl in well-differentiated squamous cell carcinoma, Positive signals were also seen at the border between involved and noninvolved skin in psoriasis vulgaris and in dyskeratotic epidermal keratinocytes in keratosis follicularis Darier. By contrast, no annexin I mRNA signals were detected in tumour cells in basal cell carcinoma. The present results suggest that annexin I expression is related to, and may play a role in, keratinization disorders.
• Shinohara K, Shinohara T, Mochizuki N, Mochizuki Y, Sawa H, Kohya T, Fujita M, Fujioka Y, Kitabatake A, Nagashima K: Expression of vascular endothelial growth factor associated with human myocardial infarction. Heart Vessels 11: 113-122, 1996*

  1996 [Not refereed][Not invited]
  
• Matsumura SCK, Koizumi H, Matsumura T, Ohkawara A, Takasu T, Furuta Y, Sawa H, Nagashima K: Localization of annexin I (lipocortin I, p35) mRNA in normal and diseased human skin by in situ hybridization. Arch Dermatol Res 288:565-569, 1996*

  1996 [Not refereed][Not invited]
  
• Takahashi HR, Sawa H, Kuroda S, Saito H, Fujita M, Fujioka Y, Fukatsu R, Nagashima K: Pathologic processes leading to cerebral hemorrhage in amyloid angiopathy. Neuropathology 16: 99-105, 1996*

  1996 [Not refereed][Not invited]
  
• Okada H, Kawaguchi H, Kudo T, Sawa H, Okamoto H, Watanabe S, Urasawa K, Murakami T, Kitabatake A: Alteration of extracellular matrix in dilated cardiomyopathic hamster heart. Mol Cell Biochem 156: 9-15, 1996*

  1996 [Not refereed][Not invited]
  
• Sawa H, Lundgren CH, Brown SL, Fujii S: Dependence of human vascular cell surface proteolysis on expression of the urokinase receptor. J Thromb Thromboly 3: 331-336, 1996*

  1996 [Not refereed][Not invited]
  
• Alteration of extracellular matrix in dilated cardiomyopathic hamster heart

  Hitoshi Okada, Hideaki Kawaguchi, Toshiyuki Kudo, Hirofumi Sawa, Hiroshi Okamoto, Shouzi Watanabe, Kazushi Urasawa, Takeshi Murakami, Akira Kitabatake

  Molecular and Cellular Biochemistry 156 (1) 9 - 15 0300-8177 1996 [Refereed][Not invited]
   

  The purpose of this study was to characterize the collagen in hereditary dilated cardiomyopathic hamster hearts, and to examine the participation of the collagen in the occurrence and progression of cardiomyopathy. BIO 53.58 hamsters (5, 10, 20 weeks old) were used as the model of dilated cardiomyopathy. Flb hamsters were used as controls. The collagen content was almost constant at any age in the Flb hamsters, but increased with age in BIO 53.58 hamsters. Type III collagen increased significantly in BIO 53.58 hamsters at 10 weeks. The acetic acid solubility of collagen decreased in BIO 53.58 hamsters as the fibrosis progressed, but was unchanged in controls. Reducible crosslinks showed a tendency to decrease progressively in BIO 53.58 hamsters. There were no differences between Flb and BIO 53.58 hamsters at 5 weeks, but its expression in BIO 53.58 hamsters at 10 and 20 weeks of age increased compared to Flb controls. These findings indicate that in the early phase of cardiomyopathy the extracellular matrix of the myocardium is rich in type III collagen. In the later phase, the matrix resembles that of hard tissues, whose collagen is mainly of type I collagen and is insoluble. These data suggest that the increased collagen synthesis may impair the cardiac function in the development of cardiomyopathy.
• EXPRESSION OF INTERCELLULAR AND VASCULAR CELL-ADHESION MOLECULES AND CLASS-II MAJOR HISTOCOMPATIBILITY ANTIGENS IN HUMAN LUNGS - LACK OF INFLUENCE BY CONDITIONS OF ORGAN PRESERVATION

  S HASEGAWA, JH RITTER, GA PATTERSON, DM OCKNER, H SAWA, T MOHANAKUMAR, JD COOPER, MR WICK

  JOURNAL OF HEART AND LUNG TRANSPLANTATION 14 (5) 897 - 905 1053-2498 1995/09 [Refereed][Not invited]
   

  Background: The expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and class II major histocompatibility complex antigens was studied in control lung tissue and preserved human donor lungs. The three controls were represented by wedge biopsy specimens taken from non-neoplastic lung surrounding bronchogenic carcinomas. Methods: Nine lungs were harvested from six brain-dead donors, flushed with Euro-Collins solution or low potassium-dextran-glucose solution, and stored at 1 degrees or 10 degrees C. Samples of the latter organs were taken at the time of surgical harvest (baseline) and after 2, 12, 24, and 48 hours of preservation time. Immunostains with monoclonal antibodies against intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and class II major histocompatibility complex molecules were performed on all samples, and the relative presence of these determinants was evaluated. Results: In both the controls and preserved lungs, intercellular adhesion molecule-1 expression was intense in the septal capillary endothelium and alveolar pneumocytes, but essentially absent in bronchial epithelium. Vascular cell adhesion molecule-1 was moderately to strongly labeled in the endothelia of large and small blood vessels of all types, and it was not seen in other cell types. Class II major histocompatibility complex antigens were variably observed in pulmonary epithelial cells, but they were not expressed by endothelia. There appeared to be no significant difference in the immunohistologic density of intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 immunostaining in allografts at the specified time points of preservation; this conclusion was confirmed by Western blot analysis. Similar findings pertained to staining results for human leukocyte DR antigens. There was likewise no significant difference in the expression of the three analytes when donor lungs perfused with Euro-Collins solution versus low potassium-dextran-glucose solution were compared; this was also true of organs preserved at 1 degrees C versus 10 degrees C. Conclusions: These results suggest that, in the immediate postharvest period, modulations in the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, or class II major histocompatibility complex antigens in pulmonary allografts are not attributable to the influences of preservation conditions.
• Mochizuki EY, Mochizuki N, Sawa H, Takada A, Okamoto H, Kawaguchi H, Nagashima K, Kitabatake A: Expression of renin and angiotensin converting enzyme in human hearts. Heart Vessels 10: 285-293, 1995*

  1995 [Not refereed][Not invited]
  
• Nordt TK, Sawa H, Fujii S, Sobel BE: Induction of plasminogen activator inhibitor type-1 (PAI-1) by proinsulin and insulin in vivo. Circulation 91: 764-770, 1995*

  1995 [Not refereed][Not invited]
  
• Motoyama N, Wang F, Roth KA, Sawa H, Nakayama Ki, Nakayama K, Negishi I, Senju S, Zhang Q, Fujii S, Loh DY: Massiva cell death of post-mitotic immature lymphocytes in Bcl-x-deficient mice. Science 267: 1506-1510, 1995*

  1995 [Not refereed][Not invited]
  
• Expression of renin and angiotensin-converting enzyme in human hearts

  Y EndoMochizuki, N Mochizuki, H Sawa, A Takada, H Okamoto, H Kawaguchi, K Nagashima, A Kitabatake

  HEART AND VESSELS 10 (6) 285 - 293 0910-8327 1995 [Refereed][Not invited]
   

  To understand the significance of the tissue renin-angiotensin system in the heart, we examined the expression of renin and angiotensin-converting enzyme (ACE) in autopsied human hearts. Samples were taken from organs obtained at autopsy from 15 patients without heart disease and 3 patients with heart disease (old myocardial infarctions, acute myocardial infarctions, and hypertrophic cardiomyopathy). We examined the expression of renin and ACE mRNA by using the reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR showed the expression of renin in the right atria in all patients. However, expression of renin mRNA in the left ventricles was not found in any of the 15 hearts without heart disease. In contrast, renin mRNA was detected in the left ventricles in hearts with heart disease. ACE mRNA was detected in both the atria and the ventricles in normal hearts, and its expression did not alter in diseased hearts. These findings suggest that renin mRNA is expressed mainly in the right atria in normal hearts, but that its expression in the left ventricle can be activated in some pathological conditions.
• Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice

  Noboru Motoyama, Fanping Wang, Kevin A. Roth, Hirofumi Sawa, Kei Ichi Nakayama, Keiko Nakayama, Izumi Negishi, Satoru Senju, Qing Zhang, Satoshi Fujii, Dennis Y. Loh

  Science 267 1506 - 1510 0036-8075 1995/01/01 [Not refereed][Not invited]
   

  Bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x. The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic cell death was evident in postmitotic immature neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic. Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished. The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability of immature cells during the development of the nervous and hematopoietic systems.
• INCREASED INTRAMURAL EXPRESSION OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 AFTER BALLOON INJURY - A POTENTIAL PROGENITOR OF RESTENOSIS

  H SAWA, C LUNDGREN, BE SOBEL, S FUJII

  JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY 24 (7) 1742 - 1748 0735-1097 1994/12 [Not refereed][Not invited]
   

  Objectives. This study was performed to determine whether altered gene expression of plasminogen activator inhibitor type 1 (PAT-I) occurs within the arterial wall after experimentally induced balloon injury. Background. PAI-1, known to inhibit fibrinolysis in the circulation and to be present within atherosclerotic vessels, may influence proteolysis in the arterial wall and neointimal formation after angioplasty. Methods. In rabbit carotid arteries subjected to balloon injury, both PAI-1 gene and protein expression were assayed sequentially with the use of Northern blotting, in situ hybridization and immunohistochemical studies. Results. In uninjured, normal vessels PAI-1 messenger ribonu cleic acid (mRNA) was not detectable by Northern blotting or in situ hybridization. However, injury was followed within 3 h by increases in PAI-1 mRNA (3.2 kb) of 5.9 fold compared with that in contralateral control carotid arteries (Northern blots). PAI-I mRNA was detectable by in situ hybridization early after injury first in adventitia; after 24 h it was particularly prominent in the media. From 1 to 4 weeks after injury it was consistently detectable and was localized in neointimal vascular smooth muscle and endothelial cells at a time when neointimal thickening was marked. Cells of both types exhibited PAI-1 protein detected immunohistochemically. In vessels maintained in organ culture after balloon injury in vivo, sustained increases in PAI-1 activity appeared in conditioned media as well. Conclusions. Our results indicate that balloon injury simulating angioplasty in patients induces intramural expression of PAI-1 in vascular smooth muscle and endothelial cells. The decreased cell surface fibrinolytic activity likely to result from the increased PAI-1 expression may initiate or exacerbate mural thrombosis. Accordingly, excessive stimulation with clot-associated mitogens may stimulate vascular smooth muscle cell proliferation, which, coupled with increased accumulation of extracellular matrix attributable to decreased plasmin-mediated degradation, may contribute to restenosis.
• MODULATION OF EXPRESSION OF MONOCYTE/MACROPHAGE PLASMINOGEN-ACTIVATOR ACTIVITY AND ITS IMPLICATIONS FOR ATTENUATION OF VASCULOPATHY

  CH LUNDGREN, H SAWA, BE SOBEL, S FUJII

  CIRCULATION 90 (4) 1927 - 1934 0009-7322 1994/10 [Not refereed][Not invited]
   

  Background The binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR) on cell surfaces has the potential to influence degradation of extracellular matrix (ECM). Thus, uPA bound to monocyte/macrophages and its interactions with plasminogen activator inhibitors types 1 and 2 (PAI-1 and PAI-2) may modify atherogenesis by altering cell-associated proteolytic activity, degradation of ECM, and neointimal formation at sites of vascular injury. Methods and Results To determine whether the expression of proteins on the surface of cells involved in fibrinolysis changes in human cells in response to mediators implicated in atherogenesis, we exposed U937 cells (an immortal human monocyte-like cell line) to transforming growth factor-beta (TGF-beta) and to thrombin. Induction of uPAR mRNA occurred with TGF-beta (5 ng/mL) in a time-dependent fashion (P=.05; n=4). Thrombin (5 National Institutes of Health [NIH] U/mL) increased uPAR mRNA by 2.8-fold above control (n=4) without altering PAI-1 mRNA or protein synthesis (n=4). The increase in uPAR gene expression in cells exposed to either TGF-beta or thrombin translated into a functional increase in cell-surface proteolytic activity. Under control conditions, U937 cells expressed PAI-2 but not PAI-1 mRNA. PAI-2 mRNA expression increased (P<.05; n=4) with thrombin (5 NIH U/mL) but was suppressed by TGF-beta (5 ng/mL). TGF-beta induced PAI-1 mRNA within 6 hours accompanied by a 9-fold increase in PAI-1 protein from 6 hours (2.3+/-1.9 ng/mL) to 24 hours (20.0+/-9.6 ng/mL, P=.005; n=3) paralleled by increased synthesis as shown in metabolic labeling experiments with S-35-methionine and immunoprecipitation of labeled PAI-1. PAI-1 mRNA and protein expression were seen in human coronary artery atherectomy specimens as well and were localized to analogous monocyte/macrophages and to smooth muscle cells as judged from results of in situ hybridization and immunocytochemistry studies. Conclusions The results indicate that there is induction of PAI-1 and uPAR in U937 cells exposed to TGF-beta and thrombin. In atheroma, analogous processes may modulate early migration of luminal monocytes into the subintimal space and proteolysis of ECM. Thus, cell surface, monocyte-directed fibrinolysis may influence atherosclerosis, restenosis, or both.
• INHIBITION OF TYPE-1 PLASMINOGEN-ACTIVATOR INHIBITOR PRODUCTION BY ANTISENSE OLIGONUCLEOTIDES IN HUMAN VASCULAR ENDOTHELIAL AND SMOOTH-MUSCLE CELLS

  H SAWA, BE SOBEL, S FUJII

  JOURNAL OF BIOLOGICAL CHEMISTRY 269 (19) 14149 - 14152 0021-9258 1994/05 [Not refereed][Not invited]
   

  We have hypothesized that type-1 plasminogen activator inhibitor (PAI-1) may exacerbate accumulation of extracellular matrix in atheroma by inhibiting local generation of plasmin and intramural proteolysis. Thus, suppression of PAI-1 expression would decrease atherogenesis. To inhibit expression of PAI-1 in cultured human umbilical vein endothelial and aortic smooth muscle cells, a 20-base antisense phosphorothioate oligonucleotide targeting specific sequences in the 3'-untranslated region of the PAI-1 gene was used. Studies with P-32-labeled oligomers verified stability in media. Secretion of PAI-1 protein assayed by enzyme-linked immunosorbent assay declined specifically and dose-dependently in cells exposed to the antisense oligonucleotide treated under basal conditions and after stimulation of PAI-1 expression with transforming growth factor beta (0.5 ng/ml for endothelial cell, 5 ng/ml for smooth muscle cell). Inhibition of expression was confirmed by immunoprecipitation of S-35-labeled PAI-1 and was paralleled by decreased steady-state levels of PAI-1 mRNA (Northern blots). Decreased PAI-1 synthesis was accompanied by augmentation of cell-associated plasmin activity. Thus, the antisense oligonucleotide down-regulated PAI-1 elaboration, an approach that may be useful in limiting obstructive vascular lesions.
• Targeted disruption of Bcl-2αβ in mice: Occurrence of gray hair, polycystic kidney disease, and lymphocytopenia

  Keiko Nakayama, Kei Ichi Nakayama, Izumi Negishi, Keisuke Kuida, Hirofumi Sawa, Dennis Y. Loh

  Proceedings of the National Academy of Sciences of the United States of America 91 3700 - 3704 0027-8424 1994/04/26 [Not refereed][Not invited]
   

  Mice carrying ablated coding regions of the bcl-2α and bcl-2β transcripts have been made. bcl-2(-/-) mutants are smaller but viable, although about half of them die by 6 weeks of age. As shown earlier with somatic bcl-2 gene-targeted mice, the number of lymphocytes markedly decreased within few weeks after birth while other hematopoietic lineages remained unaffected. Among lymphocytes, CD8+T cells disappeared most quickly followed by CD4+T cells, whereas B cells were least affected. bcl- 2(-/-) lymphocytes, however, could respond normally to various stimuli including anti-CD3, Con A, phorbol 12-myristate 13-acetate plus ionomycin, interleukin 2, lipopolysaccharide, and anti-IgM antibody. Abnormalities among nonlymphoid organs include smaller auricles, hair color turning gray at 4-5 weeks of age, and polycystic kidney disease-like change of renal tubules. These results suggest that Bcl-2 may be involved during morphogenesis where inductive interactions between epithelium and mesenchyme are important such as in the kidneys, hair follicles, and perichondrium of auricles. Surprisingly, the nervous system, intestines, and skin appear normal despite the fact that these organs show high levels of endogenous Bcl-2 expression in normal mice.
• Distribution of angiotensinogen in diseased human hearts

  Hirofumi Sawa, Hideaki Kawaguchi, Naoki Mochizuki, Yuka Endo, Toshiyuki Kudo, Fumio Tokuchi, Yasunori Fijioka, Kazuo Nagashima, Akira Kitabatake

  Molecular and Cellular Biochemistry 132 (1) 15 - 23 0300-8177 1994/03 [Refereed][Not invited]
   

  Extrahepatic synthesis and localization of angiotensinogen (ATN) have been described in animals, thus establishing the tissue renin-angiotensin (RA) system. However, there had been no reports of tissue RA systems in human organs, including the heart. In earlier, we have reported the possibility of ATN synthesis in the human heart using ribonuclease protection assay system. ATN mRNA was detected not only in the liver, but also in both the atrial and ventricular heart tissues, suggesting that ATN is synthesized in the human heart. In this report, we looked for the distribution of ATN in diseased human heart. Northern blot hybridization of cDNA with total RNA extracted from human liver, brain, kidney, atrial and ventricular tissues revealed that ATN mRNA exists in cardiac ventricule. Immunohistochemical studies using a specific antibody to ATN revealed a stronger reaction in the endocardial layer of the human left ventricle, than in the epicardial layer, and intense immunoreactivity in the conduction system and right atrium. This distribution pattern was similar to that of human atrial natriuretic peptide (hANP), which functions a smooth muscle relaxant. Double immunostaining of ATN and hANP demonstrated that all myocytes in the right atrium had immunopositive reactions to ATN, hANP or both of ATN and hANP. Double immunoelectron staining enabled us to show more detailed localization of ATN and hANP hANP only existed in the specific granules and ATN existed in the myofibril, but not in the granule. Furthermore, our experiments provide evidence of ATN in healthy human hearts and also reveal a widespread immunopositive reaction for ATN in the left ventricle of diseased hearts. © 1994 Kluwer Academic Publishers.
• Nakayama K, Nakayama K-i, Negishi I, Kuida K, Sawa H, Loh DY: Targeted disruption of Bcl-2 alpha beta in mice: occurrence of gray hair,polycystic kidney disease, and lymphocytopenia. Proc Natl Acad Sci U S A 91: 3700-3704, 1994*

  1994 [Not refereed][Not invited]
  
• Sawa H, Kawaguchi H, Mochizuki N, Endo Y, Kudo T, Tokuchi F, Fujioka Y, Nagashima K, Kitabatake A: Distribution of angiotensinogen in diseased human hearts. Mol Cell Biochem 132: 15-23, 1994*

  1994 [Not refereed][Not invited]
  
• POTENTIATION BY HYPERCHOLESTEROLEMIA OF THE INDUCTION OF AORTIC INTRAMURAL SYNTHESIS OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 BY ENDOTHELIAL INJURY

  H SAWA, BE SOBEL, S FUJII

  CIRCULATION RESEARCH 73 (4) 671 - 680 0009-7330 1993/10 [Not refereed][Not invited]
   

  Accumulation of plasminogen activator inhibitor type 1 (PAI-1) in the arterial wall may accelerate atherogenesis by inhibiting fibrinolysis, diminishing proteolysis of extracellular matrix proteins, or modifying migration of vascular smooth muscle cells. Increased intramural expression of the PAI-1 gene is induced by thrombosis. To determine Whether it occurs also in response to a sustained mechanical insult to endothelium, hypercholesterolemia, or both, rabbits were subjected to sustained aortic injury induced by implantation of indwelling polyethylene tubing, to hyperlipidemia induced by cholesterol and peanut oil feeding over a period of 8 weeks, or both. Sustained vascular injury alone did not increase plasma PAI-1. However, hypercholesterolemia with or without mechanically induced vascular injury increased plasma PAI-1 twofold. The expression of PAI-1 mRNA in aorta (Northern blots) was significantly increased when vascular injury was combined with hyperlipidemia. In situ hybridization showed that the increase with mechanical injury alone occurred in endothelial cells covering the neointima (positive for factor VIII and thrombomodulin), in abnormally differentiated vascular smooth muscle cells (positive for embryonic myosin heavy chain), and in macrophages (positive for the RAM-11 anti-macrophage antibody). Qualitatively similar but much more marked increases in PAI-1 gene expression were seen when arterial injury was accompanied by hypercholesterolemia. Neither vitronectin, known to stabilize PAI-1, nor vitronectin mRNA increased in liver. However, immunocytochemistry and Western blots demonstrated marked aortic accumulation of vitronectin protein with hyperlipidemia, particularly in subendothelial fibrotic regions, accompanied by increased neointimal vitronectin mRNA as shown by in situ hybridization. These results suggest that increased synthesis and stabilization of vascular PAI-1 may potentiate accumulation of extracellular matrix, thereby accelerating atherosclerosis.
• Fujii S, Sawa H, Sobel BE: Inhibition of endothelial cell expression of plasminogen activator inhibitor type-1 by gemfibrozil. Thromb Haemost 70: 642-647, 1993*

  1993 [Not refereed][Not invited]
  
• INDUCTION OF ENDOTHELIAL-CELL EXPRESSION OF THE PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 GENE BY THROMBOSIS INVIVO

  S FUJII, H SAWA, JE SAFFITZ, CL LUCORE, BE SOBEL

  CIRCULATION 86 (6) 2000 - 2010 0009-7322 1992/12 [Not refereed][Not invited]
   

  Background. We have shown previously that products from activated Platelets can augment synthesis of plasminogen activator inhibitor type 1 (PAI-1) in cultured endothelial and hepatoma (Hep G2) cells in vitro and increase plasma PAI-1 activity in vivo in rabbits. Accordingly, the effects of activation of platelets associated with thrombosis and thrombolysis in vivo on plasma PAI-1 activity and expression of the PAI-1 gene in endothelium, liver, and other organs were characterized. Methods and Results. Endothelial injury giving rise to platelet-rich thrombi was induced with electrical stimulation in carotid arteries in rabbits. Clot lysis and recanalization were induced subsequently with intravenous tissue-type plasminogen activator (t-PA) and verified with Doppler flow probes. Plasma PAI-1 activity (mean+/-SD) increased from 6+/-2 arbitrary units (AU)/ml to 129+/-48 AU/ml (n=15) within several hours after recanalization. When t-PA had failed to induce recanalization, the increase was much less (from 7+/-2 to 42+/-23 AU/ml, n=11). To define mechanisms responsible for these changes, PAI-1 messenger RNA (mRNA) was evaluated by Northern blot analysis and localized in tissues by in situ hybridization. Strong and consistent induction of PAI-1 mRNA was evident in aorta, heart, and fiver of animals subjected to thrombosis (twofold to threefold increases compared with values in controls), particularly in those in which thrombolysis had been induced (fourfold to sixfold). After thrombolysis, an intense, PAI-1 mRNA-specific signal was detected in endothelium of aorta, liver, and heart, with less intense signals in endothelium of lung, adrenals, and kidneys. Conclusions. The increases in plasma PAI-1 activity follow a preceding increase in endothelial cell expression of the PAI-1 gene as reflected by PAI-1 mRNA levels. Thus, increased synthesis of endothelial cell PAI-1 after thrombosis and thrombolysis may attenuate endogenous fibrinolysis early after coronary thrombolysis, thereby potentiating early, thrombotic reocclusion.
• Furuta Y, Takasu T, Asai T, Shinohara T, Sawa H, Nagashima K, Inuyama Y: Detection of human papillomavirus DNA in carcinomas of the nasal cavities and paranasal sinuses by polymerase chain reaction. Cancer 69: 353-357, 1992

  1992 [Not refereed][Not invited]
  
• Sawa H, Fujii S, Sobel BE: Augmented arterial wall expression of type-1 plasminogen activator inhibitor induced by thrombosis. Arterioscler Thromb 12: 1507-1515, 1992

  1992 [Not refereed][Not invited]
  
• Kawaguchi H, Shoki M, Sano H, Kudo T, Sawa H, Mochizuki N, Okamoto H, Endo Y, Kitabatake A: Polyphosphoinositide metabolism in hypertrophic rat heart. J Mol Cell Cardiol 24: 1003-1010, 1992*

  1992 [Not refereed][Not invited]
  
• Sawa H, Tokuchi F, Mochizuki N, Endo Y, Furuta Y, Shinohara T, Takada A, Kawaguchi H, Yasuda H, Nagashima K: Expression of the angiotensinogen gene and localization of its protein in the human heart. Circulation 86: 138-146, 1992

  1992 [Not refereed][Not invited]
  
• Shoki M, Kawaguchi H, Okamoto H, Sano H, Sawa H, Kudo T, Hirao N, Sakata Y, Yasuda H: Phosphatidylinositol and inositolphosphatide metabolism in hypertrophied rat heart. Jpn Circ J 56:142-147, 1992*

  1992 [Not refereed][Not invited]
  
• Kawaguchi H, Shoki M, Sano H, Kudo T, Sawa H, Okamoto H, Sakata Y, Yasuda H: The studies of cell damaging and cell growth factors which induce cardiomyopathy. Jpn Circ J 56: 1037-1044, 1992*

  1992 [Not refereed][Not invited]
  
• Polyphosphoinositide metabolism in hypertrophic rat heart

  Hideaki Kawaguchi, Mikako Shoki, Hitoshi Sano, Toshiyuki Kudo, Hirofumi Sawa, Naoki Mochizuki, Hiroshi Okamoto, Yuka Endo, Akira Kitabatake

  Journal of Molecular and Cellular Cardiology 24 (9) 1003 - 1010 0022-2828 1992 [Refereed][Not invited]
   

  The accumulations of inositol-1,4,5-trisphospate (IP3) and inositol-1,3,4,5-tetrakisphosphate (IP4) after hormonal stimulation may have a physiological role, possibly by alteration of Ca2+ levels in cardiac tissue. But the accumulation of inositol polyphosphate in a pathophysiological condition has not been studied. We investigated phosphatidylinositol-4,5-bisphosphate (PIP2) metabolism in hypertrophic cardiac myocytes, and clarified that the accumulations of IP3, IP4 and diacylglyceride after stimulation with norepinephrine were significantly enhanced in isolated myocytes from spontaneously hypertensive rat heart. Phospholipase C activity increased with age in SHRSP heart cells. These data suggest that PI turnover pathways, which can be mediated by both phosphatidylinositol-4,5-bisphosphate and diacylglyceride, may play an important role in development of hypertrophy in the hearts of rats with spontaneous hypertension. © 1992.
• The studies of cell damaging and cell growth factors which induce cardiomyopathy

  Hideaki Kawaguchi, Ikako Shoki, Hitoshi Sano, Toshiyuki Kudo, Hirofumi Sawa, Hiroshi Okamoto, Yoshihito Sakata, Hisakazu Yasuda

  JAPANESE CIRCULATION JOURNAL 56 (10) 1037 - 1044 0047-1828 1992 [Refereed][Not invited]
   

  We demonstrated that phosphatidylinositide-specific phospholipase C (PLC) activity was greater in cardiomyopathic hamster hearts (BIO 14.6 and BIO 53.58) then in hamster controls (Fib). Inositol trisphosphate (IP3) production was markedly greater in both of the cardiomyopathic hamsters, BIO 14.6 and BIO 53.58. We have also determined the sarcoplasmic reticulum (SR) function of heart. Calcium uptake into SR markedly increased in BIO 14.6. On the other hand, it significantly decreased in BIO 53.58 compared with Fib. It is well known that IP3 stimulates calcium release from SR. In BIO 14.6, calcium relrease from SR stimulated by IP3 increased, but its effect decreased in BIO 53.58 compared with Fib. These results suggest that PI response may produce high intracellular calcium levels in both BIO 14.6 and BIO 53.58 myocytes. In addition, in the BIO 53.58 hamster the sarcoplasmic reticulum deteriorate in function. It was concluded from these results that a prolonged high intracellular calcium level may lead to the death of BIO 53.58 myocytes. The expression of angiotensinogen mRNA was observed in the hamster heart. There was no differences in its expression level between Fib, BIO 14.6 and BIO 53.58. There was no effect of ages on its expression in these hamster hearts. We have also determined the distribution of angiotensinogen in these hamsters. At 4 weeks of age, the immunohistochemical study revealed that angiotensinogen was widely distributed in subendcardium in these hamsters. There was no difference in its distribution between Fib, BIO 14.6 and BIO 53.58. But at 20 weeks old of age its immunoreactivity decreased in BIO 53.58. There was no effect of age on its reactivity in Fib and BIO 14.6. We have detected angiotensinogen in heart, but its role is still not clear. A further study should be done to clarify its role in cardiac hypertrophy and cell damage. Qpn Circ J 1992 56: 1037-1044). © 1992, The Japanese Circulation Society. All rights reserved.
• EFFECT OF ENDOTHELIN ON ANGIOTENSIN CONVERTING ENZYME-ACTIVITY IN CULTURED PULMONARY-ARTERY ENDOTHELIAL-CELLS

  H KAWAGUCHI, H SAWA, H YASUDA

  JOURNAL OF HYPERTENSION 9 (2) 171 - 174 0263-6352 1991/02 [Not refereed][Not invited]
   

  We studied the effects of endothelin on angiotensin converting enzyme (ACE) in cultured pulmonary artery endothelial cells. ACE activity was increased 2.5-fold by the addition of 1 x 10(-8) mol/l endothelin-1. Endothelin-1 also stimulated calcium influx and phospholipase C activity in a dose-dependent manner. Calcium influx, phospholipase C and ACE activity were suppressed 60-70% in the presence of endothelin-1 (10(-10) to 10(-6) mol/l) by 50-mu-l neomycin. These results suggest that ACE was stimulated by endothelin-1 and that its activity may be closely related to phosphatidylinositol turnover stimulated by endothelin-1.
• Kawaguchi H, Shoki M, Sano H, Kudo T, Sawa H, Okamoto H, Sakata Y, Yasuda H: Phospholipid metabolism in cardiomyopathic hamster heart cells. Circ Res 69: 1015-1021, 1991*

  1991 [Not refereed][Not invited]
  
• Mochizuki N, Sawa H, Yasuda H, Shinohara T, Nagashima K, Yamaji N, Ohnuma N, Hall WW: Distribution of atrial natriuretic peptide in the conduction system and ventricular muscles of the human heart. Virchows Arch A Pathol Anat Histopathol 418: 9-16, 1991*

  1991 [Not refereed][Not invited]
  
• Phospholipid metabolism in cardiomyopathic hamster heart cells

  H. Kawaguchi, M. Shoki, H. Sano, T. Kudo, H. Sawa, H. Okamoto, Y. Sakata, H. Yasuda

  Circulation Research 69 (4) 1015 - 1021 0009-7330 1991 [Refereed][Not invited]
   

  We demonstrated that the activities of phosphatidylinositide-specific phospholipase C, inositol 1,4,5-trisphosphate (IP3) kinase, and IP3 phosphatase were enhanced in cardiomyopathic hamster hearts (BIO 14.6 and BIO 53.58) in comparison to control hamsters (F1b). Release of both arachidonic acid and prostacyclin was markedly enhanced by norepinephrine in the cardiomyopathic hamsters. Phospholipase C in heart has high substrate specificity to phosphatidylinositol. IP3 production was markedly enhanced in the cardiomyopathic hamsters. We also determined the intracellular calcium concentration, which was higher in BIO 53.58 hamsters than in BIO 14.6 hamsters at 5-20 weeks of age. There was no significant difference in the intracellular calcium level between F1b and BIO 14.6 hamsters at 5 weeks of age. These results suggest that phosphatidylinositol turnover stimulated by norepinephrine may produce high intracellular calcium levels in both BIO 14.6 and BIO 53.58 myocytes. In addition, in BIO 53.58 hamsters, some mechanism such as the sarcoplasmic reticulum, which controls the intracellular calcium level, may deteriorate in function. We concluded from these results that a prolonged high intracellular calcium level may lead to the death of BIO 53.58 myocytes.
• DISTRIBUTION OF ATRIAL-NATRIURETIC-PEPTIDE IN THE CONDUCTION SYSTEM AND VENTRICULAR MUSCLES OF THE HUMAN HEART

  N MOCHIZUKI, H SAWA, H YASUDA, T SHINOHARA, K NAGASHIMA, T YAMAJI, N OHNUMA, WW HALL

  VIRCHOWS ARCHIV A-PATHOLOGICAL ANATOMY AND HISTOPATHOLOGY 418 (1) 9 - 16 0174-7398 1991 [Refereed][Not invited]
   

  Atrial natriuretic peptide (ANP), a cardiac hormone, is known to be located in the atrial specific granules, but its presence and localization in the ventricular muscle of the human heart has not been examined fully. Using a specific antibody to human ANP, we studied the conduction system and ventricular muscle with immunohistochemical and ultrastructural methods in 30 hearts obtained at autopsy. These included 12 normal and 18 diseased hearts. In the normal hearts, ANP-positive granules, which were regularly observed in the atrial myocytes, were found in small quantities in the cells of the penetrating and branching bundles in 4 of 12, and in the cells of the ventricular free walls in 2 of the 12 hearts. In the diseased hearts, the positivity increased significantly (P < 0.05), being found in 13 of 18 (72.2%) conduction systems and 10 of 18 (55.6%) ventricular muscles. The granules were confirmed to be immunoreactive with ANP by ultrastructural examination. Furthermore, the presence of ANP mRNA in the conduction system as well as in the ventricular myocytes was demonstrated by Northern blot hybridization for which we used the complementary DNA of human ANP. Thus, a small quantity of ANP appears to be synthesized and stored in the conduction system and ventricles of some normal hearts. However, ANP was shown to be present in a large percentage of the diseased hearts.
• ENDOTHELIN STIMULATES ANGIOTENSIN-I TO ANGIOTENSIN-II CONVERSION IN CULTURED PULMONARY-ARTERY ENDOTHELIAL-CELLS

  H KAWAGUCHI, H SAWA, H YASUDA

  JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 22 (8) 839 - 842 0022-2828 1990/08 [Not refereed][Not invited]
  
• Mechanism of increased angiotensin-converting enzyme activity stimulated by platelet-activating factor

  Hideaki Kawaguchi, Hirofumi Sawa, Hisakazu Yasuda

  BBA - Molecular Cell Research 1052 (3) 503 - 508 0167-4889 1990/05/22 [Refereed][Not invited]
   

  We studied the effects of platelet activating factor (PAF) on angiotensin-converting enzyme (ACE). PAF (1 · 10-10 to 1 · 10-6 M) had a novel effect on angiotensin I conversion. Pulmonary artery endothelial cells converted 1 nmol/dish of 125I-angiotensin I to angiotensin II in the absence of PAF. ACE activity was increased to 2.5 nmol/dish by the addition of 1 · 10-6 M of PAF. To clarify the mechanism of this stimulatory effect of PAF on ACE, Ca2+ influx and inositol 1,4,5-trisphosphate (IP3) release in pulmonary artery endothelial cells were determined. PAF stimulated Ca2+ influx in a dose-dependent manner. PAF also stimulated phospholipase C (PLC) activity and released IP3. To study the relationship between PLC activity and ACE activity, neomycin was added. The Ca2+ influx and IP3 release stimulated by 10-6 M of PAF were suppressed by about 60-70%. ACE activity was also inhibited up to 70% in the presence of PAF (10-10 - 10-6 M) by 50 M of neomycin. These results suggest that ACE was stimulated by PAF, and that its activityl in endothelial cells may be mediated by the PI-turnover pathway via changes in PLC activity and IP3-mediated Ca2+ release from intracellular stores. © 1990.
• MECHANISM OF INCREASED ANGIOTENSIN-CONVERTING ENZYME-ACTIVITY STIMULATED BY PLATELET-ACTIVATING FACTOR

  H KAWAGUCHI, H SAWA, H YASUDA

  BIOCHIMICA ET BIOPHYSICA ACTA 1052 (3) 503 - 508 0006-3002 1990/05 [Not refereed][Not invited]
  
• Kawaguchi H, Sawa H, Iizuka K, Yasuda H: Platelet-activating factor stimulates angiotensin converting enzyme activity. J Hypertens 8: 173-177, 1990*

  1990 [Not refereed][Not invited]
  
• Kawaguchi H, Sawa H, Yasuda H: Effect of atrial natriuretic factor on angiotensin converting enzyme. J Hypertens 8: 749-753, 1990*

  1990 [Not refereed][Not invited]
  
• Platelet-activating factor stimulates angiotensin converting enzyme activity

  Hideaki Kawaguchi, Hirofumi Sawa, Kenji Iizuka, Hisakazu Yasuda

  Journal of Hypertension 8 (2) 173 - 177 1473-5598 1990 [Refereed][Not invited]
   

  We studied the effects of platelet-activating factor (PAF) on the conversion of angiotensin I to angiotensin II in pulmonary artery endothelial cells. PAF had a novel effect on angiotensin I conversion. The apparent Vmax and Km for angiotensin converting enzyme (ACE) were 2.5 nmol/min per dish and 50 µmol/l, respectively. This activity was enhanced by the addition of PAF to cells. When PAF was added to pulmonary artery endothelial cells, the conversion of angiotensin I to angiotensin II was enhanced about twofold at 10−6mol/l PAF. Maximal stimulation was achieved at 10˜5mol/l PAF. This stimulatory effect was suppressed by ACE inhibitors such as enalapril and PAF antagonist CV3988. When cells were incubated with 10−6mol/l PAF, the conversion of angiotensin I to angiotensin II stimulated with PAF was suppressed by CV3988. Enalapril (10−6mol/l) completely inhibited the conversion of angiotensin I to angiotensin II in the presence of PAF. Bradykinin also suppressed ACE activity, but phosphatidylcholine and lysophosphatidylcholine did not affect its activity. These results suggest that PAF may have an important role in regulating vascular tone by modulating angiotensin conversion. © Current Science Ltd.
• Effect of atrial natriuretic factor on angiotensin converting enzyme

  Hideaki Kawaguchi, Hirofumi Sawa, Hisakazu Yasuda

  Journal of Hypertension 8 (8) 749 - 753 1473-5598 1990 [Refereed][Not invited]
   

  We studied the effects of atrial natriuretic factor (ANF) on the conversion of angiotensin I to angiotensin II. Pulmonary artery endothelial cells converted 1.22nmol/min per dish [125l]angiotensin I to angiotensin II, but ANF suppressed the conversion by 0.475 nmol/min per dish. The maximum rate of inhibition of angiotensin converting enzyme (ACE) activity by ANF was 60% at 10-6mol/l ANF compared with control conditions. The amino acid fragments of ANF were studied to determine whether its specific structure was necessary for inhibiting ACE in endothelial cells. We found that atriopeptin II (fragment 5-27) inhibited ACE activity as much as ANF. Fragment 7-25 had the same rate of inhibition, but fragment 13-27 had no effect on the conversion of angiotensin I to angiotensin II. These results suggest that the amino acid sequence of Cys (7)-Cys (23) in ANF is important for the inhibition of ACE in pulmonary endothelial cells. © Current Science Ltd.
• POSITIVE TRANSMISSION OF CREUTZFELDT-JAKOB DISEASE VERIFIED BY MURINE KURU PLAQUES

  T KITAMOTO, J TATEISHI, H SAWA, K DOHURA

  LABORATORY INVESTIGATION 60 (4) 507 - 512 0023-6837 1989/04 [Refereed][Not invited]
  
• Kawaguchi H, Sawa H, Yasuda H: Effect of atrial natriuretic factor on angiotensin converting enzyme. J Mol Cell Cardiol 21: 959-961, 1989*

  1989 [Not refereed][Not invited]
  
• Visualization of liver on bone scintigraphy during treatment with iron chondroitin sulfate colloid

  S. Tanaka, H. Sawa, T. Fukuda

  Kakuigaku 20 (8) 1175 - 1181 0022-7854 1983 [Refereed][Not invited]
   

  In order to see its probable cause in the incidental liver delineation on the bone scan with 99mTc-MDP, patients and experimental rabbits were intravenously injected with Blutal (iron chondroitin sulfate) various times following the intravenous injection of 99mTc-MDP. The liver images were obtained during the early periods. An injection of Blutal-99mTc pertechnetate mixture did not result in any appreciable hepatic delineation. Stannous chloride in the MDP kit could have enticed formation of radiocolloids in the presence of iron chondroitin sulfate. Diagnostic problems can be avoided by carefully planning the bone scintigraphy in relation to Blutal administration.

MISC

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• IgA抗体の自己重合反応による四量体形成とその応用

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• Genetic characterization of various herpesviruses in bats in Zambia

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• Pathogenicity analysis of two strains of West Nile virus newly isolated from mosquito and crocodile in Zambia

  小林広子, 田畑耕史郎, 有泉拓馬, CHAMBARO Herman, PHONGPHAEW Wallaya, SIMULUNDU Edgar, 佐々木道仁, 澤洋文, 澤洋文, 澤洋文, 大場靖子, 大場靖子, 大場靖子  日本ウイルス学会学術集会プログラム・予稿集(Web)  70th-  2023
• Investigation of anti-Nairovirus compounds

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• Comparison of the viral infectivity between the fixed and street strains of Rabies virus in peripheral tissues

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• Discovery history of novel oral drug S-217622 (ensitrelvir) for COVID-19

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• Temperature-dependent regulatory mechanism of infectivity on Insect-Specific Flavivirus

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• Discovery of a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses

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• 血管内皮細胞におけるSARS-CoV-2侵入機構の解析

  桜井優弥, 間石奈湖, 藤岡容一朗, 大場雄介, 武田遼, 佐々木道仁, 大場靖子, 樋田泰浩, 澤洋文, 澤洋文, 樋田京子  日本血管生物医学会学術集会プログラム・抄録集  31st (CD-ROM)-  2023
• X-206 exhibits broad-spectrum and robust inhibition against infections by several drug-resistant isolates and variants of SARS-CoV-2.

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• Combination therapy with oral antiviral and anti-inflammatory treatments improves the delayed-therapeutic effect on COVID-19

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• Genes involved in spread of SARS-CoV-2 into the lower respiratory tract may be under adaptive selection.

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• 5-Ethynylimidazole-4-carboxamide(EICA)ヌクレオチドプロドラッグの合成と抗デングウイルス活性

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• SARS-CoV-2感染マウスを用いた肺血管内皮細胞の重症化関連遺伝子の解析

  武田遼, 武田遼, 伊藤航, 手代木孝仁, 佐々木道仁, 大場靖子, 大場靖子, 間石奈湖, 松田彩, 佐藤彰彦, 鳥羽晋輔, 李智媛, 飯村忠浩, 樋田泰浩, 北川善政, 澤洋文, 澤洋文, 澤洋文, 樋田京子  日本血管生物医学会学術集会プログラム・抄録集  30th (CD-ROM)-  2022
• 人と動物の健康を守るワンヘルスの取組み-北海道大学One Healthリサーチセンターの活動紹介-

  堀内基広, 池中良徳, 山崎淳平, 佐藤豊孝, 鈴木章夫, 直亨則, 松野啓太, 澤洋文  日本獣医師会獣医学術学会年次大会講演要旨集  2022-  2022
• SARS-CoV-2BA.2株に由来する亜株の性状解析

  木村出海, 山岨大智, 山岨大智, 田村友和, 直亨則, 直亨則, 伊東潤平, 松野啓太, 松野啓太, 松野啓太, 澤洋文, 澤洋文, 澤洋文, 福原崇介, 佐藤佳  日本ウイルス学会学術集会プログラム・予稿集(Web)  69th-  2022
• 狂犬病ウイルスL蛋白質に存在するNPYNE配列はウイルス複製に重要である

  泉郁輝, 牧野真知子, 佐々木道仁, 中川賢人, 高橋龍樹, 岡田和真, 藤井祐至, 西山祥子, 正谷達謄, 正谷達謄, 澤洋文, 杉山誠, 伊藤直人, 伊藤直人  日本ウイルス学会学術集会プログラム・予稿集(Web)  69th-  2022
• マウス感染モデルを用いたCOVID-19重症化における血管病態の解析

  積田卓也, 武田遼, 澤洋文, 佐々木道仁, 間石奈湖, 樋田泰浩, 松田彩, 佐藤彰彦, 佐藤彰彦, 鳥羽晋輔, 鳥羽晋輔, 田中宏典, 樋田京子  高血圧関連疾患モデル学会学術総会抄録集  57th (CD-ROM)-  2021
• Characterization of a Nelson Bay orthoreovirus isolated from an Egyptian fruit bat in Zambia

  播磨勇人, 佐々木道仁, 大場靖子, 邱永晋, チャングラカテンディ, 梶原将大, シムルンドゥエドガー, 谷口怜, 高田礼人, 西條政幸, ハンゴンべバーナード, 澤洋文  日本ウイルス学会学術集会プログラム・予稿集(Web)  68th-  2021
• Glycoprotein Analyses by Mass Spectrometry

  梶裕之, 富岡あづさ, 日尾野隆大, 日尾野隆大, 佐々木道仁, 大場靖子, 澤洋文, 久野敦  質量分析総合討論会講演要旨集  69th-  2021
• RIG-Iはヒト肺細胞においてシグナル伝達非依存的な抗SARS-CoV-2防御を誘導する

  山田大翔, 山田大翔, 佐藤精一, 佐藤精一, 外山雄貴, 外山雄貴, 大場靖子, 澤洋文, 山内肇, 佐々木道仁, 高岡晃教, 高岡晃教  日本インターフェロン・サイトカイン学会学術集会抄録集  85th (CD-ROM)-  2021
• G蛋白質333位のアミノ酸残基が規定する狂犬病ウイルスの病原性発現機構の解析

  板倉友香里, 田畑耕史郎, 伊藤直人, 大場靖子, 澤洋文, 佐々木道仁  日本獣医学会学術集会講演要旨集  164th (CD-ROM)-  2021
• ボリビア多民族国サンタクルス県の牛におけるBabesiaおよびAnaplasmaの感染状況

  小方昌平, PEREIRA Juan Antonio, 松野啓太, 大場靖子, 澤洋文, 川森文彦, 野中成晃, 中尾亮  日本獣医学会学術集会講演要旨集  164th (CD-ROM)-  2021
• 北海道産ヤマトヤブカにおけるジカウイルス感受性の評価

  川江詩乃, 岩本夏奈, 江下優樹, 大場靖子, 澤洋文, 村松康和, 内田玲麻  日本獣医学会学術集会講演要旨集  164th (CD-ROM)-  2021
• ヤマトヤブカ体内におけるジカウイルスのQuasispecies評価

  三木要, 岩本夏奈, 江下優樹, 大場靖子, 澤洋文, 村松康和, 内田玲麻  日本獣医学会学術集会講演要旨集  164th (CD-ROM)-  2021
• ザンビアに生息する野生げっ歯類動物における脳心筋炎ウイルス(EMCV)の疫学調査

  岸本麻衣, HANG’OMBE Bernard M., 大場靖子, 澤洋文, 佐々木道仁  日本獣医学会学術集会講演要旨集  164th (CD-ROM)-  2021
• ザンビアのブタにおける消化管ウイルスの疫学調査

  播磨勇人, 梶原将大, 佐々木道仁, SIMULUNDU Edgar, BWALYA Eugene, 邱永晋, 奥谷公亮, 大場靖子, HANG’OMBE Bernard M, MWEENE Aaron S, 高田礼人, 澤洋文  日本獣医学会学術集会講演要旨集  164th (CD-ROM)-  2021
• 質量分析とレクチンアレイを組み合わせたSARS-CoV-2スパイクタンパク質の高精度糖鎖構造解析

  日尾野隆大, 日尾野隆大, 富岡あづさ, 梶裕之, 佐々木道仁, 大場靖子, 澤洋文, 久野敦  日本獣医学会学術集会講演要旨集  164th (CD-ROM)-  2021
• Characterization of a newly emerging orthonairovirus in Hokkaido, Japan

  有泉拓馬, 山口宏樹, 澤洋文, 大場靖子, 松野啓太  日本分子生物学会年会プログラム・要旨集(Web)  44th-  2021
• Evaluation of cell-penetrating peptides for the production of intracellular invasive antiviral neutralizing antibody

  板倉友香里, 田畑耕史郎, 大場靖子, 澤洋文, 澤洋文, 佐々木道仁  日本分子生物学会年会プログラム・要旨集(Web)  44th-  2021
• A mechanism of Ig variable region-independent oligomerization of secretory IgA antibody accelerated by polymeric Ig receptor.

  田畑耕史郎, 田畑耕史郎, 鈴木忠樹, 佐野芳, 齊藤慎二, 相内章, 佐々木道仁, 長谷川秀樹, 大場靖子, 澤洋文, 澤洋文  日本分子生物学会年会プログラム・要旨集(Web)  44th-  2021
• SARS-CoV-2 variants are generated during propagation in Vero cells

  佐々木道仁, 上村健太朗, 上村健太朗, 鳥羽晋輔, 鳥羽晋輔, 佐藤彰彦, 佐藤彰彦, 板倉友香里, INTARUCK Kittiya, 岸本麻衣, 田畑耕史郎, CHAMBARO Herman, 大場靖子, 大場靖子, 澤洋文, 澤洋文  日本分子生物学会年会プログラム・要旨集(Web)  44th-  2021
• Examination of the accumulation of TDP-43 observed in neuronal cells infected with West Nile virus

  小林進太郎, 好井健太朗, 松野啓太, 大場靖子, 澤洋文, 苅和宏明  日本分子生物学会年会プログラム・要旨集(Web)  44th-  2021
• Investigation of an orthoreovirus isolated from a fruit bat in Zambia

  播磨勇人, 佐々木道仁, 大場靖子, QIU Yongjin, 梶原将大, 谷口怜, 高田礼人, 西條政幸, 澤洋文  日本分子生物学会年会プログラム・要旨集(Web)  44th-  2021
• Identification of host type II transmembrane serine proteases which promote SARS-CoV-2 infection

  岸本麻衣, 上村健太朗, 上村健太朗, 佐名木孝央, 佐名木孝央, 佐藤彰彦, 佐藤彰彦, 大場靖子, 澤洋文, 佐々木道仁  日本分子生物学会年会プログラム・要旨集(Web)  44th-  2021
• Arthropod-borne viruses (arboviruses)

  大場靖子, 澤洋文, 松野啓太, 松野啓太  ウイルス  70-  (1)  3  -14  2020  

  "Arbovirus" is a term for a virus transmitted to mammals by hematophagous arthropods; arboviruses; replicate in both mammals and arthropods. Since the life cycle of arboviruses is highly dependent on arthropods, control of the arthropods (vectors) is generally considered important for the control of arbovirus infection. Various pathogens that cause diseases in the medical and veterinary fields are grouped into arboviruses with a history of their discoveries since the early 20th century. Furthermore, because of recent advances in sequencing technology, new arboviruses have been discovered one after another. Here we would like to overview the known arboviruses and their infections.
• エンベロープタンパク質のアミノ酸変異によるウエストナイルウイルスの増殖および病態形成への影響

  小林 進太郎, 金子 知里, 川上 怜子, 長谷部 理絵, 澤 洋文, 好井 健太朗, 苅和 宏明  日本獣医学会学術集会講演要旨集  162回-  431  -431  2019/08  [Not refereed][Not invited]
  
• 豚コレラウイルスワクチン株感染による細胞死誘導と細胞変性効果

  板倉友香里, 板倉友香里, 松野啓太, 松野啓太, 伊藤麻子, 藤本悠理, 田村友和, 亀山健一郎, 岡松正敏, NICOLAS Ruggli, NICOLAS Ruggli, 喜田宏, 喜田宏, 澤洋文, 澤洋文, 迫田義博, 迫田義博  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• エンベロープタンパク質の159番目のアミノ酸のウエストナイルウイルスの増殖および病原性発現に与える影響

  小林進太郎, 金子知里, 川上怜子, 長谷部理絵, 澤洋文, 好井健太朗, 苅和宏明  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• Autopsy findings in the early stage of amyotrophic lateral sclerosis with “dropped head” syndrome

  Tanikawa Satoshi, Tanino Mishie, Wang Lei, Ishikawa Marin, Miyazaki Masaya, Tsuda Masumi, Tsuda Masumi, Tsuda Masumi, Orba Yasuko, Sawa Hirofumi, Matoba Kotarou, Nakamura Nishio, Nagashima Kazuo, Hall William W., Tanaka Shinya, Tanaka Shinya, Tanaka Shinya  Neuropathology (Web)  39-  (5)  374  -377  2019  

  Dropped head syndrome (DHS) has been rarely observed in amyotrophic lateral sclerosis (ALS), and the neuropathological findings of this condition have almost never been described. The identification of transactivation response DNA-binding protein 43 kDa (TDP-43), which binds to RNA/DNA has provided a new method for studying ALS and frontotemporal lobar degeneration (FTLD). Post-mortem examination of an adult sudden death case of a 71-year-old patient who complained of DHS exhibited severe loss of anterior motor neurons in the cervical cord (C4-6). Loss of nerve fibers of the anterior roots was striking compared with posterior roots, together with marked neurogenic atrophy of posterior muscles semispinalis cervicis. Bunina bodies were found in large neurons of Betz giant cells, but not in the motor neurons of spinal cords, or neurons of bulbar regions. Phosphorylated TDP-43 (p-TDP-43)-positive structures were detected in the residual neurons of the cervical, thoracic and lumber cords, hypoglossal nucleus, cerebellar dentate nucleus and parahippocampal cortex, together with ubiquitin-positive inclusions. Phosphorylated Tau positive structures in neuronal cytoplasm were found in the amygdala, entorhinal cortex and parahippocampal cortex, some of which co-expressed p-TDP-43. The medial zone of cervical cords may be the first onset site, and that is the cause of head drop in the early stage of ALS. In spite of detailed examination, the direct cause of sudden death was not verified. This autopsy report revealed the relation of DHS which is a rare clinical manifestation of ALS, and neuropathological findings.
• In vitroにおける四量体分泌型IgA抗体形成機構の解明

  田畑耕史郎, 田畑耕史郎, 鈴木忠樹, 佐野芳, 齊藤慎二, 相内章, 大場靖子, 長谷川秀樹, 長谷川秀樹, 澤洋文, 澤洋文  日本分子生物学会年会プログラム・要旨集(Web)  42nd-  2019
• インターフェロン受容体欠損マウスにおける狂犬病ウイルス病原性発現機構

  板倉友香里, 大場靖子, 澤洋文, 澤洋文, 佐々木道仁  日本分子生物学会年会プログラム・要旨集(Web)  42nd-  2019
• ウエストナイルウイルス感染で起こるAMP-activated protein kinaseの分解およびオートファジーの抑制による神経病態形成への影響

  小林進太郎, 好井健太朗, PHONGPAEW Wallaya, 武藤芽未, 平野港, 大場靖子, 澤洋文, 苅和宏明  日本分子生物学会年会プログラム・要旨集(Web)  42nd-  2019
• ロタウイルス感染を促進させる宿主プロテアーゼの同定

  佐々木道仁, 伊藤直人, 杉山誠, 大場靖子, 澤洋文, 澤洋文  日本分子生物学会年会プログラム・要旨集(Web)  42nd-  2019
• ESCRTタンパク質HGSはチクングニアウイルスのコードするタンパク質と相互作用し,細胞内増殖の2過程を制御する

  鳥居志保, 鳥居志保, 大場靖子, 佐々木道仁, 和田雄治, HOBSON-PETERS Jody, HALL Roy A., 澤洋文  日本分子生物学会年会プログラム・要旨集(Web)  42nd-  2019
• 蚊のゲノムに内在する多様なフラビウイルスエレメント

  大場靖子, HANG’OMBE Bernard M., MWEENE Aaron S., 佐々木道仁, 江下優樹, 澤洋文, 澤洋文, 澤洋文  日本分子生物学会年会プログラム・要旨集(Web)  42nd-  2019
• ESCRT蛋白質HGSはチクングニアウイルスと相互作用し,細胞内増殖2過程を制御する

  鳥居志保, 鳥居志保, 大場靖子, 佐々木道仁, 和田雄治, HOBSON-PETERS Jody, HALL Roy, 澤洋文, 澤洋文, 澤洋文  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• 宿主タンパク質YB-1は狂犬病ウイルスmRNAに結合し遺伝子発現を促進する

  佐々木道仁, 伊藤直人, 杉山誠, GONZALEZ Gabriel, 伊藤公人, 伊藤公人, 大場靖子, 澤洋文, 澤洋文  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• 抗デングウイルス活性をもつ低分子化合物の探索

  佐藤彰彦, 佐藤彰彦, 登治謙, 鳥羽晋輔, 鳥羽晋輔, 上村健太朗, 上村健太朗, 吉田立, 宍戸貴雄, 大場靖子, 澤洋文, 澤洋文  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• 多種の蚊に内在するフラビウイルスエレメント

  大場靖子, HANG’OMBE Bernard M., MWEENE Aaron S., 佐々木道仁, 江下優樹, 澤洋文, 澤洋文, 澤洋文  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• ザンビアのエジプトルーセットオオコウモリから検出されたマールブルグウイルスの遺伝学的解析

  梶原将大, CHANGULA Katendi, HANG’OMBE Bernard, 播磨勇人, 宮本洋子, 衛藤芳樹, 奥谷公亮, 磯野真央, 吉田玲子, 邱永晋, 森(梶原)亜紀奈, 大場靖子, 澤洋文, 澤洋文, 小川寛仁, SIMULUNDU Edgar, SQUARRE David, MUKONKA Victor, MWEENE Aaron, 高田礼人, 高田礼人  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• ザンビアのダニで発見した新規フラビウイルスはフラビウイルス属の中で独特な系統を形成するNgoye virusに遺伝的に密接に関係している

  播磨勇人, 鳥居志保, 邱永晋, 梶原将大, 衛藤芳樹, 大場靖子, 江下優樹, ハンゴンベ バーナード, 好井健太朗, シムンザ マーティン, 高田礼人, 澤洋文  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• 狂犬病ウイルスに対して強い抗ウイルス活性を示す低分子化合物の同定

  上村健太朗, 上村健太朗, 鳥羽晋輔, 鳥羽晋輔, 登治謙, 吉田立, 宍戸貴雄, 佐々木道仁, 伊藤直人, 大場靖子, 澤洋文, 澤洋文, 佐藤彰彦, 佐藤彰彦  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• LCMV感染マウスモデルを用いたCompound Xのin vivo PK/PD解析

  鳥羽晋輔, 鳥羽晋輔, 登治謙, 上村健太朗, 上村健太朗, 垰田善之, 佐藤健司, 吉田立, 宍戸貴雄, 大場靖子, 澤洋文, 澤洋文, 佐藤彰彦, 佐藤彰彦  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• マダニ中の多様なフレボウイルスは何者か?

  松野啓太, 松野啓太, 中尾亮, 草木迫浩大, 大場靖子, 澤洋文, 澤洋文  日本獣医学会学術集会講演要旨集  162nd-  2019
• ウエストナイルウイルス感染で起こるオートファジーの抑制と神経病態形成の関係

  小林 進太郎, 好井 健太朗, Wallaya Phongpaew, 武藤 芽未, 平野 港, 大場 靖子, 澤 洋文, 苅和 宏明  日本獣医学会学術集会講演要旨集  161回-  395  -395  2018/08  [Not refereed][Not invited]
  
• 日本産ヒトスジシマカのジカウイルス媒介能

  江下優樹, 江下優樹, 江下優樹, 江下優樹, 和田雄治, 林田京子, 山岸潤也, 杉本千尋, 佐々木道仁, 大場靖子, 澤洋文, 飛弾野真也, 神山長慶, 小林隆志, 高崎智彦, 加藤文博, 田島茂, 倉根一郎, LIM Chang Kweng  衛生動物  69-  (Supplement)  51  2018/03/30  [Not refereed][Not invited]
  
• ウエストナイルウイルスのカプシドタンパク質によるオートファジーの抑制による変性タンパク質の蓄積と神経病態形成についての解析

  小林 進太郎, 好井 健太朗, Phonqphaew Wallaya, 武藤 芽未, 平野 港, 大場 靖子, 澤 洋文, 苅和 宏明  生命科学系学会合同年次大会  2017年度-  [3P  -1110]  2017/12  [Not refereed][Not invited]
  
• ウイルス研究の最前線 2.ザンビア,インドネシアにおける野生動物が保有するDNAウイルスの探索

  澤洋文, 澤洋文, 佐々木道仁, 大場靖子  ウイルス  67-  (2)  151‐160  -160  2017/12  [Not refereed][Not invited]
   

  人獣共通感染症は自然界にその原因微生物が存在していることから、根絶することは不可能と考えられる。それ故、その発生を予測し、流行を防止する先回り戦略をとることが重要である。先回り戦略をとるために、病原微生物の起源と自然界における存続のメカニズム、伝播と侵入の経路および感染、発症と流行に関与する諸要因を明らかにする必要がある。我々のチームは、検査体制、医療体制が不十分であり、感染症対策の支援を必要としているザンビア、およびインドネシアにおいて、野生動物が保有するウイルスを対象とした疫学研究を推進している。本稿では、両国との共同研究により得られた研究結果の内、DNAウイルスであるオルソポックスウイルス、ポリオーマウイルス、ヘルペスウイルスを対象とした研究について得られた結果を紹介する。(著者抄録)
• 本邦における進行性多巣性白質脳症(PML)サーベイランスの現状 PMLサーベイランス委員会報告

  三浦 義治, 中道 一生, 西條 政幸, 高橋 健太, 鈴木 忠樹, 阿江 竜介, 濱口 毅, 原 由紀子, 三條 伸夫, 雪竹 基弘, 岸田 修二, 澤 洋文, 奴久妻 総一, 水澤 英洋, 山田 正仁  NEUROINFECTION  22-  (2)  246  -246  2017/09  [Not refereed][Not invited]
  
• 狂犬病ウイルスの細胞吸着に関与する宿主因子の解析

  佐々木道仁, アニンディタ パウリナ, 伊藤直人, 杉山誠, 福原秀雄, 尾瀬農之, 尾瀬農之, 前仲勝実, 澤洋文, 澤洋文  日本獣医学会学術集会講演要旨集  160th-  400  -400  2017/08/30  [Not refereed][Not invited]
  
• 狂犬病ウイルスの細胞吸着に関与する宿主因子の解析

  佐々木 道仁, アニンディ・タパウリナ, 伊藤 直人, 杉山 誠, 福原 秀雄, 尾瀬 農之, 前仲 勝実, 澤 洋文  日本獣医学会学術集会講演要旨集  160回-  400  -400  2017/08  [Not refereed][Not invited]
  
• In vitroでの狂犬病ウイルスに対する5-エチニル-1-リボフラノシルイミダゾール-4-カルボキサミド(EICAR)の抗ウイルス活性に関する検討(Examination of antiviral activity of 5-ethynyl-1-ribofuranosylimidazole-4-carboxamide(EICAR) against rabies virus in vitro)

  Anindita Paulina Duhita, 佐々木 道仁, 伊藤 直人, 杉山 誠, 南川 典昭, 周東 智, 乙黒 聡子, 市川 聡, 松田 彰, 前仲 勝実, 大場 靖子, 澤 洋文  日本獣医学会学術集会講演要旨集  160回-  390  -390  2017/08  [Not refereed][Not invited]
  
• Examination of antiviral activity of 5-ethynyl-1-ribofuranosylimidazole-4-carboxamide(EICAR) against rabies virus in vitro(和訳中)

  Anindita Paulina Duhita, 佐々木 道仁, 伊藤 直人, 杉山 誠, 南川 典昭, 周東 智, 乙黒 聡子, 市川 聡, 松田 彰, 前仲 勝実, 大場 靖子, 澤 洋文  日本獣医学会学術集会講演要旨集  160回-  390  -390  2017/08  [Not refereed][Not invited]
  
• 【One Healthの視点からみた感染症の現状と対策】 野生動物が保有するウイルスを対象とする研究

  佐々木 道仁, 大場 靖子, 澤 洋文  最新医学  72-  (4)  508  -512  2017/04  [Not refereed][Not invited]
   

  人獣共通感染症を克服するためには,病原微生物の自然宿主,自然界における存続機構と伝播経路を解明し,先回り対策を講ずることが必要である.遺伝子検出技術の進歩・普及に伴い,自然界に存在するウイルスに関する知見が急速に増加している.本稿では上記の目的に基づいて,我々が実施している野生動物の保有するウイルスを対象とした研究を紹介する.(著者抄録)
• 本邦発症の進行性多巣性白質脳症患者に対する塩酸メフロキン治療の有効性に関する検討

  三浦 義治, 池内 和彦, 岸田 修二, 中道 一生, 西條 政幸, 高橋 健太, 鈴木 忠樹, 三條 伸夫, 阿江 竜介, 中村 好一, 澤 洋文, 長嶋 和郎, 奴久妻 総一, 原 由紀子, 雪竹 基弘, 浜口 毅, 水澤 英洋, 山田 正仁  臨床神経学  56-  (Suppl.)  S292  -S292  2016/12  [Not refereed][Not invited]
  
• 進行性多巣性白質脳症の診断・治療の新展開 PMLの診断と治療

  三浦 義治, 岸田 修二, 中道 一生, 西條 政幸, 高橋 健太, 鈴木 忠樹, 三條 伸夫, 阿江 竜介, 澤 洋文, 奴久妻 聡一, 宍戸 由紀子[原], 雪竹 基弘, 浜口 毅, 水澤 英洋, 山田 正仁  NEUROINFECTION  21-  (2)  172  -172  2016/09  [Not refereed][Not invited]
  
• PMLとPMLサーベイランス

  三條 伸夫, 宍戸 由紀子[原], 中道 一生, 西條 政幸, 三浦 義治, 雪竹 基弘, 高橋 健太, 鈴木 忠樹, 岸田 修二, 澤 洋文, 中村 好一, 浜口 毅, 山田 正仁, 水澤 英洋, 横田 隆徳  NEUROINFECTION  21-  (2)  192  -192  2016/09  [Not refereed][Not invited]
  
• 本邦発症進行性多巣性白質脳症患者に対する塩酸メフロキン治療の多数例における有効性の解析

  三浦 義治, 中道 一生, 西條 政幸, 岸田 修二, 高橋 健太, 鈴木 忠樹, 三條 伸夫, 阿江 竜介, 澤 洋文, 奴久妻 聡一, 原 由紀子, 雪竹 基弘, 浜口 毅, 水澤 英洋, 山田 正仁  NEUROINFECTION  21-  (2)  229  -229  2016/09  [Not refereed][Not invited]
  
• ザンビア共和国に飛来するストローオオコウモリ(Eidolon helvum)から分離されたアデノウイルスの性状解析と分子疫学的解析

  小川寛人, 小川寛人, 小川寛人, 梶原将大, 直亨則, 丸山隼輝, 上野恵介, 石井秋宏, 石井秋宏, 藤倉大輔, HANG’OMBE Bernard, AARON Mweene, 山田雅夫, 東秀明, 東秀明, 澤洋文, 澤洋文, 高田礼人, 高田礼人  日本獣医学会学術集会講演要旨集  159th-  376  -376  2016/08/30  [Not refereed][Not invited]
  
• ウエストナイルウイルスの粒子放出過程におけるRab8bタンパク質の役割

  小林 進太郎, 鈴木 忠樹, 川口 晶, Phongphaew Wallaya, 好井 健太朗, 苅和 宏明, 大場 靖子, 澤 洋文  日本獣医学会学術集会講演要旨集  159回-  409  -409  2016/08  [Not refereed][Not invited]
  
• ヘパラン硫酸を介した狂犬病ウイルスの細胞吸着機構の解析

  佐々木道仁, アニンディタ パウリナ, 伊藤直人, 杉山誠, 福原秀雄, 尾瀬農之, 前仲勝実, 大場靖子, 澤洋文, 澤洋文  日本分子生物学会年会プログラム・要旨集(Web)  39th-  ROMBUNNO.2P‐0314 (WEB ONLY)  2016  [Not refereed][Not invited]
  
• ウエストナイルウイルス感染による変性タンパク質蓄積機構の解析

  小林 進太郎, Phongphaew Wallaya, 好井 健太朗, 平野 港, 武藤 芽未, 大場 靖子, 澤 洋文, 苅和 宏明  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  88回・38回-  [1P1135]  -[1P1135]  2015/12  [Not refereed][Not invited]
  
• 本邦発症の進行性多巣性白質脳症患者サーベイランスの現状と課題 厚労科研PML研究班PMLサーベイランス報告

  三浦 義治, 中道 一生, 岸田 修二, 西條 政幸, 高橋 健太, 鈴木 忠樹, 三條 伸夫, 阿江 竜介, 澤 洋文, 奴久妻 総一, 原 由紀子, 雪竹 基弘, 濱口 毅, 水澤 英洋, 山田 正仁  NEUROINFECTION  20-  (2)  175  -175  2015/09  [Not refereed][Not invited]
  
• 様々な血清型のListeria monocytogenesのマウス尾静脈接種モデルにおける病原性解析

  長谷部理絵, 中尾亮, 平井佑治, 鈴木章夫, 山崎剛士, 澤洋文, 高井伸二, 堀内基広  日本獣医学会学術集会講演要旨集  158th-  375  2015/08/30  [Not refereed][Not invited]
  
• Potential viral pathogens in wild rodents and bats in Africa

  石井 秋宏, 澤 洋文  医学のあゆみ  253-  (1)  37  -42  2015/04/04
• プリオン病及び遅発性ウイルス感染症の分子病態解明・治療法開発に関する研究 進行性多巣性白質脳症(PML)におけるJCウイルスタンパク質の発現様式の免疫組織学的検討

  澤洋文, 鈴木忠樹, 大場靖子  プリオン病及び遅発性ウイルス感染症の分子病態解明・治療法開発に関する研究 平成26年度 委託業務成果報告書  67  -70  2015  [Not refereed][Not invited]
  
• 北海道産マダニからの新規フレボウイルスの検出

  NAKAO RYO, KAJIWARA MASAHIRO, KYU EISHIN, MORI AKINA, NAO NAGANORI, MURAMATSU MIEKO, YOSHII KENTARO, KARIWA HIROAKI, SAWA HIROFUMI, SUGIMOTO CHIHIRO, TAKADA AYATO  日本獣医学会学術集会講演要旨集  157th-  451  2014/08/11  [Not refereed][Not invited]
  
• 次世代シーケンサーによるリステリア菌国内分離株のゲノム比較解析

  HASEBE RIE, NAKAO RYO, HIRAI YUJI, SUZUKI AKIO, YAMASAKI TAKESHI, SAWA HIROFUMI, TAKAI SHINJI, HORIUCHI MOTOHIRO  日本獣医学会学術集会講演要旨集  157th-  445  2014/08/11  [Not refereed][Not invited]
  
• Reinhard Kurth Honorary Lecture HTLV-I and Adult T Cell Leukemia/Lymphoma (ATLL) : Cellular and Molecular Basis of Transformation

  William W. Hall, Hirofumi Sawa, Hideki Hasegawa  JAIDS-JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES  65-  54  -54  2014/04  [Not refereed][Not invited]
  
• RNase内包ウイルスカプセルの作製と内部でのRNA分解反応

  杉村尚俊, 新倉謙一, 萩原恭二, 三友秀之, 伊藤嘉浩, 澤洋文, 長田義仁, 居城邦治  高分子学会予稿集(CD-ROM)  63-  (1)  2014
• ウイルスカプセルの内部空間での配列特異的RNA分解反応

  杉村尚俊, 新倉謙一, 萩原恭二, 三友秀之, 澤洋文, 居城邦治  高分子学会予稿集(CD-ROM)  63-  (2)  2014
• Involvement of EphA2-mediated tyrosine phosphorylation of Shp2 in Shp2-regulated activation of extracellular signal-regulated kinase

  K. Miura, Y. Wakayama, M. Tanino, Y. Orba, H. Sawa, M. Hatakeyama, S. Tanaka, H. Sabe, N. Mochizuki  Oncogene  32-  5292  -5301  2013/11/07  [Not refereed][Not invited]
   

  Shp2 is a positive regulator for Erk activation downstream of receptor tyrosine kinases for growth factors. It has been controversial how Shp2 induces Erk activation. We here demonstrate that EphA2 is responsible for Shp2-mediated Erk activation by phosphorylating Tyr542 and Tyr580 of Shp2 in the cells stimulated with growth factors. In NMuMG mammary epithelial cells stimulated with hepatocyte growth factor (HGF), HGF-dependent Erk phosphorylation was prolonged only in the presence of EphA2. This Erk activation paralleled the phosphorylation of Tyr542/580 of Shp2 and the association of Grb2 with Shp2, suggesting the positive signal involving Grb2 signal to activate Ras-Erk pathway. Immunohistochemical studies of mammary cancer specimens revealed that the cancer progression was associated with both Tyr580 phosphorylation of Shp2 and increased expression of EphA2, which were also correlated with increased Erk phosphorylation. Overexpression of either Shp2Thr468Met (a phosphatase- defective mutant found in Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth and sensorineural Deafness (LEOPARD) syndrome) or Shp2Asn308Asp (a phosphatase-active mutant found in Noonan syndrome) with EphA2 exhibited comparable activation of Erk and stronger activation than wild-type Shp2, suggesting the phosphatase-independent Erk activation. Expression of Shp2Thr468Met with Tyr542/580Phe mutations resulted in the suppression of Erk activation. Phosphatase-active and -inactive, and wild-type Shp2s bound equally to Grb2, suggesting that phosphorylation of Tyr542/580 of Shp2 was essential but not sufficient for Shp2-mediated Erk activation. We found that Gab1 (Grb2-associated binder 1) was involved in the mutant Shp2-mediated Erk activation. Zebrafish injected with Shp2Thr468Met mRNA showed cardiac edema, whereas those depleted of EphA2b showed less phenotype, suggesting that EphA2 might partly account for the phenotype of LEOPARD syndrome. Collectively, tyrosine phosphorylation of Shp2 by EphA2 contributes to the phosphatase-independent Shp2-mediated activation of Erk and might be involved in Shp2-associated diseases. © 2013 Macmillan Publishers Limited.
• Investigation of the role of JC virus VP1 cysteine residues

  Hirofumi Sawa  JOURNAL OF NEUROVIROLOGY  19-  (3)  301  -301  2013/06  [Not refereed][Not invited]
  
• 進行性多巣性白質脳症(PML)の病態解明への分子アプローチ

  鈴木忠樹, 澤洋文, 大場靖子, 片野晴隆, 佐藤由子, 長谷川秀樹, 長嶋和郎  日本病理学会会誌  102-  (1)  207  2013/04/26  [Not refereed][Not invited]
  
• オートファジーはウエストナイルウイルスの増殖を抑制する

  小林進太郎, 大場靖子, 山口宏樹, 佐々木道仁, 長谷部理絵, 木村享史, 澤洋文  日本ウイルス学会学術集会プログラム・抄録集  61st-  2013
• 酵素内包ウイルスカプセルの作製と微小空間での酵素反応

  杉村尚俊, 新倉謙一, 萩原恭二, 三友秀之, 永井健治, 澤洋文, 居城邦治  高分子学会予稿集(CD-ROM)  62-  (2)  2013
• 霊長類動物からの新規ポリオーマウイルスの検出

  山口宏樹, 小林進太郎, 石井秋宏, 小川寛人, 木村享史, 澤洋文, 大場靖子  日本分子生物学会年会プログラム・要旨集(Web)  36th-  2013
• インフルエンザウイルス感染におけるマウス肺の病理学的変化の観察

  澤洋文, MUTO Mton‐Akio, 寸田祐嗣, 服部ともえ, 藤倉大輔, 中山洋佑, 宮崎忠昭, 丸山光生, 木村享史  日本ウイルス学会学術集会プログラム・抄録集  60th-  334  2012/10/31  [Not refereed][Not invited]
  
• ザンビアの野生動物におけるオルソポックスウイルス感染の疫学調査

  大場靖子, 石井秋宏, トーマス 由佳, 小川寛人, 中村一郎, 木村享史, 森川茂, 西條政幸, 澤洋文  日本ウイルス学会学術集会プログラム・抄録集  60th-  443  2012/10/31  [Not refereed][Not invited]
  
• JC virus encephalopathy is associated with a novel Agnoprotein-deletion JCV variant

  Xin Dang, Christian Wuethrich, Jennifer Gordon, Hirofumi Sawa, Igor Koralnik  JOURNAL OF NEUROVIROLOGY  18-  27  -27  2012/05  [Not refereed][Not invited]
  
• Glutathione-triggered drug release from virus capsules in cells

  Kenichi Niikura, Keita Nagakawa, Yusuke Musashi, Hirofumi Sawa, Masataka Kinjo, Kuniharu Ijiro  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY  243-  2012/03  [Not refereed][Not invited]
  
• Shape dependent immunogenicity of antigen-coated gold nanomaterials

  Tatsuya Matsunaga, Kenichi Niikura, Tadaki Suzuki, Shintaro Kobayashi, Hiroki Yamaguchi, Hirofumi Sawa, Kuniharu Ijiro  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY  243-  2012/03  [Not refereed][Not invited]
  
• ウエストナイルウイルス感染神経細胞におけるユビキチン化タンパク質の蓄積

  小林進太郎, 小林進太郎, 大場靖子, 山口宏樹, 木村享史, 澤洋文, 澤洋文  日本ウイルス学会学術集会プログラム・抄録集  60th-  2012
• 形状の異なる金ナノ粒子を用いた擬似ウイルス粒子の作成とワクチン活性の形状依存性

  松永達也, 新倉謙一, 鈴木忠樹, 小林進太郎, 山口宏樹, 澤洋文, 居城邦治  日本DDS学会学術集会プログラム予稿集  28th-  2012
• 霊長類動物におけるパラインフルエンザウイルスの疫学調査

  佐々木道仁, 石井秋宏, 大場靖子, トーマス由佳, 小川寛人, 中村一郎, 木村享史, 澤洋文, 澤洋文  日本ウイルス学会学術集会プログラム・抄録集  60th-  2012
• ザンビアにおける霊長類動物からの新規ポリオーマウイルスの検出

  山口宏樹, 山口宏樹, 大場靖子, 小林進太郎, 小林進太郎, 石井秋宏, 小川寛人, 中村一郎, THOMAS由佳, 木村享史, 澤洋文, 澤洋文  日本ウイルス学会学術集会プログラム・抄録集  60th-  2012
• 霊長類動物におけるパラインフルエンザウイルス3型の疫学調査

  佐々木道仁, 石井秋宏, 大場靖子, THOMAS Yuka, 小川寛人, 中村一郎, 木村享史, 澤洋文, 澤洋文  日本分子生物学会年会プログラム・要旨集(Web)  35th-  2012
• ダチョウ肝臓に発現する異物代謝酵素の配列解析

  川合佑典, 渡邉研右, 石井秋宏, 大沼愛子, 澤洋文, 池中良徳, 石塚真由美  日本進化学会大会プログラム・講演要旨集(Web)  14th-  2012
• Identification of the plague reservoir in an endemic area of Zambia

  Bernard M. Hang'Ombe, I. Nakamura, D. Kaile, A. S. Mweene, K. L. Samui, B. S. Kilonzo, H. Sawa, C. Sugimoto, B. Wren  Onderstepoort Journal of Veterinary Research  79-  (2)  2012  [Not refereed][Not invited]
  
• ウマMHC class Iが示すウマヘルペスウイルス1型レセプター機能の解析

  佐々木道仁, 五十嵐学, 澤洋文, 伊藤公人, 福士秀人, 木村享史  日本獣医学会学術集会講演要旨集  152nd-  239  -239  2011/08/31  [Not refereed][Not invited]
  
• 糖鎖認識を利用した金属ナノ粒子の集合化によるウイルス検出

  永川桂大, 新倉謙一, 野村尚生, 松尾保孝, 鈴木忠樹, 澤洋文, 居城邦治  バイオ・高分子シンポジウム講演要旨集  21st-  29-30  2011/07/15  [Not refereed][Not invited]
  
• ウイルス粒子の再集合における金属ナノ粒子内包条件の検討

  永川桂大, 新倉謙一, 鈴木忠樹, 松尾保孝, 澤洋文, 居城邦治  ナノ学会大会講演予稿集  9th-  113  2011/06/02  [Not refereed][Not invited]
  
• ウイルス様ナノカプセルを包含した金ナノシェル型構造体の構築

  永川桂大, 野村尚生, 新倉謙一, 鈴木忠樹, 松尾保孝, 澤洋文, 居城邦治  日本化学会講演予稿集  91st-  (3)  989  2011/03/11  [Not refereed][Not invited]
  
• ザンビア共和国の霊長類動物におけるポリオーマウイルスの疫学調査

  山口宏樹, 小林進太郎, 大場靖子, 石井秋宏, 小川寛人, Thomas由佳, 木村享史, 澤洋文  日本獣医学会学術集会講演要旨集  152nd-  2011
• 特別講演 進行性多巣性白質脳症(PML)の原因ウイルスであるJCウイルスの細胞内動態 (第3回信州NeuroCPC)

  澤 洋文, 高 昌星  信州医学雑誌  59-  (3)  202  -207  2011
• Progressive multifocal leukoencephalopathy (PML)

  Hidehiro Mizusawa, Shuji Kishida, Masayuki Saijo, Motohiro Yukishita, Yukiko Shishido-Hara, Hirohumi Sawa, Kazuo Nagashima, Souichi Nukuzuma, Masahito Yamada  Clinical Neurology  51-  (11)  1051  -1057  2011  [Not refereed][Not invited]
   

  Progressive multifocal leukoencephalopathy (PML) is caused by reactivation of latently infected JCV when hosts' immune system is impaired by HIV infection, hematologic diseases, collagen diseases, immunemodulatory therapy and so on. PML was rare but HIV infection and Natalizumab have made it much more common while the prognosis is much better than other PML. PML patients present with various signs and symptoms including hemiparesis, dementia aphasia, visual disturbance, cranial nerve paresis, cerebellar signs and bladder bowel disturbance. Brain MRI reveals characteristic demyelinating lesions in the CNS white matter and CSF mild increase of protein with or without mild mononuclear pleocytosis. Detection of JCV genome from CSF is crucial for the clinical diagnosis of PML. PML was once thought to be fatal but some HIV infected PML patients showed halting progression or even recovery after introduction of HAART. In addition, anti-malarial drug mefloquine was found to be effective. Recovery of immunity may provoke some inflammatory responses known as immune reconstruction inflammatory syndrome (IRIS) which requires high dose corticosteroid. In Japan, we are providing free test of CSF-JCV genome and organized a unique system for surveillance and clinical research of PML. Using this system we hope to improve diagnosis and therapy of PML in Japan.
• 分子シャペロンCdc37/Hsp90はTaxの細胞内安定性とNF‐κB誘導活性を規定する

  池辺詠美, 川口晶, 中野聡子, 八尋隆明, 田村陽介, 松本昂, 三井孝広, 仙波和代, 緒方正男, 堀光雄, 澤洋文, 田中勇悦, 西園晃, 長谷川秀樹, 伊波英克  生化学  83回・33回-  3P  -0993  2010/12  [Not refereed][Not invited]
  
• 野生齧歯動物からの新規ポリオーマウイルスの検出

  大場靖子, 小林進太郎, 中村一郎, 石井秋宏, 木村享史, 澤洋文  日本ウイルス学会学術集会プログラム・抄録集  58th-  445  2010/10/15  [Not refereed][Not invited]
  
• A novel deletion in JC Virus agnoprotein causes productive infection of cortical pyramidal neurons

  Xin Dang, Christian Wuthrich, Jennifer Gordon, Hirofumi Sawa, Igor Koralnik  JOURNAL OF NEUROVIROLOGY  16-  24  -24  2010/10  [Not refereed][Not invited]
  
• Disruption of Intracellular Vesicular Trafficking by Agnoprotein is Essential for Viroporin Activity and JC Virus Replication

  Tadaki Suzuki, Yasuko Orba, Yoshinori Malcino, Yuki Okada, Yuji Sunden, Takashi Kimura, Hideki Hasegawa, Tetsutaro Sata, William W. Hall, Hirofumi Sawa  JOURNAL OF NEUROVIROLOGY  16-  84  -84  2010/10  [Not refereed][Not invited]
  
• Large T antigen promotes JC virus replication in G2 arrest by inducing G2 checkpoint signaling

  Yasuko Orba, Tadaki Suzuki, Takashi Kimura, Hirofumi Sawa  JOURNAL OF NEUROVIROLOGY  16-  64  -65  2010/10  [Not refereed][Not invited]
  
• Cys80 of JC Virus Capsid Protein, VP1 is Essential for Intrapentamer Disulfide Bond and Pentamer Formation

  Shintaro Kobayashi, Tadaki Suzuki, Manabu Igarashi, Noriko Ohtake, Keita Nagakawa, Kimihito Ito, Kenichi Niikura, Takashi Kimura, Harumi Kasamatsu, Hirofumi Sawa  JOURNAL OF NEUROVIROLOGY  16-  45  -46  2010/10  [Not refereed][Not invited]
  
• ウイルスのシアル酸認識を利用した有機/無機ハイブリッドナノ粒子の構築

  永川桂大, 新倉謙一, 大竹範子, 鈴木忠樹, 松尾保孝, 澤洋文, 居城邦治  高分子学会予稿集(CD−ROM)  59-  (2 Disk1)  ROMBUNNO.2PC119  2010/09/01  [Not refereed][Not invited]
  
• pHに応答した分子放出のためのウイルスナノカプセルの機能化

  大竹範子, 新倉謙一, 鈴木忠樹, 三國新太郎, 松尾保孝, 永川桂太, 金城政孝, 澤洋文, 居城邦治  日本化学会講演予稿集  90th-  (3)  888  2010/03/12  [Not refereed][Not invited]
  
• 分子シャペロン阻害剤によるTaxとTax結合蛋白質の機能相関性に対する抑制的影響

  池辺詠美, 川口晶, 川口晶, 田口慎也, 川嶋太郎, 田中勇悦, 堀光雄, 澤洋文, 西園晃, 長谷川秀樹, 伊波英克  日本ウイルス学会学術集会プログラム・抄録集  58th-  2010
• 新規エントリーレセプターを介したウマヘルペスウイルス1型の細胞内侵入機構の解析

  佐々木道仁, 長谷部理絵, 福士秀人, 谷山弘行, 澤洋文, 木村享史  日本獣医学会学術集会講演要旨集  150th-  2010
• Novel entry pathway of equine herpesvirus-1

  佐々木道仁, 長谷部理絵, 澤洋文, 福士秀人, 谷山弘行  生化学  2010
• Preparation of functionalized virus-like particles enabling pH-mediated release of target molecules

  OHTAKE Noriko, NIIKURA Kenichi, SUZUKI Tadaki, MIKUNI Shintaro, MATSUO Yasutaka, NAGAKAWA Keita, KINJO Masataka, SAWA Hirofumi, IJIRO Kuniharu  Abstr RIES Hokudai Symp  11th-  109-110  2010  [Not refereed][Not invited]
  
• Inhibition of the SDF-1F?-CXCR4 Axis by the CXCR4 Antagonist AMD3100 Suppresses the Migration of Human ATLL Cells and Murine Lymphoblastoid Cells from HTLV-I Tax Transgenic Mice

  A. Kawaguichi, Hidekatsu, I, H. Hasegawa, H. Sawa, M. Ogata, T. Tsuji, T. Kimura, T. Sata, W. W. Hall, Y. Orba  AIDS RESEARCH AND HUMAN RETROVIRUSES  25-  (11)  1212  -1212  2009/11  [Not refereed][Not invited]
  
• CXCR4アンタゴニスト(AMD3100)を用いたSCIDマウスにおけるTaxトランスジェニックマウス由来腫瘍細胞の組織浸潤抑制

  川口晶, 辻隆裕, 大場靖子, 木村享史, 相内章, 伊波英克, 緒方正男, 佐多徹太郎, 澤洋文, 長谷川秀樹  日本ウイルス学会学術集会プログラム・抄録集  57th-  445  2009/10/01  [Not refereed][Not invited]
  
• ウイルスを中心とした金ナノ粒子の三次元配列構造体とその光学応答

  永川桂大, 新倉謙一, 大竹範子, 鈴木忠樹, 松尾保孝, 澤洋文, 居城邦治  高分子学会予稿集(CD−ROM)  58-  (2 Disk1)  ROMBUNNO.1L5-12  2009/09/01  [Not refereed][Not invited]
  
• ウイルスを基盤とした金ナノ粒子の三次元配列化とその光学応答

  永川桂大, 新倉謙一, 大竹範子, 鈴木忠樹, 松尾保孝, 澤洋文, 居城邦治  高分子学会予稿集(CD−ROM)  58-  (1 Disk1)  1PD088  2009/05/12  [Not refereed][Not invited]
  
• ウイルス様ナノカプセルを鋳型とした金ナノ粒子の3次元配列化

  永川桂大, 新倉謙一, 大竹範子, 鈴木忠樹, 松尾保孝, 澤洋文, 居城邦治  日本化学会講演予稿集  89th-  (2)  1256  2009/03/13  [Not refereed][Not invited]
  
• Viroporin activity of JCV agnoprotein

  Tadaki Suzuki, Yasuko Orba, Yuji Sunden, Takashi Kimura, Hirofumi Sawa  JOURNAL OF NEUROVIROLOGY  15-  96  -97  2009  [Not refereed][Not invited]
  
• ヒトATL細胞及びTaxトランスジェニックマウス由来腫瘍細胞のSDF‐1αに対する走化性

  川口晶, 大場靖子, 木村享史, 伊波英克, 緒方正男, 辻隆裕, 佐多徹太郎, 澤洋文, 長谷川秀樹  日本ウイルス学会学術集会プログラム・抄録集  56th-  247  2008/10/01  [Not refereed][Not invited]
  
• ウイルスの糖鎖認識能を利用した金ナノ粒子の規則配列化

  永川桂大, 新倉謙一, 大竹範子, 鈴木忠樹, 松尾保孝, 澤洋文, 居城邦治  日本化学会バイオテクノロジー部会シンポジウム講演要旨集  11th-  303  2008/09/08  [Not refereed][Not invited]
  
• 光局在場を目指したウイルスカプセル表面における金ナノ粒子の規則配列化

  永川桂大, 新倉謙一, 大竹範子, 鈴木忠樹, 松尾保孝, 澤洋文, 居城邦治  応用物理学会学術講演会講演予稿集  69th-  (3)  1218  2008/09/02  [Not refereed][Not invited]
  
• ATL腫瘍細胞の走化性におけるCXCR4を介したシグナル経路の解析

  川口晶, 大場靖子, 木村享史, 伊波英克, 緒方正男, 佐多徹太郎, 澤洋文, 長谷川秀樹  補体シンポジウム講演集  45th-  179  2008/07  [Not refereed][Not invited]
  
• ウイルスカプセルを鋳型とした金ナノ粒子3Dアレイの作製

  永川桂大, 新倉謙一, 大竹範子, 鈴木忠樹, 松尾保孝, 澤洋文, 居城邦治  日本化学会講演予稿集  88th-  (2)  878  2008/03/12  [Not refereed][Not invited]
  
• ウイルスカプセルを鋳型とした金ナノ粒子3Dアレイの作製

  永川桂大, 石塚範子, 新倉謙一, 松尾保孝, 鈴木忠樹, 澤洋文, 居城邦治  高分子学会北海道支部研究発表会講演要旨集  42nd-  21  2008/01/29  [Not refereed][Not invited]
  
• Gab family scaffolding adaptor proteins are essential for postnatal maintenance of cardiac function via neuregulin-1/ErbB signaling

  Yoshikazu Nakaoka, Atsunori Kamiya, Takashi Minami, Hirofumi Sawa, Yasushi Fujio, Tatsuya Koyama, Hisao Hirota, Keiko Yamauchi-Takihara, Kinya Otsu, Masatsugu Hori, Toshio Hirano, Naoki Mochizuki  CIRCULATION  116-  (16)  5  -5  2007/10  [Not refereed][Not invited]
  
• A water-soluble hsp90 inhibitor 17-DMAG suppresses tax-mediated oncogenic signaling both in vitro and in vivo

  Hidekatsu Iha, Sin-ya Taguchi, Akira Kawaguchi, Taro Kawashima, Yuetsu Tanaka, Hirofumi Sawa, Akira Nishizono, Tetsutaro Sata, William Hall, Kuan T. Jeang, Hideki T. Hasegawa  AIDS RESEARCH AND HUMAN RETROVIRUSES  23-  (4)  594  -595  2007/04  [Not refereed][Not invited]
  
• The nuclear import of the HTLV-I tax protein is carrier and energy independent

  Takahiro Tsuji, Noreen Sheehy, Virginie Gautier, Hitoshi Hayakawa, Hirofumi Sawa, William Hall  AIDS RESEARCH AND HUMAN RETROVIRUSES  23-  (4)  614  -614  2007/04  [Not refereed][Not invited]
  
• 超分子ナノ材料としてのウイルスカプセルの作製

  永川桂大, 石塚範子, 新倉謙一, 松尾保孝, 鈴木忠樹, 澤洋文, 居城邦治, 居城邦治  高分子学会北海道支部研究発表会講演要旨集  41st-  45  2007/02/06  [Not refereed][Not invited]
  
• 【進行性多巣性白質脳症の新しい展開 PMLが治る時代へ向けて】 JCウイルスの最近の基礎的知見

  澤 洋文, 鈴木 忠樹, 大場 靖子, 寸田 祐嗣, 長嶋 和郎  BRAIN and NERVE: 神経研究の進歩  59-  (2)  101  -108  2007/02  [Not refereed][Not invited]
  
• 新規分子シャペロン阻害剤によるNF-κBシグナルの抑制と抗ガン剤への応用

  伊波英克, 川口晶, 田口慎也, 川嶋太郎, 村上真弓, 田中勇悦, 澤洋文, 佐多徹太郎, 堀光雄, 後藤和代, 西園晃, JEANG Kuan-Teh, HALL William W, 長谷川秀樹  生化学  2007
• Proteome analysis of Wig rat brain: A model for attention-deficit hyperactivity disorder [ADHD] research

  Yoshinori Masuo, Misato Hirano, Junko Shibato, Hitoshi Iwahashi, Hirofumi Sawa, Kazuo Nagashima, Randeep Rakwal  NEUROSCIENCE RESEARCH  58-  S47  -S47  2007  [Not refereed][Not invited]
  
• JC virus agnoprotein modifies plasma membrane permeability and facilitates

  Tadaki Suzuki, Yuki Okada, Yasuko Orba, Yuji Sunden, Takashi Kimura, Kazuo Nagashima, Hirofumi Sawa  JOURNAL OF NEUROVIROLOGY  13-  128  -128  2007  [Not refereed][Not invited]
  
• Pharmacological cdk inhibitor suppresses JC virus proliferation

  Yasuko Orba, Yuji Sunden, Tadaki Suzuki, Takashi Kimura, Shinya Tanaka, Kazuo Nagashima, Hirofumi Sawa  JOURNAL OF NEUROVIROLOGY  13-  109  -110  2007  [Not refereed][Not invited]
  
• Biological effects of Natalizurnab on JC virus infectivity in its permissive human neural cell lines in vitro

  Tadaki Suzuki, Satoko Yamanouchi, Yuji Sunden, Yasuko Orba, Shinya Tanaka, Takashi Kimura, Hirofumi Sawa  JOURNAL OF NEUROVIROLOGY  13-  127  -127  2007  [Not refereed][Not invited]
  
• Visualization of West Nile virus particles for examination on the cellular entry mechanism

  Yoshinori Makino, Tadaki Suzuki, Rie Hasebe, Akihiko Maeda, Hidehiro Takahashi, Takashi Kimura, Hirofumi Sawa  JOURNAL OF NEUROVIROLOGY  13-  100  -100  2007  [Not refereed][Not invited]
  
• Case of highly active anti-retroviral therapy-induced immune reconstitution inflammatory syndrome in AIDS-related progressive multifocal leukoencephalopathy.

  Rinsho Shinkeigaku  65-  1495  -1500  2007  [Not refereed][Not invited]
  
• Etiological agent and pathogenicity mechanism of PML.

  Nippon Rinsho  65-  1495  -1500  2007  [Not refereed][Not invited]
  
• Recent research on the JC virus.

  Brain Nerve  59-  101  -108  2007  [Not refereed][Not invited]
  
• Pathophysiological analysis of adult T-cell Leukemia model mouse

  Hall William W.  岡山実験動物研究会報  23-  43  -44  2006/12
• Gab family docking proteins are essential for prevention of dilated cardiomyopathy through transmitting neuregulin-ErbB signaling

  Yoshikazu Nakaoka, Keigo Nishida, Atsunori Kamiya, Hirofumi Sawa, Kinya Otsu, Hisao Hirota, Toshio Hirano, Naoki Mochizuki  JOURNAL OF CARDIAC FAILURE  12-  (8)  S159  -S159  2006/10  [Not refereed][Not invited]
  
• スリランカ津波被災地ならびにその近郊に生息する野生げっ歯類の病理学的検査

  木村享史, 沢洋文, 片倉賢, 中村一郎, 有川二郎, 松本芳嗣, RAJAPAKSE R.P.V.Jayanthe, 高島郁夫, 梅村孝司  日本獣医学会学術集会講演要旨集  140th-  72  -72  2005/08/31  [Not refereed][Not invited]
  
• Development of FRET-based probes for r-ras activation and R-Ras activation at Early/Recycling Endosomes

  Akiyuki Takaya, Takahiro Kamio, Michitaka Masuda, Naoki Mochizuki, Hirofumi Sawa, Mami Satoh, Kazuo Nagashim, Akiko Mizutani, Akira Matsuno, Michiyuki Matsuda  CELL STRUCTURE AND FUNCTION  30-  64  -64  2005/06  [Not refereed][Not invited]
  
• Development of a model of adult T-cell leukemia/lymphoma (Atll)

  H Hasegawa, H Sawa, M Lewis, Y Orba, T Sata, T Kurata, K Nagashima, N Sheehy, WW Hall  AIDS RESEARCH AND HUMAN RETROVIRUSES  21-  (5)  454  -455  2005/05  [Not refereed][Not invited]
  
• Subcellular localization of HTLV-I Tax using GFP-tagged protein

  TT Tsuji, N Sheehy, H Hayakawa, Gautier, V, H Sawa, Y Okada, W Hall  AIDS RESEARCH AND HUMAN RETROVIRUSES  21-  (5)  500  -500  2005/05  [Not refereed][Not invited]
  
• Agnoprotein plays a role in intracellular trafficking of JC virus

  Hirofumi Sawa, Tadaki Suzuki, Yuki Okada, Yasuko Orba, Yuji Sunden, Shingo Semba, Kazuo Nagashima  JOURNAL OF NEUROVIROLOGY  14-  63  -63  2005  [Not refereed][Not invited]
  
• A novel dynamin-associating molecule, formin-binding protein 17, induces tubular membrane invaginations and participates in endocytosis

  Y Kamioka, S Fukuhara, M Masuda, H Sawa, K Nagashima, M Matsuda, N Mochizuki  MOLECULAR BIOLOGY OF THE CELL  15-  79A  -79A  2004/11  [Not refereed][Not invited]
  
• JC virus agnoprotein facilitates viral propagation by removing FEZ1 from microtubules

  Tadaki Suzuki, Hirofumi Sawa, Yuki Okada, Yasuko Orba, Shingo Semba, Kazuo Nagashima  JOURNAL OF NEUROVIROLOGY  10-  131  -131  2004  [Not refereed][Not invited]
  
• Identification of cell surface molecule as a candidate receptor for JC virus

  C. Henmi, H. Sawa, Y. Orba, S. Tanaka, K. Nagashima  JOURNAL OF NEUROVIROLOGY  14-  42  -42  2004  [Not refereed][Not invited]
  
• Human polyomavirus agnoprotein disrupts the interaction between HP1 alpha and LBR

  H. Sawa, Y. Okada, T. Suzuki, Y. Orba, Y. Sunden, C. Henmi, S. Semba, H. Takahashi, S. Tanaka, K. Nagashima  JOURNAL OF NEUROVIROLOGY  14-  33  -33  2004  [Not refereed][Not invited]
  
• Desmoplastic malignant mesothelioma of the pleura: Autopsy reveals asbestos exposure (vol 53, pg 401, 2003)

  R Ishikawa, H Kikuchi, M Jin, M Fujita, T Itoh, H Sawa, K Nagashima  PATHOLOGY INTERNATIONAL  53-  (9)  652  -652  2003/09  [Not refereed][Not invited]
  
• リアルタイムRT-PCR法を用いたJCウイルスの動態解析

  山本 晋, 澤 洋文, 岩田 博司, 岡田 由紀, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  23-  90  -90  2003/05/01
• JC virusの神経親和性を規定する転写因子

  仙葉 愼吾, 長嶋 和郎, 大場 靖子, 澤 洋文  Neuropathology : official journal the Japanese Society of Neuropathology  23-  202  -202  2003/05/01
• siRNAを用いたJC virus関連蛋白質の発現抑制

  大場 靖子, 澤 洋文, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  23-  201  -201  2003/05/01
• JCウイルス外郭蛋白質VP1は糖脂質と糖蛋白質の糖鎖に結合する

  澤 洋文, 駒込 理佳, 田中 伸哉, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  23-  199  -199  2003/05/01
• Parkinsonism、顔面・体幹・四肢近位優位の筋萎縮を呈する神経原性筋萎縮症

  長嶋 淑子, 島 功二, 中馬 孝容, 真野 行生, 後藤 雄一, 西野 一三, 埜中 征哉, 澤 洋文, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  23-  315  -315  2003/05/01  [Not refereed][Not invited]
  
• 糖尿病・腎不全の高齢透析患者に生じた亜急性代謝性脳症

  長嶋 淑子, 岩崎 素之, 北村 淳, 寺坂 俊介, 布村 充, 高田 珠, 滝沢 英樹, 村上 弘則, 中馬 孝容, 真野 行生, 後藤 雄一, 西野 一三, 埜中 征哉, 澤 洋文, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  23-  (0)  305  -305  2003/05/01  [Not refereed][Not invited]
  
• The human polyomavirus Jc induces chromosomal instability in colonic cells

  L Ricciardiello, M Baglioni, MP Landini, A Ripalti, H Sawa, K Nagashima, RJ Frisque, A Goel, CR Boland, M Tognon, E Roda, F Bazzoli  GASTROENTEROLOGY  124-  (4)  A603  -A603  2003/04  [Not refereed][Not invited]
  
• An atypical form of sporadic panencephalopathic Creutzfeldt-Jakob disease in Japan [1]

  S. Yamamoto, H. Furukawa, T. Kitamoto, Y. Takamaru, N. Morita, M. Yasuda, Y. Okada, H. Sawa, K. Nagashima  Neuropathology and Applied Neurobiology  29-  (1)  77  -80  2003/02  [Not refereed][Not invited]
  
• Advanced glycation end products induce angiogenesis in vivo

  AC Stan, T Okamoto, S Tanaka, T Koike, M Kase, Z Makita, H Sawa, K Nagashima  ACTA NEUROPATHOLOGICA  104-  (5)  570  -570  2002/11  [Not refereed][Not invited]
  
• Expression of JC virus agnoprotein in progressive multifocal leukoencephalopathy brain

  AC Stan, Y Okada, H Sawa, S Endo, Y Orba, T Umemura, H Nishihara, S Tanaka, H Takahashi, K Nagashima  ACTA NEUROPATHOLOGICA  104-  (5)  570  -570  2002/11  [Not refereed][Not invited]
  
• Differential requirement for Apaf1 and Bcl-X-L in the regulation of programmed cell death during development

  H Yoshida, Y Okada, N Kinoshita, H Hara, M Sasaki, H Sawa, K Nagashima, TW Mak, K Ikeda, N Motoyama  CELL DEATH AND DIFFERENTIATION  9-  (11)  1273  -1276  2002/11  [Not refereed][Not invited]
  
• シグナル伝達アダプター分子CrkによるERMの局在変化と細胞運動能及び接着能の解析

  津田真寿美, 田中伸哉, 牧野吉倫, 西原広史, 沢洋文, 長嶋和郎  日本癌学会総会記事  61st-  317  2002/08/25  [Not refereed][Not invited]
  
• JCウイルスからみたグリアの生物科学 (特集 グリアの生物科学)

  仙葉 愼吾, 澤 洋文, 長嶋 和郎  神経研究の進歩  46-  (4)  557  -565  2002/08
• 関節拘縮・硬直脊椎・CK著高を伴うdysferlin欠損肢体型筋ジストロフィー(LGMD2B)の1例

  長嶋 淑子, 山口 康一, 村上 浩則, 篠原 敦也, 中馬 孝容, 真野 行生, 後藤 雄一, 南 成祐, 埜中 征也, 河合 祥雄, 澤 洋文, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  22-  184  -184  2002/05/01
• 神経症状から診断されたSjogren症候群の臨床病理学的検討

  長嶋 淑子, 滝沢 昌彦, 間口 四郎, 佐々木 勲, 篠原 敏也, 大場 靖子, 中馬 孝容, 真野 行生, 澤 洋文, 長嶋 和郎, 酒井 宏一郎  Neuropathology : official journal the Japanese Society of Neuropathology  22-  50  -50  2002/05/01
• 遺伝子導入ベクターとしてのJC virus(JCV)VP1の有用性

  逸見 千寿香, 岩田 博司, 駒込 理佳, 佐藤 真実, 田中 伸哉, 澤 洋文, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  22-  52  -52  2002/05/01
• 進行性多巣性白質脳症(PML)におけるJC virus Agnoproteinの発現と局在

  大場 靖子, 岡田 由紀, 遠藤 秀一, 澤 洋文, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  22-  49  -49  2002/05/01
• アダプター分子v‐CrkによるRho依存的細胞骨格制御と細胞運動能の解析

  津田真寿美, 田中伸哉, 西原広史, 沢洋文, 長嶋和郎  日本病理学会会誌  91-  (1)  159  2002/02/28  [Not refereed][Not invited]
  
• Laser capture microdissection法を用いた胆汁細胞診標本からの細胞採取とK‐ras遺伝子変異の検索

  大場靖子, 西原広史, 田中伸哉, 沢洋文, 伊藤智雄, 長嶋和郎  日本病理学会会誌  91-  (1)  298  2002/02/28  [Not refereed][Not invited]
  
• Molecular Neuropathology of JC Virus

  大場 靖子, 澤 洋文, 長嶋 和郎  Brain and nerve : 神経研究の進歩  54-  (2)  101  -109  2002/02
• DNA PolymeraseλKOマウスはKartagener症候群類似の病態を示す

  小林 洋介, 渡部 美穂, 岡田 由紀, 高井 裕之, 澤 洋文, 鈴木 宏志, 中西 真, 長嶋 和郎, 池田 恭治, 本山 昇  日本臨床分子医学会学術総会プログラム・抄録集  38回-  59  -59  2001/08  [Not refereed][Not invited]
  
• Broad distribution of the JC virus receptor contrasts with a marked cellular restriction of virus replication

  S Suzuki, H Sawa, R Komagome, Y Orba, M Yamada, Y Okada, Y Ishida, H Nishihara, S Tanaka, K Nagashima  VIROLOGY  286-  (1)  100  -112  2001/07  [Not refereed][Not invited]
   

  To investigate the early events of JC virus (JCV) infection, including attachment, penetration, transport to the nuclei, and replication of the virus, we analyzed the susceptibility of 15 different cell lines to infection using a semiquantitative polymerase chain reaction (PCR) assay, in situ hybridization, laser scanning confocal microscopy, and a viral replication assay. The cell lines examined were human permissive and nonpermissive cells as well as cells of monkey and mouse origin. JCV entry into the nuclei of the all cell lines was observed within 10 min after inoculation, demonstrating that the virus receptor is widely distributed among mammalian cells. inhibition of viral entry by an anti-JCV VP1 antibody and sialidase treatment to remove sialic acid residues, which are considered a candidate for the JCV receptor, suggested that VP1 may interact with the cellular surface sialic acid. In addition, chlorpromazine, a clathrin-dependent pathway inhibitor, significantly suppressed entry of JCV into nuclei. In spite of the broad spectrum of cells susceptible to JCV entry, replication of the virus occurred exclusively in human neuroblastoma cell lines. These results suggest that whereas JCV can enter a wide variety of cell types and localize to the nuclei, cell-specific intranuclear mechanisms are required for virus replication. (C) 2001 Academic Press.
• JC virus large T protein transforms rodent cells but is not involved in human medulloblastoma

  H Hayashi, S Endo, S Suzuki, S Tanaka, H Sawa, Y Ozaki, Y Sawamura, K Nagashima  NEUROPATHOLOGY  21-  (2)  129  -137  2001/06  [Not refereed][Not invited]
   

  JC virus (JCV) together with Simian virus 40 (SV40) and BK virus (BKV), belong to the polyomavirus group and these viruses are neuro-oncogenic to rodents by expression of large T antigen (LT), which binds to cellular p53 and pRB thus reducing the anticancer potential of the cell. The function of LT has not been clarified because small t antigen (st) is transcribed from the same start codon as the overlapping reading frame of LT, and is translated as a different protein with the same N-terminal residues (1-81 amino acids) by a splice-site variant of mRNA. To elucidate the function of LT without st, we constructed plasmids that express LT only by deleting the splicing region including the C-terminus of st, and consequently stable cell lines were established that express only JCLT, SV40LT and BKLT. The growth rates of these cells were examined in colonies on soft agar and it was found that LT alone has a transforming capacity; the order of efficiency being SV40LT, BKLT and JCLT. In addition, to verify the involvement of JCV in human medulloblastoma, eight cases of medulloblastoma, six cases of frozen material and five cases of paraffin-embedded tissues which included three cases of frozen tissues, were examined. PCR assay, genomic Southern blotting, and in situ hybridization were applied to detect the JCV genome, and LT and st were examined by immunohistochemistry; the results were compared with JCV-infected tissues as a positive control. All methods failed to detect not only JCV genome but also LT protein in medulloblastoma and it was concluded that JCV LT has transforming activities in rodent cells, but is not related to human medulloblastoma.
• HHV8-negative primary effusion lymphoma of the peritoneal cavity presenting with a distinct immunohistochemical phenotype

  S Tanaka, H Katano, K Tsukamoto, M Jin, S Oikawa, H Nishihara, H Sawa, K Sawada, M Shimizu, T Sata, Y Fujioka, K Nagashima  PATHOLOGY INTERNATIONAL  51-  (4)  293  -300  2001/04  [Not refereed][Not invited]
   

  Primary effusion lymphoma (PEL) has been recognized as a body-cavity-based lymphoma that was originally reported to be associated with human herpes virus 8 (HHV8) infection, and was frequently found in human immunodeficiency virus-positive (HIV) patients. Here we describe an autopsy case of PEL of the peritoneal cavity in an immunocompetent patient. Cytological analysis of tumor cells within ascites revealed immunocytochemical features of keratin positivity and CD45 negativity. At autopsy, the presence of a massive volume of ascites as well as diffuse tumor cell infiltrates within the serosa of the intestine and mesenterium were observed, Tumor cells were morphologically similar to anaplastic large-cell lymphoma, but were immunohistochemically positive for keratin and epithelial membrane antigen (EMA). They also showed no reactivity to representative lymphocyte surface markers including CD45, in addition to being negative for CD30 and p80(NPM/ALK). Molecular analysis of the tumor cells revealed monoclonality of the immunoglobulin heavy-chain gene rearrangement which demonstrated a lymphoma of the B-cell lineage. Furthermore, HHV8 was not detected by immunohistochemical analysis, PCR or nested PCR technique. Based on these results, we consider the present case to be an HHV8-negative PEL with keratin and EMA positivity.
• 滑膜肉腫の癌化機構におけるSYT‐SSX1キメラ蛋白の役割

  長井真人, 田中伸哉, 平賀博明, 津田真寿美, 遠藤秀一, 沢洋文, 西原広史, 長嶋和郎  日本病理学会会誌  90-  (1)  190  2001/03/01  [Not refereed][Not invited]
  
• すい癌におけるSykの発現の検討 浸潤・転移への関与について

  渡辺佳明, 西原広史, 田中伸哉, 沢洋文, 清水道生, 長嶋和郎  日本病理学会会誌  90-  (1)  292  2001/03/01  [Not refereed][Not invited]
  
• JC virus(JCV) agnoproteinの発現と機能に関する研究

  岡田由紀, 沢洋文, 遠藤秀一, 大場靖子, 西原広史, 田中伸哉, 長嶋和郎  日本病理学会会誌  90-  (1)  349  2001/03/01  [Not refereed][Not invited]
  
• Familial HTLV-I associated polyneuropathy

  T Nagashima, N Shinmyo, T Chuma, Mano, I, Y Okada, H Sawa, K Nagashima  AIDS RESEARCH AND HUMAN RETROVIRUSES  17-  S64  -S64  2001  [Not refereed][Not invited]
  
• Neural cell specific activation of the JC virus promoter in the presence of HTLV-1 Tax.

  Y Okada, H Sawa, S Tanaka, S Suzuki, H Hasegawa, WW Hall, K Nagashima  AIDS RESEARCH AND HUMAN RETROVIRUSES  17-  S56  -S56  2001  [Not refereed][Not invited]
  
• Semi-Quantitative RT-PCR analysis of Rat Brain in Methylmercury Intoxication.

  NAGASHIMA KAZUO, OIDA KENJI, NISHIHARA HIROSHI, TANAKA SHIN'YA, SAWA HIROFUMI  有機水銀の健康影響に関する研究 平成12年度 重金属等の健康影響に関する総合研究班  3-8  2001  [Not refereed][Not invited]
  
• 浮遊細胞特異的に発現するCDM‐family蛋白 DOCK2の機能の解析

  前田才恵, 西原広史, 田中伸哉, 松田道行, 沢洋文, 長嶋和郎  日本分子生物学会年会プログラム・講演要旨集  23rd-  432  2000/11/25  [Not refereed][Not invited]
  
• ヒト滑膜肉腫関連蛋白SYT‐SSX1とクロマチンリモデリング因子hBRMの結合の機能解析

  長井真人, 田中伸哉, 津田真寿美, 園部宏, 加藤宏幸, 平賀博明, 西原広史, 沢洋文, 長嶋和郎  日本分子生物学会年会プログラム・講演要旨集  23rd-  338  2000/11/25  [Not refereed][Not invited]
  
• Expression of JC virus early and late proteins in various cell lines and PML brains

  H Sawa, Y Okada, S Tanaka, Y Orba, S Endo, M Sasada, Y Hara, M Shintaku, K Nagashima  BRAIN PATHOLOGY  10-  (4)  757  -757  2000/09  [Not refereed][Not invited]
  
• 腫よう組織における前癌遺伝子産物CRKの発現と細胞増殖能に関する分子病理学的解析

  西原広史, 田中伸哉, 清水道生, 沢洋文, 松田道行, 長嶋和郎  Jpn J Cancer Res  91-  (Supplement (Sept))  499  2000/09/01  [Not refereed][Not invited]
  
• ヒト滑膜肉腫関連SYT‐SSX1遺伝子の形質転換機構

  長井真人, 田中伸哉, 沢洋文, 西原広史, 津田真寿美, 長嶋和郎  Jpn J Cancer Res  91-  (Supplement (Sept))  53  2000/09/01  [Not refereed][Not invited]
  
• JC virus 構成蛋白の発現と機能に関する研究

  岡田 由紀, 遠藤 秀一, 原 由紀子, 大場 靖子, 駒込 理佳, 田中 伸哉, 澤 洋文, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  20-  59  -59  2000/06/01
• Dendritic distribution of JC virus agnoprotein in PML brain.

  K Nagashima, H Sawa, Y Ohba, S Endo, Y Okada, S Tanaka  JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY  59-  (5)  427  -427  2000/05  [Not refereed][Not invited]
  
• HTLV-I TaxによるJC virusの活性化に関する研究

  岡田 由紀, 澤 洋文, 田中 伸哉, 伊藤 智雄, 長谷川 秀樹, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  19-  69  -69  1999/06/03
• 0499 新しい高周波広域地域探触子による血管外からの冠動脈の観察

  山田 聡, 西原 馨子, 齋藤 尚孝, 小野塚 久夫, 北畠 顕, 澤 洋文, 長嶋 和郎, 三神 大世  Japanese circulation journal  63-  (1)  274  -274  1999/03/01
• Cloning of a novel matrix metalloproteinase specifically expressed in femal reproductive tracts

  OHNISHI Junji, HIRANO Wakako, NAKANE Dai, MATSUI Hitoshi, KIMURA Atsushi, SAWA Hirofumi, NAGASHIMA Kazuo, TAKAHASHI Takayuki  日本分子生物学会年会プログラム・講演要旨集  21-  677  -677  1998/12/01
• 進行性多巣性白質脳症とJCウイルス調節領域

  澤 洋文, 古林 与志安, 平賀 博明, 田中 伸哉, 涌谷 陽介, 松本 理器, 森 雅裕, 余郷 嘉明, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  18-  182  -182  1998/05/28
• Distributional pattern of apoptotic cells in rat cerebellar vermis experimentally induced by methylmercury intoxication

  Y Kobayashi, H Sawa, H Akagi, C Itakura, Y Fujioka, K Nagashima  NEUROPATHOLOGY  18-  (1)  33  -37  1998/03  [Not refereed][Not invited]
   

  This report deals with the distributional pattern of apoptotic granule cells in rat cerebellar vermis following experimental methylmercury chloride (MMC) toxicosis. The rats administered MMC showed clinical signs of hind leg paralysis around day 19 and this developed to moribund by day 26. Pathologic examination of the moribund animals revealed that cerebellar degeneration characterized by apoptosis of the granule cells was not equally distributed in the cerebellar vermis; apoptotic granule cells were observed in the lingula, central lobule, Lobulus ascendens, pyramis vermis and culmen in the order of decreasing frequency: This selective vulnerability in MMC intoxication is similar to the granule cell loss of Minamata disease in humans and suggests that this rat model is a useful pathobiologic model for studding cerebellar degeneration in Minamata disease.
• 0822 拡張型心筋症におけるオステオポンチンの心筋線維化への関与

  渡邊 正司, 岡本 洋, 千葉 知, 熊本 秀樹, 中川 泉, 米谷 圭史, 小野塚 久夫, 北畠 顕, 川口 秀明, 三神 大世, 澤 洋文  Japanese circulation journal  62-  311  -311  1998/02/28
• Molecular cloning and characterization of a novel target of H-ras protein which belongs to RalGDS family

  N Mochizuki, T Murakami, N Makita, H Sawa, GR Post  CIRCULATION  96-  (8)  3112  -3112  1997/10  [Not refereed][Not invited]
  
• 有機水銀投与ラットでみられる小脳顆粒細胞アポトーシスの分布とアポトーシス関連蛋白/遺伝子の解析

  古林 与志安, 澤 洋文, 赤木 洋勝, 板倉 智敏, 藤岡 保範, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  17-  149  -149  1997/05/26
• 0801 Urokinase type plasminogen activator受容体(uPAR)を強制発現させた内皮細胞における線溶系の亢進

  澤 洋文, 藤井 聡, 北畠 顕  Japanese circulation journal  61-  313  -313  1997/03/05
• 1036 Plasminogen activator inhibitor-1(PAI-1)に対するアンチセンスオリゴヌクレオチドによるballoon injury後の内膜肥厚の抑制

  澤 洋文, 藤井 聡, 北畠 顕  Japanese circulation journal  61-  372  -372  1997/03/05
• Expression of amyloid precursor protein mRNA in vascular smooth muscle cells of the human brain

  RH Takahashi, H Sawa, A Takada, A Kitabatake, K Nagashima  NEUROPATHOLOGY  17-  (1)  11  -14  1997/03  [Not refereed][Not invited]
   

  Amyloid deposition in the cerebral vessels is the hallmark of cerebral amyloid angiopathy (CAA). In order to establish which type of cell produces amyloid, we performed Northern blot analysis and in situ hybridization studies on human meningeal arteries. Northern blotting showed that amyloid precursor protein (APP) mRNA was expressed not only in the cerebral cortex, but also in the meninges. An antisense RNA probe that hybridizes with APP transcripts was used for in situ hybridization. This analytic modality revealed APP messages in the tunica media of the meningeal arteries and arterioles. The presence of smooth muscle cells in these vessels was verified immunohistochemically on consecutive serial sections. These observations indicate that APP is produced by vascular smooth muscle cells.
• Pathologic processes leading to cerebral hemorrhage in amyloid angiopathy

  RH Takahashi, H Sawa, S Kuroda, H Saito, M Fujita, Y Fujioka, R Fukatsu, K Nagashima  NEUROPATHOLOGY  16-  (2)  99  -105  1996/06  [Not refereed][Not invited]
   

  This report concerns a study undertaken to elucidate the pathogenesis of arterial rupture related to amyloid deposition, For this purpose, histochemical and immunohistochemical analyses were carried out on brain tissue specimens of a case of cerebral amyloid angiopathy (CAA) with multiple cortical hemorrhages, A small amount of amyloid beta-protein was found in the tunica media vasorum in which the smooth muscle cells were well preserved, With increases in amyloid deposition, the entire arteriolar wall became concentrically thick, with amyloid largely occupying the tunica media; the number of smooth muscle cells was decreased and these cells were located in the intimal side of the vessel wall, Under these conditions, the elastic fibers and endothelial cells were relatively well preserved, In the advanced stage of amyloid deposition, the arterioles became devoid of smooth muscle cells and underwent either fibrous luminal occlusion or aneurysmal dilatation with fibrinoid necrosis and loss of elastic fibers; the latter would eventually rupture causing the hemorrhage, These findings suggest that amyloid beta-protein-related loss of arteriolar smooth muscle cells could be the initial event, with the subsequent damage of the vascular wall leading to cerebral hemorrhages in CAA patients.
• 新発色基質であるHNPPの神経疾患解析への応用 : FISH法、免疫染色法、in situ hybridization法の検討

  佐藤 真実, 高橋 文誉, 大場 靖子, 近藤 恭光, 鍵山 直人, 高橋 礼典, 澤 洋文, 長嶋 和郎  Neuropathology : official journal the Japanese Society of Neuropathology  16-  203  -203  1996/05/01
• Expression of vascular endothelial growth factor in human myocardial infarction

  K Shinohara, T Shinohara, N Mochizuki, Y Mochizukik, H Sawa, T Kohya, M Fujita, Y Fujioka, A Kitabatake, K Nagashima  HEART AND VESSELS  11-  (3)  113  -122  1996  [Not refereed][Not invited]
   

  To define mechanisms that may influence collateral circulation and angiogenesis, we investigated vascular endothelial growth factor (VEGF) mRNA expression in human hearts. In non-ischemic human hearts, VEGF mRNA was not detected in vessels, but was found in cardiomyocytes. In hearts with myocardial infarction, the intensity of the VEGF signal was much higher in smooth muscle cells of arterioles adjacent to necrosis and in infiltrating macrophages than in myocytes around the site of the necrosis. This study suggests that levels of VEGF expression are high in smooth muscle cells and macrophages around infarcted areas after myocardial infarction and that VEGF may play a role in promoting collateral circulation and angiogenesis in human ischemic hearts.
• SLEにおける肺高血圧症(PH)合併例の臨床的検討 : 日本循環器学会第62回北海道地方会

  山田 豊, 遠藤 由香, 布施川 哲, 西島 宏隆, 小林 毅, 安田 寿一, 沢 洋文, 長嶋 和郎  Japanese circulation journal  57-  492  -492  1993/05/20
• EXPRESSION OF THE RENIN GENE AND LOCALIZATION OF ITS PROTEIN IN THE HUMAN HEART

  Y ENDO, N MOCHIZUKI, H SAWA, A TAKADA, Y FURUTA, F TOKUCHI, K SHINOHARA, T SHINOHARA, H KAWAGUCHI, A KITABATAKE, K NAGASHIMA  CIRCULATION  86-  (4)  349  -349  1992/10  [Not refereed][Not invited]
  
• 2度の急性心筋梗塞を合併した肥大型心筋症の1例(日本循環器学会 第64回北海道地方会)

  横式 尚司, 米田 恵理子, 岡田 斉, 澤 洋文, 三神 大世, 小林 毅, 安田 寿一, 藤岡 保範, 長嶋 和郎  Japanese circulation journal  56-  711  -711  1992/07/20
• EXPRESSION OF THE ANGIOTENSINOGEN GENE AND LOCALIZATION OF ITS PROTEIN IN THE HUMAN HEART

  H SAWA, F TOKUCHI, N MOCHIZUKI, Y ENDO, Y FURUTA, T SHINOHARA, A TAKADA, H KAWAGUCHI, H YASUDA, K NAGASHIMA  CIRCULATION  86-  (1)  138  -146  1992/07  [Not refereed][Not invited]
   

  Background. There have been no reports on the presence of the tissue renin-angiotensin system in the human heart, although the presence of angiotensinogen has been described in the animal heart. Methods and Results. To determine whether angiotensinogen is synthesized in the human heart, we examined angiotensinogen messenger RNA (mRNA) synthesis in autopsy hearts by using ribonuclease protection assay. As a result, angiotensinogen mRNA was detected in the atrial muscle, muscles of the conduction system, and the left ventricular wall. In the left ventricular wall, mRNA expression was more prominent in the subendocardial muscles than in the midcardial or epicardial muscles. Using a monoclonal antibody to human angiotensinogen in immunoblotting experiments, we detected two closely spaced bands at approximately 70 kd in the heart, which was quite consistent with the human angiotensinogen molecule. Immunohistochemical studies with this monoclonal antibody demonstrated intense immunoreactivity in the atrial muscles, the muscles of the conduction system, and those of the subendocardial layers. Conclusions. We conclude that angiotensinogen was synthesized in the human heart. It was evident that the localization of angiotensinogen was not ubiquitous in the cardiac muscles, showing its predilection for the atrial muscles, muscles of the conduction system, and subendocardial layer of the left ventricle.
• ラット圧負荷モデルにおける心臓でのレニン, アンジオテンシノーゲンmRNAの発現について

  遠藤 由香, 望月 直樹, 澤 洋文, 川口 秀明, 安田 寿一, 篠原 敏也, 古田 康, 高田 明生, 長嶋 和郎  Japanese circulation journal  56-  397  -397  1992/03/01
• PHOSPHATIDYLINOSITOL AND INOSITOLPHOSPHATIDE METABOLISM IN HYPERTROPHIED RAT HEART

  SHOKI MIKAKO, KAWAGUCHI HIDEAKI, OKAMOTO HIROSHI, SANO HITOSHI, SAWA HIROFUMI, KUDO TOSHIYUKI, HIRAO NORIFUMI, SAKATA YOSHIHITO, YASUDA HISAKAZU  Japanese circulation journal  56-  (2)  142  -147  1992/02/20  

  The accumulation of both Inositol-(1,4,5)-trisphosphate (IP_3) and Inositol-(1,3,4,5)-tetrakisphosphate (IP_4) after hormonal stimulation has a physiological role, possibly in altering Ca^<2+> levels in cardiac tissue. However, the accumulation of inositol polyphosphate under pathophysiological conditions has not been studied. In our experiments the metabolism of phatidylinositol and IP_3 m cardiac myocytes as investigated. It was shown that basal levels of cytosolic phosphatidylinositol specific phospholipase C (PI-PLC), phosphatidylinositol-(4,5)-bisphosphate specific phospholipase C (PIP_2-PLC) activities markedly increased in stroke-prone spontaneously hypertensive rats (SHRSP) with age compared with age matched Wistar Kyoto rats (WKY). IP_3 kinase and IP_3 phosphatase activities also increased in SHRSP hearts with age. Their activities increased in WKY, but to a lesser antent than in SHRSPs. These data suggest that a PI turnover pathway such as the phosphatidylinositol 4,5-bisphosphate-IP_3-Ca^<2+> pathway or the diacylglyceride-protein kinase C pathway may have an important role in the development of hypertrophy in SHRSP heart.
• DETECTION OF HUMAN PAPILLOMAVIRUS DNA IN CARCINOMAS OF THE NASAL CAVITIES AND PARANASAL SINUSES BY POLYMERASE CHAIN-REACTION

  Y FURUTA, T TAKASU, T ASAI, T SHINOHARA, H SAWA, K NAGASHIMA, Y INUYAMA  CANCER  69-  (2)  353  -357  1992/01  [Not refereed][Not invited]
   

  The authors retrospectively searched for human papillomavirus (HPV) types 16 and 18 in 60 cases of carcinoma arising from the nasal cavities (NC) and paranasal sinuses (PS) by using the polymerase chain reaction (PCR) on DNA extracted from formalin-fixed, paraffin-embedded tissues. In cases of SCC (n = 49), the authors also compared the clinical features of patients with HPV-positive and HPV-negative results to determine the clinical significance of HPV. HPV 16 and 18 were detected in 7 of the 49 cases (14%) of SCC. In the other histologic types of carcinoma (n = 11), neither HPV 16 nor HPV 18 was detected. No significant differences in the clinical features were observed between patients with SCC with HPV-positive and HPV-negative results. The results suggest that HPV 16 and 18 are implicated in the pathogenesis of SCC arising from the NC and PS. However, the presence of HPV is not related to local progression, occurrence of metastases, or the prognosis of the patients.
• -1154-IMMUNOHISTOCHEMICAL STUDY OF ANP-C Rc(CLEARANCE RECEPTOR) : THE 54th ANNUAL SCIENTIFIC MEETING OF THE JAPANESE CIRCULATION SOCIETY

  MOCHIZUKI Naoki, SAWA Hirofumi, YASUDA Hisakazu, NAGASHIMA Kazuo, HIROSE Shigehisa  Japanese circulation journal  54-  (8)  1047  -1047  1990/08/20
• -0078-THE MECHANISM OF INCREASED ANGIO TENSIN CONVERTING ENZYME ACTIVITY STIMULATED WITH PLATELET ACTIVATING FACTOR

  Kawaguchi Hideaki, Sawa Hirofumi, Yasuda Hisakazu  Japanese circulation journal  54-  (7)  719  -719  1990/07/20
• -345-LOCALIZATION OF ATRIAL NATRIURETIC PEPTIDE IN HUMAN VENTRICULAR SYSTEM : Atrial Natriuretic Polypeptide : FREE COMMUNICATIONS(II) : PROCEEDINGS OF THE 53th ANNUAL SCIENTIFIC MEETING OF THE JAPANESE CIRCULATION SOCIETY

  Sawa Hirofumi, Mochizuki Naoki, Kawaguchi Hideaki, Yasuda Hisakazu, Sano Kimiaki, Shinohara Toshiya, Nagashima Kazuo  Japanese circulation journal  53-  (6)  579  -579  1989/06/20

Books etc

• プリオン病と遅発性ウイルス感染症

  金原出版 2010
• Molecular Biology of Tumor Virus Gene Products.

  Research Signpost 2009
• DNA viruses, Methods and Protocols.

  Humana Press 2005
• Understanding of Minamata disease. Methylmercury poisoning in Minamata and Niigata, Japan

  Japan Public Health Association 2001
• Endokrine und zellbiologische aspekte der arteriosklerose 10

  Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung 1996
• Arteriosklerose zerebraler gefäß 9

  Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung 1995

Association Memberships

• Global Virus network   分子生物学会   日本病理学会   日本内科学会   日本生化学会   日本神経病理学会   日本循環器学会   日本ウイルス学会   American Society of MicrobiologyAmerican Society for Biochemistry and Molecular BiologyInternational Society for Neurovirology   

Research Projects

• The role of MHC class I transactivator NLRC5 in COVID-19

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/04 -2025/03 

  Author : 小林 弘一, 澤 洋文, 福原 崇介, 應田 涼太
• VIrome analysis of bats in Indonesia

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2021/04 -2024/03 

  Author : 澤 洋文, 大場 靖子, 佐々木 道仁

   

  コウモリは人に致死的な肺炎、出血熱、脳炎、狂犬病 等の自然宿主と考えられている。現在の国際社会のCOVID-19の感染状況において、飛行機等による交通は制限されており、出入国の際には検疫が必要である。斯かる状況において、私達が海外において疫学活動を実施し、コウモリの検体を入手すること、および、海外からコウモリの検体を日本に輸送することは極めて困難である。 そこで本研究計画では、申請者の所属する研究施設に保管されているインドネシアのコウモリから採集した検体を使用することによって、海外に渡航する困難を克服できる。コウモリは3つの島 （スマトラ、ジャワ、スラウェシ）から採集しており、インドネシア地域でのウイルスの多様性を確認することが可能である。多くのウイルスの自然宿主であるコウモリにおいて、新規・既知のウイルスの存在を明らかにすることを計画する。公衆衛生学的に重要と考えられるコウモリ由来のウイルスの存在を明らかにすることを本研究の目的とする。 令和4年度においては我々の研究グループが2010年から2014年にかけて、スマトラ、ジャワ、スラウェシで採集したコウモリを対象とし血清(133検体）、糞便(96検体）および肺（172検体）を用いて、ネルソンベイオルソレオウイルスの血清疫学調査、ゲノムの検出、ウイルスの単離を試みた。その結果、血清(133検体）の内119検体で中和抗体が陽性であること、nested RT-PCR法を用いて、糞便96検体中6検体からウイルスゲノムを検出した。肺172検体からはゲノムは検出出来なかった。さらに、ゲノムが陽性であった糞便検体を、ウイルス感染を増強させることを目的として、セリンプロテアーゼであるTMPRSS2を強制発現させたVero細胞に接種してウイルスを単離した。最終的に単離したウイルスゲノムの分子系統学的解析、細胞での増殖の解析、マウスへの病原性の解析を実施した。
• 「ネオウイルス学」の国際活動支援

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2016/06 -2023/03 

  Author : 河岡 義裕, 朝長 啓造, 澤 洋文, 高橋 英樹, 川口 寧, 渡辺 登喜子, 松浦 善治, 鈴木 信弘, 長崎 慶三

   

  国際活動支援班では、各研究班の海外における研究活動を促進するために、 (1) 国際研究ネットワークの整備および強化、(2) 円滑な海外での研究試料の採取支援・提供、(3) 国際シンポジウムの開催および海外研究者の招聘による人的連携の促進、(4) サンプル収集のための海外への研究者の派遣の支援、(5) 最先端研究を行う先進諸国への若手研究者の中長期的派遣の支援、(6) 国際研究者ネットワークへの積極的な情報発信を行なっている。 2021年度は、国内で国際学会を1回 [第19回あわじ感染と免疫国際フォーラム]を共催し、また、同学会の直後に国際シンポジウムを主催した。これらの学会およびシンポジウムを通じて、海外から総計12名の研究者をオンライン招聘したほか、班員12名が発表した。招聘した研究者と計画研究班、および公募研究班班員はオンライン交流を通じて人的連携を構築した。また、引き続き、本領域のホームページの英語版を通して国際社会に本領域の活動を発信している。 COVID-19の感染拡大により、海外へサンプル収集に行くことが困難な状況であったため、海外からのサンプル輸入、および海外で採取したサンプルのネオウイルス学的解析、国内で採取したサンプルの解析結果との比較解析のための支援を実施した。
• ネオウイルス学：生命の源流から超個体、そしてエコ・スフィアーへ

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/04 -2022/03 

  Author : 河岡 義裕, 朝長 啓造, 澤 洋文, 松浦 善治, 川口 寧, 渡辺 登喜子, 鈴木 信弘, 高橋 英樹, 長崎 慶三

   

  「ネオウイルス学」領域内の計画研究および公募研究で得られた各班それぞれの研究テーマについて研究成果を取りまとめ、さらに班間、あるいは、「共進化」、「共生」、「多様性」ユニット間の連携テーマの重要性・意義を考慮しつつ、領域全体の研究成果を取りまとめた。業績集を作成し、冊子体として印刷して領域内外で配布することによって、本領域の研究成果を発信した。報告内容は、出来るだけ分かりやすい記述を心がけ、学術分野だけでなく、一般国民にも読んでもらえるような業績集を作成した。また、高校生、大学生、一般の国民に向けた講演会やセミナー、そして領域ホームページとTwitterによって、領域の活動内容や研究成果を報告することによって、国民に向けた研究成果の発信に努めた。また、新しい試みとして「ウイルスおりがみ」を作成した。ウイルスおりがみは、おりがみを折ることで、一般国民にウイルスの面白さを知ってもらうことを目的とした、科学コミュニケーションツールであり、公募研究班の牧野晶子助教（京都大）、デザイン・ユニットCOCHAE、合同会社SHIBUYA PUBLISHING & BOOKSELLERS、三宅文子アート・プランナーらのグループによって作成された。ウイルスおりがみには領域内の研究者による解説も加えられており、ウイルスの基本、構造の面白さ、役に立つウイルス、生き物の中にいる多様なウイルスについて学ぶことができる。ウイルスおりがみの配布の公募をしたところ2288部の応募があり、抽選で500部を無償で配布した。日本デザイン振興会が主催するグッドデザイン賞へウイルスおりがみを出品中である。
• Comprehensive investigation and functional analysis of endogenous viruses and endogenous viral elements in hematophagous arthropods and their host animals

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/06 -2021/03 

  Author : Sawa Hirofumi

   

  Blood-sucking arthropods, such as mosquitoes and ticks, have been studied as vectors of pathogens, including zoonotic viruses. In this study, we examined especially endogenous viruses in ticks and mosquitoes and investigated their roles in nature under the “dance floor” hypothesis; in blood-sucking arthropods, their indigenous microorganisms and those from human or animal blood are communicating each other. As a result, we have discovered new endogenous viruses in the mosquitoes and ticks collected worldwide and found that these viruses may have co-evolved with their hosts.
• Neo-virology: the raison d'etre of viruses

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/06 -2021/03 

  Author : Kawoaka Yoshihiro

   

  We have established a new academic field, designated as ‘Neo-virology’, in which we define a virus as a component of the global ecosystem and aim to elucidate its key roles in host organisms and the global ecosystem. We have constructed a system to support research activities, facilitate collaborations among research groups, develop human resources, plan and manage scientific meetings, and support public relations activities, all of which have resulted in accelerating this new field of research. Our efforts have produced numerous noteworthy publications and have helped develop many young investigators, who will continue the growth of Neo-Virology into the future.
• Investigation of latent pathogenic viruses in Latin America and South-eastern Asia by virome analysis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2020/03 

  Author : Sawa Hirofumi

   

  We obtained results based on three projects that aimed at; 1) Implementing epidemiological research in Latin America and South-eastern Asia, 2) Analysis of viral genome using “Virome methods”, 3) Investigation of distribution, evolution and the infection cycle of viruses. During screening of mosquito and fruit bat samples collected in Latin America and South-eastern Asia, respectively, we detected novel viruses i.e. flaviviruses from mosquitoes and gammaherpesviruses from fruit bats. Furthermore, we performed Virome analysis using pooled fecal samples obtained from 50 non-human primates, from which we isolated a new small circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses. Additionally, genome analysis of the polyomaviruses isolated from several different horseshoe bats indicated evidence of ‘host-switching’ during evolution in horseshoe bats.
• Functional analysis of equine herpesvirus-1 receptor and its application for infection prevention

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2008 -2011 

  Author : KIMURA Takashi, SAWA Hirofumi

   

  We analyzed the function of equine MHC class I molecule A68, which was cloned from an equine brain microvascular endothelial cell cDNA expression library as an EHV-1 receptor. Studies suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV-1 entry into equine cells, and also suggest that the amino acid residue at position 173 of the MHC class I molecule is involved in the efficiency of EHV-1 entry. In addition, we generated transgenic mice expressing A68, and also evaluated the usefulness of soluble forms of A68 molecules for preventing EHV-1 infection.
• ウイルスを鋳型とした金属微粒子3次元規則配列構造の構築

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2007 -2008 

  Author : 新倉 謙一, 澤 洋文, 松尾 保孝, 居城 邦治

   

  ウイルスのタンパク質が自己集合して形成するウイルス様微粒子(virus-like particle : VLP)は、ウイルスゲノムを持たずにウイルスと同じ経路で細胞内へ導入されるため、ドラッグデリバリーシステムのキャリアー等として注目されている。私たちはVLPの持つ糖鎖認識性に着目し、糖鎖の分子認識を利用してウイルスの周辺に規則的に金属微粒子を配列させることを目的に研究を進めた。まず金微粒子にシアル酸を提示させる技術を確立した。このシアル酸提示金微粒子は非常に水への分散性が高く、ウイルスと結合させても沈殿するようなことはなかった。VLPとシアル酸を修飾した金ナノ粒子の複合体形成の電子顕微鏡像(STEM)を詳細に検討すると、VLPの表層に特異的に金ナノ粒子が結合している様子が観察された。また、紫外可視吸収スペクトルの長波長シフトには、金ナノ粒子表面のシアル酸の有無によって有意な差が生じたことから、溶液中でも金ナノ粒子がVLPに結合していることが示された。さらに金ナノ粒子濃度の上昇に伴いプラズモン吸収の長波長側への大きなシフトが測定された。これはウイルスを鋳型とした金ナノ粒子の結合により、三次元的なプラズモンのカップリングが起きたことを意味している。さらにウイルスと金微粒子の結合を促進するために、デキストランを添加した。この効果は高分子クラウディング効果と言われるが、金属微粒子と生体分子(この場合はVLP)の特異的な結合をクラウディング効果で成功させた初めての例である。本研究によってウイルス-金微粒子複合体が外部の光と共鳴するプラズモン共鳴体になりうることを証明できた。
• Investigations for new pathogeneses of sudden deafness and Bell's palsy

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2007 -2008 

  Author : FURUTA Yasushi, ORIDATE Nobuhiko, TAKEICHI Norihito, SAWA Hirofumi

   

  原因不明の内耳疾患であるBell麻痺・突発性難聴において、発症時の患者の生体反応を遺伝子・蛋白質レベルで網羅的に捉えることにより、これら疾患における発症病態・新規病因を解明することを目的とし研究をおこなった。その結果、Bell麻痺においては自然免疫に関与する抗菌性小ペプチドであり、抗ウイルス作用を有することが判明しているα-defensinがベル麻痺の発症に関与していることが推定された。
• Analysis of West Nile virus neuroinvasion using in vitro blood-brain barrier models

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2006 -2007 

  Author : KIMURA Takashi, YOSHII Kentaro, SAWA Hirofumi

   

  1. The pathway used by West Nile virus (WNV) to leave the bloodstream and invade the central nervous system is poorly understood. To investigate how WNV cross the blood-brain barrier (BBB), we used confluent human umbilical vein endothelial cells (HUVEC) cultures on transwell inserts as an in vitro BBB model. 2. The structural protein genes (C-PrM-E region) of WNV 6-LP strain and Eg 101 strain were cloned into the pCMV expression vector. Sequential transfection of WNV sub-genomic replicon having an EGFP expression cassette and the vector that expressed the structural proteins led to the secretion of virus-like particles (VLPs). VLPs established a single-round of infection without production of progeny, and the physical structure of the VLPs was similar to that of infectious virions. 3. In vitro BBB model was infected with VLPs having structural proteins of high vilulent 6LP strain (6LP-VLPs) or VLPs having structural proteins of low vilurent Eg 101 strain (Eg-VLPs). By 24 h after infection, 10% of 6LP-VLPs had crossed in vitro BBB, whereas no Eg-VLPs seemd to traverse. Thus, the ability of WNV to cross BBB may correlate at least in part with viral virulence. Exposure of HUVEC to 6LP strain did not alter the localization of tight junction protein ZO-1, indicating that the passage of WNV across the BBB is not caused by the disruption of tight junction. On the other hand, infection of HUVEC with 6LP strain was inhibited by Chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. These results suggest the possibility that WNV may cross BBB by transcytosis via a clathrin-mediated endocytic pathway.
• Investigation on the pathogenesis of herpetic facial nerve paralysis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2004 -2006 

  Author : FURUTA Yasushi, SAWA Hirofumi, NAKAMARU Yuji

   

  1,Patterns of herpes virus reactivation in facial nerve paralysis We analyzed the relationship between HSV-1NZV load and onset of facial paralysis in patients with facial paralysis. The results indicate that facial paralysis can occur at various times between the early and the regression phase of HSV-1NZV reactivation, suggesting that there are variable patterns of development of facial nerve dysfunction caused by herpes virus reactivation and the progression of neuritis. 2,Gene expression analysis in patients with Bell's palsy Gene expression profile was analyzed in patients with Bell's palsy using DNA microarray. We found several genes which were up-or down-regulated at the onset of Bell's palsy. 3,Analysis of patients with Bell's palsy who showed incomplete recovery This study analyzed the causes of clinically diagnosed Bell's palsy who showed incomplete recovery of facial movement. The results indicate that VZV or HSV-1 reactivation is the major cause of Bell's palsy with poor prognosis. 4, Prevalence of HSV-1 reactivation in acute peripheral facial paralysis We analyzed the prevalence of HSV-1 reactivation in acute facial paralysis. The results suggested that in clinically diagnosed Bell's palsy 20% have ZSH and 20% have non-HSVNZV palsy, and the remaining 60% might have HSV-1 related palsy. 5, Efficacy of anti-viral therapy in herpetic facial nerve paralysis This study analyzed the HSV-1NZV load to assess the efficacy of the anti-viral therapy in patients with herpetic facial nerve paralysis. Anti-viral therapy may not be effective in all patients with HSV-1NZV reactivation because the facial paralysis can occur at various times between the early and the regression phase of HSV-1/VZV reactivation. 6, Assessments of the botulinum toxin therapy for synkinesis after facial palsy We have treated 17 patients with facial synkinesis after herpetic facial palsy with injection of botulinum toxin type A. The results indicate that the botulinum toxin injection is a safe and useful therapy for synkinesis after peripheral facial palsy.
• Analysis of human synovial sarcoma oncoprotein SYT-SSX and its therapeutic application.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2004 -2006 

  Author : TANAKA Shinya, AKAGI Tsuyoshi, SAWA Hirofumi, MINAMI Akio

   

  The cause of synovial sarcoma had been unknown for long time, and in 1994, Clark et al., identify the chimeric gene of t(X;18) and named it SYT-SSX. Then molecular analysis of SYT-SSX has been advanced and we have shown that SYT-SSX serves oncogenic potential on cells (Nagai, M., et al., PNAS, 2001). In this project, SYT-SSX was found to bind to not only one of the chromatin remodeling factors, hBRM, but to BRG (Gene Cells, 9, 2004), and also shown to induce cellular senescence by the induction of p21 (Oncogene, 24, 2005). Furthermore, IGF2 has been shown to play an important role for growth of synovial sarcoma cells (Oncogene, 25, 2006). We have shown that signaling adaptor protein Crk is indispensable for tumorigenesity of synovial sarcoma cell lines (Mol. Cancer Res., 7, 2006). One of the purpose of this project is to obtain the possible target molecules for synovial sarcoma treatment. As Crk knockdown cells is not going to die, but the malignant features were eliminated, thus, Crk may be the therapeutic target for the treatment of this sarcoma, and we will continue to analyze the mechanism of Crk for malignant character of synovial sarcoma.
• DNA damage checkpoint mechanism and its relation to aging

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2004 

  Author : MOTOYAMA Noboru, SAWA Hirofumi

   

  Recent identification of human genes responsible for premature aging syndromes has revealed that the encoded proteins function in the cellular response to DNA damage and thereby contribute to the maintenance of genomic integrity. These findings thus suggest that defects in the DNA damage response can result in aging as well as in cancer. We investigated that the mechanisms of the activation and the function of the Chk2-p53 pathway and of the FOXO pathway in cells subjected to DNA damage as well as in the relations of these signaling pathways to aging and cancer. Here we have shown that Chk2 regulates p53 protein stabilization by phosphorylating Mdm2 on Ser78 and that Chk2 regulates both transcription-independent and transcription-dependent mechanisms of p53-mediated apoptosis by stabilizing p53 and by increasing its transcriptional regulatory activity. Moreover, we have found that ATM-Chk2-p53-dependent signaling pathway triggered by DNA damage is dispensable for the activation of stress-induced premature senescence to IR or oxidative stress. These results suggest that both ATM-Chk2-p53-dependent and -independent signaling pathway, apoptosis and senescence, act as a "security gate" to prevent neoplastic transformation. However, as a side effect, hyperactivity of the ATM-Chk2-p53 pathway caused apoptosis may also compromises organism homeostasis and eventually results in aging. FOXO family regulates various biological activities, including cell cycle progression, the cellular response to oxidative stress, DNA repair, and apoptosis, suggesting that FOXO also contributes genomic integrity. Furthermore, we have shown that mammalian SIRT1 interacts physically, functionally, and physiologically with FOXO, suggesting that FOXO family protein and SIRTl may cooperate to extend life span in response to restriction of caloric intake in mammals by preventing DNA damage accumulation.
• Molecular investigation on prognostic factors and development of an tailor-made therapy for Hunt syndrome.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2002 -2003 

  Author : FURUTA Yasushi, SAWA Hirofumi, FUKUDA Satoshi

   

  1.Varicella-zoster virus DNA level and facial paralysis in Ramsay Hunt syndrome We have investigated whether the copy number of varicella-zoster virus (VZV) in saliva correlates with the clinical symptoms in patients with Ramsay Hunt syndrome (RHS). Patients with oropharyngeal zoster showed worse recovery of facial function than those without. It seems that the VZV DNA level in saliva reflects the kinetics of viral reactivation in the facial nerve as well as in the oropharyngeal epithelium in RHS patients. 2.Patterns of varicella-zoster virus reactivation in Ramsay Hunt syndrome We analyzed the relationship between VZV load and onset of facial paralysis in 42 RHS patients. The results indicate that facial paralysis in RHS can occur at various times between the early and the regression phase of VZV reactivation, suggesting that there are variable patterns of development of facial nerve dysfunction caused by VZV reactivation and the progression of neuritis. 3.Serological diagnosis of varicella-zoster virus reactivation in patients with acute peripheral facial paralysis We focused on the determination of an anti-VZV IgG antibody liter in sero-diagnosis by using an enzyme immunoassay (EIA) and investigated the diagnostic criteria for VZV reactivation by EIA. It was considered appropriate to give a diagnosis of VZV reactivation for those cases exhibiting a double-fold increasd in the VZV IgG EIA antibody titer. 4. Analysis of varicella-zoster virus load and cochleovestibular symptoms in patients with Ramsay Hunt syndrome The present study analyzed the relationship between VZV load and the onset of the cochleovestibular symptoms. The results indicate that cochleovestibular symptoms can occur in various timing from the early phase to the regression phase of VZV reactivation, suggesting that there are various patterns in the development of the eighth cranial nerve dysfunction caused by VZV reactivation and progression of neuritis. 5.Varicella-zoster virus reactivation is the major cause of acute peripheral facial paralysis m children This study analyzed the causes of acute peripheral facial paralysis in children by serological assays and PCR using paired sera and saliva samples. The results indicate that VZV reactivation is the major cause of acute peripheral facial paralysis in children, especially in ages between 6 and 15. In contrast, our data suggest that HSV-1 reactivation is not the major pathogenesis of Bell's palsy in children. 6.Analysis of patients with Bell's palsy who showed incomplete recovery This study analyzed the causes of clinically diagnosed Bell's palsy who showed incomplete recovery of facial movement. The results indicate that VZV or HSV-1 reactivation is the major cause of Bell's palsy with poor prognosis.
• 女性性殖器官に特異的な新規マトリックスメタロプロテアーゼMIFRの機能解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1999 -2003 

  Author : 大西 淳之, 澤 洋文, 大西 英理子, 松本 健一, 大西 英里子

   

  組換え体ヒトMMP-23を作製し、293細胞で発現させたところ、MMP-23は2型の膜タンパクとして合成され、細胞内においてfurin様プロセシング酵素により切断されて分泌型へ変換された。この際、furinの膜結合領域を削除するとMMP-23のプロセシングが亢進しないことから、細胞内におけるMMP-23のプロセシングは膜上で行われる必要があることが示唆された。培養培地より組換え体MMP-23を精製し、その酵素としての特性を調べたところ、組換え体MMP-23は熱変性タイプIおよびIVコラーゲンを限定分解する活性を示したが、フィブロネクチン、ラミニン、ビトロネクチン、テネイシン、フィブリノーゲン、α1-アンチトリプシンに対して何ら切断活性を示さなかった。また組換え体MMP-23によるタイプIゼラチンの切断様式は他のMMP分子(MMP-2とMMP-14)とは異なる切断様式を示した。次にMMP-23の生理的な機能を検討するために、抗MMP-23中和抗体をラット卵巣の被膜下に注入したところ、正常血清を注入したラットや未処理のコントロールラットと比較して有意に排卵が阻害された。このことはMMP-23には他のMMP分子とは異なる基質特異性があり、排卵過程においてその酵素活性が関与していることを示している。 初代培養系ラット顆粒膜細胞を用いて、卵胞刺激ホルモン(FSH)によるMMP-23の転写抑制機序について解析を行った。FSHの作用により細胞内のサイクリックAMP産生が促進され、その結果、Aキナーゼとホスファチジルイノシトール3リン酸キナーゼ(PI3キナーゼ)の両者が活性化されることがMMP-23の転写抑制に必須であるが、更にPI3キナーゼの下流に位置するAktが関与することを新たに見出した。しかし、顆粒膜細胞内で単独にPI3キナーゼ/Aktの経路を活性化させてもMMP-23の転写抑制は起こらないことから、この転写調節には同時にAキナーゼからのシグナル伝達が活性化されている必要があることが判明した。
• Analysis of transforming mechanism of human synovial sarcoma oncogene SYT-SSX

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2001 -2002 

  Author : TANAKA Shinya, AKAGI Tsuyoshi, MINAMI Akio, SAWA Hirofumi

   

  Human synovial sarcoma occurs primarily around the joint of extremities and more than 90% of cases have chromosomal translocation t(X;18) resulting the formation of chimeric gene SYT-SSX (synovial sarcoma translocation-synovial sarcoma X break point). As both of SYT and SSX did not share any homology region of known functional proteins, the transforming mechanism of SYT-SSX has not yet been elucidated. In this project, we established rat fibroblast cell line 3Y1 expressing SYT, SSX, and SYT-SSX, and found that SYT-SSX induced anchorage-independent growth and tumor formation in nude mice. In this SYT-SSX expressing cell lines, one of the SWI/SNF type of chromatin remodeling factor hBRM has been shown to bind to SYT-SSX and this association was essential for the transformation of 3Y1 cells. Especially, as the disruption of the binding of SYT-SSX and hBRM by specific peptides composed of 50 amino acids corresponding to hBRM155-206, suppressed the growth of SYT-SSX-3Y1 cells, these peptides could be one of the basic technique for the establishment of gene therapy for synovial sarcoma. We also found that in contrast to 3Y1 cells, SYT-SSX induced growth suppression of human adenocarcinoma cell line SW13. In this cell line, SYT-SSX induced p21WAF1/CIP1 expression confirmed by immunoblotting. The luciferase assay using p21 promoter-regulated construction, SYT-SSX was found to activate p21 promoter by transcription factor Sp1-dependent and p53-independent mechanism. Thus, SYT-SSX has differential effect for cell growth such as growth positive and growth negative mechanism, in different cell lines. We are currently considering the augmentation of growth negative function of SYT-SSX may induce the tumor cell suppression as a specific therapy of synovial sarcoma.
• Investigation on the pathogenesis of acute peripheral facial palsy caused by herpes simplex virus reactivation.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2001 

  Author : FURUWA Yasushi, SAWA Hirofumi

   

  1. Anti-HSV antibody in patients with peripheral facial palsy. The prevalence of antibodies to HSV among patients with Bell's palsy was significantly higher than the prevalence among those with VZV reactivation. VZV reactivation causes acute peripheral facial palsy in most patients who lack antibodies to HSV. 2. Herpes virus reactivation and serum tumor necrosis factor-α levels in atients with acute peripheral facial palsy. Tumor necrosis factor-α(TNF-α) plays an important role in several inflammatory and infectious diseases. We evaluated serum TNF-α levels of 75 patients with acute peripheral facial palsy. The results indicate that serum TNF-α levels are not affected by HSV-1 or VZV reactivation in patients with facial palsy. 3. Herpes simplex virus type 1 reactivation and antiviral therapy in patients with acute peripheral facial palsy. We sought to determine the efficacy acyclovir-prednisone therapy for patients with HSV-1 reactivation. The results suggest that early diagnosis of HSV-1 reactivation by PCR and subsequent acyclovir-prednisone therapy do not improve recovery from facial palsy. 4. Detection of the anti-ganglioside antibodies in patients with acute peripheral facial palsy. We examined 26 sera of the patients with history of over two episodes of acute facial nerve palsy for anti-ganglioside antibodtes. We detected anti-Ga1NAc-Gd1a-GD1a IgG antibody from 2 sera by ELISA method. No anti-ganglioside antibodes were detected by TLC. It is suggested that the anti-ganglioside antibody is not main pathogenesis of acute peripheral facial nerve palsy. 5. Herpes virus reactivation and gadolinium-enhanced magnetic resonance imaging in patients with facial palsy. We studied 15 patients with acute peripheral facial palsy to compare virologic tests and gadolinium-enhanced MRI findings. The results suggest that the meatal fundus or the geniculate ganglion may be affected first by virus reactivation in patients with herpetic facial palsy.
• The preventive effects of angiotensin II receptor blocker on lethal ventricular arrhythmias, a leading cause of sudden cardiac death, in experimental failing animal hearts

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2001 

  Author : KOHYA Tetsuro, SAWA Hirofumi, TAMAKI Nagara

   

  Several large prospeptive randomized studies demonstrated that treatment with angiotensin converting enzyme inhibitors (ACE-I) improved the prognosis of patients suffering from chronic heart failure (CHF). Although ACE-I exerts many pathophysiological actions, it is generally thought that inhibition of angiotensin-II (Ang-II) formation could account for the beneficial effects. However, it is still to be determined whether Ang-II receptor blocker (ARB) has the similar or superior effects on CHF patients compared to those of ACE-I. By using radionucleotide imaging and electrophyiological methods, we tried to compare the effects of ACE-I and ARB on experimental failing animal hearts, especially preventive effects on lethal ventricular arrhythmias, a leading cause of sudden cardiac death. We established a method of making myocardial infarction in rats that manifest congestive heart failure. Our first goal was to evaluate the changes in myocardial fatty acid metabolism (by BMIPP) as well as sympathetic nerve activity (by MIBG) at the different stages of heart failure, and to clarify the relation between these metabolic and autonomic alterations and arrhythmogenesis. Then, we will examine how ACE-I and ARB affect these pathophysiological processes. With the use of BMIPP, we observed, so called, "ischemic memory" in rats with myocardial infarction and investigated its mechanisms. In addition, there was an interesting relationship between "ischemic memory" and genesis of lethal ventricular arrhythmias. Although our project is still under way, these preliminary results have prompted us to further clarify the mechanisms of sudden death in patients with CHF. In the theoretical viewpoints, it is likely that ARB with high selectivity to Ang-II type 1-receptor (AT1-R) is more effective than ACE-I. We would like to continue our study in order to obtain the definitive answers.
• Investigation on CDDP resistance of Head and Neck Cancer.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1999 -2000 

  Author : INUYAMA Yukio, SAWA Hirofumi, FURUTA Yasushi, FUKUDA Satoshi

   

  a) Investigation on CDDP resistance-related genes by differential display We compared gene expression in the HNSCCs sensitive to CDDP with that in the resistant variants using a fluorescent differential display technique. We found that hCGα and human mt COII are highly expressed in the CDDP resistant variants. b) Investigation on CDDP resistance-related genes by cDNA microarray We compared gene expression in the HNSCCs sensitive to CDDP with that in the resistant variants using the cDNA microarray method. We found that five genes are differentially expressed between CDDP variants and the parental cells. c) CDDP resistance-related genes in clinical samples We compared gene expression in the biopsy materials from two cases of maxillary cancer using the cDNA microarray method. By using T7-based RNA amplification, we were able to analyze the difference in the gene expression of the small biopsy specimens.
• Analysis of mechanisms of demyelination in progressive multifocal leukoencephalopathy

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1998 -2000 

  Author : NAGASHIMA Kazuo, TANAKA Shinya, SAWA Hirofumi

   

  JC virus (JCV) causes in humans a demyelinating disease, progressive multifocal leukoencephalopathy (PML), but mechanisms of demyelination by JCV remain unknown. To investigate early events of JCV infection including attachment, penetration, and transport to the nuclei where viral replication occurs, we have analyzed the susceptibility of 15 different cell lines to infection using a semi-quantitative PCR assay, ISH, laser scanning confocal microscopy and a viral replication assay. JCV entry was observed in all cell lines within 10 min after inoculation. Inhibition of viral entry both by an anti-JCV VP1 antibody and sialidase treatment to remove sialic acid residues argues in favor that JCV VP1 functions as ligand and that JCV receptor is the cellular surface molecules containing sialic acids. In addition, chlorpromazine, a clathrin-dependent pathway inhibitor, significantly suppressed entry of JCV into nuclei, suggesting that JCV utilizes a clathrin pathway during the entry to cells. From these observations it has been proved that JCV receptor is broadly expressed in the cells originated from various tissues and species. Because the recent clinicopathological analyses have disclosed that human T-lymphotropic virus type I (HTLV-I) could be an underlying disease of PML, we have examined whether the HTLV-I encoded regulatory protein Tax activates JCV transcription. Using a dual luciferase assay and an electrophoretic mobility shift assay (EMSA), we found the expression of HTLV-I Tax activated the transcriptional potential of both early and late promoters of JCV in human neural cells in a NF-κB-dependent manner. In addition, we demonstrated the presence of Tax-bound protein(s) which were specifically present in non-neural cells. We also found that agnoprotein, one of the JCV late proteins, enhanced the JCV promoter itself. These results make it possible to establish an effective therapy against PML based on the mechanism of viral infection and multiplication in the neural cells.
• 血管内皮成長因子(VEGF)を併用した虚血心筋再潅流の研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1997 -1999 

  Author : 椎谷 紀彦, 国原 孝, 山内 英智, 若松 豊, 澤 洋文, 松崎 賢司

   

  1)New Zealand White rabbitの左前側方開胸、左冠動脈前下行枝結紮による急性心筋虚血モデルを用い、IGF-1(ソマトメジン)とvehicleの左房内投与の心筋梗塞縮小効果を検討した。検討項目としては虚血4.5時間のinfarct area/area at risk,TUNEL法によるapoptosisの同定とH-E染色による病理組織所見を用いた。JGF-11mg投与(n=4)2mg投与(n=2)4mg投与(n=1)とvehicleを比較検討したが、infarct area/area at riskはばらつきが大きく一定の差異を認めなかった。 2)New Zealand White rabbitの左前側方開胸、左冠動脈前下行枝結紮による急性心筋虚血モデルを用い、CO2レーザー心電図非同期出力200mJ、直径400μmで、虚血領域に3-5mm間隔で4-6個のtransmyocardial channelを作成した。また対照群として、虚血のみの群、同等の針穴をあけた群を設け、各群n=4施行した。虚血4.5時間のinfaect area /area at risk、TUNEL法によるapoptosisの同定とH-E染色による病理組織所見を検討したが、一定の成果は得られなかった。 以上1)2)の結果には、モデルが実験に適さない可能性と、IGF-1や心電図非同期TMLRが無効である可能性の両者があると考えられたが、予算の制約から中動物モデルへの変更は断念した。またここ2年間で、心筋虚血に関する種々GF投与の効果、TMLRとのcombination therapyの効果についての報告がいくつかみられるようになり、心筋虚血に関する実験継続の意義も低下した。そこで、脊髄の虚血障害防止に対する各種GFの応用に実験の中心をシフトした。 3)IGF-1はBBBを通過しないことが知られており、従来教室で脊髄虚血モデルとして用いてきたウサギ腎動脈下バルーン遮断モデルにおける応用を考慮して、ウサギくも膜下腔に留置カテーテルを挿入するモデルを検討した。(n=10)。しかし従来報告されている椎弓切除を伴う方法では、手術侵襲が大きく急性期死亡が多いため、本実験系には不向きであると判断した。そこで、実験動物をラットに変更し、くも膜下腔カテーテル留置慢性動物を作成した(n=3)。現在、胸部下行大動脈バルーン遮断による脊髄虚血障害モデルの確立に取り組んでいる。
• Analysis of apoptotic mechanism of cerebellar granular cells

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1997 -1999 

  Author : NAGASHIMA Kazuo, TANAKA Shinya, SAWA Hirofumi

   

  We have established a rat model of injury of cerebellar granule cells by intoxication of methylmercury chloride (MMC). To elucidate the molecular mechanisms of brain damage induced by MMC we initial ly examined the pathologic examination by H&E staining, electron microscope, TUNEL staining, and DNA fragmentation. These examination revealed that cerebellar granule cell death by organic mercury intoxication occurs via an apoptotic process and was restricted to the granule cells and that the distributional pattern of apoptotic cerebellar granule cells was specific in rat cerebellar vermis. Secondly, we investigated expression levels of apoptosis related molecules such as; BDNF, Trk B, Fas ligand, RIP, Akt, Bad, Bcl-2, Caspase 9, Caspase 3, CAS, and TIAR using Western blot and also evaluated the function of Purkinje cells by immunostaining with anti-calbindin D. Moreover, we investigated the alteration of gene expression of rat brain after MMC intoxication. These results demonstrated that expression of BDNF and Trk B were up-regulated, while RIP, CAS, Fas ligand, Bad, and Caspase 9 were down-regulated. The findings suggest that the up-regulated molecules function to suppress apoptosis at this stage. Gene expression analysis disclosed that among up-regulated genes 1) those related to apoptosis were protein kinase C γ, interleukin 13, insulin receptor substrate Z, cyclophilin, protein phosphatase 1, and RL/IF-1, 2) those related to hormone were vasopressin and oxytocin, 3) those related to enzyme were serum and glucocorticoid-regulated kinase, mast cell protease, serine protease, glycoprotein specific UDP-glucuronyl transferase, protein tyrosine phosphatase, 4) 7 miscellaneous molecules, and 5) 7 unknown genes. In contrast, down-regulated genes were 2α1 globin gene and 2 unknown genes. The results could be summarized that apoptosis suppressive genes were activated at the beginning of cerebellar apoptosis. Thus, it could be concluded that in the initial stage of MMC intoxication the expression of anti-apoptotic molecules were more enhanced to protect cell death than apoptosis-inducing molecules.
• Yeast One Hybrid法によるJC virus複製機構の検討

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1996 -1997 

  Author : 長嶋 和郎, 田中 伸哉, 澤 洋文, 篠原 敏也

   

  1.Clontech(Palo Alto,CA)社のyeast one-hybrid system)を用いてJC virus NF-1領域に特異的に結合する蛋白を同定した。NF-1配列を用いたスクリーニングで30個の陽性クローンから4個はこれまでにdata baseに登録されていない未知遺伝子であったためにこのDNA配列を確認し、NF-1/Xをcloningし、その発現が脳に多いことを示した。 2.ヒトに無症候性に感染し、常時尿に排泄されている型Archetype JC virusであるCY株(調節領域が原始型)がCOS-7細胞で増殖することを見いだし、この細胞を用いた解析系を樹立した。Mad-1(調節領域がPML型)と調節領域をCYに変えたキメラウイルスCY/Mad-1で感染性を調べ、cellular tropsimに調節領域が関与していることを示した。 3.Microinjection法により非感受性細胞であるCOS7でも細胞膜を越えてvirusを注入するとvirusの増殖が見られることから、virus感染に細胞膜因子が存在することを示唆した。 4.過去2年間に日本国内にて発症が確認されたPMLの5症例のregulatory regionを解析し、4例のPML型と1例のArchetype JCの症例を見い出し、報告した。またPMLの髄液を用いたPCRによる診断方法を確立した。

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• JCウイルスのVP-1に対するsiRNA、およびそれを含有してなる医薬組成物

  特許第4672654号
• JCウイルスagnoを対象としたPMLの治療

  特許第4840792号