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Kobayashi Shintaro

Faculty of Veterinary Medicine　Veterinary Medicine　Preventive Veterinary MedicineAssociate Professor
International Institute for Zoonosis ControlAssociate Professor
Institute for Integrated Innovations　Institute for Vaccine Research and DevelopmentAssociate Professor
One Health Research CenterAssociate Professor

Researcher basic information

■ Degree
  • 獣医学博士, 北海道大学
■ URL
researchmap URLホームページURL■ Various IDs
Researcher number
  • 00634205
J-Global ID■ Research Keywords and Fields
Research Keyword
  • zoonosis
  • molecular biology
  • virus
  • pathology
  • veterinary medicine
Research Field
  • Life Science, Experimental pathology
  • Life Science, Virology
  • Life Science, Veterinary medical science
■ Educational Organization

Career

■ Career
Career
  • Aug. 2020 - Present
    Hokkaido University, 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
  • Jun. 2016 - Jul. 2020
    Hokkaido University, Graduate School of Veterinary Medicine
  • Jun. 2015 - May 2016
    Hokkaido University, Graduate School of Veterinary Medicine, 特任助教
  • Apr. 2015 - May 2015
    Hokkaido University, Research Center for Zoonosis Control, 特任助教
  • Apr. 2012 - Mar. 2015
    Hokkaido University, Research Center for Zoonosis Control, 博士研究員
Educational Background
  • Apr. 2008 - Mar. 2012, 北海道大学大学院, 獣医学研究科
  • Apr. 2002 - Mar. 2008, Rakuno Gakuen University, School of Veterinary Medicine, Department of Veterinary Medicine
Committee Memberships
  • ウイルス学キャンプ 世話人
  • トガ・フラビ・ペスチウイルス研究会 世話人
  • 日本ウイルス学会 評議委員
  • 日本獣医学会 評議委員

Research activity information

■ Awards
  • Sep. 2025, 日本獣医学会, Veterinary Science Awards
    Studies on the pathogenesis and control of flavivirus infection
    小林進太郎
  • Sep. 2022, 日本小動物獣医学会, 令和4年度日本小動物獣医学会 北海道地区学会長賞
    人獣共通感染症の医学界との連携強化推進〜飼育犬・猫のダニ媒介性脳炎の疫学調査〜
    上田広之;小林進太郎;好井健太朗, Japan society, Japan
  • Nov. 2021, 日本農学会, 日本農学進歩賞
    フラビウイルス感染による病態形成機構の解明および診断法の開発
    小林進太郎
  • Oct. 2021, 第7回北海道大学部局横断シンポジウム ベストポスター賞
    ウエストナイルウイルス感染によるタンパク質の核内輸送の阻害と細胞死の関連の解析
  • Oct. 2020, 日本ウイルス学会, 日本ウイルス学会杉浦奨励賞
    ウエストナイルウイルスの脳炎病態の形成機構の解明
    小林 進太郎
  • Oct. 2017, トガ・フラビ・ペスチウイルス研究会, 第24回トガ・フラビ・ペスチウイルス研究会若手奨励賞
    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と編成タンパク質の蓄積および神経病態形成機構についての解析
    小林進太郎
  • Sep. 2017, 日本獣医学会, 獣医学奨励賞
    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
    小林進太郎
  • Apr. 2013, 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）
    Accumulation of ubiquitinated proteins is related to West Nile virus-induced neuronal apoptosis
    小林進太郎
■ Papers
  • Development of blood-brain barrier-penetrating antibodies for neutralizing tick-borne encephalitis virus in the brain.
    Mizuki Fukuta; Sayo Fukano; Naoya Maekawa; Shintaro Kobayashi; Shunsuke Okamoto; Minato Hirano; Junko Nio-Kobayashi; Hiroaki Kariwa; Shigeru Kawakami; Satoru Konnai; Kentaro Yoshii
    mSphere, e0018425, 07 Jul. 2025, [Peer-reviewed], [International Magazine]
    English, Scientific journal, UNLABELLED: Tick-borne encephalitis virus (TBEV) belongs to the genus Flavivirus and causes tick-borne encephalitis (TBE), a disease characterized by severe neurological symptoms with a high mortality rate. Currently, no specific antiviral treatments have been developed for TBE. The blood-brain barrier (BBB) restricts drug delivery to the central nervous system, posing a major challenge in developing effective therapies targeting TBEV in the brain. In this study, we developed recombinant anti-TBEV antibodies fused with BBB-penetrating rabies virus glycoprotein (RVG) peptides to facilitate their transport across the BBB. The fusion of RVG peptides to the C-terminus of the heavy chain of whole antibodies or single-chain variable fragment had minimal impact on their neutralizing ability against TBEV. The RVG fusion enhanced antibody binding to the surface of a human brain endothelial cell line and increased permeability in an in vitro BBB model. The RVG-fused antibodies exhibited a higher transport efficiency to the brain than the non-fused antibodies following peripheral injection in mice. Notably, the peripheral administration of the RVG-fused whole antibody after viral invasion into the brain significantly neutralized TBEV in the brains of infected mice. These findings suggest that RVG-fused antibodies represent a promising therapeutic strategy for treating TBE once the virus has entered the brain. IMPORTANCE: Tick-borne encephalitis virus is a neuroinvasive pathogen that causes severe neurologic disease, significantly affecting patients' quality of life. No specific antiviral treatment is available for tick-borne encephalitis caused by virus multiplication in the brain. The delivery of drugs to the brain via peripheral administration is often obstructed by the blood-brain barrier. To develop targeted antiviral therapies for brain infections, we engineered recombinant antibodies capable of crossing the blood-brain barrier via brain-targeted ligands. These antibodies exhibited permeability across the blood-brain barrier in both in vitro and in vivo models and notably effectively neutralized the virus within the brain following peripheral administration. This study is the first to highlight the therapeutic potential of brain-targeted recombinant antibodies after viral entry into the brain, offering a promising pathway for the development of effective antiviral treatments for tick-borne encephalitis.
  • Development of a novel plate reader-based antibody-dependent enhancement (ADE) assay for orthoflavivirus infections.
    Yannis Aklil; Hiroto Takeuchi; Gabriel Gonzalez; Atsuko Inoue; Kaito Maeda; Shintaro Kobayashi; Yukari Itakura; Shinji Saito; Anavaj Sakuntabhai; Michihito Sasaki; William W Hall; Hirofumi Sawa; Yasuko Orba; Koshiro Tabata
    Journal of virological methods, 115210, 115210, 24 Jun. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, Antibody-dependent enhancement (ADE) is one of the mechanisms associated with severe clinical outcomes in infections caused by certain viruses, including dengue virus (DENV). Several ADE assay systems have been established, including flow cytometric assays using live viruses, enzyme-linked immuno-sorbent assay (ELISA) for the detection of viral NS1, and luciferase reporter gene assays. Among these, the flow cytometric assay is the most commonly used to evaluate ADE activity; however, it has limitations such as high operational costs due to fixation and immunostaining procedures, as well as a long analysis time. Fluorescent protein-expressing single-round infectious particles (SRIPs) enables label-free detection of ADE activity, but the flow cytometric procedure still requires a long analysis time. In this study, to simplify and expedite the ADE assay using enhanced green fluorescent protein (EGFP)-expressing SRIPs, we developed a plate reader-based ADE assay as an alternative to the conventional flow cytometry-based method. To evaluate effectiveness of this assay, we measured ADE activities in K562 cells induced by pan-orthoflavivirus 4G2 and pan-dengue 4E11 monoclonal antibodies (mAbs) using both flow cytometric assays using live viruses and plate-reader-based EGFP-expressing SRIPs assays. The results showed strong correlations between the two different ADE assays with R² values of 0.92 for 4G2 mAb and 0.94 for 4E11 mAb (Pearson correlation coefficients). In summary, this newly established assay offers a high-throughput and cost-effective method for comprehensive characterization of the relationship between vaccine- or infection-induced antibodies and ADE in orthoflavivirus infections.
  • Development of a highly specific enzyme-linked immunosorbent assay for detection of antibodies to Duck Tembusu virus using subviral particles.
    Iyarath Putchong; Thaweesak Songserm; Sittinee Kulprasertsri; Shintaro Kobayashi; Preeda Lertwatcharasarakul; Wallaya Phongphaew
    PloS one, 20, 6, e0326913, 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, Duck Tembusu virus (DTMUV) belongs to the family Flaviviridae and genus Orthoflavivirus. It causes disease in ducks, affecting the nervous system and significantly reducing egg production. The first outbreak of DTMUV in Thailand was reported in 2013, with widespread cases across various regions. However, serological diagnosis of DTMUV is challenging due to antibody cross-reactivity with other flaviviruses. To address this issue, we developed an ELISA based on subviral particles. The cassette encoding the membrane precursor and envelope genes of DTMUV (strain KPS54A61) were cloned into a pCAGGS vector with an OSF-tag and transfected into HEK-293T cells to generate subviral particles. The subviral particles were detected in the supernatant of the transfected cell via immunoblotting using anti-DTMUV E protein and anti-Strep-tag antibodies, which revealed a protein band of approximately 59 kDa. An electron microscopy confirmed the presence of particles approximately 35 nm in diameter. To optimize the SP-based ELISA, checkerboard titration identified the optimal antigen concentration as 70 µg/mL and the optimal serum dilution as 1:100,000. A cut-off value was established for the assay, and testing 300 duck serum samples using the SP-based ELISA identified 41 positive samples (14%) and 259 negative samples (86%). The SP-based ELISA exhibited 100% sensitivity and specificity, achieving a perfect agreement score of 1.0 in comparison with the serum neutralization test. Additionally, specificity testing using antibodies specific to Japanese Encephalitis virus (JEV) revealed no cross-reactivity in the ELISA test. Therefore, the developed SP-based ELISA is highly effective for screening and monitoring DTMUV outbreaks in duck farms, significantly reducing the risk of viral spread and enabling the timely implementation of disease control measures.
  • Status Epilepticus Caused by Tick-borne Encephalitis: A Case Report
    Kazuhiro Horiuchi; Shuntaro Nakamura; Kazuki Yamada; Kazuya Mitsuhashi; Kei Watari; Kazuma Tamiya; Hiroki Yamaguchi; Shintaro Kobayashi
    Internal Medicine, Japanese Society of Internal Medicine, 2025, [Peer-reviewed], [Last author], [International Magazine]
    Scientific journal
  • African lineage 1a West Nile virus isolated from crocodiles exhibits low neuroinvasiveness in mice.
    Hiroko Kobayashi; Herman Chambaro; Koshiro Tabata; Takuma Ariizumi; Wallaya Phongphaew; Kunda Ndashe; Joseph Ndebe; Paul Fandamu; Shintaro Kobayashi; Naoto Ito; Michihito Sasaki; Bernard M Hang'ombe; Edgar Simulundu; Yasuko Orba; Hirofumi Sawa
    The Journal of general virology, 105, 11, Nov. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, West Nile virus (WNV) is a mosquito-borne flavivirus that causes encephalitis in humans and infects crocodiles, resulting in rashes and neurological signs. In Zambia, two distinct lineages of WNV have been detected in neighbouring areas: lineage 2 in mosquitoes and lineage 1a in farmed crocodiles. Considering the risk of direct or vector-mediated WNV transmission from crocodiles to mammals, it is necessary to elucidate the pathogenicity of WNV strains derived from crocodiles. In this study, WNV was successfully isolated from naturally infected farmed crocodiles (Croc110/2019/1/ZM, Croc110). We then investigated its proliferation and pathogenicity in mice in comparison with a WNV isolate from mosquitoes in Zambia (Zmq16) and two reference strains, including one highly pathogenic (NY99) and one low pathogenic (Eg101) strain. Although viral proliferation in Vero and mammalian neuronal cells was comparable among the strains, Croc110 exhibited low cell-to-cell transmission efficiency. In vivo, more than 70% of mice (C57BL/6) intracerebrally inoculated with Croc110 displayed neurological signs, and Croc110-infected mice exhibited similarly high mortality rates as NY99- and Zmq16-infected mice. Meanwhile, comparable virus growth was observed among the strains in the brain. However, the virulence of Croc110 was significantly lower than that of Zmq16 and NY99 following intradermal (ID) and intraperitoneal inoculation. Consistently, Croc110 displayed lower growth than Zmq16 and NY99 in the brain and peripheral tissues after ID inoculation. Our study revealed that the crocodile-derived WNV strain is less neuroinvasive in mice, and it exhibits distinct pathogenicity from the highly pathogenic mosquito-derived WNV strain circulating in Zambia.
  • Increased production of orthoflavivirus single-round infectious particles produced in mammalian cells at a suboptimal culture temperature of 28°C
    Koshiro Tabata; Shintaro Kobayashi; Yukari Itakura; Gabriel Gonzalez; Chilekwa F. Kabamba; Shinji Saito; Michihito Sasaki; William W. Hall; Hirofumi Sawa; Yasuko Orba
    Journal of Virological Methods, 329, 115007, 115007, Elsevier BV, Sep. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    Scientific journal
  • Molecular evolution of Hokkaido virus, a genotype of Orthohantavirus puumalaense, among Myodes rodents.
    Duong Thi Ngoc Thuy; Michihito Sasaki; Yasuko Orba; Passawat Thammahakin; Keisuke Maezono; Shintaro Kobayashi; Hiroaki Kariwa
    Virology, 597, 110168, 110168, 03 Jul. 2024, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Viruses in the genus Orthohantavirus within the family Hantaviridae cause human hantavirus infections and represent a threat to public health. Hokkaido virus (HOKV), a genotype of Orthohantavirus puumalaense (Puumala virus; PUUV), was first identified in Tobetsu, Hokkaido, Japan. Although it is genetically related to the prototype of PUUV, the evolutionary pathway of HOKV is unclear. We conducted a field survey in a forest in Tobetsu in 2022 and captured 44 rodents. Complete coding genome sequences of HOKVs were obtained from five viral-RNA-positive rodents (four Myodes rufocanus bedfordiae and one Apodemus speciosus). Phylogenetic analysis revealed a close relationship between the phylogenies and geographical origins of M. rufocanus-related orthohantaviruses. Comparison of the phylogenetic trees of the S segments of orthohantaviruses and the cytochrome b genes of Myodes species suggested that Myodes-related orthohantaviruses evolved in Myodes rodent species as a result of genetic isolation and host switching.
  • Rab27a promotes degradation of West Nile virus E protein in the lysosome.
    Shintaro Kobayashi; Seira Kawai; Yukine Fukuda; Haruto Eguchi; Keisuke Maezono; Passawat Thammahakin; Hirofumi Sawa; Hiroaki Kariwa
    iScience, 27, 4, 109539, 109539, 19 Apr. 2024, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
    English, Scientific journal, Rab27a, a Rab family small GTPases, plays an important role in the trafficking and secretion of the intracellular proteins and has been reported to promote various viral multiplication. However, whether Rab27a is involved in West Nile virus (WNV) multiplication is unknown. This study examined the ability of Rab27a to suppress WNV multiplication. The inhibition of Rab27a expression increased viral multiplication and the intracellular levels of WNV structural proteins, E and prM proteins. Rab27a partially colocalized with E protein, mainly in the perinuclear region, while inhibition of Rab27a expression resulted in diffuse subcellular localization of E protein. In addition, some of the perinuclear E protein colocalized with the lysosomal marker LAMP1, and inhibition of lysosomal acidification increased intracellular levels of Rab27a and E proteins. These observations suggested that Rab27a inhibits WNV multiplication by inducing the degradation of viral protein in lysosomes.
  • Ubiquitin accumulation induced by the finger and palm sub-domains of NS5 modulates the replication of West Nile virus.
    Shintaro Kobayashi; Ryoko Kawakami; Chisaki Takeda; Keisuke Maezono; Passawat Thammahakin; Haruto Eguchi; Bernard M Hang'ombe; Yasuko Orba; Hirofumi Sawa; Kentaro Yoshii; Hiroaki Kariwa
    Virology, 588, 109902, 109902, Nov. 2023, [Peer-reviewed], [Lead author, Corresponding author], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, West Nile virus (WNV) causes encephalitis in human and animals. WNV is phylogenetically classified into at least five distinct genetic lineages with different pathogenicity. The pathogenesis of West Nile encephalitis is affected by ubiquitin accumulation in infected cells, but the mechanism is unknown. In this study, the association between ubiquitin accumulation and WNV pathogenicity was investigated. Ubiquitin accumulation was detected in cells infected with NY99 strain belonging to lineage-1, but not FCG and Zmq16 strains belonging to lineage-2. Substitution of the Finger and Palm sub-domains of NS5 from lineage-1 to -2 decreased ubiquitin accumulation and viral replication. Furthermore, the survival rate was increased, and viral replication and ubiquitin accumulation in the brain were attenuated, in mice inoculated with the substituted WNV compared with lineage-1 WNV. Therefore, the intracellular ubiquitin accumulation induced by the Finger and Palm sub-domains of NS5 is linked to the differences in pathogenicity among WNV lineages.
  • Development of flavivirus subviral particles with low cross-reactivity by mutations of a distinct antigenic domain.
    Koshiro Tabata; Yukari Itakura; Takuma Ariizumi; Manabu Igarashi; Hiroko Kobayashi; Kittiya Intaruck; Mai Kishimoto; Shintaro Kobayashi; William W Hall; Michihito Sasaki; Hirofumi Sawa; Yasuko Orba
    Applied microbiology and biotechnology, 13 Oct. 2023, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, The most conserved fusion loop (FL) domain present in the flavivirus envelope protein has been reported as a dominant epitope for cross-reactive antibodies to mosquito-borne flaviviruses (MBFVs). As a result, establishing accurate serodiagnosis for MBFV infections has been difficult as anti-FL antibodies are induced by both natural infection and following vaccination. In this study, we modified the most conserved FL domain to overcome this cross-reactivity. We showed that the FL domain of lineage I insect-specific flavivirus (ISFV) has differences in antigenicity from those of MBFVs and lineage II ISFV and determined the key amino acid residues (G106, L107, or F108), which contribute to the antigenic difference. These mutations were subsequently introduced into subviral particles (SVPs) of dengue virus type 2 (DENV2), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). In indirect enzyme-linked immunosorbent assays (ELISAs), these SVP mutants when used as antigens reduced the binding of cross-reactive IgG and total Ig induced by infection of ZIKV, JEV, and WNV in mice and enabled the sensitive detection of virus-specific antibodies. Furthermore, immunization of ZIKV or JEV SVP mutants provoked the production of antibodies with lower cross-reactivity to heterologous MBFV antigens compared to immunization with the wild-type SVPs in mice. This study highlights the effectiveness of introducing mutations in the FL domain in MBFV SVPs with lineage I ISFV-derived amino acids to produce SVP antigens with low cross-reactivity and demonstrates an improvement in the accuracy of indirect ELISA-based serodiagnosis for MBFV infections. KEY POINTS: • The FL domain of Lineage I ISFV has a different antigenicity from that of MBFVs. • Mutated SVPs reduce the binding of cross-reactive antibodies in indirect ELISAs. • Inoculation of mutated SVPs induces antibodies with low cross-reactivity.
  • Detection of disease-associated microglia among various microglia phenotypes induced by West Nile virus infection in mice.
    Passawat Thammahakin; Keisuke Maezono; Naoya Maekawa; Hiroaki Kariwa; Shintaro Kobayashi
    Journal of neurovirology, 29, 4, 367, 375, Aug. 2023, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
    English, Scientific journal, West Nile virus (WNV) has emerged as a significant cause of viral encephalitis in humans and horses. However, the pathogenesis of the West Nile encephalitis remains unclear. Microglia are activated by WNV infection, and the pathogenic involvement of their phenotypes is controversial. In this study, we examined the diversity of microglia phenotypes caused by WNV infection by assessing various microglia markers and identified disease-associated microglia in WNV-infected mouse brain tissue. Cells positive for general microglia markers such as Iba1, P2RY12, or TMEM119 were detected in the control and WNV-infected brain tissue. The morphology of the positive cells in brain tissue infected by WNV was different from that of control brain tissue, indicating that WNV infection induced activation of microglia. The activated microglia were classified into various phenotypes by investigation of specific marker expression. Among the activated microglia, disease-associated microglia that were positive for CD11c and weakly positive for TMEM119 were detected close to the WNV-infected cells. These results indicate that WNV infection induces activation of diverse microglia phenotypes and that disease-associated microglia may be associated with the pathogenicity of WNV infection in the mouse brain.
  • Development of recombinant West Nile virus expressing mCherry reporter protein.
    Shintaro Kobayashi; Yukine Fukuda; Kentaro Yoshii; Passawat Thammahakin; Keisuke Maezono; Luděk Eyer; Daniel Růžek; Hiroaki Kariwa
    Journal of virological methods, 317, 114744, 114744, Jul. 2023, [Peer-reviewed], [Lead author, Corresponding author], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, West Nile virus (WNV) is transmitted to humans and animals by a mosquito and enters the central nervous system, leading to lethal encephalitis. Reporter viruses expressing fluorescent proteins enable detection of infected cells in vitro and in vivo, facilitating evaluation of the dynamics of viral infection, and the development of diagnostic or therapeutic methods. In this study, we developed a method for production of a recombinant replication-competent WNV expressing mCherry fluorescent protein. The expression of mCherry was observed in viral antigen-positive cells in vitro and in vivo, but the growth of the reporter WNV was reduced as compared to the parental WNV. The expression of mCherry was stable during 5 passages in reporter WNV-infected culture cells. Neurological symptoms were observed in mice inoculated intracranially with the reporter WNV. The reporter WNV expressing mCherry will facilitate research into WNV replication in mouse brains.
  • The ‘World Café’: Strengthening Rabies Prevention with the Government–Academia Collaboration in Japan
    Chiho Kaneko; Kentaro Yoshii; Yumi Kirino; Shintaro Kobayashi; Genki Arikawa; Akitoyo Hotta; Naoaki Misawa; Satoshi Inoue
    One Health for Dog-mediated Rabies Elimination in Asia, 47, 59, CABI, 29 Jun. 2023, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    In book
  • Morphogenesis of Bullet-Shaped Rabies Virus Particles Regulated by TSG101.
    Yukari Itakura; Koshiro Tabata; Takeshi Saito; Kittiya Intaruck; Nijiho Kawaguchi; Mai Kishimoto; Shiho Torii; Shintaro Kobayashi; Naoto Ito; Michiko Harada; Satoshi Inoue; Ken Maeda; Ayato Takada; William W Hall; Yasuko Orba; Hirofumi Sawa; Michihito Sasaki
    Journal of virology, 97, 5, e0043823, 12 Apr. 2023, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host endosomal sorting complex required for transport (ESCRT) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection and, specifically, the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a small interfering RNA (siRNA) screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. IMPORTANCE Enveloped viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT complexes. Some enveloped viruses utilize their late (L-) domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped virions, but the underlying molecular mechanisms involved remain elusive. Here, we showed that TSG101, one of the ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via the L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.
  • Necroptosis of neuronal cells is related to the neuropathology of tick-borne encephalitis.
    Dai Tsujino; Kentaro Yoshii; Misa Kajiyama; Yuji Takahashi; Naoya Maekawa; Hiroaki Kariwa; Shintaro Kobayashi
    Virus research, 321, 198914, 198914, 03 Sep. 2022, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
    English, Scientific journal, Tick-borne encephalitis virus (TBEV) is a zoonotic virus that causes tick-borne encephalitis (TBE) in humans. Infections of Sapporo-17-Io1 (Sapporo) and Oshima 5-10 (Oshima) TBEV strains showed different pathogenic effects in mice. However, the differences between the two strains are unknown. In this study, we examined neuronal degeneration and death, and activation of glial cells in mice inoculated with each strain to investigate the pathogenesis of TBE. Viral growth was similar between Sapporo and Oshima, but neuronal degeneration and death, and activation of glial cells, was more prominent with Oshima. In human neuroblastoma cells, apoptosis and pyroptosis were not observed after TBEV infection. However, the expression of the necroptosis marker, mixed lineage kinase domain-like (MLKL) protein, was upregulated by TBEV infection, and this upregulation was more pronounced in Oshima than Sapporo infections. As necroptosis is a pro-inflammatory type of cell death, differences in necroptosis induction might be involved in the differences in neuropathogenicity of TBE.
  • Dual control of tick-borne encephalitis virus replication by autophagy in mouse macrophages.
    Zuzana Beránková; Jan Kopecký; Shintaro Kobayashi; Jaroslava Lieskovská
    Virus research, 315, 198778, 198778, 02 Jul. 2022, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, Autophagy is a lysosomal degradative pathway responsible for recycling cytosolic proteins and organelles and also functions as an innate defense mechanism that host cells use against viral infection. While many viruses have evolved mechanisms to antagonize the antiviral effects of the autophagy pathway, others subvert autophagy to facilitate replication. For flaviviruses, both the positive and negative role of autophagy in virus replication has been reported. The interplay between autophagy and tick-borne encephalitis virus (TBEV) in innate immune cells is largely unknown. Here we report the relationship between an autophagy and TBEV replication in mouse macrophage cell line PMJ2-R using Hypr strain of TBEV. First, we examined the effect of Hypr infection on the autophagy pathway. We detected a mild and a temporary increase of autophagy marker LC3-II in Hypr-infected cells. The role of autophagy in TBEV replication was evaluated in autophagy related gene 5 (Atg5) knockdown cells (shAtg5). Our results showed that during an early stage of Hypr infection the viral titers were increased, while later on, at 72 hpi, the titers have declined in shAtg5 cells compared to control. Moreover, the higher number of virus-positive cells was observed in shAtg5 cells in early stage of infection and correlated with enhanced virus entry. Finally, we found an increased production of IFN-β in Hypr-infected shAtg5 cells in comparison to control at 48 and 72 hpi implicating that autophagy restricts the amount of IFN produced by TBEV-infected macrophages. To conclude, in mouse macrophages TBEV replication is controlled by autophagy in time dependent manner, having temporally an antiviral and then a pro-viral role during infection. Our study points out to a delicate and complex involvement of autophagy machinery at level of virus entry and IFN-β production when controlling TBEV infection.
  • Analysis of the relationship between replication of the Hokkaido genotype of Puumala orthohantavirus and autophagy.
    Kazuma Tamiya; Shintaro Kobayashi; Kentaro Yoshii; Hiroaki Kariwa
    Virus research, 318, 198830, 198830, 28 May 2022, [Peer-reviewed], [Corresponding author], [International Magazine]
    English, Scientific journal, Hantaviruses are potentially fatal zoonotic pathogens of the family Hantaviridae. No human infection by the Hokkaido genotype of Puumala orthohantavirus (PUUV-Hok) has been reported. However, other PUUV genotypes cause hemorrhagic fever with renal syndrome (HFRS) in humans. Autophagy is a highly conserved lysosomal degradation process in eukaryotic cells that affects the replication of various viruses. In this study, we examined the role of autophagy in PUUV-Hok replication. PUUV-Hok infection induced the expression of LC3-II, an autophagosome marker, and the nucleocapsid protein (NP) of PUUV-Hok was colocalized with punctate structures of LC3. Inhibition of autophagy using an siRNA for Atg5, an autophagy-related gene, increased the replication of PUUV-Hok, whereas an autophagy inducer decreased its replication. Inhibition of lysosomal degradation increased the expression of NP and LC3-II. In summary, autophagy was induced by PUUV-Hok infection, which inhibited PUUV-Hok replication in a manner related to the degradation of the NP in lysosomes.
  • Effect of heat exposure on the growth and developmental competence of bovine oocytes derived from early antral follicles.
    Kohei Kawano; Kenichiro Sakaguchi; Chelenga Madalitso; Nattapong Ninpetch; Shintaro Kobayashi; Eri Furukawa; Yojiro Yanagawa; Seiji Katagiri
    Scientific reports, 12, 1, 8857, 8857, 25 May 2022, [Peer-reviewed], [International Magazine]
    English, Scientific journal, In dairy cows, low fertility caused by summer heat stress continues into the cooler autumn season. This can be caused by impaired oocyte quality in small growing follicles during summer. Here, we subjected oocyte-cumulus-granulosa complexes (OCGCs) derived from early antral follicles (0.5-1 mm) to in vitro growth (IVG) culture under two different temperature settings (the control and heat shock groups), and evaluated effects of heat exposure on growth and developmental competence of oocytes, factors affecting the developmental competence of oocytes (steroidogenesis of granulosa cells, oxidative stress in oocytes, and cell-to-cell communication between oocytes and somatic cells). Oocyte diameters after culture were smaller in the heat shock group. Although nuclear maturation and cleavage rates were similar between the groups, blastocyst rates were lower in the heat shock group (0.0%) than in the control group (27.7%), and reduced glutathione (GSH) levels in oocytes were lower in the heat shock group. Supplementation of cysteine, which stimulates GSH synthesis, increased GSH level and improved blastocyst rate of heat shocked oocytes (27.9%). These results suggest that heat exposure impairs the growth and developmental competence of oocytes in early antral follicles through GSH depletion, which can induce low fertility during summer and the following autumn.
  • Y-shaped RNA secondary structure of a noncoding region in the genomic RNA of tick-borne encephalitis virus affects pathogenicity.
    Shoko Nishiyama; Minato Hirano; Memi Muto; Mao Kambara; Naoto Ito; Shintaro Kobayashi; Hiroaki Kariwa; Kentaro Yoshii
    Microbiology and immunology, 66, 5, 234, 237, May 2022, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Tick-borne encephalitis virus (TBEV) is a zoonotic virus that causes encephalitis in humans. Various deletions have been reported in a variable region of the 3' untranslated region of the TBEV genome. This study analyzed the role of a Y-shaped secondary structure in the pathogenicity of TBEV by using reverse genetics. Deletion of the structure increased the mortality rate of virus-infected mice but did not affect the virus multiplication in cultured cells and organs. The results indicate that the secondary structure is involved in the regulation of TBEV pathogenesis.
  • Characterization of tick-borne encephalitis virus isolated from tick infesting dog in central Hokkaido in 2018.
    Yuji Takahashi; Shintaro Kobayashi; Ryo Nakao; Hiroaki Kariwa; Kentaro Yoshii
    Ticks and tick-borne diseases, 13, 2, 101900, 101900, Mar. 2022, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Tick-borne encephalitis virus (TBEV) is a zoonotic virus belonging to the genus Flavivirus of the family Flaviviridae, causing meningitis or meningoencephalitis in humans. TBEV is widely distributed across the Eurasian northern regions, including Japan. Dogs have been reported to be sentinel hosts of TBEV in endemic areas, but studies of ticks infesting dogs are limited in Japan. This study isolated a novel TBEV strain from a tick (Ixodes ovatus) collected on a dog from central Hokkaido. Whole-genome sequencing revealed that the isolated strain belonged to the Far Eastern subtype of TBEV and was classified under a different subcluster of other Japanese isolates. Nanporo-18-44 showed growth properties similar to those of Oshima 5-10 both in vitro and in vivo. The pathogenicity of both viruses was similar in mice infected intracerebrally, however they showed a distinct distribution in the infected neurons of the mouse brain. Our results suggest that infections of humans and animals by unknown strains of TBEV exist in other areas of Japan. Further surveys including those conducted outside of Hokkaido, are required to elucidate the epidemiological risk of TBEV in Japan.
  • 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, 24 Sep. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, 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.
  • Duck Tembusu virus induces stronger cellular responses than Japanese encephalitis virus in primary duck neurons and fibroblasts.
    Sittinee Kulprasertsri; Shintaro Kobayashi; Keisuke Aoshima; Atsushi Kobayashi; Takashi Kimura
    Microbiology and immunology, 65, 11, 481, 491, 14 Jul. 2021, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal, Duck Tembusu virus (DTMUV) and Japanese encephalitis virus (JEV) are mosquito-borne flaviviruses. These two viruses infect ducks; however, they show different neurological outcomes. The mechanism of DTMUV- and JEV-induced neuronal death has not been well investigated. In the present study, we examined the differences in the mechanisms involved in virus-induced cell death and innate immune responses between DTMUV KPS54A61 strain and JEV JaGAr-01 strain using primary duck neurons (DN) and duck fibroblasts (CCL-141). DN and CCL-141 were permissive for the infection and replication of these two viruses, which upregulated the expression of innate immunity genes. Both DTMUV and JEV induced cell death via a caspase-3-dependent manner; however, DTMUV triggered more cell death than JEV did in both CCL-141 and DN. These findings suggest that DTMUV infection causes apoptosis in duck neurons and fibroblasts more strongly than JEV. Levels of the mRNA expression of innate immunity-related genes after DTMUV infection were generally higher than levels after JEV infection, suggesting that DTMUV-induced immune response in duck cells may exhibit toxic effect rather than protective effects. This article is protected by copyright. All rights reserved.
  • Development of a highly specific serodiagnostic ELISA for West Nile virus infection using subviral particles.
    Keisuke Maezono; Shintaro Kobayashi; Koshiro Tabata; Kentaro Yoshii; Hiroaki Kariwa
    Scientific reports, 11, 1, 9213, 9213, 28 Apr. 2021, [Peer-reviewed], [Corresponding author], [International Magazine]
    English, Scientific journal, West Nile virus (WNV), a member of the Japanese encephalitis virus (JEV) serocomplex group, causes lethal encephalitis in humans and horses. Because serodiagnosis of WNV and JEV is hampered by cross-reactivity, the development of a simple, secure, and WNV-specific serodiagnostic system is required. The coexpression of prM protein and E protein leads to the secretion of subviral particles (SPs). Deletion of the C-terminal region of E protein is reported to affect the production of SPs by some flaviviruses. However, the influence of such a deletion on the properties and antigenicity of WNV E protein is unclear. We analyzed the properties of full-length E protein and E proteins lacking the C-terminal region as novel serodiagnostics for WNV infection. Deletion of the C-terminal region of E protein suppressed the formation of SPs but did not affect the production of E protein. The sensitivity of an enzyme-linked immunosorbent assay (ELISA) using the full-length E protein was higher than that using the truncated E proteins. Furthermore, in the ELISA using full-length E protein, there was little cross-reactivity with anti-JEV antibodies, and the sensitivity was similar to that of the neutralization test.
  • Inactivation of sars-cov-2 by povidone-iodine products: Implications for effective mouth rinsing and gargling
    Hiroaki Kariwa; Hirofumi Sawa; Shintaro Kobayashi
    Japanese Journal of Veterinary Research, 69, 3, 183, 187, Hokkaido University, 2021
    English, Scientific journal
  • Development and characterization of recombinant tick-borne encephalitis virus expressing mCherry reporter protein: A new tool for high-throughput screening of antiviral compounds, and neutralizing antibody assays.
    Jan Haviernik; Ludek Eyer; Kentaro Yoshii; Shintaro Kobayashi; Jiri Cerny; Antoine Nougairède; Jean-Sélim Driouich; Jiri Volf; Martin Palus; Xavier de Lamballerie; Ernest A Gould; Daniel Ruzek
    Antiviral research, 185, 104968, 104968, 04 Nov. 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the mCherry reporter protein. The resulting TBEV reporter virus (named mCherry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the mCherry-TBEV virus produced smaller plaques. The magnitude of mCherry expression correlated well with progeny virus production but remained stable over <4 passages in cell culture. Using well-characterized antiviral compounds known to inhibit TBEV, 2'-C-methyladenosine and 2'-deoxy-2'-β-hydroxy-4'-azidocytidine (RO-9187), we demonstrated that mCherry-TBEV is suitable for high-throughput screening of antiviral drugs. Serum samples from a TBEV-vaccinated human and a TBEV-infected dog were used to evaluate the mCherry-based neutralization test. Collectively, recombinant mCherry-TBEV reporter virus described here provides a powerful tool to facilitate the identification of potential antiviral agents, and to measure levels of neutralizing antibodies in human and animal sera.
  • A Retrospective Epidemiological Study of Tick-Borne Encephalitis Virus in Patients with Neurological Disorders in Hokkaido, Japan.
    Kentaro Yoshii; Ikuko Takahashi-Iwata; Shinichi Shirai; Shintaro Kobayashi; Ichiro Yabe; Hidenao Sasaki
    Microorganisms, 8, 11, 28 Oct. 2020, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Tick-borne encephalitis (TBE) is a zoonotic disease that usually presents as a moderate febrile illness followed by severe encephalitis, and various neurological symptoms are observed depending on the distinct central nervous system (CNS) regions affected by the TBE virus (TBEV) infection. In Japan, TBE incidence is increasing and TBEV distributions are reported in wide areas, specifically in Hokkaido. However, an extensive epidemiological survey regarding TBEV has not been conducted yet. In this study, we conducted a retrospective study of the prevalence of antibodies against TBEV in patients with neurological disorders and healthy populations in a TBEV-endemic area in Hokkaido. Among 2000 patients, three patients with inflammatory diseases in the CNS had TBEV-specific IgM antibodies and neutralizing antibodies. The other four patients diagnosed clinically with other neurological diseases were positive for TBEV-specific IgG and neutralizing antibodies, indicating previous TBEV infection. In a total of 246 healthy residents in a TBEV-endemic region, one resident had TBEV-specific antibodies. These results demonstrated undiagnosed TBEV infections in Japan. Further surveys are required to reveal the actual epidemiological risk of TBE and to consider preventive measures, such as a vaccine program, for the control of TBE in Japan.
  • Amino acid 159 of the envelope protein affects viral replication and T-cell infiltration by West Nile virus in intracranial infection.
    Shintaro Kobayashi; Chisato Kaneko; Ryoko Kawakami; Rie Hasebe; Hirofumi Sawa; Kentaro Yoshii; Hiroaki Kariwa
    Scientific reports, 10, 1, 7168, 7168, 28 Apr. 2020, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
    English, Scientific journal, 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.
  • Characterization of tick-borne encephalitis virus isolated from a tick in central Hokkaido in 2017.
    Yuji Takahashi; Shintaro Kobayashi; Mariko Ishizuka; Minato Hirano; Memi Muto; Shoko Nishiyama; Hiroaki Kariwa; Kentaro Yoshii
    The Journal of general virology, 101, 5, 497, 509, 05 Mar. 2020, [Peer-reviewed], [International Magazine]
    English, Scientific journal, 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.
  • West Nile virus capsid protein inhibits autophagy by AMP-activated protein kinase degradation in neurological disease development.
    Shintaro Kobayashi; Kentaro Yoshii; Wallaya Phongphaew; Memi Muto; Minato Hirano; Yasuko Orba; Hirofumi Sawa; Hiroaki Kariwa
    PLoS pathogens, 16, 1, e1008238, Jan. 2020, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
    English, Scientific journal, 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.
  • Serological evidence of Zika virus infection in non-human primates in Zambia.
    Christida E Wastika; Michihito Sasaki; Kentaro Yoshii; Paulina D Anindita; Bernard M Hang'ombe; Aaron S Mweene; Shintaro Kobayashi; Hiroaki Kariwa; Michael J Carr; William W Hall; Yuki Eshita; Yasuko Orba; Hirofumi Sawa
    Archives of virology, 164, 8, 2165, 2170, Aug. 2019, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, 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.
  • Development of a serodiagnostic IgM-ELISA for tick-borne encephalitis virus using subviral particles with strep-tag
    Miki Nakayasu; Minato Hirano; Memi Muto; Shintaro Kobayashi; Hiroaki Kariwa; Kentaro Yoshii
    Ticks and Tick-borne Diseases, 9, 6, 1391, 1394, Elsevier BV, Sep. 2018, [Peer-reviewed], [International Magazine]
    Scientific journal
  • Serologic Evidence of Tick-Borne Encephalitis Virus Infection in a Patient with Suspected Lyme Disease in Japan.
    Kentaro Yoshii; Kozue Sato; Mariko Ishizuka; Shintaro Kobayashi; Hiroaki Kariwa; Hiroki Kawabata
    The American journal of tropical medicine and hygiene, 99, 1, 180, 181, Jul. 2018, [Peer-reviewed], [International Magazine]
    English, Scientific journal, 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.
  • Identification and analysis of host proteins that interact with the 3′-untranslated region of tick-borne encephalitis virus genomic RNA
    Memi Muto; Wataru Kamitani; Mizuki Sakai; Minato Hirano; Shintaro Kobayashi; Hiroaki Kariwa; Kentaro Yoshii
    Virus Research, 249, 52, 56, Elsevier BV, Apr. 2018, [Peer-reviewed], [International Magazine]
    Scientific journal
  • Detection of novel gammaherpesviruses from fruit bats in Indonesia
    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, Microbiology Society, 01 Mar. 2018, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • Development of a rapid and quantitative method for the analysis of viral entry and release using a NanoLuc luciferase complementation assay
    Michihito Sasaki; Paulina D. Anindita; Wallaya Phongphaew; Michael Carr; Shintaro Kobayashi; Yasuko Orba; Hirofumi Sawa
    VIRUS RESEARCH, 243, 69, 74, Jan. 2018, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • Serological survey of severe fever with thrombocytopenia syndrome virus infection in Sika deer and rodents in Japan
    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, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal
  • Shape-dependent adjuvanticity of nanoparticle-conjugated RNA adjuvants for intranasal inactivated influenza vaccines
    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, [Peer-reviewed], [International Magazine]
    English, Scientific journal
  • Targeting of severe fever with thrombocytopenia syndrome virus structural proteins to the ERGIC (endoplasmic reticulum Golgi intermediate compartment) and Golgi complex
    Tapiwa LUNDU; Yoshimi TSUDA; Ryo ITO; Kenta SHIMIZU; Shintaro KOBAYASHI; Kentaro YOSHII; Kumiko YOSHIMATSU; Jiro ARIKAWA; Hiroaki KARIWA
    Biomedical Research, 39, 1, 27, 38, Biomedical Research Press, 2018, [Peer-reviewed], [Domestic magazines]
    Scientific journal
  • Escape of Tick-Borne Flavivirus from 2'-C-Methylated Nucleoside Antivirals Is Mediated by a Single Conservative Mutation in NS5 That Has a Dramatic Effect on Viral Fitness
    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, Nov. 2017, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • Dendritic transport of tick-borne flavivirus RNA by neuronal granules affects development of neurological disease
    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, Sep. 2017, [Peer-reviewed], [International Magazine]
    English, Scientific journal
  • Discovery of a novel antiviral agent targeting the nonstructural protein 4 (nsP4) of chikungunya virus
    Yuji Wada; Yasuko Orba; Michihito Sasaki; Shintaro Kobayashi; Michael J. Carr; Haruaki Nobori; Akihiko Sato; William W. Hall; Hirofumi Sawa
    VIROLOGY, 505, 102, 112, May 2017, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • A novel reverse genetics system for production of infectious West Nile virus using homologous recombination in mammalian cells
    Shintaro Kobayashi; Kentaro Yoshii; Minato Hirano; Memi Muto; Hiroaki Kariwa
    JOURNAL OF VIROLOGICAL METHODS, 240, 14, 20, Feb. 2017, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
    English, Scientific journal
  • Valosin-containing protein (VCP/p97) plays a role in the replication of West Nile virus
    Wallaya Phongphaew; Shintaro Kobayashi; Michihito Sasaki; Michael Carr; William W. Hall; Yasuko Orba; Hirofumi Sawa
    VIRUS RESEARCH, 228, 114, 123, Jan. 2017, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • Divergent bufavirus harboured in megabats represents a new lineage of parvoviruses
    Michihito Sasaki; Gabriel Gonzalez; Yuji Wada; Agus Setiyono; Ekowati Handharyani; Ibenu Rahmadani; Siswatiana Taha; Sri Adiani; Munira Latief; Zainal Abidin Kholilullah; Mawar Subangkit; Shintaro Kobayashi; Ichiro Nakamura; Takashi Kimura; Yasuko Orba; Kimihito Ito; Hirofumi Sawa
    SCIENTIFIC REPORTS, 6, 24257, Apr. 2016, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • Rab8b Regulates Transport of West Nile Virus Particles from Recycling Endosomes
    Shintaro Kobayashi; Tadaki Suzuki; Akira Kawaguchi; Wallaya Phongphaew; Kentaro Yoshii; Tomohiko Iwano; Akihiro Harada; Hiroaki Kariwa; Yasuko Orba; Hirofumi Sawa
    JOURNAL OF BIOLOGICAL CHEMISTRY, 291, 12, 6559, 6568, Mar. 2016, [Peer-reviewed], [Lead author], [International Magazine]
    English, Scientific journal
  • Development of a serodiagnostic multi-species ELISA against tick-borne encephalitis virus using subviral particles
    Eri Inagaki; Mizuki Sakai; Minato Hirano; Memi Muto; Shintaro Kobayashi; Hiroaki Kariwa; Kentaro Yoshii
    TICKS AND TICK-BORNE DISEASES, 7, 5, 723, 729, 2016, [Peer-reviewed], [International Magazine]
    English, Scientific journal
  • Detection of coronavirus genomes in Moluccan naked-backed fruit bats in Indonesia
    Paulina Duhita Anindita; Michihito Sasaki; Agus Setiyono; Ekowati Handharyani; Yasuko Orba; Shintaro Kobayashi; Ibnu Rahmadani; Siswatiana Taha; Sri Adiani; Mawar Subangkit; Ichiro Nakamura; Hirofumi Sawa; Takashi Kimura
    ARCHIVES OF VIROLOGY, 160, 4, 1113, 1118, Apr. 2015, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • Detection of novel polyomaviruses in fruit bats in Indonesia
    Shintaro Kobayashi; Michihito Sasaki; Ryo Nakao; Agus Setiyono; Ekowati Handharyani; Yasuko Orba; Ibnu Rahmadani; Siswatiana Taha; Sri Adiani; Mawar Subangkit; Ichiro Nakamura; Takashi Kimura; Hirofumi Sawa
    ARCHIVES OF VIROLOGY, 160, 4, 1075, 1082, Apr. 2015, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • Autophagy inhibits viral genome replication and gene expression stages in West Nile virus infection
    Shintaro Kobayashi; Yasuko Orba; Hiroki Yamaguchi; Kenta Takahashi; Michihito Sasaki; Rie Hasebe; Takashi Kimura; Hirofumi Sawa
    VIRUS RESEARCH, 191, 83, 91, Oct. 2014, [Peer-reviewed], [Lead author], [International Magazine]
    English, Scientific journal
  • Isolation and Characterization of a Novel Alphaherpesvirus in Fruit Bats
    Michihito Sasaki; Agus Setiyono; Ekowati Handharyani; Shintaro Kobayashi; Ibenu Rahmadani; Siswatiana Taha; Sri Adiani; Mawar Subangkit; Ichiro Nakamura; Hirofumi Sawa; Takashi Kimura
    JOURNAL OF VIROLOGY, 88, 17, 9819, 9829, Sep. 2014, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • Role of the C-Terminal Region of Vervet Monkey Polyomavirus 1 VP1 in Virion Formation
    Hiroki Yamaguchi; Shintaro Kobayashi; Junki Maruyama; Michihito Sasaki; Ayato Takada; Takashi Kimura; Hirofumi Sawa; Yasuko Orba
    JOURNAL OF VETERINARY MEDICAL SCIENCE, 76, 5, 637, 644, May 2014, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal
  • Cysteine Residues in the Major Capsid Protein, Vp1, of the JC Virus Are Important for Protein Stability and Oligomer Formation
    Shintaro Kobayashi; Tadaki Suzuki; Manabu Igarashi; Yasuko Orba; Noriko Ohtake; Keita Nagakawa; Kenichi Niikura; Takashi Kimura; Harumi Kasamatsu; Hirofumi Sawa
    PLoS ONE, 8, 10, e76668, 09 Oct. 2013, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • Cross-Reactivity of Secondary Antibodies against African Rodents and Application for Sero-Surveillance
    Ichiro Nakamura; Bernard Mudenda Hang'ombe; Hirofumi Sawa; Shintaro Kobayashi; Yasuko Orba; Akihiro Ishii; Yuka Thomas; Rie Isozumi; Kumiko Yoshimatsu; Aaron S. Mweene; Ayato Takada; Chihiro Sugimoto; Jiro Arikawa
    JOURNAL OF VETERINARY MEDICAL SCIENCE, 75, 6, 819, 825, Jun. 2013, [Peer-reviewed], ［Internationally co-authored］, [Domestic magazines]
    English, Scientific journal
  • Identification of a novel polyomavirus from vervet monkeys in Zambia
    Hiroki Yamaguchi; Shintaro Kobayashi; Akihiro Ishii; Hirohito Ogawa; Ichiro Nakamura; Ladslav Moonga; Bernard M. Hang'ombe; Aaron S. Mweene; Yuka Thomas; Takashi Kimura; Hirofumi Sawa; Yasuko Orba
    Journal of General Virology, 94, 6, 1357, 1364, 01 Jun. 2013, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • Gold Nanoparticles as a Vaccine Platform: Influence of Size and Shape on Immunological Responses in Vitro and in Vivo
    Kenichi Niikura; Tatsuya Matsunaga; Tadaki Suzuki; Shintaro Kobayashi; Hiroki Yamaguchi; Yasuko Orba; Akira Kawaguchi; Hideki Hasegawa; Kiichi Kajino; Takafumi Ninomiya; Kuniharu Ijiro; Hirofumi Sawa
    ACS NANO, 7, 5, 3926, 3938, May 2013, [Peer-reviewed], [International Magazine]
    English, Scientific journal
  • Virus-like particles with removable cyclodextrins enable glutathione-triggered drug release in cells
    Kenichi Niikura; Naotoshi Sugimura; Yusuke Musashi; Shintaro Mikuni; Yasutaka Matsuo; Shintaro Kobayashi; Keita Nagakawa; Shuko Takahara; Chie Takeuchi; Hirofumi Sawa; Masataka Kinjo; Kuniharu Ijiro
    Molecular BioSystems, 9, 3, 501, 507, Mar. 2013, [Peer-reviewed], [International Magazine]
    English, Scientific journal
  • Role of JC virus agnoprotein in virion formation
    Tadaki Suzuki; Shingo Semba; Yuji Sunden; Yasuko Orba; Shintaro Kobayashi; Kazuo Nagashima; Takashi Kimura; Hideki Hasegawa; Hirofumi Sawa
    MICROBIOLOGY AND IMMUNOLOGY, 56, 9, 639, 646, Sep. 2012, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal
  • Accumulation of ubiquitinated proteins is related to West Nile virus-induced neuronal apoptosis
    Shintaro Kobayashi; Yasuko Orba; Hiroki Yamaguchi; Takashi Kimura; Hirofumi Sawa
    NEUROPATHOLOGY, 32, 4, 398, 405, Aug. 2012, [Peer-reviewed], [Lead author], [Domestic magazines]
    English, Scientific journal
  • Inhibitory effects of an M2-specific monoclonal antibody on different strains of influenza A virus
    Nilton Akio Muto; Reiko Yoshida; Tadaki Suzuki; Shintaro Kobayashi; Hiroichi Ozaki; Daisuke Fujikura; Rashid Manzoor; Mieko Muramatsu; Ayato Takada; Takashi Kimura; Hirofumi Sawa
    JAPANESE JOURNAL OF VETERINARY RESEARCH, 60, 2-3, 71, 83, Aug. 2012, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal
  • Detection and characterization of a novel polyomavirus in wild rodents
    Yasuko Orba; Shintaro Kobayashi; Ichiro Nakamura; Akihiro Ishii; Bernard M. Hang'ombe; Aaron S. Mweene; Yuka Thomas; Takashi Kimura; Hirofumi Sawa
    JOURNAL OF GENERAL VIROLOGY, 92, Pt 4, 789, 795, Apr. 2011, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal
  • Non-Cytopathic Bovine Viral Diarrhea Virus Infection Inhibits Differentiation of Bovine Neural Stem/progenitor Cells into Astrocytes in Vitro
    Kazuya Matsuda; Shintaro Kobayashi; Ken-ichiro Kameyama; Michiko Sato; Masateru Koiwa; Yoshihiro Sakoda; Hiroyuki Taniyama
    JOURNAL OF VETERINARY MEDICAL SCIENCE, 72, 7, 903, 907, Jul. 2010, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal
  • Systemic Candidiasis and Mesenteric Mast Cell Tumor with Multiple Metastases in a Dog
    Kazuya Matsuda; Kanako Sakaguchi; Shintaro Kobayashi; Makiko Tominaga; Kazuko Hirayama; Tsuyoshi Kadosawa; Hiroyuki Taniyama
    JOURNAL OF VETERINARY MEDICAL SCIENCE, 71, 2, 229, 232, Feb. 2009, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal
  • Tubulopapillary carcinoma with spindle cell metaplasia of the mammary gland in a cat
    Kazuya Matsuda; Shintaro Kobayashi; Misato Yamashita; Kazuko Hirayama; Tsuyoshi Kadosawa; Hiroyuki Taniyama
    JOURNAL OF VETERINARY MEDICAL SCIENCE, 70, 5, 479, 481, May 2008, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal
■ Other Activities and Achievements
■ Books and other publications
  • 獣医公衆衛生学
    獣医公衆衛生学教育研修協議会, 黄熱、日本脳炎、ウエストナイル熱、ダニ媒介性脳炎、デング熱、ジカウイルス感染症、チクングニア熱
    文永堂出版, Mar. 2024, 9784830032899, xviii, 477p, Japanese, [Contributor]
  • 北海道におけるダニ媒介脳炎の発生状況調査およびその新規診断法について
    渡 慧; 山口 宏樹; 三津橋 和也; 田宮 和真; 小林 進太郎; 好井 健太朗; 松田 麻未; 鈴木 亮介
    日本臨床ウイルス学会, Sep. 2023, Japanese
  • ダニ媒介脳炎における検査法の評価および後方視的調査結果について
    山口 宏樹; 駒込 理佳; 三好 正浩; 伊東 拓也; 後藤 明子; 三津橋 和也; 渡 慧; 山野 公明; 小林 進太郎; 苅和 宏明; 好井 健太朗
    国立感染症研究所, Aug. 2023, Japanese, 2017年6月～2023年3月までのダニ媒介脳炎の検査結果を示し、その検査法の有用性について検討した。さらに、2017年6月以前のダニ媒介感染症疑い症例における後方視的調査結果を報告した。当所に搬入された294症例369検体についてELISAおよび中和試験を実施した。その結果、新たに3症例(6検体)からダニ媒介脳炎ウイルス(TBEV)に対する特異的抗体が検出された。ELISAと中和試験の結果を比較した結果、IgM捕捉ELISAにおいて陽性の6検体では偽陰性・偽陽性はみられず、IgG-ELISAで陰性、中和試験において抗体が検出された3検体はそれぞれの回復期血清においてIgG抗体陽転が確認された。2017年6月以前に当所に搬入された88症例99検体において後方視的調査を実施した。その結果、3症例(3検体)からIgM抗体および中和抗体が検出され、新たに2名のTBEV抗体陽性症例の存在が明らかとなった。
  • One Health for dog-mediated rabies elimination in Asia : a collection of local experiences
    Chiho Kaneko; Kentaro Yoshii; Yumi Kirino; Shintaro Kobayashi; Genki Arikawa; Akitoyo Hotta; Naoaki Misawa; Satoshi Inoue, The ‘World Café’: Strengthening Rabies Prevention with the Government–Academia Collaboration in Japan
    CABI, 2023, 9781800622951, xiii, 258p., English, [Contributor]
  • 北海道獣医師会が行った飼育犬・猫のダニ媒介性脳炎の疫学調査について
    上田 広之; 小林 進太郎; 好井 健太朗
    (公社)北海道獣医師会, Aug. 2022, Japanese
  • 創薬研究者がこれだけは知っておきたい最新のウイルス学
    技術情報協会, ウエストナイルウイルスの増殖および病原性発現メカニズム
    技術情報協会, Aug. 2021, 9784861048555, 602p, Japanese, [Contributor]
  • [Elucidation of neuropathogenesis of West Nile Encephalitis].
    Shintaro Kobayashi
    2021, Japanese, West Nile virus, which causes serious encephalitis in humans and horses, infects neuronal cells and induces cell death. As the neuronal cell death leads to the induction of various inflammatory responses, elucidation of the molecular mechanism of cell death is important for development of a treatment for West Nile encephalitis. In this paper, we investigated the pathology of the neuronal cells infected with West Nile virus and summarized the mechanism of neuronal cell death and their effect on the neuropathogenesis.
  • 【グローバル化・温暖化と感染症対策】節足動物媒介感染症 ダニ媒介性脳炎
    小林 進太郎; 好井 健太朗
    (株)日本小児医事出版社, Dec. 2017, Japanese
  • 蚊媒介性ウイルス感染症理解の最前線 7.ウエストナイル熱/ウエストナイル脳炎
    小林進太郎; 好井健太朗
    (株)医薬ジャーナル社, Jul. 2017, Japanese
  • ポリオーマウイルスの疫学研究と基礎研究
    澤 洋文; 小林 進太郎; 鈴木 忠樹; 大場 靖子
    日本ウイルス学会, Jun. 2014, Japanese
■ Lectures, oral presentations, etc.
  • ウイルス研究から紐解く細胞理解
    小林 進太郎
    慶應薬学先端実学（サイヤンス）セミナー, 30 Oct. 2025
    [Invited]
  • フラビウイルス感染症の病態形成機構および制御法に関する研究
    小林 進太郎
    第168回日本獣 医学会学術集会, 04 Sep. 2025, Japanese, Invited oral presentation
    [Invited]
  • Nuclear deformation caused by West Nile virus infection affects viral replication and cellular function
    Shintaro Kobayashi
    U.S.-Japan Cooperative Medical Sciences Program (USJCMSP) Viral Diseases Panel Meeting, 15 Mar. 2025, English, Invited oral presentation
    [Invited]
  • West Nile virus Capsid protein induces nuclear membrane loss and promotes viral replication
    Shintaro Kobayashi
    Viral Diseases Panel Meeting, U.S.-Japan Cooperative Medical Sciences Program, 08 Mar. 2024, Invited oral presentation
    [Invited]
  • 細胞質で増殖するウイルスの核への影響の研究
    小林進太郎
    第2回感染症・分子シンポジウム, 16 Feb. 2024, Public discourse
    [Invited]
  • ウエストナイルウイルス感染による神経細胞の傷害メカニズム
    小林進太郎
    令和5年度第49回大学院セミナー（長崎大学）, 25 Oct. 2023, Public discourse
    [Invited]
  • ウイルス性脳炎における神経細胞の傷害メカニズム
    小林 進太郎
    第9回日本獣医病理学専門家協会学術集会, 30 Mar. 2022, Public discourse
    [Invited]
  • フラビウイルス感染による病態形成機構の解明および診断法の開発
    小林 進太郎
    第20回日本農学進歩賞受賞者講演, 26 Nov. 2021, Invited oral presentation
    [Invited]
  • ウエストナイルウイルスの脳炎病態の形成機構の解明
    小林 進太郎
    第68回日本ウイルス学会学術集会, 17 Nov. 2021
    [Invited]
  • 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
    小林進太郎
    大阪大学微生物病研究所セミナー, 31 May 2019, Japanese, Public discourse
    [Invited]
  • Role of autophagy in the pathogenesis of West Nile virus infection
    Shintaro Kobayashi; Kentaro Yoshii; Hirofumi Sawa; Hiroaki Kariwa
    Joint Czechoslovak Virology Conference 2019, 14 Feb. 2019, English, Invited oral presentation
    [Invited]
  • 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
    小林進太郎
    第160回日本獣医学会学術集会, 14 Sep. 2017, Japanese, Others
    [Invited], [Domestic Conference]
  • ウエストナイルウイルスとオートファジー
    小林進太郎
    第160回日本獣医学会学術集会, 14 Sep. 2017, Japanese, Nominated symposium
    [Invited], [Domestic Conference]
■ Syllabus
  • 獣医公衆衛生学特論, 2024年, 博士後期課程, 獣医学院
  • 獣医公衆衛生学特論, 2024年, 博士後期課程, 国際感染症学院
  • 人獣共通感染症対策専門特論, 2024年, 博士後期課程, 国際感染症学院
  • アドバンスト演習, 2024年, 学士課程, 獣医学部
  • アドバンスト演習, 2024年, 学士課程, 獣医学部
  • アドバンスト演習, 2024年, 学士課程, 獣医学部
  • 畜産食品衛生学, 2024年, 学士課程, 農学部
  • 動物衛生学実習, 2024年, 学士課程, 獣医学部
  • 獣医公衆衛生学実習, 2024年, 学士課程, 獣医学部
  • アドバンスト演習, 2024年, 学士課程, 獣医学部
■ Affiliated academic society
  • THE JAPANESE SOCIETY FOR VIROLOGY
  • THE MOLECULAR BIOLOGY SOCIETY OF JAPAN
  • JAPANESE SOCIETY OF VETERINARY SCIENCE
■ Research Themes
  • ウイルス性脳炎による神経学的後遺症に対するミクログリアの神経転換の効果の検証
    科学研究費助成事業
    27 Jun. 2025 - 31 Mar. 2028
    小林 進太郎
    日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 25K22699
  • 多分野融合AI創薬プラットフォームによるウエストナイルウイルス病原性PPI阻害薬の創製
    新興・再興感染症研究基盤創生事業（多分野融合研究領域）
    Oct. 2025 - Mar. 2028
    日本医療研究開発機構, Coinvestigator
  • Interorganelle communication between nucleus and endoplasmic reticulum in Flavivirus infection
    Grants-in-Aid for Scientific Research
    01 Apr. 2024 - 31 Mar. 2027
    小林 進太郎
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 24K01917
  • タイをフィールドとして活用する新たなフラビウイルスエコロジーの理解
    2026（令和8）年度 アジア地域重点学術助成
    Apr. 2026 - Mar. 2027
    公益財団法人 平和中島財団, Principal investigator
  • フラビウイルスの細胞内増殖における核内膜タンパク質のリン酸化の分子機構および意義の解明
    2026年度新興感染症制御研究拠点共同研究
    Apr. 2026 - Mar. 2027
    長崎大学, Principal investigator
  • フラビウイルス感染で起こる核膜消失による核と小胞体のオルガネラ間コミュニケーションの感染病理学的意義の解明
    2024年度医学系研究継続助成
    Oct. 2024 - Mar. 2027
    公益財団法人武田科学振興財団, Principal investigator
  • Development of a new serodiagnostic method for tick-borne encephalitis by immunochromatography
    Grants-in-Aid for Scientific Research
    Apr. 2024 - Mar. 2027
    苅和 宏明; 小林 進太郎
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 24K13434
  • ウイルスを用いた核と小胞体ダイナミクスの関連の解明
    2024年度助成金
    Jan. 2025 - Dec. 2026
    公益財団法人アステラス病態代謝研究会, Principal investigator
  • 核内膜タンパク質の小胞体への局在変化に着目した神経向性フラビウイルスの細胞内増殖機構の解明
    2025年度新興感染症制御研究拠点共同研究
    Apr. 2025 - Mar. 2026
    長崎大学, Principal investigator
  • ウイルスを用いた核内膜タンパク質の小胞体への局在変化の分子機構と生物学的意義の解明
    第55回（2023年度）内藤記念科学奨励金・研究助成
    Oct. 2023 - Sep. 2025
    公益財団法人内藤記念科学振興財団, Principal investigator, Competitive research funding
  • フラビウイルス性脳炎における核膜の構造変化の病理学的意義の解析
    2024年度新興感染症制御研究拠点共同研究
    Apr. 2024 - Mar. 2025
    長崎大学, Principal investigator
  • a
    Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))
    Oct. 2021 - Mar. 2025
    好井 健太朗
    マダニは多様な病原体を媒介し、中でも致死性が高いダニ媒介性人獣共通感染症ウイルスは旧大陸の広範な地域で流行しており、その流行防止対策は重要な課題となっている。そのためには、マダニにおけるウイルス感染機構を明らかにし、それを対象とした疾病制御法を確立する必要がある。しかしマダニの細胞や生体の飼育系等、解析するための実験系が確立していないため、ウイルス感染機構の詳細な解析は世界的にも困難な状況にある。そこで本研究では、マダニのバイオリソースを用いてウイルス感染モデルの構築を行い、それを活用してダニ媒介性ウイルスの感染・伝播機序の解析を行う。
    本年度の研究では、多様なマダニ由来培養細胞に対して、ダニ媒介ウイルスとしてダニ媒介性脳炎ウイルス、ランガットウイルス、ハザラウイルス等を感染させて、ウイルスの増殖性や感染による宿主細胞への影響の解析を行った。いずれのウイルスも哺乳動物由来培養細胞ではウイルスの増殖と共に細胞変性効果が認められたのに対して、マダニ由来培養細胞で効率的に増殖していたものの細胞変性効果等を示すことなく、長期間にわたってウイルスが持続感染する事が示された。ダニ媒介性脳炎ウイルスについて、ウイルス遺伝子RNA中の3’UTR領域に高度に保存されている領域に欠損を持つウイルスを感染させたところ、完全長の配列を持つウイルスと比較して、初期のウイルス増殖に立ち遅れが認められた。この領域は、マダニ等の自然界での分離株では高度に保存されており、ヒトの脳炎患者や実験動物での継代株などで欠損が認められることから、媒介動物であるマダニの中での効率的な増殖に関与している可能性が示唆される。
    Japan Society for the Promotion of Science, Fund for the Promotion of Joint International Research (Fostering Joint International Research (B)), Nagasaki University, Coinvestigator, 21KK0123
  • フラビウイルス感染による核内膜タンパク質の小胞体局在のウイルス学および病理学的意義の解明
    2023年度次世代育成支援研究助成金
    Jan. 2024 - Dec. 2024
    公益財団法人シオノギ感染症研究振興財団, Principal investigator
  • 脳炎発症につながる神経向性フラビウイルスの中枢神経組織侵入の分子機構の解明
    令和5年度北海道大学遺伝子病制御研究所共同研究（一般共同研究）
    Apr. 2023 - Mar. 2024
    北海道大学遺伝子病制御研究所, Principal investigator
  • フラビウイルス性脳炎の神経細胞死とTDP-43の細胞質内蓄積の関連の解析
    2023年度新興感染症制御研究拠点共同研究（継続分）
    Apr. 2023 - Mar. 2024
    長崎大学, Principal investigator
  • 神経向性フラビウイルス感染によるタンパク質の核内輸送の抑制が脳炎病態の形成に与える影響の解析
    研究助成ー感染症領域ー
    Jan. 2022 - Jan. 2024
    公益財団法人 MSD生命科学財団, Principal investigator
  • フラビウイルス性脳炎の神経細胞死とTDP-43の細胞質内蓄積の関連の解析
    2022年度 新興感染症制御研究拠点共同研究
    Oct. 2022 - Mar. 2023
    長崎大学, Principal investigator
  • 北海道で分離したウイルス株の比較によるダニ媒介性脳炎の病態形成機構の解明
    研究助成（一般）
    Sep. 2022 - Mar. 2023
    公益財団法人 秋山記念生命科学振興財団, 北海道大学, Principal investigator, Competitive research funding
  • ウイルスゲノム複製複合体内に局在するウイルス由来二本鎖RNAを標的にしたフラビウイルス感染症の治療法の開発
    新興・再興感染症に対する革新的医薬品等開発推進研究事業
    Jul. 2022 - Mar. 2023
    日本医療研究開発機構, 北海道大学, Principal investigator, Competitive research funding, 21fk0108567h0001
  • 神経向性フラビウイルスの脳内侵入部位および機構の解明
    令和4年度北海道大学遺伝子病制御研究所共同利用・共同研究拠点（一般共同研究）
    Apr. 2022 - Mar. 2023
    北海道大学遺伝子病制御研究所, Principal investigator
  • 神経向性フラビウイルス感染による脳炎の病態形成機構の解明につながる核-細胞質タンパク質輸送に関する研究
    2021年度 医学系研究助成
    Oct. 2021 - Mar. 2023
    公益財団法人 武田科学振興財団, Principal investigator
  • 血液脳関門を透過する新規DDSによる神経向性ウイルス感染の治療法開発
    科学研究費助成事業 基盤研究(B)
    Apr. 2020 - Mar. 2023
    好井 健太朗; 小林 進太郎; 五十嵐 学; 今内 覚
    ダニ媒介性脳炎、日本脳炎、狂犬病など神経向性の人獣共通感染症ウイルスは、脳に侵入・増殖することで重篤な神経症状を引き起こし、致死率も高い。しかし血液脳関門：BBBの存在のため、抗ウイルス分子をウイルスの増殖する脳に到達させる手法が無く、治療法が未開発であった。BBBにはトランスサイトーシスと呼ばれる物質を血流から脳内へと輸送する機構があり、近年の研究により、このBBB透過に関わる分子の機構が明らかになってきている。本研究では、このBBB透過機構に着目し、組換え抗体等の抗ウイルス分子技術と融合させることで、BBBを透過する機能を利用した抗ウイルス分子の脳内導入法を構築し、神経向性ウイルス感染に対する治療応用を試みる。
    本年度における研究では、昨年度の研究で構築した、ダニ媒介性脳炎ウイルス（TBEV）の幅広いウイルス株に対して中和効果を示すモノクローナル抗体をベースとして、BBBのトランスサイトーシスへの関与が示されている狂犬病ウイルスのG蛋白上のアミノ酸配列を融合させた組換え抗体を用いての研究を進めた。本抗体に誘導させた、狂犬病ウイルスのG蛋白上のアミノ酸配列は、神経細胞やBBBの血管内皮細胞上に発現するアセチルコリンレセプターとの結合により細胞内輸送小胞に取り込まれて、これがトランスサイトーシスに関与する事が示されている。そこで本研究ではアセチルコリンレセプターを細胞膜上に豊富に発現している神経細胞由来の培養細胞等を用いて検証した所、作製した組換え抗体分子は細胞膜表面に結合している事が示され、これはアセチルコリンレセプターを介したものである事が示唆された。さらに抗体をマウスの末梢に投与した所、マウスの脳内から抗体が検出されたことから、抗体は脳内に移行していることが示され、本研究で検証した組換え抗体分子は、脳内で増殖するウイルスに対する治療応用に有効である可能性が示唆された。
    日本学術振興会, 基盤研究(B), 長崎大学, Coinvestigator, 20H03136
  • Elucidation of the pathogenesis of flaviviral encephalitis focusing on the activation of microglia
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Apr. 2020 - Mar. 2023
    小林 進太郎
    脳内の免疫細胞であるミクログリアは変性した神経細胞に由来する分子スイッチ因子によって神経保護と神経傷害の両タイプの作用を示し、そのバランスが病態形成に重要である。 世界中で流行し、人獣共通感染症の原因となるフラビウイルスの感染によって起こるフラビウイルス性脳炎の病態形成機構の解明のために、神経向性フラビウイルス感染により産生される神経保護または神経傷害型ミクログリアへの活性化を促す分子スイッチ因子を特定し、活性化ミクログリアの脳炎病態における役割を明らかにする。これらにより、フラビウイルス性脳炎発症機構における活性化ミクログリアの重要性を明らかにし、治療法開発のための基盤形成を目指す。
    神経向性フラビウイルスであるウエストナイルウイルス(WNV)が感染したマウスの脳組織を用いた免疫組織学的解析で、WNVの感染によりIba1やTMEM119などの一般的なミクログリアマーカー陽性細胞数が増加し、またその形態も変化することが明らかになった。ミクログリアマーカー陽性細胞の細胞集団にはマーカーの発現が異なる細胞が含まれており、マーカーの発現が強い細胞はウイルス抗原陽性細胞の遠くに、弱い細胞は近傍に観察された。また、ミクログリアマーカーの発現が弱い細胞はDisease-associated microglia (DAM)のマーカーであるCD11c/ITGAX陽性であった。
    DAMの機能的な解析は進んでおらず、今後はDAMのWNV感染に対する役割や機能の解析および、ミクログリアの培養系を用いてDAMへの分化の分子機構について明らかにする予定である。
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 20K06406
  • 神経向性フラビウイルスの脳内侵入部位および機構の解明
    令和3年度遺伝子病制御研究所一般共同研究
    Apr. 2021 - Mar. 2022
    北海道大学遺伝子病制御研究所, Principal investigator
  • TDP-43の凝集体形成に着目したフラビウイルス性脳炎の分子病態の解明
    令和3年度熱帯医学研究拠点一般共同研究
    Apr. 2021 - Mar. 2022
    長崎大学熱帯医学研究所
  • シングルセルレベルでの活性化ミクログリアの解析によるフラビウイ ルス性脳炎の病態形成メカニズムの解明
    第35回（令和2年度）研究助成
    Apr. 2021 - Mar. 2022
    公益財団法人 寿原記念財団, Principal investigator
  • Elucidation of pathogenesis of flavivirus encephalitis by analysis of activated microglia at the single cell level
    第35回基礎医学医療研究助成
    Oct. 2020 - Sep. 2021
    公益財団法人 金原一郎記念医学医療振興財団, Principal investigator
  • 遺伝子組換え技術を用いたダニ媒介性脳炎ウイルスの神経病原性発現機構の解析
    研究助成
    Sep. 2020 - Mar. 2021
    公益財団法人 栗林育英学術財団, Principal investigator
  • 北海道の伴侶動物における人獣共通感染症の血清疫学的調査
    2020年度臨床研究推進研究費
    Jul. 2020 - Mar. 2021
    北海道大学大学院獣医学研究院
  • 神経向性フラビウイルスの脳内侵入部位および機構の解明
    一般共同研究
    Apr. 2020 - Mar. 2021
    北海道大学遺伝子病制御研究所
  • 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
    科学研究費補助金（若手研究）
    Apr. 2018 - Mar. 2020
    小林進太郎
    文部科学省, Principal investigator, Competitive research funding
  • Regulation of flavivirus infection by virus-derived long non-coding RNA
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Apr. 2017 - Mar. 2020
    YOSHII Kentaro
    The 3'-untranslated region (3'-UTR) of tick-borne encephalitis virus (TBEV) plays an important role in the viral pathogenesis. In this study, we investigated detailed mechanisms of virus-derived long non-coding RNA (lncRNA) from the 3'-UTR of TBEV in TBEV infection. Deletions in the 3'-UTR increased virulence in a mouse model and affected the production of virus-derived lncRNA. We identified host proteins interacting the 3'-UTR of TBEV RNA in mammalian and tick cells, and revealed that the interaction affected viral replication mechanisms. These insights are useful for understanding virus transmission and pathogenic mechanism of flavivirus.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, Coinvestigator, 17H03910
  • ウイルス性神経疾患でのオートファジーの機能解析
    平成30年度若手研究者研究加速事業
    Nov. 2018 - Mar. 2019
    小林進太郎
    北海道大学, Principal investigator, Competitive research funding
  • 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
    医学系研究奨励
    Nov. 2017 - Mar. 2019
    小林進太郎
    公益財団法人 武田科学振興財団, Principal investigator, Competitive research funding
  • 神経向性ウイルスの脳内侵入機構の解明とその応用
    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）
    Aug. 2017 - Mar. 2018
    小林進太郎
    ノーステック財団, Principal investigator, Competitive research funding
  • 神経向性フラビウイルスの脳内侵入メカニズムの解明
    研究助成（奨励）
    2016 - 2017
    小林進太郎
    秋山記念生命科学振興財団, Principal investigator, Competitive research funding
  • Analysis of release of mosquito-borne flaviviral particles
    Grants-in-Aid for Scientific Research
    2015 - 2016
    Kobayashi Shintaro
    We performed siRNA-based screening for silencing 18 Rab genes that are related to the vesicle transport from the ER to the plasma membrane. The siRNA screen revealed that KD of Rab8b significantly decreased VLP release efficiency compared with control siRNA-transfected cells. RNAi analysis revealed that Rab8b plays a role in WNV particle release. 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.
    Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, Principal investigator, Competitive research funding, 15K19069
  • 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
    グローバルCOEプログラム若手研究者研究活動経費
    May 2012 - Mar. 2013
    小林進太郎
    北海道大学, Principal investigator, Competitive research funding
  • Elucidation of the pathogenesis of West Nile encephalomyelitis that focused on autophagy
    Grants-in-Aid for Scientific Research
    2012 - 2013
    KOBAYASHI SHINTARO
    Autophagy is one of the intracellular protein degradation system that is implicated in elimination of denatured proteins and infectious microbes. It is expected that inhibition of autophagy is cause of West Nile encephalomyelitis. In this study, we found that autophagy was inhibited by NS2B/3 that is non-structural proteins of WNV. We also found autophagy inhibited West Nile virus replication at the viral genome replication and gene expression stages.
    Japan Society for the Promotion of Science, Grant-in-Aid for Research Activity Start-up, Hokkaido University, Principal investigator, Competitive research funding, 24880002
  • ウエストナイルウイルスの神経病原性発現機構の解析
    グローバルCOEプログラム若手研究者研究活動経費
    May 2011 - Mar. 2012
    小林進太郎
    北海道大学, Principal investigator, Competitive research funding
  • ポリオーマウイルスの粒子形成機構の解析
    グローバルCOEプログラム若手研究者研究活動経費
    Dec. 2010 - Mar. 2011
    小林進太郎
    北海道大学, Principal investigator, Competitive research funding
■ Industrial Property Rights
  • ロッド型ナノ粒子及び核酸からなる組成物
    Patent right, 新倉謙一; 居城邦治; 澤洋文; 大場靖子; 小林進太郎; 田崎太悠; 鈴木忠樹; 大原有樹; 中野哲郎, 国立大学法人北海道大学, 国立感染症研究所長, 協和発酵バイオ株式会社
    特願2014-252111, 12 Dec. 2014
    特開2016-113388, 23 Jun. 2016