研究者データベース

小林 進太郎(コバヤシ シンタロウ)
獣医学研究院 獣医学部門 衛生学分野
准教授

基本情報

所属

  • 獣医学研究院 獣医学部門 衛生学分野

職名

  • 准教授

学位

  • 獣医学博士(北海道大学)

ホームページURL

科研費研究者番号

  • 00634205

J-Global ID

研究キーワード

  • 人獣共通感染症   分子生物   ウイルス   病理   獣医学   

研究分野

  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学

職歴

  • 2020年08月 - 現在 北海道大学大学院 獣医学研究院 獣医学部門 衛生学分野 公衆衛生学教室 准教授
  • 2016年06月 - 2020年07月 北海道大学大学院獣医学研究科 環境獣医科学講座 公衆衛生学教室 助教
  • 2015年06月 - 2016年05月 北海道大学大学院獣医学研究科 環境獣医科学講座 公衆衛生学教室 特任助教
  • 2015年04月 - 2015年05月 北海道大学 人獣共通感染症リサーチセンター 分子病態・診断部門 特任助教
  • 2012年04月 - 2015年03月 北海道大学 人獣共通感染症リサーチセンター 分子病態・診断部門 博士研究員

学歴

  • 2008年04月 - 2012年03月   北海道大学大学院   獣医学研究科
  • 2002年04月 - 2008年03月   酪農学園大学   獣医学部   獣医学科

所属学協会

  • 日本ウイルス学会   日本分子生物学会   日本獣医学会   

研究活動情報

論文

  • Shintaro Kobayashi, Chisato Kaneko, Ryoko Kawakami, Rie Hasebe, Hirofumi Sawa, Kentaro Yoshii, Hiroaki Kariwa
    Scientific reports 10 1 7168 - 7168 2020年04月28日 [査読有り][通常論文]
     
    West Nile virus (WNV) is an important cause of viral encephalitis in birds and animals, including humans. Amino acid 159 of the envelope (E) protein is reportedly implicated in the different levels of neurovirulence in mice infected with WNV NY99 or Eg101. We investigated the role of amino acid 159 of the E protein in the pathogenesis of WNV infection. We produced recombinant WNV with the structural proteins of the NY99 or Eg101 strain (NY-WT or EgCME-WT) and mutant viruses with substitutions of amino acid 159 of the E protein (NY-E-V159I or EgCME-E-I159V). The NY-WT and NY-E-V159I or EgCME-WT and EgCME-E-I159V titers in culture supernatant were similar. The mortality rate and viral titer in the brains of mice inoculated intraperitoneally with NY-WT or NY-E-V159I were also similar. In contrast, the mortality rate and viral titer in the brains of mice inoculated intracranially with EgCME-E-I159V were significantly higher than those of mice inoculated with EgCME-WT. The numbers of CD3-positive and CD8-positive T cells were greater in brains inoculated with EgCME-E-I159V than in those inoculated with EgCME-WT. Therefore, amino acid 159 of the E protein modulates the pathogenicity of WNV by affecting viral replication and T-cell infiltration in the brain.
  • Yuji Takahashi, Shintaro Kobayashi, Mariko Ishizuka, Minato Hirano, Memi Muto, Shoko Nishiyama, Hiroaki Kariwa, Kentaro Yoshii
    The Journal of general virology 2020年03月05日 [査読有り][通常論文]
     
    Tick-borne encephalitis virus (TBEV) is a zoonotic virus in the genus Flavivirus, family Flaviviridae. TBEV is widely distributed in northern regions of the Eurasian continent, including Japan, and causes severe encephalitis in humans. Tick-borne encephalitis (TBE) was recently reported in central Hokkaido, and wild animals with anti-TBEV antibodies were detected over a wide area of Hokkaido, although TBEV was only isolated in southern Hokkaido. In this study, we conducted a survey of ticks to isolate TBEV in central Hokkaido. One strain, designated Sapporo-17-Io1, was isolated from ticks (Ixodes ovatus) collected in Sapporo city. Sequence analysis revealed that the isolated strain belonged to the Far Eastern subtype of TBEV and was classified in a different subcluster from Oshima 5-10, which had previously been isolated in southern Hokkaido. Sapporo-17-Io1 showed similar growth properties to those of Oshima 5-10 in cultured cells and mouse brains. The mortality rate of mice infected intracerebrally with each virus was similar, but the survival time of mice inoculated with Sapporo-17-Io1 was significantly longer than that of mice inoculated with Oshima 5-10. These results indicate that the neurovirulence of Sapporo-17-Io1 was lower than that of Oshima 5-10. Using an infectious cDNA clone, the replacement of genes encoding non-structural genes from Oshima 5-10 with those from Sapporo-17-Io1 attenuated the neuropathogenicity of the cloned viruses. This result indicated that the non-structural proteins determine the neurovirulence of these two strains. Our results provide important insights for evaluating epidemiological risk in TBE-endemic areas of Hokkaido.
  • Shintaro Kobayashi, Kentaro Yoshii, Wallaya Phongphaew, Memi Muto, Minato Hirano, Yasuko Orba, Hirofumi Sawa, Hiroaki Kariwa
    PLoS pathogens 16 1 e1008238  2020年01月 [査読有り][通常論文]
     
    West Nile virus (WNV) belongs to the Flaviviridae family and has emerged as a significant cause of viral encephalitis in birds and animals including humans. WNV replication directly induces neuronal injury, followed by neuronal cell death. We previously showed that accumulation of ubiquitinated protein aggregates was involved in neuronal cell death in the WNV-infected mouse brain. In this study, we attempted to elucidate the mechanisms of the accumulation of protein aggregates in the WNV-infected cells. To identify the viral factor inducing the accumulation of ubiquitinated proteins, intracellular accumulation of ubiquitinated proteins was examined in the cells expressing the viral protein. Expression of capsid (C) protein induced the accumulation, while mutations at residues L51 and A52 in C protein abrogated the accumulation. Wild-type (WT) or mutant WNV in which mutations were introduced into the residues was inoculated into human neuroblastoma cells. The expression levels of LC3-II, an autophagy-related protein, and AMP-activated protein kinase (AMPK), an autophagy inducer, were reduced in the cells infected with WT WNV, while the reduction was not observed in the cells infected with WNV with the mutations in C protein. Similarly, ubiquitination and degradation of AMPK were only observed in the cells infected with WT WNV. In the cells expressing C protein, AMPK was co-precipitated with C protein and mutations in L51 and A52 reduced the interaction. Although the viral replication was not affected, the accumulation of ubiquitinated proteins in brain and neurological symptoms were attenuated in the mouse inoculated with WNV with the mutations in C protein as compared with that with WT WNV. Taken together, ubiquitination and degradation of AMPK by C protein resulted in the inhibition of autophagy and the accumulation of protein aggregates, which contributes to the development of neurological disease.
  • Christida E Wastika, Michihito Sasaki, Kentaro Yoshii, Paulina D Anindita, Bernard M Hang'ombe, Aaron S Mweene, Shintaro Kobayashi, Hiroaki Kariwa, Michael J Carr, William W Hall, Yuki Eshita, Yasuko Orba, Hirofumi Sawa
    Archives of virology 164 8 2165 - 2170 2019年08月 [査読有り][通常論文]
     
    Zika virus (ZIKV) circulation occurs between non-human primates (NHPs) in a sylvatic transmission cycle. To investigate evidence of flavivirus infection in NHPs in Zambia, we performed a plaque reduction neutralization test (PRNT) to quantify neutralizing antibodies. PRNT revealed that sera from NHPs (African green monkeys and baboons) exhibited neutralizing activity against ZIKV (34.4%; 33/96), whereas a PRNT for yellow fever virus using NHP sera showed no neutralization activity. ZIKV genomic RNA was not detected in splenic tissues from NHPs, suggesting that the presence of anti-ZIKV neutralizing antibodies represented resolved infections. Our evidence suggests that ZIKV is maintained in NHP reservoirs in Zambia.
  • Miki Nakayasu, Minato Hirano, Memi Muto, Shintaro Kobayashi, Hiroaki Kariwa, Kentaro Yoshii
    Ticks and Tick-borne Diseases 9 6 1391 - 1394 2018年09月 [査読有り][通常論文]
  • Kentaro Yoshii, Kozue Sato, Mariko Ishizuka, Shintaro Kobayashi, Hiroaki Kariwa, Hiroki Kawabata
    The American journal of tropical medicine and hygiene 99 1 180 - 181 2018年07月 [査読有り][通常論文]
     
    Tick-borne encephalitis (TBE) is widely prevalent on the Eurasian continent, including Japan, but four cases of TBE have been reported in Japan. To inspect unconfirmed TBE cases in Japan, we conducted a retrospective seroepidemiological study of a total of 158 samples from 81 meningoencephalitis patients suspected as Lyme disease. Two serum samples from one patient showed neutralizing antibodies against TBE virus. The patient with severe and progressive encephalitis had a history of tick bite in Hokkaido in 2012. These results demonstrated that tick-borne encephalitis virus (TBEV) case was actually unconfirmed in Japan. Further seroepidemiological surveys are required to identify unconfirmed TBEV infections to consider the pros and cons of introducing specific countermeasures including vaccination in Japan.
  • Memi Muto, Wataru Kamitani, Mizuki Sakai, Minato Hirano, Shintaro Kobayashi, Hiroaki Kariwa, Kentaro Yoshii
    Virus Research 249 52 - 56 2018年04月 [査読有り][通常論文]
  • Yuji Wada, Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Ibenu Rahmadani, Siswatiana Taha, Sri Adiani, Munira Latief, Zainal Abidin Kholilullah, Mawar Subangkit, Shintaro Kobayashi, Ichiro Nakamura, Takashi Kimura, Yasuko Orba, Hirofumi Sawa
    Journal of Medical Microbiology 67 3 415 - 422 2018年03月01日 [査読有り][通常論文]
     
    Bats are an important natural reservoir of zoonotic viral pathogens. We previously isolated an alphaherpesvirus in fruit bats in Indonesia, and here establish the presence of viruses belonging to other taxa of the family Herpesviridae. We screened the same fruit bat population with pan-herpesvirus PCR and discovered 68 sequences of novel gammaherpesvirus, designated ‘megabat gammaherpesvirus’ (MgGHV). A phylogenetic analysis of approximately 3.4 kbp of continuous MgGHV sequences encompassing the glycoprotein B gene and DNA polymerase gene revealed that the MgGHV sequences are distinct from those of other reported gammaherpesviruses. Further analysis suggested the existence of co-infections of herpesviruses in Indonesian fruit bats. Our findings extend our understanding of the infectious cycles of herpesviruses in bats in Indonesia and the phylogenetic diversity of the gammaherpesviruses.
  • Michihito Sasaki, Paulina D. Anindita, Wallaya Phongphaew, Michael Carr, Shintaro Kobayashi, Yasuko Orba, Hirofumi Sawa
    VIRUS RESEARCH 243 69 - 74 2018年01月 [査読有り][通常論文]
     
    Subviral particles (SVPs) self-assemble and are released from cells transfected with expression plasmids encoding flavivirus structural proteins. Flavivirus-like particles (VLPs), consisting of flavivirus structural proteins and a subgenomic replicon, can enter cells and cause single-round infections. Neither SVPs or VLPs possess complete viral RNA genomes, therefore are replication-incompetent systems; however, they retain the capacity to fuse and bud from target cells and follow the same maturation process as whole virions. SVPs and VLPs have been previously employed in studies analyzing entry and release steps of viral life cycles. In this study, we have developed quantitative methods for the detection of cellular entry and release of SVPs and VLPs by applying a luciferase complementation assay based on the high affinity interaction between the split NanoLuc luciferase protein, LgBiT and the small peptide, HiBiT. We introduced HiBiT into the structural protein of West Nile virus and generated SVPs and VLPs harboring HiBiT (SVP-HiBiT and VLP-HiBiT, respectively). As SVP-HiBiT emitted strong luminescence upon exposure to LgBiT and its substrate, the nascently budded SVP-HiBiT in the supernatant was readily quantified by luminometry. Similarly, the cellular entry of VLP-HiBiT generated luminescence when VLP-HiBiT was infected into LgBiT-expressing cells. These methods utilizing SVP-HiBiT and VLP-HiBiT will facilitate research into life cycles of flaviviruses, including WNV.
  • Tapiwa Lundu, Kentaro Yoshii, Shintaro Kobayashi, Shigeru Morikawa, Toshio Tsubota, Naoaki Misawa, Daisuke Hayasaka, Hiroaki Kariwa
    Japanese Journal of Veterinary Research 66 1 21 - 28 2018年 [査読有り][通常論文]
     
    Severe fever with thrombocytopenia syndrome (SFTS) is a newly recognized zoonosis that occurs in China, Japan, and South Korea and is caused by the SFTS virus (SFTSV), which is in the genus Phlebovirus, family Phenuiviridae. Since its discovery in Japan in 2013, SFTS has been reported in the western parts of the country. To elucidate the distribution of SFTSV, we conducted a serological survey of deer and rodents. Serum was screened using enzyme-linked immunosorbent assay (ELISA) and suspected cases were further tested with an indirect immunofluorescence antibody (IFA) assay. Serum samples from 315 deer from Hokkaido (non-endemic area), 41 deer from Miyazaki (endemic area), and 910 rodents from six locations in Japan were tested. Of the 41 deer from Miyazaki, 2 (4.9%) had high ELISA optical density (OD) values (0.1 < OD < 0.3) and a positive IFA result. All of the deer samples from Hokkaido were negative by ELISA (OD < 0.1). No SFTSV-positive rodents were found. Our results indicate that deer in Miyazaki were exposed to SFTSV, unlike deer from Hokkaido (P < 0.05).
  • Taiyu Tazaki, Koshiro Tabata, Akira Ainai, Yuki Ohara, Shintaro Kobayashi, Takafumi Ninomiya, Yasuko Orba, Hideyuki Mitomo, Tetsuo Nakano, Hideki Hasegawa, Kuniharu Ijiro, Hirofumi Sawa, Tadaki Suzuki, Kenichi Niikura
    RSC Advances 8 30 16527 - 16536 2018年 [査読有り][通常論文]
     
    Intranasal inactivated influenza vaccines can elicit mucosal immune responses that protect against virus infection. For the development of intranasal inactivated influenza vaccines, effective adjuvants inducing minimal adverse reactions are required. Generally, however, lower toxicity adjuvants have lower adjuvanticity. In this research, we fabricated nanoparticle-based adjuvants to enhance its adjuvanticity. Herein, we focused on low-molecular-weight polyinosinic-polycytidylic acid, referred to as uPIC(40:400), as a weak and less toxic RNA adjuvant. We conjugated uPIC(40:400) with different shaped gold nanoparticles (AuNPs) electrostatically. Conjugation with gold nanorods, but not spherical AuNPs, markedly enhanced the adjuvanticity of uPIC(40:400), leading to the suppression of viral infection in mice. Notably, conjugation with gold nanorods did not increase the inflammatory cytokine production in dendritic cells. These data indicated that gold nanorods can provide a good platform for enhancing the weak adjuvanticity of uPIC(40:400) while maintaining low inflammatory cytokine production toward the development of intranasal inactivated influenza vaccines.
  • Tapiwa LUNDU, Yoshimi TSUDA, Ryo ITO, Kenta SHIMIZU, Shintaro KOBAYASHI, Kentaro YOSHII, Kumiko YOSHIMATSU, Jiro ARIKAWA, Hiroaki KARIWA
    Biomedical Research 39 1 27 - 38 2018年 [査読有り][通常論文]
  • Ludek Eyer, Hirofumi Kondo, Darina Zouharova, Minato Hirano, James J. Valdes, Memi Muto, Tomas Kastl, Shintaro Kobayashi, Jan Haviernik, Manabu Igarashi, Hiroaki Kariwa, Marketa Vaculovicova, Jiri Cerny, Rene Kizek, Andrea Kroeger, Stefan Lienenklaus, Milan Dejmek, Radim Nencka, Martin Palus, Jiri Salat, Erik De Clercq, Kentaro Yoshii, Daniel Ruzek
    JOURNAL OF VIROLOGY 91 21 2017年11月 [査読有り][通常論文]
     
    borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-C-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-C-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-C-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP- luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-C-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus. IMPORTANCE This study found that the nucleoside analog 7-deaza-2'-C-methyl-adenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-C-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.
  • Minato Hirano, Memi Muto, Mizuki Sakai, Hirofumi Kondo, Shintaro Kobayashi, Hiroaki Kariwa, Kentaro Yoshii
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 37 9960 - 9965 2017年09月 [査読有り][通常論文]
     
    Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, little is known about the detailed mechanisms of viral replication and pathogenicity in the brain. Previously, we reported that the genomic RNA of tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is transported and replicated in the dendrites of neurons. In the present study, we analyzed the transport mechanism of the viral genome to dendrites. We identified specific sequences of the 5' untranslated region of TBEV genomic RNA that act as a cis-acting element for RNA transport. Mutated TBEV with impaired RNA transport in dendrites caused a reduction in neurological symptoms in infected mice. We show that neuronal granules, which regulate the transport and local translation of dendritic mRNAs, are involved in TBEV genomic RNA transport. TBEV genomic RNA bound an RNA-binding protein of neuronal granules and disturbed the transport of dendritic mRNAs. These results demonstrated a neuropathogenic virus hijacking the neuronal granule system for the transport of viral genomic RNA in dendrites, resulting in severe neurological disease.
  • Yuji Wada, Yasuko Orba, Michihito Sasaki, Shintaro Kobayashi, Michael J. Carr, Haruaki Nobori, Akihiko Sato, William W. Hall, Hirofumi Sawa
    VIROLOGY 505 102 - 112 2017年05月 [査読有り][通常論文]
     
    Chikungunya fever (CHIKF) is caused by chikungunya virus (CHIKV) infection which is a re-emerging mosquito-borne zoonosis. At present, there are no approved therapeutics for CHIKF. Herein, we have investigated candidate compounds which can inhibit CHIKV infection. Screening of chemical compound libraries were performed and one candidate, a benzimidazole-related compound designated Compound-A was found to inhibit infection by several CHIKV strains and a Sindbis virus strain at nanomolar concentrations. To investigate the inhibitory mechanism of action, a Compound-A resistant CHIKV (res-CHIKV) was isolated and a key mutation associated with resistance was identified by reverse-genetic recombinant CHIICVs containing amino acid substitutions present in res-CHIKV. These results demonstrated that the target site of Compound-A was the M2295 residue in the nonstructural protein 4 (nsP4), which is located in one of the functional domains of RNA-dependent RNA-polymerase (RdRp). We also confirmed that Compound-A inhibits RdRp function of CHIKV by using CHIKV replicons.
  • Shintaro Kobayashi, Kentaro Yoshii, Minato Hirano, Memi Muto, Hiroaki Kariwa
    JOURNAL OF VIROLOGICAL METHODS 240 14 - 20 2017年02月 [査読有り][通常論文]
     
    Reverse genetics systems facilitate investigation of many aspects of the life cycle and pathogenesis of viruses. However, genetic instability in Escherichia coli has hampered development of a reverse genetics system for West Nile virus (WNV). In this study, we developed a novel reverse genetics system for WNV based on homologous recombination in mammalian cells. Introduction of the DNA fragment coding for the WNV structural protein together with a DNA-based replicon resulted in the release of infectious WNV. The growth rate and plague size of the recombinant virus were almost identical to those of the parent WNV. Furthermore, chimeric WNV was produced by introducing the DNA fragment coding for the structural protein and replicon plasmid derived from various strains. Here, we report development of a novel system that will facilitate research into WNV infection. (C) 2016 Elsevier B.V. All rights reserved.
  • Wallaya Phongphaew, Shintaro Kobayashi, Michihito Sasaki, Michael Carr, William W. Hall, Yasuko Orba, Hirofumi Sawa
    VIRUS RESEARCH 228 114 - 123 2017年01月 [査読有り][通常論文]
     
    Valosin-containing protein (VCP) is classified as a member of the type II AAA(+) ATPase protein family. VCP functions in several cellular processes, including protein degradation, membrane fusion, vesicular trafficking and disassembly of stress granules. Moreover, VCP is considered to play a role in the replication of several viruses, albeit through different mechanisms. In the present study, we have investigated the role of VCP in West Nile virus (WNV) infection. Endogenous VCP expression was inhibited using either VCP inhibitors or by siRNA knockdown. It could be shown that the inhibition of endogenous VCP expression significantly inhibited WNV infection. The entry assay revealed that silencing of endogenous VCP caused a significant reduction in the expression levels of WNV-RNA compared to control siRNA-treated cells. This indicates that VCP may play a role in early steps either the binding or entry steps of the WNV life cycle. Using WNV virus like particles and WNV-DNA-based replicon, it could be demonstrated that perturbation of VCP expression decreased levels of newly synthesized WNV genomic RNA. These findings suggest that VCP is involved in early steps and during genome replication of the WNV life cycle. (C) 2016 Elsevier B.V. All rights reserved.
  • Michihito Sasaki, Gabriel Gonzalez, Yuji Wada, Agus Setiyono, Ekowati Handharyani, Ibenu Rahmadani, Siswatiana Taha, Sri Adiani, Munira Latief, Zainal Abidin Kholilullah, Mawar Subangkit, Shintaro Kobayashi, Ichiro Nakamura, Takashi Kimura, Yasuko Orba, Kimihito Ito, Hirofumi Sawa
    SCIENTIFIC REPORTS 6 24257  2016年04月 [査読有り][通常論文]
     
    Bufavirus is a recently recognized member of the genus Protoparvovirus in the subfamily Parvovirinae. It has been reported that human bufavirus was detected predominantly in patients with diarrhoea in several countries. However, little is known about bufavirus or its close relatives in nonhuman mammals. In this study, we performed nested-PCR screening and identified bufavirus from 12 megabats of Pteropus spp. in Indonesia. Furthermore, we determined nearly the full genome sequence of a novel megabat-borne bufavirus, tentatively named megabat bufavirus 1. Phylogenetic analyses showed that megabat bufavirus 1 clustered with known protoparvoviruses, including human bufavirus but represented a distinct lineage of bufavirus. Our analyses also inferred phylogenetic relationships among animal-borne bufaviruses recently reported by other studies. Recombination analyses suggested that the most common recent ancestor of megabat bufavirus 1 might have arisen from multiple genetic recombination events. These results characterized megabat bufavirus 1 as the first protoparvovirus discovered from megabats and indicates the high genetic divergence of bufavirus.
  • Shintaro Kobayashi, Tadaki Suzuki, Akira Kawaguchi, Wallaya Phongphaew, Kentaro Yoshii, Tomohiko Iwano, Akihiro Harada, Hiroaki Kariwa, Yasuko Orba, Hirofumi Sawa
    JOURNAL OF BIOLOGICAL CHEMISTRY 291 12 6559 - 6568 2016年03月 [査読有り][通常論文]
     
    West Nile virus (WNV) particles assemble at and bud into the endoplasmic reticulum (ER) and are secreted from infected cells through the secretory pathway. However, the host factor related to these steps is not fully understood. Rab proteins, belonging to the Ras superfamily, play essential roles in regulating many aspects of vesicular trafficking. In this study, we sought to determine which Rab proteins are involved in intracellular trafficking of nascent WNV particles. RNAi analysis revealed that Rab8b plays a role in WNV particle release. We found that Rab8 and WNV antigen were colocalized in WNV-infected human neuroblastoma cells, and that WNV infection enhanced Rab8 expression in the cells. In addition, the amount of WNV particles in the supernatant of Rab8b-deficient cells was significantly decreased compared with that of wild-type cells. We also demonstrated that WNV particles accumulated in the recycling endosomes in WNV-infected cells. In summary, these results suggest that Rab8b is involved in trafficking of WNV particles from recycling endosomes to the plasma membrane.
  • Eri Inagaki, Mizuki Sakai, Minato Hirano, Memi Muto, Shintaro Kobayashi, Hiroaki Kariwa, Kentaro Yoshii
    TICKS AND TICK-BORNE DISEASES 7 5 723 - 729 2016年 [査読有り][通常論文]
     
    Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. A wide range of animal species could be infected with TBEV in endemic areas. A serological survey of wild animals is effective in identifying TBEV-endemic areas. Safe, simple, and reliable TBEV serodiagnostic tools are needed to test animals. In this study, ELISA was developed to detect anti-TBEV specific antibodies in multi-species of animals, using recombinant subviral particles (SPs) with an affinity tag and protein A/G. A Strep-tag was fused at the N terminus of the E protein of the plasmid coding TBEV prME. The E proteins with Strep-tag were secreted as SPs, of which Strep-tag was exposed on the surface. The tagged E proteins were associated with prM. The SPs with Strep-tag were applied as the antigen of ELISA, and TBEV-specific antibodies were detected by the protein A/G. Compared to neutralization test results, the ELISA showed 96.8% sensitivity and 97.7% specificity in rodents and 95.1% sensitivity and 96.0% specificity in humans, without cross-reactivity with antibodies to Japanese encephalitis virus. These results indicate that our ELISA would be useful to detect TBE-specific antibodies in a wide range of animal species. (C) 2016 Elsevier GmbH. All rights reserved.
  • Paulina Duhita Anindita, Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Yasuko Orba, Shintaro Kobayashi, Ibnu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Ichiro Nakamura, Hirofumi Sawa, Takashi Kimura
    ARCHIVES OF VIROLOGY 160 4 1113 - 1118 2015年04月 [査読有り][通常論文]
     
    Bats have been shown to serve as natural reservoirs for numerous emerging viruses including severe acute respiratory syndrome coronavirus (SARS-CoV). In the present study, we report the discovery of bat CoV genes in Indonesian Moluccan naked-backed fruit bats (Dobsonia moluccensis). A partial RNA-dependent RNA polymerase gene sequence was detected in feces and tissues samples from the fruit bats, and the region between the RdRp and helicase genes could also be amplified from fecal samples. Phylogenetic analysis suggested that these bat CoVs are related to members of the genus Betacoronavirus.
  • Shintaro Kobayashi, Michihito Sasaki, Ryo Nakao, Agus Setiyono, Ekowati Handharyani, Yasuko Orba, Ibnu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Ichiro Nakamura, Takashi Kimura, Hirofumi Sawa
    ARCHIVES OF VIROLOGY 160 4 1075 - 1082 2015年04月 [査読有り][通常論文]
     
    Bats are an important natural reservoir for a variety of viral pathogens, including polyomaviruses (PyVs). The aims of this study were: (i) to determine which PyVs are present in bats in Indonesia and (ii) to analyze the evolutionary relationships between bat PyVs and other known PyVs. Using broad-spectrum polymerase chain reaction (PCR)-based assays, we screened PyV DNA isolated from spleen samples from 82 wild fruit bats captured in Indonesia. Fragments of the PyV genome were detected in 10 of the 82 spleen samples screened, and eight full-length viral genome sequences were obtained using an inverse PCR method. A phylogenetic analysis of eight whole viral genome sequences showed that BatPyVs form two distinct genetic clusters within the proposed genus Orthopolyomavirus that are genetically different from previously described BatPyVs. Interestingly, one group of BatPyVs is genetically related to the primate PyVs, including human PyV9 and trichodysplasia spinulosa-associated PyV. This study has identified the presence of novel PyVs in fruit bats in Indonesia and provides genetic information about these BatPyVs.
  • Shintaro Kobayashi, Yasuko Orba, Hiroki Yamaguchi, Kenta Takahashi, Michihito Sasaki, Rie Hasebe, Takashi Kimura, Hirofumi Sawa
    VIRUS RESEARCH 191 83 - 91 2014年10月 [査読有り][通常論文]
     
    Autophagy is a lysosomal degradation pathway that is implicated in many viral infections. However, its role in West Nile virus (WNV) infection remains controversial. In the present study, we examined the relationship between WNV infection and autophagy in infected cells. We demonstrated that LC3-II expression, a molecular marker for autophagosomal membranes, was enhanced in WNV-infected cells 6 h post-infection. LC3-II expression was further enhanced in WNV-inoculated cells when treated with a lysosomal protease inhibitor. Meanwhile, WNV replication in cells lacking Atg5, an essential factor for autophagy, was increased compared with replication in wild-type cells. In addition, WNV replication was inhibited in cells lacking Atg5 when they were transfected with an ATG5 expression plasmid. These results suggest an antiviral role for autophagy in WNV-infected cells. We also examined which viral replication stages were affected by autophagy by using a Tat-beclin 1 peptide to induce autophagy and pseudo-infectious WNV reporter virus particles (WNV-RVPs) that monitor viral genome replication and gene expression stages via GFP expression. We found that autophagy induction in HeLa cells by Tat-beclin 1 peptide 3 h after WNV inoculation inhibited viral replication, and GFP expression was significantly inhibited in wild-type cells when compared with cells lacking Atg5. Taken together, these results suggest that autophagy is induced by WNV infection, and that this induction inhibits WNV replication at the viral genome replication and gene expression stages. (C) 2014 Elsevier B.V. All rights reserved.
  • Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Shintaro Kobayashi, Ibenu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Ichiro Nakamura, Hirofumi Sawa, Takashi Kimura
    JOURNAL OF VIROLOGY 88 17 9819 - 9829 2014年09月 [査読有り][通常論文]
     
    Bats are known to harbor emerging RNA viruses. Recent studies have used high-throughput sequencing technology to identify various virus species, including DNA viruses that are harbored by bats; however, little is known about the nature of these potentially novel viruses. Here, we report the characterization of a novel herpesvirus isolated from an Indonesian pteropodid bat. The virus, tentatively named fruit bat alphaherpesvirus 1 (FBAHV1), has a double-stranded DNA genome of 149,459 bp. The phylogenetic analyses suggested that FBAHV1 is phylogenetically grouped with simplexviruses within the subfamily Alphaherpesvirinae. Inoculation of FBAHV1 into laboratory mice caused a lethal infection. Virus infection was observed in lung, liver, and brain tissue. Serological and PCR screening revealed that fruit bats infected with FBAHV1 or its related virus are widely distributed in Indonesia. The identification of FBAHV1 makes a considerable contribution to our understanding of simplexviruses associated with bats. IMPORTANCE Bats are known to harbor emerging viruses, such as lyssaviruses, henipaviruses, severe acute respiratory syndrome-like coronaviruses, and filoviruses. Although alphaherpesviruses are disseminated in humans and other animals, there is little information about their distribution in bats. Here, we isolated a previously unknown alphaherpesvirus from an Indonesian fruit bat. Genome sequence analysis suggested that the virus is a member of the genus Simplexvirus within the subfamily Alphaherpesvirinae, which also includes common human viruses, such as herpes simplex virus 1 and herpes simplex virus 2. FBAHV1 is the first batderived alphaherpesvirus whose complete genome has been sequenced.
  • Hiroki Yamaguchi, Shintaro Kobayashi, Junki Maruyama, Michihito Sasaki, Ayato Takada, Takashi Kimura, Hirofumi Sawa, Yasuko Orba
    JOURNAL OF VETERINARY MEDICAL SCIENCE 76 5 637 - 644 2014年05月 [査読有り][通常論文]
     
    Recently, we detected novel vervet monkey polyomavirus 1 (VmPyV) in a vervet monkey. Among amino acid sequences of major capsid protein VP1s of other polyomaviruses, VmPyV VP1 is the longest with additional amino acid residues in the C-terminal region. To examine the role of VmPyV VP1 in virion formation, we generated virus-like particles (VLPs) of VmPyV VP1, because VLP is a useful tool for the investigation of the morphological characters of polyomavirus virions. After the full-length VmPyV VP1 was subcloned into a mammalian expression plasmid, the plasmid was transfected into human embryonic kidney 293T (HEK293T) cells. Thereafter, VmPyV VLPs were purified from the cell lysates of the transfected cells via sucrose gradient sedimentation. Electron microscopic analyses revealed that VmPyV VP1 forms VLPs with a diameter of approximately 50 nm that are exclusively localized in cell nuclei. Furthermore, we generated VLPs consisting of the deletion mutant VmPyV VP1 (Delta C VP1) lacking the C-terminal 116 amino acid residues and compared its VLP formation efficiency and morphology to those of VLPs from wild-type VmPyV VP1 (WT VP1). WT and Delta C VP1 VLPs were similar in size, but the number of Delta C VP1 VLPs was much lower than that of WT VP1 VLPs in VP1-expressing HEK293T cells. These results suggest that the length of VP1 is unrelated to virion morphology; however, the C-terminal region of VmPyV VP1 affects the efficiency of its VLP formation.
  • Shintaro Kobayashi, Tadaki Suzuki, Manabu Igarashi, Yasuko Orba, Noriko Ohtake, Keita Nagakawa, Kenichi Niikura, Takashi Kimura, Harumi Kasamatsu, Hirofumi Sawa
    PLoS ONE 8 10 e76668  2013年10月09日 [査読有り][通常論文]
     
    The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.
  • Ichiro Nakamura, Bernard Mudenda Hang'ombe, Hirofumi Sawa, Shintaro Kobayashi, Yasuko Orba, Akihiro Ishii, Yuka Thomas, Rie Isozumi, Kumiko Yoshimatsu, Aaron S. Mweene, Ayato Takada, Chihiro Sugimoto, Jiro Arikawa
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 6 819 - 825 2013年06月 [査読有り][通常論文]
     
    A total of 466 rodents were captured in the Republic of Zambia from 2006 to 2010. Based on morphological observations and phylogenetic analyses of mitochondrial gene sequences, rodents were divided into 10 groups consisting of 39 Rattus rodents, 263 multimammate rats, 18 other Murinae rodents, 95 gerbils, 11 pouched mice, 1 giant-pouched rat, 38 fat mice and 1 dormouse. Rodent antibodies except that from Rattus were examined for their cross-reactivity to commercially available antibody detection reagents. Anti-mouse immunoglobulin G (IgG) was most cross-reactive to heterologous antibodies including multimammate rat, gerbil, pouched mouse and fat mouse. Thus, anti-mouse IgG would be a useful detection tool in serological examination of the Zambian rodent population. Preliminary sero-surveillance for plague, leptospirosis and hantavirus infection was performed by ELISA.
  • Hiroki Yamaguchi, Shintaro Kobayashi, Akihiro Ishii, Hirohito Ogawa, Ichiro Nakamura, Ladslav Moonga, Bernard M. Hang'ombe, Aaron S. Mweene, Yuka Thomas, Takashi Kimura, Hirofumi Sawa, Yasuko Orba
    Journal of General Virology 94 6 1357 - 1364 2013年06月01日 [査読有り][通常論文]
     
    To examine polyomavirus (PyV) infection in wildlife, we investigated the presence of PyVs in Zambia with permission from the Zambia Wildlife Authority. We analysed 200 DNA samples from the spleens and kidneys (n5100 each) of yellow baboons and vervet monkeys (VMs) (n550 each). We detected seven PyV genome fragments in 200 DNA samples using a nested broadspectrum PCR method, and identified five full-length viral genomes using an inverse PCR method. Phylogenetic analysis of virally encoded proteins revealed that four PyVs were closely related to either African green monkey PyV or simian agent 12. Only one virus detected from a VM spleen was found to be related, with relatively low nucleotide sequence identity (74 %), to the chimpanzee PyV, which shares 48% nucleotide sequence identity with the human Merkel cell PyV identified from Merkel cell carcinoma. The obtained entire genome of this virus was 5157 bp and had large T- and small t-antigens, and VP1 and VP2 ORFs. This virus was tentatively named vervet monkey PyV 1 (VmPyV1) as a novel PyV. Comparison with other PyVs revealed that VmPyV1, like chimpanzee PyV, had a longer VP1 ORF. To examine whether the VmPyV1 genome could produce viral proteins in cultured cells, the whole genome was transfected into HEK293T cells. We detected VP1 protein expression in the transfected HEK293T cells by immunocytochemical and immunoblot analyses. Thus, we identified a novel PyV genome from VM spleen. © 2013 SGM.
  • Kenichi Niikura, Tatsuya Matsunaga, Tadaki Suzuki, Shintaro Kobayashi, Hiroki Yamaguchi, Yasuko Orba, Akira Kawaguchi, Hideki Hasegawa, Kiichi Kajino, Takafumi Ninomiya, Kuniharu Ijiro, Hirofumi Sawa
    ACS NANO 7 5 3926 - 3938 2013年05月 [査読有り][通常論文]
     
    This paper demonstrates how the shape and size of gold nanoparticles (AuNPs) affect immunological responses in vivo and in vitro for the production of antibodies for West Nile virus (WNV). We prepared spherical (20 and 40 nm in diameter), rod (40 x 10 nm), and cubic (40 x 40 x 40 nm) AuNPs as adjuvants and coated them with WNV envelope (E) protein. We measured anti-WNVE antibodies after inoculation of these WNVE-coated AuNPs (AuNP-Es) into mice. The 40 nm spherical AuNP-Es (Sphere40-Es) induced the highest level of WNVE-specific antibodies, while rod AuNP-Es (Rod-Es) induced only 50% of that of Sphere40-E. To examine the mechanisms of the shape-dependent WNVE antibody production, we next measured the efficiency of cellular uptake of AuNP-Es into RAW264.7 macrophage cells and bone-marrow-derived dendritic cells (BMDCs) and the subsequent cytokine secretion from BMDCs. The uptake of Rod-Es into the cells proceeded more efficiently than those of Sphere-Es or cubic WNVE-coated AuNPs (Cube-Es), suggesting that antibody production was not dependent on the uptake efficiency of the different AuNP-Es. Cytokine production from BMDCs treated with the AuNP-Es revealed that only Rod-E-treated cells produced significant levels of interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18), indicating that Rod-Es activated inflammasome-dependent cytokine secretion. Meanwhile, Sphere40-Es and Cube-Es both significantly induced inflammatory cytokine production, including tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-12, and granulocyte macrophage colony-stimulating factor (GM-CSF). These results suggested that AuNPs are effective vaccine adjuvants and enhance the immune response via different cytokine pathways depending on their sizes and shapes.
  • Kenichi Niikura, Naotoshi Sugimura, Yusuke Musashi, Shintaro Mikuni, Yasutaka Matsuo, Shintaro Kobayashi, Keita Nagakawa, Shuko Takahara, Chie Takeuchi, Hirofumi Sawa, Masataka Kinjo, Kuniharu Ijiro
    Molecular BioSystems 9 3 501 - 507 2013年03月 [査読有り][通常論文]
     
    The efficient delivery of hydrophobic drugs into target cells without the use of organic solvents or chemical linkage to delivery carriers is an important theme in the biomedical and pharmaceutical field. In this study, we synthesized virus-like particles (VLPs) coupled with cyclodextrins (CDs) as hydrophobic pockets through disulfide bonds inside the VLPs, where hydrophobic drugs can be incorporated. We report here the intracellular delivery of hydrophobic dyes or drugs encapsulated in VLPs through CDs with high efficiency and their subsequent release in cells in response to glutathione. As a model anticancer drug, paclitaxel (PTX)-CD complexes were encapsulated inside VLPs and the cytotoxic drug activity of PTX loaded VLPs against NIH3T3 cells was evaluated by CCK-8 assay. PTX-loaded VLPs exhibited a dose-dependent cytotoxic effect with a 20-fold smaller IC50 than that of free PTX dissolved in DMSO. These results indicate that VLPs with removable CDs afford highly promising carriers of hydrophobic drugs without chemical modification of drugs. © 2013 The Royal Society of Chemistry.
  • Tadaki Suzuki, Shingo Semba, Yuji Sunden, Yasuko Orba, Shintaro Kobayashi, Kazuo Nagashima, Takashi Kimura, Hideki Hasegawa, Hirofumi Sawa
    MICROBIOLOGY AND IMMUNOLOGY 56 9 639 - 646 2012年09月 [査読有り][通常論文]
     
    JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T' antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein-negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus-like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S-transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1-pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.
  • Shintaro Kobayashi, Yasuko Orba, Hiroki Yamaguchi, Takashi Kimura, Hirofumi Sawa
    NEUROPATHOLOGY 32 4 398 - 405 2012年08月 [査読有り][通常論文]
     
    West Nile virus (WNV) belongs to the Flaviviridae family of viruses and has emerged as a significant cause of viral encephalitis in humans, animals and birds. It has been reported that WNV replication directly induces neuronal injury, followed by neuronal cell death proven as apoptosis. Therefore, it is important to understand the mechanism of neuronal apoptosis caused by this virus to develop strategies to control its pathogenicity. Accumulation of ubiquitinated abnormal proteins has been reported to be associated with neuronal apoptosis in some pathological conditions. A lot of cellular stresses prevent cellular protein quality control mechanisms, resulting in the accumulation of ubiquitinated abnormal proteins. To obtain a better understanding of the mechanisms of WNV-induced neuronal apoptosis, we evaluated the accumulation of ubiquitinated proteins in the WNV-infected neuronal cells. We have observed that WNV infection caused massive neuronal injury in the brains of mice. Viral antigen was detected in the neuronal cytoplasm of the cells exhibiting neuronal apoptosis. Notably, ubiquitinated proteins were detected in WNV-infected neuronal cells. In addition, accumulation of ubiquitinated proteins was markedly enhanced in mouse neuroblastoma, Neuro-2a cells after WNV infection. Our histopathological and in vitro studies suggest that accumulation of ubiquitinated proteins in neuronal cells might be associated with neuronal apoptosis caused by WNV infection.
  • Nilton Akio Muto, Reiko Yoshida, Tadaki Suzuki, Shintaro Kobayashi, Hiroichi Ozaki, Daisuke Fujikura, Rashid Manzoor, Mieko Muramatsu, Ayato Takada, Takashi Kimura, Hirofumi Sawa
    JAPANESE JOURNAL OF VETERINARY RESEARCH 60 2-3 71 - 83 2012年08月 [査読有り][通常論文]
     
    New approaches to the treatment of influenza have been designed based on the highly conserved antigenicity of the M2 envelope protein among influenza A virus strains. The present study examined the anti-viral activities of an anti-M2 ectodomain (M2e) monoclonal antibody (clone rM2ss23), which binds to the M2 proteins of the influenza A virus strains A/Aichi/2/68 (H3N2) (Aichi) and A/PR/8/34 (H1N1) (PR8). The results showed that rM2ss23 bound to both Aichi and PR8 M2 proteins expressed on the cell surface. While the antibody did not prevent virus entry into cells, it significantly inhibited plaque formation by the Aichi strain in a dose-dependent manner when infected cells were cultured in the presence of the antibody. By contrast, the growth of PR8 (H1N1) was not affected by the antibody. A reverse genetics approach revealed that the inhibitory effect of rM2ss23 on the Aichi virus was abolished by replacing the genes encoding the HA and/or M proteins with those of the PR8 strain. These results suggest that rM2ss23 prevents virus release from infected cells and further suggest that the mechanisms underlying the virus budding mediates by HA and M2 proteins might differ between the Aichi and PR8 strains.
  • Yasuko Orba, Shintaro Kobayashi, Ichiro Nakamura, Akihiro Ishii, Bernard M. Hang'ombe, Aaron S. Mweene, Yuka Thomas, Takashi Kimura, Hirofumi Sawa
    JOURNAL OF GENERAL VIROLOGY 92 Pt 4 789 - 795 2011年04月 [査読有り][通常論文]
     
    To investigate polyomavirus infection in wild rodents, we analysed DNA samples from the spleens of 100 wild rodents from Zambia using a broad-spectrum PCR-based assay. A previously unknown polyomavirus genome was identified in a sample from a multimammate mouse (Mastomys species) and the entire viral genome of 4899 bp was :subsequently sequenced. This viral genome contained potential ORFs for the capsid proteins, VP1, VP2 and VP3, and early proteins, small t antigen and large T antigen. Phylogenetic analysis showed that it was a novel member of the family Polyomaviridae, and thus the virus was tentatively named mastomys polyomavirus. After transfection of the viral genome into several mammalian cell lines, transient expression of the VP1 and large T antigen proteins was confirmed by immunoblotting and immunocytochemical analyses. Comparison of large T antigen function in mastomys polyomavirus with that in rhesus monkey polyomavirus SV40 and human polyomavirus JC virus revealed that the large T antigen from mastomys polyomavirus interacted with the tumour suppressor protein pRb, but not with p53.
  • Kazuya Matsuda, Shintaro Kobayashi, Ken-ichiro Kameyama, Michiko Sato, Masateru Koiwa, Yoshihiro Sakoda, Hiroyuki Taniyama
    JOURNAL OF VETERINARY MEDICAL SCIENCE 72 7 903 - 907 2010年07月 [査読有り][通常論文]
     
    Bovine viral diarrhea virus (BVDV) causes fetal brain malformations in ruminants when the fetuses are infected transplacentally in mid-pregnancy. In both cytopathic and non-cytopatic virus infections, viral lytic infection in actively replicating cells and interruption of vascular integrity have been suggested as the pathogenesis, but functional disturbance of infected neural developing cells has been unclear. In this study, we examined the effect of infection with non-cytopathic BVDV2 on the differentiation of neural stem/precursor cells isolated from the bovine fetus. In the process of differentiation to three types of neural cells, neurons, astrocytes and oligodendrocytes, virus infection significantly and selectively inhibited the differentiation of neural stem/precursor cells into the astrocytic lineage. This inhibition is possibly important for the pathogenesis of congenital brain malformations associated with non-cytopathic BVDV infection.
  • Kazuya Matsuda, Kanako Sakaguchi, Shintaro Kobayashi, Makiko Tominaga, Kazuko Hirayama, Tsuyoshi Kadosawa, Hiroyuki Taniyama
    JOURNAL OF VETERINARY MEDICAL SCIENCE 71 2 229 - 232 2009年02月 [査読有り][通常論文]
     
    A 5-year-old female miniature dachshund presenting with persistent vomiting and diarrhea had two concurrent rare pathological conditions: systemic candidiasis and mesenteric mast cell tumor with multiorgan metastases. Neoplastic mast cells formed mass in the mesentery of the cecal-colonic region and were also found in the liver, spleen, kidneys, lungs, adrenal grands, ovaries, bone marrow and other tissues. The cells had intracytoplasmic granules With metachromasia and were immunohistochemically positive for c-kit and histamine. Granulomatous lesions with fungal organisms were present in the heart, lungs, kidneys, pancreas, subserosal and surrounding adipose tissue of the duodenum, thyroid glands and mesenteric mass, and phagocytosed organisms were detected in the liver and bone marrow. Bacteriologically and immunohistochemically, the fungi were consistent with Candida albicans.
  • Kazuya Matsuda, Shintaro Kobayashi, Misato Yamashita, Kazuko Hirayama, Tsuyoshi Kadosawa, Hiroyuki Taniyama
    JOURNAL OF VETERINARY MEDICAL SCIENCE 70 5 479 - 481 2008年05月 [査読有り][通常論文]
     
    Sarcomatous proliferation of spindle cells was present in the mammary gland and many metastatic sites in a 10-year-old female domestic cat with tubulopapillary carcinoma in the mammary gland. Transition from neoplastic tubular structures to spindle cells in the primary site and fascicular proliferation of the spindle cells with or without coexistance of tubulopapillary carcinoma in the primary and metastatic sites were observed. Most of spindle cells were positive for cytokeratin CAM5.2 as well as the normal luminal epithelium but not the myoepithelium. From these results, this case was diagnosed as tubulopapillary carcinoma with spindle cell metaplasia and it was clarified that neoplastic luminal epithelial cells can transform to sarcomatous appearence.
  • 秋田犬における悪性毛包上皮種の全身転移の1例
    下山由美子, 秋原祐子, 小林進太郎, 平山和子, 松田一哉, 岡本実, 廉澤剛, 谷山弘行
    北海道獣医師会雑誌 50 8 342  2006年08月 [査読有り][通常論文]

講演・口頭発表等

  • ウエストナイルウイルス感染によるオートファジーの抑制と病態形成への影響  [招待講演]
    小林進太郎
    大阪大学微生物病研究所セミナー 2019年05月 公開講演,セミナー,チュートリアル,講習,講義等
  • ウエストナイルウイルス感染の病態形成におけるオートファジーの役割  [招待講演]
    小林進太郎, 好井健太朗, 澤洋文, 苅和宏明
    2019年チェコスロバキア合同ウイルス学会議 2019年02月 口頭発表(招待・特別)
  • フラビウイルス感染症における脳炎発症機序の解明と新規診断法・予防法開発への応用  [招待講演]
    小林進太郎
    第160回日本獣医学会学術集会 2017年09月 その他
  • ウエストナイルウイルスとオートファジー  [招待講演]
    小林進太郎
    第160回日本獣医学会学術集会 2017年09月 シンポジウム・ワークショップパネル(指名)

その他活動・業績

  • ウエストナイルウイルスのカプシドタンパク質とAMPKの相互作用の解析
    鈴木 健矢, 小林 進太郎, 好井 健太朗, 苅和 宏明 日本獣医学会学術集会講演要旨集 162回 420 -420 2019年08月 [査読無し][通常論文]
  • 2017、2018年に北海道道央地域のヤマトマダニから分離されたダニ媒介性脳炎ウイルスの性状解析
    高橋 侑嗣, 小林 進太郎, 石塚 万里子, 中尾 亮, 苅和 宏明, 好井 健太朗 日本獣医学会学術集会講演要旨集 162回 421 -421 2019年08月 [査読無し][通常論文]
  • ダニ媒介性脳炎ウイルス由来非コードRNAと相互作用するマダニ宿主蛋白質の同定及び機能解析
    西山 祥子, 平野 港, 武藤 芽未, 小林 進太郎, 神谷 亘, 苅和 宏明, 好井 健太朗 日本獣医学会学術集会講演要旨集 162回 421 -421 2019年08月 [査読無し][通常論文]
  • エンベロープタンパク質のアミノ酸変異によるウエストナイルウイルスの増殖および病態形成への影響
    小林 進太郎, 金子 知里, 川上 怜子, 長谷部 理絵, 澤 洋文, 好井 健太朗, 苅和 宏明 日本獣医学会学術集会講演要旨集 162回 431 -431 2019年08月 [査読無し][通常論文]
  • ダニ媒介性脳炎ウイルスのウイルス様粒子を用いた新規IgM-ELISA系の開発
    中安 美樹, 小林 進太郎, 平野 港, 武藤 芽未, 苅和 宏明, 好井 健太朗 日本獣医学会学術集会講演要旨集 161回 384 -384 2018年08月 [査読無し][通常論文]
  • 2017年に北海道道央地域で分離されたダニ媒介性脳炎ウイルスの性状解析
    高橋 侑嗣, 石塚 万里子, 平野 港, 武藤 芽未, 西山 祥子, 小林 進太郎, 苅和 宏明, 好井 健太朗 日本獣医学会学術集会講演要旨集 161回 384 -384 2018年08月 [査読無し][通常論文]
  • ダニ媒介性脳炎ウイルス由来非コードRNAの感染時における機能解析
    西山 祥子, 平野 港, 武藤 芽未, 神原 真生, 小林 進太郎, 苅和 宏明, 好井 健太朗 日本獣医学会学術集会講演要旨集 161回 384 -384 2018年08月 [査読無し][通常論文]
  • ユビキチンの蓄積に着目したウエストナイルウイルスの病原性解析
    川上 怜子, 小林 進太郎, 好井 健太朗, 苅和 宏明 日本獣医学会学術集会講演要旨集 161回 384 -384 2018年08月 [査読無し][通常論文]
  • 北海道のアライグマを対象としたダニ媒介性脳炎ウイルスの血清疫学調査
    佐鹿 万里子, 石塚 万里子, 三瓶 孝男, 小林 進太郎, 西山 祥子, 苅和 宏明, 好井 健太朗 日本獣医学会学術集会講演要旨集 161回 394 -394 2018年08月 [査読無し][通常論文]
  • ウエストナイルウイルス感染で起こるオートファジーの抑制と神経病態形成の関係
    小林 進太郎, 好井 健太朗, Wallaya Phongpaew, 武藤 芽未, 平野 港, 大場 靖子, 澤 洋文, 苅和 宏明 日本獣医学会学術集会講演要旨集 161回 395 -395 2018年08月 [査読無し][通常論文]
  • 中釜 尚人, 平野 港, 武藤 芽未, 西山 祥子, 中尾 亮, 小林 進太郎, 苅和 宏明, 好井 健太朗 日本獣医学会学術集会講演要旨集 161回 383 -383 2018年08月 [査読無し][通常論文]
  • 小林 進太郎, 好井 健太朗 小児科臨床 70 (増刊) 2251 -2255 2017年12月 [査読無し][通常論文]
  • ウエストナイルウイルスのカプシドタンパク質によるオートファジーの抑制による変性タンパク質の蓄積と神経病態形成についての解析
    小林 進太郎, 好井 健太朗, Phonqphaew Wallaya, 武藤 芽未, 平野 港, 大場 靖子, 澤 洋文, 苅和 宏明 生命科学系学会合同年次大会 2017年度 [3P -1110] 2017年12月 [査読無し][通常論文]
  • ウエストナイルウイルスの脳内侵入におけるエンベロープタンパク質のアミノ酸の解析
    金子 知里, 小林 進太郎, 好井 健太朗, 苅和 宏明 日本獣医学会学術集会講演要旨集 160回 411 -411 2017年08月 [査読無し][通常論文]
  • ダニ媒介性ウイルス感染におけるダニRNAi関連因子の機能解析
    神原 真生, 平野 港, 武藤 芽未, 石塚 万里子, 小林 進太郎, 苅和 宏明, 好井 健太朗 日本獣医学会学術集会講演要旨集 160回 411 -411 2017年08月 [査読無し][通常論文]
  • ダニ媒介性脳炎ウイルスの3'非翻訳領域に関わる宿主因子の検索
    武藤 芽未, 神谷 亘, 境 瑞紀, 平野 港, 小林 進太郎, 苅和 宏明, 好井 健太朗 日本獣医学会学術集会講演要旨集 160回 411 -411 2017年08月 [査読無し][通常論文]
  • 小林 進太郎, 好井 健太朗 化学療法の領域 33 (8) 1645 -1651 2017年07月 [査読無し][通常論文]
     
    ウエストナイル熱・ウエストナイル脳炎はウエストナイルウイルスを保有した蚊の吸血によって起こる人獣共通感染症である。その病態は,急性熱性疾患であるウエストナイル熱と,脳脊髄炎などの中枢神経系の症状を示すウエストナイル脳炎に分けられる。ウイルスは自然界では蚊とトリのあいだで感染環が形成されており,世界中で流行が確認されている。わが国ではこれまでにウエストナイルウイルスの流行は確認されていないが,流行のための媒介動物である蚊や増幅動物である野鳥が存在していることから注意が必要である。本稿ではウエストナイル熱・ウエストナイル脳炎の一般的な特徴や,米国を中心とした本病の流行について概説し,わが国における対策の必要性とともに述べる。(著者抄録)
  • Hokkaidoウイルスと他のハンタウイルスの共感染による増殖性の補完に関する研究
    梶河 紗代, 岩崎 里奈, 真田 崇弘, 小林 進太郎, 好井 健太朗, 苅和 宏明 日本獣医学会学術集会講演要旨集 159回 408 -408 2016年08月 [査読無し][通常論文]
  • フラビウイルスゲノムの神経細胞内輸送機構の解析
    平野 港, 境 瑞紀, 武藤 芽未, 小林 進太郎, 苅和 宏明, 好井 健太朗 日本獣医学会学術集会講演要旨集 159回 409 -409 2016年08月 [査読無し][通常論文]
  • ウエストナイルウイルスの粒子放出過程におけるRab8bタンパク質の役割
    小林 進太郎, 鈴木 忠樹, 川口 晶, Phongphaew Wallaya, 好井 健太朗, 苅和 宏明, 大場 靖子, 澤 洋文 日本獣医学会学術集会講演要旨集 159回 409 -409 2016年08月 [査読無し][通常論文]
  • In trans補完系によるダニ媒介性脳炎ウイルスのゲノム複製機構の解析
    山内 沙也果, 小林 進太郎, 平野 港, 武藤 芽未, 石塚 万里子, 苅和 宏明, 好井 健太朗 日本獣医学会学術集会講演要旨集 159回 409 -409 2016年08月 [査読無し][通常論文]
  • ダニ媒介性脳炎ウイルスのNucleoside Inhibitor耐性変異の解析
    近藤 寛史, 平野 港, 石塚 万里子, 武藤 芽未, 小林 進太郎, 苅和 宏明, Daniel Ruzek, 好井 健太朗 日本獣医学会学術集会講演要旨集 159回 423 -423 2016年08月 [査読無し][通常論文]
  • ダニ媒介性脳炎ウイルスの神経細胞内におけるウイルスゲノムRNA輸送機構の解析
    平野 港, 境 瑞紀, 苅和 宏明, 小林 進太郎, 好井 健太朗 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [1P1134] -[1P1134] 2015年12月 [査読無し][通常論文]
  • ウエストナイルウイルス感染による変性タンパク質蓄積機構の解析
    小林 進太郎, Phongphaew Wallaya, 好井 健太朗, 平野 港, 武藤 芽未, 大場 靖子, 澤 洋文, 苅和 宏明 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [1P1135] -[1P1135] 2015年12月 [査読無し][通常論文]
  • 澤 洋文, 小林 進太郎, 鈴木 忠樹, 大場 靖子 ウイルス 64 (1) 25 -34 2014年06月 [査読無し][通常論文]
     
    ポリオーマウイルスはポリオーマウイルス科に属する哺乳類動物由来のOrthopolyomavirusとWukipolyomavirus、及び鳥類由来のAvipolyomavirusに分類されている。我々は疫学研究を通じて新規のポリオーマウイルスであるMastomys Polyoamvirus(MasPyV)及びVervet monkey Polyoamvirus-1(VmPyV-1)を単離し、そのゲノムがコードするウイルスタンパク質の機能解析を実施した。更に、ヒトポリオーマウイルスであるJC polyomavirus(JCPyV)についての基礎研究を推進し、最近、早期タンパク質であるLarge T抗原の感染細胞における機能解析、後期タンパク質であるVP1のシステイン残基の粒子形成への影響、及び後期タンパク質であるAgnoのウイルス粒子の細胞外放出機構に対する影響について知見を得たのでその内容を紹介する。(著者抄録)
  • Tatsuya Matsunaga, Kenichi Niikura, Tadaki Suzuki, Shintaro Kobayashi, Hiroki Yamaguchi, Hirofumi Sawa, Kuniharu Ijiro ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 243 2012年03月 [査読無し][通常論文]
  • 大場靖子, 小林進太郎, 中村一郎, 石井秋宏, 木村享史, 澤洋文 日本ウイルス学会学術集会プログラム・抄録集 58th 445 2010年10月15日 [査読無し][通常論文]
  • Shintaro Kobayashi, Tadaki Suzuki, Manabu Igarashi, Noriko Ohtake, Keita Nagakawa, Kimihito Ito, Kenichi Niikura, Takashi Kimura, Harumi Kasamatsu, Hirofumi Sawa JOURNAL OF NEUROVIROLOGY 16 45 -46 2010年10月 [査読無し][通常論文]

特許

受賞

  • 2017年09月 日本獣医学会 獣医学奨励賞
     
    受賞者: 小林進太郎
  • 2013年04月 日本神経病理学会 日本神経病理学会賞(実験病理学的研究)
     
    受賞者: 小林進太郎

共同研究・競争的資金等の研究課題

  • ミクログリアの活性化に着目したフラビウイルス性脳炎の発症機構の解明
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 小林 進太郎
  • ウイルス由来ノンコーディングRNAによるフラビウイルス感染制御メカニズムの解明
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 好井 健太朗, 田仲 哲也, 小林 進太郎
     
    本研究においては、ダニ媒介性フラビウイルスであるダニ媒介性脳炎ウイルス(TBEV)について、これまでの研究成果で推定された、ウイルス遺伝子RNA由来長鎖ノンコーディングRNA(lncRNA)の産生に関わると推定される領域に変異を導入したウイルスを作製し、その性状を解析した。ウイルスを感染させ、細胞内におけるウイルス由来lncRNAの産生状況について解析した所、哺乳動物細胞と節足動物由来培養細胞で産生されるlncRNAのパターンに相違があることが明らかになった。また、哺乳動物細胞においては変異による増殖性の相違は認められなかったが、節足動物細胞においては増殖の遅延が認められ、特定のウイルス由来lncRNAが、節足動物における効率の良い増殖に関与している可能性が示唆された。 またマウスモデルにウイルスを感染させ、病原性を検討した所、変異により接種時の病原性の上昇が認められた。さらにレポーターアッセイにより、ウイルス感染細胞中のインターフェロン応答を解析した所、ウイルス感染によりインターフェロンプロモーター活性の抑制が認められたが、変異によりこの抑制が増強されることが示された。これらの事よりウイルス由来lncRNAはマウス体内において、自然免疫応答の制御機構があり、これが病態発現につながっている可能性が示唆される。 以上の成果から、ウイルス由来lncRNAは感染宿主の種に応じて異なる機能を持っており、これらが自然界におけるウイルス伝播や、病態発現に関与していると考えられるため、今後詳細に解析していく必要があると考えられる。
  • フラビウイルス性脳炎の病態形成におけるタンパク質品質管理機構の関与についての解析
    文部科学省:科学研究費補助金(若手研究)
    研究期間 : 2018年04月 -2020年03月 
    代表者 : 小林進太郎
  • ウイルス性神経疾患でのオートファジーの機能解析
    北海道大学:平成30年度若手研究者研究加速事業
    研究期間 : 2018年11月 -2019年03月 
    代表者 : 小林進太郎
  • リサイクリングエンドソームに着目したウイルス感染時の病態形成機構の解析
    公益財団法人 武田科学振興財団:医学系研究奨励
    研究期間 : 2017年11月 -2019年03月 
    代表者 : 小林進太郎
  • 神経向性ウイルスの脳内侵入機構の解明とその応用
    ノーステック財団:若手研究人材・ネットワーク育成補助金(ノースタレント補助金)
    研究期間 : 2017年08月 -2018年03月 
    代表者 : 小林進太郎
  • 神経向性フラビウイルスの脳内侵入メカニズムの解明
    秋山記念生命科学振興財団:研究助成(奨励)
    研究期間 : 2016年 -2017年 
    代表者 : 小林進太郎
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2015年 -2016年 
    代表者 : 小林 進太郎
  • ウエストナイルウイルス感染における宿主因子応答の解析
    北海道大学:グローバルCOEプログラム若手研究者研究活動経費
    研究期間 : 2012年05月 -2013年03月 
    代表者 : 小林進太郎
  • 文部科学省:科学研究費補助金(研究活動スタート支援)
    研究期間 : 2012年 -2013年 
    代表者 : 小林 進太郎
     
    神経細胞に病原性を有するウエストナイルウイルスは細胞内タンパク質分解系の一つであるオートファジーを抑制する。オートファジーの低下は神経細胞死を誘導することが知られており、ウエストナイルウイルス感染によるオートファジー抑制機構を解明することを目的としている。ウエストナイルウイルスゲノムがコードする10個の遺伝子について、それぞれの塩基配列を哺乳類細胞発現ベクターにクローニングした。構築したウイルスタンパク質発現プラスミドを神経系由来細胞に導入し、オートファジーマーカーであるLC3-IIの発現をイムノブロットおよび、免疫細胞化学染色で評価している。また、変異体ウエストナイルウイルスを作出するために、米国ペンシルバニア大学のDoms教授から供与して頂いた956株のウイルスゲノムをコードしているプラスミドを鋳型として、NY99 6-LP株のウイルスゲノムに組換えたプラスミドを作製している。本年度はオートファジーに必須な因子であるATG5を欠損した細胞を用いて、オートファジーがウエストナイルウイルスの増殖に与える影響を調べた。その結果、オートファジーはウイルス感染後3-6時間で誘導され、ウイルスゲノムの複製および遺伝子発現を抑制することを見出した。これらの結果をまとめて英文雑誌Virus Researchに投稿し、現在revise中である。さらにオートファジーとウエストナイルウイルスの増殖性および神経病原性の関係を明らかにするために、理化学研究所より神経細胞特異的ATG5ノックアウトマウスを購入し、飼育・繁殖中である。必要な数のマウスを得られ次第、感染実験を実施する予定である。
  • ウエストナイルウイルスの神経病原性発現機構の解析
    北海道大学:グローバルCOEプログラム若手研究者研究活動経費
    研究期間 : 2011年05月 -2012年03月 
    代表者 : 小林進太郎
  • ポリオーマウイルスの粒子形成機構の解析
    北海道大学:グローバルCOEプログラム若手研究者研究活動経費
    研究期間 : 2010年12月 -2011年03月 
    代表者 : 小林進太郎

教育活動情報

主要な担当授業

  • 獣医科学・感染症学基礎科目 獣医公衆衛生学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医科学基礎科目 獣医公衆衛生学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院


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