研究者データベース

佐々木 道仁(ササキ ミチヒト)
人獣共通感染症国際共同研究所 分子病態・診断部門
講師

基本情報

所属

  • 人獣共通感染症国際共同研究所 分子病態・診断部門

職名

  • 講師

学位

  • 博士(獣医学)(北海道大学)

ホームページURL

科研費研究者番号

  • 70609403

J-Global ID

研究キーワード

  • 野生動物由来ウイルス   SARS-CoV-2   ロタウイルス   モデル動物解析   ウイルス病原性発現機構   新興感染症   人獣共通感染症   狂犬病ウイルス   分子生物学   ウイルス学   

研究分野

  • ライフサイエンス / ウイルス学
  • ライフサイエンス / 獣医学

職歴

  • 2021年04月 - 現在 北海道大学 人獣共通感染症国際共同研究所 講師
  • 2020年05月 - 2021年03月 北海道大学 人獣共通感染症リサーチセンター 講師
  • 2020年04月 - 2020年04月 北海道大学 人獣共通感染症リサーチセンター 助教
  • 2015年04月 - 2020年03月 北海道大学 人獣共通感染症リサーチセンター 特任助教
  • 2011年04月 - 2015年03月 北海道大学 人獣共通感染症リサーチセンター 博士研究員

学歴

  • 2007年04月 - 2011年03月   北海道大学   獣医学研究科
  • 2001年04月 - 2007年03月   酪農学園大学   獣医学部   獣医学科

所属学協会

  • 日本獣医学会   日本分子生物学会   日本ウイルス学会   

研究活動情報

論文

  • 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年09月06日 
    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.
  • 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 2022年08月18日 
    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.
  • 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年08月15日 
    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.
  • 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年08月06日 
    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.
  • 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年07月23日 
    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.
  • 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年06月 
    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.
  • 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年04月15日 
    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.
  • 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 2022年03月30日 
    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.
  • Kosuke Okuya, Takanari Hattori, Takeshi Saito, Yoshihiro Takadate, Michihito Sasaki, Wakako Furuyama, Andrea Marzi, Yoichi Ohiro, Satoshi Konno, Takeshi Hattori, Ayato Takada
    Microbiology spectrum e0155321  2022年03月23日 
    Antibody-dependent enhancement (ADE) of infection is generally known for many viruses. A potential risk of ADE in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has also been discussed since the beginning of the coronavirus disease 2019 (COVID-19) pandemic; however, clinical evidence of the presence of antibodies with ADE potential is limited. Here, we show that ADE antibodies are produced by SARS-CoV-2 infection and the ADE process can be mediated by at least two different host factors, Fcγ receptor (FcγR) and complement component C1q. Of 89 serum samples collected from acute or convalescent COVID-19 patients, 62.9% were found to be positive for SARS-CoV-2-specific IgG. FcγR- and/or C1q-mediated ADE were detected in 50% of the IgG-positive sera, whereas most of them showed neutralizing activity in the absence of FcγR and C1q. Importantly, ADE antibodies were found in 41.4% of the acute COVID-19 patients. Neutralizing activity was also detected in most of the IgG-positive sera, but it was counteracted by ADE in subneutralizing conditions in the presence of FcγR or C1q. Although the clinical importance of ADE needs to be further investigated with larger numbers of COVID-19 patient samples, our data suggest that SARS-CoV-2 utilizes multiple mechanisms of ADE. C1q-mediated ADE may particularly have a clinical impact since C1q is present at high concentrations in plasma and its receptors are ubiquitously expressed on the surfaces of many types of cells, including respiratory epithelial cells, which SARS-CoV-2 primarily infects. IMPORTANCE Potential risks of antibody-dependent enhancement (ADE) in the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been discussed and the proposed mechanism mostly depends on the Fc gamma receptor (FcγR). However, since FcγRs are exclusively expressed on immune cells, which are not primary targets of SARS-CoV-2, the clinical importance of ADE of SARS-CoV-2 infection remains controversial. Our study demonstrates that SARS-CoV-2 infection induces antibodies that increase SARS-CoV-2 infection through another ADE mechanism in which complement component C1q mediates the enhancement. Although neutralizing activity was also detected in the serum samples, it was counteracted by ADE in the presence of FcγR or C1q. Considering the ubiquity of C1q and its cellular receptors, C1q-mediated ADE may more likely occur in respiratory epithelial cells, which SARS-CoV-2 primarily infects. Our data highlight the importance of careful monitoring of the antibody properties in COVID-19 convalescent and vaccinated individuals.
  • 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年03月 
    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.
  • 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 2021年12月21日 
    Mass spectrometry 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 SARS-CoV-2 3CL protease inhibitors. Primary screening of 32,033 chemical samples was completed in 12 hours. Among the hits showing selective, dose-dependent 3CL-inhibitory activity, 8 compounds showed antiviral activity in cell-based assay.
  • 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年12月 
    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.
  • 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年11月15日 
    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.
  • 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年11月05日 
    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.
  • 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年10月22日 
    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.
  • 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年10月22日 
    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.
  • 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年09月29日 
    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.
  • Hideyuki Shimizu, Manabu Kodama, Masaki Matsumoto, Yasuko Orba, Michihito Sasaki, Akihiko Sato, Hirofumi Sawa, Keiichi I. Nakayama
    2021年09月25日 
    SUMMARYAlthough 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.
  • 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年09月07日 
    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.
  • 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 2021年08月24日 
    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.
  • 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年08月24日 
    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.
  • 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年08月24日 
    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.
  • 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年07月 
    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.
  • 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年06月23日 
    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.
  • 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 (Basel, Switzerland) 10 6 2021年06月11日 
    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.
  • Taisho Yamada, Seiichi Sato, Yuki Sotoyama, Yasuko Orba, Hirofumi Sawa, Hajime Yamauchi, Michihito Sasaki, Akinori Takaoka
    Nature immunology 22 7 820 - 828 2021年05月11日 
    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.
  • 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年04月30日 
    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.
  • Takahiro Hiono, Azusa Tomioka, Hiroyuki Kaji, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Atsushi Kuno
    2021年04月12日 
    AbstractThe 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.
  • 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  2021年04月 
    BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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%. CONCLUSIONS/SIGNIFICANCE: 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.
  • Michihito Sasaki, Yukari Itakura, Mai Kishimoto, Koshiro Tabata, Kentaro Uemura, Naoto Ito, Makoto Sugiyama, Christida E Wastika, Yasuko Orba, Hirofumi Sawa
    Journal of virology 2021年03月24日 
    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.
  • 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年03月08日 
    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.
  • 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年03月01日 
    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.
  • Mai Kishimoto, Kentaro Uemura, Takao Sanaki, Akihiko Sato, William W Hall, Hiroaki Kariwa, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki
    Viruses 13 3 2021年02月28日 
    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.
  • Mai Kishimoto, Bernard M Hang'ombe, William W Hall, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki
    The Journal of general virology 102 3 2021年02月03日 
    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.
  • 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年01月21日 
    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.
  • 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年01月08日 
    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.
  • 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年11月12日 
    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.
  • 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年11月 [査読有り][通常論文]
     
    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.
  • 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年10月12日 
    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.
  • 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年10月 [査読有り][通常論文]
     
    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.
  • 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年09月11日 [査読有り][通常論文]
     
    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.
  • Takuya Inagaki, Souichi Yamada, Hikaru Fujii, Tomoki Yoshikawa, Miho Shibamura, Shizuko Harada, Shuetsu Fukushi, Mai Quynh Le, Co Thach Nguyen, Thi Thu Thuy Nguyen, Thanh Thuy Nguyen, Thanh Thuy Nguyen, Van Tay Quach, Vu Dinh Thong, Kazuki Mori, Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Haruko Takeyama, Futoshi Hasebe, Masayuki Saijo
    Journal of virology 94 18 2020年08月31日 [査読有り][通常論文]
     
    Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.
  • 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年08月31日 [査読有り][通常論文]
     
    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.
  • 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年08月20日 [査読有り][通常論文]
     
    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.
  • Kosuke Okuya, Nao Eguchi, Rashid Manzoor, Reiko Yoshida, Shinji Saito, Tadaki Suzuki, Michihito Sasaki, Takeshi Saito, Yurie Kida, Akina Mori-Kajihara, Hiroko Miyamoto, Osamu Ichii, Masahiro Kajihara, Hideaki Higashi, Ayato Takada
    Viruses 12 7 2020年07月20日 [査読有り][通常論文]
     
    The influenza A virus (IAV) matrix-2 (M2) protein is an antigenically conserved viral envelope protein that plays an important role in virus budding together with another envelope protein, hemagglutinin (HA). An M2-specific mouse monoclonal IgG antibody, rM2ss23, which binds to the ectodomain of the M2 protein, has been shown to be a non-neutralizing antibody, but inhibits plaque formation of IAV strains. In this study, we generated chimeric rM2ss23 (ch-rM2ss23) IgG and IgA antibodies with the same variable region and compared their antiviral activities. Using gel chromatography, ch-rM2ss23 IgA were divided into three antibody subsets: monomeric IgA (m-IgA), dimeric IgA (d-IgA), and trimeric and tetrameric IgA (t/q-IgA). We found that t/q-IgA had a significantly higher capacity to reduce the plaque size of IAVs than IgG and m-IgA, most likely due to the decreased number of progeny virus particles produced from infected cells. Interestingly, HA-M2 colocalization was remarkably reduced on the infected cell surface in the presence of ch-rM2ss23 antibodies. These results indicate that anti-M2 polymeric IgA restricts IAV budding more efficiently than IgG and suggest a role of anti-M2 IgA in cross-protective immunity to IAVs.
  • 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年06月05日 [査読有り][通常論文]
     
    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.
  • Kosuke Okuya, Reiko Yoshida, Rashid Manzoor, Shinji Saito, Tadaki Suzuki, Michihito Sasaki, Takeshi Saito, Yurie Kida, Akina Mori-Kajihara, Tatsunari Kondoh, Masahiro Sato, Masahiro Kajihara, Hiroko Miyamoto, Osamu Ichii, Hideaki Higashi, Ayato Takada
    Journal of virology 94 12 2020年06月01日 [査読有り][通常論文]
     
    IgA antibodies on mucosal surfaces are known to play an important role in protection from influenza A virus (IAV) infection and are believed to be more potent than IgG for cross-protective immunity against IAVs of multiple hemagglutinin (HA) subtypes. However, in general, neutralizing antibodies specific to HA are principally HA subtype specific. Here, we focus on nonneutralizing but broadly cross-reactive HA-specific IgA antibodies. Recombinant IgG, monomeric IgA (mIgA), and polymeric secretory IgA (pSIgA) antibodies were generated based on the sequence of a mouse anti-HA monoclonal antibody (MAb) 5A5 that had no neutralizing activity but showed broad binding capacity to multiple HA subtypes. While confirming that there was no neutralizing activity of the recombinant MAbs against IAV strains A/Puerto Rico/8/1934 (H1N1), A/Adachi/2/1957 (H2N2), A/Hong Kong/483/1997 (H5N1), A/shearwater/South Australia/1/1972 (H6N5), A/duck/England/1/1956 (H11N6), and A/duck/Alberta/60/1976 (H12N5), we found that pSIgA, but not mIgA and IgG, significantly reduced budding and release of most of the viruses from infected cells. Electron microscopy demonstrated that pSIgA deposited newly produced virus particles on the surfaces of infected cells, most likely due to tethering of virus particles. Furthermore, we found that pSIgA showed significantly higher activity to reduce plaque sizes of the viruses than IgG and mIgA. These results suggest that nonneutralizing pSIgA reactive to multiple HA subtypes may play a role in intersubtype cross-protective immunity against IAVs.IMPORTANCE Mucosal immunity represented by pSIgA plays important roles in protection from IAV infection. Furthermore, IAV HA-specific pSIgA antibodies are thought to contribute to cross-protective immunity against multiple IAV subtypes. However, the mechanisms by which pSIgA exerts such versatile antiviral activity are not fully understood. In this study, we generated broadly cross-reactive recombinant IgG and pSIgA having the same antigen-recognition site and compared their antiviral activities in vitro These recombinant antibodies did not show "classical" neutralizing activity, whereas pSIgA, but not IgG, significantly inhibited the production of progeny virus particles from infected cells. Plaque formation was also significantly reduced by pSIgA, but not IgG. These effects were seen in infection with IAVs of several different HA subtypes. Based on our findings, we propose an antibody-mediated host defense mechanism by which mucosal immunity may contribute to broad cross-protection from IAVs of multiple HA subtypes, including viruses with pandemic potential.
  • 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年05月20日 [査読有り][通常論文]
     
    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. While 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 < .0001, OR = 39.3) and PCR-positive results (p < .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.
  • 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年04月 [査読有り][通常論文]
     
    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.
  • 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年02月04日 [査読有り][通常論文]
     
    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.
  • Michael Carr, Gabriel Gonzalez, Axel Martinelli, Christida E Wastika, Kimihito Ito, Yasuko Orba, Michihito Sasaki, William W Hall, Hirofumi Sawa
    Virus genes 55 5 630 - 642 2019年10月 [査読有り][通常論文]
     
    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.
  • Walter Muleya, Herman Moses Chambaro, Michihito Sasaki, Lambert Fadzai Gwenhure, Roy Mwenechanya, Masahiro Kajihara, Ngonda Saasa, Zacharia Mupila, Akina Mori-Kajihara, Yongjin Qiu, Elias Kangwa, Aaron Mweene, Boniface Namangala, Ayato Takada, Hirofumi Sawa
    Virus genes 55 5 713 - 719 2019年10月 [査読有り][通常論文]
     
    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.
  • Christida E Wastika, Michihito Sasaki, Kentaro Yoshii, Paulina D Anindita, Bernard M Hang'ombe, Aaron S Mweene, Shintaro Kobayashi, Hiroaki Kariwa, Michael J Carr, William W Hall, Yuki Eshita, Yasuko Orba, Hirofumi Sawa
    Archives of virology 164 8 2165 - 2170 2019年08月 [査読有り][通常論文]
     
    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.
  • Yongjin Qiu, Ryo Nakao, Bernard Mudenda Hang'ombe, Kozue Sato, Masahiro Kajihara, Sharon Kanchela, Katendi Changula, Yoshiki Eto, Joseph Ndebe, Michihito Sasaki, May June Thu, Ayato Takada, Hirohumi Sawa, Chihiro Sugimoto, Hiroki Kawabata
    Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 69 1 107 - 112 2019年06月18日 [査読有り][通常論文]
     
    BACKGROUND: Relapsing fever is an infectious disease previously neglected in Africa, which imposes a large public health burden in the country. We aimed to investigate and report on a case of relapsing fever borreliosis in Zambia. METHODS: A previously unknown Borrelia species was isolated from the blood of a febrile patient. Investigations of the presumptive vector ticks and natural hosts for the Borrelia species were conducted by culture isolation and/or DNA detection by Borrelia-specific polymerase chain reaction. Using culture isolates from the patient and bat specimens, genetic characterization was performed by multilocus sequence analysis based on the draft genome sequences. RESULTS: The febrile patient was diagnosed with relapsing fever. The isolated Borrelia species was frequently detected in Ornithodoros faini (n = 20/50 [40%]) and bats (n = 64/237 [27%]). Multilocus sequence analysis based on a draft genome sequence revealed that the Borrelia species isolates from the patient and presumptive reservoir host (bats) formed a monophyletic lineage that clustered with relapsing fever borreliae found in the United States. CONCLUSIONS: A febrile illness caused by a Borrelia species that was treatable with erythromycin was identified in Zambia. This is the first study to report on relapsing fever Borrelia in Zambia and suggesting the likely natural reservoir hosts of the isolated Borrelia species. Interestingly, the isolated Borrelia species was more closely related to New World relapsing fever borreliae, despite being detected in the Afrotropic ecozone.
  • 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年04月 [査読有り][通常論文]
     
    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.
  • 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 2018年11月 [査読有り][通常論文]
  • Michihito Sasaki, Masahiro Kajihara, Katendi Changula, Akina Mori-Kajihara, Hirohito Ogawa, Bernard M. Hang'ombe, Aaron S. Mweene, Martin Simuunza, Reiko Yoshida, Michael Carr, Yasuko Orba, Ayato Takada, Hirofumi Sawa
    Infection, Genetics and Evolution 63 104 - 109 2018年09月01日 [査読有り][通常論文]
     
    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.
  • 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 2018年08月 [査読有り][通常論文]
  • 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 2018年06月 [査読有り][通常論文]
     
    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.
  • Shiho Torii, Yasuko Orba, Bernard M Hang'ombe, Aaron S Mweene, Yuji Wada, Paulina D Anindita, Wallaya Phongphaew, Yongjin Qiu, Masahiro Kajihara, Akina Mori-Kajihara, Yoshiki Eto, Hayato Harima, Michihito Sasaki, Michael Carr, William W Hall, Yuki Eshita, Takashi Abe, Hirofumi Sawa
    Virus research 250 31 - 36 2018年05月02日 [査読有り][通常論文]
     
    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.
  • 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 2018年05月 [査読有り][通常論文]
  • Yuji Wada, Michihito Sasaki, 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, Hirofumi Sawa
    Journal of Medical Microbiology 67 3 415 - 422 2018年03月01日 [査読有り][通常論文]
     
    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.
  • Michihito Sasaki, Paulina D. Anindita, Wallaya Phongphaew, Michael Carr, Shintaro Kobayashi, Yasuko Orba, Hirofumi Sawa
    VIRUS RESEARCH 243 69 - 74 2018年01月 [査読有り][通常論文]
     
    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.
  • 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 2017年11月 [査読有り][通常論文]
     
    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.
  • Yuji Wada, Yasuko Orba, Michihito Sasaki, Shintaro Kobayashi, Michael J. Carr, Haruaki Nobori, Akihiko Sato, William W. Hall, Hirofumi Sawa
    VIROLOGY 505 102 - 112 2017年05月 [査読有り][通常論文]
     
    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.
  • 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 2017年04月 [査読有り][通常論文]
     
    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.
  • 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 2017年02月 [査読有り][通常論文]
     
    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.
  • Wallaya Phongphaew, Shintaro Kobayashi, Michihito Sasaki, Michael Carr, William W. Hall, Yasuko Orba, Hirofumi Sawa
    VIRUS RESEARCH 228 114 - 123 2017年01月 [査読有り][通常論文]
     
    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.
  • Gabriel Gonzalez, Michihito Sasaki, Lucy Burkitt-Gray, Tomonori Kamiya, Noriko M. Tsuji, Hirofumi Sawa, Kimihito Ito
    SCIENTIFIC REPORTS 7 40447  2017年01月 [査読有り][通常論文]
     
    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.
  • 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 2016年10月 [査読有り][通常論文]
     
    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.
  • 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  2016年04月 [査読有り][通常論文]
     
    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.
  • Paulina Duhita Anindita, Michihito Sasaki, Haruaki Nobori, Akihiko Sato, Michael Carr, Naoto Ito, Makoto Sugiyama, Yasuko Orba, Hirofumi Sawa
    VIRUS RESEARCH 215 121 - 128 2016年04月 [査読有り][通常論文]
     
    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.
  • 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 2015年07月 [査読有り][通常論文]
     
    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.
  • 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 2015年04月 [査読有り][通常論文]
     
    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.
  • 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 2015年04月 [査読有り][通常論文]
     
    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.
  • 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 2015年02月 [査読有り][通常論文]
     
    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.
  • 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 2015年02月 [査読有り][通常論文]
     
    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.
  • 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  2014年12月 [査読有り][通常論文]
     
    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.
  • Shintaro Kobayashi, Yasuko Orba, Hiroki Yamaguchi, Kenta Takahashi, Michihito Sasaki, Rie Hasebe, Takashi Kimura, Hirofumi Sawa
    VIRUS RESEARCH 191 83 - 91 2014年10月 [査読有り][通常論文]
     
    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.
  • 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 2014年09月 [査読有り][通常論文]
     
    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.
  • 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 2014年05月 [査読有り][通常論文]
     
    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.
  • 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 2014年04月 [査読有り][通常論文]
     
    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.
  • 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 95 325 - 330 2014年02月 [査読有り][通常論文]
     
    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.
  • Takashi Kimura, Megumi Okumura, Eunmi Kim, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa
    MICROBIOLOGY AND IMMUNOLOGY 57 10 723 - 731 2013年10月 [査読有り][通常論文]
     
    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.
  • 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 2013年09月 [査読有り][通常論文]
     
    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.
  • Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Ibenu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Hirofumi Sawa, Ichiro Nakamura, Takashi Kimura
    VIROLOGY JOURNAL 9 240  2012年10月 [査読有り][通常論文]
     
    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.
  • Michihito Sasaki, Eunmi Kim, Manabu Igarashi, Kimihito Ito, Rie Hasebe, Hideto Fukushi, Hirofumi Sawa, Takashi Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 286 45 39370 - 39378 2011年11月 [査読有り][通常論文]
     
    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.
  • 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 2011年06月 [査読有り][通常論文]
     
    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.
  • 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 2011年04月 [査読有り][通常論文]
     
    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.
  • Kim Eunmi, Okumura Megumi, Sasaki Michihito, Fujikura Daisuke, Miyazaki Tadaaki, Sawa Hirofumi, Kimura Takashi
    JOURNAL OF NEUROVIROLOGY 16 44  2010年10月 [査読有り][通常論文]
  • T. Kimura, M. Sasaki, M. Okumura, E. Kim, H. Sawa
    VETERINARY PATHOLOGY 47 5 806 - 818 2010年09月 [査読有り][通常論文]
     
    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.
  • Rie Hasebe, Michihito Sasaki, Hirofumi Sawa, Ryuichi Wada, Takashi Umemura, Takashi Kimura
    VIROLOGY 393 2 198 - 209 2009年10月 [査読有り][通常論文]
     
    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.

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    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 2017年度 [1P -1118] 2017年12月
  • 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年12月 [査読無し][通常論文]
     
    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)
  • 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年08月30日 [査読無し][通常論文]
  • 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 160回 391 -391 2017年08月
  • 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年08月 [査読無し][通常論文]
  • 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年08月 [査読無し][通常論文]
  • 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年08月 [査読無し][通常論文]
  • 佐々木 道仁, 大場 靖子, 澤 洋文 最新医学 72 (4) 508 -512 2017年04月 [査読無し][通常論文]
     
    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)
  • 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年 [査読無し][通常論文]
  • 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年12月
  • 野生動物が保有するウイルスの探索と性状解析
    佐々木 道仁 日本獣医学会学術集会講演要旨集 158回 132 -132 2015年08月
  • 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 158回 340 -340 2015年08月
  • 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 156回 214 -214 2013年08月
  • 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 156回 267 -267 2013年08月
  • 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 156回 283 -283 2013年08月
  • 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年
  • 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年
  • 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 2011年08月31日 [査読無し][通常論文]

受賞

  • 2021年11月 日本ウイルス学会 杉浦奨励賞
     動物由来ウイルスの探索および性状解析
  • 2017年11月 農学会 日本農学進歩賞
     動物が保有するウイルスの多様性とその起源に関する研究 
    受賞者: 佐々木道仁
  • 2015年09月 日本獣医学会 獣医学奨励賞
     野生動物が保有するウイルスの探索と性状解析 
    受賞者: 佐々木道仁

共同研究・競争的資金等の研究課題

  • 新規ウイルスを対象とするポストメタゲノム研究
    日本学術振興会:科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2021年07月 -2024年03月 
    代表者 : 佐々木 道仁
  • インドネシアのコウモリの呼吸器検体を用いたVirome解析
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 澤 洋文, 大場 靖子, 佐々木 道仁
  • 最先端イメージング技術を活用した狂犬病ウイルス野外株の末梢感染機序の解明
    日本学術振興会:科学研究費助成事業 国際共同研究加速基金(国際共同研究強化(B))
    研究期間 : 2018年10月 -2022年03月 
    代表者 : 伊藤 直人, 山田 健太郎, 佐々木 道仁, 西山 祥子
     
    2018年度は、大分大学から分与を受けた狂犬病ウイルス野外株1088株(ウッドチャック由来)の遺伝子操作系の動作確認を岐阜大学にて実施し、完全長ゲノムプラスミドから感染性を有した組換えウイルスが正常に回収できることを確認した。このウイルスを、今後の実験に使用する野生型ウイルスのストックとして保存した。また、本系を用いて、5種類のウイルスP蛋白質アイソフォームのうち、P2-5の発現能を欠損した変異株(1088dP2-5)の作出に成功し、そのストックを作製した。これらのストック中のウイルス感染価をフォーカスアッセイにより算出した。その結果、いずれのウイルスも1.0x10^5 FFU/ml以上の感染価であることが確認できた。 ドイツのフリードリヒ・レフラー研究所のステファン・フィンケ博士より、別の野外株2株、RABV-Dog株(犬由来)およびRABV-Fox株(キツネ由来)の遺伝子操作系の分与を受けた。現在、輸入のために断片化されたRABV-Dog株の完全長プラスミドの再構成を行っている。
  • 宿主因子との相互作用解析から紐解く狂犬病ウイルス感染メカニズム
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2018年04月 -2022年03月 
    代表者 : 佐々木 道仁
     
    狂犬病ウイルスは致死的な神経症状を惹き起こし、毎年55,000名以上を死に至らしめる。本研究では、狂犬病ウイルス感染時のウイルスと宿主因子の相互作用解析を通じて、狂犬病ウイルスの細胞内増殖機構およびそれに対する宿主の感染制御機構の解明を目的としている。これまでに、siRNAライブラリースクリーニングを実施し、細胞の狂犬病ウイルス感受性を変化させる28個の宿主遺伝子を同定している。 本年度は、同定した狂犬病ウイルス感染に関与する28個の遺伝子のうち27個のORFをクローニングし、発現ベクターを構築した。今後、発現ベクターを使用した遺伝子発現解析を行う。 また、上記の遺伝子群のうち狂犬病ウイルスの細胞感染を促進させる宿主転写関連因子Y-box binding protein(YB1)の解析を実施した。細胞のYB-1遺伝子発現のノックダウンにより、感染細胞におけるRABV遺伝子発現量と上清中の子孫ウイルス量が減少した。cross-linking immunoprecipitation(CLIP)およびゲルシフトアッセイ(RNA-EMSA)解析により、YB-1がウイルスmRNAの5’-UTRに結合することが判明し、その標的配列 (Y-box recognition sequence, YRS) を同定した。同定したYRSは5つのRABV遺伝子全ての5’-UTRに存在していたが、各YRSは1-2塩基の相違を認めた。そこで、各YRSのいずれかを有するRABV minigenome発現プラスミドをそれぞれ作出し、レポーターアッセイを実施した結果、NおよびP遺伝子の5’-UTRに存在するYRSを有するRABV minigenomeは、M,G,L遺伝子の5’-UTRに存在するYRSを有するRABV minigenomeに比べて強いレポーターシグナルを生じた。以上の結果から、RABVは自身のmRNAに存在するYRSとYB-1の結合を介して、ウイルス遺伝子発現量を増加させ、ウイルス増殖を亢進させることを明らかとした。
  • シングルセル遺伝子発現解析を用いた新型コロナウイルス感染症重症化分子機構の解明
    武田科学振興財団:ハイリスク新興感染症研究助成
    研究期間 : 2021年07月 
    代表者 : 佐々木道仁
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2016年04月 -2020年03月 
    代表者 : 澤 洋文, 大場 靖子, 佐々木 道仁
     
    本研究課題で計画した、1) 中南米・東南アジアでの疫学研究の実施、2) Virome解析法等による検体の解析、3) 病原性ウイルスの分布・進化・生活環の把握について以下の成果を得た。 中南米、東南アジアで採集した検体を用いて、ウイルスゲノムのスクリーニングを実施し、コウモリから新規のガンマヘルペスウイルスを検出した。また、霊長類動物の糞便50検体分をプールしてVirome解析を実施して、新規のDNAウイルスを単離した。さらに、キクガシラコウモリ科の複数種のコウモリから単離したポリオ―マウイルスのゲノムを解析することにより、ウイルスの進化の過程で宿主の乗り換えが生じていることを示す結果を得た。
  • 遺伝子導入細胞を用いた感染性ウイルスサルベージ戦略
    上原記念生命科学財団:研究奨励金
    研究期間 : 2019年 -2020年 
    代表者 : 佐々木道仁
  • ウイルス叢解析によるコウモリ保有ウイルスの疫学調査
    栗林育英学術財団:個人研究助成
    研究期間 : 2016年09月 -2017年03月 
    代表者 : 佐々木道仁
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2015年 -2017年 
    代表者 : 佐々木 道仁
     
    ウイルス感染成立と病原性発現には宿主因子が密接に関与するが、狂犬病ウイルス感染に関与する宿主因子の研究は少ない。本研究では、狂犬病ウイルス感染に関与する宿主因子を同定と狂犬病ウイルス感染機構の解明を目的した。ヒトsiRNAライブラリーを用いたスクリーニングを実施し、狂犬病ウイルス感染に関与する宿主遺伝子を28個同定した。また、細胞のヘパラン硫酸が狂犬病ウイルスの吸着因子として機能することを見出し、その詳細な分子機構を明らかにした。一連の解析を通じて、狂犬病ウイルスの感染機構の一端を解明した。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2012年04月 -2015年03月 
    代表者 : 佐々木 道仁
     
    細胞表面に発現するウマ主要組織適合遺伝子複合体(MHC)クラスIがウマヘルペスウイルス4型(EHV-4)のエンベロープ糖タンパクgDと結合し、EHV-4の細胞内侵入を誘導するレセプターであることを明らかにした。また、ウマMHCクラスIを標的とする感染阻害法がウマヘルペスウイルス1型(EHV-1)とEHV-4両ウイルス感染を同時に阻害することを明らかにした。加えて、ウサギMHCクラスIのEHV-1レセプター機能を解析し、ウマ培養細胞とウサギ培養細胞におけるEHV-1感染機構の違いについて考察した。
  • MHCクラスI分子を介したヘルペスウイルス細胞内侵入機構の解明
    文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2012年 -2015年 
    代表者 : 佐々木道仁
  • 組換え狂犬病ウイルスを用いた狂犬病ウイルス感染に関与する宿主因子の網羅的探索
    秋山記念生命科学振興財団:研究助成(奨励)
    研究期間 : 2012年 -2013年 
    代表者 : 佐々木道仁


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