Researcher Profile and Settings

Affiliation

• International Institute for Zoonosis Control　Division of Collaboration and Education

Job Title

• Professor

URL

https://kaken.nii.ac.jp/d/r/80535328.ja.html

J-Global ID

• 201501037186254321

Research Interests

• トランスクリプトーム   転写制御   転写開始点   バイオインフォマティクス   TSS-seq   原虫   次世代シーケンサー   トキソプラズマ   遺伝子診断   ステージ変換   血清学診断   マダニ媒介   Theileria   ブラディゾイト   Neospora caninum   マダニ   組換えワクチン   Babesia   弱毒化   Toxoplasma   TgSAG1   予防対策   生活環   タキゾイト   Toxoplasma gondii   疫学調査   東南アジア   病原性   発現ベクター   

Research Areas

• Life sciences / Veterinary medicine

Educational Organization

•  Doctoral (PhD) degree program　Graduate School of Infectious Diseases

Academic & Professional Experience

• 2023/10 - Today 北海道大学 人獣共通感染症国際共同研究所 教授
• 2015 Hokkaido University

Research Activities

Published Papers

• Extraction of the CDRH3 sequence of the mouse antibody repertoire selected upon influenza virus infection by subtraction of the background antibody repertoire

  Masashi Shingai, Sayaka Iida, Naoko Kawai, Mamiko Kawahara, Toshiki Sekiya, Marumi Ohno, Naoki Nomura, Chimuka Handabile, Tomomi Kawakita, Ryosuke Omori, Junya Yamagishi, Kaori Sano, Akira Ainai, Tadaki Suzuki, Kazuo Ohnishi, Kimihito Ito, Hiroshi Kida

  Journal of Virology 0022-538X 2024/02/07 

  As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.
• Upregulation of ATP6V0D2 benefits intracellular survival of Leishmania donovani in erythrocytes-engulfing macrophages

  Jing Hong, Budhaditya Mukherjee, Chizu Sanjoba, Junya Yamagishi, Yasuyuki Goto

  Frontiers in Cellular and Infection Microbiology 14 2024/01/31 

  Visceral leishmaniasis (VL) is the most severe type of leishmaniasis which is caused by infection of Leishmania donovani complex. In the BALB/c mouse model of VL, multinucleated giant cells (MGCs) with heavy parasite infection consist of the largest population of hemophagocytes in the spleen of L. donovani-infected mice, indicating that MGCs provide the parasites a circumstance beneficial for their survival. Although ATP6V0D2 is a demonstrated factor inducing the formation of hemophagocytic MGCs during L. donovani infection, functions of this protein in shaping the infection outcome in macrophages remain unclear. Here we evaluated the influence of upregulated ATP6V0D2 on intracellular survival of the parasites. L. donovani infection-induced hemophagocytosis of normal erythrocytes by macrophages was suppressed by RNAi-based knockdown of Atp6v0d2. The knockdown of Atp6v0d2 did not improve the survival of amastigotes within macrophages when the cells were cultured in the absence of erythrocytes. On the other hand, reduced intracellular survival of amastigotes in macrophages by the knockdown was observed when macrophages were supplemented with antibody-opsonized erythrocytes before infection. There, increase in cytosolic labile iron pool was observed in the L. donovani-infected knocked-down macrophages. It suggests that ATP6V0D2 plays roles not only in upregulation of hemophagocytosis but also in iron trafficking within L. donovani-infected macrophages. Superior access to iron in macrophages may be how the upregulated expression of the molecule brings benefit to Leishmania for their intracellular survival in the presence of erythrocytes.
• Combination therapy with oral antiviral and anti-inflammatory drugs improves the efficacy of delayed treatment in a COVID-19 hamster model.

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

  EBioMedicine 99 104950 - 104950 2023/12/29 

  BACKGROUND: Pulmonary infection with SARS-CoV-2 stimulates host immune responses and can also result in the progression of dysregulated and critical inflammation. Throughout the pandemic, the management and treatment of COVID-19 has been continuously updated with a range of antiviral drugs and immunomodulators. Monotherapy with oral antivirals has proven to be effective in the treatment of COVID-19. However, treatment should be initiated in the early stages of infection to ensure beneficial therapeutic outcomes, and there is still room for further consideration on therapeutic strategies using antivirals. METHODS: We studied the therapeutic effects of monotherapy with the oral antiviral ensitrelvir or the anti-inflammatory corticosteroid methylprednisolone and combination therapy with ensitrelvir and methylprednisolone in a delayed dosing model of hamsters infected with SARS-CoV-2. FINDINGS: Combination therapy with ensitrelvir and methylprednisolone improved respiratory conditions and reduced the development of pneumonia in hamsters even when the treatment was started after 2 days post-infection. The combination therapy led to a differential histological and transcriptomic pattern in comparison to either of the monotherapies, with reduced lung damage and down-regulation of expression of genes involved in the inflammatory response. Furthermore, we found that the combination treatment is effective in case of infection with either the highly pathogenic delta or circulating omicron variants. INTERPRETATION: Our results demonstrate the advantage of combination therapy with antiviral and corticosteroid drugs in COVID-19 treatment from the perspective of lung pathology and host inflammatory responses. FUNDING: Funding bodies are described in the Acknowledgments section.
• Whole-Genome Investigation of Zoonotic Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 Isolated from Pigs and Humans in Thailand.

  Pawarut Narongpun, Pattrarat Chanchaithong, Junya Yamagishi, Jeewan Thapa, Chie Nakajima, Yasuhiko Suzuki

  Antibiotics (Basel, Switzerland) 12 (12) 2023/12/16 

  Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been widespread globally in pigs and humans for decades. Nasal colonization of LA-MRSA is regarded as an occupational hazard to people who are regularly involved in livestock production. Our previous study suggested pig-to-human transmission caused by LA-MRSA clonal complex (CC) 398, using traditional molecular typing methods. Instead, this study aimed to investigate the zoonotic transmission of LA-MRSA CC398 using whole genome sequencing (WGS) technologies. A total of 63 LA-MRSA isolates were identified and characterized in Thailand. Further, the 16 representatives of LA-MRSA CC9 and CC398, including porcine and worker isolates, were subjected to WGS on the Illumina Miseq platform. Core-genome single nucleotide polymorphism (SNP)-based analyses verify the zoonotic transmission caused by LA-MRSA CC398 in two farms. WGS-based characterization suggests the emergence of a novel staphylococcal cassette chromosome (SCC) mec type, consisting of multiple cassette chromosome recombinase (ccr) gene complexes via genetic recombination. Additionally, the WGS analyses revealed putative multi-resistant plasmids and several cross-resistance genes, conferring resistance against drugs of last resort used in humans such as quinupristin/dalfopristin and linezolid. Significantly, LA-MRSA isolates, in this study, harbored multiple virulence genes that may become a serious threat to an immunosuppressive population, particularly for persons who are in close contact with LA-MRSA carriers.
• Surveys of eleven species of wild and zoo birds and feeding experiments in white-tailed eagles reveal differences in the composition of the avian gut microbiome based on dietary habits between and within species.

  Kohei Ogasawara, Naoki Yamada, Shouta Mm Nakayama, Yukiko Watanabe, Keisuke Saito, Akane Chiba, Yoshitaka Uchida, Kaoru Ueda, Yasunori Takenaka, Kentaro Kazama, Mami Kazama, Junya Yamagishi, Hazuki Mizukawa, Yoshinori Ikenaka, Mayumi Ishizuka

  The Journal of veterinary medical science 2023/10/31 

  The composition of the gut microbiome varies due to dietary habits. We investigated influences of diet on the composition of the gut microbiome using the feces of 11 avian species, which consumed grain-, fish- and meat-based diets. We analyzed gut microbiome diversity and composition by next-generation sequencing (NGS) of 16S ribosomal RNA. The grain-diet group had higher gut microbiome diversity than the meat- and fish-diet group. The ratio of Bacteroidetes and Firmicutes phyla was higher in the grain-diet group than in the meat- and fish-diet groups. The grain-diet group had a higher ratio of Veillonellaceae than the meat-diet group and a higher ratio of Eubacteriaceae than the fish-diet habit group. To clarify the influence of diet within the same species, white-tailed eagles (Haliaeetus albicilla, n=6) were divided into two groups, and given only deer meat or fish for approximately one month. The composition of the gut microbiome of individuals in both groups were analyzed by NGS. There were indications of fluctuation in the levels of some bacteria (Lactobacillus, Coriobacteriales, etc.) in each diet group. Moreover, one individual for each group which switched each diet in last week changed to each feature of composition of bacterial flora. The above results show that the composition of the gut microbiome differ depending on diet, even within the same species.
• Bovine Piroplasma Populations in the Philippines Characterized Using Targeted Amplicon Deep Sequencing.

  Eloiza May Galon, Adrian Miki Macalanda, Tatsuki Sugi, Kyoko Hayashida, Naoko Kawai, Taishi Kidaka, Rochelle Haidee Ybañez, Paul Franck Adjou Moumouni, Aaron Edmond Ringo, Hang Li, Shengwei Ji, Junya Yamagishi, Adrian Ybañez, Xuenan Xuan

  Microorganisms 11 (10) 2023/10/18 

  Molecular assays and capillary electrophoresis sequencing have been used to identify parasites in livestock. The low sample capacity, which increases labor and processing time, is one drawback. Targeted amplicon sequencing (Ampliseq) uses the fast and large sample capacity platform to identify parasites in the target host, overcoming this limitation. DNA was extracted from 162 whole blood samples collected from cattle in three provinces in the Philippines. Using Illumina's Miseq platform, the V4 hypervariable region of the piroplasma 18S rRNA gene was amplified and sequenced. The AMPtk pipeline was used to obtain distinct amplicon sequence variants (ASVs) and the NCBI BLAST non-redundant database was used to assign taxonomy. In total, 95 (58.64%) samples were positive for piroplasma. Using the AMPTk pipeline, 2179 ASVs were obtained. A total of 79 distinct ASVs were obtained after clustering and filtering, which belonged to genera Babesia (n = 58), Theileria (n = 17), Hepatozoon (n = 2), and Sarcocystis (n = 2). The ASV top hits were composed of 10 species: Babesia bovis, B. bigemina, Theileria orientalis, Babesia sp., Hepatozoon canis, Sarcocystis cruzi, T. annulata, T. equi, T. mutans, and Theileria sp. Thung Song. The results generated in this study demonstrated the applicability of Ampliseq in detecting piroplasmid parasites infecting cattle in the Philippines.
• Prevalence and Genomic Characterization of Rotavirus A from Domestic Pigs in Zambia: Evidence for Possible Porcine–Human Interspecies Transmission

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

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

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

  Akihiro Ochi, Taishi Kidaka, Hassan Hakimi, Masahito Asada, Junya Yamagishi

  BMC genomics 24 (1) 483 - 483 2023/08/24 

  BACKGROUND: Babesia caballi is an intraerythrocytic parasite from the phylum Apicomplexa, capable of infecting equids and causing equine piroplasmosis. However, since there is limited genome information available on B. caballi, molecular mechanisms involved in host specificity and pathogenicity of this species have not been fully elucidated yet. RESULTS: Genomic DNA from a B. caballi subclone was purified and sequenced using both Illumina and Nanopore technologies. The resulting assembled sequence consisted of nine contigs with a size of 12.9 Mbp, rendering a total of 5,910 protein-coding genes. The phylogenetic tree of Apicomplexan species was reconstructed using 263 orthologous genes. We identified 481 ves1-like genes and named "ves1c". In contrast, expansion of the major facilitator superfamily (mfs) observed in closely related B. bigemina and B. ovata species was not found in B. caballi. A set of repetitive units containing an open reading frame with a size of 297 bp was also identified. CONCLUSIONS: We present a chromosome-level genome assembly of B. caballi. Our genomic data may contribute to estimating gene expansion events involving multigene families and exploring the evolution of species from this genus.
• Draft genome sequence data of Haemaphysalis longicornis Oita strain

  Rika Umemiya-Shirafuji, Xuenan Xuan, Kozo Fujisaki, Junya Yamagishi

  Data in Brief 2023/08
• 一酪農家におけるクリプトスポリジウム症の清浄化への試み

  高橋 和瑛, 松尾 智英, 山岸 潤也, 芝原 友幸, 笹井 和美, 松林 誠

  家畜診療 (公社)全国農業共済協会 70 (7) 407 - 416 0287-0754 2023/07
• Surveillance, Isolation, and Genetic Characterization of Bat Herpesviruses in Zambia.

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

  Viruses 15 (6) 2023/06/13 

  Bats are of significant interest as reservoirs for various zoonotic viruses with high diversity. During the past two decades, many herpesviruses have been identified in various bats worldwide by genetic approaches, whereas there have been few reports on the isolation of infectious herpesviruses. Herein, we report the prevalence of herpesvirus infection of bats captured in Zambia and genetic characterization of novel gammaherpesviruses isolated from striped leaf-nosed bats (Macronycteris vittatus). By our PCR screening, herpesvirus DNA polymerase (DPOL) genes were detected in 29.2% (7/24) of Egyptian fruit bats (Rousettus aegyptiacus), 78.1% (82/105) of Macronycteris vittatus, and one Sundevall's roundleaf bat (Hipposideros caffer) in Zambia. Phylogenetic analyses of the detected partial DPOL genes revealed that the Zambian bat herpesviruses were divided into seven betaherpesvirus groups and five gammaherpesvirus groups. Two infectious strains of a novel gammaherpesvirus, tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1), were successfully isolated from Macronycteris vittatus bats, and their complete genomes were sequenced. The genome of MaGHV1 encoded 79 open reading frames, and phylogenic analyses of the DNA polymerase and glycoprotein B demonstrated that MaGHV1 formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses. Our findings provide new information regarding the genetic diversity of herpesviruses maintained in African bats.
• タイにおける豚からヒトへのLA-MRSAの人獣共通感染の全ゲノム解析(Whole Genome Analysis of Zoonotic Transmission of LA-MRSA from Pigs to Humans in Thailand)

  Narongpun Pawarut, チャンチャイトン・パッタラット, 山岸 潤也, 中島 千絵, 鈴木 定彦

  日本細菌学雑誌 78 (1) 109 - 109 0021-4930 2023/02
• Current status and molecular epidemiology of rabies virus from different hosts and regions in Malawi.

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

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

  Although rabies is endemic in Malawi, there have been no studies in which rabies virus was systematically investigated and characterized in multiple animal hosts in that country. In order to provide molecular epidemiological data on rabies virus in Malawi, 683 suspected rabies case reports from 2008 to 2021 were examined, and 46 (dog = 40, cow = 5, and cat = 1) viable rabies-positive brain samples archived at the Central Veterinary Laboratory (CVL), Lilongwe, Malawi, were analyzed genetically. The results showed an increase in the submission of brain samples from 2008 to 2010, with the highest number of submissions observed in 2020. Of the 683 case reports analyzed for the period under review, 38.1% (260/683) (CI: 34.44 - 41.84) were confirmed by direct fluorescent antibody test. Among the confirmed cases, 65.4% (170/260) (CI: 59.23 - 71.09) were canine rabies. Further, phylogenetic analysis revealed that sequences from different animal hosts clustered together within the Africa 1b lineage, suggesting that the strains circulating in livestock are similar to those in domestic dogs. This finding supports the hypothesis that canine rabies is spilling over to livestock and emphasizes the need for further studies to provide data for effective control of rabies in Malawi.
• 一酪農場におけるクリプトスポリジウム症の清浄化への試み

  高橋 和瑛, 松尾 智英, 山岸 潤也, 芝原 友幸, 笹井 和美, 松林 誠

  家畜診療 (公社)全国農業共済協会 70 (1) 47 - 48 0287-0754 2023/01
• Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.

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

  PloS one 18 (5) e0285861  2023 

  A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.
• Cloning, Expression and Evaluation of Thioredoxin Peroxidase-1 Antigen for the Serological Diagnosis of Schistosoma mekongi Human Infection.

  Atcharaphan Wanlop, Jose Ma M Angeles, Adrian Miki C Macalanda, Masashi Kirinoki, Yuma Ohari, Aya Yajima, Junya Yamagishi, Kevin Austin L Ona, Shin-Ichiro Kawazu

  Diagnostics (Basel, Switzerland) 12 (12) 2022/12/07 

  Schistosoma mekongi, a blood fluke that causes Asian zoonotic schistosomiasis, is distributed in communities along the Mekong River in Cambodia and Lao People's Democratic Republic. Decades of employing numerous control measures including mass drug administration using praziquantel have resulted in a decline in the prevalence of schistosomiasis mekongi. This, however, led to a decrease in sensitivity of Kato-Katz stool microscopy considered as the gold standard in diagnosis. In order to develop a serological assay with high sensitivity and specificity which can replace Kato-Katz, recombinant S. mekongi thioredoxin peroxidase-1 protein (rSmekTPx-1) was expressed and produced. Diagnostic performance of the rSmekTPx-1 antigen through ELISA for detecting human schistosomiasis was compared with that of recombinant protein of S. japonicum TPx-1 (rSjTPx-1) using serum samples collected from endemic foci in Cambodia. The sensitivity and specificity of rSmekTPx-1 in ELISA were 89.3% and 93.3%, respectively, while those of rSjTPx-1 were 71.4% and 66.7%, respectively. In addition, a higher Kappa value of 0.82 calculated between rSmekTPx-1 antigen ELISA and Kato-Katz confirmed better agreement than between rSjTPx-1 antigen ELISA and Kato-Katz (Kappa value 0.38). These results suggest that ELISA with rSmekTPx-1 antigen can be a potential diagnostic method for detecting active human S. mekongi infection.
• Leishmania infection-induced multinucleated giant cell formation via upregulation of ATP6V0D2 expression

  Jing Hong, Chizu Sanjoba, Wataru Fujii, Junya Yamagishi, Yasuyuki Goto

  Frontiers in Cellular and Infection Microbiology 12 2022/09/23 

  Leishmaniasis is caused by infection with protozoan parasites of the genus Leishmania. In both clinical and experimental visceral leishmaniasis, macrophage multinucleation is observed in parasitized tissues. However, the feature and the mechanism of macrophage multinucleation remained unclear. Here, we report that infection of Leishmania donovani, a causative agent of visceral leishmaniasis, induces multinucleation of bone marrow-derived macrophages (BMDMs) in vitro. When these infection-induced multinucleated macrophages were compared with cytokine-induced multinucleated giant cells, the former had higher phagocytic activity on red blood cells but no apparent changes on phagocytosis of latex beads. BMDMs infected with L. donovani had increased expression of ATP6V0D2, one of the components of V-ATPase, which was also upregulated in the spleen of infected mice. Infection-induced ATP6V0D2 localized in a cytoplasmic compartment, which did not overlap with the mitochondria, endoplasmic reticulum, or lysosomes. When ATP6V0D2 expression was recombinantly induced in BMDMs, the formation of multinucleated macrophages was induced as seen in the infected macrophages. Taken together, L. donovani infection induces multinucleation of macrophages via ATP6V0D2 upregulation leading to a unique metamorphosis of the macrophages toward hemophagocytes.
• Single-cell analytical technologies: uncovering the mechanisms behind variations in immune responses.

  Yukie Kashima, Patrick Reteng, Yasuhiko Haga, Junya Yamagishi, Yutaka Suzuki

  The FEBS journal 2022/09/09 

  The immune landscape varies among individuals. It determines the immune response and results in surprisingly diverse symptoms, even in response to similar external stimuli. However, the detailed mechanisms underlying such diverse immune responses have remained mostly elusive. The utilization of recently developed single-cell multimodal analysis platforms has started to answer this question. Emerging studies have elucidated several molecular networks that may explain diversity with respect to age or other factors. An elaborate interplay between inherent physical conditions and environmental conditions has been demonstrated. Furthermore, the importance of modifications by the epigenome resulting in transcriptome variation among individuals is gradually being revealed. Accordingly, epigenomes and transcriptomes are direct indicators of the medical history and dynamic interactions with environmental factors. COVID-19 has recently become one of the most remarkable examples of the necessity of in-depth analyses of diverse responses with respect to various factors to improve treatment in severe cases and to prevent viral transmission from asymptomatic carriers. In fact, determining why some patients develop serious symptoms is still a pressing issue. Here, we review the current "state of the art" in single-cell analytical technologies and their broad applications to healthy individuals and representative diseases, including COVID-19.
• SICA-mediated cytoadhesion of Plasmodium knowlesi-infected red blood cells to human umbilical vein endothelial cells.

  Huai Chuang, Miako Sakaguchi, Amuza Byaruhanga Lucky, Junya Yamagishi, Yuko Katakai, Satoru Kawai, Osamu Kaneko

  Scientific reports 12 (1) 14942 - 14942 2022/09/02 

  Zoonotic malaria due to Plasmodium knowlesi infection in Southeast Asia is sometimes life-threatening. Post-mortem examination of human knowlesi malaria cases showed sequestration of P. knowlesi-infected red blood cells (iRBCs) in blood vessels, which has been proposed to be linked to disease severity. This sequestration is likely mediated by the cytoadhesion of parasite-iRBCs to vascular endothelial cells; however, the responsible parasite ligands remain undetermined. This study selected P. knowlesi lines with increased iRBC cytoadhesion activity by repeated panning against human umbilical vein endothelial cells (HUVECs). Transcriptome analysis revealed that the transcript level of one gene, encoding a Schizont Infected Cell Agglutination (SICA) protein, herein termed SICA-HUVEC, was more than 100-fold increased after the panning. Transcripts of other P. knowlesi proteins were also significantly increased, such as PIR proteins exported to the iRBC cytosol, suggesting their potential role in increasing cytoadhesion activity. Transgenic P. knowlesi parasites expressing Myc-fused SICA-HUVEC increased cytoadhesion activity following infection of monkey as well as human RBCs, confirming that SICA-HUVEC conveys activity to bind to HUVECs.
• Advances in understanding red blood cell modifications by Babesia.

  Hassan Hakimi, Junya Yamagishi, Shin-Ichiro Kawazu, Masahito Asada

  PLoS pathogens 18 (9) e1010770  2022/09 

  Babesia are tick-borne protozoan parasites that can infect livestock, pets, wildlife animals, and humans. In the mammalian host, they invade and multiply within red blood cells (RBCs). To support their development as obligate intracellular parasites, Babesia export numerous proteins to modify the RBC during invasion and development. Such exported proteins are likely important for parasite survival and pathogenicity and thus represent candidate drug or vaccine targets. The availability of complete genome sequences and the establishment of transfection systems for several Babesia species have aided the identification and functional characterization of exported proteins. Here, we review exported Babesia proteins; discuss their functions in the context of immune evasion, cytoadhesion, and nutrient uptake; and highlight possible future topics for research and application in this field.
• Behavior and toxic effects of Pb in a waterfowl model with oral exposure to Pb shots: Investigating Pb exposure in wild birds.

  Hiroshi Sato, Chihiro Ishii, Shouta M M Nakayama, Takahiro Ichise, Keisuke Saito, Yukiko Watanabe, Kohei Ogasawara, Ryota Torimoto, Atsushi Kobayashi, Takashi Kimura, Yukiko Nakamura, Junya Yamagishi, Yoshinori Ikenaka, Mayumi Ishizuka

  Environmental pollution (Barking, Essex : 1987) 308 119580 - 119580 2022/09/01 

  Among wild birds, lead (Pb) exposure caused by ingestion of ammunition is a worldwide problem. We aimed to reveal the behavior and toxic effect of Pb caused by ingesting Pb shots in waterfowl. Four male, eight-week old Muscovy ducks (Cairina moschata) were given three Pb shots (approximately 240 mg in total) orally and then fed for 29 days after exposure, simulating a low-dose Pb exposure in wild waterfowl. During the breeding period, blood samples were collected 10 times, and fecal samples every day. Additionally, 22 fresh tissue and 6 bone samples were obtained from each duck through the dissection. Although there were no gross abnormalities, the maximum blood Pb concentration of each duck ranged from 0.6 to 3.7 mg/L, reaching a threshold concentration indicative of clinical symptoms (>0.5 mg/L). δ-aminolevulinic acid dehydratase declined one day after exposure and remained low throughout the feeding period. Hematocrit also tended to decrease, indicating signs of anemia. The highest Pb accumulation was observed in the bones, followed by the kidneys, intestinal tracts, and liver. High Pb accumulation in the bones, which are known to have a long Pb half-life, suggested that Pb would remain in the body and possibly affect bird health beyond 28 days after exposure. Gene expression analysis showed a significant increase in the expression of the toll-like receptor-3 gene, which is involved in virus discrimination in the liver, suggesting a disruption of the immune system. Microbiota analyses showed a correlation between the blood Pb concentration and the abundances of Lachnospiraceae and Ruminococcaceae, suggesting that Pb affects lipid metabolism. These results provide fundamental data on Pb exposure in wild birds and a new perspective on the damage such exposure causes.
• Circular Whole-Transcriptome Amplification (cWTA) and mNGS Screening Enhanced by a Group Testing Algorithm (mEGA) Enable High-Throughput and Comprehensive Virus Identification.

  Patrick Reteng, Linh Nguyen Thuy, Mizanur Rahman, Ana Maria Bispo de Filippis, Kyoko Hayashida, Tatsuki Sugi, Gabriel Gonzalez, William W Hall, Lan Anh Nguyen Thi, Junya Yamagishi

  mSphere e0033222  2022/08/25 

  Metagenomic next-generation sequencing (mNGS) offers a hypothesis-free approach for pathogen detection, but its applicability in clinical diagnosis, in addition to other factors, remains limited due to complicated library construction. The present study describes a PCR-free isothermal workflow for mNGS targeting RNA, based on a multiple displacement amplification, termed circular whole-transcriptome amplification (cWTA), as the template is circularized before amplification. The cWTA approach was validated with clinical samples and nanopore sequencing. Reads homologous to dengue virus 2 and chikungunya virus were detected in clinical samples from Bangladesh and Brazil, respectively. In addition, the practicality of a high-throughput detection system that combines mNGS and a group testing algorithm termed mNGS screening enhanced by a group testing algorithm (mEGA) was established. This approach enabled significant library size reduction while permitting trackability between samples and diagnostic results. Serum samples of patients with undifferentiated febrile illnesses from Vietnam (n = 43) were also amplified with cWTA, divided into 11 pools, processed for library construction, and sequenced. Dengue virus 2, hepatitis B virus, and parvovirus B19 were successfully detected without prior knowledge of their existence. Collectively, cWTA with the nanopore platform opens the possibility of hypothesis-free on-site comprehensive pathogen diagnosis, while mEGA contributes to the scaling up of sample throughput. IMPORTANCE Given the breadth of pathogens that cause infections, a single approach that can detect a wide range of pathogens is ideal but is impractical due to the available tests being highly specific to a certain pathogen. Recent developments in sequencing technology have introduced mNGS as an alternative that provides detection of a wide-range of pathogens by detecting the presence of their nucleic acids in the sample. However, sequencing library preparation is still a bottleneck, as it is complicated, costly, and time-consuming. In our studies, alternative approaches to optimize library construction for mNGS were developed. This included isothermal nucleic acid amplification and expansion of sample throughput with a group testing algorithm. These methods can improve the utilization of mNGS as a diagnostic tool and can serve as a high-throughput screening system aiding infectious disease surveillance.
• Genomic Surveillance of SARS-CoV-2 in the Southern Province of Zambia: Detection and Characterization of Alpha, Beta, Delta, and Omicron Variants of Concern.

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

  Viruses 14 (9) 2022/08/24 

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

  Thillaiampalam Sivakumar, Bumduuren Tuvshintulga, Davaajav Otgonsuren, Enkhbaatar Batmagnai, Believe Ahedor, Hemal Kothalawala, Singarayar Caniciyas Vimalakumar, Seekkuge Susil Priyantha Silva, Junya Yamagishi, Naoaki Yokoyama

  Parasites & vectors 15 (1) 299 - 299 2022/08/24 

  BACKGROUND: The recently discovered Babesia sp. Mymensingh, which causes clinical bovine babesiosis, has a wide geographical distribution. We investigated the phylogenetic position of Babesia sp. Mymensingh using its mitochondrial, plastid, and nuclear genes. Based on morphological and molecular data, Babesia sp. Mymensingh is a unique species and we named it as Babesia naoakii n. sp. METHODS: A blood DNA sample from a Babesia sp. Mymensingh-infected cow was subjected to genome sequencing to obtain the sequences of mitochondrial, plastid, and nuclear genes. Six phylogenetic trees were then constructed with (1) concatenated amino acid sequences of cytochrome oxidase subunit I, cytochrome oxidase subunit III, and cytochrome b genes of the mitochondrial genome; (2) 16S rRNA of the plastid genome; (3) nucleotide sequences of the elongation factor Tu gene of the plastid genome; (4) ITS1-5.8S rRNA-ITS2; (5) concatenated nucleotide sequences of 89 nuclear genes; and (6) concatenated amino acid sequences translated from the 89 nuclear genes. RESULTS: In all six phylogenetic trees, B. naoakii n. sp. formed a sister clade to the common ancestor of Babesia bigemina and B. ovata. The concatenated nuclear genes of B. naoakii n. sp. and their translated amino acid sequences shared lower identity scores with the sequences from B. bigemina (82.7% and 84.7%, respectively) and B. ovata (83.5% and 85.5%, respectively) compared with the identity scores shared between the B. bigemina and B. ovata sequences (86.3% and 87.9%, respectively). CONCLUSIONS: Our study showed that B. naoakii n. sp. occupies a unique phylogenetic position distinct from existing Babesia species. Our findings, together with morphological differences, identify B. naoakii n. sp. as a distinct parasite species.
• Inactivated whole influenza virus particle vaccines induce neutralizing antibodies with an increase in immunoglobulin gene subclones of B-lymphocytes in cynomolgus macaques.

  Masanori Shiohara, Saori Suzuki, Shintaro Shichinohe, Hirohito Ishigaki, Misako Nakayama, Naoki Nomura, Masashi Shingai, Toshiki Sekiya, Marumi Ohno, Sayaka Iida, Naoko Kawai, Mamiko Kawahara, Junya Yamagishi, Kimihito Ito, Ryotarou Mitsumata, Tomio Ikeda, Kenji Motokawa, Tomoyoshi Sobue, Hiroshi Kida, Kazumasa Ogasawara, Yasushi Itoh

  Vaccine 40 (30) 4026 - 4037 2022/06/26 [Refereed][Not invited]
   

  The All-Japan Influenza Vaccine Study Group has been developing a more effective vaccine than the current split vaccines for seasonal influenza virus infection. In the present study, the efficacy of formalin- and/or β-propiolactone-inactivated whole virus particle vaccines for seasonal influenza was compared to that of the current ether-treated split vaccines in a nonhuman primate model. The monovalent whole virus particle vaccines or split vaccines of influenza A virus (H1N1) and influenza B virus (Victoria lineage) were injected subcutaneously into naïve cynomolgus macaques twice. The whole virus particle vaccines induced higher titers of neutralizing antibodies against H1N1 influenza A virus and influenza B virus in the plasma of macaques than did the split vaccines. At challenge with H1N1 influenza A virus or influenza B virus, the virus titers in nasal swabs and the increases in body temperatures were lower in the macaques immunized with the whole virus particle vaccine than in those immunized with the split vaccine. Repertoire analyses of immunoglobulin heavy chain genes demonstrated that the number of B-lymphocyte subclones was increased in macaques after the 1st vaccination with the whole virus particle vaccine, but not with the split vaccine, indicating that the whole virus particle vaccine induced the activation of vaccine antigen-specific B-lymphocytes more vigorously than did the split vaccine at priming. Thus, the present findings suggest that the superior antibody induction ability of the whole virus particle vaccine as compared to the split vaccine is attributable to its stimulatory properties on the subclonal differentiation of antigen-specific B-lymphocytes.
• Autochthonous Leishmania infantum in Dogs, Zambia, 2021.

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

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

  Leishmaniases are neglected tropical diseases of humans and animals. We detected Leishmania infantum in 3 mixed-breed dogs in Zambia that had no travel history outside the country. Our findings suggest presence of and probable emergence of leishmaniasis in Zambia, indicating the need for physicians and veterinarians to consider the disease during diagnosis.
• Global research alliance in infectious disease: a collaborative effort to combat infectious diseases through dissemination of portable sequencing.

  Lucky R Runtuwene, Nuankanya Sathirapongsasuti, Raweewan Srisawat, Narumon Komalamisra, Josef S B Tuda, Arthur E Mongan, Gabriel O Aboge, Victoria Shabardina, Wojciech Makalowski, Dela Ria Nesti, Wayan T Artama, Lan Anh Nguyen-Thi, Kiew-Lian Wan, Byoung-Kuk Na, William Hall, Arnab Pain, Yuki Eshita, Ryuichiro Maeda, Junya Yamagishi, Yutaka Suzuki

  BMC research notes 15 (1) 44 - 44 2022/02/12 

  OBJECTIVE: To disseminate the portable sequencer MinION in developing countries for the main purpose of battling infectious diseases, we found a consortium called Global Research Alliance in Infectious Diseases (GRAID). By holding and inviting researchers both from developed and developing countries, we aim to train the participants with MinION's operations and foster a collaboration in infectious diseases researches. As a real-life example in which resources are limited, we describe here a result from a training course, a metagenomics analysis from two blood samples collected from a routine cattle surveillance in Kulan Progo District, Yogyakarta Province, Indonesia in 2019. RESULTS: One of the samples was successfully sequenced with enough sequencing yield for further analysis. After depleting the reads mapped to host DNA, the remaining reads were shown to map to Theileria orientalis using BLAST and OneCodex. Although the reads were also mapped to Clostridium botulinum, those were found to be artifacts derived from the cow genome. An effort to construct a consensus sequence was successful using a reference-based approach with Pomoxis. Hence, we concluded that the asymptomatic cow might be infected with T. orientalis and showed the usefulness of sequencing technology, specifically the MinION platform, in a developing country.
• TSS-seq of Toxoplasma gondii sporozoites revealed a novel motif in stage-specific promoters.

  Taishi Kidaka, Tatsuki Sugi, Kyoko Hayashida, Yutaka Suzuki, Xuenan Xuan, Jitender P Dubey, Junya Yamagishi

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 98 105213 - 105213 2022/01/15 

  Toxoplasma gondii is one of the most common zoonotic protozoan parasites. It has three major infectious stages: rapidly multiplying tachyzoites (Tz), slowly replicating bradyzoites (Bz) and a resting/free-living stage, sporozoites (Sz). The regulatory mechanisms governing stage-specific gene expression are not fully understood. Few transcriptional start sites (TSS) are known for Sz. In this study, we obtained TSS of Sz using an oligo-capping method and RNA-seq analysis. We identified 1,043,503 TSS in the Sz transcriptome. These defined 38,973 TSS clusters, of which, 11,925 were expressed in Sz and 1535 TSS differentially expressed in Sz. Based on these data, we defined promoter regions and novel sporozoite stage-specific motifs using MEME. TGTANNTACA was distributed around -55 to -75 regions from each TSS. Interestingly, the same motif was reported in another apicomplexan, Plasmodium berghei, as a cis-element of female-specific gametocyte genes, implying the presence of common regulatory machinery. Further comparative analysis should better define the distribution and function of these elements in other members of this important parasitic phylum.
• COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens.

  Tatsuki Sugi, Mizanur Rahman, Rummana Rahim, Abu Hasan, Naoko Kawai, Kyoko Hayashida, Junya Yamagishi

  Interdisciplinary perspectives on infectious diseases 2022 2109641 - 2109641 2022 

  With an increasing number of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) sequences gathered worldwide, we recognize that deletion mutants and nucleotide substitutions that may affect whole-genome sequencing are accumulating. Here, we propose an additional strategy for tiling PCR for whole-genome resequencing, which can make the pipeline robust for mutations at the primer annealing site by a redundant amplicon scheme. We further demonstrated that subtracting overrepresented amplicons from the multiplex PCR products reduced the bias of the next-generation sequencing (NGS) library, resulting in decreasing required sequencing reads per sample. We applied this sequencing strategy to clinical specimens collected in Bangladesh. More than 80% out of the 304 samples were successfully sequenced. Less than 5% were ambiguous nucleotides, and several known variants were detected. With the additional strategies presented here, we believe that whole-genome resequencing of SARS-CoV-2 from clinical samples can be optimized.
• Single Cell Transcriptomes of In Vitro Bradyzoite Infected Cells Reveals Toxoplasma gondii Stage Dependent Host Cell Alterations.

  Tatsuki Sugi, Tadakimi Tomita, Taishi Kidaka, Naoko Kawai, Kyoko Hayashida, Louis M Weiss, Junya Yamagishi

  Frontiers in cellular and infection microbiology 12 848693 - 848693 2022 

  Toxoplasma gondii bradyzoites establish chronic infections within their host cells. Recent studies have demonstrated that several parasite effector proteins are translocated to host cells during the bradyzoite stage of chronic infection. To understand the interaction between host cells and bradyzoites at the transcriptomic landscape level, we utilized single-cell RNA-sequencing (scRNA-Seq) to characterize the bradyzoite-induced host cell response. Distinct gene expression profiles were observed in infected host, cells with low parasite mapped reads, and mock (non-exposed) control cells. Gene set enrichment analysis showed that c-Myc and NF-κB signaling and energy metabolic pathways were upregulated by infection. Type I and II interferon response pathways were upregulated in cells with low parasite mapped reads compared to the non-exposed host control cells, and this upregulation effect was reversed in infected cells. Differences were observed in the host cells depending on the differentiation status of the parasites, as determined by BAG1 and SAG1 expression. NF-κB, inflammatory response pathways, and IFN-γ response pathways were downregulated in host cells containing T. gondii BAG1+/SAG1-, whereas this downregulation effect was reversed in case of T. gondii BAG1-/SAG1+. We also identified two distinct host cell subsets that contained T. gondii BAG1+/SAG1-, one of which displayed distinct transcriptomes with upregulated c-Myc expression. Overall, these data clearly demonstrate that host cell transcriptional alteration by bradyzoite infection is different from that of tachyzoite infection, indicating fine-tuning of the host immune response.
• SARS-CoV-2 Genome Sequences, Including One with an ORF7a Deletion, Obtained from Patients in Bangladesh.

  Mizanur Rahman, Rummana Rahim, Abu Hasan, Naoko Kawai, Lavel Chinyama Moonga, Tatsuki Sugi, Kyoko Hayashida, Junya Yamagishi

  Microbiology resource announcements 10 (49) e0076421  2021/12/09 

  Genomic sequences from a complete SARS-CoV-2 open reading frame (ORF) were obtained from 24 patients diagnosed in May 2020 in Dhaka, Bangladesh. All sequences belonged to clade 20A or 20B, and none were variants of concern. Interestingly, one sequence showed a 161-nucleotide deletion in ORF7a.
• Structural Requirements in the Hemagglutinin Cleavage Site-Coding RNA Region for the Generation of Highly Pathogenic Avian Influenza Virus.

  Yurie Kida, Kosuke Okuya, Takeshi Saito, Junya Yamagishi, Aiko Ohnuma, Takanari Hattori, Hiroko Miyamoto, Rashid Manzoor, Reiko Yoshida, Naganori Nao, Masahiro Kajihara, Tokiko Watanabe, Ayato Takada

  Pathogens (Basel, Switzerland) 10 (12) 2021/12/09 

  Highly pathogenic avian influenza viruses (HPAIVs) with H5 and H7 hemagglutinin (HA) subtypes are derived from their low pathogenic counterparts following the acquisition of multiple basic amino acids in their HA cleavage site. It has been suggested that consecutive adenine residues and a stem-loop structure in the viral RNA region that encodes the cleavage site are essential for the acquisition of the polybasic cleavage site. By using a reporter assay to detect non-templated nucleotide insertions, we found that insertions more frequently occurred in the RNA region (29 nucleotide-length) encoding the cleavage site of an H5 HA gene that was predicted to have a stem-loop structure containing consecutive adenines than in a mutated corresponding RNA region that had a disrupted loop structure with fewer adenines. In virus particles generated by using reverse genetics, nucleotide insertions that created additional codons for basic amino acids were found in the RNA region encoding the cleavage site of an H5 HA gene but not in the mutated RNA region. We confirmed the presence of virus clones with the ability to replicate without trypsin in a plaque assay and to cause lethal infection in chicks. These results demonstrate that the stem-loop structure containing consecutive adenines in HA genes is a key molecular determinant for the emergence of H5 HPAIVs.
• Evidence of Borrelia theileri in Wild and Domestic Animals in the Kafue Ecosystem of Zambia.

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

  Microorganisms 9 (11) 2021/11/22 

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

  Kota Maeda, Sonya Sadoughi, Ayako Morimoto, Kazuyuki Uchida, James K Chambers, Chizu Sanjoba, Junya Yamagishi, Yasuyuki Goto

  Pathogens (Basel, Switzerland) 10 (11) 2021/10/20 

  Visceral leishmaniasis (VL) is the most severe form of leishmaniasis caused by protozoan parasites of the genus Leishmania. Hepatomegaly is one of the most frequent clinical manifestations of VL, whereas immunopathology of the symptom has not been well investigated. Using our chronic model of experimental VL, we examined the influence of Leishmania donovani infection on the liver by clinical, histological, and biochemical analyses. The infected mice showed increased liver weight 24 weeks post-infection. Although an increase in serum ALT and inflammatory cell accumulation were observed in the livers of infected mice, no apparent parenchymal necrosis or fibrosis was observed. Tissue water content analyses demonstrated that increased liver weight was predominantly due to an increase in water weight. Together with the finding of hepatic sinusoidal dilation, these results suggested that edema associated with sinusoidal dilation causes hepatomegaly in L. donovani infection. Immunostaining of platelets and erythrocytes showed no thrombus formation or damage to the sinusoidal endothelium in the liver of infected mice. Taken together, these results suggest that hepatomegaly during experimental VL is caused by non-obstructive sinusoidal dilation.
• Rickettsia lusitaniae in Ornithodoros Porcinus Ticks, Zambia.

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

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

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

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

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

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

  Lavel C Moonga, Kyoko Hayashida, Namwiinga R Mulunda, Yukiko Nakamura, James Chipeta, Hawela B Moonga, Boniface Namangala, Chihiro Sugimoto, Zephaniah Mtonga, Mable Mutengo, Junya Yamagishi

  Emerging infectious diseases 27 (8) 2237 - 2239 2021/08 [Refereed]
   

  Rickettsia asembonensis is a flea-related Rickettsia with unknown pathogenicity to humans. We detected R. asembonensis DNA in 2 of 1,153 human blood samples in Zambia. Our findings suggest the possibility of R. asembonensis infection in humans despite its unknown pathogenicity.
• DNA methylation landscape of 16 canine somatic tissues by methylation-sensitive restriction enzyme-based next generation sequencing.

  Jumpei Yamazaki, Yuki Matsumoto, Jaroslav Jelinek, Teita Ishizaki, Shingo Maeda, Kei Watanabe, Genki Ishihara, Junya Yamagishi, Mitsuyoshi Takiguchi

  Scientific reports 11 (1) 10005 - 10005 2021/05/11 [Refereed]
   

  DNA methylation plays important functions in gene expression regulation that is involved in individual development and various diseases. DNA methylation has been well studied in human and model organisms, but only limited data exist in companion animals like dog. Using methylation-sensitive restriction enzyme-based next generation sequencing (Canine DREAM), we obtained canine DNA methylation maps of 16 somatic tissues from two dogs. In total, we evaluated 130,861 CpG sites. The majority of CpG sites were either highly methylated (> 70%, 52.5-64.6% of all CpG sites analyzed) or unmethylated (< 30%, 22.5-28.0% of all CpG sites analyzed) which are methylation patterns similar to other species. The overall methylation status of CpG sites across the 32 methylomes were remarkably similar. However, the tissue types were clearly defined by principle component analysis and hierarchical clustering analysis with DNA methylome. We found 6416 CpG sites located closely at promoter region of genes and inverse correlation between DNA methylation and gene expression of these genes. Our study provides basic dataset for DNA methylation profiles in dogs.
• Detection of B.1.351 SARS-CoV-2 Variant Strain - Zambia, December 2020.

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

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

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

  Yukiko Nakamura, Kyoko Hayashida, Victoire Delesalle, Yongjin Qiu, Ryosuke Omori, Martin Simuunza, Chihiro Sugimoto, Boniface Namangala, Junya Yamagishi

  Frontiers in veterinary science 8 599815 - 599815 2021 

  We clarified the genetic diversity of Trypanosoma spp. within the Kafue ecosystem, using PCR targeting the internal transcribed spacer 1 and the cathepsin L-like cysteine protease (CatL) sequences. The overall prevalence of Trypanosoma spp. in cattle and tsetse flies was 12.65 and 26.85%, respectively. Cattle positive for Trypanosoma vivax had a significantly lower packed cell volume, suggesting that T. vivax is the dominant Trypanosoma spp. causing anemia in this area. Among the 12 operational taxonomic units (OTUs) of T. vivax CatL sequences detected, one was from a known T. vivax lineage, two OTUs were from known T. vivax-like lineages, and nine OTUs were considered novel T. vivax-like lineages. These findings support previous reports that indicated the extensive diversity of T. vivax-like lineages. The findings also indicate that combining CatL PCR with next generation sequencing is useful in assessing Trypanosoma spp. diversity, especially for T. vivax and T. vivax-like lineages. In addition, the 5.42% prevalence of Trypanosoma brucei rhodesiense found in cattle raises concern in the community and requires careful monitoring of human African trypanosomiasis.
• Investigation of the piroplasm diversity circulating in wildlife and cattle of the greater Kafue ecosystem, Zambia.

  David Squarre, Yukiko Nakamura, Kyoko Hayashida, Naoko Kawai, Herman Chambaro, Boniface Namangala, Chihiro Sugimoto, Junya Yamagishi

  Parasites & vectors 13 (1) 599 - 599 2020/11/30 

  BACKGROUND: Piroplasms are vector-borne intracellular hemoprotozoan parasites that infect wildlife and livestock. Wildlife species are reservoir hosts to a diversity of piroplasms and play an important role in the circulation, maintenance and evolution of these parasites. The potential for likely spillover of both pathogenic and non-pathogenic piroplasm parasites from wildlife to livestock is underlined when a common ecological niche is shared in the presence of a competent vector. METHOD: To investigate piroplasm diversity in wildlife and the cattle population of the greater Kafue ecosystem, we utilized PCR to amplify the 18S rRNA V4 hyper-variable region and meta-barcoding strategy using the Illumina MiSeq sequencing platform and amplicon sequence variant (ASV)-based bioinformatics pipeline to generate high-resolution data that discriminate sequences down to a single nucleotide difference. RESULTS: A parasite community of 45 ASVs corresponding to 23 species consisting of 4 genera of Babesia, Theileria, Hepatozoon and Colpodella, were identified in wildlife and the cattle population from the study area. Theileria species were detected in buffalo, impala, hartebeest, sable antelope, sitatunga, wild dog and cattle. In contrast, Babesia species were only observed in cattle and wild dog. Our results demonstrate possible spillover of these hemoprotozoan parasites from wildlife, especially buffalo, to the cattle population in the wildlife-livestock interface. CONCLUSION: We demonstrated that the deep amplicon sequencing of the 18S rRNA V4 hyper-variable region for wildlife was informative. Our results illustrated the diversity of piroplasma and the specificity of their hosts. They led us to speculate a possible ecological cycle including transmission from wildlife to domestic animals in the greater Kafue ecosystem. Thus, this approach may contribute to the establishment of appropriate disease control strategies in wildlife-livestock interface areas.
• Development of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Method for Simultaneous Detection of Spotted Fever Group Rickettsiae and Malaria Parasites by Dipstick DNA Chromatography.

  Lavel Chinyama Moonga, Kyoko Hayashida, Naoko Kawai, Ryo Nakao, Chihiro Sugimoto, Boniface Namangala, Junya Yamagishi

  Diagnostics (Basel, Switzerland) 10 (11) 2020/11/02 

  Spotted fever group (SFG) rickettsiae causes febrile illness in humans worldwide. Since SFG rickettsiosis's clinical presentation is nonspecific, it is frequently misdiagnosed as other febrile diseases, especially malaria, and complicates proper treatment. Aiming at rapid, simple, and simultaneous detection of SFG Rickettsia spp. and Plasmodium spp., we developed a novel multiple pathogen detection system by combining a loop-mediated isothermal amplification (LAMP) method and dipstick DNA chromatography technology. Two primer sets detecting SFG Rickettsia spp. and Plasmodium spp. were mixed, and amplified products were visualized by hybridizing to dipstick DNA chromatography. The multiplex LAMP with dipstick DNA chromatography distinguished amplified Rickettsia and Plasmodium targeted genes simultaneously. The determined sensitivity using synthetic nucleotides was 1000 copies per reaction for mixed Rickettsia and Plasmodium genes. When genomic DNA from in vitro cultured organisms was used, the sensitivity was 100 and 10 genome equivalents per reaction for Rickettsia monacensis and Plasmodium falciparum, respectively. Although further improvement will be required for more sensitive detection, our developed simultaneous diagnosis technique will contribute to the differential diagnosis of undifferentiated febrile illness caused by either SFG Rickettsia spp. or Plasmodium spp. in resource-limited endemic areas. Importantly, this scheme is potentially versatile for the simultaneous detection of diverse infectious diseases.
• The Lethal(2)-Essential-for-Life [L(2)EFL] Gene Family Modulates Dengue Virus Infection in Aedes aegypti.

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

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

  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.
• Novel Babesia bovis exported proteins that modify properties of infected red blood cells.

  Hassan Hakimi, Thomas J Templeton, Miako Sakaguchi, Junya Yamagishi, Shinya Miyazaki, Kazuhide Yahata, Takayuki Uchihashi, Shin-Ichiro Kawazu, Osamu Kaneko, Masahito Asada

  PLoS pathogens 16 (10) e1008917  2020/10 

  Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.
• Development of a bio-inkjet printed LAMP test kit for detecting human African trypanosomiasis.

  Kyoko Hayashida, Peter Nambala, Nick Van Reet, Philippe Büscher, Naoko Kawai, Mable Mwale Mutengo, Janelisa Musaya, Boniface Namangala, Chihiro Sugimoto, Junya Yamagishi

  PLoS neglected tropical diseases 14 (10) e0008753  2020/10 

  Human African trypanosomiasis (HAT) is one of the neglected tropical diseases in sub-Saharan Africa. Early diagnosis and treatment prior to disease progression are crucial for the survival of HAT patients. We had previously established a loop-mediated isothermal amplification (LAMP) method for HAT diagnosis in which the reagents were dried for field-use purposes. In this study, we used a semi-automated process to produce the test tubes using a bio-inkjet printer to achieve an accurate production. The performance of the inkjet printer-produced dried LAMP test (CZC-LAMP) was found to be stable after storage for up to 180 days at 30 °C. The diagnostic accuracy of CZC-LAMP HAT was evaluated using DNA samples that were extracted from 116 Trypanosoma brucei gambiense patients and 66 T. b. rhodesiense patients. The sensitivity was 72% for T. b. gambiense (95%CI: 63%-80%) and 80% for T. b. rhodesiense (95%CI: 69%-89%). The specificity determined using DNA from 116 endemic control DNA samples was 95% (95%CI: 89%-98%). The performance of the CZC-LAMP HAT and CZC-LAMP rHAT were also evaluated using 14 crude blood lysate samples obtained from T. b. rhodesiense patients and endemic control samples collected from Rumphi District in Malawi. The sensitivity and specificity were both 100% (95%CI: 77%-100%). As the developed CZC-LAMP test does not require a cold chain or a sophisticated laboratory, it holds promise for use as a routine simple molecular tool for point-of-care HAT diagnosis in endemic areas.
• Diversity of trypanosomes in wildlife of the Kafue ecosystem, Zambia.

  David Squarre, Kyoko Hayashida, Alex Gaithuma, Herman Chambaro, Naoko Kawai, Ladslav Moonga, Boniface Namangala, Chihiro Sugimoto, Junya Yamagishi

  International journal for parasitology. Parasites and wildlife 12 34 - 41 2020/08 

  The Kafue ecosystem is a vast conservation protected area comprising the Kafue National Park (KNP) and the Game Management Areas (GMA) that act as a buffer around the national park. The KNP has been neglected as a potential foci for rhodesiense sleeping sickness despite the widespread presence of the tsetse vector and abundant wildlife reservoirs. The aim of this study was to generate information on circulating trypanosomes and their eminent threat/risk to public health and livestock production of a steadily growing human and livestock population surrounding the park. We detected various trypanosomes circulating in different mammalian wildlife species in KNP in Zambia by applying a high throughput ITS1-polymerase chain reaction (PCR)/nanopore sequencing method in combination with serum resistant associated-PCR/Sanger sequencing method. The prevalence rates of trypanosomes in hartebeest, sable antelope, buffalo, warthog, impala and lechwe were 6.4%, 37.2%, 13.2%, 11.8%, 2.8% and 11.1%, respectively. A total of six trypanosomes species or subspecies were detected in the wildlife examined, including Trypanosoma brucei brucei, T. godfreyi, T. congolense, T. simiae and T. theileri. Importantly we detected human infective T. b. rhodesiense in buffalo and sable antelope with a prevalence of 9.4% and 12.5%, respectively. In addition, T. b. rhodesiense was found in the only vervet monkey analyzed. The study thus reaffirmed that the Kafue ecosystem is a genuine neglected and re-emerging foci for human African trypanosomiasis. This is the first assessment of the trypanosome diversity circulating in free-ranging wildlife of the KNP.
• Morphological and molecular identification of Eimeria spp. in breeding chicken farms of Japan.

  Makoto Matsubayashi, Tomoyuki Shibahara, Tomohide Matsuo, Toshimitsu Hatabu, Junya Yamagishi, Kazumi Sasai, Takashi Isobe

  The Journal of veterinary medical science 82 (5) 516 - 519 2020/05/30 [Refereed][Not invited]
   

  There have been no reports of the prevalence of Eimeria spp. in poultry breeding farms in Japan unlike those of broiler farms. From 2017 to 2018, we examined the prevalence of Eimeria spp. on breeding farms in Japan by oocyst morphology and PCR analyses. A total of 143 samples was collected from 37 breeding farms in 21 prefectures of Japan. We detected oocysts of seven species at 34 of 37 breeding farms by PCR, and we identified E. brunetti at 51.5% of farms found to be positive for Eimeria. The differences in the identification of Eimeria spp. between the morphology and PCR assay methods of oocysts were pronounced for E. maxima and E. necatrix. We confirmed that molecular tools were more suitable for accurately estimating prevalence of Eimeria spp., and these findings suggest that E. brunetti could be widespread in Japan.
• Draft Genome Sequence of Leishmania tarentolae Parrot Tar II, Obtained by Single-Molecule Real-Time Sequencing.

  Yasuyuki Goto, Akihiro Kuroki, Kengo Suzuki, Junya Yamagishi

  Microbiology resource announcements 9 (21) 2020/05/21 

  Leishmania tarentolae is a protozoan parasite of lizards and is nonpathogenic to mammals. Genome information for the nonpathogenic species will facilitate an understanding of the pathology caused by species pathogenic to mammals. Here, we report resequencing of the L. tarentolae genome with a single-molecule real-time (SMRT) sequencer to provide a more complete genome assembly.
• Longitudinal plasma amino acid profiling with maternal genomic background throughout human pregnancy

  Matsuyuki Shirota, Daisuke Saigusa, Riu Yamashita, Yasutake Kato, Mitsuyo Matsumoto, Junya Yamagishi, Noriko Ishida, Kazuki Kumada, Yuji Oe, Hisaaki Kudo, Junji Yokozawa, Yoko Kuroki, Ikuko Motoike, Fumiki Katsuoka, Masao Nagasaki, Seizo Koshiba, Keiko Nakayama, Osamu Tanabe, Jun Yasuda, Shigeo Kure, Kengo Kinoshita, Hirohito Metoki, Shinichi Kuriyama, Nobuo Yaegashi, Masayuki Yamamoto, Junichi Sugawara

  Medical Mass Spectrometry 4 (1) 36 - 49 2020/04/16 [Refereed]
  
• Blood meal sources and bacterial microbiome diversity in wild-caught tsetse flies.

  Alex Gaithuma, Junya Yamagishi, Kyoko Hayashida, Naoko Kawai, Boniface Namangala, Chihiro Sugimoto

  Scientific reports 10 (1) 5005 - 5005 2020/03/19 

  Tsetse flies are the vectors of African trypanosomiasis affecting 36 sub-Saharan countries. Both wild and domestic animals play a crucial role in maintaining the disease-causing parasites (trypanosomes). Thus, the identification of animal reservoirs of trypanosomes is vital for the effective control of African trypanosomiasis. Additionally, the biotic and abiotic factors that drive gut microbiome diversity in tsetse flies are primarily unresolved, especially under natural, field conditions. In this study, we present a comprehensive DNA metabarcoding approach for individual tsetse fly analysis in the identification of mammalian blood meal sources and fly bacterial microbiome composition. We analyzed samples from two endemic foci, Kafue, Zambia collected in June 2017, and Hurungwe, Zimbabwe sampled in April 2014 (pilot study) and detected DNA of various mammals including humans, wild animals, domestic animals and small mammals (rat and bat). The bacterial diversity was relatively similar in flies with different mammalian species DNA, trypanosome infected and uninfected flies, and female and male flies. This study is the first report on bat DNA detection in wild tsetse flies. This study reveals that small mammals such as bats and rats are among the opportunistic blood meal sources for tsetse flies in the wild, and the implication on tsetse biology and ecology needs to be studied.
• Genetic and Biological Diversity of Porcine Sapeloviruses Prevailing in Zambia.

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

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

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

  Megasari Marsela, Kyoko Hayashida, Ryo Nakao, Elisha Chatanga, Alex Kiarie Gaithuma, Kawai Naoko, Janelisa Musaya, Chihiro Sugimoto, Junya Yamagishi

  Parasite (Paris, France) 27 46 - 46 2020 

  This study aimed to identify trypanosomes infecting cattle in Malawi in order to understand the importance of cattle in the transmission dynamics of Human African Trypanosomiasis (HAT) and Animal African Trypanosomosis (AAT). A total of 446 DNA samples from cattle blood from three regions of Malawi were screened for African trypanosomes by ITS1 PCR. The obtained amplicons were sequenced using a portable next-generation sequencer, MinION, for validation. Comparison of the results from ITS1 PCR and MinION sequencing showed that combining the two methods provided more accurate species identification than ITS1 PCR alone. Further PCR screening targeting the serum resistance-associated (SRA) gene was conducted to detect Trypanosoma brucei rhodesiense. Trypanosoma congolense was the most prevalent Trypanosoma sp., which was found in Nkhotakota (10.8%; 20 of 185), followed by Kasungu (2.5%; 5 of 199). Of note, the prevalence of T. b. rhodesiense detected by SRA PCR was high in Kasungu and Nkhotakota showing 9.5% (19 of 199) and 2.7% (5 of 185), respectively. We report the presence of animal African trypanosomes and T. b. rhodesiense from cattle at the human-livestock-wildlife interface for the first time in Malawi. Our results confirmed that animal trypanosomes are important causes of anemia in cattle and that cattle are potential reservoirs for human African trypanosomiasis in Malawi.
• Legionella pneumophila Infection Rewires the Acanthamoeba castellanii Transcriptome, Highlighting a Class of Sirtuin Genes.

  Pengfei Li, Dane Vassiliadis, Sze Ying Ong, Vicki Bennett-Wood, Chihiro Sugimoto, Junya Yamagishi, Elizabeth L Hartland, Shivani Pasricha

  Frontiers in cellular and infection microbiology 10 428 - 428 2020 

  Legionella pneumophila is an environmental bacterium that has evolved to survive predation by soil and water amoebae such as Acanthamoeba castellanii, and this has inadvertently led to the ability of L. pneumophila to survive and replicate in human cells. L. pneumophila causes Legionnaire's Disease, with human exposure occurring via the inhalation of water aerosols containing both amoebae and the bacteria. These aerosols originate from aquatic biofilms found in artifical water sources, such as air-conditioning cooling towers and humidifiers. In these man-made environments, A. castellanii supports L. pneumophila intracellular replication, thereby promoting persistence and dissemination of the bacteria and providing protection from external stress. Despite this close evolutionary relationship, very little is known about how A. castellanii responds to L. pneumophila infection. In this study, we examined the global transcriptional response of A. castellanii to L. pneumophila infection. We compared A. castellanii infected with wild type L. pneumophila to A. castellanii infected with an isogenic ΔdotA mutant strain, which is unable to replicate intracellularly. We showed that A. castellanii underwent clear morphological and transcriptional rewiring over the course of L. pneumophila infection. Through improved annotation of the A. castellanii genome, we determined that these transcriptional changes primarily involved biological processes utilizing small GTPases, including cellular transport, signaling, metabolism and replication. In addition, a number of sirtuin-encoding genes in A. castellanii were found to be conserved and upregulated during L. pneumophila infection. Silencing of sirtuin gene, sir6f (ACA1_153540) resulted in the inhibition of A. castellanii cell proliferation during infection and reduced L. pneumophila replication. Overall our findings identified several biological pathways in amoebae that may support L. pneumophila replication and A. castellanii proliferation in environmental conditions.
• Molecular characterization of Cryptosporidium spp. from patients with diarrhoea in Lusaka, Zambia.

  Namwiinga Rozaria Mulunda, Kyoko Hayashida, Junya Yamagishi, Sandie Sianongo, Gilbert Munsaka, Chihiro Sugimoto, Mable Mwale Mutengo

  Parasite (Paris, France) 27 53 - 53 2020 

  Cryptosporidium is a major etiological agent of diarrhoeal diseases among children and immune-compromised individuals in sub-Saharan African countries. We conducted a study to determine the prevalence and genetic characteristics of Cryptosporidium spp. in stool samples from patients with diarrhoea who presented at the University Teaching Hospital in Lusaka, Zambia. Cryptosporidium species and subtypes from 71 microscopically confirmed cryptosporidiosis stool samples collected between 2017 and 2019 were determined by polymerase chain reaction followed by partial sequencing of the small subunit rRNA and 60-kDa glycoprotein (gp60) gene. Additionally, data for the period between 2014 and 2019 were reviewed and analysed for cryptosporidiosis seasonal and age distribution. Cryptosporidium was more prevalent in the rainy season. The highest number of cases was reported among the 1-4 year age group. By sequence analysis of the 71 positive isolates, Cryptosporidium hominis (n = 42; 59.2%), C. parvum (n = 27; 38%), C. felis (n = 1; 1.4%), and C. meleagridis (n = 1; 1.4%) were identified. Four C. hominis subtype families (Ia, Ib, Id, and Ie) and three C. parvum subtype families (IIc, IIe, and IIs) were identified. The most frequent subtypes were IeA11G3T3 (n = 20; 28.2%), IIcA5G3 (n = 12; 16.9%), IIeA12G1 (n = 11; 15.5%) and IaA30R3 (n = 10; 14.1%). The observed species/subtypes of C. hominis and C. parvum indicated that the infection was mainly transmitted through the anthroponotic route. The identification of C. felis and C. meleagridis suggests that an atypical zoonotic transmission cycle also exists.
• Complete genome and bimodal genomic structure of the amoebal symbiont Neochlamydia strain S13 revealed by ultra-long reads obtained from MinION.

  Yamagishi J, Hayashida K, Matsuo J, Okubo T, Kuroda M, Nagai H, Sekizuka T, Yamaguchi H, Sugimoto C

  Journal of human genetics 65 (1) 41 - 48 1434-5161 2019/11 [Refereed][Not invited]
  
• Rapid sequencing-based diagnosis of infectious bacterial species from meningitis patients in Zambia.

  Nakagawa S, Inoue S, Kryukov K, Yamagishi J, Ohno A, Hayashida K, Nakazwe R, Kalumbi M, Mwenya D, Asami N, Sugimoto C, Mutengo MM, Imanishi T

  Clinical & Translational Immunology 8 (11) e01087  2019/11 [Refereed][Not invited]
   

  Objectives: We have developed a portable system for the rapid determination of bacterial composition for the diagnosis of infectious diseases. Our system comprises of a nanopore technology-based sequencer, MinION, and two laptop computers. To examine the accuracy and time efficiency of our system, we provided a proof-of-concept for the detection of the causative bacteria of 11 meningitis patients in Zambia. Methods: We extracted DNA from cerebrospinal fluid samples of each patient and amplified the 16S rRNA gene regions. The sequencing library was prepared, and the sequenced reads were simultaneously processed for bacterial composition determination using the minimap2 software and the representative prokaryote genomes. Results: The sequencing results of four of the six culture-positive samples were consistent with those of conventional culture-based methods. The dominant bacterial species in each of these samples were identified from the sequencing data within only 3 min. Although the major bacterial species were also detected from the other two culture-positive samples and five culture-negative samples, their presence could not be confirmed. Moreover, as a whole, although the number of sequencing reads obtained within a short sequencing run was small, there was no change in the major bacterial species over time with prolonged sequencing. In addition, the processing time strongly correlated with the number of sequencing reads used for the analysis. Conclusion: Our results suggest that time-effective analysis could be achieved by determining the number of sequencing reads required for the rapid diagnosis of infectious bacterial species depending on the complexity of bacterial species in a sample.
• Transcriptome analysis of the effect of C-C chemokine receptor 5 deficiency on cell response to Toxoplasma gondii in brain cells.

  Kobayashi K, Umeda K, Ihara F, Tanaka S, Yamagishi J, Suzuki Y, Nishikawa Y

  BMC genomics 20 (1) 705  2019/09 [Refereed][Not invited]
   

  Background: Infection with Toxoplasma gondii is thought to damage the brain and be a risk factor for neurological and psychotic disorders. The immune response-participating chemokine system has recently been considered vital for brain cell signaling and neural functioning. Here, we investigated the effect of the deficiency of C-C chemokine receptor 5 (CCR5), which is previously reported to be associated with T. gondii infection, on gene expression in the brain during T. gondii infection and the relationship between CCR5 and the inflammatory response against T. gondii infection in the brain. Results: We performed a genome-wide comprehensive analysis of brain cells from wild-type and CCR5-deficient mice. Mouse primary brain cells infected with T. gondii were subjected to RNA sequencing. The expression levels of some genes, especially in astrocytes and microglia, were altered by CCR5-deficiency during T. gondii infection, and the gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed an enhanced immune response in the brain cells. The expression levels of genes which were highly differentially expressed in vitro were also investigated in the mouse brains during the T. gondii infections. Among the genes tested, only Saa3 (serum amyloid A3) showed partly CCR5-dependent upregulation during the acute infection phase. However, analysis of the subacute phase showed that in addition to Saa3, Hmox1 may also contribute to the protection and/or pathology partly via the CCR5 pathway. Conclusions: Our results indicate that CCR5 is involved in T. gondii infection in the brain where it contributes to inflammatory responses and parasite elimination. We suggest that the inflammatory response by glial cells through CCR5 might be associated with neurological injury during T. gondii infection to some extent.
• Complete Genome Sequence of Rickettsia asiatica Strain Maytaro1284, a Member of Spotted Fever Group Rickettsiae Isolated from an Ixodes ovatus Tick in Japan.

  Thu MJ, Qiu Y, Yamagishi J, Kusakisako K, Ogata S, Moustafa MAM, Isoda N, Sugimoto C, Katakura K, Nonaka N, Nakao R

  Microbiology resource announcements 8 (37) 2019/09 [Refereed][Not invited]
   

  This is the first report of the complete genome sequence of Rickettsia asiatica strain Maytaro1284, isolated from an Ixodes ovatus tick in Japan. The genome contains a 1,344,324-bp circular chromosome and one plasmid of 74,761 bp. There was no outer membrane protein A ( ompA ) gene encoded in the genome.
• Transitions in morphological forms and rapid development of the asexual schizonts of Eimeria tenella through serial passaging in chicks.

  Matsubayashi M, Yamaguchi H, Hatta T, Kawahara F, Hatabu T, Iseki H, Yamagishi J, Isobe T, Teramoto I, Kaneko A, Kita K, Tsuji N, Sasai K

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 75 103993  1567-1348 2019/08 [Refereed][Not invited]
   

  Attenuated strains of avian Eimeria parasites, generated by the selection of precocious lines through serial passaging in chicks, have been used widely as live vaccines. Detailed morphological transitions including their life cycle depending on the passages remain poorly understood. Here, we showed early development and acceleration of transitions in morphological forms of the asexual schizonts of E. tenella that had been attenuated for virulence by serial passaging. Our results may be helpful in understanding parasitism, facilitating further molecular analyses such as comparative genomic or transcriptomic tests.
• Initiated Babesia ovata Sexual Stages under In Vitro Conditions Were Recognized by Anti-CCp2 Antibodies, Showing Changes in the DNA Content by Imaging Flow Cytometry.

  Nguyen TT, Dang-Trinh MA, Higuchi L, Mosqueda J, Hakimi H, Asada M, Yamagishi J, Umemiya-Shirafuji R, Kawazu SI

  Pathogens (Basel, Switzerland) 8 (3) 2019/07 [Refereed][Not invited]
   

  Sexual stage induction under in vitro conditions is useful for biological and molecular studies of Babesia parasites. Therefore, in the present study, we induced B. ovata tick stages using the chemical inducers: xanthurenic acid (XA), dithiothreitol (DTT) and tris (2-carboxyethyl) phosphine (TCEP) at 27 °C or 37 °C conditions. Cultures at low temperature (27 °C) or treated with XA/TCEP induced a large number of extra-erythrocytic merozoites, which transformed into round shape cells at 12-24 h post-induction (pi). However, typical forms of tick stages (aggregation forms and the spiky forms/ray bodies) were only observed in the cultures treated with 40 mM or 60 mM of DTT during 3-6 h pi. The induced cells were recognized by anti-CCp2 rabbit antisera. DNA content of the cell population treated with 40 mM of DTT was analyzed by imaging flow cytometry at 0, 12 and 48 h pi. The results indicated that the parasite population with diploid-like double DNA content increased at 48 h pi. Our observations on morphological and changes in the DNA content provide useful information for understanding the life cycle of B. ovata under in vitro conditions, which will facilitate further studies on basic biology and the development of transmission blocking vaccines against bovine babesiosis.
• Genetic diversity and population structure of Glossina morsitans morsitans in the active foci of human African trypanosomiasis in Zambia and Malawi.

  Nakamura Y, Yamagishi J, Hayashida K, Osada N, Chatanga E, Mweempwa C, Chilongo K, Chisi J, Musaya J, Inoue N, Namangala B, Sugimoto C

  PLoS neglected tropical diseases 13 (7) e0007568  1935-2727 2019/07 [Refereed][Not invited]
  
• Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing.

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

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

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

  Moonga LC, Hayashida K, Nakao R, Lisulo M, Kaneko C, Nakamura I, Eshita Y, Mweene AS, Namangala B, Sugimoto C, Yamagishi J

  Parasites & vectors 12 (1) 168  2019/04 [Refereed][Not invited]
  
• Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing.

  Lin Sun, Naoko Kono, Hiroyuki Toh, Hanbing Xue, Kaori Sano, Tadaki Suzuki, Akira Ainai, Yasuko Orba, Junya Yamagishi, Hideki Hasegawa, Yoshimasa Takahashi, Shigeyuki Itamura, Kazuo Ohnishi

  Journal of visualized experiments : JoVE (145) 2019/03/15 [Refereed][Not invited]
   

  The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5'-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird's-eye view of the antibody repertoire, and our computational methods.
• A single test approach for accurate and sensitive detection and taxonomic characterization of Trypanosomes by comprehensive analysis of internal transcribed spacer 1 amplicons.

  Gaithuma AK, Yamagishi J, Martinelli A, Hayashida K, Kawai N, Marsela M, Sugimoto C

  PLoS neglected tropical diseases 13 (2) e0006842  1935-2727 2019/02 [Refereed][Not invited]
  
• Draft Genome Sequence of Trypanosoma equiperdum Strain IVM-t1.

  Batdorj Davaasuren, Junya Yamagishi, Daiki Mizushima, Sandagdorj Narantsatsral, Davaajav Otgonsuren, Punsantsogvoo Myagmarsuren, Badgar Battsetseg, Banzragch Battur, Noboru Inoue, Keisuke Suganuma

  Microbiology resource announcements 8 (9) 2576-098X 2019/02 [Refereed][Not invited]
   

  Trypanosoma equiperdum primarily parasitizes the genital organs and causes dourine in equidae. We isolated a new T. equiperdum strain, T. equiperdum IVM-t1, from the urogenital tract of a horse definitively diagnosed as having dourine in Mongolia. Here, we report the whole-genome sequence, the predicted gene models, and their annotations.
• Hemophagocytosis induced by Leishmania donovani infection is beneficial to parasite survival within macrophages

  Ayako Morimoto, Kazuyuki Uchida, James K. Chambers, Kai Sato, Jing Hong, Chizu Sanjoba, Yoshitsugu Matsumoto, Junya Yamagishi, Yasuyuki Goto

  PLoS Neglected Tropical Diseases 13 (11) 1935-2727 2019 

  © 2019 Morimoto et al. Visceral leishmaniasis (VL) is caused by parasitic protozoa of the genus Leishmania and is characterized by clinical manifestations such as fever, hepatosplenomegaly and anemia. Hemophagocytosis, the phenomenon of phagocytosis of blood cells by macrophages, is found in VL patients. In a previous study we established an experimental model of VL, reproducing anemia in mice for the first time, and identified hemophagocytosis by heavily infected macrophages in the spleen as a possible cause of anemia. However, the mechanism for parasite-induced hemophagocytosis or its role in parasite survival remained unclear. Here, we established an in vitro model of Leishmania-induced hemophagocytosis to explore the molecules involved in this process. In contrast to naive RAW264.7 cells (mouse macrophage cell line) which did not uptake freshly isolated erythrocytes, RAW264.7 cells infected with L. donovani showed enhanced phagocytosis of erythrocytes. Additionally, for hemophagocytes found both in vitro and in vivo, the expression of signal regulatory protein α (SIRPα), one of the receptors responsible for the edonft-eat-mef signal was suppressed by post-transcriptional control. Furthermore, the overlapped phagocytosis of erythrocytes and Leishmania parasites within a given macrophage appeared to be beneficial to the parasites; the in vitro experiments showed a higher number of parasites within macrophages that had been induced to engulf erythrocytes. Together, these results suggest that Leishmania parasites may actively induce hemophagocytosis by manipulating the expression of SIRPα in macrophages/hemophagocytes, in order to secure their parasitism.
• Consensus and variations in cell line specificity among human metapneumovirus strains.

  Nao N, Sato K, Yamagishi J, Tahara M, Nakatsu Y, Seki F, Katoh H, Ohnuma A, Shirogane Y, Hayashi M, Suzuki T, Kikuta H, Nishimura H, Takeda M

  PloS one 14 (4) e0215822  2019 [Refereed][Not invited]
   

  Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPVGFP). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b180nt-dup strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b180nt-dup strains, and six recombinant HMPVGFP strains, including the newly generated recombinant HMPV A2b180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPVGFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.
• Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella.

  Matsubayashi M, Inaoka DK, Komatsuya K, Hatta T, Kawahara F, Sakamoto K, Hikosaka K, Yamagishi J, Sasai K, Shiba T, Harada S, Tsuji N, Kita K

  Genes 10 (1) 2019/01 [Refereed][Not invited]
   

  Eimeria tenella is an intracellular apicomplexan parasite, which infects cecal epithelial cells from chickens and causes hemorrhagic diarrhea and eventual death. We have previously reported the comparative RNA sequence analysis of the E. tenella sporozoite stage between virulent and precocious strains and showed that the expression of several genes involved in mitochondrial electron transport chain (ETC), such as type II NADH dehydrogenase (NDH-2), complex II (succinate:quinone oxidoreductase), malate:quinone oxidoreductase (MQO), and glycerol-3-phosphate dehydrogenase (G3PDH), were upregulated in virulent strain. To study E. tenella mitochondrial ETC in detail, we developed a reproducible method for preparation of mitochondria-rich fraction from sporozoites, which maintained high specific activities of dehydrogenases, such as NDH-2 followed by G3PDH, MQO, complex II, and dihydroorotate dehydrogenase (DHODH). Of particular importance, we showed that E. tenella sporozoite mitochondria possess an intrinsic ability to perform fumarate respiration (via complex II) in addition to the classical oxygen respiration (via complexes III and IV). Further analysis by high-resolution clear native electrophoresis, activity staining, and nano-liquid chromatography tandem-mass spectrometry (nano-LC-MS/MS) provided evidence of a mitochondrial complex II-III-IV supercomplex. Our analysis suggests that complex II from E. tenella has biochemical features distinct to known orthologues and is a potential target for the development of new anticoccidian drugs.
• Establishment of a mouse-tick infection model for Theileria orientalis and analysis of its transcriptome.

  Kyoko Hayashida, Rika Umemiya-Shirafuji, Thillaiampalam Sivakumar, Junya Yamagishi, Yutaka Suzuki, Chihiro Sugimoto, Naoaki Yokoyama

  International journal for parasitology 48 (12) 915 - 924 0020-7519 2018/10 [Refereed][Not invited]
   

  Oriental theileriosis caused by Theileria orientalis is an economically significant disease in cattle farming. The lack of laboratory animal models and in vitro culture systems is a major obstacle in the drive to better understand the biology of this parasite. Notably, research on the sporozoite stage of T. orientalis has rarely been undertaken, although such investigations are of paramount importance for vaccine development based on blocking sporozoite invasion of its host animals. In the present study, we established a mouse-tick infection model for propagating T. orientalis in mice and for producing the sporozoite stage in tick salivary glands. Splenectomized severe combined immunodeficient mice transfused with bovine erythrocytes were infected with T. orientalis. The larval ticks of Haemaphysalis longicornis were then fed on the T. orientalis-infected mice. The piroplasm and sporozoite stages were microscopically observed in the mouse blood and nymphal salivary glands, respectively. The transcriptomics data generated from the piroplasm and sporozoite stages revealed a stage-specific expression pattern for the parasite genes. The mouse-tick infection model and the transcriptomics data it has provided will contribute to a better understanding of T. orientalis biology and will also provide much needed information for the design of effective control measures targeting oriental theileriosis.
• Targeting of RNA Polymerase II by a nuclear Legionella pneumophila Dot/Icm effector SnpL.

  Schuelein R, Spencer H, Dagley LF, Li PF, Luo L, Stow JL, Abraham G, Naderer T, Gomez-Valero L, Buchrieser C, Sugimoto C, Yamagishi J, Webb AI, Pasricha S, Hartland EL

  Cellular microbiology 20 (9) e12852  1462-5814 2018/09 [Refereed][Not invited]
  
• Eimeria tenella 弱毒株の作出と比較ゲノム解析による弱毒化分子機構の解明

  松林 誠, 川原 史也, 山岸 潤也, 八田 岳士, 畑生 俊光, 山口 浩貴, 寺本 勲, 金子 明, 磯部 尚, 北 潔, 辻 尚利, 笹井 和美

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 161回 316 - 316 1347-8621 2018/08
• ザンビア、マラウイにおけるツェツェバエの集団遺伝学解析 ツェツェバエ媒介性アフリカトリパノソーマ症対策への応用

  中村 有紀子, 林田 京子, 山岸 潤也, 杉本 千尋

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 161回 320 - 320 1347-8621 2018/08
• Genetic analysis of Babesia isolates from cattle with clinical babesiosis in Sri Lanka.

  Sivakumar T, Tuvshintulga B, Zhyldyz A, Kothalawala H, Yapa PR, Kanagaratnam R, Vimalakumar SC, Abeysekera TS, Weerasingha AS, Yamagishi J, Igarashi I, Silva SSP, Yokoyama N

  Journal of clinical microbiology 56 (11) 0095-1137 2018/08 [Refereed][Not invited]
   

  Bovine babesiosis is a serious threat to the cattle industry. We prepared blood DNA samples from 13 cattle with clinical babesiosis from the Badulla (n = 8), Jaffna (n = 3), and Kilinochchi (n = 2) districts in Sri Lanka. These DNA samples tested positive in PCR assays specific for Babesiabovis (n = 9), Babesia bigemina (n = 9), and Babesiaovata (n = 1). Twelve cattle were positive for B. bovis and/or B. bigemina One cow was negative for the tested Babesia species but was positive for Babesia on microscopic examination; the phylogenetic positions of 18S rRNA and cytochrome oxidase subunit III gene sequences suggested that the cow was infected with Babesia sp. Mymensingh, which was recently reported from a healthy cow in Bangladesh. We then developed a novel Babesia sp. Mymensingh-specific PCR assay and obtained positive results for one other sample. Analysis of gene sequences from the cow with positive B. ovata-specific PCR results demonstrated that the animal was infected not with B. ovata but with Babesia sp. Hue-1, which was recently reported from asymptomatic cattle in Vietnam. The virulence of Babesia sp. Hue-1 is unclear, as the cow was coinfected with B. bovis and B. bigemina However, Babesia sp. Mymensingh probably causes severe clinical babesiosis, as it was the sole Babesia species detected in a clinical case. The present study revealed the presence of two bovine Babesia species not previously reported in Sri Lanka, plus the first case of severe bovine babesiosis caused by a Babesia species other than B. bovis, B. bigemina, and Babesiadivergens.
• Development and validation of direct dry loop mediated isothermal amplification for diagnosis of Trypanosoma evansi.

  Salim B, Hayashida K, Mossaad E, Nakao R, Yamagishi J, Sugimoto C

  Veterinary parasitology 260 53 - 57 0304-4017 2018/08 [Refereed][Not invited]
  
• An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer.

  Kazuo Imai, Norihito Tarumoto, Lucky Ronald Runtuwene, Jun Sakai, Kyoko Hayashida, Yuki Eshita, Ryuichiro Maeda, Josef Tuda, Hideaki Ohno, Takashi Murakami, Shigefumi Maesaki, Yutaka Suzuki, Junya Yamagishi, Takuya Maeda

  Malaria journal 17 (1) 217 - 217 2018/05/29 [Refereed][Not invited]
   

  BACKGROUND: The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. RESULTS: A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. CONCLUSIONS: An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.
• Nanopore sequencing of drug-resistance-associated genes in malaria parasites, Plasmodium falciparum.

  Lucky R Runtuwene, Josef S B Tuda, Arthur E Mongan, Wojciech Makalowski, Martin C Frith, Mallika Imwong, Suttipat Srisutham, Lan Anh Nguyen Thi, Nghia Nguyen Tuan, Yuki Eshita, Ryuichiro Maeda, Junya Yamagishi, Yutaka Suzuki

  Scientific reports 8 (1) 8286 - 8286 2018/05/29 [Refereed][Not invited]
   

  Here, we report the application of a portable sequencer, MinION, for genotyping the malaria parasite Plasmodium falciparum. In the present study, an amplicon mixture of nine representative genes causing resistance to anti-malaria drugs is diagnosed. First, we developed the procedure for four laboratory strains (3D7, Dd2, 7G8, and K1), and then applied the developed procedure to ten clinical samples. We sequenced and re-sequenced the samples using the obsolete flow cell R7.3 and the most recent flow cell R9.4. Although the average base-call accuracy of the MinION sequencer was 74.3%, performing >50 reads at a given position improves the accuracy of the SNP call, yielding a precision and recall rate of 0.92 and 0.8, respectively, with flow cell R7.3. These numbers increased significantly with flow cell R9.4, in which the precision and recall are 1 and 0.97, respectively. Based on the SNP information, the drug resistance status in ten clinical samples was inferred. We also analyzed K13 gene mutations from 54 additional clinical samples as a proof of concept. We found that a novel amino-acid changing variation is dominant in this area. In addition, we performed a small population-based analysis using 3 and 5 cases (K13) and 10 and 5 cases (PfCRT) from Thailand and Vietnam, respectively. We identified distinct genotypes from the respective regions. This approach will change the standard methodology for the sequencing diagnosis of malaria parasites, especially in developing countries.
• Establishment of a stable transfection system for genetic manipulation of Babesia gibsoni

  Mingming Liu, Paul Franck Adjou Moumouni, Masahito Asada, Hassan Hakimi, Tatsunori Masatani, Patrick Vudriko, Seung-Hun Lee, Shin-Ichiro Kawazu, Junya Yamagishi, Xuenan Xuan

  Parasites and Vectors 11 (1) 260  1756-3305 2018/04/23 [Refereed][Not invited]
   

  Background: Genetic manipulation techniques, such as transfection, have been previously reported in many protozoan parasites. In Babesia, stable transfection systems have only been established for bovine Babesia parasites. We recently reported a transient transfection system and the selection of promoter candidates for Babesia gibsoni. The establishment of a stable transfection system for B. gibsoni is considered to be urgent to improve our understanding of the basic biology of canine Babesia parasites for a better control of babesiosis. Results: GFP-expressing parasites were observed by fluorescence microscopy as early as two weeks after drug selection, and consistently expressed GFP for more than 3 months without drug pressure. Genome integration was confirmed by PCR, sequencing and Southern blot analysis. Conclusions: We present the first successful establishment of a stable transfection system for B. gibsoni. This finding will facilitate functional analysis of Babesia genomes using genetic manipulation and will serve as a foundation for the development of tick-Babesia and host-Babesia infection models.
• Comparative genomics and proteomic analyses between lethal and nonlethal strains of Plasmodium berghei

  Mamoru Niikura, Shin Ichi Inoue, Toshiyuki Fukutomi, Junya Yamagishi, Hiroko Asahi, Fumie Kobayashi

  Experimental Parasitology 185 1 - 9 0014-4894 2018/02 [Refereed][Not invited]
   

  © 2018 Elsevier Inc. Plasmodium berghei (Pb) XAT, a rodent malaria parasite, is an irradiation-attenuated variant derived from the lethal strain Pb NK65. Differences in genome sequence, protein structure and function between Pb XAT and Pb NK65 are currently unknown. In this study, to investigate genetic alterations in Pb XAT, we performed comparative genomics and proteomics analyses of nonlethal and lethal strains of Pb. We found mutations, such as a deletion mutation in rhoptry-associated protein (rap) 1, and deletion of rap2/3 and skeleton-binding protein 1 (sbp1), in Pb XAT. RAP1 is required for targeting of RAP2 to the rhoptries. However, the contribution of RAP2/3 to the lethality of Plasmodium is unclear. Therefore, we generated RAP1- and RAP2/3-deficient mutants of Pb ANKA, a reference strain of P. berghei. Furthermore, we investigated the effect of RAP1 and RAP2/3 deficiency on the outcome of infection. The parasitemia in mice infected with RAP1-deficient parasites was increased compared to that in control parasite-infected mice during the early phase of infection. However, mice infected with RAP1-deficient parasites survived longer than did control parasite-infected mice. Moreover, mice infected with RAP2/3-deficient parasites showed low levels of parasitemia and ultimately recovered from the infection The aim of this study was to investigate the effect of RAP2/3 expression on the outcome of infection with Pb XAT using a RAP2/3-expressing Pb XAT. Results showed that complementation of RAP2/3 expression in Pb XAT partially restored virulence. Our findings suggest that RAP1 and RAP2/3 contribute to virulence and a decrease in their expression explains the loss of virulence of the Pb XAT strain.
• 【どこでも 誰でも より長く ナノポアシークエンサーが研究の常識を変える!】フィールドでの熱帯病原性微生物タイピング

  山岸 潤也, Runtuwene Lucky, 林田 京子, 鈴木 穣

  実験医学 (株)羊土社 36 (1) 27 - 31 0288-5514 2018/01 

  シンガポールではデングウイルス感染者が日々発生しているが、それがニュースになることはない。一方、本邦では2015年のデングウイルスの国内感染例が大いに世間を騒がせた。これは日本人にとって熱帯感染症がそれだけ遠い世界の物語である(と思い込んでいる)ことを示していることの証左といえる。しかしながら目を世界に転じれば、そこには熱帯感染症が満ち溢れている。本稿では、近年のポータブルシークエンサーMinIONの熱帯感染症病原性微生物、特にその野外株についてのゲノム解析の応用について概説する。on siteでのシークエンスがより普及すれば、さらに多くの多型あるいは新規の病原性微生物が同定されるかもしれない。(著者抄録)
• Transcriptional profiling of Toll-like receptor 2-deficient primary murine brain cells during Toxoplasma gondii infection

  Kousuke Umeda, Sachi Tanaka, Fumiaki Ihara, Junya Yamagishi, Yutaka Suzuki, Yoshifumi Nishikawa

  PLOS ONE 12 (11) e0187703  1932-6203 2017/11 [Refereed][Not invited]
   

  Background Toxoplasma gondii is capable of persisting in the brain, although it is efficiently eliminated by cellular immune responses in most other sites. While Toll-like receptor 2 (TLR2) reportedly plays important roles in protective immunity against the parasite, the relationship between neurological disorders induced by T. gondii infection and TLR2 function in the brain remains controversial with many unknowns. In this study, primary cultured astrocytes, microglia, neurons, and peritoneal macrophages obtained from wild-type and TLR2-deficient mice were exposed to T. gondii tachyzoites. To characterize TLR2-dependent functional pathways activated in response to T. gondii infection, gene expression of different cell types was profiled by RNA sequencing. Results During T. gondii infection, a total of 611, 777, 385, and 1105 genes were upregulated in astrocytes, microglia, neurons, and macrophages, respectively, while 163, 1207, 158, and 1274 genes were downregulated, respectively, in a TLR2-dependent manner. Overrepresented Gene Ontology (GO) terms for TLR2-dependently upregulated genes were associated with immune and stress responses in astrocytes, immune responses and developmental processes in microglia, metabolic processes and immune responses in neurons, and metabolic processes and gene expression in macrophages. Overrepresented GO terms for downregulated genes included ion transport and behavior in astrocytes, cell cycle and cell division in microglia, metabolic processes in neurons, and response to stimulus, signaling and cell motility in macrophages. Conclusions To our knowledge, this is the first transcriptomic study of TLR2 function across different cell types during T. gondii infection. Results of RNA-sequencing demonstrated roles for TLR2 varied by cell type during T. gondii infection. Our findings facilitate understanding of the detailed relationship between TLR2 and T. gondii infection, and elucidate mechanisms underlying neurological changes during infection.
• Identification and characterization of interchangeable cross-species functional promoters between Babesia gibsoni and Babesia bovis.

  Liu M, Adjou Moumouni PF, Cao S, Asada M, Wang G, Gao Y, Guo H, Li J, Vudriko P, Efstratiou A, Ringo AE, Lee SH, Hakimi H, Masatani T, Sunaga F, Kawazu SI, Yamagishi J, Jia L, Inoue N, Xuan X

  Ticks and tick-borne diseases 1877-959X 2017/11 [Refereed][Not invited]
  
• Whole-genome assembly of Babesia ovata and comparative genomics between closely related pathogens

  Junya Yamagishi, Masahito Asada, Hassan Hakimi, Takeshi Q. Tanaka, Chihiro Sugimoto, Shin-ichiro Kawazu

  BMC GENOMICS 18 (1) 832  1471-2164 2017/10 [Refereed][Not invited]
   

  Background: Babesia ovata, belonging to the phylum Apicomplexa, is an infectious parasite of bovids. It is not associated with the manifestation of severe symptoms, in contrast to other types of bovine babesiosis caused by B. bovis and B. bigemina; however, upon co-infection with Theileria orientalis, it occasionally induces exacerbated symptoms. Asymptomatic chronic infection in bovines is usually observed only for B. ovata. Comparative genomic analysis could potentially reveal factors involved in these distinguishing characteristics; however, the genomic and molecular basis of these phenotypes remains elusive, especially in B. ovata. From a technical perspective, the current development of a very long read sequencer, MinION, will facilitate the obtainment of highly integrated genome sequences. Therefore, we applied next-generation sequencing to acquire a high-quality genome of the parasite, which provides fundamental information for understanding apicomplexans. Results: The genome was assembled into 14,453,397 bp in size with 5031 protein-coding sequences (91 contigs and N50 = 2,090,503 bp). Gene family analysis revealed that ves1 alpha and beta, which belong to multigene families in B. bovis, were absent from B. ovata, the same as in B. bigemina. Instead, ves1a and ves1b, which were originally specified in B. bigemina, were present. The B. ovata and B. bigemina ves1a configure one cluster together even though they divided into two sub-clusters according to the spp. In contrast, the ves1b cluster was more dispersed and the overlap among B. ovata and B. bigemina was limited. The observed redundancy and rapid evolution in sequence might reflect the adaptive history of these parasites. Moreover, same candidate genes which potentially involved in the distinct phenotypes were specified by functional analysis. An anamorsin homolog is one of them. The human anamorsin is involved in hematopoiesis and the homolog was present in B. ovata but absent in B. bigemina which causes severe anemia. Conclusions: Taking these findings together, the differences demonstrated by comparative genomics potentially explain the evolutionary history of these parasites and the differences in their phenotypes. Besides, the draft genome provides fundamental information for further characterization and understanding of these parasites.
• Transient transfection of intraerythrocytic Babesia gibsoni using elongation factor-1 alpha promoter

  Mingming Liu, Masahito Asada, Shinuo Cao, Paul Franck Adjou Moumouni, Patrick Vudriko, Artemis Efstratiou, Hassan Hakimi, Tatsunori Masatani, Fujiko Sunaga, Shin-ichiro Kawazu, Junya Yamagishi, Xuenan Xuan

  MOLECULAR AND BIOCHEMICAL PARASITOLOGY 216 56 - 59 0166-6851 2017/09 [Refereed][Not invited]
   

  The development of gene manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not yet been established for Babesia gibsoni. Here, we report for the first time, the successful transient transfection of B. gibsoni. The plasmid containing the firefly luciferase reporter gene (pBS-ELA) was transfected into B. gibsoni by an AMAXA 4D Nudeofector (TM) device. Transfection using program FA113 and Lonza buffer SF showed the highest luciferase expression. Twenty micrograms of plasmid produced the highest relative transfection efficiency. The fluorescent protein-expressing parasites were determined by GFP-containing plasmid (pBS-EGA) at 48 and 72 h post transfection. This finding is the first step towards a stable transfection method for B. gibsoni, which may contribute to a better understanding of the biology of the parasite.
• A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION (TM) nanopore sequencer

  Kazuo Imai, Norihito Tarumoto, Kazuhisa Misawa, Lucky Ronald Runtuwene, Jun Sakai, Kyoko Hayashida, Yuki Eshita, Ryuichiro Maeda, Josef Tuda, Takashi Murakami, Shigefumi Maesaki, Yutaka Suzuki, Junya Yamagishi, Takuya Maeda

  BMC INFECTIOUS DISEASES 17 (1) 621  1471-2334 2017/09 [Refereed][Not invited]
   

  Background: A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION (TM) nanopore sequencer. Methods: We generated specific LAMP primers targeting the 18S-rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION (TM) nanopore sequencer to identify each Plasmodium species. Results: Our LAMP method allowed amplification of all targeted 18S-rRNA genes of the reference plasmids with detection limits of 10-100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION (TM) was consistent with the reference plasmid sequences and the results of nested PCR. Conclusions: Our diagnostic method combined with LAMP and MinION (TM) could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.
• Eimeria tenella弱毒株の作出および病理組織学的比較解析

  山口 浩貴, 松林 誠, 川原 史也, 八田 岳士, 畑生 俊光, 山岸 潤也, 磯部 尚, 寺本 勲, 金子 明, 北 潔, 辻 尚利, 笹井 和美

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 160回 336 - 336 1347-8621 2017/08
• PEGylation of a TLR2-agonist-based vaccine delivery system improves antigen trafficking and the magnitude of ensuing antibody and CD8(+) T cell responses

  Toshiki Sekiya, Junya Yamagishi, John Henry V. Gray, Paul G. Whitney, Axel Martinelli, Weiguang Zeng, Chinn Yi Wong, Chihiro Sugimoto, David C. Jackson, Brendon Y. Chua

  BIOMATERIALS 137 61 - 72 0142-9612 2017/08 [Refereed][Not invited]
   

  The lipopeptide R4Pam2Cys is an agonist for toll-like receptor-2 (TLR2), a key pathogen-associated molecular pattern receptor expressed on many antigen-presenting cells such as dendritic cells (DCs). Electrostatic association of R4Pam2Cys with soluble protein antigens significantly enhances their immunogenicity and there is evidence to suggest that reducing the size of suitably adjuvanted-antigen complexes in solution may further improve their immunostimulatory capabilities. In this study, we investigated how incorporation of polyethylene glycol (PEG) into R4Pam2Cys affects the size, activity and efficacy of formed antigen-lipopeptide complexes. The presence of PEG was shown to increase solubility with a concomitant reduction in the particle size of vaccine formulations that was dependent on the length of PEG used. When compared to non-PEGylated R4Pam2Cys, vaccination of animals with antigen-complexed PEGylated R4Pam2Cys resulted not only in improvements in antibody production but significantly higher antigen-specific CD8(+) T cell responses. Both lipopeptides exhibited similar in vitro capabilities to induce DC maturation, facilitate antigen uptake and presentation to T cells. Moreover, analyses of the transcriptomes obtained from DCs treated with either lipopeptide revealed a large number of commonly induced genes with similar transcript expression levels, suggesting that common signalling pathways and processes were engaged following activation by either lipopeptide. In vivo analysis however revealed that vaccination with antigen-complexed PEGylated R4Pam2Cys resulted in improved antigen presentation to T cells. These heightened responses were not attributed to prolonged antigen persistence but rather due to more rapid transportation of antigen from the injection site into the draining lymph nodes over a short period of time. Our results indicate that reducing the size of formed antigen-TLR2-agonist complexes by PEGylation does not compromise the activity of the agonist but in fact enhances its trafficking in vivo ultimately leading to improved humoral and cell-mediated immune responses. (C) 2017 Elsevier Ltd. All rights reserved.
• Use of the Oxford Nanopore MinION sequencer for MLST genotyping of vancomycin-resistant enterococci.

  N Tarumoto, J Sakai, K Sujino, T Yamaguchi, M Ohta, J Yamagishi, L R Runtuwene, T Murakami, Y Suzuki, T Maeda, S Maesaki

  The Journal of hospital infection 96 (3) 296 - 298 2017/07 [Refereed]
  
• Use of the Oxford Nanopore MinION sequencer for MLST genotyping of vancomycin-resistant enterococci.

  Tarumoto N, Sakai J, Sujino K, Yamaguchi T, Ohta M, Yamagishi J, Runtuwene LR, Murakami T, Suzuki Y, Maeda T, Maesaki S

  J Hosp Infect 96 (3) 296 - 298 2017/06 [Refereed][Not invited]
  
• Serotyping dengue virus with isothermal amplification and a portable sequencer

  Junya Yamagishi, Lucky R. Runtuwene, Kyoko Hayashida, Arthur E. Mongan, Lan Anh Nguyen Thi, Linh Nguyen Thuy, Cam Nguyen Nhat, Kriengsak Limkittikul, Chukiat Sirivichayakul, Nuankanya Sathirapongsasuti, Martin Frith, Wojciech Makalowski, Yuki Eshita, Sumio Sugano, Yutaka Suzuki

  SCIENTIFIC REPORTS 7 (1) 3510  2045-2322 2017/06 [Refereed][Not invited]
   

  The recent development of a nanopore-type portable DNA sequencer has changed the way we think about DNA sequencing. We can perform sequencing directly in the field, where we collect the samples. Here, we report the development of a novel method to detect and genotype tropical disease pathogens, using dengue fever as a model. By combining the sequencer with isothermal amplification that only requires a water bath, we were able to amplify and sequence target viral genomes with ease. Starting from a serum sample, the entire procedure could be finished in a single day. The analysis of blood samples collected from 141 Indonesian patients demonstrated that this method enables the clinical identification and serotyping of the dengue virus with high sensitivity and specificity. The overall successful detection rate was 79%, and a total of 58 SNVs were detected. Similar analyses were conducted on 80 Vietnamese and 12 Thai samples with similar performance. Based on the obtained sequence information, we demonstrated that this approach is able to produce indispensable information for etiologically analyzing annual or regional diversifications of the pathogens.
• A TLR3-Specific Adjuvant Relieves Innate Resistance to PD-L1 Blockade without Cytokine Toxicity in Tumor Vaccine Immunotherapy

  Yohei Takeda, Keisuke Kataoka, Junya Yamagishi, Seishi Ogawa, Tsukasa Seya, Misako Matsumoto

  CELL REPORTS 19 (9) 1874 - 1887 2211-1247 2017/05 [Refereed][Not invited]
   

  Cancer patients having anti-programmed cell death-1 (PD-1)/PD ligand 1 (L1)-unresponsive tumors may benefit from advanced immunotherapy. Double-stranded RNA triggers dendritic cell (DC) maturation to cross-prime antigen-specific cytotoxic T lymphocytes (CTLs) via Toll-like receptor 3 (TLR3). The TLR3-specific RNA agonist, ARNAX, can induce anti-tumor CTLs without systemic cytokine/interferon (IFN) production. Here, we have developed a safe vaccine adjuvant for cancer that effectively implements anti-PD-L1 therapy. Co-administration of ARNAX with a tumor-associated antigen facilitated tumor regression in mouse models, and in combination with anti-PD-L1 antibody, activated tumor-specific CTLs in lymphoid tissues, enhanced CTL infiltration, and overcame anti-PD-1 resistance without cytokinemia. The TLR3-TICAM-1-interferon regulatory factor (IRF) 3-IFN-beta axis in DCs exclusively participated in CD8(+) T cell cross-priming. ARNAX therapy established Th1 immunity in the tumor microenvironment, upregulating genes involved in DC/T cell/natural killer (NK) cell recruitment and functionality. Human ex vivo studies disclosed that ARNAX+ antigen induced antigen-specific CTL priming and proliferation in peripheral blood mononuclear cells (PBMCs), supporting the feasibility of ARNAX for potentiating anti-PD-1/PD-L1 therapy in human vaccine immunotherapy.
• Genetic polymorphisms in Plasmodium falciparum chloroquine resistance genes, pfcrt and pfmdr1, in North Sulawesi, Indonesia

  Patrick Reteng, Visia Vrisca, Inka Sukarno, Ilham Habib Djarkoni, Jane Angela Kalangi, George Eduardo Jacobs, Lucky Ronald Runtuwene, Yuki Eshita, Ryuichiro Maeda, Yutaka Suzuki, Arthur Elia Mongan, Sarah Maria Warouw, Junya Yamagishi, Josef Tuda

  BMC Research Notes 10 (1) 147  1756-0500 2017/04/04 [Refereed][Not invited]
   

  Background: Malaria still poses one of the major threats to human health. Development of effective antimalarial drugs has decreased this threat however, the emergence of drug-resistant Plasmodium falciparum, a cause of Malaria, is disconcerting. The antimalarial drug chloroquine has been effectively used, but resistant parasites have spread worldwide. Interestingly, the withdrawal of the drug reportedly leads to an increased population of susceptible parasites in some cases. We examined the prevalence of genomic polymorphisms in a malaria parasite P. falciparum, associated with resistance to an antimalarial drug chloroquine, after the withdrawal of the drug from Indonesia. Results: Blood samples were collected from 95 malaria patients in North Sulawesi, Indonesia, in 2010. Parasite DNA was extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for pfcrt and pfmdr1. In parallel, multiplex amplicon sequencing for the same genes was carried out with Illumina MiSeq. Of the 59 cases diagnosed as P. falciparum infection by microscopy, PCR-RFLP analysis clearly identified the genotype 76T in pfcrt in 44 cases. Sequencing analysis validated the identified genotypes in the 44 cases and demonstrated that the haplotype in the surrounding genomic region was exclusively SVMNT. Results of pfmdr1 were successfully obtained for 51 samples, where the genotyping results obtained by the two methods were completely consistent. In pfmdr1, the 86Y mutant genotype was observed in 45 cases (88.2%). Conclusions: Our results suggest that the prevalence of the mutated genotypes remained dominant even 6 years after the withdrawal of chloroquine from this region. Diversified haplotype of the resistance-related locus, potentially involved in fitness costs, unauthorized usage of chloroquine, and/or a short post-withdrawal period may account for the observed high persistence of prevalence.
• Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin

  Naganori Nao, Junya Yamagishi, Hiroko Miyamoto, Manabu Igarashi, Rashid Manzoor, Aiko Ohnuma, Yoshimi Tsuda, Wakako Furuyama, Asako Shigeno, Masahiro Kajihara, Noriko Kishida, Reiko Yoshida, Ayato Takada

  MBIO 8 (1) 2150-7511 2017/01 [Refereed][Not invited]
   

  Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes. IMPORTANCE Influenza A viruses are divided into subtypes based on the antigenicity of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. This study shows the potential role of the viral RNA secondary structure for nucleotide insertions and demonstrates a key mechanism explaining why the acquisition of the polybasic HA cleavage site is restricted to particular HA subtypes in nature. Our findings will contribute to better understanding of the ecology of influenza A viruses and will also be useful for the development of genetically modified vaccines against H5 and H7 influenza A viruses with increased stability.
• MinIONを利用したLAMP法による革新的マラリア診断系の作成

  今井 一男, 三沢 和央, 樽本 憲人, 前崎 繁文, 前田 卓哉, 鈴木 穰, 山岸 潤也, 林田 京子

  日本臨床微生物学雑誌 (一社)日本臨床微生物学会 27 (Suppl.) 255 - 255 0917-5059 2016/12
• Identification and functional analysis of a novel mitochondria-localized 2-Cys peroxiredoxin, BbTPx-2, from Babesia bovis

  Tatsunori Masatani, Masahito Asada, Hassan Hakimi, Kei Hayashi, Junya Yamagishi, Shin-ichiro Kawazu, Xuenan Xuan

  PARASITOLOGY RESEARCH 115 (8) 3139 - 3145 0932-0113 2016/08 [Refereed][Not invited]
   

  Cysteine-based peroxidases, known as peroxiredoxins (Prx) or thioredoxin peroxidases (TPx), are important antioxidant enzymes that prevent oxidative damage caused by reactive oxygen species (ROS). In this study, we identified a novel mitochondrial 2-Cys Prx, BbTPx-2, from a bovine Babesia parasite, B. bovis. BbTPx-2 complementary DNA (cDNA) encodes a polypeptide of 254 amino acid residues. This protein has a mitochondrial targeting peptide at the N-terminus and two conserved cysteine residues of the typical 2-Cys Prx. By using a thiol mixed-function oxidation assay, the antioxidant activity of recombinant BbTPx-2 was revealed, and its antioxidant activity was comparable to that of a cytosolic 2-Cys Prx from B. bovis, BbTPx-1. Notably, we confirmed that BbTPx-2 was expressed in the mitochondrion of B. bovis merozoites. Taken together, the results suggest that the mitochondrial BbTPx-2 is an antioxidative enzyme for scavenging ROS in B. bovis.
• Mycophenolic Acid and Its Derivatives as Potential Chemotherapeutic Agents Targeting Inosine Monophosphate Dehydrogenase in Trypanosoma congolense

  Keisuke Suganuma, Albertus Eka Yudistira Sarwono, Shinya Mitsuhashi, Marcin Jakalski, Tadashi Okada, Molefe Nthatisi, Junya Yamagishi, Makoto Ubukata, Noboru Inoue

  ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 60 (7) 4391 - 4393 0066-4804 2016/07 [Refereed][Not invited]
   

  This study aimed to evaluate the trypanocidal activity of mycophenolic acid (MPA) and its derivatives for Trypanosoma congolense. The proliferation of T. congolense was completely inhibited by adding <1 mu M MPA and its derivatives. In addition, the IMP dehydrogenase in T. congolense was molecularly characterized as the target of these compounds. The results suggest that MPA and its derivatives have the potential to be new candidates as novel trypanocidal drugs.
• Transcriptional profiles of virulent and precocious strains of Eimeria tenella at sporozoite stage; novel biological insight into attenuated asexual development

  Makoto Matsubayashi, Fumiya Kawahara, Takeshi Hatta, Junya Yamagishi, Takeharu Miyoshi, Anisuzzaman, Kazumi Sasai, Takashi Isobe, Kiyoshi Kita, Naotoshi Tsuji

  INFECTION GENETICS AND EVOLUTION 40 54 - 62 1567-1348 2016/06 [Refereed][Not invited]
   

  Chicken coccidiosis is caused by Eimeria spp., particularly Eimeria tenella, and is characterized by watery or hemorrhagic diarrhea, resulting in death in severe cases. Precociously attenuated live vaccines are widely used to control the disease, and these are produced by serially passaging virulent strains through chickens, and the collection of oocysts from feces at progressively earlier time points during oocyst shedding. Sporozoites of the precocious strain rapidly enter the intestinal mucosa, and their subsequent asexual development reduces their growth. However, there have been few detailed genetic or transcriptional analyses of the strains. Here, we used RNA sequencing to gain novel biological insight into the pathogenicity and precocity of E. tenella. We compared the differential transcription in the sporozoites (the initial stage of endogenous development) of virulent and precocious strains by mapping the sequence reads onto the draft genome of E. tenella. About 90% of the reads from both strains were mapped to the genome, and 16,630 estimated transcript regions were identified. Using Gene Ontology slim and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and the annotation of the estimated transcripts with Blastx, we found that the expression of some genes involved in carbohydrate metabolism were expressed two-fold more strongly in the virulent strain than in the precocious strain. Characteristically, genes related to proteins secreted from the apical complex, proteases, cell attachment proteins, mitochondrial proteins, and transporters were most strongly upregulated in the virulent strain. Interestingly, the expression of genes associated with cell survival, development, or proliferation was strongly upregulated in the precocious strain. These findings suggest that virulent strains survive long before invasion and invade actively/successfully into host cells, whereas proliferative processes appear to affect precocity. (c) 2016 Elsevier B.V. All rights reserved.
• Characterization of an epimastigote -stage-specific hemoglobin receptor of Trypanosoma congolense

  Shino Yamasaki, Keisuke Suganuma, Junya Yamagishi, Masahito Asada, Naoaki Yokoyama, Shin-ichiro Kawazu, Noboru Inoue

  PARASITES & VECTORS 9 (1) 299  1756-3305 2016/05 [Refereed][Not invited]
   

  Background: Since Trypanosorna spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life -cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T brucei Hp-Hb complex receptor (TbHpHbR).Methods: Trypanosorna congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay.Results: We found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (K,A) =2.1x10(-8) M) but free-Hp with low affinity (Kd = 2.2x10(-7) M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome).Conclusion: We conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T congolense EMF-specific Hb receptor (TcEpHbR).
• Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes

  Junya Yamagishi, Yukuto Sato, Natsuko Shinozaki, Bin Ye, Akito Tsuboi, Masao Nagasaki, Riu Yamashita

  PLOS ONE 11 (4) e0154389  1932-6203 2016/04 [Refereed][Not invited]
   

  The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study.
• Establishment of transient and stable transfection systems for Babesia ovata

  Hassan Hakimi, Junya Yamagishi, Yuto Kegawa, Osamu Kaneko, Shin-ichiro Kawazu, Masahito Asada

  PARASITES & VECTORS 9 171  1756-3305 2016/03 [Refereed][Not invited]
   

  Background: Bovine babesiosis is a tick-borne disease caused by several species of Babesia which produce acute and fatal disease in cattle and affect livestock industry worldwide. Babesia ovata is a benign species widespread in east Asian countries and causes anemia, particularly in cattle which are co-infected with Theileria orientalis. The development of genetic manipulation methods is necessary to improve our understanding of the basic biology of protozoan pathogens toward a better control of disease. Such tools have not been developed for B. ovata, and are the aim of this study. Methods: In this study we transfected constructs that were designed to evaluate the ability of several B. ovata promoter candidates to drive expression of a reporter luciferase. We found that the elongation factor-1 alpha intergenic region (ef-1 alpha IG) and the actin 5' non-coding region (NR) had highest promoter activities. To establish a stable transfection system, we generated a plasmid construct in which the ef-1 alpha IG promoter drives gfp expression, and the actin 5' NR mediates expression of the selectable marker hdhfr. The plasmid was designed for episomal transfection, as well as to integrate by double cross-over homologous recombination into the ef-1 alpha locus. Circular or linearized plasmid was transfected by electroporation into in vitro cultured B. ovata and retention of the plasmid was facilitated by drug selection with 5 nM WR99210 initiated 48 h after transfection. Results: After one-week cultivation with WR99210, GFP-expressing parasites were observed by fluorescence microscopy. Integration of the plasmid construct into the ef-1 alpha locus was confirmed by PCR, Southern blot analysis, and sequencing of recombination sites. These results confirm successful development of a stable transfection system for B. ovata. Conclusion: The current study provides a fundamental molecular tool to aid in molecular and cellular studies of B. ovata.
• Characterization of Toxoplasma gondii glyoxalase 1 and evaluation of inhibitory effects of curcumin on the enzyme and parasite cultures

  Youn-Kyoung Goo, Junya Yamagishi, Akio Ueno, Mohamad Alaa Terkawi, Gabriel Oluga Aboge, Dongmi Kwak, Yeonchul Hong, Dong-Il Chung, Makoto Igarashi, Yoshifumi Nishikawa, Xuenan Xuan

  PARASITES & VECTORS 8 654  1756-3305 2015/12 [Refereed][Not invited]
   

  Background: The glyoxalase pathway, which includes two enzymes, glyoxalase 1 and 2 (Glo1 and Glo2), is a ubiquitous cellular system responsible for the removal of cytotoxic methylglyoxal produced during glycolysis. Protozoan parasites, including Toxoplasma gondii (T. gondii) tachyzoites, produce methylglyoxal because of increased glycolytic fluxes. A Glo1 inhibitor such as curcumin could be considered a drug candidate for anti-protozoan, anti-inflammatory, and anti-cancer therapy. Methods: The T. gondii Glo1 gene (TgGlo1) was cloned and the recombinant protein was produced. Enzyme kinetics of TgGlo1 and five mutants were evaluated by adding methylglyoxal and glutathione to a reaction mixture. Finally, the inhibitory effects of various concentrations of curcumin on recombinant TgGlo1 were evaluated using in vitro cultures of T. gondii. Results: Active recombinant TgGlo1 was successfully produced and the active sites (E166 and E251) of TgGlo1 were verified by point mutagenesis. Curcumin at the tested doses inhibited the enzymatic activity of recombinant TgGlo1 as well as the parasitic propagation of in vitro-cultured T. gondii. The K-i and IC50 were 12.9 +/- 0.5 mu M and 38.3 +/- 0.9 mu M, respectively. Conclusion: The inhibitory effect of curcumin on the enzymatic activity of TgGlo1 and parasitic propagation of T. gondii could be explored in the potential development of a potent drug for the treatment of toxoplasmosis. However, considering the fact that curcumin is known to have many effects on other molecules in the micromolar range, further elucidation of curcumin's direct inhibition of the glyoxalase system of T. gondii will be needed.
• Eimeria tenellaのミトコンドリア呼吸鎖酵素活性の測定と阻害剤による虫体殺滅効果

  松林 誠, 稲岡 ダニエル健, 小松谷 啓介, 八田 岳士, 三好 猛晴, 磯部 尚, 川原 史也, 山岸 潤也, 彦坂 健児, 佐藤 暖, 志波 智生, 原田 繁春, 北 潔, 辻 尚利

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 158回 305 - 305 1347-8621 2015/08
• Inter-Individual Differences in the Oral Bacteriome Are Greater than Intra-Day Fluctuations in Individuals

  Yukuto Sato, Junya Yamagishi, Riu Yamashita, Natsuko Shinozaki, Bin Ye, Takuji Yamada, Masayuki Yamamoto, Masao Nagasaki, Akito Tsuboi

  PLOS ONE 10 (6) e0131607  1932-6203 2015/06 [Refereed][Not invited]
   

  Given the advent of massively parallel DNA sequencing, human microbiome is analyzed comprehensively by metagenomic approaches. However, the inter-and intra-individual variability and stability of the human microbiome remain poorly characterized, particularly at the intra-day level. This issue is of crucial importance for studies examining the effects of microbiome on human health. Here, we focused on bacteriome of oral plaques, for which repeated, time-controlled sampling is feasible. Eighty-one supragingival plaque subjects were collected from healthy individuals, examining multiple sites within the mouth at three time points (forenoon, evening, and night) over the course of 3 days. Bacterial composition was estimated by 16S rRNA sequencing and species-level profiling, resulting in identification of a total of 162 known bacterial species. We found that species compositions and their relative abundances were similar within individuals, and not between sampling time or tooth type. This suggests that species-level oral bacterial composition differs significantly between individuals, although the number of subjects is limited and the intra-individual variation also occurs. The majority of detected bacterial species (98.2%; 159/162), however, did not fluctuate over the course of the day, implying a largely stable oral microbiome on an intra-day time scale. In fact, the stability of this data set enabled us to estimate potential interactions between rare bacteria, with 40 co-occurrences supported by the existing literature. In summary, the present study provides a valuable basis for studies of the human microbiome, with significant implications in terms of biological and clinical outcomes.
• DB-AT: a 2015 update to the Full-parasites database brings a multitude of new transcriptomic data for apicomplexan parasites

  Marcin Jakalski, Hiroyuki Wakaguri, Tabea G. Kischka, Yoshifumi Nishikawa, Shin-ichiro Kawazu, Makoto Matsubayashi, Fumiya Kawahara, Naotoshi Tsuji, Shinuo Cao, Fujiko Sunaga, Xuenan Xuan, Kazuhiro Okubo, Ikuo Igarashi, Josef Tuda, Arthur E. Mongan, Yuki Eshita, Ryuichiro Maeda, Wojciech Makalowski, Yutaka Suzuki, Junya Yamagishi

  NUCLEIC ACIDS RESEARCH 43 (D1) D631 - D636 0305-1048 2015/01 [Refereed][Not invited]
   

  The previous release of our Full-parasites database ( ext-link-type="uri" xlink:href="http://fullmal.hgc.jp/" xlink:type="simple">http://fullmal.hgc.jp/) brought enhanced functionality, an expanded full-length cDNA content, and new RNA-Seq datasets from several important apicomplexan parasites. The 2015 update witnesses the major shift in the databases content with focus on diverse transcriptomes of the apicomplexan parasites. The content of the database was substantially enriched with transcriptome information for new apicomplexan parasites. The latest version covers a total of 17 species, with addition of our newly generated RNA-Seq data of a total of 909 150 388 tags. Moreover, we have generated and included two novel and unique datasets, which represent diverse nature of transcriptomes in individual parasites in vivo and in vitro. One is the data collected from 116 Indonesian patients infected with Plasmodium falciparum. The other is a series of transcriptome data collected from a total of 38 single cells of P. falciparum cultured in vitro. We believe that with the recent advances our database becomes an even better resource and a unique platform in the analysis of apicomplexan parasites and their interaction with their hosts. To adequately reflect the recent modifications and the current content we have changed the database name to DB-AT-DataBase of Apicomplexa Transcriptomes.
• Transcriptome and Histopathological Changes in Mouse Brain Infected with Neospora caninum

  Maki Nishimura, Sachi Tanaka, Fumiaki Ihara, Yoshikage Muroi, Junya Yamagishi, Hidefumi Furuoka, Yutaka Suzuki, Yoshifumi Nishikawa

  SCIENTIFIC REPORTS 5 7936  2045-2322 2015/01 [Refereed][Not invited]
   

  Neospora caninum is a protozoan parasite that causes neurological disorders in dogs and cattle. It can cause nonsuppurative meningoencephalitis and a variety of neuronal symptoms are observed, particularly in dogs. However, the pathogenic mechanism, including the relationship between the parasite distribution and the clinical signs, is unclear. In this study, to understand the pathogenic mechanism of neosporosis, parasite distribution and lesions were assessed in the brain of mice infected with N. caninum (strain Nc-1). Host gene expression was also analyzed with RNA sequencing (RNA-Seq). The histopathological lesions in the frontal lobe and the medulla oblongata were significantly more severe in symptomatic mice than in asymptomatic mice, although no association between the severity of the lesions and parasite numbers was found. In infected mice, the expression of 772 mouse brain genes was upregulated. A GOstat analysis predicted that the upregulated genes were involved in the host immune response. Genes whose expression correlated positively and negatively with parasite numbers were involved in the host immune response, and neuronal morphogenesis and lipid metabolic processes, respectively. These results suggest that changes in the gene expression profile associated with neuronal functions as well as immune responses can contribute to the pathogenesis in N. caninum-infected animals.
• Cloning and characterization of aspartic protease 3 of Toxoplasma gondii.

  Kamyingkird K, Goo YK, Cao S, Adjou Moumouni PF, Aboge GO, Yamagishi J, Terkawi MA, Masatani T, Yu L, Nishikawa Y, Xuan X

  Journal of Protozoology Research National Research Center for Protozoan Diseases 24 (1) 18 - 25 2014/12 [Not refereed][Not invited]
  
• Defining the roles of the baculovirus regulatory proteins IE0 and IE1 in genome replication and early gene transactivation

  Nadia Sokal, Yingchao Nie, Leslie G. Willis, Junya Yamagishi, Gary W. Blissard, Mark R. Rheault, David A. Theilmann

  VIROLOGY 468 160 - 171 0042-6822 2014/11 [Refereed][Not invited]
   

  IE0 and IE1 of the baculovirus Autographa californica multiple nucleopolyhedrovirus are essential transregulatory proteins required for both viral DNA replication and transcriptional transactivation. IE0 is identical to IE1 except for 54 amino acids at the N-terminus but the functional differences between these two proteins remain unclear. The purpose of this study was to determine the separate roles of these critical proteins in the virus life cycle. Unlike prior studies, IE0 and IE1 were analyzed using viruses that expressed ie0 and ie1 from an identical promoter so that the timing and levels of expression were comparable. IE0 and IE1 were found to equally support viral DNA replication and budded virus (BV) production. However, specific viral promoters were selectively transactivated by IE0 relative to IE1 but only when expressed at low levels. These results indicate that IE0 preferentially transactivates specific viral genes at very early times post-infection enabling accelerated replication and BV production. Crown Copyright (C) 2014 Published by Elsevier Inc. All rights reserved.
• Interactive transcriptome analysis of malaria patients and infecting Plasmodium falciparum

  Junya Yamagishi, Anna Natori, Mohammed E. M. Tolba, Arthur E. Mongan, Chihiro Sugimoto, Toshiaki Katayama, Shuichi Kawashima, Wojciech Makalowski, Ryuichiro Maeda, Yuki Eshita, Josef Tuda, Yutaka Suzuki

  GENOME RESEARCH 24 (9) 1433 - 1444 1088-9051 2014/09 [Refereed][Not invited]
   

  To understand the molecular mechanisms of parasitism in vivo, it is essential to elucidate how the transcriptomes of the human hosts and the infecting parasites affect one another. Here we report the RNA-seq analysis of 116 Indonesian patients infected with the malaria parasite Plasmodium falciparum(Pf). We extracted RNAs from their peripheral blood as a mixture of host and parasite transcripts and mapped the RNA-seq tags to the human and Pf reference genomes to separate the respective tags. We were thus able to simultaneously analyze expression patterns in both humans and parasites. We identified human and parasite genes and pathways that correlated with various clinical data, which may serve as primary targets for drug developments. Of particular importance, we revealed characteristic expression changes in the human innate immune response pathway genes including TLR2 and T1CAM2 that correlated with the severity of the malaria infection. We also found a group of transcription regulatory factors, JUND, for example, and signaling molecules, TNFAIP3, for example, that were strongly correlated in the expression patterns of humans and parasites. We also identified several genetic variations in important anti-malaria drug resistance-related genes. Furthermore, we identified the genetic variations which are potentially associated with severe malaria symptoms both in humans and parasites. The newly generated data should collectively lay a unique foundation for understanding variable behaviors of the field malaria parasites, which are far more complex than those observed under laboratory conditions.
• Eimeria tenellaスポロゾイトのミトコンドリア精製法の確立と呼吸鎖酵素活性

  松林 誠, 稲岡 ダニエル健, 小松谷 啓介, 八田 岳士, 三好 猛晴, 磯部 尚, 川原 史也, 山岸 潤也, 彦坂 健児, 佐藤 暖, 志波 智生, 原田 繁春, 北 潔, 辻 尚利

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 157回 355 - 355 1347-8621 2014/08
• The Babesia bovis gene and promoter model: an update from full-length EST analysis

  Junya Yamagishi, Hiroyuki Wakaguri, Naoaki Yokoyama, Riu Yamashita, Yutaka Suzuki, Xuenan Xuan, Ikuo Igarashi

  BMC GENOMICS 15 678  1471-2164 2014/08 [Refereed][Not invited]
   

  Background: Babesia bovis is an apicomplexan parasite that causes babesiosis in infected cattle. Genomes of pathogens contain promising information that can facilitate the development of methods for controlling infections. Although the genome of B. bovis is publically available, annotated gene models are not highly reliable prior to experimental validation. Therefore, we validated a preproposed gene model of B. bovis and extended the associated annotations on the basis of experimentally obtained full-length expressed sequence tags (ESTs). Results: From in vitro cultured merozoites, 12,286 clones harboring full-length cDNAs were sequenced from both ends using the Sanger method, and 6,787 full-length cDNAs were assembled. These were then clustered, and a nonredundant referential data set of 2,115 full-length cDNA sequences was constructed. The comparison of the preproposed gene model with our data set identified 310 identical genes, 342 almost identical genes, 1,054 genes with potential structural inconsistencies, and 409 novel genes. The median length of 5' untranslated regions (UTRs) was 152 nt. Subsequently, we identified 4,086 transcription start sites (TSSs) and 2,023 transcriptionally active regions (TARs) by examining 5' ESTs. We identified ATGGGG and CCCCAT sites as consensus motifs in TARs that were distributed around -50 bp from TSSs. In addition, we found ACACA, TGTGT, and TATAT sites, which were distributed periodically around TSSs in cycles of approximately 150 bp. Moreover, related periodical distributions were not observed in mammalian promoter regions. Conclusions: The observations in this study indicate the utility of integrated bioinformatics and experimental data for improving genome annotations. In particular, full-length cDNAs with one-base resolution for TSSs enabled the identification of consensus motifs in promoter sequences and demonstrated clear distributions of identified motifs. These observations allowed the illustration of a model promoter composition, which supports the differences in transcriptional regulation frameworks between apicomplexan parasites and mammals.
• Transcriptome Analysis of Mouse Brain Infected with Toxoplasma gondii

  Sachi Tanaka, Maki Nishimura, Fumiaki Ihara, Junya Yamagishi, Yutaka Suzuki, Yoshifumi Nishikawa

  INFECTION AND IMMUNITY 81 (10) 3609 - 3619 0019-9567 2013/10 [Refereed][Not invited]
   

  Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. Chronic infections with T. gondii become established in the tissues of the central nervous system, where the parasites may directly or indirectly modulate neuronal function. However, the mechanisms underlying parasite-induced neuronal disorder in the brain remain unclear. This study evaluated host gene expression in mouse brain following infection with T. gondii. BALB/c mice were infected with the PLK strain, and after 32 days of infection, histopathological lesions in the frontal lobe were found to be more severe than in other areas of the brain. Total RNA extracted from infected and uninfected mouse brain samples was subjected to transcriptome analysis using RNA sequencing (RNA-seq). In the T. gondii-infected mice, 935 mouse brain genes were upregulated, whereas 12 genes were downregulated. GOstat analysis predicted that the upregulated genes were primarily involved in host immune responses and cell activation. Positive correlations were found between the numbers of parasites in the infected mouse brains and the expression levels of genes involved in host immune responses. In contrast, genes that had a negative correlation with parasite numbers were predicted to be involved in neurological functions, such as small-GTPase-mediated signal transduction and vesicle-mediated transport. Furthermore, differential gene expression was observed between mice exhibiting the clinical signs of toxoplasmosis and those that did not. Our findings may provide insights into the mechanisms underlying neurological changes during T. gondii infection.
• Actin polymerization mediated by Babesia gibsoni aldolase is required for parasite invasion

  Youn-Kyoung Goo, Akio Ueno, Mohamad Alaa Terkawi, G. Oluga Aboge, Yamagishi Junya, Makoto Igarashi, Jung-Yeon Kim, Yeon-Chul Hong, Dong-Il Chung, Yoshifumi Nishikawa, Xuenan Xuan

  EXPERIMENTAL PARASITOLOGY 135 (1) 42 - 49 0014-4894 2013/09 [Refereed][Not invited]
   

  Host cell invasion by apicomplexan parasites driven by gliding motility and empowered by actin-based movement is essential for parasite survival and pathogenicity. The parasites share a conserved invasion process: actin-based motility led by the coordination of adhesin-cytoskeleton via aldolase. A number of studies of host cell invasion in the Plasmodium species and Toxoplasma gondii have been performed. However, the mechanisms of host cell invasion by Babesia species have not yet been studied. Here, we show that Babesia gibsoni aldolase (BgALD) forms a complex with B. gibsoni thrombospondin-related anonymous protein (BgTRAP) and B. gibsoni actin (BgACT), depending on tryptophan-734 (W-734) in BgTRAP. In addition, actin polymerization is mediated by BgALD. Moreover, cytochalasin D, which disrupts actin polymerization, suppressed B. gibsoni parasite growth and inhibited the host cell invasion by parasites, indicating that actin dynamics are essential for erythrocyte invasion by B. gibsoni. This study is the first molecular approach to determine the invasion mechanisms of Babesia species. (C) 2013 Elsevier Inc. All rights reserved.
• Adenosine-uridine-rich element is one of the required cis-elements for epimastigote form stage-specific gene expression of the congolense epimastigote specific protein

  Keisuke Suganuma, Kennedy Miyoro Mochabo, Hassan Hakimi, Shino Yamasaki, Junya Yamagishi, Masahito Asada, Shin-ichiro Kawazu, Noboru Inoue

  MOLECULAR AND BIOCHEMICAL PARASITOLOGY 191 (1) 36 - 43 0166-6851 2013/09 [Refereed][Not invited]
   

  It is known that gene expression in kinetoplastida is regulated post-transcriptionally. Several previous studies have shown that stage-specific gene expression in trypanosomes is regulated by cis-elements located in the 3' untranslated region (UTR) of each mRNA and also by RNA binding proteins. Our previous study revealed that gene expression of congolense epimastigote specific protein (cesp) was regulated by cis-elements located in the 3'UTR In the present study, we identified the adenosine and uridine rich region in the cesp 3'UTR. Using transgenic trypanosome cell lines with different egfp expression cassettes, we showed that this adenosine and uridine rich region is one of the regulatory elements for epimastigote form (EMF) stage-specific gene expression via the regulatory cis-element of the eukaryotic AU rich element (ARE). Therefore this required element within the cesp 3'UTR was designated as T. congolense ARE. This required cis-element might selectively stabilize mRNA in the EMF stage and destabilize mRNA in other stages. By RNA electro mobility shift assay, unknown stage-specific RNA binding proteins (RBPs) whose sequences specifically interacted with the required cis-element were found. These results indicate that EMF stage specific cis-element and REP complexes might specifically stabilize cesp mRNA in EMF. (C) 2013 Elsevier B.V. All rights reserved.
• TgGRA23, a novel Toxoplasma gondii dense granule protein associated with the parasitophorous vacuole membrane and intravacuolar network

  Tatsunori Masatani, Tomohide Matsuo, Tetsuya Tanaka, Mohamad Alaa Terkawi, Eung-Goo Lee, Youn-Kyoung Goo, Gabriel Oluga Aboge, Junya Yamagishi, Kei Hayashi, Kyohko Kameyama, Shinuo Cao, Yoshifumi Nishikawa, Xuenan Xuan

  PARASITOLOGY INTERNATIONAL 62 (4) 372 - 379 1383-5769 2013/08 [Refereed][Not invited]
   

  Toxoplasma gondii is an intracellular protozoan parasite, which relies on a specialized compartment, the parasitophorous vacuole (PV), to survive within host cells. Dense granules within the parasite release a large variety of proteins to maintain the integrity of the vacuole structure. Here, we identified a novel dense granule protein in T. gondii, TgGRA23, which is a homolog of the Sarcocystis muris dense granule protein, SmDG32. Recombinant TgGRA23 (rTgGRA23) expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein was used to raise antisera in mice and rabbits. Immunoblotting showed that antisera from the immunized mice and rabbits reacted with parasite lysates to yield a 21-kDa native protein. In addition, immuno-electron microscopic examination showed that TgGRA23 resides in the dense granules, PV membrane and intravacuolar network of the parasite. To confirm the precise subcellular localization of TgGRA23 in T. gondii, an immunofluorescent antibody test was performed using dense granule markers. Notably, TgGRA23 co-localized with other dense granule proteins including TgGRA4 and TgGRA7, in the extracellular-stage parasites. Biochemical experiments indicated that TgGRA23 is insoluble and may form an electrostatic complex that is resistant to non-ionic detergents. Furthermore, specific antibodies to TgGRA23 were detected during the chronic stage of Toxoplasma infection in mice. Our results suggest that TgGRA23 is an as yet unknown member of the T. gondii dense granule proteins, and that it may be involved in remodeling or maintenance of the PV. (C) 2013 Elsevier Ireland Ltd. All rights reserved.
• Epidemiological survey of Babesia bovis and Babesia bigemina infections of cattle in Philippines

  Longzheng Yu, Mohmad Alaa Terkawi, Mary Jane Cruz-Flores, Florencia G. Claveria, Gabriel Oluga Aboge, Junya Yamagishi, Youn-Kyoung Goo, Shinuo Cao, Tatsunori Masatani, Yoshifumi Nishikawa, Xuenan Xuan

  Journal of Veterinary Medical Science 75 (7) 995 - 998 0916-7250 2013 [Refereed][Not invited]
   

  A total of 250 blood samples were collected from clinically healthy cattle in five provinces of Philippines. DNA was extracted from the samples and analyzed by nested PCR assays for an epidemiological survey of Babesia bovis and Babesia bigemina infections. Out of the 250 samples, 27 (10.8%) and 16 (6.4%) were positive for B. bovis infection and B. bigemina infection, respectively. Mixed infections were detected in a total of 4 samples (1.6%). Our data provide baseline information regarding the epidemiology of B. bovis and B. bigemina infections in cattle in Philippines, which can be utilized in developing proper strategies for disease control and management. © 2013 The Japanese Society of Veterinary Science.
• Protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing TgAMA1 as vaccines against Toxoplasma gondii infection in mice

  Longzheng Yu, Junya Yamagishi, Shoufa Zhang, Chunmei Jin, Gabriel Oluga Aboge, Houshuang Zhang, Guohong Zhang, Tetsuya Tanaka, Kozo Fujisaki, Yoshifumi Nishikawa, Xuenan Xuan

  PARASITOLOGY INTERNATIONAL 61 (3) 481 - 486 1383-5769 2012/09 [Refereed][Not invited]
   

  A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection. (C) 2012 Elsevier Ireland Ltd. All rights reserved.
• Molecular detection and identification of Babesia bovis and Babesia bigemina in cattle in northern Thailand

  Shinuo Cao, Gabriel Oluga Aboge, Mohamad Alaa Terkawi, Longzheng Yu, Ketsarin Kamyingkird, Yuzi Luo, Yan Li, Youn-Kyoung Goo, Junya Yamagishi, Yoshifumi Nishikawa, Naoaki Yokoyama, Hiroshi Suzuki, Ikuo Igarashi, Ryuichiro Maeda, Tawin Inpankaew, Sathaporn Jittapalapong, Xuenan Xuan

  PARASITOLOGY RESEARCH 111 (3) 1259 - 1266 0932-0113 2012/09 [Refereed][Not invited]
   

  Although Babesia bovis and Babesia bigemina infections cause economic losses in the cattle industry in northern Thailand, there is inadequate information on Babesia isolates present in the area. Therefore, to determine the prevalence and genetic relationship between Babesia isolates, we screened 200 blood samples of cattle from Chiang Rai, Chiang Mai, and Lumpang provinces of northern Thailand. A nested polymerase chain reaction using primers targeting B. bovis spherical body protein 2 (BboSBP2) and B. bigemina rhoptry-associated protein 1a (BbiRAP-1a) genes revealed a prevalence of 12 and 21 % for B. bovis and B. bigemina, respectively, while that of mixed infections was 6.5 % samples. The prevalences of B. bovis in Chiang Rai, Chiang Mai, and Lumpang were 9.5, 3.7, and 25.5 %, respectively. For B. bigemina, the prevalences were 15.8, 12.9, and 39.2 % in Chiang Rai, Chiang Mai, and Lumpang, respectively. Mixed infections with B. bovis and B. bigemina were 6.3 % in Chiang Rai, 1.9 % in Chiang Mai, and 13.7 % in Lumpang. The identical sequences of either BboSBP2 gene or BbiRAP-1a gene were shared among the Babesia isolates in the three provinces of northern Thailand. Further analysis using the internal transcribed spacer gene revealed at least four genotypes for B. bovis and five genotypes for B. bigemina in northern Thailand, while the sequences present great genetic diversities in the different isolates. Overall, we have demonstrated a high prevalence and polymorphism of Babesia parasites in northern Thailand calling for the need to design effective control programs for bovine babesiosis.
• Four promising antigens, BgP32, BgP45, BgP47, and BgP50, for serodiagnosis of Babesia gibsoni infection were classified as B. gibsoni merozoite surface protein family

  Youn-Kyoung Goo, Gabriel Oluga Aboge, M. Alaa Terkawi, Honglin Jia, Junya Yamagishi, Fujiko Sunaga, Kazuhiko Namikawa, Se-Yeoun Cha, Hyung-Kwan Jang, Suk Kim, Yoshifumi Nishikawa, Xuenan Xuan

  PARASITOLOGY INTERNATIONAL 61 (2) 364 - 368 1383-5769 2012/06 [Refereed][Not invited]
   

  We determined the molecular characteristics of four proteins, BgP32, BgP45, BgP47, and BgP50, of Babesia gibsoni. Localization by subcellular fractionations followed by Western blotting revealed that the corresponding native proteins belong to merozoite surface protein family of B. gibsoni (BgMSP). Moreover, antisera against either rBgP45 or rBgP47 cross-reacted with all the proteins of the BgMSP family on ELISA and IFAT analyses. Of the four candidate antigens, ELISA with rBgP45 yielded high sensitivity, and ELISA with rBgP32 resulted in high specificity and in concordance with IFAT results. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
• Characterization of Toxoplasma gondii 5’ UTR with encyclopedic TSS information

  Yamagishi J, Watanabe J, Goo YK, Masatani T, Suzuki Y, Xuan X

  The Journal of Parasitology American Society of Parasitologists 98 (2) 445 - 447 2012/02 [Refereed][Not invited]
   

  The 5′ UTR is widely involved in gene expression via post-transcriptional regulation. However, a detailed profile of the 5′ UTR for Toxoplasma gondii has not yet been demonstrated. To investigate the issue, we compared the predicted open reading frames (ORFs) and transcription start sites (TSSs) of T. gondii obtained by TSS-seq, a method that enables analysis of encyclopedic TSSs with next-generation sequencers. As a result, it was demonstrated that the mode length of the 5′ UTR is between 120 and 140 nucleotides (nts) when a subset of genes with predicted signal peptides was examined. However, when genes without the signal peptide were examined, the length was extended to approximately 600 nts. Because additional information on the predicted signal peptide generates increased reliability to the 5′ end estimation of each ORF, we believe that the former value was more reliable as a representative of the 5′ UTR length of T. gondii. The discrepancy suggests that current predictions of the 5′ end of the ORF were less accurate and considerably more discordant with the natural status. The 5′ untranslated region (5′ UTR) is defined as that between the 5′ end of the transcripts and just in front of a start codon of an ORF. Therefore, the 5′ UTR does not contain any information for a protein sequence; however, it is involved in the control of protein expression via the modulation of translational efficiency (Kozak, 1991b; Hughes, 2006).http://www.journalofparasitology.org/doi/abs/10.1645/GE-2864.1
• Macrophages Are Critical for Cross-Protective Immunity Conferred by Babesia microti against Babesia rodhaini Infection in Mice

  Yan Li, Mohamad Alaa Terkawi, Yoshifumi Nishikawa, Gabriel Oluga Aboge, Yuzi Luo, Hideo Ooka, Youn-Kyoung Goo, Longzheng Yu, Shinuo Cao, Yongfeng Sun, Junya Yamagishi, Tatsunori Masatani, Naoaki Yokoyama, Ikuo Igarashi, Xuenan Xuan

  INFECTION AND IMMUNITY 80 (1) 311 - 320 0019-9567 2012/01 [Refereed][Not invited]
   

  Although primary infection of mice with Babesia microti has been shown to protect mice against subsequent lethal infection by Babesia rodhaini, the mechanism behind the cross-protection is unknown. To unravel this mechanism, we investigated the influence of primary infection of mice with nonlethal B. microti using different time courses on the outcome of subsequent lethal B. rodhaini infection. Simultaneous infections of mice with these parasites resulted in rapid increases in parasitemia, with 100% mortality in BALB/c mice, as observed with control mice infected with B. rodhaini alone. In contrast, mice with acute, resolving, and chronic-phase B. microti infections were completely protected against B. rodhaini, resulting in low parasitemia and no mortalities. Mice immunized with dead B. microti were not protected from B. rodhaini infection, although high antibody responses were induced. Interestingly, the protected mice had significantly decreased levels of antibody response, cytokines (including gamma interferon [IFN-gamma], interleukin-2 [IL-2], IL-8, IL-10, and IL-12), and nitric oxide levels after infection with B. rodhaini. SCID mice and IFN-gamma-deficient mice with chronic B. microti infections demonstrated protective responses comparable to those of immunocompetent mice. Likewise, in vivo NK cell depletion did not significantly impair the protective responses. Conversely, macrophage depletion resulted in increased susceptibility to B. rodhaini infection associated with changes in their antibody and cytokines profiles, indicating that macrophages contribute to the protection against this challenge infection. We conclude that future development of vaccines against Babesia should include a strategy that enhances the appropriate activation of macrophages.
• Epidemiological Survey of Theileria Parasite Infection of Cattle in Northeast China by Allele-Specific PCR

  Longzheng Yu, Shoufa Zhang, Wanfeng Liang, Chunmei Jin, Lijun Ji, Yuzi Luo, Yan Li, Shinuo Cao, Junya Yamagishi, Yoshifumi Nishikawa, Suguru Kawano, Kozo Fujisaki, Xuenan Xuan

  JOURNAL OF VETERINARY MEDICAL SCIENCE 73 (11) 1509 - 1512 0916-7250 2011/11 [Refereed][Not invited]
   

  An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.
• Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test

  Yuzi Luo, Honglin Jia, M. Alaa Terkawi, Youn-Kyoung Goo, Suguru Kawano, Hideo Ooka, Yan Li, Longzheng Yu, Shinuo Cao, Junya Yamagishi, Kozo Fujisaki, Yoshifumi Nishikawa, Atsuko Saito-Ito, Ikuo Igarashi, Xuenan Xuan

  PARASITOLOGY INTERNATIONAL 60 (2) 119 - 125 1383-5769 2011/06 [Refereed][Not invited]
   

  Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
• Construction and analysis of full-length cDNA library of Cryptosporidium parvum

  Junya Yamagishi, Hiroyuki Wakaguri, Sumio Sugano, Suguru Kawano, Kozo Fujisaki, Chihiro Sugimoto, Junichi Watanabe, Yutaka Suzuki, Isao Kimata, Xuenan Xuan

  PARASITOLOGY INTERNATIONAL 60 (2) 199 - 202 1383-5769 2011/06 [Refereed][Not invited]
   

  A full-length cDNA library was constructed from the sporozoite of Cryptosporidium parvum. Normalized clones were subjected to Solexa shotgun sequencing, and then complete sequences for 1066 clones were reconfigured. Detailed analyses of the sequences revealed that 13.5% of the transcripts were spliced; the average and median 5' UTR lengths were 213.5 and 122 nucleotides, respectively. There were 148 inconsistencies out of 562 examined genes between the experimentally described cDNA sequence and the predicted sequence from its genome. In addition, we identified 118 sequences that had little homology against annotated genes of C. parvum as prospective candidates for addable genes. These observations should improve the reliability of C. parvum transcriptome and provide a versatile resource for further studies. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
• Babesia microti: Molecular and antigenic characterizations of a novel 94-kDa protein (BmP94)

  Hideo Ooka, Mohamad Alaa Terkawi, Youn-Kyoung Goo, Yuzi Luo, Yan Li, Junya Yamagishi, Yoshifumi Nishikawa, Ikuo Igarashi, Xuenan Xuan

  EXPERIMENTAL PARASITOLOGY 127 (1) 287 - 293 0014-4894 2011/01 [Refereed][Not invited]
   

  A novel gene. BmP94, encoding 94-kDa protein of Babesia micron was identified by immunoscreening of the cDNA expression library. The full-length of BmP94 was expressed in Escherichia coli (rBmP94), which resulted in insoluble form with low yield, and the truncated hydrophilic C-terminus region of the gene was expressed as a soluble protein (rBmP94/CT) with improved productivity. Antiserum raised against rBmP94/CT recognized the 94-kDa native protein in the parasite extract by Western blot analysis. Next, an ELISA using rBmP94/CT was evaluated for diagnostic use, and it demonstrated high sensitivity and specificity when tested with the sera from mice experimentally infected with B. microti and closely related parasites. Moreover, the immunoprotective property of rBmP94/CT as a subunit vaccine was evaluated in BALB/c mice against a B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmP94 could be a promising candidate for diagnostic use for human babesiosis. (C) 2010 Elsevier Inc. All rights reserved.
• Full-parasites: database of full-length cDNAs of apicomplexa parasites, 2010 update

  Josef Tuda, Arthur E. Mongan, Mohammed E. M. Tolba, Mihoko Imada, Junya Yamagishi, Xuenan Xuan, Hiroyuki Wakaguri, Sumio Sugano, Chihiro Sugimoto, Yutaka Suzuki

  NUCLEIC ACIDS RESEARCH 39 (Database issue) D625 - D631 0305-1048 2011/01 [Refereed][Not invited]
   

  Full-Parasites (http://fullmal.hgc.jp/) is a transcriptome database of apicomplexa parasites, which include Plasmodium and Toxoplasma species. The latest version of Full-Parasites contains a total of 105 786 EST sequences from 12 parasites, of which 5925 full-length cDNAs have been completely sequenced. Full-Parasites also contain more than 30 million transcription start sites (TSS) for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg), which were identified using our novel oligo-capping-based protocol. Various types of cDNA data resources were interconnected with our original database functionalities. Specifically, in this update, we have included two unique RNA-Seq data sets consisting of 730 million mapped RNA-Seq tags. One is a dataset of 16 time-lapse experiments of cultured bradyzoite differentiation for Tg. The other dataset includes 31 clinical samples of Pf. Parasite RNA was extracted together with host human RNA, and the extracted mixed RNA was used for RNA sequencing, with the expectation that gene expression information from the host and parasite would be simultaneously represented. By providing the largest unique full-length cDNA and dynamic transcriptome data, Full-Parasites is useful for understanding host-parasite interactions and will help to eventually elucidate how monophyletic organisms have evolved to become parasites by adopting complex life cycles.
• Construction of Neospora caninum stably expressing TgSAG1 and evaluation of its protective effects against Toxoplasma gondii infection in mice

  Guohong Zhang, Xiaohong Huang, Damdinsuren Boldbaatar, Banzragch Battur, Badgar Battsetseg, Houshuang Zhang, Longzheng Yu, Yan Li, Yuzi Luo, Shinuo Cao, Youn-Kyong Goo, Junya Yamagishi, Jinlin Zhou, Shoufa Zhang, Hiroshi Suzuki, Ikuo Igarashi, Takeshi Mikami, Yoshifumi Nishikawa, Xuenan Xuan

  VACCINE 28 (45) 7243 - 7247 0264-410X 2010/10 [Refereed][Not invited]
   

  Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites. The surface antigen 1 of T. gondii (TgSAG1) is a major immunodominant antigen and, therefore, is considered to be a good candidate for the development of an effective recombinant vaccine against toxoplasmosis. In this study, N. caninum stably expressing the TgSAG1 gene (Nc/TgSAG1) was constructed using pyrimethamine-resistant DHFR-TS and GFP genes as double-selection markers. The expression level, molecular weight, and antigenic property of recombinant TgSAG1 expressed by the Nc/TgSAG1 were similar to those of the native TgSAG1. The mice immunized with Nc/TgSAG1 induced TgSAG1-specific Th1-dominant immune responses and protected the mice from a lethal challenge infection with T. gondii. These results indicate that N. caninum may provide a new tool for the production of a live recombinant vector vaccine against toxoplasmosis in animals. To our knowledge, this is the first report to evaluate the usefulness of N. caninum-based live vaccine. (C) 2010 Elsevier Ltd. All rights reserved.
• High-Resolution Characterization of Toxoplasma gondii Transcriptome with a Massive Parallel Sequencing Method

  Junya Yamagishi, Hiroyuki Wakaguri, Akio Ueno, Youn-Kyoung Goo, Mohammed Tolba, Makoto Igarashi, Yoshifumi Nishikawa, Chihiro Sugimoto, Sumio Sugano, Yutaka Suzuki, Junichi Watanabe, Xuenan Xuan

  DNA RESEARCH 17 (4) 233 - 243 1340-2838 2010/08 [Refereed][Not invited]
   

  For the last couple of years, a method that permits the collection of precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high-throughput manner was established. We applied this novel method, 'tss-seq', to elucidate the transcriptome of tachyzoites of the Toxoplasma gondii, which resulted in the identification of 124 000 TSSs, and they were clustered into 10 000 transcription regions (TRs) with a statistics-based analysis. The TRs and annotated ORFs were paired, resulting in the identification of 30% of the TRs and 40% of the ORFs without their counterparts, which predicted undiscovered genes and stage-specific transcriptions, respectively. The massive data for TSSs make it possible to execute the first systematic analysis of the T. gondii core promoter structure, and the information showed that T. gondii utilized an initiator-like motif for their transcription in the major and novel motif, the downstream thymidine cluster, which was similar to the Y patch observed in plants. This encyclopaedic analysis also suggested that the TATA box, and the other well-known core promoter elements were hardly utilized.
• Serodiagnosis of ovine toxoplasmosis in Mongolia by an enzyme-linked immunosorbent assay with recombinant Toxoplasma gondii matrix antigen 1

  Buyannemekh Tumurjav, Mohamad Alaa Terkawi, Houshuang Zhang, Guohong Zhang, Honglin Jia, Youn-Kyoung Goo, Junya Yamagishi, Yoshifumi Nishikawa, Ikuo Igarashi, Chihiro Sugimoto, Xuenan Xuan

  JAPANESE JOURNAL OF VETERINARY RESEARCH 58 (2) 111 - 119 0047-1917 2010/08 [Refereed][Not invited]
   

  Toxoplasma gondii matrix antigen 1 (TgMAG1), known as the 65-kDa protein, which is abundantly expressed in both bradyzoites and tachyzoites, was evaluated as a candidate for the development of a diagnostic reagent for ovine toxoplasmosis. The TgMAG1 gene was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), and the recombinant TgMAG1 (rTgMAG1) was tested in an enzyme-linked immunosorbent assay (ELISA). The ELISA with rTgMAG1 showed a highly specific reaction with sera from mice experimentally infected with T. gondii but not with the closely related Neospora caninum. The antibodies to TgMAG1 were detectable from the acute to the chronic infectious stages in a mouse model. A total of 175 serum samples collected from sheep in 7 provinces of Mongolia were examined for the serodiagnosis of T. gondii infection by the ELISA with rTgMAG1, and the results were compared with those from the commercialized latex agglutination test (LAT). Of 175 serum samples analyzed, 42 (24.00%) and 29 (16.57%) samples were positive by the ELISA and LAT, respectively. Of 29 LAT-positive samples, 27 (93.10%) were positive by the ELISA. These results suggest that rTgMAG1 could be used as a reliable antigen for the detection of T. gondii infection in sheep.
• Neospora caninum: Application of apical membrane antigen 1 encapsulated in the oligomannose-coated liposomes for reduction of offspring mortality from infection in BALB/c mice

  Houshuang Zhang, Yoshifumi Nishikawa, Junya Yamagishi, Jinlin Zhou, Yuzuru Ikehara, Naoya Kojima, Naoaki Yokoyama, Xuenan Xuan

  EXPERIMENTAL PARASITOLOGY 125 (2) 130 - 136 0014-4894 2010/06 [Refereed][Not invited]
   

  Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidy-lethanolamine (M3-DPPE), referred to as M3-DPPE liposomes, have been shown to induce cellular immunity against antigens encapsulated therein. To evaluate whether these M3-DPPE liposomes have an adjuvant capacity against Neospora caninum infection, a novel immunization method utilizing soluble N. caninum apical membrane antigen 1 (NcAMA1) encapsulated in the M3-DPPE liposomes (M3-NcAMA1) was employed. The results revealed that a significant amount of interferon (IFN)-gamma production was detected in culture supernatants of NcAMA1 protein- or N. caninum lysate-stimulated spleen cells obtained from the mice one week after the third immunization with M3-NcAMA1. The parasite burden in the dams' brain tissue was decreased and the survival rate of offspring increased significantly in M3-NcAMA1-immunized mice. Thus, a parasite-specific Th1 immune response was successfully induced in the pregnant mice immunized with M3-NcAMA1, and an effective reduction of offspring mortality from N. caninum infection was triggered. (C) 2010 Elsevier Inc. All rights reserved.
• Characterization of a leucine aminopeptidase from Toxoplasma gondii

  Honglin Jia, Yoshifumi Nishikawa, Yuzi Luo, Junya Yamagishi, Chihiro Sugimoto, Xuenan Xuan

  MOLECULAR AND BIOCHEMICAL PARASITOLOGY 170 (1) 1 - 6 0166-6851 2010/03 [Refereed][Not invited]
   

  The M17 family leucine aminopeptidase (LAP) hydrolyzes amino acids from the N-terminus of peptides. Many LAPS from parasitic protozoa, including Plasmodium, Trypanosoma, and Leishmania, have been intensely investigated because of their crucial roles in parasite biology. In this study, the functional recombinant Toxoplasma gondii LAP (rTgLAP) was expressed in Escherichia coli, and its enzymatic activity against synthetic substrates for aminopeptidase, as well as cellular localization, was determined. The activity was strongly dependent on metal divalent cations, and was inhibited by bestatin, which is an inhibitor for metalloprotease. Our results indicated that TgLAP is a functional aminopeptidase in the cytoplasm of T. gondii. (C) 2009 Elsevier B.V. All rights reserved.
• Molecular characterization and expression of a 47-kDa merozoite surface protein of Babesia gibsoni for serodiagnosis by enzyme-linked immunosorbent assay.

  Aboge, G. O, Batbaatar, V, Goo, Y-K, Yamagishi, J, Nishikawa, Y, Sunaga, F, Namikawa, K, Igarashi, I, Fujisaki, K, Suzuki, H, Xuan, X

  J. Protozool. Res. 20 59 - 69 2010 [Refereed][Not invited]
  
• Babesia gibsoni: Identification, expression, localization, and serological characterization of a Babesia gibsoni 22-kDa protein

  Youn-Kyoung Goo, Honglin Jia, Mohamad Alaa Terkawi, Gabriel Oluga Aboge, Junya Yamagishi, Yoshifumi Nishikawa, Suk Kim, Hyung-Kwan Jang, Kozo Fujisaki, Xuenan Xuan

  EXPERIMENTAL PARASITOLOGY 123 (3) 273 - 276 0014-4894 2009/11 [Refereed][Not invited]
   

  Babesia gibsoni causes canine babesiosis. Here, we describe the identification and characterization of a novel gene, BgP22, containing an open reading frame of 621 bp and encoding a 22-kDa protein from B. gibsoni, as a serodiagnostic candidate. The recombinant BgP22 (rBgP22) was expressed and used as an antigen to produce anti-rBgP22 sera in mice. Using these anti-rBgP22 sera, a native 22-kDa protein was recognized by Western blot analysis and observed in the membrane of the parasites by immunofluorescent antibody tests (IFAT). The enzyme-linked immunosorbent assay (ELISA) using the rBgP22 detected specific antibodies to this protein in the sera of dogs experimentally and naturally infected with B. gibsoni in chronic stage. Furthermore, it did not show a cross reaction with the closely related apicomplexan parasites, indicating that the rBgP22 could be used as a diagnostic antigen for a detection of the chronic carrier stages of B. gibsoni infection. (C) 2009 Elsevier Inc. All rights reserved.
• Molecular characterizations of three distinct Babesia gibsoni rhoptry-associated protein-1s (RAP-1s).

  Terkawi MA, Amornthep A, Ooka H, Aboge G, Jia H, Goo YK, Nelson B, Yamagishi J, Nishikawa Y, Igarashi I, Kawazu SI, Fujisaki K, Xuan X

  Parasitology 10 136 (10) 1147 - 1160 0031-1820 2009/09 [Refereed][Not invited]
   

  Three cDNAs encoding rhoptry-associated protein 1 (RAP-1) homologues were found in the Babesia gibsoni EST database. Based on similarities to BgRAP-1a, which was identified previously by serological screening of a cDNA merozoite library, the two new genes were designated BgRAP-1b (33.7%) and BgRAP-1c (57%). Mice antiserum raised against each recombinant protein reacted specifically with B. gibsoni parasites as determined by Western blotting, which showed native molecular sizes of the BgRAP-1a (51 kDa), BgRAP-1b (53 kDa) and BgRAP-1c (47 kDa) consistent with predictable molecular weights. Immunofluoresence using these antibodies revealed localization of all BgRAP-1s within the matrix of merozoites; however, BgRAP-1a appeared to diverge from the other two when it was found secreted into the cytoplasm of infected erythrocytes. Apical localization of all 3 BgRAP-1s during the extracellular stage of the parasite combined with their ability to bind a canine erythrocyte membrane fraction was suggestive of a role for these proteins in erythrocyte attachment. Lastly, the ability of these recombinant proteins to be used as diagnostic reagents was tested by ELISA and the sensitivities of BgRAP-1a and BgRAP-1c were found increased through N-terminal truncation. Taken together, our data suggest divergent roles for the 3 BgRAP-1s in the merozoite stage of B. gibsoni.
• Characterization of a leucine aminopeptidase of Babesia gibsoni.

  Jia H, Terkawi MA, Aboge GO, Goo YK, Luo Y, Li Y, Yamagishi J, Nishikawa Y, Igarashi I, Sugimoto C, Fujisaki K, Xuan X

  Parasitology 9 136 (9) 945 - 952 0031-1820 2009/08 [Refereed][Not invited]
   

  Peptidases of parasitic protozoa are currently under intense investigation in order to identify novel virulence factors, drug targets, and vaccine candidates, except in Babesia. Leucine aminopeptidases in protozoa, such as Plasmodium and Leishmania, have been identified to be involved in free amino acid regulation. We report here the molecular and enzymatic characterization, as well as the localization of a leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from B. gibsoni (BgLAP). A functional recombinant BgLAP (rBgLAP) expressed in Escherichia coli efficiently hydrolysed synthetic substrates for aminopeptidase, a leucine substrate. Enzyme activity of the rBgLAP was found to be optimum at pH 8.0 and at 37 degrees C. The substrate profile was slightly different from its homologue in P. falciprum. The activity was also strongly dependent on metal divalent cations, and was inhibited by bestatin, which is a specific inhibitor for metalloprotease. These results indicated that BgLAP played an important role in free amino acid regulation.
• Molecular and immunological characterization of Babesia gibsoni and Babesia microti heat shock protein-70.

  Terkawi MA, Aboge G, Jia H, Goo YK, Ooka H, Yamagishi J, Nishikawa Y, Yokoyama N, Igarashi I, Kawazu SI, Fujisaki K, Xuan X

  Parasite immunology 6 31 (6) 328 - 340 0141-9838 2009/06 [Refereed][Not invited]
   

  Serological immunoscreening was used to identify a gene encoding heat shock protein-70 from Babesia gibsoni (BgHSP-70) that showed high homology with HSP-70s from other apicomplexan parasites. This gene corresponded to a full-length cDNA containing an open reading frame of 1968 bp predicted to result in a 70-kDa mature protein consisting of 656 amino acids. Analysis of the expression levels of BgHSP-70 indicated elevated transcription from cultured parasites incubated at 40 degrees C for 1 h, but not at 30 degrees C. Interestingly, antiserum raised against recombinant BgHSP-70 protein reacted specifically not only with a 70-kDa protein of B. gibsoni but also with a corresponding native protein of B. microti (BmHSP-70), indicating the high degree of conservation of this protein. The BmHSP-70 gene was then isolated and characterized and the immunoprotective properties of recombinant BgHSP-70 (rBgHSP-70) and rBmHSP-70 were compared in vitro and in vivo. Both proteins had potent mitogenic effects on murine and canine mononuclear cells as evidenced by high proliferative responses and IFN-gamma production after stimulation. Immunization regimes in BALB/c and C57BL/6 mice using rBgHSP-70 and rBmHSP-70 elicited high antibody levels, with concurrent significant reductions in peripheral parasitaemias. Taken together, these results emphasize the potential of HSP-70s as a molecular adjuvant vaccine.
• Toxoplasma gondii: Sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method

  Houshuang Zhang, Oriel M. M. Thekisoe, Gabriel O. Aboge, Hisako Kyan, Junya Yamagishi, Noboru Inoue, Yoshifumi Nishikawa, Satoshi Zakimi, Xuenan Xuan

  EXPERIMENTAL PARASITOLOGY 122 (1) 47 - 50 0014-4894 2009/05 [Refereed][Not invited]
   

  Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity and rapidity. In this study, we used a conserved sequence in the 200- to 300-fold repetitive 529 bp gene of Toxoplasma gondii to design primers for LAMP test. Detection limit of T. gondii LAMP assay with the primers is 1 pg/mu L of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. Furthermore, LAMP and conventional PCR methods were applied for amplification of the T. gondii DNA extracted from the lymph nodes taken from pigs which were suspected to be Toxoplasma infection. As a result, 76.9% (70/91) and 85.7% (78/91) of the samples were positive on PCR and LAMP analyzes, respectively. Therefore, the LAMP has a potential to be applied as an alternative molecular diagnostic tool for detection of T gondii infection from veterinary samples. This is the first study, which applies the LAMP method to diagnose Toxoplasma from veterinary samples. (C) 2009 Elsevier Inc. All rights reserved.
• Characterization of the Babesia gibsoni 12-kDa protein as a potential antigen for the serodiagnosis

  Youn-Kyoung Goo, Honglin Jia, G. Oluga Aboge, M. Alaa Terkawi, Eung-Goo Lee, Junya Yamagishi, Yoshifumi Nishikawa, Hyung-Kwan Jang, Fujiko Sunaga, Kazuhiko Namikawa, Kozo Fujisaki, Xuenan Xuan

  PARASITOLOGY INTERNATIONAL 58 (1) 55 - 60 1383-5769 2009/03 [Refereed][Not invited]
   

  A novel gene, BgP12, encoding a 12-kDa protein was identified from Babesia gibsoni. The full-length cDNA of BgP12 contains an open reading frame of 378 bp, corresponding to 126 amino acid (aa) residues consisting of a putative 26 aa signal peptide and a 100 aa mature protein. The recombinant BgP12 (rBgP12) lacking the N-terminal signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein (rBgP12) that produced all anti-rBgP12 serum in mice after immunization. Using this anti-rBgP12 serum, a native 12-kDa protein in B. gibsoni was recognized by Western blot analysis. Immunofluorescent antibody tests (IFAT) revealed that BgP12 was mainly seen during the ring stage of B. gibsoni trophozoite. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP12 detected specific antibodies in the sequential sera of a dog experimentally infected with B. gibsoni beginning 10 days post-infection to 442 clays post-infection, even when the dog became chronically infected and showed a low level of parasitemia, Moreover, the antigen did not show cross-reaction with antibodies to the closely related apicomplexan parasites, indicating that the rBgP12 might be all immunodominant antigen for B. gibsoni infection that Could be used as a diagnostic antigen for B. gribsoni infection with high specificity and sensitivity. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
• The AcMNPV pp31 gene is not essential for productive AcMNPV replication or late gene transcription but appears to increase levels of most viral transcripts

  Junya Yamagishi, Erik D. Burnett, Steven H. Harwood, Gary W. Blissard

  VIROLOGY 365 (1) 34 - 47 0042-6822 2007/08 [Refereed][Not invited]
   

  The pp31 gene of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes a phosphorylated DNA binding protein that associates with virogenic stroma in the nuclei of infected cells. Prior studies of pp31 by transient late expression assays suggested that pp3l may play an important role in transcription of AcMNPV late genes [Todd, J. W., Passarelli, A. L., and Miller, L. K. (1995). Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. J. Virol. 69, 968-974] although genetic studies of the closely related BmNPV pp3l gene suggested that pp31 may be dispensable [Gomi, S., Zhou, C. E., Yih, W., Majima, K., and Maeda, S. (1997). Deletion analysis of four of eighteen late gene expression factor gene homologues of the baculovirus, BmNPV. Virology 230 (1), 35-47]. In the current study, we examined the role of the pp3l gene in the context of the AcMNPV genome during infection. We used a BACmid-based system to generate a pp3l knockout in the AcMNPV genome. The pp3l knockout was subsequently rescued by reinserting the pp3l gene into the polyhedrin locus of the same virus genome. We found that pp3l was not essential for viral replication although the absence of pp3l resulted in a lower viral titer. Analysis of viral DNA replication in the absence of pp3l showed that the kinetics of viral DNA replication were unaffected. An AcMNPV oligonucleotide microarray was used to compare gene expression from all AcMNPV genes in the presence or absence of pp31. In the absence of pp31, a modest reduction in transcripts was detected for many viral genes (99 genes) while no substantial increase or decrease was observed for 43 genes. Transcripts from 6 genes (p6.9, ORF 97, ORF 60, ORF 98, ORF 102 and chitinase) were reduced by 66% or more compared to the levels detected from the control virus. Microarray results were further examined by qPCR analysis of selected genes. In combination, these data show that deletion of the pp3l gene was not lethal and did not appear to affect viral DNA replication but resulted in an apparent modest down-regulation of a subset of AcMNPV genes that included both early and late genes. (c) 2007 Elsevier Inc. All rights reserved.
• Expression of Autographa californica multiple nucleopolyhedrovirus genes in mammalian cells and Upregulation of the host beta-actin gene

  R Fujita, T Matsuyama, J Yamagishi, K Sahara, S Asano, H Bando

  JOURNAL OF VIROLOGY 80 (5) 2390 - 2395 0022-538X 2006/03 [Refereed][Not invited]
   

  The gene expression of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was examined in two types of mammalian cells, human HeLa14 and hamster BHK cells. DNA microarray analysis followed by reverse transcription-PCR identified at least 12 viral genes transcribed in both HeLa14 cells and BHK cells inoculated with AcMNPV. 5' rapid amplification of cDNA ends was carried out to examine the transcriptional fidelity of these genes in HeLa14 cells. The transcription of ie-1, ie-0 and gp64 was initiated at a baculovirus early gene motif, CAGT, accompanied by a TATA motif. In addition, the same splicing observed for ie-0 mRNA in Sf9 cells occurred in HeLa14 cells. While the transcription initiation sites for pe38 and p6.9 were not located in the CAGT motif, most of them were in a typical eukaryotic RNA polymerase II promoter structure (a conventional TATA motif and/or an initiator). Interestingly, the expression of beta-actin was upregulated in the mammalian cells inoculated with AcMNPV. Subsequent experiments using UV-inactivated virus confirmed the upregulation, suggesting that de novo synthesis of viral products is not required for the event. These results indicated that the AcMNPV genome acts as a template for transcription in mammalian cells through the usual infection pathway, though there is no evidence for the functional expression of viral genes at present.
• The use of a recombinant baculovirus expressing a chitinase from the hard tick Haemaphysalis longicornis and its potential application as a bioacaricide for tick control

  SP Assenga, M You, CH Shy, J Yamagishi, T Sakaguchi, JL Zhou, MK Kibe, XN Xuan, K Fujisaki

  PARASITOLOGY RESEARCH 98 (2) 111 - 118 0932-0113 2006/01 [Refereed][Not invited]
   

  Baculoviruses are specific insect pathogens used as selective biological insecticides on lepidopteran insects. We have tested a recombinant baculovirus expressing a chitinase gene for its efficacy as a tick bioacaricide. The recombinant Autographa californica multiple nuclear polyhedrosis virus expressing a chitinase enzyme (AcMNPV-CHT1) from the hard tick, Haemaphysalis longicornis, was constructed and found to have a novel bioacaricidal effect against ticks. The recombinant baculovirus was used to express the chitinase enzyme in Spodoptera frugiperda (Sf9) insect cells. Topical application of the supernatant harvested from the insect cell culture was found to cause mortality in nymphal ticks of H. longicornis. High temperature (> 30 degrees C) and infrared radiation affected the chitinase enzyme activity and recombinant baculovirus infectivity by reducing the speed of tick killing by 60%. A mixture of recombinant virus and chitinase was found to kill ticks faster (p < 0.01) than pure chitinase and recombinant virus alone. Thus, the recombinant virus showed a synergistic effect with the foreign chitinase gene. In order to reduce the excessive use and cost of acaricides, it was found that a mixture of recombinant virus and flumethrin could halve the dose of the chemical acaricide used. These findings are important for the safe use of the recombinant virus expressing chitinase as a bioacaricide against ticks.
• [Baculovirus vector--gene transfer into mammalian cells].

  Tani H, Abe T, Limn CK, Mochizuki R, Yamagishi J, Kitagawa Y, Watanabe R, Moriishi K, Matsuura Y

  Uirusu 53 (2) 185 - 193 0042-6857 2003/12 [Refereed][Not invited]
  
• DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines

  J Yamagishi, R Isobe, T Takebuchi, H Bando

  ARCHIVES OF VIROLOGY 148 (3) 587 - 597 0304-8608 2003/03 [Refereed][Not invited]
   

  We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef-6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.
• Genome organization and mRNA structure of Periplaneta fuliginosa densovirus imply alternative splicing involvement in viral gene expression

  J Yamagishi, Y Hu, J Zheng, H Bando

  ARCHIVES OF VIROLOGY 144 (11) 2111 - 2124 0304-8608 1999 [Refereed][Not invited]
   

  We determined the complete nucleotide sequence of an infectious clone of the cockroach small spherical virus (CSSV) genome. Analysis of the genome organization and the predicted viral protein sequences showed clearly that this virus should be classified as a new member of the subfamily Densovirinae, genus Densovirus, and should be designated as PfDNV. However, our data revealed some differences between the gene expression strategies used by PfDNV and other DNVs. An internal promoter, in addition to the promoter (p3) at the genome terminus, was observed at map unit 18 (p18), implying transcriptional regulation of generation of the nonstructural proteins of PfDNV. Furthermore, the structural analysis of cDNAs complementary to mRNAs from the region coding for structural proteins suggested alternative splicing and polyadenylation as means for generation of the structural proteins of PfDNV.

MISC

• Combination therapy with oral antiviral and anti-inflammatory treatments improves the delayed-therapeutic effect on COVID-19

  佐々木道仁, 杉達紀, 飯田俊, 平田雄一郎, 日下部伸治, 日下部伸治, 小西慧, 小西慧, 板倉友香里, 板倉友香里, 田畑耕史郎, 田畑耕史郎, 岸本麻衣, 岸本麻衣, 小林広子, 有泉拓馬, KITTIYA Intaruck, 登治謙, 鳥羽晋輔, 鳥羽晋輔, 佐藤彰彦, 佐藤彰彦, 佐藤彰彦, 松野啓太, 松野啓太, 山岸潤也, 山岸潤也, 鈴木忠樹, 大場靖子, 大場靖子, 澤洋文, 澤洋文, 澤洋文  日本ウイルス学会学術集会プログラム・予稿集(Web)  70th-  2023
• Whole-genome sequence of Babesia caballi

  山岸潤也, 越智章仁, 木高大志, HAKIMI Hassan, 麻田正仁  日本寄生虫学会大会プログラム・抄録集  92nd-  2023
• Identification of genes of Coxiella-like endosymbiont in Haemaphysalis longicornis tick involved in metabolism of B vitamins and cofactors

  草木迫浩大, 山本佳代子, 今里裕平, 山岸潤也, 白藤(梅宮)梨可, 玄学南, 野中成晃, 中尾亮  日本寄生虫学会大会プログラム・抄録集  89th-  2020
• Cattle are potential animal reservoirs of sleeping sickness in Malawi

  MARSELA Megasari, 林田京子, 中尾亮, CHATANGA Elisha, GAITHUMA Alex Kiari, 河合直子, MUSAYA Janelisa, 杉本千尋, 山岸潤也  日本寄生虫学会大会プログラム・抄録集  89th-  2020
• Corrigendum: Interactive transcriptome analysis of malaria patients and infecting Plasmodium falciparum.

  Junya Yamagishi, Anna Natori, Mohammed E M Tolba, Arthur E Mongan, Chihiro Sugimoto, Toshiaki Katayama, Shuichi Kawashima, Wojciech Makalowski, Ryuichiro Maeda, Yuki Eshita, Josef Tuda, Yutaka Suzuki  Genome research  28-  (8)  1253  -1253  2018/08  [Not refereed][Not invited]
  
• 日本産ヒトスジシマカのジカウイルス媒介能

  江下優樹, 江下優樹, 江下優樹, 江下優樹, 和田雄治, 林田京子, 山岸潤也, 杉本千尋, 佐々木道仁, 大場靖子, 澤洋文, 飛弾野真也, 神山長慶, 小林隆志, 高崎智彦, 加藤文博, 田島茂, 倉根一郎, LIM Chang Kweng  衛生動物  69-  (Supplement)  51  2018/03/30  [Not refereed][Not invited]
  
• Investigating the roles of IFN gamma and IFN gamma-stimulated GTPases during Legionella pneumophila replication in alveolar macrophages and monocyte-derived cells

  Chao Yang, Shivani Pasricha, Sze Ying Ong, Andrew Stephen Brown, Junya Yamagishi, Chihiro Sugimoto, Sammy Bedoui, Ian R. van Driel, Elizabeth L. Hartland  CYTOKINE  100-  149  -149  2017/12  [Not refereed][Not invited]
  
• Theileria orientalisのマウスとマダニを用いた感染実験系の確立と遺伝子発現解析

  林田京子, 林田京子, 白藤梨可, SIVAKUMAR Thillaiampalam, 山岸潤也, 鈴木穣, 杉本千尋, 横山直明  日本寄生虫学会大会プログラム・抄録集  86th-  89  2017  [Not refereed][Not invited]
  
• Babesia ovataのゲノム・トランスクリプトーム解析

  山岸潤也, 麻田正仁, HASSAN Hakimi, 田中健, 田中健, 杉本千尋, 河津信一郎  日本獣医学会学術集会講演要旨集  159th-  334  -334  2016/08/30  [Not refereed][Not invited]
  
• 2種類のインビトロ培養条件下における大型ピロプラズマ原虫Babesia ovataの比較トランスクリプトーム解析

  田中健, 山岸潤也, HASSAN Hakimi, 麻田正仁, 河津信一郎  日本獣医学会学術集会講演要旨集  158th-  304  -304  2015/08/30  [Not refereed][Not invited]
  
• Babesia ovataの短時間かつ安定的な遺伝子導入系の確立(Establishment of transient and stable transfection system for Babesia ovata)

  Hakimi Hassan, 山岸 潤也, 外川 裕人, 田中 健, 金子 修, 河津 信一郎, 麻田 正仁  日本獣医学会学術集会講演要旨集  158回-  304  -304  2015/08  [Not refereed][Not invited]
  
• 1‐Cys型ペルオキシレドキシン遺伝子欠損Plasmodium bergheiの赤内型RNA‐seq解析

  薄井美帆, 増田(菅沼)裕乃, 元岡大祐, 中村昇太, 山岸潤也, 福本晋也, 井上昇, 堀井俊宏, 河津信一郎  日本寄生虫学会大会プログラム・抄録集  84th-  79  2015/02  [Not refereed][Not invited]
  
• 8.一過性的にデングウイルス血症をマウスに起こして、定量的感染蚊を大量に作る新規の方法(一般講演(1),都市有害生物管理学会第35回大会講演要旨)

  江下 優樹, Runtuwene Lucky R, 牧野 芳大, 野口 香緒里, 川上 絵理, 福田 昌子, 小林 隆志, 小西 英二, 山中 敦史, Srisawat Raweewan, Komalamisra Narumon, 成田 弘成, 牛島 廣治, Mon-gan Arthur E, 今田 美穂子, 山岸 潤也, 鈴木 穣, 中井 謙太, 前田 龍一郎, 杉本 千尋, 倉根 一郎, 高崎 智彦  都市有害生物管理  4-  (2)  120  -121  2014/12/20  [Not refereed][Not invited]
  
• Eimeria tenellaスポロゾイトのミトコンドリア精製法の確立と呼吸鎖酵素活性

  稲岡 ダニエル健, 松林 誠, 小松谷 啓介, 八田 岳士, 三好 猛晴, 磯部 尚, 川原 史也, 山岸 潤也, 彦坂 健児, 佐藤 暖, 志波 智生, 原田 繁春, 辻 尚利, 北 潔  日本生化学会大会プログラム・講演要旨集  87回-  [4T09p  -15]  2014/10  [Not refereed][Not invited]
  
• 歯垢検体の保存温度および保存期間による細菌叢への影響

  SHINOZAKI NATSUKO, YAMAGISHI JUN'YA, SATO YUKUTO, YAMASHITA RIU, YAMADA TAKUJI, NAGASAKI MASAO, TSUBOI AKITO  日本ゲノム微生物学会年会要旨集  8th-  66  2014  [Not refereed][Not invited]
  
• Babesia gibsoniのドラフトゲノム解析

  山岸 潤也, 麻田 正仁, 正谷 達謄, 于 龍政, 曹 世諾, 須永 藤子, 玄 学南  日本獣医学会学術集会講演要旨集  156回-  238  -238  2013/08  [Not refereed][Not invited]
  
• Validation of 40S ribosomal protein 17S as internal control for qRT-PCR of dengue-infected Aedes aegypti.

  RUNTUWENE LUCKY R, KAWASHIMA SHUICHI, YAMAGISHI JUNYA, SUZUKI YUTAKA, SUGANO SUMIO, NAKAI KENTA, MAEDA RYUICHIRO, SUGIMOTO CHIHIRO, TAKASAKI TOMOHIKO, KURANE ICHIRO, KOBAYASHI TAKASHI, ESHITA YUKI  衛生動物  64-  (2)  138  2013/06/15  [Not refereed][Not invited]
  
• Trypanosoma congolenseエピマスティゴート型原虫のヘモグロビン取込みに関する研究

  山崎詩乃, 菅沼啓輔, 麻田正仁, NGASAMAN Ruttayaporn, 周末, 山岸潤也, 河津信一郎, 井上昇  日本獣医学会学術集会講演要旨集  155th-  216  -216  2013/03/04  [Not refereed][Not invited]
  
• Trypanosoma congolense EMFステージ特異的遺伝子の発現調節機構解析

  菅沼啓輔, 山崎詩乃, 山岸潤也, 麻田正仁, 河津信一郎, 井上昇  日本獣医学会学術集会講演要旨集  155th-  118  -118  2013/03/04  [Not refereed][Not invited]
  
• congolense epimastigote specific protein(CESP)のEMF特異的発現は3'非翻訳領域によって調節されている

  菅沼 啓輔, 山崎 詩乃, 山岸 潤也, 麻田 正仁, 河津 信一郎, 井上 昇  獣医寄生虫学会誌  11-  (1)  49  -49  2012/10  [Not refereed][Not invited]
  
• Trypanosoma congolenseハプトグロビン-ヘモグロビン複合体レセプター様タンパク質(TcHpHbR)に関する研究

  山崎 詩乃, 菅沼 啓輔, 山岸 潤也, 麻田 正仁, 河津 信一郎, 井上 昇  獣医寄生虫学会誌  11-  (1)  50  -50  2012/10  [Not refereed][Not invited]
  
• アピコンプレクス門原虫のプロモーター構造比較

  山岸潤也, 鈴木穣, 五十嵐郁男, 河津信一郎, 玄学南  日本熱帯医学会大会プログラム抄録集  53rd-  56  -57  2012/09  [Not refereed][Not invited]
  
• congolense epimastigote specific protein(CESP)のEMF特異的発現は3’非翻訳領域によって調節されている

  菅沼啓輔, 山崎詩乃, 山岸潤也, 麻田正仁, 河津信一郎, 井上昇  日本獣医学会学術集会講演要旨集  153rd-  224  -224  2012/03/01  [Not refereed][Not invited]
  
• Trypanosoma congolenseハプトグロビン‐ヘモグロビン複合体レセプター様タンパク質(TcHpHbR)に関する研究

  山崎詩乃, 菅沼啓輔, 山岸潤也, 麻田正仁, 河津信一郎, 井上昇  日本獣医学会学術集会講演要旨集  153rd-  225  -225  2012/03/01  [Not refereed][Not invited]
  
• トキソプラズマのステージ間比較トランスクリプトーム解析

  山岸潤也, 若栗浩幸, 河津信一郎, 鈴木穣, XUAN Xuenan  日本寄生虫学会大会プログラム・抄録集  81st-  87  2012/02  [Not refereed][Not invited]
  
• アピコンプレクス門原虫のプロモーター構造種間比較

  山岸潤也, 山下理宇, 鈴木穣, 五十嵐郁男, 河津信一郎, XUAN Xuenan  日本分子生物学会年会プログラム・要旨集(Web)  35th-  1P-0029 (WEB ONLY)  2012  [Not refereed][Not invited]
  
• Babesia bovisメロゾイト滑走運動のバイオイメージング解析

  麻田正仁, 山岸潤也, 後藤康之, 横山直明, 井上昇, 河津信一郎  日本寄生虫学会東日本支部大会プログラム・講演要旨  72nd-  19  2012  [Not refereed][Not invited]
  
• ヒトと熱帯熱マラリア原虫のトランスクリプトーム相互作用データの収集と解析

  名取杏奈, TUDA Josef, MONGAN Arthur, TOLBA Mohammed, TOLBA Mohammed, 今田美穂子, 山岸潤也, XUAN Xuenan, 若栗浩幸, 菅野純夫, 杉本千尋, 鈴木穣  日本分子生物学会年会プログラム・要旨集(Web)  35th-  WEB ONLY 4P-0211  2012  [Not refereed][Not invited]
  
• Identification of an immunodominant Babesia gibsoni 47-kDa antigen

  Goo Jia Y-K, Aboge G. O, Terkawi M. A, Yamagishi J, Nishikawa Y, Igarashi I, Xuan X  The Journal of protozoology research  19-  (2)  16  -21  2009/12
• Identification of a novel B. gibsoni 27-kDa protein as a serodiagnostic antigen

  Terkawi M. A., Aboge G., Jia H., Goo Y-K., Ooka H., Yamagishi Junya, Nishikawa Yoshifumi, Kawazu Shin-ichiro, Fujisaki K., Xuan Xuenan  The Journal of Protozoology Research  18-  (2)  48  -56  2008/12  

  A novel gene encoding 27-kDa protein was identified by the screening of Babesia gibsoni cDNA library with acutely infected dog serum. The BgP27 is a single copy gene with a predicted open reading frame of 762 bp and 254 amino acids. The phylogenic analysis of the deduced amino acid of BgP27 demonstrated considerable identities with members of Plasmodium berghei circumsporozoite protein family that ranged between 18.4% and 22.8%. The BgP27 was expressed as a glutathione S-transferase fusion protein in Escherichia coli. The serum raised in mice against the recombinant protein specifically reacted with a 27-kDa protein in the extracts of B. gibsoni parasites. Confocal laser scanning microscopic observation showed high fluorescent reactivity with both extracellular and intracellular merozoite. Furthermore, recombinant BgP27 was used as an antigen in an enzyme-linked immunosorbent assay (ELISA). Thus, the kinetics of the anti-BgP27 antibody was detected in serial serum samples collected from a B. gibsoni-infected dog. IgG levels were high throughout the course of infection. In addition, the ELISA was able to differentiate between B. gibsoni-infected dog serum and B. canis subspecies-infected dog serum or normal dog serum. The diagnostic performance of BgP27-ELISA revealed the potential use of the antigen for detection of infection in dogs.
• 【ウイルスベクター 最新の話題】 バキュロウイルスベクター 哺乳動物細胞への遺伝子導入

  谷 英樹, 阿部 隆之, 林 昌宏, 望月 理加, 山岸 潤也, 北川 善紀, 渡辺 理恵, 宮本 大伸, 森石 恆司, 松浦 善治  ウイルス  53-  (2)  185  -193  2003/12  [Not refereed][Not invited]
  
• PfDNV nonstructural proteins suppress viral RNA splicing

  YAMAGISHI JUNYA, ASANO SHINICHIRO, SAHARA KEN, IIZUKA TOSHIHIKO, BANDO HISANORI  日本蠶絲學雜誌  69-  (4)  271  -276  2000/08/31  [Not refereed][Not invited]
  
• PfDNV nonstructural proteins suppress viral RNA splicing

  Junya Yamagishi, Shinichiro Asano, Ken Sahara, Toshihiko Iizuka, Hisanori Bando  Journal of Sericultural Science of Japan  69-  (4)  271  -276  2000  [Not refereed][Not invited]
   

  Alternative splicing observed in the mRNAs for the structural proteins of PfDNV seems to be necessary to generate five structural proteins from two separated ORFs. Involvement of several splicing donor sites and acceptor sites in the alternative splicing complicates understanding of the splicing mechanism of the PfDNV. First we developed a method for detection of spliced RNA molecules using the combination of the different two techniques, RT-PCR and primer extension. This splicing-detection method demonstrated that the sites of alternative splicing occurred within the PfDNV RNA was also recognized in S2 cells (a Drosophila cell line). Interestingly, a drastic suppression of the splicing and the accumulation of the unspliced RNA molecules were observed in S2 cells transfected with the PfDNV non-structural proteins (γ and β)-expression plasmids. These results suggested that the cellular factors play an important role on the selection of the specific splicing sites and that the viral nonstruc ural proteins may regulate it in a suppressive manner. © 2000, The Japanese Society of Sericultural Science. All rights reserved.

Research Grants & Projects

• Analysis of host defense mechanism against canine babesiosis and development of novel vaccines

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/04 -2025/03 

  Author : 玄 学南, 麻田 正仁, 正谷 達謄, 山岸 潤也
• Immunopathology of visceral leishmaniasis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2021/04 -2024/03 

  Author : 後藤 康之, 藤井 渉, 片岡 直行, 山岸 潤也

   

  内臓型リーシュマニア症（VL）はリーシュマニア原虫の感染により引き起こされる人獣共通感染症である。本症の化学療法はさまざまな問題点を抱えており、宿主免疫を適切に調節することで効果を発揮する免疫療法は新たなVLの治療法として期待される。一方、発熱、貧血、肝脾腫といったVLの症状が免疫応答に起因することから、不適切な免疫刺激による逆効果も予想される。つまり、VLに対する効果的な免疫療法の確立にはその感染・発症機序を詳細にとらえる必要がある。本研究では、発生工学を駆使したマウスモデルと、ヒト患者やイヌ由来材料を用いた解析を有機的に組み合わせることで、VLにおける病態免疫を明らかにし、症状の改善を促す免疫療法の開発を目指している。 2021年度は特に貧血の原因となる脾臓での血球貪食に焦点を当てて研究を進めた。血球貪食が亢進する感染マウスの脾臓では多核化マクロファージ（MGC）の出現が顕著になる。そこで本年度はin vitroにて感染誘導性マクロファージ多核化現象の構築を行った。その結果、L. donovani感染によって引き起こされる多核化はGM-CSFなどのサイトカインによって誘導される多核化とは異なり、血球貪食能力を特に引き上げることが明らかとなった。感染マウスでの血球貪食が肝臓では見られず脾臓で見られることから、感染臓器トランスクリプトーム解析により脾臓のみで上昇する遺伝子の探索を行ったところ、その一つがin vitro誘導MGCでも顕著に上昇することが明らかとなった。プラスミドを用いてマクロファージにその遺伝子を過発現させたところ、感染なしでもマクロファージの多核化が誘導された。つまり、原虫感染による当該遺伝子発現の上昇は、血球貪食型のMGCを誘導することが示唆された。
• Comprehensive analysis of Babesia tick-stage antigens: Establishing of a base for development the TBV

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/04 -2022/03 

  Author : 河津 信一郎, 白藤 梨可, 麻田 正仁, 山岸 潤也

   

  本研究はバベシアのマダニ体内発育ステージに特異的な細胞表在性蛋白質を網羅的に検出し、その機能を解析するとともに伝搬阻止ワクチン（TBV）抗原としての性能を評価することを目的とする。 【材料と方法】Babesia ovata（三宅株）赤血球内発育ステージ（Blood stage）をin vitro誘導法（PubMed ID: 1549158）によりマダニ体内発育ステージ（Tick stage）へと誘導し、RNA-sequence法（RNA-seq.）を用いてTick stage分子の探索を行った。まず、誘導後何時間の原虫をRNA-seq.に用いるか検討した。次に、特徴的な形態が観察され、またRT-PCR法でTick stage遺伝子の発現が確認出来た分画をパーコール密度勾配遠心法で濃縮した。得られた原虫からRNAを抽出し、RNA-seq.に供した。次世代シークエンシング（NGS)でデータを取得し、同時に実施したBlood stage RNA-seq.データとの間で比較解析を行った。【結果】Tick stage誘導後の原虫は様々な形態が認められた。特に誘導後6時間では核を１つまたは２つ有し、短い突起を持つray bodyと呼ばれる特徴的な形態が確認された。また、Tick stageに発現すると予想された遺伝子群の発現が認められた。そこで、TBV標的に適した細胞外発育ステージに相当する、誘導後6時間のTick stage原虫をRNA-seq.に用いることにした。分画濃縮法にて、ウシ赤血球が除かれたTick stage原虫分画を精製し、RNA-seq.に供した。NGS RNA-seq.データをBlood stage/Tick stage間で比較解析したところ、Blood stageよりも Tick stageで発現量が2倍以上大きい遺伝子は413個あり、特にTick stageにおいて十分な転写産物発現量があると考えられる遺伝子は253個あった。【考察】Tick stage原虫では、DNAの複製に関連する遺伝子の発現上昇が認められ、このステージでの活発な細胞分裂（原虫増殖）が裏付けられた。
• Analysis of the life cycle of Babesia in ticks and development of transmission-blocking vaccines

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2022/03 

  Author : 玄 学南, 白藤 梨可, 正谷 達謄, 山岸 潤也

   

  バベシア原虫はマダニによって媒介される病原体であり、種々の動物に重篤な貧血を引き起こし、家畜・ペット産業に重大な損害をもたらしている。本研究は、実験室レベルで全ライフサイクル解析が可能なバベシア原虫とフタトゲチマダ二を材料とし、マダニ体内におけるバベシア原虫発育の分子基盤解明と、それに基づくバベシア原虫伝播阻止ワクチンの開発を目指す。当該年度に得られた成果は、下記の通りである。１）in vitro培養系におけるB. gibsoniの有性生殖期虫体の誘導方法（キサンツレン酸の添加による誘導法）を確立した。２）有性生殖期マーカー分子とされるCCp1、CCp2、CCp3の発現が確認された。３）CCp1、CCp２、CCp3遺伝子をクローニングし、組換えタンパク質を作製した。４）組換えCCp1、CCp2、CCp3をマウスに免疫し、特異抗体を作製した。５）蛍光抗体法により、CCp1、CCp2、CCp3の特異抗体は、有性生殖期虫体と特異的反応を示すことを確認した。
• Studies on development of the Babesia parasites that express cytokine for non-specific immune activation

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/06 -2021/03 

  Author : KAWAZU Shin-ichiro

   

  The object of this study was to express cytokines such as interferon in haemoprotozoan Babesia parasite that causes asymptomatic infection in cattle. The parasite which expresses cytokine can modify immunity of the host animal accordingly regardless the types of pathogens such as viruses, bacteria, and parasites to reduce the damage caused by chronic debilitating infections caused by these pathogens. In order to develop a basic technology to produce Babesia parasite which expreses cytokines, an experimental system for editing the parasite genome with the CRISPR / Cas9 system and a knockdown experimental system applying the glucosamine (GlcN)-induced glmS ribozyme were established in non-domestic parasite species Babesia bovis.
• Immunopathology of visceral leishmaniasis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2021/03 

  Author : GOTO Yasuyuki

   

  Visceral leishmaniasis (VL) is a zoonosis caused by infection with the protozoan Leishmania species. In this study, we used a mouse model that reproduce symptoms in human VL patients including anemia and hepatosplenomegaly, to clarify the immune responses that affect these conditions. For anemia, we found that Leishmania infection altered the expression of SIRPα by macrophages and induced hemophagocytosis. This hemophagocytosis contributes to the increase in the number of Leishmania parasites inside the macrophages, suggesting that the parasites create an environment favorable for their own survival by controlling host molecules. We also found that the host inflammatory factor MRP14 is involved in this anemia and splenomegaly.
• Establishing a system in protozoan single cell transcriptomics

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2017/04 -2021/03 

  Author : Yamagishi Junya

   

  Protozoan parasites are diverse in size, morphology, and parasitic mode, and it was necessary to establish a suitable technical basis for single-cell transcriptome analysis, which has been increasingly used in recent years. In this study, we evaluated available systems in scRNA-seq library construction and found that BD Rhapsody was suitable. Furthermore, scRNA-seq analysis was conducted using tachyzoite to bradyzoite stage conversion in Toxoplasma gondii as a model, and it was found that the IFN alpha pathway is activated in the bystander cells surrounding the infected cells, whereas the pathway in the infected cells is conversely suppressed.
• Immunopathology of liver injury during malaria

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2018/03 

  Author : Goto Yasuyuki, FUJII Wataru, YAMAGISHI Junya

   

  Hepatic dysfunction is one of the clinical features in severe malaria. However, the mechanism of hepatic injury during malaria is still unknown. MRP14 is abundantly expressed by myeloid cells and involved in various inflammatory diseases. In order to verify whether extracellular MRP14 is involved in the pathology of hepatic injury during rodent malaria, we intravenously administrated recombinant MRP14 (rMRP14) to mice infected with Plasmodium berghei. The administration of rMRP14 exacerbated the hepatic injury during the infection, and their serum concentration of hepatic enzymes increased significantly more than PBS-treated controls. More MRP14+ macrophages accumulated in rMRP14-treated mice than PBS-treated controls after infection. The results indicate that MRP14 promotes the accumulation of MRP14+ cells and the up-regulation of pro-inflammatory molecules, which amplify inflammatory cascade leading to hepatic injury.
• Detection of Pathogens in unknown fever cases using Nanopore Sequencer

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2015/04 -2018/03 

  Author : Suzuki Yutaka

   

  In this study, we have attempted to develop a method to detect pathogens in patients having unknown fevers. Even though we were not able to develop the method based on the random-primer amplification due to the high-level noises, we successfully develop the method, by which representative viral, bacterial and parasite pathogens, such as dengue viruses, 16S rRNA sequencing for pan-bacterial detections, and malaria parasites, respectively, could be robustly detected. We actually applied the developed method to diagnose the feverish patients in Manado, Indonesia. We have reported the successful diagnosis from more than 50 cases regarding their infecting pathogens. Moreover, it was possible to identify the SNPs in their genomes. Especially, we found that the SNPs residing in the drug-resistance related genes should have particularly important clinical relevance, when the therapeutic strategy of the patients should be decided.
• Development of rapid diagnosis methods based on pathogen genome and their validation using clinical sample

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2015/04 -2018/03 

  Author : Yamagishi Junya

   

  To develop novel system for rapid diagnosis of infectious diseases based on pathogen genome, we had attempted to integrate 1) MinION, a rapid real-time portable next generation sequencer available in the market recently, and isothermal amplification system such as 2) Loop-mediated Isothermal Amplification (LAMP) method and 3) non- or semi-specific multiple displacement amplification (MDA) method in this study. The whole system including sequencing and library preparation is totally portable; therefore, it is executable in any place in standalone. We had proved the concept of the system at the lab then successfully demonstrated that it is feasible even at clinical site including Indonesia where various infectious are endemic.
• Epidemiology of zoonotic protozoan diseases and development of diagnosis system for social implementation in Indonesia

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : Nishikawa Yoshifumi, SUZUKI Yutaka, YAMAGISHI Jyunya, SHIMODA Naomi, UMEDA Kousuke

   

  This study focused on epidemiology of zoonotic protozoan diseases and development of diagnosis system for social implementation in Indonesia. Infection risk of protozoan parasites such as Toxoplasma and Cryptosporidium was found in Sulawesi and Java, Indonesia. Especially, infection risk of Toxoplasma in human increased at ages 10-20, suggesting the infection from meat or environment. In fact, Toxoplasma infection in cattle and pig was confirmed in these regions. Additionally, useful diagnosis system of Cryptosporidium was developed for future epidemiological study. For development of human resources based on collaborative research, seminar and technical workshop were organized.
• Reverse vaccinology for cell-mediated immunity

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2014/03 

  Author : GOTO Yasuyuki, MATSUMOTO Yoshitsugu, YAMAGISHI Junya

   

  Identification of parameters influencing the effectiveness of vaccine antigens for leishmaniasis was searched in this study. Statistical analyses revealed amino acid composition, expression level, and localization to exosome were influential parameters. A formula based on the three parameters discriminated the previously characterized vaccine antigens from the whole proteome. Furthermore, novel antigens identified using the formula were as antigenic as the known antigens. These results suggest the in silico approach developed is useful to identify candidate vaccine antigens for leishmaniasis.
• High resolution transcriptome analysis in protozoan parasites

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2010 -2012 

  Author : YAMAGISHI Junya

   

  By using RNA-seq and TSS-seq which are one of the application of next generation sequencers, it becomes possible to obtain transcriptomes of wide variety of living organisms. We applied the methods on representative three stages which consist of lifecycle of an obligate, intracellular, parasitic protozoan, Toxoplasma gondii. As a result, we demonstrated gene expression profiles of the three stages as well as putative DNA cis-elements which regulate the gene expression.
• トキソプラズマ原虫の発育ステージ変換機構の解明と新規次世代型ワクチンの開発

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2010 -2010 

  Author : 玄 学南, 山岸 潤也

   

  本研究は、トキソプラズマ原虫の発育ステージ(急性感染期のタキゾイト→慢性感染期のブラディゾイト)変換の分子機構の解明と新規次世代型ワクチン開発を目指して実施する。本年度に実施した研究内容と得られた研究成果は以下の通りである。 1. 試験管内ステージ変換系を確立した。試験管内で人為的にタキゾイトからブラディゾイトへのステージ変換を促し、大量のブラディゾイト虫体を得ることができた。 2. ブラディゾイト虫体の完全長cDNAラブラリーとESTデータベースを作成した。ブラディゾイト虫体のRNAを抽出し、オリゴキャッピング法にて完全長cDNAライブラリーを構築した後、約10,000クローンの全塩基配列を解読し、ESTデータベースを作成した。 3. トランスクリプトーム解析を行った。以前当研究グループで構築したタキゾイトESTデータベースと2)で作成したブラディゾイトESTデータベースの比較により、タキゾイト或いはブラディゾイト単一ステージのみに特異的に発現する遺伝子を網羅的に探索した。その結果、タキゾイトとブラディゾイト特異遺伝子がそれぞれ424個と759個が同定された。そのうち転写制御因子と推定されるAP2遺伝子がそれぞれ4個と6個が含まれていた。また.ブラディゾイト特異的に発現するAP2はCCAGTGモチーフに結合することで、ステージ特異的な転写制御が行われている可能性が示唆された。
• 異種抗原発現用原虫ベクターの構築と次世代型組換えワクチン開発への応用

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2009 -2010 

  Author : 玄 学南, 山岸 潤也

   

  本研究は異種抗原発現用新規原虫ベクターの構築と組換えワクチン開発への応用を目指して実施する。本年度に実施した研究内容と得られた研究成果は以下の通りである。 1. 過年度に異種抗原発現用ネオスポラ原虫ベクターを構築し、トキソプラズマ原虫ワクチン候補遺伝子TgSAG1の発現(Nc/TgSAG1)に成功した。 2. 今年度はまずNc/TgSAG1をBALB/cマウスに接種し、TgSAG1に対する特異抗体反応を誘導することを確認した。特異抗体のサブクラスを調べたところ、Th1型優勢免疫が誘導されていることが示唆された。また、Nc/TgSAG1を接種したマウスにおいてはIFN-γの産生がベクターのみを接種した対照群と比べ有意に高いことが示された。なお、IL-4の産生には対照群と比べ有意な変化がなかった。 3. 次に、Nc/TgSAG1にて免疫したマウスに致死量のトキソプラズマ原虫を接種したところ、約80%のマウスが生残した。これらの結果より、異種抗原発現用原虫ベクターは次世代型ワクチン開発に新しいツールを提供しうることが示唆された。
• 東南アジアにおけるダニ媒介性動物原虫感染症の流行実態の解明と予防対策の確立

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2009 -2010 

  Author : 玄 学南, 山岸 潤也

   

  本研究は東南アジアにおけるダニ媒介性動物原虫感染症の流行実態の解明と予防対策の確立を目指して実施する。本年度に実施した研究内容と得られた研究成果は以下の通りである。 1. 昨年度に引き続きタイ国北部地域における牛のマダニ媒介性原虫感染症の流行実態を調べた。計200頭の牛の血液サンプルを採集し、全DNAを抽出した。ウシバベシア原虫特異PCR法を用いて、血液サンプル中の原虫DNAの検出を行ったところ、Babesia bovisとBabesia bigeminaの陽性率がそれぞれ12%(24/200)と21%(42/200)であった。この結果より、ウシバベシア症はタイ国北部地域広く流行しており、本症の制圧対策は当地域の牛の生産性向上に重要であることが示唆された。 2. フィリピンマニラ周辺地域における牛のマダニ媒介性原虫感染症の流行実態を調べた。計250頭の牛より血液サンプルを採集し、全DNAを抽出した。ウシバベシア原虫特異PCR法とウシタイレリア原虫特異PCR法を用いて予備実験を行ったところ、2種類のウシバベシア原虫(Babesia bovisとBabesia bigemina)と1種類のウシタイレリア原虫(Theileria orientalis)DNAが高率に検出された。これらの結果により、フィリピンマニラ周辺地域の牛にはマダニ媒介性バベシア原虫とタイレリア原虫感染症が高率に流行していることが示唆された。

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