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研究者情報

マスター

アカウント（マスター）

  • 氏名

    五十嵐　学(イガラシ　マナブ), イガラシ　マナブ

所属（マスター）

  • 人獣共通感染症国際共同研究所　国際疫学部門

所属（マスター）

  • 人獣共通感染症国際共同研究所　国際疫学部門

独自項目

syllabus

  • 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, Bioinformatics, Computational Biology
  • 2021, 生物統計学特論, Advanced Lecture on Biostatistics, 博士後期課程, 獣医学院
  • 2021, 生物統計学特論, Advanced Lecture on Biostatistics, 博士後期課程, 国際感染症学院
  • 2021, 情報科学特論, Advanced Lecture on Information Science, 博士後期課程, 獣医学院
  • 2021, 情報科学特論, Advanced Lecture on Information Science, 博士後期課程, 国際感染症学院
  • 2021, 製剤開発特論, Advanced Lecture on Pharmaceutical Development, 博士後期課程, 国際感染症学院

researchmap

プロフィール情報

学位

  • 博士（医学）（北海道大学）

プロフィール情報

  • 姓

    五十嵐, イガラシ

  • 名

    学, マナブ

  • ID各種

    201301011296456953

対象リソース

業績リスト

研究キーワード

  • 抗ウイルス薬   分子モデリング   フィロウイルス   分子動力学   蛋白質   分子シミュレーション   生体生命情報学   人獣共通感染症   薬剤耐性   ウイルス   インフルエンザ   インシリコ創薬   計算科学   

研究分野

  • ライフサイエンス / 獣医学

経歴

  • 2015年04月 - 現在 北海道大学電子科学研究所附属社会創造数学研究センター 准教授（兼務）
  • 2014年07月 - 現在 北海道大学人獣共通感染症リサーチセンター 准教授
  • 2011年01月 - 2014年06月 北海道大学人獣共通感染症リサーチセンター 特任助教
  • 2005年12月 - 2010年12月 北海道大学人獣共通感染症リサーチセンター 博士研究員
  • 2004年04月 - 2005年11月 北海道大学大学院医学研究科 博士研究員
  • 2001年04月 - 2004年03月 日本学術振興会特別研究員

学歴

  • 2000年04月 - 2004年03月   北海道大学大学院   医学研究科   生体機能学
  • 1997年04月 - 2000年03月   北海道大学大学院   工学研究科   分子化学
  •         - 1997年03月   北海道大学   工学部   合成化学工学

委員歴

  • 2021年04月 - 現在   日本ウイルス学会   ウイルス学将来構想検討委員

論文

  • Zihan Zhang, Toru Takenaga, Sarah Katharina Fehling, Manabu Igarashi, Takatsugu Hirokawa, Yukiko Muramoto, Koji Yamauchi, Chiho Onishi, Masahiro Nakano, Shuzo Urata, Allison Groseth, Thomas Strecker, Takeshi Noda
    Journal of virology 98 7 e0071424  2024年07月23日 
    Lassa virus (LASV) is the causative agent of human Lassa fever which in severe cases manifests as hemorrhagic fever leading to thousands of deaths annually. However, no approved vaccines or antiviral drugs are currently available. Recently, we screened approximately 2,500 compounds using a recombinant vesicular stomatitis virus (VSV) expressing LASV glycoprotein GP (VSV-LASVGP) and identified a P-glycoprotein inhibitor as a potential LASV entry inhibitor. Here, we show that another identified candidate, hexestrol (HES), an estrogen receptor agonist, is also a LASV entry inhibitor. HES inhibited VSV-LASVGP replication with a 50% inhibitory concentration (IC50) of 0.63 µM. Importantly, HES also inhibited authentic LASV replication with IC50 values of 0.31 µM-0.61 µM. Time-of-addition and cell-based membrane fusion assays suggested that HES inhibits the membrane fusion step during virus entry. Alternative estrogen receptor agonists did not inhibit VSV-LASVGP replication, suggesting that the estrogen receptor itself is unlikely to be involved in the antiviral activity of HES. Generation of a HES-resistant mutant revealed that the phenylalanine at amino acid position 446 (F446) of LASVGP, which is located in the transmembrane region, conferred resistance to HES. Although mutation of F446 enhanced the membrane fusion activity of LASVGP, it exhibited reduced VSV-LASVGP replication, most likely due to the instability of the pre-fusion state of LASVGP. Collectively, our results demonstrated that HES is a promising anti-LASV drug that acts by inhibiting the membrane fusion step of LASV entry. This study also highlights the importance of the LASVGP transmembrane region as a target for anti-LASV drugs.IMPORTANCELassa virus (LASV), the causative agent of Lassa fever, is the most devastating mammarenavirus with respect to its impact on public health in West Africa. However, no approved antiviral drugs or vaccines are currently available. Here, we identified hexestrol (HES), an estrogen receptor agonist, as the potential antiviral candidate drug. We showed that the estrogen receptor itself is not involved in the antiviral activity. HES directly bound to LASVGP and blocked membrane fusion, thereby inhibiting LASV infection. Through the generation of a HES-resistant virus, we found that phenylalanine at position 446 (F446) within the LASVGP transmembrane region plays a crucial role in the antiviral activity of HES. The mutation at F446 caused reduced virus replication, likely due to the instability of the pre-fusion state of LASVGP. These findings highlight the potential of HES as a promising candidate for the development of antiviral compounds targeting LASV.
  • Yannick Munyeku-Bazitama, Takeshi Saito, Takanari Hattori, Hiroko Miyamoto, Boniface Pongombo Lombe, Akina Mori-Kajihara, Masahiro Kajihara, Jean-Jacques Muyembe-Tamfum, Manabu Igarashi, Eun-sil Park, Shigeru Morikawa, Sheila Makiala-Mandanda, Ayato Takada
    Journal of Virology 2024年07月02日 
    ABSTRACT Tibroviruses are novel rhabdoviruses detected in humans, cattle, and arthropods. Four tibroviruses are known to infect humans: Bas-Congo virus (BASV), Ekpoma virus 1 (EKV-1), Ekpoma virus 2, and Mundri virus. However, since none of them has been isolated, their biological properties are largely unknown. We aimed to characterize the human tibrovirus glycoprotein (G), which likely plays a pivotal role in viral tropism and pathogenicity. Human tibrovirus Gs were found to share some primary structures and display 14 conserved cysteine residues, although their overall amino acid homology was low (29%–48%). Multiple potential glycosylation sites were found on the G molecules, and endoglycosidase H- and peptide-N-glycosidase F-sensitive glycosylation was confirmed. AlphaFold-predicted three-dimensional (3D) structures of human tibrovirus Gs were overall similar. Membrane fusion mediated by these tibrovirus Gs was induced by acidic pH. The low pH-induced conformational change that triggers fusion was reversible. Virus-like particles (VLPs) were produced by transient expression of Gs in cultured cells and used to produce mouse antisera. Using vesicular stomatitis Indiana virus pseudotyped with Gs, we found that the antisera to the respective tibrovirus VLPs showed limited cross-neutralizing activity. It was also found that human C-type lectins and T-cell immunoglobulin mucin 1 acted as attachment factors for G-mediated entry into cells. Interestingly, BASV-G showed the highest ability to utilize these molecules. The viruses infected a wide range of cell lines with preferential tropism for human-derived cells whereas the preference of EKV-1 was unique compared with the other human tibroviruses. These findings provide fundamental information to understand the biological properties of the human tibroviruses. IMPORTANCE Human tibroviruses are poorly characterized emerging rhabdoviruses associated with either asymptomatic infection or severe disease with a case fatality rate as high as 60% in humans. However, the extent and burden of human infection as well as factors behind differences in infection outcomes are largely unknown. In this study, we characterized human tibrovirus glycoproteins, which play a key role in virus-host interactions, mainly focusing on their structural and antigenic differences and cellular tropism. Our results provide critical information for understanding the biological properties of these novel viruses and for developing appropriate preparedness interventions such as diagnostic tools, vaccines, and effective therapies.
  • Yannick Munyeku-Bazitama, Patient Okitale-Talunda, Takanari Hattori, Takeshi Saito, Boniface Pongombo Lombe, Hiroko Miyamoto, Akina Mori-Kajihara, Masahiro Kajihara, Agathe Bikupe Nkoy, Augustin Tshibwabwa Twabela, Justin Masumu, Steve Ahuka-Mundeke, Jean-Jacques Muyembe-Tamfum, Manabu Igarashi, Eun-sil Park, Shigeru Morikawa, Sheila Makiala-Mandanda, Ayato Takada
    The Lancet Microbe 2024年03月
  • Yuta Tsukamoto, Manabu Igarashi, Hiroki Kato
    Cell chemical biology 2023年12月05日 
    Methylation is one of the critical modifications that regulates numerous biological processes. Guanine capping and methylation at the 7th position (m7G) have been shown to mature mRNA for increased RNA stability and translational efficiency. The m7G capped cap0 RNA remains immature and requires additional methylation at the first nucleotide (N1-2'-O-Me), designated as cap1, to achieve full maturation. This cap1 RNA with N1-2'-O-Me prevents its recognition by innate immune sensors as non-self. Viruses have also evolved various strategies to produce self-like capped RNAs with the N1-2'-O-Me that potentially evades the antiviral response and establishes an efficient replication. In this review, we focus on the importance of the presence of N1-2'-O-Me in viral RNAs and discuss the potential for drug development by targeting host and viral N1-2'-O-methyltransferases.
  • 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 107 24 7515 - 7529 2023年10月13日 
    Abstract 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.
  • H13亜型鳥インフルエンザウイルスはオオセグロカモメの呼吸器上皮に分布するフコシル化α2,3シアル酸糖鎖を認識する
    原田 里桜, 日尾野 隆大, 小林 大樹, 伴 日向子, 五十嵐 学, 磯田 典和, 迫田 義博
    日本獣医学会学術集会講演要旨集 166回 130 - 130 (公社)日本獣医学会 2023年09月
  • Manabu Igarashi, Takatsugu Hirokawa, Ayato Takada
    The Journal of Infectious Diseases 228 Supplement_7 S479 - S487 2023年04月29日 
    Abstract Background Our previous study demonstrated that the fruit bat (Yaeyama flying fox)-derived cell line FBKT1 showed preferential susceptibility to Ebola virus (EBOV), whereas the human cell line HEK293T was similarly susceptible to EBOV and Marburg virus (MARV). This was due to 3 amino acid differences of the endosomal receptor Niemann-Pick C1 (NPC1) between FBKT1 and HEK293T (ie, TET and SGA, respectively, at positions 425–427), as well as 2 amino acid differences at positions 87 and 142 of the viral glycoprotein (GP) between EBOV and MARV. Methods/Results To understand the contribution of these amino acid differences to interactions between NPC1 and GP, we performed molecular dynamics simulations and binding free energy calculations. The average binding free energies of human NPC1 (hNPC1) and its mutant having TET at positions 425–427 (hNPC1/TET) were similar for the interaction with EBOV GP. In contrast, hNPC1/TET had a weaker interaction with MARV GP than wild-type hNPC1. As expected, substitutions of amino acid residues at 87 or 142 in EBOV and MARV GPs converted the binding affinity to hNPC1/TET. Conclusions Our data provide structural and energetic insights for understanding potential differences in the GP-NPC1 interaction, which could influence the host tropism of EBOV and MARV.
  • Yuta Tsukamoto, Takahiro Hiono, Shintaro Yamada, Keita Matsuno, Aileen Faist, Tobias Claff, Jianyu Hou, Vigneshwaran Namasivayam, Anja Vom Hemdt, Satoko Sugimoto, Jin Ying Ng, Maria H Christensen, Yonas M Tesfamariam, Steven Wolter, Stefan Juranek, Thomas Zillinger, Stefan Bauer, Takatsugu Hirokawa, Florian I Schmidt, Georg Kochs, Masayuki Shimojima, Yi-Shuian Huang, Andreas Pichlmair, Beate M Kümmerer, Yoshihiro Sakoda, Martin Schlee, Linda Brunotte, Christa E Müller, Manabu Igarashi, Hiroki Kato
    Science (New York, N.Y.) 379 6632 586 - 591 2023年02月10日 
    Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.
  • Takanari Hattori, Takeshi Saito, Hiroko Miyamoto, Masahiro Kajihara, Manabu Igarashi, Ayato Takada
    Viruses 14 10 2124 - 2124 2022年09月26日 
    Human T-cell immunoglobulin mucin 1 (hTIM-1) is known to promote cellular entry of enveloped viruses. Previous studies suggested that the polymorphisms of hTIM-1 affected its function. Here, we analyzed single nucleotide variants (SNVs) of hTIM-1 to determine their ability to promote cellular entry of viruses using pseudotyped vesicular stomatitis Indiana virus (VSIV). We obtained hTIM-1 sequences from a public database (Ensembl genome browser) and identified 35 missense SNVs in 3 loops of the hTIM-1 immunoglobulin variable (IgV) domain, which had been reported to interact with the Ebola virus glycoprotein (GP) and phosphatidylserine (PS) in the viral envelope. HEK293T cells transiently expressing wildtype hTIM-1 or its SNV mutants were infected with VSIVs pseudotyped with filovirus or arenavirus GPs, and their infectivities were compared. Eleven of the thirty-five SNV substitutions reduced the efficiency of hTIM-1-mediated entry of pseudotyped VSIVs. These SNV substitutions were found not only around the PS-binding pocket but also in other regions of the molecule. Taken together, our findings suggest that some SNVs of the hTIM-1 IgV domain have impaired ability to interact with PS and/or viral GPs in the viral envelope, which may affect the hTIM-1 function to promote viral entry into cells.
  • Valter Bergant, Shintaro Yamada, Vincent Grass, Yuta Tsukamoto, Teresa Lavacca, Karsten Krey, Maria-Teresa Mühlhofer, Sabine Wittmann, Armin Ensser, Alexandra Herrmann, Anja Vom Hemdt, Yuriko Tomita, Shutoku Matsuyama, Takatsugu Hirokawa, Yiqi Huang, Antonio Piras, Constanze A Jakwerth, Madlen Oelsner, Susanne Thieme, Alexander Graf, Stefan Krebs, Helmut Blum, Beate M Kümmerer, Alexey Stukalov, Carsten B Schmidt-Weber, Manabu Igarashi, Thomas Gramberg, Andreas Pichlmair, Hiroki Kato
    The EMBO journal 41 17 e111608  2022年09月01日 
    The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.
  • Haruhiko Kamiki, Shin Murakami, Takashi Nishikaze, Takahiro Hiono, Manabu Igarashi, Yuki Furuse, Hiromichi Matsugo, Hiroho Ishida, Misa Katayama, Wataru Sekine, Yasushi Muraki, Masateru Takahashi, Akiko Takenaka-Uema, Taisuke Horimoto
    Journal of virology e0041622  2022年07月18日 [査読有り]
     
    Avian or human influenza A viruses bind preferentially to avian- or human-type sialic acid receptors, respectively, indicating that receptor tropism is an important factor for determining the viral host range. However, there are currently no reliable methods for analyzing receptor tropism biologically under physiological conditions. In this study, we established a novel system using MDCK cells with avian- or human-type sialic acid receptors and with both sialic acid receptors knocked out (KO). When we examined the replication of human and avian influenza viruses in these KO cells, we observed unique viral receptor tropism that could not be detected using a conventional solid-phase sialylglycan binding assay, which directly assesses physical binding between the virus and sialic acids. Furthermore, we serially passaged an engineered avian-derived H4N5 influenza virus, whose PB2 gene was deleted, in avian-type receptor KO cells stably expressing PB2 to select a mutant with enhanced replication in KO cells; however, its binding to human-type sialylglycan was undetectable using the solid-phase binding assay. These data indicate that a panel of sialic acid receptor KO cells could be a useful tool for determining the biological receptor tropism of influenza A viruses. Moreover, the PB2KO virus experimental system could help to safely and efficiently identify the mutations required for avian influenza viruses to adapt to human cells that could trigger a new influenza pandemic. IMPORTANCE The acquisition of mutations that allow avian influenza A virus hemagglutinins to recognize human-type receptors is mandatory for the transmission of avian viruses to humans, which could lead to a pandemic. In this study, we established a novel system using a set of genetically engineered MDCK cells with knocked out sialic acid receptors to biologically evaluate the receptor tropism for influenza A viruses. Using this system, we observed unique receptor tropism in several virus strains that was undetectable using conventional solid-phase binding assays that measure physical binding between the virus and artificially synthesized sialylglycans. This study contributes to elucidation of the relationship between the physical binding of virus and receptor and viral infectivity. Furthermore, the system using sialic acid knockout cells could provide a useful tool to explore the sialic acid-independent entry mechanism. In addition, our system could be safely used to identify mutations that could acquire human-type receptor tropism.
  • Takanari Hattori, Takeshi Saito, Kosuke Okuya, Yuji Takahashi, Hiroko Miyamoto, Masahiro Kajihara, Manabu Igarashi, Ayato Takada
    Microbiology Spectrum 2022年07月11日 
    SARS-CoV and SARS-CoV-2 are known to cause severe pneumonia in humans. The S protein of these CoVs binds to the ACE2 molecule on the plasma membrane and mediates virus entry into cells.
  • Boniface Pongombo Lombe, Takeshi Saito, Hiroko Miyamoto, Akina Mori-Kajihara, Masahiro Kajihara, Masayuki Saijo, Justin Masumu, Takanari Hattori, Manabu Igarashi, Ayato Takada
    Viruses 14 3 2022年03月06日 
    Crimean-Congo hemorrhagic fever virus (CCHFV), a nairovirus, is a tick-borne zoonotic virus that causes hemorrhagic fever in humans. The CCHFV nucleoprotein (NP) is the antigen most used for serological screening of CCHFV infection in animals and humans. To gain insights into antibody epitopes on the NP molecule, we produced recombinant chimeric NPs between CCHFV and Nairobi sheep disease virus (NSDV), which is another nairovirus, and tested rabbit and mouse antisera/immune ascites, anti-NP monoclonal antibodies, and CCHFV-infected animal/human sera for their reactivities to the NP antigens. We found that the amino acids at positions 161-320 might include dominant epitopes recognized by anti-CCHFV IgG antibodies, whereas cross-reactivity between anti-CCHFV and anti-NSDV antibodies was limited. Their binding capacities were further tested using a series of synthetic peptides whose sequences were derived from CCHFV NP. IgG antibodies in CCHFV-infected monkeys and patients were reactive to some of the synthetic peptide antigens (e.g., amino acid residues at positions 131-150 and 211-230). Only a few peptides were recognized by IgG antibodies in the anti-NSDV serum. These results provide useful information to improve NP-based antibody detection assays as well as antigen detection tests relying on anti-NP monoclonal antibodies.
  • Yoko Fujita-Fujiharu, Yukihiko Sugita, Yuki Takamatsu, Kazuya Houri, Manabu Igarashi, Yukiko Muramoto, Masahiro Nakano, Yugo Tsunoda, Ichiro Taniguchi, Stephan Becker, Takeshi Noda
    Nature communications 13 1 1191 - 1191 2022年03月04日 
    The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP-RNA complex. The NP-RNA complex constitutes the core structure for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP-RNA complex determined using cryo-electron microscopy at a resolution of 3.1 Å. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for helical assembly and subsequent viral RNA synthesis. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV.
  • Mohini Yadav, Manabu Igarashi, Norifumi Yamamoto
    PeerJ Physical Chemistry 3 e19 - e19 2021年11月22日 
    The substitution of Ile to Val at residue 117 (I117V) of neuraminidase (NA) reduces the susceptibility of the A/H5N1 influenza virus to oseltamivir (OTV). However, the molecular mechanism by which the I117V mutation affects the intermolecular interactions between NA and OTV has not been fully elucidated. In this study, we performed molecular dynamics (MD) simulations to analyze the characteristic conformational changes that contribute to the reduced binding affinity of NA to OTV after the I117V mutation. The results of MD simulations revealed that after the I117V mutation in NA, the changes in the secondary structure around the mutation site had a noticeable effect on the residue interactions in the OTV-binding site. In the case of the WT NA-OTV complex, the positively charged side chain of R118, located in the β-sheet region, frequently interacted with the negatively charged side chain of E119, which is an amino acid residue in the OTV-binding site. This can reduce the electrostatic repulsion of E119 toward D151, which is also a negatively charged residue in the OTV-binding site, so that both E119 and D151 simultaneously form hydrogen bonds with OTV more frequently, which greatly contributes to the binding affinity of NA to OTV. After the I117V mutation in NA, the side chain of R118 interacted with the side chain of E119 less frequently, likely because of the decreased tendency of R118 to form a β-sheet structure. As a result, the electrostatic repulsion of E119 toward D151 is greater than that of the WT case, making it difficult for both E119 and D151 to simultaneously form hydrogen bonds with OTV, which in turn reduces the binding affinity of NA to OTV. Hence, after the I117V mutation in NA, influenza viruses are less susceptible to OTV because of conformational changes in residues of R118, E119, and D151 around the mutation site and in the binding site.
  • Nodoka Kasajima, Keita Matsuno, Hiroko Miyamoto, Masahiro Kajihara, Manabu Igarashi, Ayato Takada
    Viruses 13 11 2021年11月20日 
    Viral protein 35 (VP35) of Ebola virus (EBOV) is a multifunctional protein that mainly acts as a viral polymerase cofactor and an interferon antagonist. VP35 interacts with the viral nucleoprotein (NP) and double-stranded RNA for viral RNA transcription/replication and inhibition of type I interferon (IFN) production, respectively. The C-terminal portion of VP35, which is termed the IFN-inhibitory domain (IID), is important for both functions. To further identify critical regions in this domain, we analyzed the physical properties of the surface of VP35 IID, focusing on hydrophobic patches, which are expected to be functional sites that are involved in interactions with other molecules. Based on the known structural information of VP35 IID, three hydrophobic patches were identified on its surface and their biological importance was investigated using minigenome and IFN-β promoter-reporter assays. Site-directed mutagenesis revealed that some of the amino acid substitutions that were predicted to disrupt the hydrophobicity of the patches significantly decreased the efficiency of viral genome replication/transcription due to reduced interaction with NP, suggesting that the hydrophobic patches might be critical for the formation of a replication complex through the interaction with NP. It was also found that the hydrophobic patches were involved in the IFN-inhibitory function of VP35. These results highlight the importance of hydrophobic patches on the surface of EBOV VP35 IID and also indicate that patch analysis is useful for the identification of amino acid residues that directly contribute to protein functions.
  • Taksoo Kim, Loc Tan Huynh, Shizuka Hirose, Manabu Igarashi, Takahiro Hiono, Norikazu Isoda, Yoshihiro Sakoda
    Viruses 13 8 2021年08月23日 
    The GPE- strain is a live attenuated vaccine for classical swine fever (CSF) developed in Japan. In the context of increasing attention for the differentiating infected from vaccinated animals (DIVA) concept, the achievement of CSF eradication with the GPE- proposes it as a preferable backbone for a recombinant CSF marker vaccine. While its infectious cDNA clone, vGPE-, is well characterized, 10 amino acid substitutions were recognized in the genome, compared to the original GPE- vaccine seed. To clarify the GPE- seed availability, this study aimed to generate and characterize a clone possessing the identical amino acid sequence to the GPE- seed. The attempt resulted in the loss of the infectious GPE- seed clone production due to the impaired replication by an amino acid substitution in the viral polymerase NS5B. Accordingly, replication-competent GPE- seed variant clones were produced. Although they were mostly restricted to propagate in the tonsils of pigs, similarly to vGPE-, their type I interferon-inducing capacity was significantly lower than that of vGPE-. Taken together, vGPE- mainly retains ideal properties for the CSF vaccine, compared with the seed variants, and is probably useful in the development of a CSF marker vaccine.
  • Manabu Igarashi, Takatsugu Hirokawa, Yoshihiro Takadate, Ayato Takada
    Viruses 13 5 913 - 913 2021年05月14日 
    Filoviruses, including marburgviruses and ebolaviruses, have a single transmembrane glycoprotein (GP) that facilitates their entry into cells. During entry, GP needs to be cleaved by host proteases to expose the receptor-binding site that binds to the endosomal receptor Niemann-Pick C1 (NPC1) protein. The crystal structure analysis of the cleaved GP (GPcl) of Ebola virus (EBOV) in complex with human NPC1 has demonstrated that NPC1 has two protruding loops (loops 1 and 2), which engage a hydrophobic pocket on the head of EBOV GPcl. However, the molecular interactions between NPC1 and the GPcl of other filoviruses remain unexplored. In the present study, we performed molecular modeling and molecular dynamics simulations of NPC1 complexed with GPcls of two ebolaviruses, EBOV and Sudan virus (SUDV), and one marburgvirus, Ravn virus (RAVV). Similar binding structures were observed in the GPcl–NPC1 complexes of EBOV and SUDV, which differed from that of RAVV. Specifically, in the RAVV GPcl–NPC1 complex, the tip of loop 2 was closer to the pocket edge comprising residues at positions 79–88 of GPcl; the root of loop 1 was predicted to interact with P116 and Q144 of GPcl. Furthermore, in the SUDV GPcl–NPC1 complex, the tip of loop 2 was slightly closer to the residue at position 141 than those in the EBOV and RAVV GPcl–NPC1 complexes. These structural differences may affect the size and/or shape of the receptor-binding pocket of GPcl. Our structural models could provide useful information for improving our understanding the differences in host preference among filoviruses as well as contributing to structure-based drug design.
  • Kazuki Takeda, Yoshinori Ikenaka, Denis Fourches, Kazuyuki D Tanaka, Shouta M M Nakayama, Dhoha Triki, Xinhao Li, Manabu Igarashi, Tsutomu Tanikawa, Mayumi Ishizuka
    Pesticide biochemistry and physiology 173 104774 - 104774 2021年03月 
    Well-known 4-hydroxycoumarin derivatives, such as warfarin, act as inhibitors of the vitamin K epoxide reductase (VKOR) and are used as anticoagulants. Mutations of the VKOR enzyme can lead to resistance to those compounds. This has been a problem in using them as medicine or rodenticide. Most of these mutations lie in the vicinity of potential warfarin-binding sites within the ER-luminal loop structure (Lys30, Phe55) and the transmembrane helix (Tyr138). However, a VKOR mutation found in Tokyo in warfarin-resistant rats does not follow that pattern (Leu76Pro), and its effect on VKOR function and structure remains unclear. We conducted both in vitro kinetic analyses and in silico docking studies to characterize the VKOR mutant. On the one hand, resistant rats (R-rats) showed a 37.5-fold increased IC50 value to warfarin when compared to susceptible rats (S-rats); on the other hand, R-rats showed a 16.5-fold lower basal VKOR activity (Vmax/Km). Docking calculations exhibited that the mutated VKOR of R-rats has a decreased affinity for warfarin. Molecular dynamics simulations further revealed that VKOR-associated warfarin was more exposed to solvents in R-rats and key interactions between Lys30, Phe55, and warfarin were less favored. This study concludes that a single mutation of VKOR at position 76 leads to a significant resistance to warfarin by modifying the types and numbers of intermolecular interactions between the two.
  • Mohini Yadav, Manabu Igarashi, Norifumi Yamamoto
    PeerJ 9 e11552  2021年 
    BACKGROUND: Oseltamivir (OTV)-resistant influenza virus exhibits His-to-Tyr mutation at residue 274 (H274Y) in N1 neuraminidase (NA). However, the molecular mechanisms by which the H274Y mutation in NA reduces its binding affinity to OTV have not been fully elucidated. METHODS: In this study, we used dynamic residue interaction network (dRIN) analysis based on molecular dynamics simulation to investigate the correlation between the OTV binding site of NA and its H274Y mutation site. RESULTS: dRIN analysis revealed that the OTV binding site and H274Y mutation site of NA interact via the three interface residues connecting them. H274Y mutation significantly enhanced the interaction between residue 274 and the three interface residues in NA, thereby significantly decreasing the interaction between OTV and its surrounding loop 150 residues. Thus, we concluded that such changes in residue interactions could reduce the binding affinity of OTV to NA, resulting in drug resistant influenza viruses. Using dRIN analysis, we succeeded in understanding the characteristic changes in residue interactions due to H274Y mutation, which can elucidate the molecular mechanism of reduction in OTV binding affinity to influenza NA. Finally, the dRIN analysis used in this study can be widely applied to various systems such as individual proteins, protein-ligand complexes, and protein-protein complexes, to characterize the dynamic aspects of the interactions.
  • Kisho Noda, Yoshimi Tsuda, Fumiya Kozawa, Manabu Igarashi, Kenta Shimizu, Jiro Arikawa, Kumiko Yoshimatsu
    Viruses 13 1 2020年12月27日 
    Severe fever with thrombocytopenia syndrome virus subclone B7 shows strong plaque formation and cytopathic effect induction compared with other subclones and the parental strain YG1. Compared to YG1 and the other subclones, only B7 possesses a single substitution in the L protein at the amino acid position 1891, in which N is changed to K (N1891K). In this study, we evaluate the effects of this mutation on L protein activity via a cell-based minigenome assay. Substitutions of N with basic amino acids (K or R) enhanced polymerase activity, while substitutions with an acidic amino acid (E) decreased this activity. Mutation to other neutral amino acids showed no significant effect on activity. These results suggest that the characteristic of the amino acid at position 1891 of the L protein are critical for its function, especially with respect to the charge status. Our data indicate that this C-terminal domain of the L protein may be crucial to its functions in genome transcription and viral replication.
  • Hiroki Atarashi, Wikum Harshana Jayasinghe, Joon Kwon, Hangil Kim, Yosuke Taninaka, Manabu Igarashi, Kotaro Ito, Tetsuya Yamada, Chikara Masuta, Kenji S. Nakahara
    Frontiers in Microbiology 11 2020年12月10日 
    Eukaryotic translation initiation factors, including eIF4E, are susceptibility factors for viral infection in host plants. Mutation and double-stranded RNA-mediated silencing of tomato eIF4E genes can confer resistance to viruses, particularly members of the Potyvirus genus. Here, we artificially mutated the eIF4E1 gene on chromosome 3 of a commercial cultivar of tomato (Solanum lycopersicum L.) by using CRISPR/Cas9. We obtained three alleles, comprising two deletions of three and nine nucleotides (3DEL and 9DEL) and a single nucleotide insertion (1INS), near regions that encode amino acid residues important for binding to the mRNA 5' cap structure and to eIF4G. Plants homozygous for these alleles were termed 3DEL, 9DEL, and 1INS plants, respectively. In accordance with previous studies, inoculation tests with potato virus Y (PVY; type member of the genus Potyvirus) yielded a significant reduction in susceptibility to the N strain (PVYN), but not to the ordinary strain (PVYO), in 1INS plants. 9DEL among three artificial alleles had a deleterious effect on infection by cucumber mosaic virus (CMV, type member of the genus Cucumovirus). When CMV was mechanically inoculated into tomato plants and viral coat accumulation was measured in the non-inoculated upper leaves, the level of viral coat protein was significantly lower in the 9DEL plants than in the parental cultivar. Tissue blotting of microperforated inoculated leaves of the 9DEL plants revealed significantly fewer infection foci compared with those of the parental cultivar, suggesting that 9DEL negatively affects the initial steps of infection with CMV in a mechanically inoculated leaf. In laboratory tests, viral aphid transmission from an infected susceptible plant to 9DEL plants was reduced compared with the parental control. Although many pathogen resistance genes have been discovered in tomato and its wild relatives, no CMV resistance genes have been used in practice. RNA silencing of eIF4E expression has previously been reported to not affect susceptibility to CMV in tomato. Our findings suggest that artificial gene editing can introduce additional resistance to that achieved with mutagenesis breeding, and that edited eIF4E alleles confer an alternative way to manage CMV in tomato fields.
  • Mao Isono, Wakako Furuyama, Makoto Kuroda, Tatsunari Kondoh, Manabu Igarashi, Masahiro Kajihara, Reiko Yoshida, Rashid Manzoor, Kosuke Okuya, Hiroko Miyamoto, Heinz Feldmann, Andrea Marzi, Masahiro Sakaitani, Asuka Nanbo, Ayato Takada
    Antiviral research 183 104932 - 104932 2020年11月 
    Ebolaviruses and marburgviruses, members of the family Filoviridae, are known to cause fatal diseases often associated with hemorrhagic fever. Recent outbreaks of Ebola virus disease in West African countries and the Democratic Republic of the Congo have made clear the urgent need for the development of therapeutics and vaccines against filoviruses. Using replication-incompetent vesicular stomatitis virus (VSV) pseudotyped with the Ebola virus (EBOV) envelope glycoprotein (GP), we screened a chemical compound library to obtain new drug candidates that inhibit filoviral entry into target cells. We discovered a biaryl sulfonamide derivative that suppressed in vitro infection mediated by GPs derived from all known human-pathogenic filoviruses. To determine the inhibitory mechanism of the compound, we monitored each entry step (attachment, internalization, and membrane fusion) using lipophilic tracer-labeled ebolavirus-like particles and found that the compound efficiently blocked fusion between the viral envelope and the endosomal membrane during cellular entry. However, the compound did not block the interaction of GP with the Niemann-Pick C1 protein, which is believed to be the receptor of filoviruses. Using replication-competent VSVs pseudotyped with EBOV GP, we selected escape mutants and identified two EBOV GP amino acid residues (positions 47 and 66) important for the interaction with this compound. Interestingly, these amino acid residues were located at the base region of the GP trimer, suggesting that the compound might interfere with the GP conformational change required for membrane fusion. These results suggest that this biaryl sulfonamide derivative is a novel fusion inhibitor and a possible drug candidate for the development of a pan-filovirus therapeutic.
  • Yoshihiro Takadate, Rashid Manzoor, Takeshi Saito, Yurie Kida, Junki Maruyama, Tatsunari Kondoh, Hiroko Miyamoto, Hirohito Ogawa, Masahiro Kajihara, Manabu Igarashi, Ayato Takada
    Microorganisms 8 10 2020年10月05日 
    Lloviu virus (LLOV), a bat-derived filovirus that is phylogenetically distinct from human pathogenic filoviruses such as Ebola virus (EBOV) and Marburg virus (MARV), was discovered in Europe. However, since infectious LLOV has never been isolated, the biological properties of this virus remain poorly understood. We found that vesicular stomatitis virus (VSV) pseudotyped with the glycoprotein (GP) of LLOV (VSV-LLOV) showed higher infectivity in one bat (Miniopterus sp.)-derived cell line than in the other bat-derived cell lines tested, which was distinct from the tropism of VSV pseudotyped with EBOV (VSV-EBOV) and MARV GPs. We then focused on the interaction between GP and Niemann-Pick C1 (NPC1) protein, one of the cellular receptors of filoviruses. We introduced the Miniopterus bat and human NPC1 genes into NPC1-knockout Vero E6 cells and their susceptibilities to the viruses were compared. The cell line expressing the bat NPC1 showed higher susceptibility to VSV-LLOV than that expressing human NPC1, whereas the opposite preference was seen for VSV-EBOV. Using a site-directed mutagenesis approach, amino acid residues involved in the differential tropism were identified in the NPC1 and GP molecules. Our results suggest that the interaction between GP and NPC1 is an important factor in the tropism of LLOV to a particular bat species.
  • Ankhanbaatar Ulaankhuu, Enkhbold Bazarragchaa, Masatoshi Okamatsu, Takahiro Hiono, Khishgee Bodisaikhan, Tsolmon Amartuvshin, Jargalsaikhan Tserenjav, Tsogtbaatar Urangoo, Khanui Buyantogtokh, Keita Matsuno, Takanari Hattori, Tatsunari Kondoh, Masahiro Sato, Yoshihiro Takadate, Shiho Torii, Mao Isono, Kosuke Okuya, Takeshi Saito, Nodoka Kasajima, Yurie Kida, Junki Maruyama, Manabu Igarashi, Ayato Takada, Hiroshi Kida, Damdinjav Batchuluun, Yoshihiro Sakoda
    Virus genes 56 4 472 - 479 2020年08月 [査読有り][通常論文]
     
    The circulation of highly pathogenic avian influenza viruses (HPAIVs) of various subtypes (e.g., H5N1, H5N6, H5N8, and H7N9) in poultry remains a global concern for animal and public health. Migratory waterfowls play important roles in the transmission of these viruses across countries. To monitor virus spread by wild birds, active surveillance for avian influenza in migratory waterfowl was conducted in Mongolia from 2015 to 2019. In total, 5000 fecal samples were collected from lakesides in central Mongolia, and 167 influenza A viruses were isolated. Two H5N3, four H7N3, and two H7N7 viruses were characterized in this study. The amino acid sequence at hemagglutinin (HA) cleavage site of those isolates suggested low pathogenicity in chickens. Phylogenetic analysis revealed that all H5 and H7 viruses were closely related to recent H5 and H7 low pathogenic avian influenza viruses (LPAIVs) isolated from wild birds in Asia and Europe. Antigenicity of H7Nx was similar to those of typical non-pathogenic avian influenza viruses (AIVs). While HPAIVs or A/Anhui/1/2013 (H7N9)-related LPAIVs were not detected in migratory waterfowl in Mongolia, sporadic introductions of AIVs including H5 and H7 viruses into Mongolia through the wild bird migration were identified. Thus, continued monitoring of H5 and H7 AIVs in both domestic and wild birds is needed for the early detection of HPAIVs spread into the country.
  • Yoshihiro Takadate, Tatsunari Kondoh, Manabu Igarashi, Junki Maruyama, Rashid Manzoor, Hirohito Ogawa, Masahiro Kajihara, Wakako Furuyama, Masahiro Sato, Hiroko Miyamoto, Reiko Yoshida, Terence E Hill, Alexander N Freiberg, Heinz Feldmann, Andrea Marzi, Ayato Takada
    Cell reports 30 2 308 - 319 2020年01月14日 [査読有り][通常論文]
     
    Fruit bats are suspected to be natural hosts of filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV). Interestingly, however, previous studies suggest that these viruses have different tropisms depending on the bat species. Here, we show a molecular basis underlying the host-range restriction of filoviruses. We find that bat-derived cell lines FBKT1 and ZFBK13-76E show preferential susceptibility to EBOV and MARV, respectively, whereas the other bat cell lines tested are similarly infected with both viruses. In FBKT1 and ZFBK13-76E, unique amino acid (aa) sequences are found in the Niemann-Pick C1 (NPC1) protein, one of the cellular receptors interacting with the filovirus glycoprotein (GP). These aa residues, as well as a few aa differences between EBOV and MARV GPs, are crucial for the differential susceptibility to filoviruses. Taken together, our findings indicate that the heterogeneity of bat NPC1 orthologs is an important factor controlling filovirus species-specific host tropism.
  • Tamura T, Igarashi M, Enkhbold B, Suzuki T, Okamatsu M, Ono C, Mori H, Izumi T, Sato A, Fauzyah Y, Okamoto T, Sakoda Y, Fukuhara T, Matsuura Y
    Journal of virology 93 22 2019年11月15日 [査読有り][通常論文]
     
    Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals.IMPORTANCEIn vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.
  • Hideki Tani, Kengo Kawachi, Miyuki Kimura, Satoshi Taniguchi, Masayuki Shimojima, Shuetsu Fukushi, Manabu Igarashi, Shigeru Morikawa, Masayuki Saijo
    Virology 535 102 - 110 2019年09月 [査読有り][通常論文]
     
    Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease with a high fatality rate, caused by SFTS virus (SFTSV). Because little is known about the nature of SFTSV, basic studies are required for the developments of vaccines and effective therapies. In the present study, we identified the amino acid residue important for membrane fusion induced by the SFTSV glycoprotein (GP). Syncytium formations were observed in cells expressing the GPs of SFTSV Japanese strain (YG-1 and SPL030). In contrast, no or only weak syncytium formations were induced in cells expressing GP of SFTSV Chinese strain (HB29). The replacement of arginine at amino acid residue 962 with serine in HB29 GP (R962S) induced membrane fusion, while the replacement of serine at residue 962 with arginine in YG1 GP (S962R) did not. These data indicate that serine at residue 962 in the SFTSV-GP is critical for inducing membrane fusion and viral infection.
  • Kojima S, Sato R, Yanai M, Komatsu Y, Horie M, Igarashi M, Tomonaga K
    Journal of virology 93 5 2019年03月 [査読有り][通常論文]
  • Masahiro Sato, Junki Maruyama, Tatsunari Kondoh, Naganori Nao, Hiroko Miyamoto, Yoshihiro Takadate, Wakako Furuyama, Masahiro Kajihara, Hirohito Ogawa, Rashid Manzoor, Reiko Yoshida, Manabu Igarashi, Ayato Takada
    Scientific reports 9 1 1158 - 1158 2019年02月04日 [査読有り][通常論文]
     
    Two novel influenza A virus-like genomes were detected in fruit bats in Central and South America. However, the biological properties of these bat-derived influenza viruses (BatIVs) are still largely unknown since infectious viral particles have never been isolated from the infected host species. In this study, a reverse genetics approach was used to generate infectious BatIV particles entirely from plasmids encoding full-length sequences in eight gene segments. We inoculated BatIV particles into various cell cultures including bat-derived cell lines and found that BatIVs infected particular bat-derived cells efficiently but not the other cell lines tested. Reassortant viruses between the two BatIVs were also successfully generated and their replication in the susceptible bat cell lines was confirmed. These findings suggest a limited host range and reassortment potential of BatIVs in nature, providing fundamental information for understanding of the ecology of BatIVs.
  • Salim B, Amin M, Igarashi M, Ito K, Jongejan F, Katakura K, Sugimoto C, Nakao R
    Gene 683 216 - 224 2019年01月 [査読有り][通常論文]
  • Tatsunari Kondoh, Michael Letko, Vincent J Munster, Rashid Manzoor, Junki Maruyama, Wakako Furuyama, Hiroko Miyamoto, Asako Shigeno, Daisuke Fujikura, Yoshihiro Takadate, Reiko Yoshida, Manabu Igarashi, Heinz Feldmann, Andrea Marzi, Ayato Takada
    The Journal of infectious diseases 218 suppl_5 S397-S402  2018年11月22日 [査読有り][通常論文]
     
    Niemann-Pick C1 (NPC1), a host receptor involved in the envelope glycoprotein (GP)-mediated entry of filoviruses into cells, is believed to be a major determinant of cell susceptibility to filovirus infection. It is known that proteolytically digested Ebola virus (EBOV) GP interacts with 2 protruding loops in domain C of NPC1. Using previously published structural data and the National Center for Biotechnology Information Single-Nucleotide Polymorphism (SNP) database, we identified 10 naturally occurring missense SNPs in human NPC1. To investigate whether these SNPs affect cell susceptibility to filovirus infection, we generated Vero E6 cell lines stably expressing NPC1 with SNP substitutions and compared their susceptibility to vesicular stomatitis virus pseudotyped with filovirus GPs and infectious EBOV. We found that some of the substitutions resulted in reduced susceptibility to filoviruses, as indicated by the lower titers and smaller plaque/focus sizes of the viruses. Our data suggest that human NPC1 SNPs may likely affect host susceptibility to filoviruses.
  • Moriyama M, Igarashi M, Koshiba T, Irie T, Takada A, Ichinohe T
    Journal of virology 92 19 2018年10月 [査読有り][通常論文]
     
    The nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) sequesters TANK-binding kinase 1 (TBK1) into NSs-induced cytoplasmic structures to inhibit the phosphorylation and nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3) and subsequent interferon beta (IFN-β) production. Although the C-terminal region of SFTSV NSs (NSs66-249) has been linked to the formation of NSs-induced cytoplasmic structures and inhibition of host IFN-β responses, the role of the N-terminal region in antagonizing host antiviral responses remains to be defined. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the SFTSV and heartland virus (HRTV) NSs are essential for suppression of IRF3 phosphorylation and IFN-β mRNA expression following infection with SFTSV or recombinant influenza virus lacking the NS1 gene. Surprisingly, formation of SFTSV/HRTV NSs-induced cytoplasmic structures is not essential for inhibition of host antiviral responses. Rather, an association between SFTSV/HRTV NSs and TBK1 is required for suppression of mitochondrial antiviral signaling protein (MAVS)-mediated activation of IFN-β promoter activity. Although SFTSV NSs did not prevent the ubiquitination of TBK1, it associates with TBK1 through its N-terminal kinase domain (residues 1 to 307) to block the autophosphorylation of TBK1. Furthermore, we found that both wild-type NSs and the 21/23A mutant (NSs in which residues at positions 21 and 23 were replaced with alanine) of SFTSV suppressed NLRP3 inflammasome-dependent interleukin-1β (IL-1β) secretion, suggesting that the importance of these residues is restricted to TBK1-dependent IFN signaling. Together, our findings strongly implicate the two conserved amino acids at positions 21 and 23 of SFTSV/HRTV NSs in the inhibition of host interferon responses.IMPORTANCE Recognition of viruses by host innate immune systems plays a critical role not only in providing resistance to viral infection but also in the initiation of antigen-specific adaptive immune responses against viruses. Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by the SFTS phlebovirus (SFTSV), a highly pathogenic tick-borne phlebovirus. The 294-amino-acid nonstructural protein (NSs) of SFTSV associates with TANK-binding kinase 1 (TBK1), a key regulator of host innate antiviral immunity, to inhibit interferon beta (IFN-β) production and enhance viral replication. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the NSs of SFTSV and heartland virus, another tick-borne phlebovirus, are essential for association with TBK1 and suppression of IFN-β production. Our results provide important insight into the molecular mechanisms by which SFTSV NSs helps to counteract host antiviral strategies.
  • Milligan JC, Parekh DV, Fuller KM, Igarashi M, Takada A, Saphire EO
    The Journal of infectious diseases 219 3 415 - 419 2018年09月 [査読有り][通常論文]
  • Keita Matsuno, Masahiro Kajihara, Ryo Nakao, Naganori Nao, Akina Mori-Kajihara, Mieko Muramatsu, Yongjin Qiu, Shiho Torii, Manabu Igarashi, Nodoka Kasajima, Keita Mizuma, Kentaro Yoshii, Hirofumi Sawa, Chihiro Sugimoto, Ayato Takada, Hideki Ebihara
    mSphere 3 3 2018年06月27日 [査読有り][通常論文]
     
    The recent emergence of novel tick-borne RNA viruses has complicated the epidemiological landscape of tick-borne infectious diseases, posing a significant challenge to public health systems that seek to counteract tick-borne diseases. The identification of two novel tick-borne phleboviruses (TBPVs), severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV), as causative agents of severe illness in humans has accelerated the investigation and discoveries of novel TBPVs. In the present study, we isolated a novel TBPV designated Mukawa virus (MKWV) from host-questing Ixodes persulcatus females captured in Japan. Genetic characterization revealed that MKWV is a member of the genus Phlebovirus in the family Phenuiviridae Interestingly, MKWV is genetically distinct from other known TBPVs and shares a most recent common ancestor with mosquito/sandfly-borne (insect-borne) phleboviruses. Despite its genetic similarity to insect-borne phleboviruses, the molecular footprints of its viral proteins and its biological characteristics define MKWV as a tick-borne virus that can be transmitted to mammals. A phylogenetic ancestral-state reconstruction for arthropod vectors of phleboviruses including MKWV based on viral L segment sequences indicated that ticks likely harbored ancestral phleboviruses that evolved into both the tick-borne and MKWV/insect-borne phlebovirus lineages. Overall, our findings suggest that most of the phlebovirus evolution has occurred in hard ticks to generate divergent viruses, which may provide a seminal foundation for understanding the mechanisms underlying the evolution and emergence of pathogenic phleboviruses, such as Rift Valley fever virus and SFTSV/HRTV.IMPORTANCE The emergence of novel tick-borne RNA viruses causing severe illness in humans has complicated the epidemiological landscape of tick-borne diseases, requiring further investigation to safeguard public health. In the present study, we discovered a novel tick-borne phlebovirus from Ixodes persulcatus ticks in Japan. While its viral RNA genome sequences were similar to those of mosquito/sandfly-borne viruses, molecular and biological footprints confirmed that this is a tick-borne virus. The unique evolutionary position of the virus allowed us to estimate the ancestral phlebovirus vector, which was likely a hard tick. Our findings may provide a better understanding of the evolution and emergence of phleboviruses associated with emerging infectious diseases, such as severe fever with thrombocytopenia syndrome (SFTS) and Heartland virus disease.
  • Rashid Manzoor, Manabu Igarashi, Ayato Takada
    International Journal of Molecular Sciences 18 12 2017年12月07日 [査読有り][通常論文]
     
    Influenza A virus (IAV) matrix protein 2 (M2) is among the smallest bona fide, hence extensively studied, ion channel proteins. The M2 ion channel activity is not only essential for virus replication, but also involved in modulation of cellular homeostasis in a variety of ways. It is also the target for ion channel inhibitors, i.e., anti-influenza drugs. Thus far, several studies have been conducted to elucidate its biophysical characteristics, structure-function relationships of the ion channel, and the M2-host interactome. In this review, we discuss M2 protein synthesis and assembly into an ion channel, its roles in IAV replication, and the pathophysiological impact on the host cell.
  • 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.
  • Tatsunari Kondoh, Rashid Manzoor, Naganori Nao, Junki Maruyama, Wakako Furuyama, Hiroko Miyamoto, Asako Shigeno, Makoto Kuroda, Keita Matsuno, Daisuke Fujikura, Masahiro Kajihara, Reiko Yoshida, Manabu Igarashi, Ayato Takada
    PLOS ONE 12 10 e0186450  2017年10月 [査読有り][通常論文]
     
    It has been proposed that some non-retroviral RNA virus genes are integrated into vertebrate genomes. Endogenous filovirus-like elements (EFLs) have been discovered in some mammalian genomes. However, their potential roles in ebolavirus infection are unclear. A filovirus VP35-like element (mlEFL35) is found in the little brown bat (Myotis lucifugus) genome. Putative mlEFL35-derived protein (mlEFL35p) contains nearly full-length amino acid sequences corresponding to ebolavirus VP35. Ebola virus VP35 has been shown to bind double-stranded RNA, leading to inhibition of type I interferon (IFN) production, and is also known as a viral polymerase cofactor that is essential for viral RNA transcription/replication. In this study, we transiently expressed mlEFL35p in human kidney cells and investigated its biological functions. We first found that mlEFL35p was coimmunoprecipitated with itself and ebolavirus VP35s but not with the viral nucleoprotein. Then the biological functions of mlEFL35p were analyzed by comparing it to ebolavirus VP35s. We found that the expression of mlEFL35p significantly inhibited human IFN-beta promoter activity as well as VP35s. By contrast, expression of mlEFL35p did not support viral RNA transcription/replication and indeed slightly decrease the reporter gene expression in a minigenome assay. These results suggest that mlEFL35p potentially acts as an IFN antagonist but not a polymerase cofactor.
  • Takasuke Fukuhara, Tomokazu Tamura, Chikako Ono, Mai Shiokawa, Hiroyuki Mori, Kentaro Uemura, Satomi Yamamoto, Takeshi Kurihara, Toru Okamoto, Ryosuke Suzuki, Kentaro Yoshii, Takeshi Kurosu, Manabu Igarashi, Hiroshi Aoki, Yoshihiro Sakoda, Yoshiharu Matsuura
    PLOS PATHOGENS 13 6 e1006475  2017年06月 [査読有り][通常論文]
     
    Amphipathic alpha-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the Flaviviridae members, pestivirus and flavivirus possess a viral structural protein E-rns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while Hepacivirus including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic alpha-helices of E-rns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded E-rns and NS1 in the formation of infectious particles. We examined whether E-rns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and nonhepatic 293T cells. We found that exogenous expression of either E-rns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an E-rns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic alpha-helices of E-rns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host-and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of Flaviviridae, especially in the maturation of infectious particles, while E-rns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene.
  • Naganori Nao, Junya Yamagishi, Hiroko Miyamoto, Manabu Igarashi, Rashid Manzoor, Aiko Ohnuma, Yoshimi Tsuda, Wakako Furuyama, Asako Shigeno, Masahiro Kajihara, Noriko Kishida, Reiko Yoshida, Ayato Takada
    MBIO 8 1 2017年01月 [査読有り][通常論文]
     
    Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes. IMPORTANCE Influenza A viruses are divided into subtypes based on the antigenicity of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. This study shows the potential role of the viral RNA secondary structure for nucleotide insertions and demonstrates a key mechanism explaining why the acquisition of the polybasic HA cleavage site is restricted to particular HA subtypes in nature. Our findings will contribute to better understanding of the ecology of influenza A viruses and will also be useful for the development of genetically modified vaccines against H5 and H7 influenza A viruses with increased stability.
  • Yoshimi Tsuda, Manabu Igarashi, Ryo Ito, Sanae Nishio, Kenta Shimizu, Kumiko Yoshimatsu, Jiro Arikawa
    BIOMEDICAL RESEARCH-TOKYO 38 2 89 - 97 2017年 [査読有り][通常論文]
     
    Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus responsible for causing an emerging zoonotic disease. We previously established subclones from SFTSV strain YG1 based on differences in low-pH-dependent cell fusion activities and found two amino acid substitutions, Y328H and R624W, in the envelope glycoprotein (GP) of high fusion subclones. In this study, we show that transiently expressed GP with the R624W mutation, but not the Y328H mutation, induced cell fusion under acidic conditions. GP possessing either tryptophan, serine, glycine or aspartic acid at position 624 induced cell fusion, whereas GP possessing basic amino acids such as arginine or lysine did not induce cell fusion. These results indicated that the amino acid at position 624 has an important role for inducing low-pH-dependent cell fusion.
  • Furuyama W, Marzi A, Carmody AB, Maruyama J, Kuroda M, Miyamoto H, Nanbo A, Manzoor R, Yoshida R, Igarashi M, Feldmann H, Takada A
    PLoS pathogens 12 12 e1006139  2016年12月 [査読有り][通常論文]
     
    Antibody-dependent enhancement (ADE) of Ebola virus (EBOV) infection has been demonstrated in vitro, raising concerns about the detrimental potential of some anti-EBOV antibodies. ADE has been described for many viruses and mostly depends on the cross-linking of virus-antibody complexes to cell surface Fc receptors, leading to enhanced infection. However, little is known about the molecular mechanisms underlying this phenomenon. Here we show that Fc gamma-receptor IIa (Fc gamma RIIa)-mediated intracellular signaling through Src family protein tyrosine kinases (PTKs) is required for ADE of EBOV infection. We found that deletion of the Fc gamma RIIa cytoplasmic tail abolished EBOV ADE due to decreased virus uptake into cellular endosomes. Furthermore, EBOV ADE, but not non-ADE infection, was significantly reduced by inhibition of the Src family protein PTK pathway, which was also found to be important to promote phagocytosis/macropinocytosis for viral uptake into endosomes. We further confirmed a significant increase of the Src phosphorylation mediated by ADE. These data suggest that antibody-EBOV complexes bound to the cell surface Fc gamma RIIa activate the Src signaling pathway that leads to enhanced viral entry into cells, providing a novel perspective for the general understanding of ADE of virus infection.
  • Takahiro Hiono, Masatoshi Okamatsu, Manabu Igarashi, Ryan McBride, Robert P. de Vries, Wenjie Peng, James C. Paulson, Yoshihiro Sakoda, Hiroshi Kida
    ARCHIVES OF VIROLOGY 161 2 307 - 316 2016年02月 [査読有り][通常論文]
     
    Influenza viruses isolated from ducks are rarely able to infect chickens; it is therefore postulated that these viruses need to adapt in some way to be able to be transmitted to chickens in nature. Previous studies revealed that sialyl Lewis X (3'SLeX), which is fucosylated alpha 2,3 sialoside, was predominantly detected on the epithelial cells of the chicken trachea, whereas this glycan structure is not found in the duck intestinal tract. To clarify the mechanisms of the interspecies transmission of influenza viruses between ducks and chickens, we compared the receptor specificity of low-pathogenic avian influenza viruses isolated from these two species. Glycan-binding analysis of the recombinant hemagglutinin (HA) of a chicken influenza virus, A/chicken/Ibaraki/1/2005 (H5N2), revealed a binding preference to alpha 1,3 fucosylated sialosides. On the other hand, the HA of a duck influenza virus, A/duck/Mongolia/54/2001 (H5N2) (Dk/MNG), particularly bound to non-fucosylated alpha 2,3 sialosides such as 3'-sialyllactosamine (3'SLacNAc). Computational analysis along with binding analysis of the mutant HAs revealed that this glycan-binding specificity of the HA was determined by amino acid residues at positions 222 and 227. Inconsistent with the glycan-binding specificity of the recombinant HA protein, virions of Dk/MNG bound to both 3'SLacNAc and 3'SLeX. Glycan-binding analysis in the presence of a neuraminidase (NA) inhibitor revealed that the NA conferred binding to 3'SLeX to virions of Dk/MNG. The present results reveal the molecular basis of the interaction between fucosylated alpha 2,3 sialosides and influenza viruses.
  • Wakako Furuyama, Andrea Marzi, Asuka Nanbo, Elaine Haddock, Junki Maruyama, Hiroko Miyamoto, Manabu Igarashi, Reiko Yoshida, Osamu Noyori, Heinz Feldmann, Ayato Takada
    SCIENTIFIC REPORTS 6 20514  2016年02月 [査読有り][通常論文]
     
    During the latest outbreak of Ebola virus disease in West Africa, monoclonal antibody therapy (e.g., ZMapp) was utilized to treat patients. However, due to the antigenic differences among the five ebolavirus species, the current therapeutic monoclonal antibodies are only effective against viruses of the species Zaire ebolavirus. Although this particular species has indeed caused the majority of human infections in Central and, recently, West Africa, other ebolavirus species (e.g., Sudan ebolavirus and Bundibugyo ebolavirus) have also repeatedly caused outbreaks in Central Africa and thus should not be neglected in the development of countermeasures against ebolaviruses. Here we report the generation of an ebolavirus glycoprotein-specific monoclonal antibody that effectively inhibits cellular entry of representative isolates of all known ebolavirus species in vitro and show its protective efficacy in mouse models of ebolavirus infections. This novel neutralizing monoclonal antibody targets a highly conserved internal fusion loop in the glycoprotein molecule and prevents membrane fusion of the viral envelope with cellular membranes. The discovery of this highly cross-neutralizing antibody provides a promising option for broad-acting ebolavirus antibody therapy and will accelerate the design of improved vaccines that can selectively elicit cross-neutralizing antibodies against multiple species of ebolaviruses.
  • Junki Maruyama, Naganori Nao, Hiroko Miyamoto, Ken Maeda, Hirohito Ogawa, Reiko Yoshida, Manabu Igarashi, Ayato Takada
    VIROLOGY 488 43 - 50 2016年01月 [査読有り][通常論文]
     
    Recently found bat-derived influenza viruses (BatlVs) have hemagglutinin (HA) and neuraminidase (NA) gene segments distinct from those of previously known influenza A viruses. However, pathogenicities of these BatlVs remain unknown since infectious virus strains have not been isolated yet. To gain insight into the biological properties of BatlVs, we generated vesicular stomatitis viruses (VSVs) pseudotyped with the BatIV HA and NA. We found that VSVs pseudotyped with BatIV HAs and NAs efficiently infected particular bat cell lines but not those derived from primates, and that proteolytic cleavage with a trypsinlike protease was necessary for HA-mediated virus entry. Treatment of the susceptible bat cells with some enzymes and inhibitors revealed that BatIV HAs might recognize some cellular glycoproteins as receptors rather than the sialic acids used for the other known influenza viruses. These data provide fundamental information on the mechanisms underlying the cellular entry and host restriction of BatIVs. (C) 2015 Elsevier Inc. All rights reserved.
  • Hirohito Ogawa, Hiroko Miyamoto, Eri Nakayama, Reiko Yoshida, Ichiro Nakamura, Hirofumi Sawa, Akihiro Ishii, Yuka Thomas, Emiko Nakagawa, Keita Matsuno, Masahiro Kajihara, Junki Maruyama, Naganori Nao, Mieko Muramatsu, Makoto Kuroda, Edgar Simulundu, Katendi Changula, Bernard Hang'ombe, Boniface Namangala, Andrew Nambota, Jackson Katampi, Manabu Igarashi, Kimihito Ito, Heinz Feldmann, Chihiro Sugimoto, Ladslav Moonga, Aaron Mweene, Ayato Takada
    JOURNAL OF INFECTIOUS DISEASES 212 S101 - S108 2015年10月 [査読有り][通常論文]
     
    Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.
  • Tomokazu Tamura, Nicolas Ruggli, Naofumi Nagashima, Masatoshi Okamatsu, Manabu Igarashi, Junki Mine, Martin A. Hofmann, Matthias Liniger, Artur Summerfield, Hiroshi Kida, Yoshihiro Sakoda
    JOURNAL OF GENERAL VIROLOGY 96 9 2623 - 2635 2015年09月 [査読有り][通常論文]
     
    Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE(-) vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE(-)-derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE- replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic a-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.
  • Naganori Nao, Masahiro Kajihara, Rashid Manzoor, Junki Maruyama, Reiko Yoshida, Mieko Muramatsu, Hiroko Miyamoto, Manabu Igarashi, Nao Eguchi, Masahiro Sato, Tatsunari Kondoh, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida, Ayato Takada
    PLOS ONE 10 9 e0137989  2015年09月 [査読有り][通常論文]
     
    Two highly pathogenic avian influenza virus strains, A/duck/Hokkaido/WZ83/2010 ( H5N1) (WZ83) and A/duck/Hokkaido/WZ101/2010 (H5N1) (WZ101), which were isolated from wild ducks in Japan, were found to be genetically similar, with only two amino acid differences in their M1 and PB1 proteins at positions 43 and 317, respectively. We found that both WZ83 and WZ101 caused lethal infection in chickens but WZ101 killed them more rapidly than WZ83. Interestingly, ducks experimentally infected with WZ83 showed no or only mild clinical symptoms, whereas WZ101 was highly lethal. We then generated reassortants between these viruses and found that exchange of the M gene segment completely switched the pathogenic phenotype in both chickens and ducks, indicating that the difference in the pathogenicity for these avian species between WZ83 and WZ101 was determined by only a single amino acid in the M1 protein. It was also found that WZ101 showed higher pathogenicity than WZ83 in mice and that WZ83, whose M gene was replaced with that of WZ101, showed higher pathogenicity than wild-type WZ83, although this reassortant virus was not fully pathogenic compared to wild-type WZ101. These results suggest that the amino acid at position 43 of the M1 protein is one of the factors contributing to the pathogenicity of H5N1 highly pathogenic avian influenza viruses in both avian and mammalian hosts.
  • Yasushi Itoh, Shintaro Shichinohe, Misako Nakayama, Manabu Igarashi, Akihiro Ishii, Hirohito Ishigaki, Hideaki Ishida, Naoko Kitagawa, Takako Sasamura, Masanori Shiohara, Michiko Doi, Hideaki Tsuchiya, Shinichiro Nakamura, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida, Kazumasa Ogasawara
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 59 8 4962 - 4973 2015年08月 [査読有り][通常論文]
     
    The number of patients infected with H7N9 influenza virus has been increasing since 2013. We examined the efficacy of neuraminidase (NA) inhibitors and the efficacy of a vaccine against an H7N9 influenza virus, A/Anhui/1/2013 (H7N9), isolated from a patient in a cynomolgus macaque model. NA inhibitors (oseltamivir and peramivir) barely reduced the total virus amount because of the emergence of resistant variants with R289K or 1219T in NA [residues 289 and 219 in N9 of A/Anhui/1/2013 (H7N9) correspond to 292 and 222 in N2, respectively] in three of the six treated macaques, whereas subcutaneous immunization of an inactivated vaccine derived from A/duck/Mongolia/119/2008 (H7N9) prevented propagation of A/Anhui/1/2013 (H7N9) in all vaccinated macaques. The percentage of macaques in which variant H7N9 viruses with low sensitivity to the NA inhibitors were detected was much higher than that of macaques in which variant H5N1 highly pathogenic influenza virus was detected after treatment with one of the NA inhibitors in our previous study. The virus with R289K in NA was reported in samples from human patients, whereas that with 1219T in NA was identified for the first time in this study using macaques, though no variant H7N9 virus was reported in previous studies using mice. Therefore, the macaque model enables prediction of the frequency of emerging H7N9 virus resistant to NA inhibitors in vivo. Since H7N9 strains resistant to NA inhibitors might easily emerge compared to other influenza viruses, monitoring of the emergence of variants is required during treatment of H7N9 influenza virus infection with NA inhibitors.
  • Makoto Ozawa, Aya Matsuu, Kouki Yonezawa, Manabu Igarashi, Kosuke Okuya, Toshiko Kawabata, Kimihito Ito, Kyoko Tsukiyama-Kohara, Akira Taneno, Eisaburo Deguchi
    JOURNAL OF CLINICAL MICROBIOLOGY 53 4 1331 - 1338 2015年04月 [査読有り][通常論文]
     
    The control of swine influenza virus (SIV) infection is paramount for increasing the productivity of pig farming and minimizing the threat of pandemic outbreaks. Thus, SIV surveillance should be conducted by region and on a regular basis. Here, we established a microneutralization assay specific for SIV seroprevalence surveillance by using reporter gene-expressing recombinant influenza viruses. Growth-based SIV seroprevalence revealed that most sows and piglets were positive for neutralizing antibodies against influenza viruses. In contrast, the 90-day-old growing pigs exhibited limited neutralizing activity in their sera, suggesting that this particular age of population is most susceptible to SIV infection and thus is an ideal age group for SIV isolation. From nasal swab specimens of healthy pigs in this age population, we were able to isolate SIVs at a higher incidence (5.3%) than those of previous reports. Nucleotide sequencing and phylogenetic analysis of the hemagglutinin (HA) genes revealed that the isolated SIVs have circulated and evolved in pigs but not have been recently introduced from humans, implying that a large number of SIV lineages may remain "undiscovered" in the global porcine populations. Wepropose that the 90-day-old growing pig-targeted nasal swab collection presented in this study facilitates global SIV surveillance and contributes to the detection and control of SIV infection.
  • Yasushi Itoh, Reiko Yoshida, Shintaro Shichinohe, Megumi Higuchi, Hirohito Ishigaki, Misako Nakayama, Van Loi Pham, Hideaki Ishida, Mitsutaka Kitano, Masahiko Arikata, Naoko Kitagawa, Yachiyo Mitsuishi, Kazumasa Ogasawara, Hideaki Tsuchiya, Takahiro Hiono, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida, Mutsumi Ito, Le Quynh Mai, Yoshihiro Kawaoka, Hiroko Miyamoto, Mari Ishijima, Manabu Igarashi, Yasuhiko Suzuki, Ayato Takada
    PLOS PATHOGENS 10 6 e1004192  2014年06月 [査読有り][通常論文]
     
    Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.
  • Kentaro Yoshii, Yuji Sunden, Kana Yokozawa, Manabu Igarashi, Hiroaki Kariwa, Michael R. Holbrook, Ikuo Takashima
    JOURNAL OF VIROLOGY 88 10 5406 - 5420 2014年05月 [査読有り][通常論文]
     
    Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) are highly pathogenic tick-borne flaviviruses; TBEV causes neurological disease in humans, while OHFV causes a disease typically identified with hemorrhagic fever. Although TBEV and OHFV are closely related genetically, the viral determinants responsible for these distinct disease phenotypes have not been identified. In this study, chimeric viruses incorporating components of TBEV and OHFV were generated using infectious clone technology, and their pathological characteristics were analyzed in a mouse model to identify virus-specific determinants of disease. We found that only four amino acids near the C terminus of the NS5 protein were primarily responsible for the development of neurological disease. Mutation of these four amino acids had no effect on viral replication or histopathological features, including inflammatory responses, in mice. These findings suggest a critical role for NS5 in stimulating neuronal dysfunction and degeneration following TBEV infection and provide new insights into the molecular mechanisms underlying the pathogenesis of tick-borne flaviviruses.
  • Yonezawa Kouki, Igarashi Manabu, Ohara Yasuo, Ito Kimihito
    Genes & Genetic Systems 89 6 313  2014年 [査読有り][通常論文]
  • Ryo Nakao, Yongjin Qiu, Manabu Igarashi, Joseph W Magona, Lijia Zhou, Kimihito Ito, Chihiro Sugimoto
    Ticks and tick-borne diseases 4 6 506 - 12 2013年12月 [査読有り][通常論文]
     
    The spotted fever group (SFG) rickettsiae are obligate intracellular bacteria transmitted by ticks that cause several tick-borne rickettsioses in humans worldwide. This study was intended to determine the prevalence of SFG rickettsiae in Amblyomma variegatum from 7 districts across Uganda. In addition to sequencing of gltA and ompA genes, identification of Rickettsia species based on the sizes of highly variable intergenic spacers, namely, dksA-xerC, mppA-purC, and rpmE-tRNA(fMet) was carried out. Application of multiplex PCR for simultaneous amplification of 3 spacers combined with capillary electrophoresis separation allowed simple, accurate, and high-throughput fragment sizing with considerable time and cost savings. Rickettsia genus-specific real-time PCR detected 136 positives out of 140 samples, giving an overall prevalence of 97.1%. Most samples (n=113) had a size combination of 225, 195, and 341 bp for dksA-xerC, mppA-purC, and rpmE-tRNA(fMet), respectively, which was identical to that of R. africae, a causative agent of African tick bite fever. In addition, several samples had size variants in either dksA-xerC or rpmE-tRNA(fMet). Nonetheless, the partial sequences of gltA and ompA genes of samples of all size combinations showed the greatest similarity to R. africae (99.3-100% for gltA and 98.1-100% for ompA). Given these results, it is highly possible that the tested ticks were infected with R. africae or closely related species. This is a first report on molecular genetic detection of R. africae and its high endemicity in Uganda. Clinicians in this country should be aware of this pathogen as a cause of non-malarial febrile illness. This study provided a starting point for the development of Rickettsia species identification based on the sizes of intergenic spacers. The procedure is simple, rapid, and cost-effective to perform; hence it might be particularly well suited for preliminary species identification in epidemiological investigations. The results may be more detailed and reliable when simultaneous sequencing analysis is performed.
  • Osamu Noyori, Keita Matsuno, Masahiro Kajihara, Eri Nakayama, Manabu Igarashi, Makoto Kuroda, Norikazu Isoda, Reiko Yoshida, Ayato Takada
    VIROLOGY 446 1-2 152 - 161 2013年11月 [査読有り][通常論文]
     
    The viral envelope glycoprotein (GP) is thought to play important roles in the pathogenesis of filovirus infection. It is known that GP expressed on the cell surface forms a steric shield over host proteins such as major histocompatibility complex class I and integrin pi, which may result in the disorder of cell-to-cell contacts and/or inhibition of the immune response. However, it is not clarified whether this phenomenon contributes to the pathogenicity of filoviruses. In this study, we found that the steric shielding efficiency differed among filovirus strains and was correlated with the difference in their relative pathogenicities. While the highly glycosylated mucin-like region of GP was indispensable, the differential shielding efficiency did not necessarily depend on the primary structure of the mucin-like region, suggesting the importance of the overall properties (e.g., flexibility and stability) of the GP molecule for efficient shielding of host proteins. (C) 2013 Elsevier Inc. All rights reserved.
  • 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.
  • Yurie Motohashi, Manabu Igarashi, Masatoshi Okamatsu, Takeshi Noshi, Yoshihiro Sakoda, Naoki Yamamoto, Kimihito Ito, Ryu Yoshida, Hiroshi Kida
    VIROLOGY JOURNAL 10 118  2013年04月 [査読有り][通常論文]
     
    Background: The hemagglutinin (HA) of influenza viruses is a possible target for antiviral drugs because of its key roles in the initiation of infection. Although it was found that a natural compound, Stachyflin, inhibited the growth of H1 and H2 but not H3 influenza viruses in MDCK cells, inhibitory activity of the compound has not been assessed against H4-H16 influenza viruses and the precise mechanism of inhibition has not been clarified. Methods: Inhibitory activity of Stachyflin against H4-H16 influenza viruses, as well as H1-H3 viruses was examined in MDCK cells. To identify factors responsible for the susceptibility of the viruses to this compound, Stachyflin-resistant viruses were selected in MDCK cells and used for computer docking simulation. Results: It was found that in addition to antiviral activity of Stachyflin against influenza viruses of H1 and H2 subtypes, it inhibited replication of viruses of H5 and H6 subtypes, as well as A(H1N1)pdm09 virus in MDCK cells. Stachyflin also inhibited the virus growth in the lungs of mice infected with A/WSN/1933 (H1N1) and A/chicken/Ibaraki/1/2005 (H5N2). Substitution of amino acid residues was found on the HA2 subunit of Stachyflin-resistant viruses. Docking simulation indicated that D37, K51, T107, and K121 are responsible for construction of the cavity for the binding of the compound. In addition, 3-dimensional structure of the cavity of the HA of Stachyflin-susceptible virus strains was different from that of insusceptible virus strains. Conclusion: Antiviral activity of Stachyflin was found against A(H1N1) pdm09, H5, and H6 viruses, and identified a potential binding pocket for Stachyflin on the HA. The present results should provide us with useful information for the development of HA inhibitors with more effective and broader spectrum.
  • Masahiro Kajihara, Eri Nakayama, Andrea Marzi, Manabu Igarashi, Heinz Feldmann, Ayato Takada
    JOURNAL OF GENERAL VIROLOGY 94 4 876 - 883 2013年04月 [査読有り][通常論文]
     
    Marburg virus (MARV) and Ebola virus, members of the family Filoviridae, cause lethal haemorrhagic fever in humans and non-human primates. Although the outbreaks are concentrated mainly in Central Africa, these viruses are potential agents of imported infectious diseases and bioterrorism in non-African countries. Recent studies demonstrated that non-human primates passively immunized with virus-specific antibodies were successfully protected against fatal filovirus infection, highlighting the important role of antibodies in protective immunity for this disease. However, the mechanisms underlying potential evasion from antibody mediated immune pressure are not well understood. To analyse possible mutations involved in immune evasion in the MARV envelope glycoprotein (GP) which is the major target of protective antibodies, we selected escape mutants of recombinant vesicular stomatitis virus (rVSV) expressing MARV GP (rVSV Delta G/MARVGP) by using two GP-specific mAbs, AGP127-8 and MGP72-17, which have been previously shown to inhibit MARV budding. Interestingly, several rVSV Delta G/MARVGP variants escaping from the mAb pressure-acquired amino acid substitutions in the furin-cleavage site rather than in the mAb-specific epitopes, suggesting that these epitopes are recessed, not exposed on the uncleaved GP molecule, and therefore inaccessible to the mAbs. More surprisingly, some variants escaping mAb MGP72-17 lacked a large proportion of the mucin-like region of GP, indicating that these mutants efficiently escaped the selective pressure by deleting the mucin-like region including the mAb-specific epitope. Our data demonstrate that MARV GP possesses the potential to evade antibody mediated immune pressure due to extraordinary structural flexibility and variability.
  • Kouki Yonezawa, Manabu Igarashi, Keisuke Ueno, Ayato Takada, Kimihito Ito
    PLoS ONE 8 2 e57684  2013年02月27日 [査読有り][通常論文]
     
    A large number of nucleotide sequences of various pathogens are available in public databases. The growth of the datasets has resulted in an enormous increase in computational costs. Moreover, due to differences in surveillance activities, the number of sequences found in databases varies from one country to another and from year to year. Therefore, it is important to study resampling methods to reduce the sampling bias. A novel algorithm-called the closest-neighbor trimming method-that resamples a given number of sequences from a large nucleotide sequence dataset was proposed. The performance of the proposed algorithm was compared with other algorithms by using the nucleotide sequences of human H3N2 influenza viruses. We compared the closest-neighbor trimming method with the naive hierarchical clustering algorithm and k-medoids clustering algorithm. Genetic information accumulated in public databases contains sampling bias. The closest-neighbor trimming method can thin out densely sampled sequences from a given dataset. Since nucleotide sequences are among the most widely used materials for life sciences, we anticipate that our algorithm to various datasets will result in reducing sampling bias. © 2013 Yonezawa et al.
  • Ryo Takano, Maki Kiso, Manabu Igarashi, Quynh Mai Le, Masakazu Sekijima, Kimihito Ito, Ayato Takada, Yoshihiro Kawaoka
    JOURNAL OF INFECTIOUS DISEASES 207 1 89 - 97 2013年01月 [査読有り][通常論文]
     
    Background. The neuraminidase (NA) inhibitor oseltamivir is widely used to treat patients infected with influenza viruses. An Ile-to-Val change at position 117 in influenza A virus subtype H5N1 NA (NA-I117V) confers a reduction in susceptibility to oseltamivir carboxylate. However, the in vivo relevance and molecular basis of the decreased sensitivity mediated by this mutation are poorly understood. Methods. We created single-point-mutant viruses with 3 genetically different backgrounds (ie, 1 belonging to clade 1 and 2 belonging to clade 2.3.4) and evaluated the effects of the I117V mutation on oseltamivir susceptibility in vitro, in vivo, and in silico. Results. The NA-I117V mutation conferred a slight reduction in susceptibility to oseltamivir in vitro (1.3- to 6.3-fold changes), although it did not substantially compromise NA enzymatic activity. Mice infected with I117V virus exhibited reduced susceptibility to oseltamivir and decreased survival in 2 of 3 virus pairs tested. Molecular dynamics simulations revealed that I117V caused the loss of hydrogen bonds between an arginine at position 118 and the carboxyl group of oseltamivir, leading to a lower binding affinity for oseltamivir. Conclusions. Our findings provide new insight into the mechanism of NA-I117V-mediated oseltamivir resistance in highly pathogenic H5N1 avian influenza viruses.
  • Chairul A. Nidom, Eri Nakayama, Reviany V. Nidom, Mohamad Y. Alamudi, Syafril Daulay, Indi N. L. P. Dharmayanti, Yoes P. Dachlan, Mohamad Amin, Manabu Igarashi, Hiroko Miyamoto, Reiko Yoshida, Ayato Takada
    PLOS ONE 7 7 e40740  2012年07月 [査読有り][通常論文]
     
    Ebola virus (EBOV) and Marburg virus (MARV) belong to the family Filoviridae and cause severe hemorrhagic fever in humans and nonhuman primates. Despite the discovery of EBOV (Reston virus) in nonhuman primates and domestic pigs in the Philippines and the serological evidence for its infection of humans and fruit bats, information on the reservoirs and potential amplifying hosts for filoviruses in Asia is lacking. In this study, serum samples collected from 353 healthy Bornean orangutans (Pongo pygmaeus) in Kalimantan Island, Indonesia, during the period from December 2005 to December 2006 were screened for filovirus-specific IgG antibodies using a highly sensitive enzyme-linked immunosorbent assay (ELISA) with recombinant viral surface glycoprotein (GP) antigens derived from multiple species of filoviruses (5 EBOV and 1 MARV species). Here we show that 18.4% (65/353) and 1.7% (6/353) of the samples were seropositive for EBOV and MARV, respectively, with little cross-reactivity among EBOV and MARV antigens. In these positive samples, IgG antibodies to viral internal proteins were also detected by immunoblotting. Interestingly, while the specificity for Reston virus, which has been recognized as an Asian filovirus, was the highest in only 1.4% (5/353) of the serum samples, the majority of EBOV-positive sera showed specificity to Zaire, Sudan, Cote d'Ivoire, or Bundibugyo viruses, all of which have been found so far only in Africa. These results suggest the existence of multiple species of filoviruses or unknown filovirus-related viruses in Indonesia, some of which are serologically similar to African EBOVs, and transmission of the viruses from yet unidentified reservoir hosts into the orangutan populations. Our findings point to the need for risk assessment and continued surveillance of filovirus infection of human and nonhuman primates, as well as wild and domestic animals, in Asia.
  • Kenji S. Nakahara, Chikara Masuta, Syouta Yamada, Hanako Shimura, Yukiko Kashihara, Tomoko S. Wada, Ayano Meguro, Kazunori Goto, Kazuki Tadamura, Kae Sueda, Toru Sekiguchi, Jun Shao, Noriko Itchoda, Takeshi Matsumura, Manabu Igarashi, Kimihito Ito, Richard W. Carthew, Ichiro Uyeda
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 25 10113 - 10118 2012年06月 [査読有り][通常論文]
     
    RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM-mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.
  • Andrea Marzi, Reiko Yoshida, Hiroko Miyamoto, Mari Ishijima, Yasuhiko Suzuki, Megumi Higuchi, Yukie Matsuyama, Manabu Igarashi, Eri Nakayama, Makoto Kuroda, Masayuki Saijo, Friederike Feldmann, Douglas Brining, Heinz Feldmann, Ayato Takada
    PLOS ONE 7 4 e36192  2012年04月 [査読有り][通常論文]
     
    Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed to protect nonhuman primates from lethal disease. In this study, we used two clones of human-mouse chimeric MAbs (ch133 and ch226) with strong neutralizing activity against ZEBOV and evaluated their protective potential in a rhesus macaque model of EHF. Reduced viral loads and partial protection were observed in animals given MAbs ch133 and ch226 combined intravenously at 24 hours before and 24 and 72 hours after challenge. MAbs circulated in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum MAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. Accordingly, the rapid decrease of serum MAbs was clearly associated with increased viremia in non-survivors. Our results indicate that EBOV neutralizing antibodies, particularly in combination with other therapeutic strategies, might be beneficial in reducing viral loads and prolonging disease progression during EHF.
  • Keita Nagakawa, Kenichi Niikura, Tadaki Suzuki, Yasutaka Matsuo, Manabu Igarashi, Hirofumi Sawa, Kuniharu Ijiro
    CHEMISTRY LETTERS 41 1 113 - 115 2012年01月 [査読有り][通常論文]
     
    This manuscript describes the synthesis of virus capsid protein-coated Au nanoparticle (VP-AuNP) without the use of the inherent self-assembly of virus proteins into virus particles. Covalent binding between Au and cysteines in the virus proteins keep the cell-surface binding sites on the external surface. Based on this method, various sizes of VP-AuNP can be created in a similar manner to native virus particles. We clarified the optimum size of the VP-AuNP for internalization into cells.
  • Kentaro Yoshii, Manabu Igarashi, Osamu Ichii, Kana Yokozawa, Kimihito Ito, Hiroaki Kariwa, Ikuo Takashima
    JOURNAL OF GENERAL VIROLOGY 93 1 27 - 38 2012年01月 [査読有り][通常論文]
     
    Flaviviruses are assembled to bud into the lumen of the endoplasmic reticulum (ER) and are secreted through the vesicle transport pathway, but the details of the molecular mechanism of virion assembly remain largely unknown. In this study, a highly conserved region in the prM protein was identified among flaviviruses. In the subviral particle (SP) system of tick-borne encephalitis virus (TBEV) and Japanese encephalitis virus, secretion of SPs was impaired by a mutation in the conserved region in the prM protein. Viral proteins were sparse in the Golgi complex and accumulated in the ER. Ultrastructural analysis revealed that long filamentous structures, rather than spherical SPs, were observed in the lumen of the ER as a result of the mutation. The production of infectious virions derived from infectious cDNA of TBEV was also reduced by mutations in the conserved region. Molecular modelling analysis suggested that the conserved region is important for the association of prM-envelope protein heterodimers in the formation of a spike of immature virion. These results are the first demonstration that the conserved region in the prM protein is a molecular determinant for the flavivirus assembly process.
  • Michihito Sasaki, Eunmi Kim, Manabu Igarashi, Kimihito Ito, Rie Hasebe, Hideto Fukushi, Hirofumi Sawa, Takashi Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 286 45 39370 - 39378 2011年11月 [査読有り][通常論文]
     
    Equine herpesvirus-1 (EHV-1), an alpha-herpesvirus of the family Herpesviridae, causes respiratory disease, abortion, and encephalomyelitis in horses. EHV-1 utilizes equine MHC class I molecules as entry receptors. However, hamster MHC class I molecules on EHV-1-susceptible CHO-K1 cells play no role in EHV-1 entry. To identify the MHC class I molecule region that is responsible for EHV-1 entry, domain exchange and site-directed mutagenesis experiments were performed, in which parts of the extracellular region of hamster MHC class I (clone C5) were replaced with corresponding sequences from equine MHC class I (clone A68). Substitution of alanine for glutamine at position 173 (Q173A) within the alpha 2 domain of the MHC class I molecule enabled hamster MHC class I C5 to mediate EHV-1 entry into cells. Conversely, substitution of glutamine for alanine at position 173 (A173Q) in equine MHC class I A68 resulted in loss of EHV-1 receptor function. Equine MHC class I clone 3.4, which possesses threonine at position 173, was unable to act as an EHV-1 receptor. Substitution of alanine for threonine at position 173 (T173A) enabled MHC class I 3.4 to mediate EHV-1 entry into cells. These results suggest that the amino acid residue at position 173 of the MHC class I molecule is involved in the efficiency of EHV-1 entry.
  • Kimihito Ito, Manabu Igarashi, Yutaka Miyazaki, Teiji Murakami, Syaka Iida, Hiroshi Kida, Ayato Takada
    PLOS ONE 6 10 e25953  2011年10月 [査読有り][通常論文]
     
    Human influenza A viruses undergo antigenic changes with gradual accumulation of amino acid substitutions on the hemagglutinin (HA) molecule. A strong antigenic mismatch between vaccine and epidemic strains often requires the replacement of influenza vaccines worldwide. To establish a practical model enabling us to predict the future direction of the influenza virus evolution, relative distances of amino acid sequences among past epidemic strains were analyzed by multidimensional scaling (MDS). We found that human influenza viruses have evolved along a gnarled evolutionary pathway with an approximately constant curvature in the MDS-constructed 3D space. The gnarled pathway indicated that evolution on the trunk favored multiple substitutions at the same amino acid positions on HA. The constant curvature was reasonably explained by assuming that the rate of amino acid substitutions varied from one position to another according to a gamma distribution. Furthermore, we utilized the estimated parameters of the gamma distribution to predict the amino acid substitutions on HA in subsequent years. Retrospective prediction tests for 12 years from 1997 to 2009 showed that 70% of actual amino acid substitutions were correctly predicted, and that 45% of predicted amino acid substitutions have been actually observed. Although it remains unsolved how to predict the exact timing of antigenic changes, the present results suggest that our model may have the potential to recognize emerging epidemic strains.
  • Masahiro Kajihara, Keita Matsuno, Edgar Simulundu, Mieko Muramatsu, Osamu Noyori, Rashid Manzoor, Eri Nakayama, Manabu Igarashi, Daisuke Tomabechi, Reiko Yoshida, Masatoshi Okamatsu, Yoshihiro Sakoda, Kimihito Ito, Hiroshi Kida, Ayato Takada
    JAPANESE JOURNAL OF VETERINARY RESEARCH 59 2-3 89 - 100 2011年08月 [査読有り][通常論文]
     
    In 2010, an H5N1 highly pathogenic avian influenza virus (HPAIV) was isolated from feces of apparently healthy ducks migrating southward in Hokkaido, the northernmost prefecture of Japan. The H5N1 HPAIVs were subsequently detected in domestic and wild birds at multiple sites corresponding to the flyway of the waterfowl having stopovers in the Japanese archipelago. The Hokkaido isolate was genetically nearly identical to H5N1 HPAIVs isolated from swans in the spring of 2009 and 2010 in Mongolia, but less pathogenic in experimentally infected ducks than the 2009 Mongolian isolate. These findings suggest that H5N1 HPAIVs with relatively mild pathogenicity might be selected and harbored in the waterfowl population during the 2009-2010 migration seasons. Our data provide "early warning" signals for preparedness against the unprecedented situation in which the waterfowl reservoirs serve as perpetual sources and disseminators of HPAIVs.
  • Edgar Simulundu, Akihiro Ishii, Manabu Igarashi, Aaron S. Mweene, Yuka Suzuki, Bernard M. Hang'ombe, Boniface Namangala, Ladslav Moonga, Rashid Manzoor, Kimihito Ito, Ichiro Nakamura, Hirofumi Sawa, Chihiro Sugimoto, Hiroshi Kida, Chuma Simukonda, Wilbroad Chansa, Jack Chulu, Ayato Takada
    JOURNAL OF GENERAL VIROLOGY 92 6 1416 - 1427 2011年06月 [査読有り][通常論文]
     
    Although the quest to clarify the role of wild birds in the spread of the highly pathogenic H5N1 avian influenza virus (AIV) has yielded considerable data on AIVs in wild birds worldwide, information regarding the ecology and epidemiology of AIVs in African wild birds is still very limited. During AIV surveillance in Zambia (2008-2009), 12 viruses of distinct subtypes (H3N8, H4N6, H6N2, H9N1 and H11N9) were isolated from wild waterfowl. Phylogenetic analyses demonstrated that all the isolates were of the Eurasian lineage. Whilst some genes were closely related to those of AIVs isolated from wild and domestic birds in South Africa, intimating possible AIV exchange between wild birds and poultry in southern Africa, some gene segments were closely related to those of AIVs isolated in Europe and Asia, thus confirming the inter-regional AIV gene flow among these continents. Analysis of the deduced amino acid sequences of internal proteins revealed that several isolates harboured particular residues predominantly observed in human influenza viruses. Interestingly, the isolates with human-associated residues exhibited higher levels of virus replication in the lungs of infected mice and caused more morbidity as measured by weight loss than an isolate lacking such residues. This study stresses the need for continued monitoring of AIVs in wild and domestic birds in southern Africa to gain a better understanding of the emergence of strains with the potential to infect mammals.
  • Manabu Igarashi, Kimihito Ito, Reiko Yoshida, Daisuke Tomabechi, Hiroshi Kida, Ayato Takada
    INFLUENZA AND OTHER RESPIRATORY VIRUSES 5 402 - 404 2011年05月 [査読有り][通常論文]
  • Katsuaki Usami, Keita Matsuno, Manabu Igarashi, Kaori Denda-Nagai, Ayato Takada, Tatsuro Irimura
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 407 1 74 - 78 2011年04月 [査読有り][通常論文]
     
    Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species. (C) 2011 Elsevier Inc. All rights reserved.
  • Kentaro Yoshii, Manabu Igarashi, Kimihito Ito, Hiroaki Kariwa, Michael R. Holbrook, Ikuo Takashima
    VIRUS RESEARCH 155 1 61 - 68 2011年01月 [査読有り][通常論文]
     
    Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis serocomplex of flaviviruses, and causes hemorrhagic disease in humans. In this study, an infectious cDNA of OHFV was constructed to investigate the molecular mechanisms involved in OHFV pathogenesis for the first time. Our cDNA clone was capable of producing infectious virus which is genetically identical to the parental Guriev strain, and the recombinant virus showed similar biological properties to the parental virus including growth kinetics and virulence characteristics. While characterizing the cDNAs, fortuitous mutations at NS2A position 46 and NS5 position 836 were found to affect viral production. By using a viral replicon expressing luciferase, it was shown that both of the mutations produced a defect in RNA replication and that the NS5 mutation induced a temperature-sensitive phenotype, indicating the importance of these residues in RNA replication. This infectious cDNA will be a useful tool to study the replication and pathogenesis of OHFV. (C) 2010 Elsevier B.V. All rights reserved.
  • Yuko Uchida, Katsushi Kanehira, Masaji Mase, Nobuhiro Takemae, Chiaki Watanabe, Tatsufumi Usui, Yoshikazu Fujimoto, Toshihiro Ito, Manabu Igarashi, Kimihito Ito, Ayato Takada, Yoshihiro Sakoda, Masatoshi Okamatsu, Yu Yamamoto, Kikuyasu Nakamura, Hiroshi Kida, Yasuaki Hiromoto, Tomoyuki Tsuda, Takehiko Saito
    VETERINARY MICROBIOLOGY 147 1-2 1 - 10 2011年01月 [査読有り][通常論文]
     
    From February to March 2009, six strains of H7N6 subtype avian influenza virus were isolated from quails in three farms in Aichi prefecture in Japan. The isolates were shown to be low pathogenic for chicken by the examination performed using the "Manual of Standards for Diagnostic Tests and Vaccines" by World organisation for Animal Health (OIE). The deduced amino acid sequence at the cleavage site was PE (I/Q/L) PKRR (nucleotide sequences were cct gaa (a/c) (t/a) a cc (a/g) aaa aga aga), suggesting persistence in domestic poultry for some time. The direct putative ancestor strain could not be elucidated by phylogenetic analysis of all genome segments of the quail isolates. Diverged date from a putative common ancestor in a non-rooted phylogenetic tree among quail viruses was estimated between March 2002 and July 2004. Three putative N-linked glycosylation sites resided in the vicinity of the receptor binding pocket of HA1 region. They are considered to decrease the reactivity of neutralizing antibody against the virus. Experiments for the infectivity and pathogenicity of a quail strain to poultry indicated that the quail isolate had higher infectivity to quails than chickens and ducks. Direct and dust-borne and/or droplet-borne transmissions among quail were proven in quails with and without direct contact with experimentally infected quails. The virus is seldom transmitted among chickens either directly or indirectly, and indirect transmission from infected quails to chickens was not observed. The pathogenicity of the quail strain for mammalian, pig and mouse was low, although it could replicate in those animals. (C) 2010 Elsevier B.V. All rights reserved.
  • Eri Nakayama, Ayaka Yokoyama, Hiroko Miyamoto, Manabu Igarashi, Noriko Kishida, Keita Matsuno, Andrea Marzi, Heinz Feldmann, Kimihito Ito, Masayuki Saijo, Ayato Takada
    CLINICAL AND VACCINE IMMUNOLOGY 17 11 1723 - 1728 2010年11月 [査読有り][通常論文]
     
    Several enzyme-linked immunosorbent assays (ELISAs) for the detection of filovirus-specific antibodies have been developed. However, diagnostic methods to distinguish antibodies specific to the respective species of filoviruses, which provide the basis for serological classification, are not readily available. We established an ELISA using His-tagged secreted forms of the transmembrane glycoproteins (GPs) of five different Ebola virus (EBOV) species and one Marburg virus (MARV) strain as antigens for the detection of filovirus species-specific antibodies. The GP-based ELISA was evaluated by testing antisera collected from mice immunized with virus-like particles as well as from humans and nonhuman primates infected with EBOV or MARV. In our ELISA, little cross-reactivity of IgG antibodies was observed in most of the mouse antisera. Although sera and plasma from some patients and monkeys showed notable cross-reactivity with the GPs from multiple filovirus species, the highest reactions of IgG were uniformly detected against the GP antigen homologous to the virus species that infected individuals. We further confirmed that MARV-specific IgM antibodies were specifically detected in specimens collected from patients during the acute phase of infection. These results demonstrate the usefulness of our ELISA for diagnostics as well as ecological and serosurvey studies.
  • Rie Hasebe, Tadaki Suzuki, Yoshinori Makino, Manabu Igarashi, Satoko Yamanouchi, Akihiko Maeda, Motohiro Horiuchi, Hirofumi Sawa, Takashi Kimura
    BMC MICROBIOLOGY 10 165  2010年06月 [査読有り][通常論文]
     
    Background: West Nile virus (WNV) causes viremia after invasion to the hosts by mosquito bite. Endothelial cells could play an important role in WNV spread from the blood stream into the central nervous system and peripheral tissues. Here, we analyzed the capacity of virus-like particles (VLPs) of the highly virulent NY99 6-LP strain (6-LP VLPs) and the low virulence Eg101 strain (Eg VLPs) to cross cultured human endothelial cells. Results: 6-LP VLPs were transported from the apical to basolateral side of endothelial cells, whereas Eg VLPs were hardly transported. The localization of tight junction marker ZO-1 and the integrity of tight junctions were not impaired during the transport of 6-LP VLPs. The transport of 6-LP VLPs was inhibited by treatment with filipin, which prevents the formation of cholesterol-dependent membrane rafts, suggesting the involvement of raft-associated membrane transport. To determine the amino acid residues responsible for the transport of VLPs, we produced mutant VLPs, in which residues of E protein were exchanged between the 6-LP and Eg strains. Double amino acid substitution of the residues 156 and 159 greatly impaired the transport of VLPs. Conclusion: Our results suggest that a transcellular pathway is associated with 6-LP VLPs transport. We also showed that the combination of the residues 156 and 159 plays an important role in the transport of VLPs across endothelial cells.
  • Keita Matsuno, Noriko Kishida, Katsuaki Usami, Manabu Igarashi, Reiko Yoshida, Eri Nakayama, Masayuki Shimojima, Heinz Feldmann, Tatsuro Irimura, Yoshihiro Kawaoka, Ayato Takada
    JOURNAL OF VIROLOGY 84 10 5140 - 5147 2010年05月 [査読有り][通常論文]
     
    The glycoproteins (GPs) of filoviruses are responsible for virus entry into cells. It is known that GP interacts with cellular C-type lectins for virus attachment to cells. Since primary target cells of filoviruses express C-type lectins, C-type lectin-mediated entry is thought to be a possible determinant of virus tropism and pathogenesis. We compared the efficiency of C-type lectin-mediated entry between Marburg virus strains Angola and Musoke by using a vesicular stomatitis virus (VSV) pseudotype system. VSV pseudotyped with Angola GP (VSV-Angola) infected K562 cells expressing the C-type lectin, human macrophage galactose-type C-type lectin (hMGL), or dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) more efficiently than VSV pseudotyped with Musoke GP (VSV-Musoke). Unexpectedly, the binding affinity of the C-type lectins to the carbohydrates on GPs did not correlate with the different efficiency of C-type lectin-mediated entry. Site-directed mutagenesis identified the amino acid at position 547, which switched the efficiency of C-type lectin-mediated entry. In a three-dimensional model of GP, this amino acid was in close proximity to the putative site of cathepsin processing. Interestingly, the cathepsin inhibitors reduced the infectivity of VSV-Angola less efficiently than that of VSV-Musoke in C-type lectin-expressing K562 cells, whereas only a limited difference was found in control cells. The amino acid at position 547 was critical for the different effects of the inhibitors on the virus infectivities. These results suggest that the efficiency of C-type lectin-mediated entry of filoviruses is controlled not only by binding affinity between C-type lectins and GP but also by mechanisms underlying endosomal entry, such as proteolytic processing by the cathepsins.
  • Yasuko Watanabe, Wakako Hiraoka, Manabu Igarashi, Kimihito Ito, Yuhei Shimoyama, Motohiro Horiuchi, Tohru Yamamoria, Hironobu Yasui, Mikinori Kuwabara, Fuyuhiko Inagaki, Osamu Inanami
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 394 3 522 - 528 2010年04月 [査読有り][通常論文]
     
    To explore Cu(II) ion coordination by His(186) in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique) Six moPrP mutants. moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C). and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1). moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1) This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the rutroxide spin probe, suggesting that each interspin distance was within 20 angstrom The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), inoPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12 1 angstrom, 18 1 angstrom, 107 angstrom, and 84 angstrom, respectively In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP(C) (C) 2010 Elsevier Inc All rights reserved
  • Manabu Igarashi, Kimihito Ito, Reiko Yoshida, Daisuke Tomabechi, Hiroshi Kida, Ayato Takada
    PLOS ONE 5 1 e8553  2010年01月 [査読有り][通常論文]
     
    The pandemic influenza virus (2009 H1N1) was recently introduced into the human population. The hemagglutinin (HA) gene of 2009 H1N1 is derived from "classical swine H1N1'' virus, which likely shares a common ancestor with the human H1N1 virus that caused the pandemic in 1918, whose descendant viruses are still circulating in the human population with highly altered antigenicity of HA. However, information on the structural basis to compare the HA antigenicity among 2009 H1N1, the 1918 pandemic, and seasonal human H1N1 viruses has been lacking. By homology modeling of the HA structure, here we show that HAs of 2009 H1N1 and the 1918 pandemic virus share a significant number of amino acid residues in known antigenic sites, suggesting the existence of common epitopes for neutralizing antibodies cross-reactive to both HAs. It was noted that the early human H1N1 viruses isolated in the 1930s-1940s still harbored some of the original epitopes that are also found in 2009 H1N1. Interestingly, while 2009 H1N1 HA lacks the multiple N-glycosylations that have been found to be associated with an antigenic change of the human H1N1 virus during the early epidemic of this virus, 2009 H1N1 HA still retains unique three-codon motifs, some of which became N-glycosylation sites via a single nucleotide mutation in the human H1N1 virus. We thus hypothesize that the 2009 H1N1 HA antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in humans. Indeed, amino acid substitutions predicted here are occurring in the recent 2009 H1N1 variants. The present study suggests that antibodies elicited by natural infection with the 1918 pandemic or its early descendant viruses play a role in specific immunity against 2009 H1N1, and provides an insight into future likely antigenic changes in the evolutionary process of 2009 H1N1 in the human population.
  • Reiko Yoshida, Manabu Igarashi, Hiroichi Ozaki, Noriko Kishida, Daisuke Tomabechi, Hiroshi Kida, Kimihito Ito, Ayato Takada
    PLOS PATHOGENS 5 3 e1000350  2009年03月 [査読有り][通常論文]
     
    The hemagglutinin (HA) of influenza A viruses has been classified into sixteen distinct subtypes (H1-H16) to date. The HA subtypes of influenza A viruses are principally defined as serotypes determined by neutralization or hemagglutination inhibition tests using polyclonal antisera to the respective HA subtypes, which have little cross-reactivity to the other HA subtypes. Thus, it is generally believed that the neutralizing antibodies are not broadly cross-reactive among HA subtypes. In this study, we generated a novel monoclonal antibody (MAb) specific to HA, designated MAb S139/1, which showed heterosubtypic cross-reactive neutralization and hemagglutination inhibition of influenza A viruses. This MAb was found to have broad reactivity to many other viruses (H1, H2, H3, H5, H9, and H13 subtypes) in enzyme-linked immunosorbent assays. We further found that MAb S139/1 showed neutralization and hemagglutination-inhibition activities against particular strains of H1, H2, H3, and H13 subtypes of influenza A viruses. Mutant viruses that escaped neutralization by MAb S139/1 were selected from the A/Aichi/2/68 (H3N2), A/Adachi/2/57 (H2N2), and A/WSN/33 (H1N1) strains, and sequence analysis of the HA genes of these escape mutants revealed amino acid substitutions at positions 156, 158, and 193 (H3 numbering). A molecular modeling study showed that these amino acids were located on the globular head of the HA and formed a novel conformational epitope adjacent to the receptor-binding domain of HA. Furthermore, passive immunization of mice with MAb S139/1 provided heterosubtypic protection. These results demonstrate that MAb S139/1 binds to a common antigenic site shared among a variety of HA subtypes and neutralizes viral infectivity in vitro and in vivo by affecting viral attachment to cells. The present study supports the notion that cross-reactive antibodies play some roles in heterosubtypic immunity against influenza A virus infection, and underscores the potential therapeutic utility of cross-reactive antibodies against influenza.
  • Manabu Igarashi, Kimihito Ito, Ayato Takada
    Glycoforum 2009年
  • Manabu Igarashi, Kimihito Ito, Hiroshi Kida, Ayato Takada
    VIROLOGY 376 2 323 - 329 2008年07月 [査読有り][通常論文]
     
    The addition of oligosaccharide side chains to influenza virus hemagglutinin (HA) is believed to facilitate viral escape from immune pressure in the human population. To determine the implicit potentials for acquisition of N-linked glycosylation, we analyzed the genetic background of 16 subtypes of avian influenza virus, some of which may be potential pandemic viruses in the future. We found a significant difference among HA subtypes in their genomic sequences to produce N-glycosylation sites. Notably, recently circulating avian influenza viruses of the H5 and H9 subtypes may have rather greater capacities to undergo mutations associated with glycosylation of HA than past pandemic viruses. We hypothesize that influenza viruses maintained in natural reservoirs could have different potentials for sustained circulation, depending on their HA subtypes, if introduced into the human population. (C) 2008 Elsevier Inc. All rights reserved.
  • Ito K, Igarashi M, Takada A
    Knowledge Media Technologies 21 154 - 158 TU Ilmenau 2006年07月 [査読有り][通常論文]
     
    The hemagglutinin (HA) of influenza viruses undergoes antigenic drift to escape from antibody-mediated immune pressure. In order to predict possible structural changes of the HA molecules in future, it is important to understand the patterns of amino acid mutations and structural changes in the past. We performed a retrospective and comprehensive analysis of structural changes in H3 hemagglutinins of human influenza viruses isolated during 1968 to 2006. Amino acid sequence data of more than 2000 strains have been collected from NCBI Influenza virus resources. Information theoretic analysis of the collected sequences revealed a number of simultaneous mutations of amino acids at two or more different positions (correlated mutations). We also calculated the net charge of the HA1 subunit, based on thenumber of charged amino acid residues, and found that the net charge increased linearly from 1968 to 1984 and, after then, has been saturated. This level of the net charge may be an upper limit for H3 HA to be functional. It is noted that "correlated mutations" with the conversion of acidic and basic amino acid residues between two different positions were frequently found after 1984, suggesting that these mutations contributed to counterbalancing effect to keep the net charge of the HA . These approaches may open the way to find the direction of future antigenic drift of influenza viruses.
  • Tachikawa H, Igarashi M
    Chemical Physics 324 2-3 639 - 646 2006年05月 [査読有り][通常論文]
     
    Direct ab initio molecular dynamics (MD) calculations have been applied to a S(N)2 reaction OH- + CH3Cl -> CH3OH + Cl-. The collision dynamics with non-zero impact parameters were treated in the present study, and the results are compared with the near collinear collision dynamics previously reported by us [H. Tachikawa, M. Igarashi, T. Ishibashi, J. Phys. Chem. A 106 (2002) 10977]. The collision energy was fixed to 25 kcal/mol. The product state distribution obtained for the non-zero impact parameter collision dynamics was slightly different from that of the collinear collision. The distribution of relative translational energy between products Cl- and CH3OH in the non-zero impact parameter collision dynamics was shifted to higher energy region from that of collinear collision. Also, it was found that the mean translational energy of the product has a maximum at non-zero impact parameter (b = 0.6-1.2 angstrom). The reaction mechanism is discussed on the basis of theoretical results. (c) 2005 Elsevier B.V. All rights reserved.
  • Gino C. Matibag, Manabu Igarashi, Ron E. La Porte, Hiko Tamashiro
    Environmental Health and Preventive Medicine 10 5 273 - 281 2005年09月 [査読有り][通常論文]
     
    This paper discusses the history of emerging infectious diseases, risk communication and perception, and the Supercourse lectures as means to strengthen the concepts and definition of risk management and global governance of zoonosis. The paper begins by outlining some of the key themes and issues in infectious diseases, highlighting the way which historical analysis challenges ideas of the 'newness' of some of these developments. It then discusses the role of risk communication to public accountability. The bulk of the paper presents an overview of developments of the Internet-based learning system through the Supercourse lectures that may prove to be a strong arm for the promotion of the latest medical information particularly to developing countries.
  • Shunichi Akazawa, Manabu Igarashi, Hirofumi Sawa, Hiko Tamashiro
    Environmental Health and Preventive Medicine 10 5 282 - 285 2005年09月 [査読有り][通常論文]
     
    Information security and assurance are an increasingly critical issue in health research. Whether health research be in genetics, new drugs, disease outbreaks, biochemistry, or effects of radiation, it deals with information that is highly sensitive and which could be targeted by rogue individuals or groups, corporations, national intelligence agencies, or terrorists, looking for financial, social, or political gains. The advents of the Internet and advances in recent information technologies have also dramatically increased opportunities for attackers to exploit sensitive and valuable information. Government agencies have deployed legislative measures to protect the privacy of health information and developed information security guidelines for epidemiological studies. However, risks are grossly underestimated and little effort has been made to strategically and comprehensively protect health research information by institutions, governments and international communities. There is a need to enforce a set of proactive measures to protect health research information locally and globally. Such measures should be deployed at all levels but will be successful only if research communities collaborate actively, governments enforce appropriate legislative measures at national level, and the international community develops quality standards, concluding treaties if necessary, at the global level. Proactive measures for the best information security and assurance would be achieved through rigorous management process with a cycle of "plan, do, check, and act". Each health research entity, such as hospitals, universities, institutions, or laboratories, should implement this cycle and establish an authoritative security and assurance organization, program and plan coordinated by a designated Chief Security Officer who will ensure implementation of the above process, putting appropriate security controls in place, with key focus areas such as policies and best practices, enforcement and certification, risk assessment and audit, monitoring and incident response, awareness and training, and modern protection method and architecture. Governments should enforce a comprehensive scheme, and international health research communities should adopt standardized innovative methods and approaches.
  • Gino C. Matibag, Manabu Igarashi, Hiko Tamashiro
    Environmental Health and Preventive Medicine 10 5 303 - 314 2005年09月 [査読有り][通常論文]
     
    Since the advent of bovine spongiform encephalopathy (BSE) in the United Kingdom in 1986, new BSE cases have recently become rare. However, in Japan and the United States, positive cases have started to be seen recently. The rise in BSE cases paved the way for the human form of this disease, the variant Creutzfeldt-Jakob disease (vCJD). The observed trends in the UK may be attributed to effective implementation of public health policies coupled with increased vigilance through advancement in science and technology, or they may well be a reflection of the natural disease progression. We aim to discuss the BSE chronology of events, and compare examination methods, costs and cost-efficiency, management, and public policies of Japan, Europe, and the USA.
  • H Tachikawa, M Igarashi, J Nishihira, T Ishibashi
    JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 79 1 11 - 23 2005年04月 [査読有り][通常論文]
     
    Ab initio molecular orbital (MO) and hybrid density functional theory (DFT) calculations have been applied to the initial step of the acylation reaction catalyzed by acetylcholinesterase (AChE), which is the nucleophiric addition of Ser200 in catalytic triads to a neurotransmitter acetylcholine (ACh). We focus our attention mainly on the effects of oxyanion hole and Glu327 on the potential energy surfaces (PESs) for the proton transfer reactions in the catalytic triad Ser200-His440-G1027. The activation barrier for the addition reaction of Ser200 to ACh was calculated to be 23.4 kcal/mol at the B3LYP/6-31G(d)//HF/3-21G(d) level of theory. The barrier height under the existence of oxyanion hole, namely, Ser200-His440-Glu327-ACh-(oxyanion hole) system, decreased significantly to 14.2 kcal/mol, which is in reasonable agreement with recent experimental value (12.0 kcal/mol). Removal of Glu327 from the catalytic triad caused destabilization of both energy of transition state for the reaction and tetrahedral intermediate (product). PESs calculated for the proton transfer reactions showed that the first proton transfer process is the most important in the stabilization of tetrahedral intermediate complex. The mechanism of addition reaction of ACh was discussed on the basis of theoretical results. (c) 2004 Elsevier B.V. All rights reserved.
  • Energetics of Catalytic Reaction of Acetylcholinesterase (AChE) with Acetylcholine (ACh): Role of Oxyanion Hole
    Igarashi M, Ishibashi T, Nishihira J, Tachikawa H
    Internet Electronic Journal of Molecular Design 2 712 - 722 2003年11月 [査読有り][通常論文]
  • H Tachikawa, M Igarashi, T Ishibashi
    JOURNAL OF PHYSICAL CHEMISTRY A 107 38 7505 - 7513 2003年09月 [査読有り][通常論文]
     
    Ionization dynamics of trans-formanilide-water 1:1 complexes FA(H2O) have been investigated by means of direct ab initio trajectory method. From the static ab initio calculations, three conformers of the FA(H2O) complexes were obtained as stable structures: namely, these are the N-H site, the C=O carbonyl site and the bridge site, which are different in the positions of H2O around FA. In the N-H and C=O sites, a water molecule binds to the hydrogen and oxygen atoms of the peptide (-NH-CO), respectively. In the bridge site, the hydrogen and oxygen atoms of H2O bind to the C=O carbonyl and a hydrogen of benzene ring (o-position) of FA, respectively. The trajectories from the vertical ionization points of these three structures were calculated by means of full dimensional direct ab initio trajectory method. It was found that the H2O molecule in the N-H site is still remained in its site after ionization, i.e., the strong complex cation where H2O binds to the N-H site of FA(+) is directly formed. On the other hand, in the cases of the ionization from both CO and bridge sites, the water molecule was moved easily around both the benzene ring and the C=O carbonyl group. The mechanism of the ionization of FA(H2O) was discussed on theoretical results.
  • Tachikawa H, Igarashi M, Ishibashi T
    J. Phys. Chem. A 106 46 10977 - 10984 2002年11月 [査読有り][通常論文]
     
    Direct ab initio trajectory calculations have been applied to a S(N)2 reaction, OH- + CH3Cl --> CH3OH + Cl-. First, static ab initio molecular orbital (MO) calculations with several basis sets were examined to select the most convenient and best fit basis set to that of high-quality calculations. As a result of the static ab initio calculations, it was found that the Hartree-Fock (HF)/3-21+G(d) calculation reasonably represents a potential energy surface calculated at the MP2/6-311++G(2df,2pd) level. Next, direct ab initio dynamics calculations using the 3-21+G(d) basis set were carried out for the S(N)2 reaction. A full dimensional potential energy surface including all degrees of freedom was used in the dynamics calculation. The collision energies chosen were E-coll = 5 and 25 kcal/mol. In the collisions at E-coll = 5 and 25 kcal/mol, 48% and 63% of the total available energies were, respectively, partitioned into the relative translational mode between CH3OH and Cl-. Also, it was predicted that the C-H stretching mode of the product CH3OH is excited after the S(N)2 reaction, which is not detected in the case of the halogen-atom exchange S(N)2 reaction. The reaction mechanism was discussed on the basis of theoretical results.
  • M Igarashi, T Ishibashi, H Tachikawa
    JOURNAL OF MOLECULAR STRUCTURE-THEOCHEM 594 61 - 69 2002年10月 [査読有り][通常論文]
     
    Temperature and solvation effects on the hyperfine coupling constants (HFCCs) of methyl radical have been investigated by means of direct ab initio molecular dynamics (MD) method. The complexes composed of methyl radical and H2O molecules, CH3(H2O)(n) (n = 0-2), were chosen as models of the solvation systems. The geometry optimizations of CH3(H2O) showed that the hydrogen of H2O molecule orients toward the carbon of CH3, and it is weakly bound to the carbon atom of CH3. The binding energies for n = 1 and n = 2 were calculated to be 1.50 and 2.82 kcal/mol at the MP2/6-311 + + G(d,p) level, respectively. The direct ab initio MD calculations indicated large temperature dependence of HFCCs: hydrogen-HFCC of CH3 decreases with increasing temperature. This large change is due to the fact that the structure of the complex is flexible and is significantly varied by thermal activation. Mechanism of the temperature dependence of HFCCs was discussed on the basis of theoretical results. (C) 2002 Elsevier Science B.V. All rights reserved.
  • H Tachikawa, M Igarashi, T Ishibashi
    CHEMICAL PHYSICS LETTERS 363 3-4 355 - 361 2002年09月 [査読有り][通常論文]
     
    Direct ab initio trajectory calculations have been applied to a typical microsolvated S(N)2 reaction F- (H2O) + CH3Cl at hyperthermal collision energies. The branching ratios for the product channels, F- (H2O) + CH3Cl --> Cl- + H2O + CH3F channel I, --> Cl- (H2O) + CH3F channel II --> Cl- + CH3F-H2O channel III, were calculated as a function of center-of-mass collision energy (E-coll). It was found that branching ratios of the product channels were drastically changed by the increase of E-coll. At the collision energy of 30 kcal/mol, the branching ratios of channels I:II:III were calculated to be 0.37, 0.53, and 0.10, respectively. The branching ratio for channel II became 0.69 at collision energy of 40 kcal/mol, meaning that channel II is dominant at higher collision energies, although the ratio of channel II was close to zero at thermal energy. These results suggested that the product channels in the microsolvated S(N)2 reaction are significantly affected by the collision energy. (C) 2002 Elsevier Science B.V. All rights reserved.
  • H Tachikawa, M Igarashi, T Ishibashi
    CHEMICAL PHYSICS LETTERS 352 1-2 113 - 119 2002年01月 [査読有り][通常論文]
     
    Temperature effects on the hyperfine coupling constants (hfcc's) of the methyl radical have been investigated by means of direct ab initio molecular dynamics (MD) method. The calculations showed that the hydrogen-hfcc (H-hfcc) of CH increases with increasing temperature. Also, it was indicated that hfcc of the carbon atom of CH3 increases as temperature is increased. The H-hfcc's of CH3 were varied from -22.94 to -22.77 G in the temperature ranges 10-300 K. These features were in good agreement with electron paramagnetic resonance (EPR) experiments. The effects of temperature on H-hfcc were discussed on the basis of theoretical results. (C) 2001 Elsevier Science B.V. All rights reserved.
  • H Tachikawa, M Igarashi, T Ishibashi
    PHYSICAL CHEMISTRY CHEMICAL PHYSICS 3 15 3052 - 3056 2001年 [査読有り][通常論文]
     
    Structure and vibrational frequencies of the benzene-water complex cation [BzH(2)O](+) have been calculated by means of density functional theory (B3LYP calculation). A planar structure with a C-s symmetry, in which all heavy atoms are located on a molecular plane, was obtained as the most stable form of the [BzH(2)O](+) cation. Hydrogen atoms of the water molecule are located above and below the molecular plane. From the present calculations, it was predicted that vibrational frequencies of symmetric and asymmetric O-H stretching modes of H2O (nu (1) and nu (3) modes, respectively) are red-shifted from those of a free H2O by the formation of a complex, whereas the H-O-H bending mode (nu (2) mode) is blue-shifted. Infrared intensities of the three modes of H2O were significantly increased by the complex formation. Similar features were obtained for the ethylene-H2O complex cation [C2H4-H2O](+). The origin of the frequency-shifts is discussed on the basis of theoretical results. The hydrogen-hyperfine coupling constants of the complex cations were also predicted theoretically.
  • 阿部和厚, 五十嵐学
    高等教育ジャーナル─高等教育と生涯学習─ 8 79 - 84 北海道大学 2000年03月 [査読有り][通常論文]
  • M Igarashi, H Tachikawa
    INTERNATIONAL JOURNAL OF MASS SPECTROMETRY 197 243 - 252 2000年02月 [査読有り][通常論文]
     
    Direct ab initio dynamics calculations on the photoelectron detachment processes of H3O- anion have been carried out by using HF/6-311G** full dimensional potential energy surface (PES). Total energy and the energy gradient of atoms were calculated in each time step. The excess energy was assumed to be zero at the starting point of the trajectory. The H3O- anion is composed of two isomers, H-(H2O) and OH-(H-2) in which H- and OH- anions are solvated by H2O and H-2 molecules, respectively. The present calculations indicated that the available energy, yielded by the electron detachment of H-(H2O), is almost distributed into the relative translational energy between fragments. On the other hand, the available energy in an OH-(H-2) isomer is Partitioned into both the relative translational mode and internal modes of the fragments, although the latter energy is minor. The excitation of internal modes of the products were observed for only the detachment process of the OH-(H-2) complex. These are due to the fact that the structure of solvent molecules (H2O and H-2) in each complex is significantly close to that of free molecules whereas the PES of the neutral state is always repulsive. The mechanism of the electron detachment process of H3O- is discussed on the basis of theoretical results. (C) 2000 Elsevier Science B.V.
  • Tachikawa H, Igarashi M
    Chem. Phys. Lett. 303 1-2 81 - 86 1999年04月 [査読有り][通常論文]
     
    Direct ab-initio dynamics calculations have been applied to a gas phase S(N)2 reaction F- + CH3Cl --> CH3F + Cl-. An ab-initio potential energy surface including all degrees of freedom was used. Total energies and gradients were calculated at each time step. Results for near-collinear collision are reported. The initial geometrical configurations of (F- + CH3Cl) at time zero were randomly generated in the range 180 +/- 3 degrees for the collision angle angle F-C-Cl and of r(F--C) = 6.0-6.5 Angstrom. The vibrational phase of CH3Cl was generated to take a temperature of 10 K. It was found that almost all available energy is partitioned into the relative translational mode between the products (similar to 43%) and the C-F stretching mode (similar to 57%) at zero collision energy. The other internal modes of CH3F remain in the ground state. The lifetimes of early- and late-complexes F- ... CH3Cl and FCH3 ... Cl- were short enough to dissociate directly to products, while the energy was not completely distributed within the lifetime. It is concluded that the S(N)2 reaction F- ... CH3Cl proceeds non-statistically via a direct mechanism in the case of near-collinear collision. (C) 1999 Elsevier Science B.V. All rights reserved.
  • Igarashi M, Tachikawa H
    J. Mass Spectrom. 181 151 - 157 1998年12月 [査読有り][通常論文]
     
    Ab-initio molecular orbital (MO) and direct ab initio dynamics calculations have been applied to the gas phase S(N)2 reaction F- + CH3Cl --> CH3F + Cl-. Several basis sets were examined in order to select the most convenient and best fitted basis set to that of high-quality calculations. The Hartree-Fock (HF) 3-21+G(d) calculation reasonably represents a potential energy surface calculated at the MP2/6-311++G(2df,2pd) level. A direct ab initio dynamics calculation at the HF/3 - 21 + G(d) level was carried out for the S(N)2 reaction. A full dimensional ab initio potential energy surface including all degrees of freedom was used in the dynamics calculation. Total energies and gradients were calculated at each time step. Two initial configurations at time zero were examined in the direct dynamics calculations: one is a near collinear collision, and the other is a side-attack collision. It was found that in the near collinear collision almost all total available energy is partitioned into two modes: the relative translational mode between the products (similar to 40%) and the C - F stretching mode (similar to 60%). The other internal modes of CH3F were still in the ground state. The lifetimes of the early- and late-complexes F-... CH3Cl and FCH3...Cl- are significantly short enough to dissociate directly to the products. On the other hand, in the side-attack collision, the relative translation energy was about 20% of total available energy. (Int J Mass Spectrom 181 (1998) 151-157) (C) 1998 Elsevier Science B.V.
  • Tachikawa H, Igarashi M
    J. Phys. Chem. A 102 45 8648 - 8656 1998年10月 [査読有り][通常論文]
  • Tachikawa H, Hamabayashi T, Igarashi M
    J. Mol. Struct. (THEOCHEM) 453 1-3 191 - 196 1998年10月 [査読有り][通常論文]
     
    Quenching probabilities (P-Q values) in the energy transfer reaction S(D-1) + CO --> S(P-3) + CO(v,J) have been calculated as functions of both center of mass collision energy (E-coll) and spin-orbit coupling (H-12) by means of the surface-hopping trajectory method using ab initio fitted potential energy surfaces (PESs). Two ab initio fitted potential energy surfaces (singlet and triplet PESs) obtained at the MP2/6-31G* level were employed in the quenching process. The surface hopping was treated as the Landau-Zener model in the crossing seam between the singlet and triplet PESs. The calculations showed that the reaction is composed of three reaction channels: one is a complex channel in which the reaction proceeds via a long-lived complex [SCO], and the second one is a direct channel in which the reaction proceeds without the long-lived complex. The third one is a mixed channel composed of direct and complex channels. The P-Q decreases with increasing E-coll and increases linearly with increasing spin-coupling value H12 The collision energy dependence of P-Q is due to the fact that branching ratio of direct channels to complex ones is varied as a function of collision energy. (C) 1998 Elsevier Science B.V. All rights reserved.
  • H Tachikawa, M Igarashi, K Komaguchi
    INTERNATIONAL JOURNAL OF MASS SPECTROMETRY 177 1 17 - 21 1998年08月 [査読有り][通常論文]
     
    The structures and electronic states of the CCl4- anion in the condensed phase have been studied by direct ab initio dynamics calculation. The calculation showed that two stationary points were obtained as stable forms of CCl4- which correspond to elongated and compressed forms of CCl4-. The elongated form has an elongated C-Cl bond, whereas the compressed form has a shorter C-Cl bond in CCl4-. The MP4SDQ/6-31G* calculation indicated that the elongated form is energetically more favored than the compressed form. However, the contact spin density for the elongated form disagrees with that measured by electron spin resonance spectroscopy in the condensed phase. By contrast, the spin density for the compressed form is in reasonable agreement with the experiment. The formation mechanism of the compressed form in the condensed phase is discussed on the basis of direct ab initio dynamics calculation. The present results strongly indicated that the CCl4- has the compressed form in condensed form. (Int J Mass Spectrom 177 (1998) 17-21). (C) 1998 Elsevier Science B.V.

MISC

担当経験のある科目（授業）

  • 製剤開発特論北海道大学
  • 生物統計学特論北海道大学
  • 全学講義「感染症と免疫」北海道大学
  • 情報科学特論北海道大学
  • ケミカルハザード特論北海道大学

所属学協会

  • インフルエンザ研究者交流の会   情報計算化学生物学会   日本ウイルス学会   

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2023年04月 -2027年03月 
    代表者 : 園下 将大, 五十嵐 学, 小沼 剛, 市川 聡, 合田 圭介
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2023年04月 -2027年03月 
    代表者 : 園下 将大, 市川 聡, 五十嵐 学, 小沼 剛
  • 代謝制御因子を標的とする新規膵がん治療法の開発
    日本医療研究開発機構(AMED)：次世代がん医療加速化研究事業
    研究期間 : 2023年07月 -2026年03月 
    代表者 : 園下将大, 合田圭介, 小松崎民樹, 市川聡, 五十嵐学, 小沼剛
  • 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 : 2023年04月 -2026年03月 
    代表者 : 五十嵐 学, 広川 貴次, 阿部 貴志, 松野 啓太
  • 宿主RNAメチルトランスフェラーゼを標的とした抗インフルエンザウイルス薬の探索
    日本学術振興会：科学研究費助成事業 国際共同研究加速基金(国際共同研究強化(B))
    研究期間 : 2022年10月 -2026年03月 
    代表者 : 五十嵐 学, 松野 啓太, 日尾野 隆大
  • 高精度立体構造予測を活用した新規ウイルスの探索：ダークマター配列への挑戦
    日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2022年06月 -2025年03月 
    代表者 : 五十嵐 学, 堀江 真行
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2022年04月 -2025年03月 
    代表者 : 迫田 義博, 五十嵐 学, 青木 博史
     
    豚熱（CSF）ウイルスおよび非定型豚ペスチ（APP）ウイルスの病原性、特に持続感染を引き起こす分子基盤を解明するために、2つのウイルスが属するフラビウイルス科ペスチウイルス属において病原性因子として細胞の内・外からその機能が発揮できるウイルス構造蛋白質Ernsに着目する。ウイルスがブタに持続感染し、結果として胎子や新生子豚に病原性を発揮するきっかけとなる宿主免疫からの回避と制御された細胞死の抑制に関するErns蛋白質の機能を調べる。 まず、Erns蛋白質の三次元構造と自然免疫の抑制に重要なRNase活性およびⅠ型インターフェロンの産生調節の比較するために、近年国内で分離された中程度の病原性のCSFウイルス野外株、古典的な野外強毒株、およびワクチン株GPE－株のErns遺伝子を真核細胞発現プラスミドにクローニングした。同様に、APPウイルスのErns遺伝子を真核細胞発現プラスミドにクローニングした。これらのプラスミドから発現させた組換えErns蛋白質の自然免疫の抑制に関与するRNase活性とⅠ型インターフェロンの産生抑制能をウイルス株間で比較するための実験系を構築した。またAPPウイルスErns蛋白質の構造を解析し、CSFウイルスのそれと比較してRNase活性ドメインのモチーフに特有の配列があることがわかった。さらに、APPウイルス国内分離株をブタに接種し、臨床症状の観察に加え、ウイルスの生体での検出期間、各臓器における組織病変を確認した。その結果、豚での増殖は非常に限定的であることがわかった。
  • 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 : 2022年04月 -2025年03月 
    代表者 : 迫田 義博, 五十嵐 学, 青木 博史
  • 革新的生体イメージング技術によるフラビウイルス感染動態を解析できる動物モデルの開発
    日本医療研究開発機構(AMED)：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
    研究期間 : 2024年05月 -2025年03月 
    代表者 : 田村友和, 五十嵐学
  • 構造類似性から紐解くクリミア・コンゴ出血熱ウイルスの創薬標的の探索
    長崎大学（AMED）：長崎大学感染症共同研究拠点「国際的に脅威となる一類感染症の研究及び高度 安全実験施設（BSL-4）を活用する人材の育成」補助事業 にかかる研究課題（研究分担者）
    研究期間 : 2023年07月 -2025年03月 
    代表者 : 五十嵐学, 松野啓太
  • レポーター技術を駆使したデング熱新規診断法の開発
    日本医療研究開発機構(AMED)：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
    研究期間 : 2023年10月 -2024年03月 
    代表者 : 田村友和, 五十嵐学
  • 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 : 2020年04月 -2023年03月 
    代表者 : 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：科学研究費助成事業 基盤研究(B)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 五十嵐 学, 広川 貴次, 阿部 貴志, 松野 啓太
  • 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 : 2017年04月 -2023年03月 
    代表者 : 五十嵐 学
     
    本研究では、抗体の親和性・特異性を制御するための論理的分子設計手法の確立を目指している。具体的に本研究課題では、インフルエンザウイルスの表面糖蛋白質ヘマグルチニン（HA）と中和モノクローナル抗体S139/1との複合体構造を用いて、分子動力学（MD）計算から得られる結合自由エネルギーを指標に抗体の結合能を改変し、エスケープ変異株や複数のHA亜型のウイルスを中和する抗体の設計を試みている。このような立体構造を基盤に既存抗体の親和性・特異性を改変する技術は、様々な人獣共通感染症の診断・治療研究へ幅広く応用されることが想定される。
    これまで、S139/1存在下でA/Victoria/3/75 (H3N2)（Vic株）を培養すると、HAの158番目にアミノ酸変異を持つエスケープ変異体（HA_vic158）が分離されることを明らかにした。本年度も、昨年度に引き続き、S139/1がHA_vic158に再結合できるよう、S139/1の改変を試みた。HA_vic158の158番目の残基は、S139/1のL鎖CDR3と物理的に衝突する。この衝突を回避できるL鎖CDR3を、公共データベース（PDB）に対して検索した。はじめに、PDBに登録されている抗体構造のL鎖CDR3をすべて抽出した。次に、これらをS139/1のL鎖CDR3に移植し、改変S139/1の構造モデルを計算機上で網羅的に構築した。つづいて、これらの改変S139/1とHA_vic158との親和性を結合自由エネルギー計算により評価したが、現在まで強い相互作用を示す改変S139/1は得られていない。現在、HA_vic158との衝突が小さい改変S139/1モデルを選別し、これらの改変S139/1の結合領域にアミノ酸置換を導入することで、強い相互作用を示す改変S139/1の取得を試みている。
  • 新規膵がん動物モデルに立脚した膵がん組み合わせ療法の開発
    日本医療研究開発機構(AMED)：次世代がん医療創生研究事業
    研究期間 : 2020年04月 -2022年03月 
    代表者 : 園下 将大, 大塩 貴子, 市川 聡, 五十嵐 学
  • フラビウイルス科ウイルスの粒子形成に関わる蛋白質の構造生物学的解析
    大阪大学微生物病研究所：共同利用・共同研究拠点「微生物病共同研究拠点」事業
    研究期間 : 2019年04月 -2020年03月 
    代表者 : 五十嵐学
  • 立体構造情報を活用した高病原性ウイルスの蛋白質機能探索
    日本医療研究開発機構(AMED)：感染症研究革新イニシアティブ(J-PRIDE)
    研究期間 : 2017年08月 -2020年03月 
    代表者 : 五十嵐 学, 松野 啓太, 高田 礼人
  • 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 : 2012年04月 -2017年03月 
    代表者 : 高田 礼人, 伊藤 公人, 五十嵐 学, 吉田 玲子, 村松 美笑子, 宮本 洋子
     
    インフルエンザAウイルスのヘマグルチニン(HA)亜型間交差感染防御免疫における抗体の役割を、新たな抗ウイルス活性（ウイルス出芽阻害）を測定する手法によって明らかにした。特に、HA分子上の同じエピトープを認識するモノクローナルIgGおよびIgA抗体の比較によるIgAの優位性の実証に至った。また、構造情報を基にした分子間相互作用シミュレーション技術と組換え抗体作出技術を用いて、抗体の特異性を改変する技術の開発を試みた。
  • 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 : 2013年04月 -2016年03月 
    代表者 : 伊藤 公人, 高田 礼人, 五十嵐 学
     
    インフルエンザの予防にはワクチン接種が有効であるが，人の免疫圧による選択淘汰を受けてウイルスの遺伝子が変異し続けるため，ワクチン株を頻繁に更新しなければならない。そこで，本研究では，ワクチン株を先回りして準備するために，文字列の集合に対する逐次データ同化により，ウイルス遺伝子配列の実データから，近い将来におこるウイルス遺伝子上の変異を予測する手法を研究した。その結果，人の集団免疫と感染およびウイルスの遺伝子変異の過程を表すモデルが構築され，文字列集合からの逐次データ同化により，インフルエンザウイルスの抗原変異を予測する研究基盤を構築した。
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2012年04月 -2014年03月 
    代表者 : 高田 礼人, 伊藤 公人, 五十嵐 学
     
    マールブルグウイルス (MARV) の表面糖蛋白質(GP) 特異的モノクローナル抗体を作出し、ウイルス出芽阻害活性の有無を解析した。その結果、通常の中和活性は持たない抗体の中に、MARV粒子の感染細胞からの出芽・放出を顕著に抑制する抗体が存在することが明らかとなった。これらの抗体存在下で増殖可能なエスケープ変異体を選択し、GPに導入されたアミノ酸変異を調べた結果、ムチン様領域の大規模な欠損またはfurin開裂部位の点変異が認められた。以上の結果より、抗体によるMARV感染抑制の新規メカニズムとMARVのユニークな免疫逃避メカニズムが明らかとなった。
  • インフルエンザウイルスと亜型間交差反応性抗体の相互作用解析と薬剤設計への応用
    公益財団法人 秋山記念生命科学振興財団：研究助成（奨励）
    研究期間 : 2013年 
    代表者 : 五十嵐 学
  • 文部科学省：科学研究費補助金(基盤研究(C))
    研究期間 : 2013年 
    代表者 : 五十嵐 学
  • 文部科学省：科学研究費補助金(若手研究(B))
    研究期間 : 2011年 -2012年 
    代表者 : 五十嵐 学
     
    インフルエンザの流行を効率よく制御するためには、抗原変異の機序を理解し、予測することが必要である。本研究では、インフルエンザウイルスの主要抗原であるヘマグルチニン（HA）とそのモノクローナル抗体（mAb）に焦点をあて、ウイルスがmAbからエスケープする際に起こるHA上のアミノ酸置換の特徴を明らかにすることを目的とした。具体的には、アミノ酸置換に伴うHA-mAb複合体の相互作用変化、およびHAの機能や構造安定性への影響を分子シミュレーション等の計算科学的手法により解析し、実際にエスケープ変異株で観測されるアミノ酸と比較した。
  • 文部科学省：科学研究費補助金(若手研究(B))
    研究期間 : 2009年 -2010年 
    代表者 : 五十嵐 学
     
    インフルエンザの流行を効率よく制御するためには、抗原変異の機序を理解し、予測することが必要である。本研究では、インフルエンザウイルスの主要抗原であるヘマグルチニン(HA)の立体構造に焦点をあて、構造バイオインフォマティクス的手法により、抗原変異に伴うHAの抗原構造の変遷を解析した。また結果に基づき、パンデミック2009(H1N1)ウイルスの抗原変異に伴う今後のアミノ酸置換を予測した。
  • 文部科学省：科学研究費補助金(若手研究(B))
    研究期間 : 2007年 -2008年 
    代表者 : 五十嵐 学
     
    ヒトに馴化したインフルエンザウイルスは、抗原性を毎年少しずつ変化させて、ヒトの免疫機構を巧みに回避し、流行を繰り返す。もし、この変異の過程を予測することが出来れば、インフルエンザを先回りして制御することが出来る。本研究では、計算科学的手法を用いてヒトインフルエンザウイルスの抗原変異に伴う抗原構造の変遷を解析し、将来いずれかのHA 亜型の鳥インフルエンザウイルスがヒトの新型インフルエンザウイルスとして出現した場合の予測方法を検討した。
  • 量子動力学シミュレーションによる神経伝達物質とレセプターとの相互作用の解明
    文部科学省: 科学研究費補助金(特別研究員奨励費)：
    研究期間 : 2001年 -2003年 
    代表者 : 五十嵐学

産業財産権

  • PCT/JP2023/036277:抗インフルエンザウイルス用組成物、医薬、飲食品、サプリメント、農薬、飼料および化粧料  2023年10月04日
    五十嵐学, 加藤博己, 塚本雄太  国立大学法人北海道大学
  • 増田 健一, 齊藤 隆, 石井 保之, 高田 礼人, 五十嵐 学, 丸山 隼輝, 齋藤 祐介, 奈良 拓也  国立研究開発法人理化学研究所, 動物アレルギー検査株式会社, 国立大学法人北海道大学  201803010563100282
  • 増田 健一, 齊藤 隆, 石井 保之, 高田 礼人, 五十嵐 学, 丸山 隼輝, 齋藤 祐介, 奈良 拓也  国立研究開発法人理化学研究所, 動物アレルギー検査株式会社, 国立大学法人北海道大学  201803015653821541
  • 特願2009-275367:Ｈ５亜型インフルエンザウイルスに対する抗体およびその利用  
    樋口恵, 鈴木定彦, 高田礼人, 喜田宏, 吉田玲子, 五十嵐学, 宮本洋子

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