Researcher Database

Hiroshi KIDA
International Institute for Zoonosis Control
University Professor

Researcher Profile and Settings


  • International Institute for Zoonosis Control

Job Title

  • University Professor


J-Global ID

Research Interests

  • インフルエンザウイルス   ヘマグルチニン   ワクチン   ニュ-カッスル病ウイルス   病原性   渡り鴨   予測   ブタ   シベリア   ヘルペスウイルス   営巣地   オーエスキー病ウイルス   糞便   カモ   H5N1   香港   新型ウイルス   インフルエンザ   ウイルスの存続   渡りガモ   血管内皮細胞   アジア   新型インフルエンザウイルス   渡り鳥   ニワトリ   凍結湖沼水   ニューキャッスル病ウイルス   レセプター特異性   疫学調査   血管内血液凝固   

Research Areas

  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Virology
  • Life sciences / Laboratory animal science
  • Life sciences / Experimental pathology

Academic & Professional Experience

  • 2017/04 - Today 長崎大学 感染症共同研究拠点長
  • 2016/04 - Today Hokkaido University Research Center for Zoonosis Control
  • 2016/04 - Today Hokkaido University
  • 2012/04 - Today Hokkaido University Research Center for Zoonosis Control
  • 2012/04 - Today Hokkaido University
  • 2007/12 - Today 日本学士院 会員
  • 2012/04 - 2014/03 北海道大学大学院・獣医学研究科 特任教授
  • 2005/04 - 2012/03 Hokkaido University Research Center for Zoonosis Control
  • 1995/04 - 2012/03 北海道大学 大学院・獣医学研究科 教授
  • 2001/05 - 2005/04 Hokkaido University Graduate School of Veterinary Medicine
  • 1995/06 - 2001/04 Hokkaido University
  • 1989/01 - 1989/03 ザンビア大学 教授(JICA派遣専門家)
  • 1986/01 - 1987/01 St. Jude Children's Research Hospital 客員教授(Karnofsky 賞招聘研究員)
  • 1980/12 - 1981/11 St. Jude Children's Research Hospital 客員科学者


  • 1967/04 - 1968/03  北海道大学大学院
  •        - 1967/03  Hokkaido University  School of Veterinary Medicine  Veterinary Medicine

Association Memberships


Research Activities

Published Papers

  • Enkhbold Bazarragchaa, Masatoshi Okamatsu, Ankhanbaatar Ulaankhuu, Augustin Tshibwabwa Twabela, Keita Matsuno, Hiroshi Kida, Yoshihiro Sakoda
    Journal of virological methods 265 121 - 125 2019/03 [Refereed][Not invited]
    Rapid and accurate diagnosis of influenza virus infection is essential for quick responses for both human and animal health. The Alere™ i Influenza A&B is a novel isothermal nucleic acid amplification kit that can detect and differentiate between influenza A and B viruses in human specimens in approximately 15 min. In the present study, the performance of the Alere™ i Influenza A&B kit was evaluated for its ability to detect avian influenza virus in chickens. The kit was able to detect representative avian influenza virus strains (hemagglutinin subtypes H1-H16, including the recently isolated H5 and H7 highly pathogenic avian influenza viruses), and the detection limit of the kit for these viruses varied between 10-1.4-102.1 50% egg-infective dose per test, which is higher than the analytical sensitivity of the antigen detection immunochromatography kit ESPLINE® A INFLUENZA. In experimentally infected chickens inoculated with a highly pathogenic avian influenza virus strain A/chicken/Hokkaido/002/2016 (H5N6), viral RNA was detected in the tracheal and cloacal swabs. These results indicate that this kit has the potential to be used as a rapid screening test of influenza A virus infection in chickens.
  • Takeshi Noshi, Mitsutaka Kitano, Keiichi Taniguchi, Atsuko Yamamoto, Shinya Omoto, Keiko Baba, Takashi Hashimoto, Kayo Ishida, Yukihiro Kushima, Kazunari Hattori, Makoto Kawai, Ryu Yoshida, Masanori Kobayashi, Tomokazu Yoshinaga, Akihiko Sato, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida, Takao Shishido, Akira Naito
    Antiviral research 160 109 - 117 2018/12 [Refereed][Not invited]
    Cap-dependent endonuclease (CEN) resides in the PA subunit of the influenza virus and mediates the critical "cap-snatching" step of viral RNA transcription, which is considered to be a promising anti-influenza target. Here, we describe in vitro characterization of a novel CEN inhibitor, baloxavir acid (BXA), the active form of baloxavir marboxil (BXM). BXA inhibits viral RNA transcription via selective inhibition of CEN activity in enzymatic assays, and inhibits viral replication in infected cells without cytotoxicity in cytopathic effect assays. The antiviral activity of BXA is also confirmed in yield reduction assays with seasonal type A and B viruses, including neuraminidase inhibitor-resistant strains. Furthermore, BXA shows broad potency against various subtypes of influenza A viruses (H1N2, H5N1, H5N2, H5N6, H7N9 and H9N2). Additionally, serial passages of the viruses in the presence of BXA result in isolation of PA/I38T variants with reduced BXA susceptibility. Phenotypic and genotypic analyses with reverse genetics demonstrate the mechanism of BXA action via CEN inhibition in infected cells. These results reveal the in vitro characteristics of BXA and support clinical use of BXM to treat influenza.
  • Akihiro Shibata, Masatoshi Okamatsu, Riho Sumiyoshi, Keita Matsuno, Zu-Jyun Wang, Hiroshi Kida, Hiroyuki Osaka, Yoshihiro Sakoda
    Virology 524 10 - 17 2018/11 [Refereed][Not invited]
    H7N9 highly and low pathogenic avian influenza viruses (HPAIV and LPAIV, respectively) have been isolated from duck meat products that were brought illegally into Japan by flight passengers in their hand luggage. These H7N9 virus isolates were phylogenetically closely related to those prevailing in China. Antigenic analysis revealed that the hemagglutinin of the H7N9 HPAIV isolate was slightly different from those of the H7N9 LPAIV and older H7 strains. These meat products contaminated with AIVs repeatedly brought into Japan lead to increased risks of poultry and public health. Continuous border disease control based on the detection and culling of infected poultry and meat products is, thus, essential for the prevention of introduction and spread of AIVs.
  • Zu-Jyun Wang, Yuto Kikutani, Lam Thanh Nguyen, Takahiro Hiono, Keita Matsuno, Masatoshi Okamatsu, Scott Krauss, Richard Webby, Youn-Jeong Lee, Hiroshi Kida, Yoshihiro Sakoda
    Virus genes 54 (4) 543 - 549 2018/08 [Refereed][Not invited]
    Among 16 haemagglutinin (HA) subtypes of avian influenza viruses (AIVs), H13 AIVs have rarely been isolated in wild waterfowl. H13 AIVs cause asymptomatic infection and are maintained mainly in gull and tern populations; however, the recorded antigenic information relating to the viruses has been limited. In this study, 2 H13 AIVs, A/duck/Hokkaido/W345/2012 (H13N2) and A/duck/Hokkaido/WZ68/2012 (H13N2), isolated from the same area in the same year in our surveillance, were genetically and antigenically analyzed with 10 representative H13 strains including a prototype strain, A/gull/Maryland/704/1977 (H13N6). The HA genes of H13 AIVs were phylogenetically divided into 3 groups (I, II, and III). A/duck/Hokkaido/W345/2012 (H13N2) was genetically classified into Group III. This virus was distinct from a prototype strain, A/gull/Maryland/704/1977 (H13N6), and the virus, A/duck/Hokkaido/WZ68/2012 (H13N2), both belonging to Group I. Antigenic analysis indicated that the viruses of Group I were antigenically closely related to those of Group II, but distinct from those of Group III, including A/duck/Hokkaido/W345/2012 (H13N2). In summary, our study indicates that H13 AIVs have undergone antigenic diversification in nature.
  • Daisuke Fujikura, Daisuke Muramatsu, Kochi Toyomane, Satoko Chiba, Takuji Daito, Atsushi Iwai, Takahisa Kouwaki, Masaaki Okamoto, Hideaki Higashi, Hiroshi Kida, Hiroyuki Oshiumi
    Journal of Biochemistry 163 (1) 31 - 38 1756-2651 2018/01/01 [Refereed][Not invited]
    Several microbial molecules with pathogen-associated molecular patterns stimulate host innate immune responses. The innate immune system plays a crucial role in activating acquired immune response via cytokine production and antigen presentation. Previous studies have shown that Aureobasidium pullulans-cultured fluid (AP-CF), which contains b-glucan, exhibits adjuvant activity and renders mice resistance to influenza A virus infection however, the underlying mechanism remains elusive. In this study, we investigated the innate immune response to AP-CF. We found that intraperitoneal administration of AP-CF increased the serum level of IL-18 and the number of splenic IFN-c producing CD4+ cells during influenza A virus infection. The adjuvant effect of AP-CF was distinct from that of alum, which is known to have the ability to stimulate a Th2 immune response. In addition, AP-CF injection barely increased the number of peritoneal neutrophils and inflammatory macrophages, whereas alum injection markedly increased the number of neutrophils and inflammatory macrophages, suggesting that AP-CF is a weak inducer of inflammation compared to alum. APCF induced IL-18 production by DC2.4 cells, a dendritic cell line, and by peritoneal exudate cells that include peritoneal macrophages. Collectively, our findings indicate that AP-CF is an adjuvant that promotes the Th1 response during influenza A virus infection.
  • Daisuke Muramatsu, Mitsuyasu Okabe, Akinori Takaoka, Hiroshi Kida, Atsushi Iwai
    Scientific Reports 7 (1) 2831  2045-2322 2017/12/01 [Refereed][Not invited]
    Black yeast, Aureobasidium pullulans is extracellularly produced β-(1,3), (1,6)-D-glucan (β-glucan) under certain conditions. In this study, using Glycine max cv. Kurosengoku (Kurosengoku soybeans), the production of β-glucan through fermentation of A. pullulans was evaluated, and the effects of A. pullulans cultured fluid (AP-CF) containing β-glucan made with Kurosengoku soybeans (kAP-CF) on a human monocyte derived cell line, Mono Mac 6 cells were investigated. Concentration of β-glucan in kAP-CF reached the same level as normal AP-CF. An anti-angiogenic protein, Thrombospondin-1 (THBS1) was effectively induced after the stimulation with kAP-CF for comparison with AP-CF. The THBS1 is also induced after stimulation with hot water extract of Kurosengoku soybeans (KS-E), while the combined stimulation of β-glucan with KS-E more effectively induced THBS1 than that with KS-E alone. These results suggest effects of A. pullulans-produced β-glucan on the enhancement of Kurosengoku soybean-induced THBS1 expression.
  • D. -H. Chu, M. A. Stevenson, L. V. Nguyen, N. Isoda, S. M. Firestone, T. N. Nguyen, L. T. Nguyen, K. Matsuno, M. Okamatsu, H. Kida, Y. Sakoda
    TRANSBOUNDARY AND EMERGING DISEASES 64 (6) 1991 - 1999 1865-1674 2017/12 [Refereed][Not invited]
    In Vietnam, live bird markets are found in most populated centres, providing the means by which fresh poultry can be purchased by consumers for immediate consumption. Live bird markets are aggregation points for large numbers of poultry, and therefore, it is common for a range of avian influenza viruses to be mixed within live bird markets as a result of different poultry types and species being brought together from different geographical locations. We conducted a cross-sectional study in seven live bird markets in four districts of Thua Thien Hue Province in August and December, 2014. The aims of this study were to (i) document the prevalence of avian influenza in live bird markets (as measured by virus isolation); and (ii) quantify individual bird-, seller- and market-level characteristics that rendered poultry more likely to be positive for avian influenza virus at the time of sale. A questionnaire soliciting details of knowledge, attitude and avian influenza practices was administered to poultry sellers in study markets. At the same time, swabs and faecal samples were collected from individual poultry and submitted for isolation of avian influenza virus. The final data set comprised samples from 1,629 birds from 83 sellers in the seven live bird markets. A total of 113 birds were positive for virus isolation; a prevalence of 6.9 (95% CI 5.8-8.3) avian influenza virus-positive birds per 100 birds submitted for sale. After adjusting for clustering at the market and individual seller levels, none of the explanatory variables solicited in the questionnaire were significantly associated with avian influenza virus isolation positivity. The proportions of variance at the individual market, seller and individual bird levels were 6%, 48% and 46%, respectively. We conclude that the emphasis of avian influenza control efforts in Vietnam should be at the individual seller level as opposed to the market level.
  • Lam Thanh Nguyen, Tatsuya Nishi, Shintaro Shichinohe, Duc-Huy Chu, Takahiro Hiono, Keita Matsuno, Masatoshi Okamatsu, Hiroshi Kida, Yoshihiro Sakoda
    VIROLOGY 510 252 - 261 0042-6822 2017/10 [Refereed][Not invited]
    Vaccination-primed immunity in poultry has been suggested for selection of antigenically drifted highly pathogenic avian influenza viruses (HPAIVs). In this study, we performed two consecutive passage studies of an H5N1 HPAIV in vaccinated chickens, namely, study-I and study-II, to select antigenic variants under immune pressure from the vaccination. In study-I, nine consecutive passages of a wild-type H5N1 HPAIV were carried out in chickens vaccinated with the homologous challenge strain. Antigenically drifted variants with mutations at position 179 in the hemagglutinin (HA) were selected after three passages. Similarly, in study-II, a vaccination-mediated antigenic variant isolated in study-I was used as the vaccine and challenge strain to confirm further antigenic drift after updating the vaccine; after the third passage, additional antigenic variants with a mutation at position 256 in the HA were selected. Thus, our study demonstrated the contribution of vaccination in the selection of antigenic variants of H5 HPAIVs in chickens.
  • Takahiro Hiono, Masatoshi Okamatsu, Keita Matsuno, Atsushi Haga, Ritsuko Iwata, Lam Thanh Nguyen, Mizuho Suzuki, Yuto Kikutani, Hiroshi Kida, Manabu Onuma, Yoshihiro Sakoda
    MICROBIOLOGY AND IMMUNOLOGY 61 (9) 387 - 397 0385-5600 2017/09 [Refereed][Not invited]
    On 15 November 2016, a black swan that had died in a zoo in Akita prefecture, northern Japan, was strongly suspected to have highly pathogenic avian influenza (HPAI); an HPAI virus (HPAIV) belonging to the H5N6 subtype was isolated from specimens taken from the bird. After the initial report, 230 cases of HPAI caused by H5N6 viruses from wild birds, captive birds, and domestic poultry farms were reported throughout the country during the winter season. In the present study, 66 H5N6 HPAIVs isolated from northern Japan were further characterized. Phylogenetic analysis of the hemagglutinin gene showed that the H5N6 viruses isolated in northern Japan clustered into Group C of Clade together with other isolates collected in Japan, Korea and Taiwan during the winter season of 2016-2017. The antigenicity of the Japanese H5N6 isolate differed slightly from that of HPAIVs isolated previously in Japan and China. The virus exhibited high pathogenicity and a high replication capacity in chickens, whereas virus growth was slightly lower in ducks compared with that of an H5N8 HPAIV isolate collected in Japan in 2014. Comprehensive analyses of Japanese isolates, including those from central, western, and southern Japan, as well as rapid publication of this information are essential for facilitating greater control of HPAIVs.
  • Lam Thanh Nguyen, Kazunari Nakaishi, Keiko Motojima, Ayako Ohkawara, Erina Minato, Junki Maruyama, Takahiro Hiono, Keita Matsuno, Masatoshi Okamatsu, Takashi Kimura, Ayato Takada, Hiroshi Kida, Yoshihiro Sakoda
    PLOS ONE 12 (8) e0182228  1932-6203 2017/08 [Refereed][Not invited]
    Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade H5 HPAIVs in domestic and wild birds.
  • Ayako Ohkawara, Masatoshi Okamatsu, Makoto Ozawa, Duc-Huy Chu, Lam Thanh Nguyen, Takahiro Hiono, Keita Matsuno, Hiroshi Kida, Yoshihiro Sakoda
    MICROBIOLOGY AND IMMUNOLOGY 61 (5) 149 - 158 0385-5600 2017/05 [Refereed][Not invited]
    H5 highly pathogenic avian influenza viruses (HPAIV) have spread in both poultry and wild birds since late 2003. Continued circulation of HPAIV in poultry in several regions of the world has led to antigenic drift. In the present study, we analyzed the antigenic properties of H5 HPAIV isolated in Asia using four neutralizing mAbs recognizing hemagglutinin, which were established using A/chicken/Kumamoto/1-7/2014 (H5N8), belonging to clade and also using polyclonal antibodies. Viruses of clades 1.1,, 2.3.4, and had different reactivity patterns to the panel of mAbs, thereby indicating that the antigenicity of the viruses of clade were similar but differed from the other clades. In particular, the antigenicity of the viruses of clade differed from those of the viruses of clades 2.3.4 and, which suggests that the recent H5 HPAIV have further evolved antigenically divergent. In addition, reactivity of antiserum suggests that the antigenicity of viruses of clade differed slightly among groups A, B, and C. Vaccines are still used in poultry in endemic countries, so the antigenicity of H5 HPAIV should be monitored continually to facilitate control of avian influenza. The panel of mAbs established in the present study will be useful for detecting antigenic drift in the H5 viruses that emerge from the current strains.
  • Akihiro Nakatsukasa, Koji Kuruma, Masatoshi Okamatsu, Takahiro Hiono, Mizuho Suzuki, Keita Matsuno, Hiroshi Kida, Takayoshi Oyamada, Yoshihiro Sakoda
    VACCINE 35 (21) 2855 - 2861 0264-410X 2017/05 [Refereed][Not invited]
    Transdermal vaccination using a microneedle (MN) confers enhanced immunity compared with subcutaneous (SC) vaccination. Here we developed a novel dissolving MN patch for the influenza vaccine. The potencies of split virion and whole virus particle (WVP) vaccines prepared from A/Puerto Rico/8/1934 (H1N1) and A/duck/Hokkaido/Vac-3/2007 (H5N1), respectively, were evaluated. MN vaccination induced higher neutralizing antibody responses than SC vaccination in mice. Moreover, MN vaccination with a lower dose of antigens conferred protective immunity against lethal challenges of influenza viruses than SC vaccination in mice. These results suggest that the WVP vaccines administered using MN are an effective combination for influenza vaccine to be further validated in humans. (C) 2017 Elsevier Ltd. All rights reserved.
  • Masatoshi Okamatsu, Makoto Ozawa, Kosuke Soda, Hiroki Takakuwa, Atsushi Haga, Takahiro Hiono, Aya Matsuu, Yuko Uchida, Ritsuko Iwata, Keita Matsuno, Masakazu Kuwahara, Toshiyo Yabuta, Tatsufumi Usui, Hiroshi Ito, Manabu Onuma, Yoshihiro Sakoda, Takehiko Saito, Koichi Otsuki, Toshihiro Ito, Hiroshi Kida
    EMERGING INFECTIOUS DISEASES 23 (4) 691 - 695 1080-6040 2017/04 [Refereed][Not invited]
    Highly pathogenic avian influenza viruses (HPAIVs) A(H5N6) were concurrently introduced into several distant regions of Japan in November 2016. These viruses were classified into the genetic clade and were genetically closely related to H5N6 HPAIVs recently isolated in South Korea and China. In addition, these HPAIVs showed further antigenic drift.
  • Masanori Kobayashi, Makoto Kodama, Takeshi Noshi, Ryu Yoshida, Takushi Kanazu, Naoki Nomura, Kosuke Soda, Norikazu Isoda, Masatoshi Okamatsu, Yoshihiro Sakoda, Yoshinori Yamano, Akihiko Sato, Hiroshi Kida
    ANTIVIRAL RESEARCH 139 41 - 48 0166-3542 2017/03 [Refereed][Not invited]
    High morbidity and mortality associated with human cases of highly pathogenic avian influenza (HPAI) viruses, including H5N1 influenza virus, have been reported. The purpose of the present study was to evaluate the antiviral effects of peramivir against HPAI viruses. In neuraminidase (NA) inhibition and virus replication inhibition assays, peramivir showed strong inhibitory activity against H5N1, H7N1 and H7N7 HPAI viruses with sub-nanomolar activity in enzyme assays. In H5N1 viruses containing the NA H275Y mutation, the antiviral activity of peramivir against the variant was lower than that against the wild-type. Evaluation of the in vivo antiviral activity showed that a single intravenous treatment of peramivir (10 mg/kg) prevented lethality in mice infected with wild-type H5N1 virus and also following infection with H5N1 virus with the H275Y mutation after a 5 day administration of peramivir (30 mg/kg). Furthermore, mice injected with peramivir showed low viral titers and low levels of proinflammatory cytokines in the lungs. These results suggest that peramivir has therapeutic activity against HPAI viruses even if the virus harbors the NA H275Y mutation. (C) 2016 Elsevier B.V. All rights reserved.
  • Misako Nakayama, Hiroichi Ozaki, Yasushi Itoh, Kosuke Soda, Hirohito Ishigaki, Masatoshi Okamatsu, Yoshihiro Sakoda, Chun-Ho Park, Hideaki Tsuchiya, Hiroshi Kida, Kazumasa Ogasawara
    PATHOLOGY INTERNATIONAL 66 (12) 678 - 686 1320-5463 2016/12 [Refereed][Not invited]
    H9N2 avian influenza virus causes sporadic human infection. Since humans do not possess acquired immunity specific to this virus, we examined the pathogenicity of an H9N2 virus isolated from a human and then analyzed protective effects of a vaccine in cynomolgus macaques. After intranasal challenge with A/Hong Kong/1073/1999 (H9N2) (HK1073) isolated from a human patient, viruses were isolated from nasal and tracheal swabs in unvaccinated macaques with mild fever and body weight loss. A formalin-inactivated H9N2 whole particle vaccine derived from our virus library was subcutaneously inoculated to macaques. Vaccination induced viral antigen-specific IgG and neutralization activity in sera. After intranasal challenge with H9N2, the virus was detected only the day after inoculation in the vaccinated macaques. Without vaccination, many bronchus-associated lymphoid tissues (BALTs) were formed in the lungs after infection, whereas the numbers of BALTs were smaller and the cytokine responses were weaker in the vaccinated macaques than those in the unvaccinated macaques. These findings indicate that the H9N2 avian influenza virus HK1073 is pathogenic in primates but seems to cause milder symptoms than does H7N9 influenza virus as found in our previous studies and that a formalin-inactivated H9N2 whole particle vaccine induces protective immunity against H9N2 virus.
  • Fumihiko Yasui, Yasushi Itoh, Ai Ikejiri, Masahiro Kitabatake, Nobuo Sakaguchi, Keisuke Munekata, Shintaro Shichinohe, Yukiko Hayashi, Hirohito Ishigaki, Misako Nakayama, Yoshihiro Sakoda, Hiroshi Kida, Kazumasa Ogasawara, Michinori Kohara
    SCIENTIFIC REPORTS 6 37915  2045-2322 2016/11 [Refereed][Not invited]
    H5N1 highly pathogenic avian influenza (H5N1 HPAI) virus causes elevated mortality compared with seasonal influenza viruses like H1N1 pandemic influenza (H1N1 pdm) virus. We identified a mechanism associated with the severe symptoms seen with H5N1 HPAI virus infection. H5N1 HPAI virus infection induced a decrease of dendritic cell number in the splenic extrafollicular T-cell zone and impaired formation of the outer layers of B-cell follicles, resulting in insufficient levels of antibody production after infection. However, in animals vaccinated with a live recombinant vaccinia virus expressing the H5 hemagglutinin, infection with H5N1 HPAI virus induced parafollicular dendritic cell accumulation and efficient antibody production. These results indicate that a recombinant vaccinia encoding H5 hemagglutinin gene does not impair dendritic cell recruitment and can be a useful vaccine candidate.
  • Masatoshi Okamatsu, Takahiro Hiono, Hiroshi Kida, Yoshihiro Sakoda
    VETERINARY JOURNAL 215 82 - 86 1090-0233 2016/09 [Refereed][Not invited]
    The diagnosis of influenza A virus infections in poultry or wild birds is difficult due to variations in the pathogenicity of the viruses in different avian hosts and also the antigenic and genetic diversity of the virus, particularly the recent H5 highly pathogenic avian influenza viruses. A classical standard laboratory technique is virus isolation prior to subtyping and pathotyping. This diagnostic technique is crucial for further virological analyses, particularly during an initial outbreak; however, delays in diagnosis have thwarted effective disease control in recent years. Recent developments in molecular biological techniques provide an accelerated diagnosis. Such technologies, which include real-time reverse transcriptase PCR, isothermal nucleic acid amplification, next-generation sequencing and immunochromatography, contribute to simpler and more rapid diagnosis. The advantages of each of these diagnostic techniques should be considered for effective control of avian influenza. (C) 2016 Elsevier Ltd. All rights reserved.
  • Masatoshi Okamatsu, Yurie Motohashi, Takahiro Hiono, Tomokazu Tamura, Kazuki Nagaya, Keita Matsuno, Yoshihiro Sakoda, Hiroshi Kida
    ARCHIVES OF VIROLOGY 161 (8) 2235 - 2242 0304-8608 2016/08 [Refereed][Not invited]
    Influenza viruses isolated from wild ducks do not replicate in chickens. This fact is not explained solely by the receptor specificity of the hemagglutinin (HA) from such viruses for target host cells. To investigate this restriction in host range, the fusion activities of HA molecules from duck and chicken influenza viruses were examined. Influenza viruses A/duck/Mongolia/54/2001 (H5N2) (Dk/MNG) and A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR), which replicate only in their primary hosts, were used. The optimal pH for membrane fusion of Ck/IBR was 5.9, higher than that of Dk/MNG at 4.9. To assess the relationship between the optimal pH for fusion and the host range of avian influenza viruses, the optimal pH for fusion of 55 influenza virus strains isolated from ducks and chickens was examined. No correlation was found between the host range and optimal pH for membrane fusion by the viruses, and this finding applied also to the H5N1 highly pathogenic avian influenza viruses. The optimal pH for membrane fusion for avian influenza viruses was shown to not necessarily be correlated with their host range or pathogenicity in ducks and chickens.
  • Chu DH, Okamatsu M, Matsuno K, Hiono T, Ogasawara K, Nguyen LT, Van Nguyen L, Nguyen TN, Nguyen TT, Van Pham D, Nguyen DH, Nguyen TD, To TL, Van Nguyen H, Kida H, Sakoda Y
    Vet Microbiol 192 194 - 203 2016/07 [Refereed][Not invited]
  • Hiromi Takaki, Haruko Sato, Riho Kurata, Hirokazu Hikono, Takahiro Hiono, Hiroshi Kida, Misako Matsumoto, Takehiko Saito, Tsukasa Seya
    Microbiology and immunology 60 (7) 511 - 5 2016/07 [Refereed][Not invited]
    Eye spray influenza vaccines for chickens are increasingly available; however, how to enhance cellular and antibody responses to them remains undetermined. Here, eye-drops containing the immune-enhancing adjuvants Pam2CSK4 or polyI:C were assessed in chickens. Application of these TLR agonists to chicken conjunctiva resulted in up-regulation of IL-1β, but not other cytokines, including IFN and IL-6, in the spleen, lung and Harderian gland. Thus, responses to adjuvant applied to the conjunctival mucosa of chickens differ from those expected from the responses to intra-nasal adjuvants in mammals. Identifying an appropriate delivery route for adjuvants is crucial for evoking immune responses in chickens.
  • Taisho Yamada, Hiromasa Horimoto, Takeshi Kameyama, Sumio Hayakawa, Hiroaki Yamato, Masayoshi Dazai, Ayato Takada, Hiroshi Kida, Debbie Bott, Angela C. Zhou, David Hutin, Tania H. Watts, Masahiro Asaka, Jason Matthews, Akinori Takaoka
    NATURE IMMUNOLOGY 17 (6) 687 - + 1529-2908 2016/06 [Refereed][Not invited]
    Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic activity of many environmental xenobiotics. However, its role in innate immune responses during viral infection is not fully understood. Here we demonstrate that constitutive AHR signaling negatively regulates the type I interferon (IFN-I) response during infection with various types of virus. Virus-induced IFN-beta production was enhanced in AHR-deficient cells and mice and resulted in restricted viral replication. We found that AHR upregulates expression of the ADP-ribosylase TIPARP, which in turn causes downregulation of the IFN-I response. Mechanistically, TIPARP interacted with the kinase TBK1 and suppressed its activity by ADP-ribosylation. Thus, this study reveals the physiological importance of endogenous activation of AHR signaling in shaping the IFN-I-mediated innate response and, further, suggests that the AHR-TIPARP axis is a potential therapeutic target for enhancing antiviral responses.
  • Hiroshi Yamada, Satoshi Nagase, Kazuo Takahashi, Yoshihiro Sakoda, Hiroshi Kida, Shigefumi Okamoto
    ANTIVIRAL RESEARCH 129 81 - 92 0166-3542 2016/05 [Refereed][Not invited]
    The most effective drugs available to treat influenza are neuraminidase (NA) inhibitors, which provide important additional measures for the control of influenza virus infections. However, since the emergence of NA inhibitor-resistant viruses may compromise the clinical utility of this class of anti-influenza agents, it is very important to develop new anti-influenza agents which target a different region in NA responsible for its sensitivity from that for NA inhibitors and could be used to treat NA inhibitors resistant isolates. The oligodeoxynucleotide D35, multimerized and aggregated, suppressed replication of influenza A viruses except A/WSN/33 (WSN). The suppressive viral replication by D35 depended on G-terad and multimer formation. The range of the suppressive viral replication at the late stage, including virus assembly and release from infected cells, was much larger than that at the initial stage, viral attachment and entry. D35 suppressed NA activity of influenza A viruses. Furthermore, replacing the NA gene of A/Puerto Rico/8/34 (PR8), in which viral replication was inhibited by D35 at the late stage, with the NA gene from WSN, in which viral replication was not inhibited, eliminated the D35-dependent suppression. D35 showed an additive anti-influenza effect with oseltamivir. It was also effective in vivo. These results suggest that the influenza virus NA mainly contributse to the D35-suppressible virus release from infected cells at the late stage. In addition, because administration of D35 into the virus-infected mice suppressed viral replication and weight loss, clinical application of D35 could be considered. (C) 2016 Elsevier B.V. All rights reserved.
  • Shichinohe S, Itoh Y, Nakayama M, Ozaki H, Soda K, Ishigaki H, Okamatsu M, Sakoda Y, Kida H
    Virology (493) 31 - 38 2016/03 [Refereed][Not invited]
  • 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 0304-8608 2016/02 [Refereed][Not invited]
    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.
  • Takahiro Hiono, Masatoshi Okamatsu, Naoki Yamamoto, Kohei Ogasawara, Mayumi Endo, Saya Kuribayashi, Shintaro Shichinohe, Yurie Motohashi, Duc-Huy Chu, Mizuho Suzuki, Takaya Ichikawa, Tatsuya Nishi, Yuri Abe, Keita Matsuno, Kazuyuki Tanaka, Tsutomu Tanigawa, Hiroshi Kida, Yoshihiro Sakoda
    VETERINARY MICROBIOLOGY 182 108 - 115 0378-1135 2016/01 [Refereed][Not invited]
    Highly pathogenic avian influenza viruses (HPAIVs) have spread in both poultry and wild birds. Determining transmission routes of these viruses during an outbreak is essential for the control of avian influenza. It has been widely postulated that migratory ducks play crucial roles in the widespread dissemination of HPAIVs in poultry by carrying viruses along with their migrations; however close contacts between wild migratory ducks and poultry are less likely in modern industrial poultry farming settings. Therefore, we conducted experimental infections of HPAIVs and low pathogenic avian influenza viruses (LPAIVs) to chickens, domestic ducks, tree sparrows, jungle crows, and black rats to evaluate their roles in virus transmission. The results showed that chickens, ducks, sparrows, and crows were highly susceptible to HPAIV infection. Significant titers of virus were recovered from the sparrows and crows infected with HPAIVs, which suggests that they potentially play roles of transmission of HPAIVs to poultry. In contrast, the growth of LPAIVs was limited in each of the animals tested compared with that of HPAIVs. The present results indicate that these common synanthropes play some roles in influenza virus transmission from wild birds to poultry. (C) 2015 Elsevier B.V. All rights reserved.
  • Koichiro Gamoh, Mari Nakamizo, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida, Shoko Suzuki
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 (1) 139 - 142 0916-7250 2016/01 [Refereed][Not invited]
    H5 highly pathogenic avian influenza (HPAI) viruses have spread worldwide, and antigenic variants of different clades have been selected. In this study, the national stockpiled vaccine prepared from A/duck/HoldcaidoNac-1/2004 (H5N1) strain was evaluated for the protective efficacy against H5N8 HPAI virus isolated in Kumamoto prefecture, Japan, in April 2014. In the challenge test, all of the vaccinated chickens survived without showing any clinical signs and reduced virus shedding. It was concluded that the present stockpiled vaccine was effective against the H5N8 HPAI virus.
  • Yuri Abe, Tomokazu Tamura, Shiho Torii, Shiho Wakamori, Makoto Nagai, Kazuya Mitsuhashi, Junki Mine, Yuri Fujimoto, Naofumi Nagashima, Fumi Yoshino, Yukihiko Sugita, Takushi Nomura, Masatoshi Okamatsu, Hiroshi Kida, Yoshihiro Sakoda
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 (1) 61 - 70 0916-7250 2016/01 [Refereed][Not invited]
    In our previous study, we genetically analyzed bovine viral diarrhea viruses (BVDVs) isolated from 2000 to 2006 in Japan and reported that subgenotype 1b viruses were predominant. In the present study, 766 BVDVs isolated from 2006 to 2014 in Hokkaido, Japan, were genetically analyzed to understand recent epidemics. Phylogenetic analysis based on nucleotide sequences of the 5'-untranslated region of viral genome revealed that 766 isolates were classified as genotype 1 (BVDV-1; 544 isolates) and genotype 2 (BVDV-2; 222). BVDV-1 isolates were further divided into BVDV-la (93), 1b (371) and 1c (80) subgenotypes, and all BVDV-2 isolates were grouped into BVDV-2a subgenotype (222). Further comparative analysis was performed with BVDV-1a, 1b and 2a viruses isolated from 2001 to 2014. Phylogenetic analysis based on nucleotide sequences of the viral glycoprotein E2 gene, a major target of neutralizing antibodies, revealed that BVDV-1a, 1b and 2a isolates were further classified into several clusters. Cross-neutralization tests showed that BVDV-1b isolates were antigenically different from BVDV-1a isolates, and almost BVDV-1a, 1b and 2a isolates were antigenically similar among each subgenotype and each E2 cluster. Taken together, BVDV-1b viruses are still predominant, and BVDV-2a viruses have increased recently in Hokkaido, Japan. Field isolates of BVDV-1a, 1b and 2a show genetic diversity on the E2 gene with antigenic conservation among each subgenotype during the last 14 years.
  • 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 0022-1317 2015/09 [Refereed][Not invited]
    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  1932-6203 2015/09 [Refereed][Not invited]
    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.
  • Takahiro Hiono, Ayako Ohkawara, Kohei Ogasawara, Masatoshi Okamatsu, Tomokazu Tamura, Duc-Huy Chu, Mizuho Suzuki, Saya Kuribayashi, Shintaro Shichinohe, Ayato Takada, Hirohito Ogawa, Reiko Yoshida, Hiroko Miyamoto, Naganori Nao, Wakako Furuyama, Junki Maruyama, Nao Eguchi, Gerelmaa Ulziibat, Bazarragchaa Enkhbold, Munkhduuren Shatar, Tserenjav Jargalsaikhan, Selenge Byambadorj, Batchuluun Damdinjav, Yoshihiro Sakoda, Hiroshi Kida
    VIRUS GENES 51 (1) 57 - 68 0920-8569 2015/08 [Refereed][Not invited]
    Migratory water birds are the natural reservoir of influenza A viruses. H5 and H7 influenza viruses are isolated over the world and also circulate among poultry in Asia. In 2010, two H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from fecal samples of water birds on the flyway of migration from Siberia, Russia to the south in Hokkaido, Japan. H7N9 viruses are sporadically isolated from humans and circulate in poultry in China. To monitor whether these viruses have spread in the wild bird population, we conducted virological surveillance of avian influenza in migratory water birds in autumn from 2010 to 2014. A total of 8103 fecal samples from migratory water birds were collected in Japan and Mongolia, and 350 influenza viruses including 13 H5 and 19 H7 influenza viruses were isolated. A phylogenetic analysis revealed that all isolates are genetically closely related to viruses circulating among wild water birds. The results of the antigenic analysis indicated that the antigenicity of viruses in wild water birds is highly stable despite their nucleotide sequence diversity but is distinct from that of HPAIVs recently isolated in Asia. The present results suggest that HPAIVs and Chinese H7N9 viruses were not predominantly circulating in migratory water birds; however, continued monitoring of H5 and H7 influenza viruses both in domestic and wild birds is recommended for the control of avian influenza.
  • 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 0066-4804 2015/08 [Refereed][Not invited]
    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.
  • Junki Mine, Tomokazu Tamura, Kazuya Mitsuhashi, Masatoshi Okamatsu, Sujira Parchariyanon, Wasana Pinyochon, Nicolas Ruggli, Jon-Duri Tratschin, Hiroshi Kida, Yoshihiro Sakoda
    JOURNAL OF GENERAL VIROLOGY 96 1746 - 1756 0022-1317 2015/07 [Refereed][Not invited]
    The viral protein N-pro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, N-pro suppresses type I IFN (IFN-alpha/beta) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the N-pro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of N-pro coordinating zinc by means of the amino acid residues 0112, 0134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-alpha/beta induction despite the presence of an intact TRASH domain. Through systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of N-pro of these four isolates were related to the lack of IRF-3-degrading activity. restoring a histidine at position 40 or both a proline at position 17 and a lysine at position 61 based on the sequence of a functional N-pro contributed to higher stability of the reconstructed N-pro compared with the N-pro from the Thai isolate. This led to enhanced interaction of N-pro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of N-pro are involved in the stability of N-pro, in interaction of N-pro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-alpha/beta production.
  • Hirohito Ogawa, Nobuo Koizumi, Aiko Ohnuma, Alisheke Mutemwa, Bernard M. Hang'ombe, Aaron S. Mweene, Ayato Takada, Chihiro Sugimoto, Yasuhiko Suzuki, Hiroshi Kida, Hirofumi Sawa
    INFECTION GENETICS AND EVOLUTION 32 143 - 147 1567-1348 2015/06 [Refereed][Not invited]
    The role played by bats as a potential source of transmission of Leptospira spp. to humans is poorly understood, despite various pathogenic Leptospira spp. being identified in these mammals. Here, we investigated the prevalence and diversity of pathogenic Leptospira spp. that infect the straw-colored fruit bat (Eidolon helvum). We captured this bat species, which is widely distributed in Africa, in Zambia during 2008-2013. We detected the flagellin B gene (flaB) from pathogenic Leptospira spp. in kidney samples from 79 of 529 E. helvum (14.9%) bats. Phylogenetic analysis of 70 flaB fragments amplified from E. helvum samples and previously reported sequences, revealed that 12 of the fragments grouped with Leptospira borgpetersenii and Leptospira kirschneri; however, the remaining 58 flaB fragments appeared not to be associated with any reported species. Additionally, the 16S ribosomal RNA gene (rrs) amplified from 27 randomly chosen flaB-positive samples was compared with previously reported sequences, including bat-derived Leptospira spp. All 27 rrs fragments clustered into a pathogenic group. Eight fragments were located in unique branches, the other 19 fragments were closely related to Leptospira spp. detected in bats. These results show that rrs sequences in bats are genetically related to each other without regional variation, suggesting that Leptospira are evolutionarily well-adapted to bats and have uniquely evolved in the bat population. Our study indicates that pathogenic Leptospira spp. in E. helvum in Zambia have unique genotypes. (C) 2015 The Authors. Published by Elsevier B.V.
  • Akira Sakurai, Katsuyoshi Takayama, Namiko Nomura, Naoki Kajiwara, Masatoshi Okamatsu, Naoki Yamamoto, Tsuruki Tamura, Jitsuho Yamada, Masako Hashimoto, Yoshihiro Sakoda, Yoshihiko Suda, Yukuharu Kobayashi, Hiroshi Kida, Futoshi Shibasaki
    PLOS ONE 10 (2) e0116715  1932-6203 2015/02 [Refereed][Not invited]
    Lateral flow tests also known as Immunochromatography (IC) is an antigen-detection method conducted on a nitrocellulose membrane that can be completed in less than 20 min. IC has been used as an important rapid test for clinical diagnosis and surveillance of influenza viruses, but the IC sensitivity is relatively low (approximately 60%) and the limit of detection (LOD) is as low as 10(3) pfu per reaction. Recently, we reported an improved IC assay using antibodies conjugated with fluorescent beads (fluorescent immunochromatography; FLIC) for subtyping H5 influenza viruses (FLIC-H5). Although the FLIC strip must be scanned using a fluorescent reader, the sensitivity (LOD) is significantly improved over that of conventional IC methods. In addition, the antibodies which are specific against the subtypes of influenza viruses cannot be available for the detection of other subtypes when the major antigenicity will be changed. In this study, we established the use of FLIC to type seasonal influenza A and B viruses (FLIC-AB). This method has improved sensitivity to 100-fold higher than that of conventional IC methods when we used several strains of influenza viruses. In addition, FLIC-AB demonstrated the ability to detect influenza type A and influenza type B viruses from clinical samples with high sensitivity and specificity (Type A: sensitivity 98.7% (74/75), specificity 100% (54/54), Type B: sensitivity 100% (90/90), specificity 98.2% (54/55) in nasal swab samples) in comparison to the results of qRT-PCR. And furthermore, FLIC-AB performs better in the detection of early stage infection (under 13h) than other conventional IC methods. Our results provide new strategies to prevent the early-stage transmission of influenza viruses in humans during both seasonal outbreaks and pandemics.
  • Kozasa T, Abe Y, Mitsuhashi K, Tamura T, Aoki H, Ishimaru M, Nakamura S, Okamatsu M, Kida H, Sakoda Y
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 0916-7250 2014/12 [Refereed][Not invited]
  • Akira Sakurai, Katsuyoshi Takayama, Namiko Nomura, Naoki Yamamoto, Yoshihiro Sakoda, Yukuharu Kobayashi, Hiroshi Kida, Futoshi Shibasaki
    JOURNAL OF VIROLOGICAL METHODS 209 62 - 68 0166-0934 2014/12 [Refereed][Not invited]
    Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza viruses because of its rapid turnaround and ease of use. Despite the usefulness of IC, the limit of detection of common IC kits is as high as 10(3)-10(4) plaque forming units (pfu) per reaction, resulting in their limited sensitivities. Early diagnosis within 24 h would provide more appropriate timing of treatment. In this study, a multi-colored NanoAct(TM) bead IC was established to detect seasonal influenza viruses. This method has approximately 10-fold higher sensitivity than that of colloidal gold or colored latex bead IC assays, and does not require specific instruments. More notably, NanoAct(Tm) bead IC can distinguish influenza A and B viruses from clinical samples with a straightforward readout composed of colored lines. Our results will provide new strategies for the diagnosis, treatment, and a chance to survey of influenza viruses in developing countries and in the field research. (C) 2014 Elsevier B.V. All rights reserved.
  • Ahmad M. Haredy, Hiroshi Yamada, Yoshihiro Sakoda, Masatoshi Okamatsu, Naoki Yamamoto, Takeshi Omasa, Yasuko Mori, Hiroshi Kida, Shigefumi Okamoto, Yoshinobu Okuno, Koichi Yamanishi
    JOURNAL OF GENERAL VIROLOGY 95 2365 - 2371 0022-1317 2014/11 [Refereed][Not invited]
    Whole-virus (WV) vaccines from influenza A/duck/Hokkaido/77 (H3N2), and its reassortant strains H3N4, H3N5 and H3N7, which have the same haemagglutinin (HA) gene but different neuraminidase (NA) genes, were prepared from our influenza virus library. Mice were intranasally immunized with equivalent doses of each vaccine (1-0.01 mu g per mouse). All of the mice that received the highest dose of each vaccine (1 mu g per mouse) showed equivalent high HA-inhibiting (HI) antibody titres and survived the H3N2 challenge viruses. However, mice that received lower doses of vaccine (0.1 or 0.01 mu g per mouse) containing a heterologous NA had lower survival rates than those given the H3N2-based vaccine. The lungs of mice challenged with H3N2 virus showed a significantly higher virus clearance rate when the vaccine contained the homologous NA (N2) versus a heterologous NA, suggesting that NA contributed to the protection, especially when the HI antibody level was low. These results suggested that, even if vaccines prepared for a possible upcoming pandemic do not induce sufficient HI antibodies, WV vaccines can still be effective through other matched proteins such as NA.
  • Tatsuya Nishi, Yoshihiro Sakoda, Masatoshi Okamatsu, Hiroshi Kida
    ARCHIVES OF VIROLOGY 159 (10) 2567 - 2574 0304-8608 2014/10 [Refereed][Not invited]
    H6 influenza viruses are prevalent in domestic and wild birds in Eurasian countries and have been isolated from pigs and a human. To prepare for an influenza pandemic, we have established an influenza virus library consisting of more than 1,300 influenza virus strains, including 144 combinations of 16 hemagglutinin and 9 neuraminidase subtypes. H6 viruses in the library were classified into Early, Group II, Group III, and W312 sublineages and the North America lineage on the basis of their phylogenetic features. Chicken antisera to A/duck/Hong Kong/960/1980 (H6N2) of the Early sublineage broadly reacted with viruses of different sublineages in a hemagglutinin inhibition test. A whole inactivated virus particle vaccine was prepared from A/duck/Hong Kong/960/1980 (H6N2) which was stocked in the influenza virus library. The potency of this vaccine against A/duck/Vietnam/OIE-0033/2012 (H6N2), which belongs to a different sublineage, was evaluated in mice. The test vaccine was sufficiently potent to induce an immune response that reduced the impact of disease caused by a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice. The present results indicate that the whole inactivated virus particle vaccine prepared from a virus strain in the influenza virus library is useful as a vaccine against pandemic influenza.
  • Takuji Kumagai, Tetsuo Nakayama, Yoshinobu Okuno, Tetsuo Kase, Naoko Nishimura, Takao Ozaki, Akiko Miyata, Eitaro Suzuki, Teruo Okafuji, Takao Okafuji, Hitoshi Ochiai, Nobuo Nagata, Hiroyuki Tsutsumi, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida, Toshiaki Ihara
    VIRAL IMMUNOLOGY 27 (8) 368 - 374 0882-8245 2014/10 [Refereed][Not invited]
    The 2009 pandemic H1N1 mainly affected adolescents and children, and most of the elderly in Japan escaped clinical illness. To clarify the role of humoral immunity in the infection, the time kinetics of hemagglutination inhibition (HI), neutralization (NT), and IgG subclass antibody response directed against influenza A(H1N1)pdm2009 were analyzed in three consecutive specimens obtained from 51 young adults and children (group 1) who contracted pandemic influenza and from 74 pediatric clinic employees (group 2) inoculated with pandemic monovalent vaccine. In group 1 patients, 6 and 30 patients had lower HI and NT antibody in the acute phase respectively. Thereafter, HI and NT antibody titers increased fourfold or more in 50 patients with peak response in the third specimens obtained four weeks after the onset. IgG1 in 45 patients, IgG3 in 18 patients, and IgG4 in 29 patients showed elevated responses. Forty (54%) and 70 (95%) subjects in group 2 had positive HI and NT antibodies in the prevaccination samples, with increased antibody responses in the follow-up peaking in the second specimens. Forty of those vaccinated had increased IgG1 responses peaking in the third specimens, whereas elevated IgG3 was observed in 22 recipients with the highest level in the second samples. IgG4 did not show any increase in subjects in group 2. A few participants showed an IgG2 response in both groups. An immunologically naive population contracted influenza with apparent clinical symptoms. However, already primed subjects through subclinical infection elicited the unique pattern of IgG subclass responses by vaccination, which differed from those of naive populations.
  • Makoto Kodama, Ryu Yoshida, Takahiro Hasegawa, Masaaki Izawa, Mitsutaka Kitano, Kaoru Baba, Takeshi Noshi, Takahiro Seki, Kenichi Okazaki, Masakatsu Tsuji, Takushi Kanazu, Hiroshi Kamimori, Tomoyuki Homma, Masanori Kobayashi, Yoshihiro Sakoda, Hiroshi Kida, Akihiko Sato, Yoshinori Yamano
    ANTIVIRAL RESEARCH 109 110 - 115 0166-3542 2014/09 [Refereed][Not invited]
    The purpose of this study was to investigate the relationship between pharmacokinetic (PK) parameters of intravenous (IV) peramivir and in vivo antiviral activity pharmacodynamic (PD) outcomes in a mouse model of influenza virus infection. Peramivir was administrated to mice in three dosing schedules; once, twice and four times after infection of A/WS/33 (H1N1). The survival rate at day 14 after virus infection was employed as the antiviral activity outcome for analysis. The relationship between day 14 survival and PK parameters, including area under the concentration-time curve (AUC), maximum concentration (C-max) and time that drug concentration exceeds IC95 (T>(IC95)), was estimated using a logistic regression model, and model fitness was evaluated by calculation of the Akaike information criterion (AIC) index. The AIC indices of AUC, C-max and T->IC95 were about 114, 151 and 124, respectively. The AIC of AUC and T->IC95 were smaller than that of C-max. Therefore, both AUC and T->IC95 were the PK parameters that correlated best with the antiviral activity of peramivir IV against influenza virus infection in mice. (C) 2014 Elsevier B.V. All rights reserved.
  • Makoto Nagai, Hiroshi Aoki, Yoshihiro Sakoda, Takashi Kozasa, Kaho Tominaga-Teshima, Junki Mine, Yuri Abe, Tomokazu Tamura, Tsubasa Kobayashi, Kaoru Nishine, Kentaro Tateishi, Yudai Suzuki, Mai Fukuhara, Keitaro Ohmori, Reiko Todaka, Kazuhiko Katayama, Tetsuya Mizutani, Shigeyuki Nakamura, Hiroshi Kida, Junsuke Shirai
    JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION 26 (4) 547 - 552 1040-6387 2014/07 [Refereed][Not invited]
    In the current study, molecular, biological, and antigenic analyses were performed to characterize Border disease virus (BDV) strain FNK2012-1 isolated from a pig in 2012 in Japan. The complete genome comprises 12,327 nucleotides (nt), including a large open reading frame of 11,685 nt. Phylogenetic analysis revealed that FNK2012-1 was clustered into BDV genotype 1 with ovine strains. FNK2012-1 grew in porcine, bovine, and ovine primary cells and cell lines, but grew better in bovine and ovine cells than in porcine cells. Specific pathogen-free pigs inoculated with FNK2012-1 did not show any clinical signs. Noninoculated contact control pigs also did not show clinical signs and did not seroconvert. The results suggest that FNK2012-1 may be of ruminant origin and is poorly adapted to pigs. Such observations can provide important insights into evidence for infection and transmission of BDV, which may be of ruminant origin, among pigs.
  • Hasegawa H, van Reit E, Kida H
    Current topics in microbiology and immunology 0070-217X 2014/07 [Refereed][Not invited]
  • 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  1553-7366 2014/06 [Refereed][Not invited]
    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.
  • Duc-Huy Chu, Yoshihiro Sakoda, Tatsuya Nishi, Takahiro Hiono, Shintaro Shichinohe, Masatoshi Okamatsu, Hiroshi Kida
    VACCINE 32 (28) 3473 - 3479 0264-410X 2014/06 [Refereed][Not invited]
    H7N9 influenza virus infection in humans was reported in China on March 31, 2013. Humans are immunologically naive to the H7N9 subtype, for which the seasonal influenza vaccine is not effective. Thus, the development of an H7N9 influenza virus vaccine is an urgent issue. To prepare for the emergence of an influenza pandemic, we have established a library comprising more than 1300 influenza virus strains with 144 different combinations of 16 HA and 9 NA subtypes. An H7N9 virus strain isolated from a 35-year-old woman, A/Anhui/1/2013 (H7N9), was found to be antigenically similar to H7N9 influenza viruses isolated from migratory ducks. In the present study, the potency of an inactivated whole virus particle vaccine prepared from an H7N9 low pathogenic avian influenza virus, A/duck/Mongolia/119/2008 (H7N9), selected from the library, was assessed by a challenge with A/Anhui/1/2013 (H7N9). The results indicate that the test vaccine was potent enough to induce sufficient immunity to reduce the impact of disease caused by the challenge with A/Anhui/1/2013 (H7N9) in mice. The present results indicate that an inactivated whole virus particle vaccine prepared from an influenza virus strain stored in the library could be useful as a vaccine strain in case of an influenza pandemic. (C) 2014 Published by Elsevier Ltd.
  • Takahiro Hiono, Masatoshi Okamatsu, Shoko Nishihara, Sayaka Takase-Yoden, Yoshihiro Sakoda, Hiroshi Kida
    VIROLOGY 456 131 - 138 0042-6822 2014/05 [Refereed][Not invited]
    Influenza viruses recognize sialoglycans as receptors. Although viruses isolated form chickens preferentially bind to sialic acid alpha 2,3 galactose (SA alpha 2,3Gal) glycans as do those of ducks, chickens were not experimentally infected with viruses isolated from ducks. A chicken influenza virus, A/chicken/Ibaraki/1/ 2005 (H5N2) (Ck/IBR) bound to fucose-branched SAa2,3Gal glycans, whereas the binding towards linear SAa2,3Gal glycans was weak. On the epithelial cells of the upper respiratory tracts of chickens, fucose-branched SA alpha 2,3Gal glycans were detected, but not linear SA alpha 2,3Gal glycans. The growth of Ck/IBR in MDCK-FUT cells, which were genetically prepared to express fucose-branched SA alpha 2,3Gal glycans, was significantly higher than that in the parental MDCK cells. The present results indicate that fucosebranched SA alpha 2,3Gal glycans existing on the epithelial cells lining the upper respiratory tracts of chickens are critical for recognition by Ck/IBR. (c) 2014 Elsevier Inc. All rights reserved.
  • Tomokazu Tamura, Naofumi Nagashima, Nicolas Ruggli, Artur Summerfield, Hiroshi Kida, Yoshihiro Sakoda
    VETERINARY RESEARCH 45 47  0928-4249 2014/04 [Refereed][Not invited]
    Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein N-pro of CSFV interferes with alpha-and beta-interferon (IFN-alpha/beta) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE(-), N-pro acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-alpha/beta induction. In a previous study, we showed that the GPE(-) vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE(-) vaccine virus in pigs, the IRF3-degrading function of N-pro was not recovered. Therefore, we examined whether restoring the ability of N-pro to block IFN-alpha/beta induction of both the avirulent and moderately virulent GPE(-)-derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in N-pro regained the ability to degrade IRF3 and suppress IFN-alpha/beta induction in vitro. In pigs, functional N-pro significantly reduced the local IFN-alpha mRNA expression in lymphoid organs while it increased quantities of IFN-alpha/beta in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional N-pro influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.
  • Daisuke Muramatsu, Koji Kawata, Shiho Aoki, Hirofumi Uchiyama, Mitsuyasu Okabe, Tadaaki Miyazaki, Hiroshi Kida, Atsushi Iwai
    SCIENTIFIC REPORTS 4 4777  2045-2322 2014/04 [Refereed][Not invited]
    A beta-(1,3),(1,6)-D-glucan produced by A. pullulans (AP-PG) is known to be an immune stimulating agent. In this study, we demonstrate that the stimulation with AP-PG effectively induces the interferon (IFN) stimulated genes (ISGs) in macrophage-like cell lines. The ISGs, Mx1, ISG15, and viperin mRNAs were significantly increased in RAW264.7 cells after stimulation with AP-PG. The stimulation with AP-PG transiently induced IFN-beta mRNA. However, the expression of viperin mRNA was also increased after stimulation with AP-PG even when new protein synthesis was completely blocked by treatment with cycloheximide. Further, in IFN-alpha receptor knockdown RAW264.7 cells, AP-PG stimulation more effectively induced viperin mRNA compared with that of IFN-alpha stimulation. The phosphorylation of Ser 727 in STAT1 involved in the enhancement of STAT1 activation was immediately increased after stimulation with AP-PG. In addition, viperin mRNA expression induced after stimulation with IFN-alpha was significantly increased by combined stimulation with AP-PG. These results suggest that stimulation with AP-PG effectively induces the ISGs through the induction of IFN and the enhancement of STAT1-mediated transcriptional activation.
  • Rashid Manzoor, Kazumichi Kuroda, Reiko Yoshida, Yoshimi Tsuda, Daisuke Fujikura, Hiroko Miyamoto, Masahiro Kajihara, Hiroshi Kida, Ayato Takada
    JOURNAL OF BIOLOGICAL CHEMISTRY 289 (11) 7599 - 7614 0021-9258 2014/03 [Refereed][Not invited]
    Background: It has been shown that heat shock protein 70 (Hsp70) plays a role in influenza A virus replication. Results: A correlation between viral replication/transcription activities and nuclear/cytoplasmic shuttling of Hsp70 was observed. Conclusion: Hsp70 modulates the influenza A virus polymerase activity. Significance: This study, for the first time, suggests that Hsp70 may actually assist in influenza A virus replication. The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.
  • Tomomi Ichimiya, Shoko Nishihara, Sayaka Takase-Yoden, Hiroshi Kida, Kiyoko Aoki-Kinoshita
    BIOINFORMATICS 30 (5) 706 - 711 1367-4803 2014/03 [Refereed][Not invited]
    Motivation: It is well known influenza viruses recognize and bind terminal sialic acid (SA) on glycans that are found on the cell surface. In this work, we used a data mining technique to analyze the glycan array data of influenza viruses to find novel glycan structures other than SA that may be involved in viral infection. Results: In addition to SA structures noted previously, we noted the sulfated structures in the mining results. For verification, we overexpressed the sulfotransferase that is involved in synthesizing these structures, and we performed a viral infection experiment to assess changes in infection in these cells. In our results, we found that there is a 70-fold increase in these cells compared with the control. Thus, we have found a novel pattern in glycan structures that may be involved in viral infection.
  • Haru Ogiwara, Fumihiko Yasui, Keisuke Munekata, Asako Takagi-Kamiya, Tsubasa Munakata, Namiko Nomura, Futoshi Shibasaki, Kazuhiko Kuwahara, Nobuo Sakaguchi, Yoshihiro Sakoda, Hiroshi Kida, Michinori Kohara
    AMERICAN JOURNAL OF PATHOLOGY 184 (1) 171 - 183 0002-9440 2014/01 [Refereed][Not invited]
    Patients infected with highly pathogenic avian influenza A H5N1 viruses (H5N1 HPAIV) show diffuse alveolar damage. However, the temporal progression of tissue damage and repair after viral infection remains poorly defined. Therefore, we assessed the sequential histopathological characteristics of mouse lung after intranasal infection with H5N1 HPAIV or H1N1 2009 pandemic influenza virus (H1N1 pdm). We determined the amount and localization of virus in the Lung through IHC staining and in situ hybridization. IHC used antibodies raised against the virus protein and antibodies specific for macrophages, type II pneumocytes, or proliferating cell nuclear antigen. In situ hybridization used RNA probes against both viral RNA and mRNA encoding the nucleoprotein and the hemagglutinin protein. H5N1 HPAIV infection and replication were observed in multiple lung cell types and might result in rapid progression of lung injury. Both type II pneumocytes and macrophages proliferated after H5N1 HPAIV infection. However, the abundant macrophages failed to block the viral attack, and proliferation of type II pneumocytes failed to restore the damaged alveoli. In contrast, mice infected with H1N1 pdm exhibited modest proliferation of type II pneumocytes and macrophages and slight alveolar damage. These results suggest that the virulence of H5N1 HPAIV results from the wide range of cell tropism of the virus, excessive virus replication, and rapid development of diffuse alveolar damage.
  • Tatsuya Nishi, Masatoshi Okamatsu, Kenji Sakurai, Huy Due Chu, Long Pham Thanh, Long van Nguyen, Nam van Hoang, Diep Nguyen Thi, Yoshihiro Sakoda, Hiroshi Kida
    JOURNAL OF VETERINARY MEDICAL SCIENCE 76 (1) 85 - 87 0916-7250 2014/01 [Refereed][Not invited]
    In August 2012, A/chicken/Vietnam/OIE-2215/2012 (H5N2) was isolated from a chicken in a live bird market (LBM) in Northern Vietnam. Intravenous pathogenicity test revealed that this virus is highly pathogenic in chickens. The PA, HA, NP and M, PB2 and NA, and PB1 and NS genes of the isolate were phylogenetically closely related to those of A/duck/Vietnam/OIE-2202/2012 (H5N1) of Glade, A/chicken/Vietnam/OIE-1611/2012 (H9N2) and A/chicken/Vietnam/OIE-2468/2012 (H9N2), respectively. All of these viruses were isolated from birds in LBMs in the same province. These results indicate that A/chicken/Vietnam/OIE-2215/2012 (H5N2) is a genetic reassortant and that surveillance of avian influenza in LBMs and stamping out policy are essential for the eradication of highly pathogenic avian influenza viruses from Asia.
  • Tomoko Fujiyuki, Misako Yoneda, Fumihiko Yasui, Takeshi Kuraishi, Shosaku Hattori, Hyun-jeong Kwon, Keisuke Munekata, Yuri Kiso, Hiroshi Kida, Michinori Kohara, Chieko Kai
    PLOS ONE 8 (12) e83551  1932-6203 2013/12 [Refereed][Not invited]
    Highly pathogenic avian influenza virus (HPAIV) continues to threaten human health. Non-human primate infection models of human influenza are desired. To establish an animal infection model with more natural transmission and to determine the pathogenicity of HPAIV isolated from a wild water bird in primates, we administered a Japanese isolate of HPAIV (A/whooper swan/Hokkaido/1/2008, H5N1 clade to rhesus and cynomolgus monkeys, in droplet form, via the intratracheal route. Infection of the lower and upper respiratory tracts and viral shedding were observed in both macaques. Inoculation of rhesus monkeys with higher doses of the isolate resulted in stronger clinical symptoms of influenza. Our results demonstrate that HPAIV isolated from a water bird in Japan is pathogenic in monkeys by experimental inoculation, and provide a new method for HPAIV infection of non-human primate hosts, a good animal model for investigation of HPAIV pathogenicity.
  • Misako Nakayama, Shintaro Shichinohe, Yasushi Itoh, Hirohito Ishigaki, Mitsutaka Kitano, Masahiko Arikata, Van Loi Pham, Hideaki Ishida, Naoko Kitagawa, Masatoshi Okamatsu, Yoshihiro Sakoda, Takaya Ichikawa, Hideaki Tsuchiya, Shinichiro Nakamura, Quynh Mai Le, Mutsumi Ito, Yoshihiro Kawaoka, Hiroshi Kida, Kazumasa Ogasawara
    PLOS ONE 8 (12) e82740  1932-6203 2013/12 [Refereed][Not invited]
    H5N1 highly pathogenic avian influenza virus (HPAIV) infection has been reported in poultry and humans with expanding clade designations. Therefore, a vaccine that induces immunity against a broad spectrum of H5N1 viruses is preferable for pandemic preparedness. We established a second H5N1 vaccine candidate, A/duck/Hokkaido/Vac-3/2007 (Vac-3), in our virus library and examined the efficacy of inactivated whole particles of this strain against two clades of H5N1 HPAIV strains that caused severe morbidity in cynomolgus macaques. Virus propagation in vaccinated macaques infected with either of the H5N1 HPAIV strains was prevented compared with that in unvaccinated macaques. This vaccine also prevented propagation of a pandemic (H1N1) 2009 virus in macaques. In the vaccinated macaques, neutralization activity, which was mainly shown by anti-hemagglutinin antibody, against H5N1 HPAIVs in plasma was detected, but that against H1N1 virus was not detected. However, neuraminidase inhibition activity in plasma and T-lymphocyte responses in lymph nodes against H1N1 virus were detected. Therefore, cross-clade and heterosubtypic protective immunity in macaques consisted of humoral and cellular immunity induced by vaccination with Vac-3.
  • Naoki Yamamoto, Yoshihiro Sakoda, Masatoshi Okamatsu, Hiroshi Kida
    VIRUS RESEARCH 178 (2) 404 - 410 0168-1702 2013/12 [Refereed][Not invited]
    Influenza virus A/Hong Kong/483/97 (H5N1) (HK483-K) has the PB2 with lysine at position 627 (PB2-627K) and is highly pathogenic in chickens and mice. On the other hand, the pathogenicity of mutant virus (HK483-E), which was generated by substituting lysine with glutamic acid at the position of the PB2, is lower than that of HK483-K in mice, but is highly pathogenic in chickens. The PB2 is one of the components of heterotrimeric polymerase complex, which plays roles in the transcription and replication of virus genes. Cell-free polymerase assay revealed that intrinsic transcription activity of the polymerase complex with PB2-627K is higher than that of glutamic acid (PB2-627E). In chicken cells, transcription efficiency of the polymerase complex with PB2-627E was not lower than those with PB2-627K, indicating that transcription of virus genes is modulated by some host factors in chicken cells, resulting in high growth. Polymerase complex with PB2-627K efficiently transcribes and replicates virus polymerase genes in mouse cells, leading to high growth of HK483-K compared with that of HK483-E. The results of our experiments clearly suggest that efficient transcription and replication of virus genes by polymerase complex result in the higher pathogenicity in mice. (C) 2013 Elsevier B.V. All rights reserved.
  • Junki Maruyama, Masatoshi Okamatsu, Kosuke Soda, Yoshihiro Sakoda, Hiroshi Kida
    ARCHIVES OF VIROLOGY 158 (12) 2473 - 2478 0304-8608 2013/12 [Refereed][Not invited]
    Highly pathogenic avian influenza viruses have poly-basic amino acid sequences at the cleavage site in their hemagglutinin (HA). Although this poly-basic region is a prerequisite factor for pathogenicity in chickens, not much is known about additional factors responsible for the acquisition of pathogenicity of the duck influenza virus in chickens. Here, we introduced multiple basic amino acid residues into the HA cleavage site of the A/duck/Hokkaido/Vac-2/2004 (H7N7) strain of avian influenza virus, which has low pathogenicity in chickens; the resultant Vac2sub-P0 strain was not intravenously pathogenic in chickens. In contrast, the Vac2sub-P3 strain, which was recovered from three consecutive passages of Vac2sub-P0 in chicks, was intravenously pathogenic in chickens. Six amino acid substitutions were identified by comparison of the Vac2sub-P3 and Vac2sub-P0 genomic sequences: Lys123Glu in PB2, Asn16Asp in PB1, Glu227Gly and Ile388Thr in HA, Gly228Arg in M1, and Leu46Pro in M2. The results of intravenous inoculations of chickens with recombinant virus indicated that all six amino acid substitutions were required to varying degrees for Vac2sub-P3 pathogenicity, with Glu227Gly and Ile388Thr in HA being particularly essential. These results reveal the roles of additional viral factors in the acquisition of pathogenicity in addition to the previously characterized role of the poly-basic amino acid sequences at the HA cleavage site.
  • Akira Sakurai, Katsuyoshi Takayama, Namiko Nomura, Tsubasa Munakata, Naoki Yamamoto, Tsuruki Tamura, Jitsuho Yamada, Masako Hashimoto, Kazuhiko Kuwahara, Yoshihiro Sakoda, Yoshihiko Suda, Yukuharu Kobayashi, Nobuo Sakaguchi, Hiroshi Kida, Michinori Kohara, Futoshi Shibasaki
    PLoS ONE 8 (11) e76753  1932-6203 2013/11/06 [Refereed][Not invited]
    Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza virus because the protocol is short time and easy to use. Despite the usability of IC, the sensitivity is approximately 103 pfu per reaction. In addition, antigen-antibody interaction-based method cannot be used for the detection of influenza viruses with major antigenic change. In this study, we established the use of fluorescent immunochromatography (FLIC) to detect a broad spectrum of H5 subtype influenza A viruses. This method has improved sensitivity 10-100 fold higher than traditional IC because of the use of fluorescent conjugated beads. Our Type-E FLIC kit detected all of the H5 subtype influenza viruses that were examined, as well as recombinant hemagglutinin (HA) proteins (rHAs) belonging to the Eurasian H5 subtype viruses and the Type-N diagnosed North American H5 subtype influenza A viruses. Thus, this kit has the improved potential to detect H5 subtype influenza viruses of different clades with both Type-E and Type-N FLIC kits. Compared with PCR-based diagnosis, FLIC has a strong advantage in usability, because the sample preparation required for FLIC is only mix-and-drop without any additional steps such as RNA extraction. Our results can provide new strategies against the spread and transmission of HPAI H5N1 viruses in birds and mammals including humans. Copyright: © 2013 Sakurai et al.
  • Yoshikazu Fujimoto, Kinuyo Ozaki, Masahiro Maeda, Ken-ichi Nishijima, Hiroki Takakuwa, Koichi Otsuki, Hiroshi Kida, Etsuro Ono
    VETERINARY JOURNAL 198 (2) 487 - 493 1090-0233 2013/11 [Refereed][Not invited]
    The polymerase basic 2 (PB2) protein is one of four proteins that make up the influenza A virus replication complex, which is responsible for viral gene transcription and replication. To assess the antiviral potential of an anti-PB2 monoclonal antibody that inhibits RNA transcription of influenza A viruses, Mardin-Darby canine kidney (MDCK) cells were transformed with two transgenes that encode the light and heavy chains of the monoclonal antibody. The transformed cell lines expressing this monoclonal antibody displayed resistance to several subtypes of influenza A virus infection. In the transformed cell lines infected with influenza A virus, the level of viral RNA transcription was decreased and the effective nuclear transportation of the PB2 protein was also inhibited. These results demonstrate that the anti-PB2 intrabody is potentially able to interfere with the effective nuclear transportation of PB2 protein, resulting in the observed resistance to influenza A virus infection in vitro. (C) 2013 Elsevier Ltd. All rights reserved.
  • Masatoshi Okamatsu, Tatsuya Nishi, Naoki Nomura, Naoki Yamamoto, Yoshihiro Sakoda, Kenji Sakurai, Huy Duc Chu, Long Pham Thanh, Long Van Nguyen, Nam Van Hoang, Tien Ngoc Tien, Reiko Yoshida, Ayato Takada, Hiroshi Kida
    VIRUS GENES 47 (2) 317 - 329 0920-8569 2013/10 [Refereed][Not invited]
    To estimate the prevalence of avian influenza virus infection in Vietnam, surveillance was conducted in domestic and wild birds from households, live-bird markets, slaughtering sites, and bird sanctuaries in Vietnam between October 2010 and October 2012. Of the 4,550 samples collected, 226 influenza A virus isolates were obtained from domestic ducks, muscovy ducks, and chickens. Of these, 25 and 22 H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from apparently healthy domestic ducks in live-bird markets and slaughtering sites in northern and southern Vietnam, respectively. The HA genes of H5 viruses isolated from birds in northern Vietnam phylogenetically belonged to the genetic clade and those in southern Vietnam belonged to the genetic clade 1.1. In addition, 39 H3, 12 H4, 1 H5, 93 H6, 2 H7, 18 H9, 3 H10, and 11 H11 viruses were isolated. Phylogenetic and antigenic analyses of the H6 and H9 viruses revealed that they were closely related to the isolates obtained from domestic poultry in China. Phylogenetic analyses of internal gene segments of these isolates revealed that these viruses were circulating in both domestic and wild birds in Asia and reassortment events had occurred frequently. Therefore, it will be important to continue the surveillance and strict controls over the movement and trade of poultry and poultry products in order to eradicate H5N1 HPAIV from Asia.
  • Van Loi Pham, Misako Nakayama, Yasushi Itoh, Hirohito Ishigaki, Mitsutaka Kitano, Masahiko Arikata, Hideaki Ishida, Naoko Kitagawa, Shintaro Shichinohe, Masatoshi Okamatsu, Yoshihiro Sakoda, Hideaki Tsuchiya, Shinichiro Nakamura, Hiroshi Kida, Kazumasa Ogasawara
    PLOS ONE 8 (9) e75910  1932-6203 2013/09 [Refereed][Not invited]
    Pandemic (H1N1) 2009 influenza virus spread throughout the world since most people did not have immunity against the virus. In the post pandemic phase when many humans might possess immunity against the pandemic virus, one of the concerns is infection in immunocompromised people. Therefore, we used an immunosuppressed macaque model to examine pathogenicity of the pandemic (H1N1) 2009 virus under an immunocompromised condition. The virus in nasal samples of immunosuppressed macaques infected with the pandemic (H1N1) 2009 virus was detected longer after infection than was the virus in nasal samples of immunocompetent macaques. As expected, not only virus amounts but also virus propagation sites in the immunosuppressed macaques were larger than those in lungs of the immunocompetent macaques when they were infected with the pandemic virus. Immunosuppressed macaques possessed low levels of immune cells producing cytokines and chemokines, but levels of inflammatory cytokines/chemokine interleukin (IL)-6, IL-18, and monocyte chemotactic protein (MCP)-1 in lungs of the immunosuppressed macaques were higher than those in lungs of the immunocompetent macaques, though the differences were not statistically significant. Therefore, under an immunosuppressive condition, the pandemic influenza (H1N1) 2009 virus might cause more severe morbidity with high cytokine/chemokine production by the host innate immune system than that seen in macaques under the immunocompetent condition.
  • Shintaro Shichinohe, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida
    VIROLOGY 444 (1-2) 404 - 408 0042-6822 2013/09 [Refereed][Not invited]
    Avian influenza viruses possess hemagglutinin (HA) which preferentially bind to the sialic acid alpha 2,3-galactose sialyloligosaccharides (SA alpha 2,3Gal) receptor. In contrast, human influenza viruses bind to sialic acid alpha 2,6-galactose sialyloligosaccharides (SA alpha 2,6Gal). The A/Hong Kong/68 (H3N2) virus preferentially binds to SA alpha 2,6Gal, although its HA gene was derived from an avian influenza virus strain. To elucidate the mechanisms behind acquisition of binding specificity for the human-type receptor, the avian influenza virus, A/duck/Hokkaido/5/77 (H3N2), which carries the HA with SA alpha 2,3Gal receptor specificity, was consecutively passaged in pigs. Viruses that preferentially bind to the SA alpha 2,6Gal receptor were predominantly recovered from the nasal swabs of pigs after three passages. The present results indicate that avian influenza Viruses can acquire the potential to infect humans after multiple infections in a pig population. Intensive surveillance of swine influenza is, thus, important for the preparedness for the future pandemics. (C) 2013 Elsevier Inc. All rights reserved.
  • Tokiko Watanabe, Maki Kiso, Satoshi Fukuyama, Noriko Nakajima, Masaki Imai, Shinya Yamada, Shin Murakami, Seiya Yamayoshi, Kiyoko Iwatsuki-Horimoto, Yoshihiro Sakoda, Emi Takashita, Ryan McBride, Takeshi Noda, Masato Hatta, Hirotaka Imai, Dongming Zhao, Noriko Kishida, Masayuki Shirakura, Robert P. de Vries, Shintaro Shichinohe, Masatoshi Okamatsu, Tomokazu Tamura, Yuriko Tomita, Naomi Fujimoto, Kazue Goto, Hiroaki Katsura, Eiryo Kawakami, Izumi Ishikawa, Shinji Watanabe, Mutsumi Ito, Yuko Sakai-Tagawa, Yukihiko Sugita, Ryuta Uraki, Reina Yamaji, Amie J. Eisfeld, Gongxun Zhong, Shufang Fan, Jihui Ping, Eileen A. Maher, Anthony Hanson, Yuko Uchida, Takehiko Saito, Makoto Ozawa, Gabriele Neumann, Hiroshi Kida, Takato Odagiri, James C. Paulson, Hideki Hasegawa, Masato Tashiro, Yoshihiro Kawaoka
    NATURE 501 (7468) 551 - + 0028-0836 2013/09 [Refereed][Not invited]
    Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission(1), and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.
  • Saya Kuribayashi, Yoshihiro Sakoda, Takeshi Kawasaki, Tomohisa Tanaka, Naoki Yamamoto, Masatoshi Okamatsu, Norikazu Isoda, Yoshimi Tsuda, Yuji Sunden, Takashi Umemura, Noriko Nakajima, Hideki Hasegawa, Hiroshi Kida
    PLOS ONE 8 (7) e68375  1932-6203 2013/07 [Refereed][Not invited]
    Highly pathogenic avian influenza viruses (HPAIVs) cause lethal infection in chickens. Severe cases of HPAIV infections have been also reported in mammals, including humans. In both mammals and birds, the relationship between host cytokine response to the infection with HPAIVs and lethal outcome has not been well understood. In the present study, the highly pathogenic avian influenza viruses A/turkey/Italy/4580/1999 (H7N1) (Ty/Italy) and A/chicken/Netherlands/2586/2003 (H7N7) (Ck/NL) and the low pathogenic avian influenza virus (LPAIV) A/chicken/Ibaraki/1/2005 (H5N2) (Ck/Ibaraki) were intranasally inoculated into chickens. Ty/Italy replicated more extensively than Ck/NL in systemic tissues of the chickens, especially in the brain, and induced excessive mRNA expression of inflammatory and antiviral cytokines (IFN-gamma, IL-1 beta, IL-6, and IFN-alpha) in proportion to its proliferation. Using in situ hybridization, IL-6 mRNA was detected mainly in microglial nodules in the brain of the chickens infected with Ty/Italy. Capillary leakage assessed by Evans blue staining was observed in multiple organs, especially in the brains of the chickens infected with Ty/Italy, and was not observed in those infected with Ck/NL. In contrast, LPAIV caused only local infection in the chickens, with neither apparent cytokine expression nor capillary leakage in any tissue of the chickens. The present results indicate that an excessive cytokine response is induced by rapid and extensive proliferation of HPAIV and causes fatal multiple organ failure in chickens.
  • Haredy AM, Takenaka N, Yamada H, Sakoda Y, Okamatsu M, Yamamoto N, Omasa T, Ohtake H, Mori Y, Kida H, Yamanishi K, Okamoto S
    Clinical and vaccine immunology : CVI 1556-6811 2013/05 [Refereed][Not invited]
  • Naoki Yamamoto, Kosuke Soda, Yoshihiro Sakoda, Masatoshi Okamatsu, Hiroshi Kida
    VIRUS RESEARCH 173 (2) 294 - 298 0168-1702 2013/05 [Refereed][Not invited]
    Influenza virus rgVac1 sub-P0 (H5N1) (rgVac1-P0), in which a pair of dibasic,amino acid residues was introduced at the cleavage site of the HA of a reassortant of H5N2 and H7N1 viruses of duck origin, was low pathogenic in chickens. Vac1 sub-P3 (H5N1) (Vac1-P3) was selected as a highly pathogenic avian influenza virus by 3 consecutive passages in chickens from low pathogenic strain rgVac1-P0. Comparison of amino acid sequences of the virus proteins and experimental infection of chickens with a series of recombinant viruses demonstrated that in addition to the HA, each of the PA, NP, M1, and M2 of Vac1-P3 are responsible for the acquisition of pathogenicity in chickens. These 4 proteins of Vac1-P3 synergistically contributed to efficient virus replication in chickens. (C) 2013 Elsevier B.V. All rights reserved.
  • Shintaro Shichinohe, Masatoshi Okamatsu, Naoki Yamamoto, Yu Noda, Yuka Nomoto, Takashi Honda, Noriyasu Takikawa, Yoshihiro Sakoda, Hiroshi Kida
    VETERINARY MICROBIOLOGY 164 (1-2) 39 - 45 0378-1135 2013/05 [Refereed][Not invited]
    Antigenic variants of H5N1 highly pathogenic avian influenza virus (HPAIV) have selected and are prevailing in poultry populations in Asia. In the present study, the potency of inactivated influenza vaccine prepared from a non-pathogenic H5N1 avian influenza virus, A/duck/Hokkaido/Vac-3/2007 (H5N1), was assessed by challenging with H5N1 HPAIV variants, A/muscovy duck/Vietnam/OIE-559/2011 (H5N1), A/whooper swan/Hokkaido/4/2011 (H5N1), and A/peregrine falcon/Hong Kong/810/2009 (H5N1) belonging to clades 1, 23.2.1, and 2.3.4, respectively. All chickens immunized with the Vac-3 vaccine survived without showing any clinical signs after intranasal challenge either with A/whooper swan/Hokkaido/4/2011 (H5N1) or A/muscovy duck/Vietnam/OIE-559/2011 (H5N1). After challenge with A/peregrine falcon/Hong Kong/810/2009 (H5N1), 10 out of 12 vaccinated chickens survived and the other 2 died on 4 or 7 post-challenge days. The Vac-3 vaccine of 2.4-fold antigen concentration conferred complete protective immunity in chickens against challenge with A/peregrine falcon/Hong Kong/810/2009 (H5N1). (C) 2013 Elsevier B.V. All rights reserved.
  • Yurie Motohashi, Manabu Igarashi, Masatoshi Okamatsu, Takeshi Noshi, Yoshihiro Sakoda, Naoki Yamamoto, Kimihito Ito, Ryu Yoshida, Hiroshi Kida
    VIROLOGY JOURNAL 10 118  1743-422X 2013/04 [Refereed][Not invited]
    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.
  • Feng F, Sakoda Y, Ohyanagi T, Nagahori N, Shibuya H, Okamastu M, Miura N, Kida H, Nishimura SI
    Antiviral therapy 1359-6535 2013/02 [Refereed][Not invited]
  • Masahiro Kajihara, Yoshihiro Sakoda, Kosuke Soda, Kenji Minari, Masatoshi Okamatsu, Ayato Takada, Hiroshi Kida
    VIROLOGY JOURNAL 10 45  1743-422X 2013/02 [Refereed][Not invited]
    Background: Wild ducks are the natural hosts of influenza A viruses. Duck influenza, therefore, has been believed inapparent infection with influenza A viruses, including highly pathogenic avian influenza viruses (HPAIVs) in chickens. In fact, ducks experimentally infected with an HPAIV strain, A/Hong Kong/483/1997 (H5N1) (HK483), did not show any clinical signs. Another HPAIV strain, A/whooper swan/Mongolia/3/2005 (H5N1) (MON3) isolated from a dead swan, however, caused neurological dysfunction and death in ducks. Method: To understand the mechanism whereby MON3 shows high pathogenicity in ducks, HK483, MON3, and twenty-four reassortants generated between these two H5N1 viruses were compared for their pathogenicity in domestic ducks. Results: None of the ducks infected with MON3-based single-gene reassortants bearing the PB2, NP, or NS gene segment of HK483 died, and HK483-based single-gene reassortants bearing PB2, NP, or NS genes of MON3 were not pathogenic in ducks, suggesting that multiple gene segments contribute to the pathogenicity of MON3 in ducks. All the ducks infected with the reassortant bearing PB2, PA, HA, NP, and NS gene segments of MON3 died within five days post-inoculation, as did those infected with MON3. Each of the viruses was assessed for replication in ducks three days post-inoculation. MON3 and multi-gene reassortants pathogenic in ducks were recovered from all of the tissues examined and replicated with high titers in the brains and lungs. Conclusion: The present results indicate that multigenic factors are responsible for efficient replication of MON3 in ducks. In particular, virus growth in the brain might correlate with neurological dysfunction and the disease severity.
  • Masatoshi Okamatsu, Yoshihiro Sakoda, Takahiro Hiono, Naoki Yamamoto, Hiroshi Kida
    VIROLOGY JOURNAL 10 47  1743-422X 2013/02 [Refereed][Not invited]
    Background: The pandemic 2009 (H1N1) influenza virus has spread throughout the world and is now causing seasonal influenza. To prepare for the emergence of pandemic influenza, we have established a library of virus strains isolated from birds, pigs, and humans in global surveillance studies. Methods: Inactivated whole virus particle (WV) and ether-split (ES) vaccines were prepared from an influenza virus strain, A/swine/Hokkaido/2/1981 (H1N1), from the library and from A/Narita/1/2009 (H1N1) pandemic strain. Each of the vaccines was injected subcutaneously into mice and their potencies were evaluated by challenge with A/Narita/1/2009 (H1N1) virus strain in mice. Results: A/swine/Hokkaido/2/81 (H1N1), which was isolated from the lung of a diseased piglet, was selected on the basis of their antigenicity and growth capacity in embryonated chicken eggs. Two injections of the WV vaccine induced an immune response in mice, decreasing the impact of disease caused by the challenge with A/Narita/1/2009 (H1N1), as did the vaccine prepared from the homologous strain. Conclusion: The WV vaccine prepared from an influenza virus in the library is useful as an emergency vaccine in the early phase of pandemic influenza.
  • Masatoshi Okamatsu, Fei Feng, Tatsuya Ohyanagi, Noriko Nagahori, Kazuhiko Someya, Yoshihiro Sakoda, Nobuaki Miura, Shin-Ichiro Nishimura, Hiroshi Kida
    JOURNAL OF VIROLOGICAL METHODS 187 (2) 390 - 394 0166-0934 2013/02 [Refereed][Not invited]
    Attachment of influenza virus to susceptible cells is mediated by viral protein hemagglutinin (HA), which recognizes cell surface glycoconjugates that terminate in alpha-sialosides. To develop anti-influenza drugs based on inhibition of HA-mediated infection, novel fluorescent nanoparticles displaying multiple biantennary N-glycan chains with alpha-sialosides (A2-PC-QDs) that have high affinity for the HA were designed and constructed. The A2-PC-QDs enabled an easy and efficient fluorescence polarization (FP) assay for detection of interaction with the HA and competitive inhibition even by small molecule compounds against A2-PC-QDs-HA binding. The quantum dot (QD)-based FP assay established in the present study is a useful tool for high-throughput screening and to accelerate the development of novel and more effective blockers of the viral attachment of influenza virus. (C) 2012 Elsevier B.V. All rights reserved.
  • Toshiyuki Minami, Takashi Kijima, Satoshi Kohmo, Hisashi Arase, Yasushi Otani, Izumi Nagatomo, Ryo Takahashi, Kotaro Miyake, Masayoshi Higashiguchi, Osamu Morimura, Shoichi Ihara, Kazuyuki Tsujino, Haruhiko Hirata, Koji Inoue, Yoshito Takeda, Hiroshi Kida, Isao Tachibana, Atsushi Kumanogoh
    Scientific reports 3 2669 - 2669 2013 [Refereed][Not invited]
    Small-cell lung cancer (SCLC) easily recurs with a multidrug resistant phenotype. However, standard therapeutic strategies for relapsed SCLC remain unestablished. We found that human epidermal growth factor receptor 2 (HER2) is not only expressed in pretreated human SCLC specimens, but is also upregulated when HER2-positive SCLC cells acquire chemoresistance. Trastuzumab induced differential levels of antibody-dependent cell-mediated cytotoxicity (ADCC) to HER2-positive SCLC cells. Furthermore, as a mechanism of the differential levels of ADCC, we have revealed that coexpression of intracellular adhesion molecule (ICAM)-1 on SCLC cells is essential to facilitate and accelerate the trastuzumab-mediated ADCC. Although SN-38-resistant SCLC cells lacking ICAM-1 expression were still refractory to trastuzumab, their in vivo growth was significantly suppressed by bevacizumab treatment due to dependence on their distinctive and abundant production of vascular endothelial growth factor. Collectively, stepwise treatment with trastuzumab and bevacizumab is promising for the treatment of chemoresistant SCLC.
  • Fei Feng, Yoshihiro Sakoda, Tatsuya Ohyanagi, Noriko Nagahori, Hitomi Shibuya, Masatoshi Okamastu, Nobuaki Miura, Hiroshi Kida, Shin-Ichiro Nishimura
    Antiviral Chemistry and Chemotherapy 23 (2) 59 - 65 0956-3202 2013 [Refereed][Not invited]
    Background: The purpose of this study was to develop a new class of influenza A virus haemagglutinin (HA) blockers by tethering thiosialoside molecules to metal nanoparticles and producing glycoclusters that enhance the affinity of HA binding by N-acetylneuraminic acid. Methods: Oxygen of the glycoside bond of sialoside was replaced with sulfur to prevent hydrolytic digestion of the N-acetylneuraminic acid residue by viral neuraminidase. Two novel thiosialosides, α-2-S-[p-(N-levulinyl) aminophenyl]-5-N-acetylneuraminic acid (Neu5Ac-S-Lev) and α-2-S-[m-(N-levulinyl)aminobenzyl]-5-Nacetylneuraminic acid (Neu5Ac-S-CH 2-Lev), were tethered onto the surface of metal nanoparticles via an aminooxy functionalized thiol linker in a glycoblotting reaction. Gold (Au) and silver (Ag) nanoparticles were coated simultaneously with 11-mercaptoundecyl phosphorylcholine to reduce non-specific adsorption of proteins. Phosphorylcholine self-assembled monolayercoated metals displaying clustered Neu5Ac (Neu5Ac-PCSAM-Au and Neu5Ac-PCSAM-Ag) were subjected to haemagglutination inhibition (HI) assays using the influenza A virus strain A/PR/8/1934 (H1N1). Results: Glyconanoparticles with thiosialosides had potent HI activities. In particular, Neu5Ac-PCSAM-Au with a diameter of 20 nm corresponding to 9.8 μM monosaccharide Neu5Ac was the most potent HA inhibitor. The versatility of this strategy was demonstrated by similar submicromolar HI activities of Neu5Ac-PCSAM--Ag with diameters of 50 nm and 150 nm. Conclusions: Glycosylated metal nanoparticles were designed and synthesized as potent influenza A virus HA blockers. This study may contribute to the acceleration of the discovery of a new class of nanoparticle antiinfluenza drugs. © 2013 International Medical Press.
  • Norikazu Isoda, Yoshimi Tsuda, Shingo Asakura, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida
    ARCHIVES OF VIROLOGY 157 (12) 2257 - 2264 0304-8608 2012/12 [Refereed][Not invited]
    Avian influenza viruses A/duck/Mongolia/47/2001 (H7N1) (47/01) and A/duck/Mongolia/867/2002 (H7N1) (867/02) were defined as low-pathogenic avian influenza viruses (LPAIVs) using an intravenous pathogenicity test in chickens. On the other hand, the intracerebral pathogenicity indices of 47/01 and 867/02 were 1.30 and 0.00, respectively. A series of reassortant viruses were generated between 47/01 and 867/02, and their intracerebral pathogenicity was compared in one-day-old chicks to identify the protein(s) responsible for the intracerebral pathogenicity of 47/01. The results indicate that the amino acids at positions 50 and 98 of the nucleoprotein are related to the pathogenicity of 47/01 in chicks by intracerebral inoculation. A significant association was found between mortality of the chicks inoculated intracerebrally with 47/01 and virus replication in the lungs and/or brain. These results indicate that the NP of avian influenza viruses may be responsible for intracerebral pathogenicity in the host.
  • Tomokazu Tamura, Yoshihiro Sakoda, Fumi Yoshino, Takushi Nomura, Naoki Yamamoto, Yuka Sato, Masatoshi Okamatsu, Nicolas Ruggli, Hiroshi Kida
    JOURNAL OF VIROLOGY 86 (16) 8602 - 8613 0022-538X 2012/08 [Refereed][Not invited]
    Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious disease of pigs. There are numerous CSFV strains that differ in virulence, resulting in clinical disease with different degrees of severity. Low-virulent and moderately virulent isolates cause a mild and often chronic disease, while highly virulent isolates cause an acute and mostly lethal hemorrhagic fever. The live attenuated vaccine strain GPE(-) was produced by multiple passages of the virulent ALD strain in cells of swine, bovine, and guinea pig origin. With the aim of identifying the determinants responsible for the attenuation, the GPE- vaccine virus was readapted to pigs by serial passages of infected tonsil homogenates until prolonged viremia and typical signs of CSF were observed. The GPE(-)/P-11 virus isolated from the tonsils after the 11th passage in vivo had acquired 3 amino acid substitutions in E2 (T830A) and NS4B (V2475A and A2563V) compared with the virus before passages. Experimental infection of pigs with the mutants reconstructed by reverse genetics confirmed that these amino acid substitutions were responsible for the acquisition of pathogenicity. Studies in vitro indicated that the substitution in E2 influenced virus spreading and that the changes in NS4B enhanced the viral RNA replication. In conclusion, the present study identified residues in E2 and NS4B of CSFV that can act synergistically to influence virus replication efficiency in vitro and pathogenicity in pigs.
  • Yoshihiro Sakoda, Hiroaki Wakamoto, Tehpin Tamura, Takushi Nomura, Michiko Naito, Hiroshi Aoki, Hiroshi Morita, Hiroshi Kida, Akio Fukusho
    JAPANESE JOURNAL OF VETERINARY RESEARCH 60 (2-3) 85 - 94 0047-1917 2012/08 [Refereed][Not invited]
    An indirect enzyme-linked immunosorbent assay (ELISA) was developed for a screening test to detect antibodies against classical swine fever virus (CSFV). Viral glycoproteins, which were purified from swine kidney cells infected with CSFV ALD/A76 strain by the immunoaffinity purification using monoclonal antibody against E2 protein, were adsorbed on a microtiter plate as the antigen for the antibody detection. Each antibody titer of serum sample was expressed as a sample per positive value calculated with optical absorbance of each sample and that of a positive control. The advantage of this ELISA is its higher sensitivity: most sera containing more than 4 neutralization titers were determined to be positive. This ELISA is unable to discriminate between antibodies against CSFV and those against other ruminant pestiviruses, therefore positive sera in this ELISA should be evaluated by a cross-neutralization test using CSFV, bovine viral diarrhea virus, and border disease virus. Taken together, the indirect ELISA developed in this study is useful screening tool to detect antibodies against CSFV for the large-scale monitoring of classical swine fever.
  • Yoshihiro Sakoda, Michiko Naito, Mutsumi Ito, Yuki Ito, Norikazu Isoda, Tomohisa Tanaka, Takashi Umemura, Hiroshi Kida
    JOURNAL OF VETERINARY MEDICAL SCIENCE 74 (7) 955 - 958 0916-7250 2012/07 [Refereed][Not invited]
    Leptospira interrogans serovar Manilae strain UP-MMC was inoculated into miniature pigs to assess its pathogenicity. Leptospires were recovered from the whole blood, kidneys, and livers in the acute phase without showing any clinical signs. Under immunosuppressive conditions by dexamethasone, leptospires were recovered from the kidneys and their genes were detected from the urine in the chronic phase. These results indicate that leptospires persisted in the kidneys until the chronic phase, and excretion of leptospires in the urine was enhanced under immunosuppressive conditions, resulting in horizontal transmission among pigs on farms.
  • Yoshihiro Sakoda, Masatoshi Okamatsu, Norikazu Isoda, Naoki Yamamoto, Koichi Ozaki, Yasuto Umeda, Shigeyuki Aoyama, Hiroshi Kida
    MICROBIOLOGY AND IMMUNOLOGY 56 (7) 490 - 495 0385-5600 2012/07 [Refereed][Not invited]
    Affinity chromatography using sulfated, spherical cellulose beads (Cellufine Sulfate) was assessed for purification of influenza A and influenza B viruses. Recovery rates of viruses eluted from the beads were high for all tested virus strains. This method was also useful for removing chicken egg-derived impurities from allantoic fluids containing influenza viruses; the hemagglutination activity per amount of protein in the eluted sample was significantly higher than that in the applied sample. These results suggest that use of Cellufine Sulfate is a practical method for primary purification of influenza viruses in the process of influenza vaccine production.
  • Masahiko Arikata, Yasushi Itoh, Masatoshi Okamatsu, Toshinaga Maeda, Takashi Shiina, Keiko Tanaka, Shingo Suzuki, Misako Nakayama, Yoshihiro Sakoda, Hirohito Ishigaki, Ayato Takada, Hideaki Ishida, Kosuke Soda, Van Loi Pham, Hideaki Tsuchiya, Shinichiro Nakamura, Ryuzo Torii, Takeshi Shimizu, Hidetoshi Inoko, Iwao Ohkubo, Hiroshi Kida, Kazumasa Ogasawara
    PLOS ONE 7 (5) e37220  1932-6203 2012/05 [Refereed][Not invited]
    We made an H1N1 vaccine candidate from a virus library consisting of 144 (= 16 HAx9 NA) non-pathogenic influenza A viruses and examined its protective effects against a pandemic (2009) H1N1 strain using immunologically naive cynomolgus macaques to exclude preexisting immunity and to employ a preclinical study since preexisting immunity in humans previously vaccinated or infected with influenza virus might make comparison of vaccine efficacy difficult. Furthermore, macaques carrying a major histocompatibility complex class I molecule, Mafa-A1*052:02, were used to analyze peptide-specific CD8(+) T cell responses. Sera of macaques immunized with an inactivated whole particle formulation without addition of an adjuvant showed higher neutralization titers against the vaccine strain A/Hokkaido/2/1981 (H1N1) than did sera of macaques immunized with a split formulation. Neutralization activities against the pandemic strain A/Narita/1/2009 (H1N1) in sera of macaques immunized twice with the split vaccine reached levels similar to those in sera of macaques immunized once with the whole particle vaccine. After inoculation with the pandemic virus, the virus was detected in nasal samples of unvaccinated macaques for 6 days after infection and for 2.67 days and 5.33 days on average in macaques vaccinated with the whole particle vaccine and the split vaccine, respectively. After the challenge infection, recall neutralizing antibody responses against the pandemic virus and CD8(+) T cell responses specific for nucleoprotein peptide NP262-270 bound to Mafa-A1*052:02 in macaques vaccinated with the whole particle vaccine were observed more promptly or more vigorously than those in macaques vaccinated with the split vaccine. These findings demonstrated that the vaccine derived from our virus library was effective for pandemic virus infection in macaques and that the whole particle vaccine conferred more effective memory and broader cross-reactive immune responses to macaques against pandemic influenza virus infection than did the split vaccine.
  • Naoki Nomura, Yoshihiro Sakoda, Kosuke Soda, Masatoshi Okamatsu, Hiroshi Kida
    JOURNAL OF VETERINARY MEDICAL SCIENCE 74 (4) 441 - 447 0916-7250 2012/04 [Refereed][Not invited]
    H9N2 influenza viruses circulate in wild birds and poultry in Eurasian countries, and have been isolated from pigs and humans in China. H9N2 viruses isolated from birds, pigs and humans have been classified into three sublineages based on antigenic and genetic features. Chicken antisera to H9N2 viruses of the Korean sublineage reacted with viruses of different sublineages by the hemagglutination-inhibition test. A test vaccine prepared from a non-pathogenic A/duck/Hokkaido/49/1998 (H9N2) strain of the Korean sublineage, obtained from our influenza virus library, induced immunity in mice to reduce the impact of disease caused by the challenge with A/Hong Kong/1073/1999 (H9N2), which is of a different sublineage. The present results indicate that an inactivated whole virus vaccine prepared from a non-pathogenic influenza virus from the library could be used as an emergency vaccine during the early stage of a pandemic caused by H9N2 infection.
  • Yoshihiro Sakoda, Hiroshi Ito, Yuko Uchida, Masatoshi Okamatsu, Naoki Yamamoto, Kosuke Soda, Naoki Nomura, Saya Kuribayashi, Shintaro Shichinohe, Yuji Sunden, Takashi Umemura, Tatsufumi Usui, Hiroichi Ozaki, Tsuyoshi Yamaguchi, Toshiyuki Murase, Toshihiro Ito, Takehiko Saito, Ayato Takada, Hiroshi Kida
    JOURNAL OF GENERAL VIROLOGY 93 (3) 541 - 550 0022-1317 2012/03 [Refereed][Not invited]
    H5N1 highly pathogenic avian influenza virus (HPAIV) was reintroduced and caused outbreaks in chickens in the 2010-2011 winter season in Japan, which had been free from highly pathogenic avian influenza (HPAI) since 2007 when HPAI outbreaks occurred and were controlled. On 14 October 2010 at Lake Ohnuma, Wakkanai, the northernmost part of Hokkaido, Japan, H5N1 HPAIVs were isolated from faecal samples of ducks flying from their nesting lakes in Siberia. Since then, in Japan, H5N1 HPAIVs have been isolated from 63 wild birds in 17 prefectures and caused HPAI outbreaks in 24 chicken farms in nine prefectures by the end of March in 2011. Each of these isolates was genetically closely related to the HPAIV isolates at Lake Ohnuma, and those in China, Mongolia, Russia and Korea, belonging to genetic clade In addition, these isolates were genetically classified into three groups, suggesting that the viruses were transmitted by migratory water birds through at least three different routes from their northern territory to Japan. These isolates were antigenic variants, which is consistent with selection in poultry under the immunological pressure induced by vaccination. To prevent the perpetuation of viruses in the lakes where water birds nest in summer in Siberia, prompt eradication of HPAIVs in poultry is urgently needed in Asian countries where HPAI has not been controlled.
  • Naoki Nomura, Yoshihiro Sakoda, Mayumi Endo, Hiromi Yoshida, Naoki Yamamoto, Masatoshi Okamatsu, Kenji Sakurai, Nam Van Hoang, Long Van Nguyen, Huy Duc Chu, Tien Ngoc Tien, Hiroshi Kida
    ARCHIVES OF VIROLOGY 157 (2) 247 - 257 0304-8608 2012/02 [Refereed][Not invited]
    In the surveillance of avian influenza in Vietnam, 26 H9N2, 1 H3N2, 1 H3N8, 7 H4N6, 3 H11N3, and 1 H11N9 viruses were isolated from tracheal and cloacal swab samples of 300 domestic ducks in April 2009, and 1 H9N6 virus from 300 bird samples in March 2010. Out of the 27 H9 virus isolates, the hemagglutinins of 18 strains were genetically classified as belonging to the sublineage G1, and the other nine belonged to the Korean sublineage. Phylogenetic analysis revealed that one of the 27 H9 viruses was a reassortant in which the PB2 gene belonged to the Korean sublineage and the other seven genes belonged to the G1 sublineage. Three representative H9N2 viruses were intranasally inoculated into ducks, chickens, pigs, and mice. On the basis of experimental infection studies, it was found that each of the three viruses readily infected pigs and replicated in their upper respiratory tracts, and they infected chickens with slight replication. Viruses were recovered from the lungs of mice inoculated with two of the three isolates. The present results reveal that H9 avian influenza viruses are prevailing and genetic reassortment occurs among domestic ducks in Vietnam. It is recommended that careful surveillance of swine influenza with H9 viruses should be performed to prepare for pandemic influenza.
  • Shinya Yamada, Kyoko Shinya, Ayato Takada, Toshihiro Ito, Takashi Suzuki, Yasuo Suzuki, Quynh Mai Le, Masahito Ebina, Noriyuki Kasai, Hiroshi Kida, Taisuke Horimoto, Pierre Rivailler, Li Mei Chen, Ruben O. Donis, Yoshihiro Kawaoka
    JOURNAL OF VIROLOGY 86 (3) 1411 - 1420 0022-538X 2012/02 [Refereed][Not invited]
    Quail are thought to serve as intermediate hosts of influenza A viruses between aquatic birds and terrestrial birds, such as chickens, due to their high susceptibility to aquatic-bird viruses, which then adapt to replicate efficiently in their new hosts. However, does replication of aquatic-bird influenza viruses in quail similarly result in their efficient replication in humans? Using sialic acid-galactose linkage-specific lectins, we found both avian (sialic acid-alpha 2-3-galactose [Sia alpha 2-3Gal] linkages on sialyloligosaccharides)- and human (Sia alpha 2-6Gal)-type receptors on the tracheal cells of quail, consistent with previous reports. We also passaged a duck H3N2 virus in quail 19 times. Sequence analysis revealed that eight mutations accumulated in hemagglutinin (HA) during these passages. Interestingly, many of the altered HA amino acids found in the adapted virus are present in human seasonal viruses, but not in duck viruses. We also found that stepwise stalk deletion of neuraminidase occurred during passages, resulting in reduced neuraminidase function. Despite some hemagglutinin mutations near the receptor binding pocket, appreciable changes in receptor specificity were not detected. However, reverse-genetics-generated viruses that possessed the hemagglutinin and neuraminidase of the quail-passaged virus replicated significantly better than the virus possessing the parent HA and neuraminidase in normal human bronchial epithelial cells, whereas no significant difference in replication between the two viruses was observed in duck cells. Further, the quail-passaged but not the original duck virus replicated in human bronchial epithelial cells. These data indicate that quail can serve as intermediate hosts for aquatic-bird influenza viruses to be transmitted to humans.
  • Kayoko Sato, Atsushi Iwai, Yosuke Nakayama, Junko Morimoto, Ayato Takada, Mitsuo Maruyama, Hiroshi Kida, Toshimitsu Uede, Tadaaki Miyazaki
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 417 (1) 274 - 279 0006-291X 2012/01 [Refereed][Not invited]
    Osteopontin (OPN) is involved in exacerbating various inflammatory diseases. A severe pulmonary inflammation is frequently found in lethal influenza A virus (IAV) infection. However, the function of OPN against the infection was poorly understood. Here, we demonstrate an importance of OPN on immune response and disease severity after IAV infection. We found that the expression level of OPN was increased in mice infected with IAV. The OPN knockout (KO) mice exhibited a severe pathological phenotype and the survival rate decreased after the lethal IAV infection, compared to the wild type mice, while the survival rate increased in OPN transgenic (Tg) mice. The population of natural killer (NK) cells significantly decreased in OPN KO mice at day 5 after the infection, whereas, it increased in OPN Tg mice. These results suggest that OPN plays an important role in host defense against IAV infection through the regulation of NK cell population. (C) 2011 Elsevier Inc. All rights reserved.
  • Akira Sakurai, Namiko Nomura, Reiko Nanba, Takayuki Sinkai, Tsunehito Iwaki, Taminori Obayashi, Kazuhiro Hashimoto, Michiya Hasegawa, Yoshihiro Sakoda, Akihiro Naito, Yoshihito Morizane, Mitsugu Hosaka, Kunio Tsuboi, Hiroshi Kida, Akemi Kai, Futoshi Shibasaki
    JOURNAL OF VIROLOGICAL METHODS 178 (1-2) 75 - 81 0166-0934 2011/12 [Refereed][Not invited]
    The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2 h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18 s/cycle; 40 cycles in less than 20 min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10(-1) plaque-forming unit/reaction for viruses in culture supernatants during 20 min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks. (C) 2011 Published by Elsevier B.V.
  • Atsuhiko Wada, Yoshihiro Sakoda, Takayoshi Oyamada, Hiroshi Kida
    JOURNAL OF VIROLOGICAL METHODS 178 (1-2) 82 - 86 0166-0934 2011/12 [Refereed][Not invited]
    H5N1, a highly pathogenic avian influenza virus (HPAIV), has become a serious epizootic threat to the poultry population in Asia. In addition, significant numbers of human cases of HPAIV infection have been reported to date. To prevent the spread of HPAIV among humans and to allow for timely medical intervention, a rapid and high sensitive method is needed to detect and subtype the causative HPAIVs. In the present study, a silver amplification technique used in photographic development was combined with immunochromatography technologies and a highly sensitive and rapid diagnostic test to detect the hemagglutinin of H5 influenza viruses was developed. The sensitivity of the test kit was increased 500 times by silver amplification. The sensitivity of the method was more than 10 times higher than those of conventional rapid influenza diagnostic tests, which detect viral nucleoproteins. The diagnostic system developed in the present study can therefore provide rapid and highly sensitive results and will be useful for diagnosis of H5 HPAIV infection in humans and animals. (C) 2011 Elsevier B.V. All rights reserved.
  • Shigeru Kohno, Muh-Yong Yen, Hee-Jin Cheong, Nobuo Hirotsu, Tadashi Ishida, Jun-ichi Kadota, Masashi Mizuguchi, Hiroshi Kida, Jingoro Shimada
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 55 (11) 5267 - 5276 0066-4804 2011/11 [Refereed][Not invited]
    Antiviral medications with activity against influenza viruses are important in controlling influenza. We compared intravenous peramivir, a potent neuraminidase inhibitor, with oseltamivir in patients with seasonal influenza virus infection. In a multinational, multicenter, double-blind, double-dummy randomized controlled study, patients aged >= 20 years with influenza A or B virus infection were randomly assigned to receive either a single intravenous infusion of peramivir (300 or 600 mg) or oral administration of oseltamivir (75 mg twice a day [b.i.d.] for 5 days). To demonstrate the noninferiority of peramivir in reducing the time to alleviation of influenza symptoms with hazard model analysis and a noninferiority margin of 0.170, we planned to recruit 1,050 patients in South Korea, Japan, and Taiwan. A total of 1,091 patients (364 receiving 300 mg and 362 receiving 600 mg of peramivir; 365 receiving oseltamivir) were included in the intent-to-treat infected population. The median durations of influenza symptoms were 78.0, 81.0, and 81.8 h in the groups treated with 300 mg of peramivir, 600 mg of peramivir, and oseltamivir, respectively. The hazard ratios of the 300- and 600-mg-peramivir groups compared to the oseltamivir group were 0.946 (97.5% confidence interval [CI], 0.793, 1.129) and 0.970 (97.5% CI, 0.814, 1.157), respectively. Both peramivir groups were noninferior to the oseltamivir group (97.5% CI, <1.170). The overall incidence of adverse drug reactions was significantly lower in the 300-mg-peramivir group, but the incidence of severe reactions in either peramivir group was not different from that in the oseltamivir group. Thus, a single intravenous dose of peramivir may be an alternative to a 5-day oral dose of oseltamivir for patients with seasonal influenza virus infection.
  • Kimihito Ito, Manabu Igarashi, Yutaka Miyazaki, Teiji Murakami, Syaka Iida, Hiroshi Kida, Ayato Takada
    PLOS ONE 6 (10) e25953  1932-6203 2011/10 [Refereed][Not invited]
    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.
  • Masayuki Motoshima, Masatoshi Okamatsu, Shingo Asakura, Saya Kuribayashi, Sugar Sengee, Damdinjav Batchuluun, Mika Ito, Yukiko Maeda, Mariko Eto, Yoshihiro Sakoda, Ruuragchaa Sodnomdarjaa, Hiroshi Kida
    ARCHIVES OF VIROLOGY 156 (8) 1379 - 1385 0304-8608 2011/08 [Refereed][Not invited]
    A/equine/Kanazawa/1/2007 (H3N8), A/equine/Hokkaido/I828/2008 (H3N8) and A/equine/Mongolia/1/2008 (H3N8) were isolated from infected horses. A/equine/Yokohama/aq19/2009 (H3N8) and A/equine/Yokohama/aq13/2010 (H3N8) were isolated from horses imported from Canada and Belgium examined at the Animal Quarantine Service in Yokohama, Japan. In the present study, these five isolates were genetically and antigenically analyzed. Phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes showed that three isolates from horses in Japan and imported from Canada belonged to the same branch, clade 1 of the Florida sublineage, while the isolates from horses in Mongolia and imported from Belgium belonged to another branch, clade 2 of the Florida sublineage. Reactivity patterns of a panel of monoclonal antibodies to the HA of A/equine/Kanazawa/1/2007 (H3N8) with the five isolates indicate that the HAs of these viruses were antigenically similar to each other and to the reference strains A/equine/La Plata/1/1993 (H3N8) and A/equine/Avesta/1/1993 (H3N8). The present findings indicate that extensive antigenic variation has not accumulated among H3N8 influenza viruses in horses.
  • 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 0047-1917 2011/08 [Refereed][Not invited]
    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.
  • Yoshida H, Sakoda Y, Endo M, Motoshima M, Yoshino F, Yamamoto N, Okamatsu M, Soejima T, Senba S, Kanda H, Kida H
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 6 73 (6) 753 - 758 0916-7250 2011/06 [Refereed][Not invited]
  • Soda K, Cheng MC, Yoshida H, Endo M, Lee SH, Okamatsu M, Sakoda Y, Wang CH, Kida H
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 6 73 (6) 767 - 772 0916-7250 2011/06 [Refereed][Not invited]
  • 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 0022-1317 2011/06 [Refereed][Not invited]
    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.
  • Shigeru Kohno, Hiroshi Kida, Masashi Mizuguchi, Nobuo Hirotsu, Tadashi Ishida, Junichi Kadota, Jingoro Shimada
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 55 (6) 2803 - 2812 0066-4804 2011/06 [Refereed][Not invited]
    Influenza virus infections are known to persist longer in patients with underlying diseases, including respiratory tract diseases, and tend to become complicated by secondary influenza-associated infections, such as pneumonia. To assess the efficacy and safety of the novel anti-influenza virus drug peramivir in high-risk patients, we conducted a clinical trial of patients with diabetes or chronic respiratory tract diseases and patients being treated with drugs that suppress immune function. In this multicenter, uncontrolled, randomized, double-blind study, peramivir was intravenously administered at 300 or 600 mg/day for 1 to 5 days, as needed. Efficacy was investigated in 37 patients (300 mg, n = 18 patients; 600 mg, n = 19 patients). The median durations of influenza illness were 68.6 h (90% confidence interval, 41.5 to 113.4 h) overall, 114.4 h (90% confidence interval, 40.2 to 235.3 h) in the 300-mg group, and 42.3 h (90% confidence interval, 30.0 to 82.7 h) in the 600-mg group. The hazard ratio for the 600-mg group compared to the 300-mg group was 0.497 (90% confidence interval, 0.251 to 0.984), and the duration of influenza illness was significantly shorter in the 600-mg group than in the 300-mg group. Among the 42 patients in the safety analysis set, adverse events occurred in 73.8% and adverse drug reactions in 33.3%. No adverse events were particularly problematic clinically, and all patients recovered quickly from all events. The measured blood drug concentrations showed no tendency toward accumulation. Drug accumulation with repeated doses was thus considered to be of little concern. Intravenous peramivir appears to offer a potentially useful treatment for high-risk patients in the future.
  • Kaori Nakanishi, Yoshito Takeda, Satoshi Tetsumoto, Takeo Iwasaki, Kazuyuki Tsujino, Hanako Kuhara, Yingji Jin, Izumi Nagatomo, Hiroshi Kida, Sho Goya, Takashi Kijima, Norikazu Maeda, Tohru Funahashi, Iichiro Shimomura, Isao Tachibana, Ichiro Kawase
    AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE 183 (9) 1164 - 1175 1073-449X 2011/05 [Refereed][Not invited]
    Rationale: Chronic obstructive pulmonary disease is frequently complicated with comorbidities, such as cardiovascular disease, osteoporosis, and body weight loss, but the causal link remains unclear. Objectives: To investigate the role of adiponectin in the pathogenesis of chronic obstructive pulmonary disease and its potential use in therapy. Methods: Adiponectin localization and dynamics in the lung were analyzed in an elastase-induced emphysema model. Next, the lung of adiponectin-knockout mice, extrapulmonary effects, and the underlying mechanism were investigated. Finally, we tested whether exogenous adiponectin could ameliorate the emphysematous change in adiponectin-knockout mice. Measurements and Main Results: Adiponectin expression in lung vasculature and plasma concentration of adiponectin were reduced after elastase-instillation. Notably, adiponectin-knockout mice showed progressive alveolar enlargement and increased lung compliance. They further exhibited not only systemic inflammation, but also extrapulmonary phenotype, such as body weight loss, fat atrophy, and osteoporosis. Moreover, endothelial apoptosis was enhanced in the lungs of adiponectin-knockout mice, as evidenced by caspase-3 activity. Consistent with this, expressions of vascular endothelial growth factor receptor-2 and platelet endothelial cell adhesion molecule-1 on endothelial cells were decreased in the adiponectin-knockout mice. Finally, adenovirus-mediated adiponectin supplementation ameliorated the emphysematous phenotype. Conclusions: Adiponectin-knockout mice develop progressive chronic obstructive pulmonary disease-like phenotype with systemic inflammation and extrapulmonary phenotypes. Hypoadiponectinemia could thus play a critical role in the progression of chronic obstructive pulmonary disease and concomitant comorbidities through endothelial dysfunction. Together, adiponectin could be a novel target for chronic obstructive pulmonary disease therapy.
  • Eiji Miyagawa, Hiroyuki Kogaki, Yoshiaki Uchida, Nobuyuki Fujii, Takashi Shirakawa, Yoshihrio Sakoda, Hiroshi Kida
    JOURNAL OF VIROLOGICAL METHODS 173 (2) 213 - 219 0166-0934 2011/05 [Refereed][Not invited]
    Three anti-H5 influenza virus monoclonal antibody (mAb) clones, IFH5-26, IFH5-115 and IFH5-136, were obtained by immunising a BALB/C mouse with inactivated A/duck/Hokkaido/Vac-1/04 (H5N1). These mAbs were found to recognise specifically the haemagglutinin (HA) epitope of the influenza H5 subtypes by western blotting with recombinant HAs; however, these mAbs have no neutralising activity for A/duck/Hokkaido/84/02 (H5N3) or A/Puerto Ric/8/34 (H1N1). Each epitope of these mAbs was a conformational epitope that was formed from the regions located between 46 to 60 amino acids (aa) and 312 to 322 aa for IFH5-115, from 101 to 113 aa and 268 to 273 aa for IFH5-136 and from 61 to 80 aa and 290 to 300 aa for IFH5-26. The epitopes were located in the loop regions between the receptor region and alpha-helix structure in haemagglutinin 1 (HA1). Influenza A virus H5-specific rapid immunochromatographic test kits were tested as solid phase antibody/alkaline phosphate-conjugated mAb in the following three combinations: IFH5-26/IFH5-115, IFH5-136/IFH5-26 and IFH5-136/IFH5-115. In every combination, only influenza A H5 subtypes were detected. For effective clinical application, rapid dual discrimination immunochromatographic test kits in combination with H5 HA-specific mAb, IFA5-26 and IFA5-115 and the influenza A NP NP-specific mAb, FVA2-11, were developed. The dual discrimination immunochromatographic tests kits detected influenza A virus H5 subtypes as H5 line-positive and all influenza A subtypes as A line-positive simultaneously. The dual discrimination immunochromatographic test kits may be useful for discriminating highly pathogenic avian influenza A H5N1 viruses from seasonal influenza A virus, as well as for confirming influenza infection status in human, avian and mammalian hosts. (C) 2011 Elsevier B.V. All rights reserved.
  • Norikazu Isoda, Yoshihiro Sakoda, Masatoshi Okamatsu, Yoshimi Tsuda, Hiroshi Kida
    ARCHIVES OF VIROLOGY 156 (4) 557 - 563 0304-8608 2011/04 [Refereed][Not invited]
    The avian influenza vaccine strain A/duck/Hokkaido/Vac-1/2004 (H5N1) (Vac-1) was found to be pathogenic in chicken embryos (CEs). In order to decrease the pathogenicity of Vac-1 in CEs, a series of reassortant viruses was generated between Vac-1 and A/Puerto Rico/8/1934 (H1N1) (PR8), and their pathogenicity and growth potential were compared in CEs. The results indicated that either the PB1 or PA protein was responsible for the pathogenicity of Vac-1 in CEs. The HA titers of the allantoic fluids of CEs inoculated with the recombinant H5N1 viruses, of which pathogenicity was lower than that of the recombinant Vac-1 prepared by reverse genetics in CEs, were equivalent to those of CEs inoculated with the recombinant Vac-1. One of the reassortant viruses, rg-PR8-PA/Vac-1 (H5N1), in which the PA gene was replaced with the corresponding gene of PR8, yielded allantoic fluids with the same HA titer as that of Vac-1, indicating that this reassortant should be a good candidate as an improved vaccine strain.
  • Takuya Shiozaki, Atsushi Iwai, Yoshihiro Kawaoka, Ayato Takada, Hiroshi Kida, Tadaaki Miyazaki
    JOURNAL OF GENERAL VIROLOGY 92 (2) 315 - 325 0022-1317 2011/02 [Refereed][Not invited]
    Infection with influenza A virus causes acute respiratory tract infections in humans and may lead to lethal diseases including pneumonia. Identifying host factors that are involved in the severity of infectious diseases caused by influenza A virus is considered important for the prevention and treatment of these viral infections. This report demonstrated that Siva-1 is crucial for the induction of apoptosis caused by infection with influenza A virus and is involved in virus replication. Susceptibility to apoptosis induced by influenza A virus infection was increased in human lung-derived A549 cells, which stably express Siva-1. In addition, induction of apoptosis after influenza A virus infection was strongly inhibited by knockdown of Siva-1 expression. Furthermore, the replication of influenza A virus was significantly suppressed in A549 cells in which Siva-1 expression was inhibited and the effect of Siva-1 knockdown was eliminated by treatment with Z-VAD-FMK. These findings suggest that the caspase-dependent pathway for induction of apoptosis is involved in Siva-1-mediated influenza A virus replication.
  • Kosuke Soda, Shingo Asakura, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida
    VIROLOGY JOURNAL 8 64  1743-422X 2011/02 [Refereed][Not invited]
    Background: Outbreaks of avian influenza (AI) caused by infection with low pathogenic H9N2 viruses have occurred in poultry, resulting in serious economic losses in Asia and the Middle East. It has been difficult to eradicate the H9N2 virus because of its low pathogenicity, frequently causing in apparent infection. It is important for the control of AI to assess whether the H9N2 virus acquires pathogenicity as H5 and H7 viruses. In the present study, we investigated whether a non-pathogenic H9N2 virus, A/chicken/Yokohama/aq-55/2001 (Y55) (H9N2), acquires pathogenicity in chickens when a pair of di-basic amino acid residues is introduced at the cleavage site of its HA molecule. Results: rgY55sub (H9N2), which had four basic amino acid residues at the HA cleavage site, replicated in MDCK cells in the absence of trypsin after six consecutive passages in the air sacs of chicks, and acquired intravenous pathogenicity to chicken after four additional passages. More than 75% of chickens inoculated intravenously with the passaged virus, rgY55sub-P10 (H9N2), died, indicating that it is pathogenic comparable to that of highly pathogenic avian influenza viruses (HPAIVs) defined by World Organization for Animal Health (OIE). The chickens inoculated with the virus via the intranasal route, however, survived without showing any clinical signs. On the other hand, an avirulent H5N1 strain, A/duck/Hokkaido/Vac-1/2004 (Vac1) (H5N1), acquired intranasal pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the HA, followed by two passages by air sac inoculation in chicks. Conclusion: The present results demonstrate that an H9N2 virus has the potential to acquire intravenous pathogenicity in chickens although the morbidity via the nasal route of infection is lower than that of H5N1 HPAIV.
  • Naoki Yamamoto, Yoshihiro Sakoda, Masayuki Motoshima, Fumi Yoshino, Kosuke Soda, Masatoshi Okamatsu, Hiroshi Kida
    VIROLOGY JOURNAL 8 65  1743-422X 2011/02 [Refereed][Not invited]
    Background: Infection with H5N1 highly pathogenic avian influenza viruses (HPAIVs) of domestic poultry and wild birds has spread to more than 60 countries in Eurasia and Africa. It is concerned that HPAIVs may be perpetuated in the lakes in Siberia where migratory water birds nest in summer. To monitor whether HPAIVs circulate in migratory water birds, intensive surveillance of avian influenza has been performed in Mongolia and Japan in autumn each year. Until 2008, there had not been any H5N1 viruses isolated from migratory water birds that flew from their nesting lakes in Siberia. In autumn 2009, A/mallard/Hokkaido/24/09 (H5N1) (Mal/Hok/24/09) was isolated from a fecal sample of a mallard (Anas platyrhynchos) that flew from Siberia to Hokkaido, Japan. The isolate was assessed for pathogenicity in chickens, domestic ducks, and quails and analyzed antigenically and phylogenetically. Results: No clinical signs were observed in chickens inoculated intravenously with Mal/Hok/24/09 (H5N1). There was no viral replication in chickens inoculated intranasally with the isolate. None of the domestic ducks and quails inoculated intranasally with the isolate showed any clinical signs. There were no multiple basic amino acid residues at the cleavage site of the hemagglutinin (HA) of the isolate. Each gene of Mal/Hok/24/09 (H5N1) is phylogenetically closely related to that of influenza viruses isolated from migratory water birds that flew from their nesting lakes in autumn. Additionally, the antigenicity of the HA of the isolate was similar to that of the viruses isolated from migratory water birds in Hokkaido that flew from their northern territory in autumn and different from those of HPAIVs isolated from birds found dead in China, Mongolia, and Japan on the way back to their northern territory in spring. Conclusion: Mal/Hok/24/09 (H5N1) is a non-pathogenic avian influenza virus for chickens, domestic ducks, and quails, and is antigenically and genetically distinct from the H5N1 HPAIVs prevailing in birds in Eurasia and Africa. H5 viruses with the HA gene of HPAIV had not been isolated from migratory water birds in the surveillance until 2009, indicating that H5N1 HPAIVs had not become dominant in their nesting lakes in Siberia until 2009.
  • Rozanah Asmah Abdul Samad, Yoshihiro Sakoda, Yoshimi Tsuda, Edgar Simulundu, Rashid Manzoor, Masatoshi Okamatsu, Kimihito Ito, Hiroshi Kida
    JAPANESE JOURNAL OF VETERINARY RESEARCH 59 (1) 15 - 22 0047-1917 2011/02 [Refereed][Not invited]
    Recent introduction of H5N1 highly pathogenic avian influenza virus (HPAIV) in wild birds from poultry in Eurasia signaled the possibility that this virus may perpetuate in nature. Surveillance of avian influenza especially in migratory birds, therefore, has been conducted to provide information on the viruses brought by them to Hokkaido, Japan, from their nesting lakes in Siberia in autumn. During 2008-2009, 62 influenza viruses of 21 different combinations of hemagglutinin (HA) and neuraminidase (NA) subtypes were isolated. Up to September 2010, no HPAIV has been found, indicating that H5N1 HPAIV has not perpetuated at least dominantly in the lakes where ducks nest in summer in Siberia. The PB2 genes of 54 influenza viruses out of 283 influenza viruses isolated in Hokkaido in 2000-2009 were phylogenetically analysed. None of the genes showed close relation to those of H5N1 HPAIVs that were detected in wild birds found dead in Eurasia on the way back to their northern territory in spring.
  • Rozanah Asmah Abdul Samad, Naoki Nomura, Yoshimi Tsuda, Rashid Manzoor, Masahiro Kajihara, Daisuke Tomabechi, Takashi Sasaki, Norihide Kokumai, Toshiaki Ohgitani, Masatoshi Okamatsu, Ayato Takada, Yoshihiro Sakoda, Hiroshi Kida
    JAPANESE JOURNAL OF VETERINARY RESEARCH 59 (1) 23 - 29 0047-1917 2011/02 [Refereed][Not invited]
    Inactivated influenza virus vaccine prepared from a non-pathogenic influenza virus strain A/duck/Hokkaido/Vac-1/2004 (H5N1) from the virus library conferred protective immunity to chickens against the challenge of antigenically drifted highly pathogenic avian influenza virus (HPAIV), A/whooper swan/Hokkaido/1/2008 (H5N1). The efficacy of the vaccine was comparable to that prepared from genetically modified HPAIV strain Delta RRRRK rg-A/whooper swan/Mongolia/3/2005 (H5N1), which is more antigenically related to the challenge virus strain, in chickens.
  • Natsumi Takeyama, Kenji Minari, Masahiro Kajihara, Norikazu Isoda, Ryuichi Sakamoto, Takashi Sasaki, Norihide Kokumai, Noriyasu Takikawa, Rikiya Shiraishi, Masaji Mase, Junko Hagiwara, Toshiaki Kodama, Takashi Imamura, Masashi Sakaguchi, Toshiaki Ohgitani, Akira Sawata, Masatoshi Okamatsu, Masatake Muramatsu, Kenji Tsukamoto, Zhifeng Lin, Kotaro Tuchiya, Yoshihiro Sakoda, Hiroshi Kida
    VETERINARY MICROBIOLOGY 147 (3-4) 283 - 291 0378-1135 2011/01 [Refereed][Not invited]
    H5 and H7 highly pathogenic avian influenza virus (HPAIV) represent a major global concern in poultries and human health. Avian influenza (AI) vaccines are available but not preferred for field applications, primarily because vaccination interferes with sero-surveillances of AIV infection. To overcome the problem, ELISA systems using nonstructural protein 1 (NS1) of AIV as antigens (NS1-ELISA) have been developed to measure anti-NS1 antibodies that are raised in AIV-infected but not in vaccinated chickens. However, some AI-vaccinated chickens having a weak anti-virus immune response may subsequently be infected with AIV and spread the virus. This raises a concern for the validity of NS1-ELISA to detect AIV infection in previously vaccinated chickens. In this study, we developed NS1-ELISA and assessed its feasibility to detect HPAIV infection in chickens previously immunized with H5 or H7 AI vaccines. The results indicated that the NS1-ELISA could identify HPAIV infection in both unvaccinated and vaccinated chickens at 1 week after infection in correlation with results from time-consuming virus isolation tests. Taken together, the NS1-ELISA system would be valuable tool to define HPAIV infection when AI vaccine program is in place. (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 0378-1135 2011/01 [Refereed][Not invited]
    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.
  • Takeshi Tanaka, Hiromi Inui, Hiroshi Kida, Takeshi Kodama, Takuya Okamoto, Aki Takeshima, Yoshimitsu Tachi, Yoshiki Morimoto
    CHEMICAL COMMUNICATIONS 47 (10) 2949 - 2951 1359-7345 2011 [Refereed][Not invited]
    The characteristic indeno-tetrahydropyridine core of cytotoxic haouamine B (2) was efficiently synthesized featuring the diastereoselective construction of a diaryl-substituted stereogenic quaternary center by an intramolecular Pd-catalyzed alpha-C-arylation and subsequent direct conversion of the vinylogous imide function into the C2-C25 double bond by TsNHNH(2).
  • Masatoshi Okamatsu, Tomohisa Tanaka, Naoki Yamamoto, Yoshihiro Sakoda, Takashi Sasaki, Yoshimi Tsuda, Norikazu Isoda, Norihide Kokumai, Ayato Takada, Takashi Umemura, Hiroshi Kida
    VIRUS GENES 41 (3) 351 - 357 0920-8569 2010/12 [Refereed][Not invited]
    In April and May 2008, whooper swans (Cygnus cygnus) were found dead in Hokkaido in Japan. In this study, an adult whooper swan found dead beside Lake Saroma was pathologically examined and the identified H5N1 influenza virus isolates were genetically and antigenically analyzed. Pathological findings indicate that the swan died of severe congestive edema in the lungs. Phylogenetic analysis of the HA genes of the isolates revealed that they are the progeny viruses of isolates from poultry and wild birds in China, Russia, Korea, and Hong Kong. Antigenic analyses indicated that the viruses are distinguished from the H5N1 viruses isolated from wild birds and poultry before 2007. The chickens vaccinated with A/duck/Hokkaido/Vac-1/2004 (H5N1) survived for 14 days after challenge with A/whooper swan/Hokkaido/1/2008 (H5N1), although a small amount of the challenge virus was recovered from the tissues of the birds. These findings indicate that H5N1 highly pathogenic avian influenza viruses are circulating in wild birds in addition to domestic poultry in Asia and exhibit antigenic variation that may be due to vaccination.
  • Shigeru Kohno, Hiroshi Kida, Masashi Mizuguchi, Jingoro Shimada
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 54 (11) 4568 - 4574 0066-4804 2010/11 [Refereed][Not invited]
    Peramivir, a sialic acid analogue, is a selective inhibitor of neuraminidases produced by influenza A and B viruses. We evaluated the efficacy and safety of a single intravenous dose of peramivir in outpatients with uncomplicated seasonal influenza virus infection. A total of 300 previously healthy adult subjects aged 20 to 64 years with a positive influenza virus rapid antigen test were recruited within 48 h of the onset of influenza symptoms and randomized to three groups: single intravenous infusion of either 300 mg peramivir per kg of body weight, 600 mg peramivir, or matching placebo on study day 1. Influenza symptoms and body temperature were self-assessed for 14 days. Nasal and pharyngeal swabs were collected to determine the viral titer. The primary endpoint was the time to alleviation of symptoms. Of the 300 subjects, 296 were included in the intent-to-treat infected population (300 mg peramivir, n = 99; 600 mg peramivir, n = 97; and placebo, n = 100). Peramivir significantly reduced the time to alleviation of symptoms at both 300 mg (hazard ratio, 0.681) and 600 mg (hazard ratio, 0.666) compared with placebo (adjusted P value, 0.0092 for both comparisons). No serious adverse events were reported. Peramivir was well tolerated, and its adverse-event profile was similar to that of placebo. A single intravenous dose of peramivir is effective and well tolerated in subjects with uncomplicated seasonal influenza virus infection.
  • Yoshihiro Sakoda, Sengee Sugar, Damdinjav Batchluun, Tseren-Ochir Erdene-Ochir, Masatoshi Okamatsu, Norikazu Isoda, Kosuke Soda, Hiroki Takakuwa, Yoshimi Tsuda, Naoki Yamamoto, Noriko Kishida, Keita Matsuno, Eri Nakayama, Masahiro Kajihara, Ayaka Yokoyama, Ayato Takada, Ruuragchaa Sodnomdarjaa, Hiroshi Kida
    VIROLOGY 406 (1) 88 - 94 0042-6822 2010/10 [Refereed][Not invited]
    H5N1 highly pathogenic avian influenza (HPAI) viruses were isolated from dead wild waterfowl at Khunt, Erkhel, Doityn Tsagaan, Doroo, and Ganga Lakes in Mongolia in July 2005, May 2006, May 2009, July 2009, and May 2010, respectively. The isolates in 2005 and 2006 were classified into genetic clade 2.2, and those in 2009 and 2010 into clade 2.3.2. A/whooper swan/Mongolia/6/2009 (H5N1) experimentally infected ducks and replicated systemically with higher mortality than that of the isolates in 2005 and 2006. Intensive surveillance of avian influenza in migratory waterfowl flying from their nesting lakes in Siberia to Mongolia in every autumn indicate that HPAI viruses have not perpetuated at their nesting lakes until 2009. The present results demonstrate that wild waterfowl were sporadically infected with H5N1 HPAI viruses prevailing in domestic poultry in the southern Asia and died in Mongolia on the way back to their northern territory in spring. (C) 2010 Elsevier Inc. All rights reserved.
  • Atsushi Iwai, Takuya Shiozaki, Taro Kawai, Shizuo Akira, Yoshihiro Kawaoka, Ayato Takada, Hiroshi Kida, Tadaaki Miyazaki
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 (42) 32064 - 32074 0021-9258 2010/10 [Refereed][Not invited]
    Type I interferons (IFNs) are known to be critical factors in the activation of host antiviral responses and are also important in protection from influenza A virus infection. Especially, the RIG-I- and IPS-1-mediated intracellular type I IFN-inducing pathway is essential in the activation of antiviral responses in cells infected by influenza A virus. Previously, it has been reported that influenza A virus NS1 is involved in the inhibition of this pathway. We show in this report that the influenza A virus utilizes another critical inhibitory mechanism in this pathway. In fact, the viral polymerase complex exhibited an inhibitory activity on IFN beta promoter activation mediated by RIG-I and IPS-1, and this activity was not competitive with the function of NS1. Co-immunoprecipitation analysis revealed that each polymerase subunit bound to IPS-1 in mammalian cells, and each subunit inhibited the activation of IFN beta promoter by IPS-1 independently. In addition, by a combinational expression of each polymerase subunit, IPS-1-induced activation of IFN beta promoter was more efficiently inhibited by the expression of PB2 or PB2-containing complex. Moreover, the expression of PB2 inhibited the transcription of the endogenous IFN beta gene induced after influenza A virus infection. These findings demonstrate that the viral polymerase plays an important role for regulating host anti-viral response through the binding to IPS-1 and inhibition of IFN beta production.
  • Sasaki T, Kokumai N, Ohgitani T, Imamura T, Sawata A, Lin Z, Sakoda Y, Kida H
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 6 72 (6) 819 - 821 0916-7250 2010/06 [Refereed][Not invited]
  • Fei Feng, Nobuaki Miura, Norikazu Isoda, Yoshihiro Sakoda, Masatoshi Okamatsu, Hiroshi Kida, Shin-Ichiro Nishimura
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 20 (12) 3772 - 3776 0960-894X 2010/06 [Refereed][Not invited]
    We designed and synthesized novel trivalent anti-influenza reagents. Sialyllactose was located at the terminal of each valence which aimed to block each receptor-binding site of the hemagglutinin (HA) trimer on the surface of the virus. Structural analyses were carried out with a model which was constructed with a computer simulation. A previously reported cyclic glycopeptide blocker [Ohta, T.; Miura, N.; Fujitani, N.; Nakajima, F.; Niikura, K.; Sadamoto, R.; Guo, C.-T.; Suzuki, T.; Suzuki, Y.; Monde, K.; Nishimura, S.-I. Angew. Chem. Int. Ed., 2003, 42, 5186] bound to the HA in the model. The analyses suggest that the glutamine residue in the cyclic peptide bearing Neu5A alpha 2,3Gal beta 1,4Glc trisaccharide via a linker interacts with the Gln189 in HA through hydrogen bonding. The present anti-influenza reagents likely interact with a glutamine residue included in the vicinity of Gln189. A plague reduction assay of the influenza virus, A/PR/8/1934 (H1N1),was performed in MDCK cells to evaluate for the synthesized compounds to inhibit viral replication. One of the compounds showed approximately 85% inhibition at the concentration of 400 mu M at 4 degrees C. (C) 2010 Elsevier Ltd. All rights reserved.
  • Ming-Chu Cheng, Kosuke Soda, Ming-Shiuh Lee, Shu-Hwae Lee, Yoshihiro Sakoda, Hiroshi Kida, Ching-Ho Wang
    AVIAN DISEASES 54 (2) 885 - 893 0005-2086 2010/06 [Refereed][Not invited]
    During the surveillance of avian influenza, an H5N2 influenza A virus was isolated from a cloacal swab sample of an apparently healthy chicken in Taiwan in October 2008. It was found that the HA of the virus had a pair of dibasic amino acid residues at the cleavage site, which might be a marker of highly pathogenic avian influenza virus. However, the intravenous pathogenicity index of the isolate was 0.89, indicating that the virus was approaching high pathogenicity in chickens. Virus isolation was negative in 2916 birds from 146 farms in a 3-km radius around the farm where the virus was isolated. Genetic analysis of the eight segments of the isolate indicated that the isolated virus was a reassortant whose HA and NA gene segments belonged to the American lineage and internal genes to the Eurasian lineage.
  • Kida H
    Uirusu 1 60 (1) 17 - 20 0042-6857 2010/06 [Refereed][Not invited]
  • Tsunekun R, Ito H, Kida H, Otsuki K, Ito T
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 4 72 (4) 453 - 457 0916-7250 2010/04 [Refereed][Not invited]
  • Ryota Tsunekuni, Hiroshi Ito, Koichi Otsuki, Hiroshi Kida, Toshihiro Ito
    VIRUS GENES 40 (2) 252 - 255 0920-8569 2010/04 [Refereed][Not invited]
    Avirulent Newcastle disease viruses (NDV) harbored by waterfowl have the potential to become virulent after transmission to and circulation within chicken populations. In order to investigate how virulent viruses are selected from an avirulent background, we compared the complete sequences of the avirulent NDV isolate Goose/Alaska/415/91 and its virulent variant strain 9a5b, which was obtained by nine and five passages in the chick air sac and brain, respectively. Seven amino acid substitutions were detected in the M, F, and HN proteins. Two were detected between variants 9a3b and 9a5b (128P to H and 495E to K in HN protein) that were passed through the brain. Pathogenicity determined by the MDT and IVPI tests also differed between 9a3b and 9a5b. These results suggest that in addition to the F cleavage site sequence, these two amino acids in HN protein are also related to the pathogenicity of NDV in chickens.
  • Tomohisa Tanaka, Yuji Sunden, Yoshihiro Sakoda, Hiroshi Kida, Kenji Ochiai, Takashi Umemura
    JOURNAL OF NEUROVIROLOGY 16 (2) 125 - 132 1355-0284 2010/04 [Refereed][Not invited]
    Influenza virus-associated encephalopathy (IAE) is a highly mortal neural complication of influenza A virus (IAV) infection, mostly affecting children younger than 5 years old, and the brain pathology of IAE is characterized by peracute brain edema with evidence of an impaired blood-brain barrier. The pathogenesis of IAE is unknown, but hypercytokinemia of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and IL-6 is suspected of playing a central role in the development of IAE. Because the brain pathology of IAE is similar to that of septic encephalopathy due to endotoxemia, the effect of combined treatment of IAV and lipopolysaccharide (LPS) was tested using suckling mice. The results show that pulmonary infection with non-neurotropic IAV enhanced the neuropathogenicity of LPS and induced encephalopathy that was similar to IAE with respect to the occurrence of central nervous system (CNS) histopathology and the absence of direct infection of IAV in the brain. Influenza A virus also increased blood-brain barrier (BBB) permeability and induced inflammatory cytokines in the blood. These results suggested that the mice treated with IAV+LPS are possible animal models of IAE, and that hypercytokinemia and/or the involvement of endotoxemia in IAV infection are possible causes of IAE.
  • Taichiro Miyake, Kosuke Soda, Yasushi Itoh, Yoshihiro Sakoda, Hirohito Ishigaki, Tomoya Nagata, Hideaki Ishida, Misako Nakayama, Hiroichi Ozaki, Hideaki Tsuchiya, Ryuzo Torii, Hiroshi Kida, Kazumasa Ogasawara
    JOURNAL OF MEDICAL PRIMATOLOGY 39 (1) 58 - 70 0047-2565 2010/02 [Refereed][Not invited]
    Background Highly pathogenic avian influenza virus (HPAIV) infection has a high mortality rate in humans. Secondary bacterial pneumonia with HPAIV infection has not been reported in human patients, whereas seasonal influenza viruses sometimes enhance bacterial pneumonia, resulting in substantial morbidity and mortality. Therefore, if HPAIV infection were accompanied by bacterial infection, an increase in mortality would be expected. We examined whether a vaccine against HPAIV prevents severe morbidity caused by mixed infection with HPAIV and bacteria. Methods H7N7 subtype of HPAIV and Streptococcus pneumoniae were inoculated into cynomolgus macaques with or without vaccination of inactivated whole virus particles. Results Vaccination against H7N7 HPAIV decreased morbidity caused by HPAIV and pneumonia caused by S. pneumoniae. Bacterial replication in lungs was decreased by vaccination against HPAIV, although the reduction in bacterial colonies was not significant. Conclusions Vaccination against HPAIV reduces pneumonia caused by bacterial superinfection and may improve prognosis of HPAIV-infected patients.
  • Yasushi Itoh, Hiroichi Ozaki, Hirohito Ishigaki, Yoshihiro Sakoda, Tomoya Nagata, Kosuke Soda, Norikazu Isoda, Taichiro Miyake, Hideaki Ishida, Kiyoko Okamoto, Misako Nakayama, Hideaki Tsuchiya, Ryuzo Torii, Hiroshi Kida, Kazumasa Ogasawara
    VACCINE 28 (3) 780 - 789 0264-410X 2010/01 [Refereed][Not invited]
    Development of H7N7 highly pathogenic avian influenza virus (HPAIV) vaccines is an urgent issue since human cases of infection with this subtype virus have been reported and most humans have no immunity against H7N7 viruses. We made an H7N7 vaccine combining components from an influenza virus library of non-pathogenic type A influenza viruses Antibody and T cell recall responses specific against the vaccine strain were elicited by subcutaneous inoculation with the whole virus particle vaccine with or without alum as an adjuvant in cynomolgus macaques. No significant difference was observed in magnitude of antibody responses between vaccination with alum and vaccination without alum, though vaccination with alum induced longer recall responses of CD8(+) T cells than did vaccination without alum. After challenge with a subtype of H7N7 HPAIV, the virus was detected in nasal swabs of unvaccinated macaques for 8 days but only for I day in the animals vaccinated either with or without alum, although the macaques vaccinated with alum showed elevated body temperature more briefly after infection. These findings demonstrated that this H7N7 HPAIV strain is pathogenic to macaques and that the vaccine conferred protective immunity to macaques against H7N7 HPAIV infection. (C) 2009 Elsevier Ltd. All rights reserved.
  • Manabu Igarashi, Kimihito Ito, Reiko Yoshida, Daisuke Tomabechi, Hiroshi Kida, Ayato Takada
    PLOS ONE 5 (1) e8553  1932-6203 2010/01 [Refereed][Not invited]
    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.
  • Yoshitaka Kashima, Mizuho Ikeda, Yasushi Itoh, Yoshihiro Sakoda, Tomoya Nagata, Taichiro Miyake, Kosuke Soda, Hiroichi Ozaki, Misako Nakayama, Hitomi Shibuya, Masatoshi Okamatsu, Hirohito Ishigaki, Hideaki Ishida, Toshihiro Sawai, Yoshihiro Kawaoka, Hiroshi Kida, Kazumasa Ogasawar
    VACCINE 27 (52) 7402 - 7408 0264-410X 2009/12 [Refereed][Not invited]
    Outbreaks of highly pathogenic avian influenza viruses (HPAIVs) would cause disasters worldwide. Various strategies against HPAIVs are required to control damage. it is thought that the use of non-pathogenic avian influenza viruses as live vaccines will be effective in an emergency, even though there might be some adverse effects, because small amounts of live vaccines will confer immunity to protect against HPAIV infection. Therefore, live vaccines have the advantage of being able to be distributed worldwide soon after an outbreak. In the present study, we found that intranasal administration of a live H5N1 subtype non-pathogenic virus induced antibody and cytotoxic T lymphocyte responses and protected mice against H5N1 HPAIV infection. In addition, it was found that a small amount (100 PFU) of the live vaccine was as effective as 100 mu g (approximately 10(10-11) PFU of virus particles)of the inactivated whole particle vaccine in mice. Consequently, the use of live virus vaccines might be one strategy for preventing pandemics of HPAIVs in an emergency. (C) 2009 Elsevier Ltd. All rights reserved.
  • Shin JH, Sakoda Y, Yano S, Ochiai K, Kida H, Umemura T
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 10 71 (10) 1331 - 1336 0916-7250 2009/10 [Refereed][Not invited]
  • Edgar Simulundu, Aaron S. Mweene, Daisuke Tomabechi, Bernard M. Hang'ombe, Akihiro Ishii, Yuka Suzuki, Ichiro Nakamura, Hirofumi Sawa, Chihiro Sugimoto, Kimihito Ito, Hiroshi Kida, Lewis Saiwana, Ayato Takada
    ARCHIVES OF VIROLOGY 154 (9) 1517 - 1522 0304-8608 2009/09 [Refereed][Not invited]
    We characterized an influenza virus isolated from a great white pelican in Zambia. Phylogenetic analysis showed that all of its gene segments belonged to the Eurasian lineage and that they appear to have evolved in distinct geographical regions in Europe, Asia, and Africa, suggesting reassortment of virus genes maintained in wild aquatic birds whose flyways overlap across these continents. It is notable that this virus might possess some genes of the same origin as those of highly pathogenic H7 and H5 viruses isolated in Eurasia. The present study underscores the need for continued monitoring of avian influenza viruses in Eurasia and Africa.
  • Takashi Sasaki, Norihide Kokumai, Toshiaki Ohgitani, Ryuichi Sakamoto, Noriyasu Takikawa, Zhifeng Lin, Masatoshi Okamatsu, Yoshihiro Sakoda, Hiroshi Kida
    VACCINE 27 (38) 5174 - 5177 0264-410X 2009/08 [Refereed][Not invited]
    An influenza vaccine was prepared from inactivated whole particles of the non-pathogenic strain A/duck/Hokkaido/Vac-1/04 (H5N1) virus using an oil adjuvant containing anhydromannitol-octaclecenoate-ether (AMOE). The vaccine was injected intramuscularly into five 4-week-old chickens, and 138 weeks after vaccination, they were challenged intranasally with 100 times 50% chicken lethal dose of the highly pathogenic avian influenza (HPAI) virus A/chicken/Yamaguchi/7/04 (H5N1). All 5 chickens survived without exhibiting clinical signs of influenza, although 2 days post-challenge, 3 vaccinated chickens shed limited titres of viruses in laryngopharyngeal swabs. (C) 2009 Elsevier Ltd. All rights reserved.
  • Yasushi Itoh, Kyoko Shinya, Maki Kiso, Tokiko Watanabe, Yoshihiro Sakoda, Masato Hatta, Yukiko Muramoto, Daisuke Tamura, Yuko Sakai-Tagawa, Takeshi Noda, Saori Sakabe, Masaki Imai, Yasuko Hatta, Shinji Watanabe, Chengjun Li, Shinya Yamada, Ken Fujii, Shin Murakami, Hirotaka Imai, Satoshi Kakugawa, Mutsumi Ito, Ryo Takano, Kiyoko Iwatsuki-Horimoto, Masayuki Shimojima, Taisuke Horimoto, Hideo Goto, Kei Takahashi, Akiko Makino, Hirohito Ishigaki, Misako Nakayama, Masatoshi Okamatsu, Kazuo Takahashi, David Warshauer, Peter A. Shult, Reiko Saito, Hiroshi Suzuki, Yousuke Furuta, Makoto Yamashita, Keiko Mitamura, Kunio Nakano, Morio Nakamura, Rebecca Brockman-Schneider, Hiroshi Mitamura, Masahiko Yamazaki, Norio Sugaya, M. Suresh, Makoto Ozawa, Gabriele Neumann, James Gern, Hiroshi Kida, Kazumasa Ogasawara, Yoshihiro Kawaoka
    NATURE 460 (7258) 1021 - U110 0028-0836 2009/08 [Refereed][Not invited]
    Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses(1). Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 ( H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S- OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S- OIV pandemic.
  • Kanako Moritoh, Hideto Yamauchi, Atsushi Asano, Kentaro Yoshii, Hiroaki Kariwa, Ikuo Takashima, Norikazu Isoda, Yoshihiro Sakoda, Hiroshi Kida, Nobuya Sasaki, Takashi Agui
    JAPANESE JOURNAL OF VETERINARY RESEARCH 57 (2) 89 - 99 0047-1917 2009/08 [Refereed][Not invited]
    Mx1 (Myxovirus resistance protein) and Oas1b (Oligoadenylate synthetase-1), induced by type 1 interferon (IFN), play a role in early antiviral innate immunity by inhibiting the replication of viruses. In mice, Mx1 and Oas1b confer resistance to the infection of orthomyxoviruses including influenza viruses and flaviviruses including West Nile viruses, respectively. Laboratory mice have been used to study the mechanisms of the pathogenesis of these virus infections; however, it is possible that they are not a suitable model system to study these viruses, since most of the inbred laboratory mouse strains lack both genes. It has been reported that feral mouse-derived inbred strains show resistance to the infection of these viruses due to the presence of intact both genes. In this study, we generated congenic strains in which the Mx or Oas locus of the MSM/Ms (MSAI) mouce was introduced to the most widely used mouse strain, C57BL/6J (136). B6.MSM-Mx mice showed resistance to the infection of influenza virus but not of West Nile virus. On the other hand, B6.MSM-Oas mice showed resistance to the infection of West Nile virus but not of influenza virus. Our results indicate that Mx1 and Oas1b show highly antiviral specificity in mice possessing the same genetic background. Therefore, these congenic mice are useful for not only infection study but also investigation of host defense mechanism to these viruses.
  • Mayumi Suzuki, Isao Tachibana, Yoshito Takeda, Ping He, Seigo Minami, Takeo Iwasaki, Hiroshi Kida, Sho Goya, Takashi Kijima, Mitsuhiro Yoshida, Toru Kumagai, Tadashi Osaki, Ichiro Kawase
    JOURNAL OF IMMUNOLOGY 182 (10) 6485 - 6493 0022-1767 2009/05 [Refereed][Not invited]
    Tetraspanins facilitate the formation of multiple molecular complexes at specialized membrane microdomains and regulate cell activation and motility. In the present study, the role of tetraspanin CD9 in LPS-induced macrophage activation and lung inflammation was investigated in vitro and in vivo. When CD9 function was ablated with mAb treatment, small interfering RNA transfection, or gene knockout in RAW264.7 cells or bone marrow-derived macrophages, these macrophages produced larger amounts of TNF-alpha, matrix metalloproteinase-2, and -9 upon stimulation with LPS in vitro, when compared with control cells. Sucrose gradient analysis revealed that CD9 partly colocalized with the LPS-induced signaling mediator, CD14, at low-density light membrane fractions. In CD9 knockout macrophages, CD14 expression, CD14 and TLR4 localization into the lipid raft, and their complex formation were increased whereas I kappa B alpha expression was decreased when compared with wild-type cells, suggesting that CD9 prevents the formation of LPS receptor complex. Finally, deletion of CD9 in mice enhanced macrophage infiltration and TNF-alpha production in the lung after intranasal administration of LPS in vivo, when compared with wild-type mice. These results suggest that macrophage CD9 negatively regulates LPS response at lipid-enriched membrane microdomains. The Journal of Immunology, 2009, 182: 6485-6493.
  • Yoshimi Tsuda, Norikazu Isoda, Yoshihiro Sakoda, Hiroshi Kida
    VIRUS RESEARCH 140 (1-2) 194 - 198 0168-1702 2009/03 [Refereed][Not invited]
    Many influenza A viruses form plaques on Madin-Darby canine kidney (MDCK) cells in the presence of trypsin. A/duck/Siberia/272/1998 (H13N6) (Sib272), however, does not form plaque on MDCK cells. After three blind passages of the strain on MDCK cells, plaque-forming variant was obtained and designated as A/duck/Siberia/272PF/1998 (H13N6) (Sib272PF). Genetic and functional analyses of Sib272 and Sib272PF revealed that amino acid substitutions, F3L of the HA2 subunit and T379K of the PB1, were responsible for plaque formation of Sib272PF by enhancing fusion and polymerase activities, respectively. (c) 2009 Elsevier B.V. All rights reserved.
  • Reiko Yoshida, Manabu Igarashi, Hiroichi Ozaki, Noriko Kishida, Daisuke Tomabechi, Hiroshi Kida, Kimihito Ito, Ayato Takada
    PLOS PATHOGENS 5 (3) e1000350  1553-7366 2009/03 [Refereed][Not invited]
    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.
  • Rashid Manzoor, Yoshihiro Sakoda, Naoki Nomura, Yoshimi Tsuda, Hiroichi Ozaki, Masatoshi Okamatsu, Hiroshi Kida
    JOURNAL OF VIROLOGY 83 (4) 1572 - 1578 0022-538X 2009/02 [Refereed][Not invited]
    It has been shown that not all but most of the avian influenza viruses replicate in the upper respiratory tract of pigs (H. Kida et al., J. Gen. Virol. 75: 2183-2188, 1994). It was shown that A/chicken/Yamaguchi/7/2004 (H5N1) [Ck/Yamaguchi/04 (H5N1)] did not replicate in pigs (N. Isoda et al., Arch. Virol. 151: 1267-1279, 2006). In the present study, the genetic basis for this host range restriction was determined using reassortant viruses generated between Ck/Yamaguchi/04 (H5N1) and A/swine/Hokkaido/2/1981 (H1N1) [Sw/Hokkaido/81 (H1N1)]. Two in vivo-generated single-gene reassortant virus clones of the H5N1 subtype (virus clones 1 and 2), whose PB2 gene was of Sw/Hokkaido/81 (H1N1) origin and whose remaining seven genes were of Ck/Yamaguchi/04 (H5N1) origin, were recovered from the experimentally infected pigs. The replicative potential of virus clones 1 and 2 was further confirmed by using reassortant virus (rg-Ck-Sw/PB2) generated by reverse genetics. Interestingly, the PB2 gene of Ck/Yamaguchi/04 (H5N1) did not restrict the replication of Sw/Hokkaido/81 (H1N1), as determined by using reassortant virus rg-Sw-Ck/PB2. The rg-Sw-Ck/PB2 virus replicated to moderate levels and for a shorter duration than parental Sw/Hokkaido/81 (H1N1). Sequencing of two isolates recovered from the pigs inoculated with rg-Sw-Ck/PB2 revealed either the D256G or the E627K amino acid substitution in the PB2 proteins of the isolates. The D256G and E627K mutations enhanced viral polymerase activity in the mammalian cells, correlating with replication of virus in pigs. These results indicate that the PB2 protein restricts the growth of Ck/Yamaguchi/04 (H5N1) in pigs.
  • Takashi Sasaki, Norikazu Isoda, Kosuke Soda, Ryuichi Sakamoto, Kazue Saijo, Junko Hagiwara, Norihide Kokumai, Toshiaki Ohgitani, Takashi Imamura, Akira Sawata, Zhifeng Lin, Yoshihiro Sakoda, Hiroshi Kida
    JAPANESE JOURNAL OF VETERINARY RESEARCH 56 (4) 189 - 198 0047-1917 2009/02 [Refereed][Not invited]
    Test vaccines comprised of inactivated water-in-oil emulsions containing various antigen levels were prepared using a non-pathogenic H5N1 avian influenza (AI) virus, A/duck/Hokkaido/Vac-1/04 (H5N1). The potencies of these test vaccines were evaluated by two experiments. In the first experiment, the triangular relationship among the antigen levels of test vaccines, the hemagglutination inhibition (HI) antibody response, and the protective effect against challenge with a highly pathogenic avian influenza (HPAI) virus, A/chicken/Yamaguchi/7/04 (H5N1), was confirmed. Then lasting immunity of chickens after a single-shot vaccination was confirmed in the second experiment. As a result, complete protection after the challenge was observed in chickens immunized by test vaccines with an antigen level of 160 HA units/dose or higher. Thus, it was ascertained that the minimum antigen level in the AI vaccine was 160 RA units/dose, and the minimum HI antibody titer that could protect chickens from HPAI virus infection-related death was considered to be 1:16. Dose-dependent III antibody responses were observed in chickens after the vaccination. Thus, 640 RA units/dose was thought to be similar to the optimal antigen level. Alternatively, the HI antibody titers of chickens, injected with the vaccine containing 640 HA units/dose, were maintained at 1:181 or higher for 100 weeks after the single-shot vaccination.
  • Masatoshi Okamatsu, Yoshihiro Sakoda, Noriko Kishida, Norikazu Isoda, Hiroshi Kida
    ARCHIVES OF VIROLOGY 153 (12) 2189 - 2195 0304-8608 2008/12 [Refereed][Not invited]
    The hemagglutinins (HAs) of H9 influenza viruses isolated from birds and mammals of different species were antigenically and genetically analyzed. Antigenic variants were selected from A/swine/Hong Kong/10/98 (H9N2) and A/duck/Hokkaido/13/00 (H9N2) in the presence of monoclonal antibodies (MAbs). Based on the reactivity patterns of these mutants with a panel of MAbs, at least five non-overlapping antigenic sites were defined using eight MAbs which recognized seven distinct epitopes on the H9 HA molecule. Based on the reactivity patterns with the panel of monoclonal antibodies, 21 H9N2 virus strains isolated from birds and mammals were divided into 7 antigenically distinct groups. The present findings indicate that it is important to monitor the antigenic variation in H9 influenza viruses. The panel of MAbs in the present study, thus, should be useful for detailed antigenic analysis of the H9 HAs for epidemiological studies, the selection of vaccine strains, and diagnosis.
  • Hiroshi Kida, Yuri Sugano, Ryo Iizuka, Masahiro Fujihashi, Masafurni Yohda, Kunio Miki
    JOURNAL OF MOLECULAR BIOLOGY 383 (3) 465 - 474 0022-2836 2008/11 [Refereed][Not invited]
    Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of alpha and beta subunits and forms a "jellyfish-like" structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two alpha and two beta subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of a (alpha 1 and alpha 2) and beta subunits (beta 1 and beta 2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD beta 1 subunit at 1.9 angstrom resolution and its functional analysis. TsPFD beta 1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The beta hairpin linkers of beta 1 subunits assemble to form a beta barrel "body" around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the beta 1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric beta 1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD beta 1 subunits act as molecular chaperones in living cells of some archaea. (C) 2008 Elsevier Ltd. All rights reserved.
  • Shin Murakami, Ayaka Iwasa, Kiyoko Iwatsuki-Horimoto, Mutsumi Ito, Maki Kiso, Hiroshi Kida, Ayato Takada, Chairul A. Nidom, Le Quynh Mai, Shinya Yamada, Hirotaka Imai, Yuko Sakai-Tagawa, Yoshihiro Kawaoka, Taisuke Horimoto
    VACCINE 26 (50) 6398 - 6404 0264-410X 2008/11 [Refereed][Not invited]
    H5N1 highly pathogenic avian influenza viruses evolved into several clades, leading to appreciably distinct antigenicities of their hemagglutinins. As such, candidate H5N1 pre-pandemic vaccines for human use should be sought. Here, to evaluate fundamental immunogenic variations between H5N1 vaccines, we prepared four inactivated H5N1 test vaccines from different phylogenetic Glades (clade 1, 2.1, 2.2, and 2.3.4) in accordance with the WHO recommendation, and tested their cross-Glade immunity in a mouse model by vaccination followed by challenge with heterologous virulent viruses. All H5N1 vaccines tested provided full or partial cross-clade protective immunity, except one Glade 2.2-based vaccine, which did not protect mice from clade 2.3.4 virus challenge. Among the test vaccines, a clade 2.1-based vaccine possessed the broadest-spectrum cross-immunity. These results suggest that currently stockpiled pre-pandemic vaccines, especially clade 2.1-based vaccines, will likely be useful as backup vaccines in a pandemic situation, even one involving antigenic-drifted viruses. (c) 2008 Elsevier Ltd. All rights reserved.
  • Kosuke Soda, Hiroichi Ozaki, Yoshihiro Sakoda, Norikazu Isoda, Yoshinari Haraguchi, Saori Sakabe, Noritaka Kuboki, Noriko Kishida, Ayato Takada, Hiroshi Kida
    ARCHIVES OF VIROLOGY 153 (11) 2041 - 2048 0304-8608 2008/11 [Refereed][Not invited]
    In order to prepare H5N1 influenza virus vaccine, the hemagglutinins (HAs) of 14 H5 virus isolates from water birds in Asia were antigenically and genetically analyzed. Phylogenetic analysis of the H5 HA genes revealed that 13 isolates belong to Eurasian and the other one to North American lineages. Each of the deduced amino acid sequences of the HAs indicated a non-pathogenic profile. Antigenic analysis using a panel of monoclonal antibodies recognizing six different epitopes on the HA of A/duck/Pennsylvania/10218/1984 (H5N2) and chicken antiserum to an H5N1 reassortant strain generated between A/duck/Mongolia/54/2001 (H5N2) and A/duck/Mongolia/47/2001 (H7N1), [R(Dk/Mong-Dk/Mong) (H5N1)] showed that the HAs of highly pathogenic avian influenza (HPAI) viruses currently circulating in Asia were antigenically closely related to those of the present isolates from water birds. Mice subcutaneously injected with formalin-inactivated R(Dk/Mong-Dk/Mong) were protected from challenge with 100 mouse lethal dose of A/Viet Nam/1194/2004 (H5N1). The present results support the notion that the H5 isolates and the reassortant H5N1 strain should be useful for vaccine preparation.
  • Rashid Manzoor, Yoshihiro Sakoda, Aaron Mweene, Yoshimi Tsuda, Noriko Kishida, Gui-Rong Bai, Ken-Ichiro Kameyama, Norikazu Isoda, Kosuke Soda, Michiko Naito, Hiroshi Kida
    VIRUS GENES 37 (2) 144 - 152 0920-8569 2008/10 [Refereed][Not invited]
    During 2000-2007, 218 influenza viruses of 28 different combinations of HA (H1-H13) and NA (N1-N9) subtypes were isolated from fecal samples of free-flying water birds at two distant lakes in Hokkaido, Japan. Phylogenic analysis of the matrix (M) genes of 67 strains, selected on the basis of their subtype combinations, revealed that A/duck/Hokkaido/W95/2006 (H10N8) was a reassortant whose M gene belonged to North American non-gull-avian and the other seven genes to Eurasian non-gull-avian lineages. The M genes of other 65 strains belonged to Eurasian non-gull-avian and the one to Eurasian-gull lineages. The M genes of 65 strains were grouped into three different sublineages, indicating that influenza viruses circulating in different populations of free-flying water birds have evolved independently in nature.
  • Shiroh Miura, Hiroki Shibata, Hiroshi Kida, Kazuhito Noda, Katsuro Tomiyasu, Ken Yamamoto, Akiko Iwaki, Mitsuyoshi Ayabe, Hisarnichi Aizawa, Takayuki Taniwaki, Yasuyuki Fukumaki
    JOURNAL OF THE NEUROLOGICAL SCIENCES 273 (1-2) 88 - 92 0022-510X 2008/10 [Refereed][Not invited]
    We Studied a four-generation pedigree of a Japanese family with hereditary neuropathy to elucidate the genetic basis of this disease. Twelve members of the family were enrolled in this study. The clinical features were neurogenic muscle weakness with proximal dominancy in the lower extremities, sensory involvement, areflexia, fine postural tremors, painful Muscle cramps, elevated creatine kinase levels, recurrent paroxysmal dry cough, and neurogenic bladder. We performed a genome-wide search using genetic loci spaced at about 13 Mb intervals. Although nine chromosomes (1, 3, 4, 5, 6, 10, 17, 19, and 22) had at least one region in which the logarithm of odds (LOD) Score was over 1.0, no loci fulfilled the Criteria for significant evidence of linkage. Moreover, we analyzed an extra 14 markers on 3p12-q13 (the locus of hereditary Motor and sensory neuropathy, proximal dominant form) and an extra five markers on 3p22-p24 (the locus of hereditary sensory neuropathy with chronic cough) and observed LOD scores of <-3 on both 3p12-q13 and 3p22-p24. Mutation scanning of the entire coding regions of the MPZ and PMP22 genes revealed no mutations. We conclude that the disorder described here is a newly classified hereditary motor and sensory neuropathy with autosomal dominant inheritance. (c) 2008 Elsevier B.V. All rights reserved.
  • Norikazu Isoda, Yoshihiro Sakoda, Noriko Kishida, Kosuke Soda, Saori Sakabe, Ryuichi Sakamoto, Takashi Imamura, Masashi Sakaguchi, Takashi Sasaki, Norihide Kokumai, Toshiaki Ohgitani, Kazue Saijo, Akira Sawata, Junko Hagiwara, Zhifeng Lin, Hiroshi Kida
    ARCHIVES OF VIROLOGY 153 (9) 1685 - 1692 0304-8608 2008/09 [Refereed][Not invited]
    A reassortant influenza virus, A/duck/Hokkaido/Vac-1/2004 (H5N1) (Dk/Vac-1/04), was generated between non-pathogenic avian influenza viruses isolated from migratory ducks in Asia. Dk/Vac-1/04 (H5N1) virus particles propagated in embryonated chicken eggs were inactivated with formalin and adjuvanted with mineral oil to form a water-in-oil emulsion. The resulting vaccine was injected intramuscularly into chickens. The chickens were challenged with either of the highly pathogenic avian influenza virus strains A/chicken/Yamaguchi/7/2004 (H5N1) or A/swan/Mongolia/3/2005 (H5N1) at 21 days post-vaccination (p. v.), when the geometric mean serum HI titers of the birds was 64 with the challenge virus strains. The vaccinated chickens were protected from manifestation of disease signs upon challenge with either of the highly pathogenic avian influenza viruses. However, challenge virus was recovered at low titers from the birds at 2 and 4 days post-challenge (p.c.). All 3 chickens challenged at 6 days p.v. died, whereas 3 chickens challenged at 8 days p.v. survived. These results indicate that the present vaccine confers clinical protection and reduction of virus shedding against highly pathogenic avian influenza virus challenge and should be useful as an optional tool in emergency cases.
  • Ito M, Nagai M, Hayakawa Y, Komae H, Murakami N, Yotsuya S, Asakura S, Sakoda Y, Kida H
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 9 70 (9) 899 - 906 0916-7250 2008/09 [Refereed][Not invited]
  • Noriko Kishida, Yoshihiro Sakoda, Mai Shiromoto, Gui-Rong Bai, Norikazu Isoda, Ayato Takada, Graeme Laver, Hiroshi Kida
    VIRUS GENES 37 (1) 16 - 21 0920-8569 2008/08 [Refereed][Not invited]
    To investigate the prevalence of influenza viruses in feral water birds in the Southern Hemisphere, fecal samples of terns were collected on Heron Island, Australia, in December 2004. Six H2N5 influenza viruses were isolated. This is the first report of the isolation of the H2 subtype from shore birds in Australia. Phylogenetic analysis revealed that the M gene belonged to the American lineage of avian influenza viruses and the other genes belonged to the Eurasian lineages, indicating that genetic reassortment occurs between viruses of Eurasian and American lineages in free flying birds in nature.
  • Manabu Igarashi, Kimihito Ito, Hiroshi Kida, Ayato Takada
    VIROLOGY 376 (2) 323 - 329 0042-6822 2008/07 [Refereed][Not invited]
    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.
  • Toshihiro Sawai, Yasushi Itoh, Hiroichi Ozaki, Norikazu Isoda, Kiyoko Okamoto, Yoshitaka Kashima, Yoshihiro Kawaoka, Yoshihiro Takeuchi, Hiroshi Kida, Kazumasa Ogasawara
    IMMUNOLOGY 124 (2) 155 - 165 0019-2805 2008/06 [Refereed][Not invited]
    We investigated whether a vaccine derived from an apathogenic reassortant type A H5N1 influenza strain could induce immune responses in vivo that mediated protection from highly pathogenic avian influenza virus infection in mice. After two subcutaneous immunizations with formalin-inactivated H5N1 whole virus particles (whole particle vaccine), significant killing specific for cells presenting a nucleoprotein peptide from the vaccine strain of the virus was observed. Similar vaccination with viruses treated with ether and formalin, which are commonly used for humans as ether-split vaccines, induced little or no cytotoxic T-cell response. Furthermore, whole particle vaccines of the apathogenic H5N1 strain were more effective than ether-split vaccines at inducing antibody production able to neutralize a highly pathogenic H5N1 strain. Finally, whole particle vaccines of H5N1 protected mice against infection by an H5N1 highly pathogenic avian influenza virus more effectively than did ether-split vaccines. These results suggest that formalin-inactivated virus particles of apathogenic strains are effective for induction of both cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza viruses in vivo, resulting in protection from infection by a highly pathogenic H5N1 virus.
  • Manzoor R, Sakoda Y, Sakabe S, Mochizuki T, Namba Y, Tsuda Y, Kida H
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 6 70 (6) 557 - 562 0916-7250 2008/06 [Refereed][Not invited]
  • Ken-ichiro Kameyama, Yoshihiro Sakoda, Keita Matsuno, Asako Ito, Motoshi Tajima, Shigeyuki Nakamura, Hiroshi Kida
    MICROBIOLOGY AND IMMUNOLOGY 52 (5) 277 - 282 0385-5600 2008/05 [Refereed][Not invited]
    The NS2-3 of BVDV is cleaved in cultured cells infected with cp BVDV but not in those infected with ncp BVDV when tested more than 10 hours post infection. However, it is not known whether cleavage of NS2-3 occurs in vivo. In the present study, cleavage of NS2-3 in cattle persistently infected with BVDV was investigated. All BVDV isolated from PI animals were of the ncp biotype, and NS2-3 proteins were detected in bovine fetal muscular cells infected with these viruses. On the other hand, in the leukocytes of those PI animals, NS3 proteins, products of the cleavage of NS2-3 proteins, were detected. In addition, the NS3 proteins were also detected in leukocytes artificially infected with ncp BVDV. These results reveal that the NS2-3 protein of BVDV is cleaved in leukocytes. Furthermore, NS3 proteins were detected in many tissues of PI cattle, such as lymphoid tissue, brain, thyroid, lung, and kidney. These results suggest that the NS2-3 protein of ncp BVDV cleaves in vivo.
  • Saori Sakabe, Yoshihiro Sakoda, Yoshinari Haraguchi, Norikazu Isoda, Kosuke Soda, Hiroki Takakuwa, Kazue Saijo, Akira Sawata, Katsumi Kume, Junko Hagiwara, Kotaro Tuchiya, Zhifeng Lin, Ryuichi Sakamoto, Takashi Imamura, Takashi Sasaki, Norihide Kokumai, Yoshihiro Kawaoka, Hiroshi Kida
    VACCINE 26 (17) 2127 - 2134 0264-410X 2008/04 [Refereed][Not invited]
    During 2001-2004, 41 H7 influenza viruses (2 H7N1 and 39 H7N7 strains) were isolated from fecal samples of migratory ducks that flew from Siberia in the autumn of each year to Japan and Mongolia. A phylogenetic analysis of the hemagglutinin (HA) genes of the nine representative isolates revealed that they belonged to the Eurasian Lineage and the deduced amino acid sequence at the cleavage site of the HAs represented apathogenic profiles. One of the H7 isolates A/duck/Mongolia/736/02 (H7N7) was chosen from these H7 isolates for the preparation of the test vaccine. To improve the growth potential of A/duck/Mongolia/ 736/02 (H7N7) in chicken embryos, A/duck/Hokkaido/Vac-2/04 (H7N7) was generated by genetic reassortment between A/duck/Mongolia/736/02 (H7N7) as the donor of the PB2, PB1, PA, HA, NA, and NS genes and A/duck/Hokkaido/49/98 (H9N2) as that of NP and M genes. The test vaccine was prepared as follows; A/duck/Hokkaido/Vac-2/04 (H7N7) was propagated in chicken embryos and the virus in the allantoic fluid was inactivated and adjuvanted to form an oil-in-water emulsion. The test vaccine conferred immunity to chickens, completely protecting the manifestation of clinical signs against the challenge with lethal dose of H7 highly pathogenic avian influenza virus. These results indicate that influenza viruses isolated from natural reservoirs are useful for vaccine strains. (C) 2008 Elsevier Ltd. All rights reserved.
  • Makoto Nagai, Michiko Hayashi, Mika Itou, Toyoko Fukutomi, Hiroomi Akashi, Hiroshi Kida, Yoshihiro Sakoda
    VIRUS GENES 36 (1) 135 - 139 0920-8569 2008/02 [Refereed][Not invited]
    A part of the nucleotide sequence of the 5' untranslated region (5'UTR) and E-rns region, and the genomic regions encoding for N-pro, Core, and E2 of So-like isolates and IS25CP/01 strain which belong to bovine viral diarrhea virus genotype 1 (BVDV-1) were determined and genetic comparisons were made with sequences of other BVDV subgenotypes. Phylogenetic analysis using the 5'UTR and N-pro revealed that So-like isolates and IS25CP/01 branched into independent phylogenetic branch. So-like isolates were clustered with Korean BVDV strains taken from DDBL/EMBL/GenBank in the 5'UTR. An additional two amino acid residues were found at the C terminal of the Core region of IS25CP/01. The similarity of amino acid sequence of E2 of So-like isolates and IS25CP/01 to the BVDV-1 reference strain NADL were 78.0-78.5 and 79.0, respectively. Cross-neutralization tests showed significant antigenic differences between So-like isolates and the others (Antigenic similarity (R) value: 2.2-8.8), and IS25CP/01 and the others (R value: 1.6-8.8). So-like viruses and IS25CP/01 differed from the thirteenth subgenotypes (1a-1m) reported by Jackova et al. (2007) and were classified as new genetic subtypes, BVDV-1n (So-like) and 1o (IS25CP/01).
  • Akashi Ohtaki, Hiroshi Kida, Yusuke Miyata, Naoki Ide, Akihiro Yonezawa, Takatoshi Arakawa, Ryo Iizuka, Keiichi Noguchi, Akiko Kita, Masafumi Odaka, Kunio Miki, Masafumi Yohda
    JOURNAL OF MOLECULAR BIOLOGY 376 (4) 1130 - 1141 0022-2836 2008/02 [Refereed][Not invited]
    Prefoldin (PFD) is a heterohexameric molecular chaperone complex in the eukaryotic cytosol and archaea with a jellyfish-like structure containing six long coiled-coil tentacles. PFDs capture protein folding intermediates or unfolded polypeptides and transfer them to group II chaperonins for facilitated folding. Although detailed studies on the mechanisms for interaction with unfolded proteins or cooperation with chaperonins of archaeal PFD have been performed, it is still unclear how PFD captures the unfolded protein. In this study, we determined the X-ray structure of Pyrococcus horikoshii OT3 PFD (PhPFD) at 3.0 angstrom resolution and examined the molecular mechanism for binding and recognition of nonnative substrate proteins by molecular dynamics (MD) simulation and mutation analyses. PhPFD has a jellyfish-like structure with six long coiled-coil tentacles and a large central cavity. Each subunit has a hydrophobic groove at the distal region where an unfolded substrate protein is bound. During MD simulation at 330 K, each coiled coil was highly flexible, enabling it to widen its central cavity and capture various normative proteins. Docking MD simulation of PhPFD with unfolded insulin showed that the beta subunit is essentially involved in substrate binding and that the alpha subunit modulates the shape and width of the central cavity. Analyses of mutant PhPFDs with amino acid replacement of the hydrophobic residues of the P subunit in the hydrophobic groove have shown that beta Ile107 has a critical role in forming the hydrophobic groove. (C) 2007 Elsevier Ltd. All rights reserved.
  • Yasushi Itoh, Hiroichi Ozaki, Hideaki Tsuchiya, Kiyoko Okamoto, Ryuzo Torii, Yoshihiro Sakoda, Yoshihiro Kawaoka, Kazumasa Ogasawara, Hiroshi Kida
    Vaccine 26 (4) 562 - 572 0264-410X 2008/01/24 [Refereed][Not invited]
    In order to prepare for the emergence of pandemic influenza viruses, we have established an influenza virus library that contains non-pathogenic influenza A virus strains with 135 combinations of 15 hemagglutinin and 9 neuraminidase subtypes. In this study, we developed a vaccine against H5N1 highly pathogenic avian influenza (HPAI) virus infection in humans using a virus strain selected from the library. We examined its immunogenic potency using cynomolgus macaques as a primate model. Virus antigen-specific antibodies were elicited by intranasal or subcutaneous administration of inactivated whole virus particle vaccines. After challenge with an H5N1 HPAI virus isolate obtained from a Vietnamese patient, the virus was detected only on next day following inoculation in the nasal and/or tracheal swabs of vaccinated macaques that were asymptomatic. On the other hand, the viruses were isolated from nasal and tracheal swabs from non-vaccinated macaques until day 5 and day 7 after inoculation of the H5N1 HPAI virus, respectively. Although six non-vaccinated macaques developed a high body temperature, and two of them lost their appetite after HPAI virus infection, they recovered by the end of the 12-day observation period and did not show the severe symptoms that have been reported in human H5N1 virus infection cases. This demonstrates that the vaccine prepared with the non-pathogenic H5N1 virus from our influenza virus library conferred protective immunity against H5N1 HPAI virus infection to macaques. © 2007 Elsevier Ltd. All rights reserved.
  • Kosuke Soda, Yoshihiro Sakoda, Norikazu Isoda, Masahiro Kajihara, Yoshinari Haraguchi, Hitomi Shibuya, Hiromi Yoshida, Takashi Sasaki, Ryuichi Sakamoto, Kazue Saijo, Junko Hagiwara, Hiroshi Kida
    JAPANESE JOURNAL OF VETERINARY RESEARCH 55 (2-3) 93 - 98 0047-1917 2008/01 [Refereed][Not invited]
    To establish vaccine strains of H5 and H7 influenza viruses, A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokkaido/Vac-3/07 (H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/ Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] were generated from non-pathogenic avian influenza viruses isolated from migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1 viruses as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant obtained using H7N7 and H9 N2 viruses to give high growth character of the H9N2 virus in chicken embryonated eggs. The results of sequence analyses and experimental infections revealed that these H5N1 and H7N7 reassortant viruses were non-pathogenic in chickens and embryos, and had good growth potential in embryonated eggs. These viruses should be useful to develop vaccines against H5 and H7 highly pathogenic avian influenza viruses.
  • Chao-Tan Guo, Noriko Takahashi, Hirokazu Yagi, Koichi Kato, Tadanobu Takahashi, Shuang-Qin Yi, Yong Chen, Toshihiro Ito, Koichi Otsuki, Hiroshi Kida, Yoshihiro Kawaoka, Kazuya I-P Jwa Hidari, Daisei Miyamoto, Takashi Suzuki, Yasuo Suzuki
    Glycobiology 17 (7) 713 - 24 0959-6658 2007/07 [Refereed][Not invited]
    The receptor specificity of influenza viruses is one factor that allows avian influenza viruses to cross the species barrier. The recent transmissions of avian H5N1 and H9N2 influenza viruses from chickens and/or quails to humans indicate that avian influenza viruses can directly infect humans without an intermediate host, such as pigs. In this study, we used two strains of influenza A virus (A/PR/8/34, which preferentially binds to an avian-type receptor, and A/Memphis/1/71, which preferentially binds to a human-type receptor) to probe the receptor specificities in host cells. Epithelial cells of both quail and chicken intestines (colons) could bind both avian- and human-type viruses. Infected cultured quail colon cells expressed viral protein and allowed replication of the virus strain A/PR/8/34 or A/Memphis/1/71. To understand the molecular basis of these phenomena, we further investigated the abundance of sialic acid (Sia) linked to galactose (Gal) by the alpha2-3 linkage (Siaalpha2-3Gal) and Siaalpha2-6Gal in host cells. In glycoprotein and glycolipid fractions from quail and chicken colon epithelial cells, there were some bound components of Sia-Gal linkage-specific lectins, Maackia amurensis agglutinin (specific for Siaalpha2-3 Gal) and Sambucus nigra agglutinin (specific for Siaalpha2-6Gal), indicating that both Siaalpha2-3Gal and Siaalpha2-6Gal exist in quail and chicken colon cells. Furthermore, we demonstrated by fluorescence high-performance liquid chromatography (HPLC) analysis that 5-N-acetylneuraminic acid was the main molecular species of Sia, and we demonstrated by multi-dimensional HPLC mapping and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis that bi-antennary complex-type glycans alpha2-6 sialylated at the terminal Gal residue(s) are major (more than 79%) sialyl N-glycans expressed by intestinal epithelial tissues in both the chicken and quail. Taken together, these results indicate that quails and chickens have molecular characterization as potential intermediate hosts for avian influenza virus transmission to humans and could generate new influenza viruses with pandemic potential.
  • Matsuno K, Sakoda Y, Kameyama K, Tamai K, Ito A, Kida H
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 5 69 (5) 515 - 520 0916-7250 2007/05 [Refereed][Not invited]
  • Fujii K, Kakumoto C, Kobayashi M, Saito S, Kariya T, Watanabe Y, Sakoda Y, Kida H, Suzuki M
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 3 69 (3) 259 - 263 0916-7250 2007/03 [Refereed][Not invited]
  • Seiji Takeda, Hiroichi Ozaki, Satoshi Hattori, Atsushi Ishii, Hiroshi Kida, Koichi Mukasa
    JOURNAL OF NANOSCIENCE AND NANOTECHNOLOGY 7 (3) 752 - 756 1533-4880 2007/03 [Refereed][Not invited]
    Carbon nanotube sensors were capable of detecting hemagglutinin binding to anti-hemagglutinin antibody immobilized on the sensor. The carbon nanotube sensors were fabricated by chemical vapor deposition method and it showed field effect transistor property. Anti-hemagglutinin antibody was immobilized by cross-linking on the reverse surface of the carbon nanotube sensor. The current between the source and the drain was measured after incubation of various concentration of hemagglutinin antigen with immobilized anti-hemagglutinin antibody. I-V-gate curve was obtained by plotting the current as a function of the potential applied to the back gate. The I-V-gate curve showed a positive shift in a manner dependent on the hemagglutinin antigen concentration after immobilization of anti-hemagglutinin while no shift was observed without immobilization of anti-hemagglutinin antibody on the surface. The sensitivity of the CNT sensor was higher than that of the QCM method even without controlling the orientation of the anti-hemagglutinin antibody This method constitutes a new tool to analyze interactions among biomolecules on a substrate.
  • Hiroichi Ozaki, Hiroshi Kida
    MICROBIOLOGY AND IMMUNOLOGY 51 (5) 577 - 580 0385-5600 2007 [Refereed][Not invited]
    We previously showed that modified A/Puerto Rico/8/34 (PR8) influenza master strain had improved viral rescue and growth properties in African green monkey kidney (Vero) cell line by introducing NS gene of Vero-adapted A/England/1/53 (vaEng53). In the present study, it was found that the NS1 protein derived from vaEng53 was extensively accumulated in the nuclei than that of PR8. This accumulation was caused by 7 amino acid differences in C-terminal region of NS1 protein. These results suggest that specific accumulation of NS1 protein may contribute to efficient viral replication in Vero cells.
  • Michiko Naito, Yoshihiro Sakoda, Takayuki Kamikawa, Yoshiki Nitta, Kazuhiko Hirose, Mitsuaki Sakashita, Satoru Kurokawa, Hiroshi Kida
    MICROBIOLOGY AND IMMUNOLOGY 51 (6) 593 - 599 0385-5600 2007 [Refereed][Not invited]
    Serum samples were collected from 938 pigs of 24 farms in Hokkaido, Kagoshima, and Okinawa prefectures in Japan in 2001-2005. Enzyme-linked immunosorbent assay (ELISA) was used for the detection of antibodies to LipL32 antigen which is common to Leptospira interrogans. Samples positive in ELISA were then investigated by microscopic agglutination test for the identification of causal leptospires. Antibodies specific to leptospires of serovars Copenhageni, Bratislava, Australis and Javanica were detected in serum samples of pigs from each of the three districts. In addition, antibodies to leptospires of serovars Autummalis and Thrassovi were predominantly detected in those from Kagoshima. The present study, thus, revealed that leptospires belonging to different serovars prevail in the pig population in Japan. In addition, it is the first detection of antibodies to leptospires belonging to serovars Javanica and Thrassovi in pigs in Japan.
  • Yoshimi Tsuda, Yoshihiro Sakoda, Saori Sakabe, Tsuyoshi Mochizuki, Yasuharu Namba, Hiroshi Kida
    MICROBIOLOGY AND IMMUNOLOGY 51 (9) 903 - 907 0385-5600 2007 [Refereed][Not invited]
    Highly pathogenic avian influenza (HPAI) caused by the H5N1 subtype has given rise to serious damage in poultry industries in Asia. The virus has expanded its geographical range to Europe and Africa, posing a great risk to human health as well. For the control of avian influenza, a rapid diagnosis by detecting the causative virus and identifying its subtype is essential. In the present study, a rapid diagnosis kit combining immunochromatography with enzyme immunoassay which detects the H5 HA antigen of influenza A virus was developed using newly established anti-H5 HA monoclonal antibodies. The present kit specifically detected all of the H5 influenza viruses tested, and did not react with the other HA subtypes. H5 HA antigens were detected from swabs and tissue homogenates of chickens infected with HPAI virus strain A/chicken/Yamaguchi/7/04 (H5N1) from 2 days post inoculation. The kit showed enough sensitivity and specificity for the rapid diagnosis of HPAI.
  • Mumeka Malamo, Yoshihiro Sakoda, Hiroichi Ozaki, Hiroshi Kida
    JAPANESE JOURNAL OF VETERINARY RESEARCH 54 (2-3) 99 - 107 0047-1917 2006/11 [Refereed][Not invited]
    To develop ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the carboxyl termini of the 34 kDa proteins of M. paratuberculosis and Mycobacterium avium subsp. avium (M. avium) were expressed in Escherichia cola expression system. Antibodies specific to M. paratuberculosis were detected with the truncated 34 kDa protein of M. paratuberculosis in ELISA after pre-absorption of serum samples with the truncated 34 kDa protein of M. avium. All the serum samples from cattle confirmed to be infected with M. paratuberculosis were positive and those from healthy cattle were negative in the present ELISA system. These results indicate that the established ELISA detects antibodies specific to M. paratuberculosis with high specificity and sensitivity and is an useful tool for the screening of Johne's disease.
  • Kei Fujii, Hiroki Sato, Chiharu Kakumoto, Mari Kobayashi, Sachiko Saito, Tatsuya Kariya, Yukiko Watanabe, Yoshihiro Sakoda, Chieko Kai, Hiroshi Kida, Masatsugu Suzuki
    JAPANESE JOURNAL OF VETERINARY RESEARCH 54 (2-3) 109 - 117 0047-1917 2006/11 [Refereed][Not invited]
    Serological analysis was performed to detect morbillivirus infection in Kuril harbor seals in Hokkaido, Japan. Serum samples were collected from the seals at Nosappu (231 sera), Akkeshi (16), and Erimo (75) between 1998 and 2005. Antibodies to phocine distemper virus (PDV) were detected by ELISA in seals from Nosappu and Erimo. Antibodies to PDV were found in 56% (5/9) of the sampled seals from Nosappu in 1998, versus only 5% (3/66) for 2003, 1% (1/79) for 2004, and 1% (1/77) for 2005. These suggest epidemic caused by the virus in or before 1998. As antibody-positive seals included juvenile seals in 2003 and 2005, sporadic infections of the virus are thought to have occurred in recent years. In Erimo, antibodies to PDV were found in 50% (14/28) of sampled seals in 2004, versus only 13% (1/8) for 1999, 7 (1/15) for 2003, and 0 % (0/24) for 2005. These suggest sporadic infection by the virus before 2003 and the epizootic between after autumn in 2003, when samples of 2003 were collected, and 2004. Since antibodies to canine distemper virus (CDV) were detected in one adult seal from Nosappu in each year from 2003 to 2005, sporadic infections of the virus were suggested. There were no difference in incidence of seals with. antibodies to the viruses between males and females and between juveniles and adults.
  • Takeshi Noda, Hideki Ebihara, Yukiko Muramoto, Ken Fujii, Ayato Takada, Hiroshi Sagara, Jin Hyun Kim, Hiroshi Kida, Heinz Feldmann, Yoshihiro Kawaoka
    PLOS PATHOGENS 2 (9) 864 - 872 1553-7366 2006/09 [Refereed][Not invited]
    Ebolavirus is responsible for highly lethal hemorrhagic fever. Like all viruses, it must reproduce its various components and assemble them in cells in order to reproduce infectious virions and perpetuate itself. To generate infectious Ebolavirus, a viral genome-protein complex called the nucleocapsid (NC) must be produced and transported to the cell surface, incorporated into virions, and then released from cells. To further our understanding of the Ebolavirus life cycle, we expressed the various viral proteins in mammalian cells and examined them ultrastructurally and biochemically. Expression of nucleoprotein alone led to the formation of helical tubes, which likely serve as a core for the NC. The matrix protein VP40 was found to be critical for transport of NCs to the cell surface and for the incorporation of NCs into virions, where interaction between nucleoprotein and the matrix protein VP40 is likely essential for these processes. Examination of virus-infected cells revealed that virions containing NCs mainly emerge horizontally from the cell surface, whereas empty virions mainly bud vertically, suggesting that horizontal budding is the major mode of Ebolavirus budding. These data form a foundation for the identification and development of potential antiviral agents to combat the devastating disease caused by this virus.
  • Muramoto Y, Le TQ, Phuong LS, Nguyen T, Nguyen TH, Sakai-Tagawa Y, Horimoto T, Kida H, Kawaoka Y
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 7 68 (7) 735 - 737 0916-7250 2006/07 [Refereed][Not invited]
  • HL Wei, GR Bai, AS Mweene, YC Zhou, YL Cong, J Pu, SH Wang, H Kida, JH Liu
    VIRUS GENES 32 (3) 261 - 267 0920-8569 2006/06 [Refereed][Not invited]
    Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry and resulted in human deaths in Thailand and Viet Nam in 2004. Rapid typing and subtyping of H5N1 viruses, especially from clinical specimens, are desirable for taking prompt control measures to prevent the spread of the disease. Here, we developed a set of oligonucleotide primers able to detect, type and subtype H5 and N1 influenza viruses in a single step multiplex reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from allantoic fluid or from specimens with guanidinium isothiocyanate reagent. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A, subtype H5 and N1 in a single reaction system under identical conditions. The amplified DNA fragments were analyzed by agarose gel electrophoresis. All the H5N1 viruses tested in the study and the experimental specimens presented three specific bands by the method established here. The results presented here suggest that the method described below is rapid and specific and, therefore, could be valuable in the rapid detection of H5N1 influenza viruses in clinics.
  • Muramoto Y, Le TQ, Phuong LS, Nguyen T, Nguyen TH, Sakai-Tagawa Y, Iwatsuki-Horimoto K, Horimoto T, Kida H, Kawaoka Y
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 5 68 (5) 527 - 531 0916-7250 2006/05 [Refereed][Not invited]
  • Hetron M. Munang'andu, Victor M. Siamudaala, Andrew Nambota, John M. Bwalya, Musso Munyeme, Aaron S. Mweene, Ayato Takada, Hiroshi Kida
    JAPANESE JOURNAL OF VETERINARY RESEARCH 54 (1) 3 - 13 0047-1917 2006/05 [Refereed][Not invited]
    Eco-tourism depending on wildlife is becoming increasingly profitable and landowners are beginning to favor game farming and ecotourism. In these areas, large-scale translocation of wildlife involves a diversity of species and large populations. The African buffalo (Syncerus caffer) is one of the major tourist attractions in Zambia. It accounts for 8.7% and 12.4% of the total animal species hunted in the Game Management Areas and the total hunting revenue earned in Zambia, respectively. It is ecologically an important animal species essential for the purpose of habitat control and facilitating the provision of suitable grazing pastures. However, the rearing of the African buffalo on game ranches has been hampered by its carrier state of the Southern Africa Terroritory (SAT) serotypes of foot and mouth disease virus (FMD). The African buffalo is also known to be a carrier of Theileria parva lawrencei, the causative agent of corridor disease (CD) that continues to have devastating effects on the livestock industry in Zambia. In addition, the importation of buffaloes from countries with populations endemic to bovine tuberculosis is highly restricted. Veterinary regulations in Zambia, strongly advocate against the translocation of buffaloes from protected areas to private ranches for disease control purposes thereby mounting a considerable constraint on the economic and ecological viability of the industry. It is hoped that this review will motivate the relevant government authorities in exploiting ways in which this animal species play a central role in eco-tourism.
  • Victor M. Siamudaala, John M. Bwalya, Hetron M. Munang'andu, Peter G. Sinyangwe, Fred Banda, Aaron S. Mweene, Ayato Takada, Hiroshi Kida
    JAPANESE JOURNAL OF VETERINARY RESEARCH 54 (1) 15 - 23 0047-1917 2006/05 [Refereed][Not invited]
    Anthrax is endemic in Western and North-western Provinces of Zambia. The disease occurs throughout the year and impacts negatively on the economy of the livestock industry and public health in Zambia. During 1989-1995, there were 1, 626 suspected cases of anthrax in cattle in Western province and of these 51 were confirmed. There were 220 cases of human anthrax cases in 1990 alone and 248 cases during 1991-1998 with 19.1% and 7.7% case fatality rates, respectively. Interplay of the ecology of affected areas and anthropogenic factors seem to trigger anthrax epidemics. Anthrax has drawn considerable attention in recent years due to its potential use as a biological weapon. In this paper, the history, current status and approaches towards the control of the disease in Zambia are discussed. Quarantine measures restrict trade of livestock and exchange of animals for draught power resulting in poor food security at household levels. Challenges of anthrax control are complex and comprise of socio-political, economical, environmental and cultural factors. Inadequate funding, lack of innovative disease control strategies and lack of cooperation from stakeholders are the major constraints to the control of the disease. It is hoped that the information provided here will stimulate continued awareness for the veterinary and medical authorities to maintain their surveillance and capabilities against the disease. This may lead to a culminating positive impact on livestock and human health in the southern African region.
  • Y Muramoto, A Takada, K Fujii, T Noda, K Iwatsuki-Horimoto, S Watanabe, T Horimoto, H Kida, Y Kawaoka
    JOURNAL OF VIROLOGY 80 (5) 2318 - 2325 0022-538X 2006/03 [Refereed][Not invited]
    The genome of influenza A viruses comprises eight negative-strand RNA segments. Although all eight segments must be present in cells for efficient viral replication, the mechanism(s) by which these viral RNA (vRNA) segments are incorporated into virions is not fully understood. We recently found that sequences at both ends of the coding regions of the HA, NA, and NS vRNA segments of A/WSN/33 play important roles in the incorporation of these vRNAs into virions. In order to similarly identify the regions of the PB2, PB1, and PA vRNAs of this strain that are critical for their incorporation, we generated a series of mutant vRNAs that possessed the green fluorescent protein gene flanked by portions of the coding and noncoding regions of the respective segments. For all three polymerase segments, deletions at the ends of their coding regions decreased their virion incorporation efficiencies. More importantly, these regions not only affected the incorporation of the segment in which they reside, but were also important for the incorporation of other segments. This effect was most prominent with the PB2 vRNA. These findings suggest a hierarchy among vRNA segments for virion incorporation and may imply intersegment association of vRNAs during virus assembly.
  • K Okazaki, S Fujii, A Takada, H Kida
    VIRUS RESEARCH 115 (2) 105 - 111 0168-1702 2006/02 [Refereed][Not invited]
    In order to address the neutralization epitope on bovine herpesvirus 1 (BHV 1) glycoprotein B (gB), a panel of monoclonal antibodies (MAbs), a series of truncation forms of the glycoprotein and an MAb-escape mutant were used in this study. Immunocytochemistry on the truncations using MAbs against the glycoprotein revealed that the neutralization epitopes recognized by the MAbs lay between residues 1 and 52 of mature gB. Comparison of the sequences among the mutant, parent, and revertant viruses demonstrated that the amino-terminal residue of mature gB of the escape mutant was changed from Arg to Gln. These findings indicate that the amino-terminal residue of gB is critical for neutralization of BHV1. (c) 2005 Elsevier B.V. All rights reserved.
  • Y Muramoto, H Ozaki, A Takada, CH Park, Y Sunden, T Umemura, Y Kawaoka, H Matsuda, H Kida
    MICROBIOLOGY AND IMMUNOLOGY 50 (1) 73 - 81 0385-5600 2006 [Refereed][Not invited]
    Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus-infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.
  • T Noda, H Sagara, A Yen, A Takada, H Kida, RH Cheng, Y Kawaoka
    NATURE 439 (7075) 490 - 492 0028-0836 2006/01 [Refereed][Not invited]
    In viruses, as in eukaryotes, elaborate mechanisms have evolved to protect the genome and to ensure its timely replication and reliable transmission to progeny. Influenza A viruses are enveloped, spherical or filamentous structures, ranging from 80 to 120 nm in diameter(1). Inside each envelope is a viral genome consisting of eight single-stranded negative- sense RNA segments of 890 to 2,341 nucleotides each(1). These segments are associated with nucleoprotein and three polymerase subunits, designated PA, PB1 and PB2; the resultant ribonucleoprotein complexes (RNPs) resemble a twisted rod ( 10 - 15 nm in width and 30 - 120 nm in length) that is folded back and coiled on itself(2-4). Late in viral infection, newly synthesized RNPs are transported from the nucleus to the plasma membrane, where they are incorporated into progeny virions capable of infecting other cells. Here we show, by transmission electron microscopy of serially sectioned virions, that the RNPs of influenza A virus are organized in a distinct pattern ( seven segments of different lengths surrounding a central segment). The individual RNPs are suspended from the interior of the viral envelope at the distal end of the budding virion and are oriented perpendicular to the budding tip. This finding argues against random incorporation of RNPs into virions(5), supporting instead a model in which each segment contains specific incorporation signals that enable the RNPs to be recruited and packaged as a complete set(6-12). A selective mechanism of RNP incorporation into virions and the unique organization of the eight RNP segments may be crucial to maintaining the integrity of the viral genome during repeated cycles of replication.
  • Bai GR, Sakoda Y, Mweene AS, Fujii N, Minakawa H, Kida H
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 1 68 (1) 35 - 40 0916-7250 2006/01 [Refereed][Not invited]
  • H Ito, T Ito, M Hikida, J Yashiro, A Otsuka, H Kida, K Otsuki
    DERMATOLOGY 212 115 - 118 1018-8665 2006 [Refereed][Not invited]
    Objectives: On January 12, 2004, an outbreak of highly pathogenic avian influenza, caused by the H5N1 strain, occurred in a one-layer flock in Yamaguchi Prefecture, Japan. It had been 79 years since the last outbreak of avian influenza was confirmed in Japan. By February, 3 additional outbreaks had occurred (1 in Oita Prefecture and 2 in Kyoto Prefecture). Influenza viruses are enveloped viruses and are relatively sensitive to inactivation by lipid solvents, such as detergents. Infectivity is also rapidly destroyed by ether, sodium hypochlorite, povidone-iodine (PVP-I), peracetic acid and alcohol. However, these antiviral effects were only tested against human influenza A viruses. In the present study, the antiviral activity of PVP-I products against H5, H7 and H9 avian influenza A viruses, which had recently been transmitted to humans, were investigated. Methods:The in vitro antiviral activity of PVP-I products (2% PVP-I solution, 0.5% PVP-I scrub, 0.25% PVP-I palm, 0.23% PVP-I gargle, 0.23% PVP-I throat spray and 2% PVP-I solution for animals) against avian influenza A viruses [a highly pathogenic avian influenza virus, A/crow/Kyoto/T2/04 (H5N1; 10(6.5) EID50/0.1 ml), and 3 low pathogenic avian influenza A viruses, A/whistling swan/Shimane/499/838 (H5N3; 10(4.8) EID50/0.1 ml), A/whistling swan/Shimane/42/80 (H7N7; 10(5.5) EID50/0-1 ml) and A/duck/Hokkaido/26/99 (H9N2; 10(4.8) EID50/0-1 ml)] were investigated using embryonated hen's eggs. Results/Discussion: Viral infectious titers were reduced to levels below the detection limits by incubation for only 10 s with the PVP-I products used in this study. These results indicate that PVP-I products have virucidal activity against avian influenza A viruses. Therefore, the PVP-I products are useful in the prevention and control of human infection by avian influenza A viruses. Copyright (c) 2006 S. Karger AG, Basel.
  • Jae-Ho Shin, Yoshihiro Sakoda, Jae Hoon Kim, Tomohisa Tanaka, Hiroshi Kida, Takashi Kimura, Kenji Ochiai, Takashi Umemura
    MICROBIOLOGY AND IMMUNOLOGY 50 (10) 823 - 830 0385-5600 2006 [Refereed][Not invited]
    To evaluate the efficacy of intracerebral (IQ immunization, mice were immunized with formalin-inactivated pseudorabies virus (PRV) by either subcutaneous (SQ or IC injection, and then 10(6) plaque-forming units of PRV were introduced into the hindleg of the immunized or non-immunized mice by intramuscular injection. The antibody titer in serum was elevated and boosted by additional immunization via both the SC and IC routes, but was higher after IC immunization. Intracerebrally immunized mice were completely protected from mortality and neurological signs, whereas all the non-immunized and 80% of the subcutaneously immunized mice died after developing neurological signs. In mouse models, IC immunization is more effective at inducing a protective immune response against the transneural spread of PRV than SC immunization.
  • Nakamura Y, Yoshizawa H, Hirasawa M, Kida H, Matsumoto Y, Ueyama T
    Circulation journal : official journal of the Japanese Circulation Society 9 69 (9) 1079 - 1083 1346-9843 2005/09 [Refereed][Not invited]
  • S Takeda, A Sbagyo, Y Sakoda, A Ishii, M Sawamura, K Sueoka, H Kida, K Mukasa, K Matsumoto
    BIOSENSORS & BIOELECTRONICS 21 (1) 201 - 205 0956-5663 2005/07 [Refereed][Not invited]
    Carbon nanotube sensors detected anti-hemagglutinin binding to immobilized hemagglutinins. An ultra-sensitive detection method for antibodies or antigens in serum is required. Hemagglutinins were immobilized on the reverse side of a carbon nanotube, thereby producing a source and a drain. Electrode pads covered each edge of the nanotube. The 1-V curves between the source and the drain were measured after incubation of anti-hemagglutinins with immobilized hemagglutinins in a buffered solution on the reverse side of the nanotube. The sensitivity of the CNT sensor was higher than that of an ELISA system. This method constitutes a new tool to analyze interaction among biomolecules on a substrate. (c) 2004 Elsevier B.V. All rights reserved.
  • K Matsuda, T Shibata, Y Sakoda, H Kida, T Kimura, K Ochiai, T Umemura
    JOURNAL OF GENERAL VIROLOGY 86 (4) 1131 - 1139 0022-1317 2005/04 [Refereed][Not invited]
    Neural involvement following infections of influenza viruses can be serious. The neural transport of influenza viruses from the periphery to the central nervous system has been indicated by using mouse models. However, no direct evidence for neuronal infection has been obtained in vitro and the mechanisms of neural transmission of influenza viruses have not been reported. In this study, the transneural transmission of a neurotropic influenza A virus was examined using compartmentalized cultures of neurons from mouse dorsal root ganglia, and the results were compared with those obtained using the pseudorabies virus, a virus with well-established neurotransmission. Both viruses reached the cell bodies of the neurons via the axons. This is the first report on axonal transport of influenza A virus in vitro. In addition, the role of the cytoskeleton (microtubules, microfilaments and intermediate filaments) in the neural transmission of influenza virus was investigated by conducting cytoskeletal perturbation experiments. The results indicated that the transport of avian influenza A virus in the neurons was independent of microtubule integrity but was dependent on the integrity of intermediate filaments, whereas pseudorabies virus needed both for neural spread.
  • Noda T, Aoyama K, Sagara H, Kida H, Kawaoka Y
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 3 67 (3) 325 - 328 0916-7250 2005/03 [Refereed][Not invited]
  • H Kida, M Yoshida, S Hoshino, K Inoue, Y Yano, M Yanagita, T Kumagai, T Osaki, Tachibana, I, Y Saeki, Kawase, I
    AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY 288 (2) L342 - L349 1040-0605 2005/02 [Refereed][Not invited]
    The goal of this study was to examine whether IL-6 could directly protect lung resident cells, especially alveolar epithelial cells, from reactive oxygen species (ROS)-induced cell death. ROS induced IL-6 gene expression in organotypic lung slices of wild-type (WT) mice. ROS also induced IL-6 gene expression in mouse primary lung fibroblasts, dose dependently. The organotypic lung slices of WT were more resistant to ROS-induced DNA fragmentation than those of IL-6-deficient (IL-6-/-) mice. WT resistance against ROS was abrogated by treatment with anti-IL-6 antibody. TdT-mediated dUTP nick end labeling stain and electron microscopy revealed that DNA fragmented cells in the IL-6-/- slice included alveolar epithelial cells and endothelial cells. In vitro studies demonstrated that IL-6 reduced ROS-induced A549 alveolar epithelial cell death. Together, these data suggest that IL-6 played an antioxidant role in the lung by protecting lung resident cells, especially alveolar epithelial cells, from ROS-induced cell death.
  • N Odontsetseg, AS Mweene, H Kida
    JAPANESE JOURNAL OF VETERINARY RESEARCH 52 (4) 151 - 162 0047-1917 2005/02 [Refereed][Not invited]
    This review focuses on the status of infectious diseases that are serious for animal health and have adverse economic effects in Mongolia. Data presented here are limited due to the lack of published or other easily available documents. Foot-and-mouth disease continues to cause substantial economic losses as exemplified by the outbreak of infection with serotype O PanAsia lineage virus. In the case of the 2001 outbreak, a 65% reduction in export revenues was recorded. In order to ascertain the free status of Mongolia from rinderpest, sero-epidemiological surveillance has been carried out since 2001. In 2004, Mongolia was certified free from rinderpest by Office International des Epizooties (OIE). A sharp rise in both animal and human brucellosis incidence has become a serious problem. Rabies and anthrax remain endemic with occasional human cases. Other prevailing infectious diseases are contagious pustular dermatitis, contagious agalactia, enterotoxemia and pasteurellosis. The current programs for the control of infectious diseases in livestock in Mongolia lack a definite policy that would enable rapid implementation. A large-scale surveillance of infectious diseases in animals and management of appropriate preventive measures are urgently required in Mongolia.
  • N Odontsetseg, Y Sakoda, H Kida
    MICROBIOLOGY AND IMMUNOLOGY 49 (9) 865 - 869 0385-5600 2005 [Refereed][Not invited]
    Serum samples were collected randomly from Mongol breed cattle in three geographically distinct provinces of Mongolia. Antibodies specific to Leptospira interrogans serovar Hardjo were detected by microscopic agglutination test (MAT) at titres of 100 or more in 80.4% (86/107) of the samples from Dornod Province, but in only 28.9% (13/45) in Arkhangai and 23.5% (12/51) in Khuvsgul Provinces, respectively. Treatment of 9 serum samples from Dornod, positive to serovar Hardjo in MAT and negative in the homologous immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), with 2-mercaptoethanol (2-ME) indicated that those animals were recently infected.
  • GR Bai, Y Sakoda, AS Mweene, N Kishida, T Yamada, H Minakawa, H Kida
    MICROBIOLOGY AND IMMUNOLOGY 49 (12) 1063 - 1067 0385-5600 2005 [Refereed][Not invited]
    The ESPLINE(R) INFLUENZA A&B-N kit was evaluated for its applicability to the rapid diagnosis of influenza in chickens and pigs. The kit specifically detected viral antigens in tracheal swabs and tissue homogenates of the trachea, liver, spleen, and colon of chickens inoculated with a highly pathogenic avian influenza virus strain, A/chicken/Yamaguchi/7/04 (H5N1), at 48 hr post-inoculation (p.i.) as well as in the tracheal and cloacal swabs and tissue homogenates of dead chickens. For those infected with a low pathogenic strain, A/chicken/aq-Y-55/01 (H9N2), antigens were detected only in the samples from tracheal swabs and organs 1-4 days p.i. The kit also detected viral antigens in the nasal swabs of miniature pigs infected with swine and avian influenza viruses. The kit was found to be sensitive and specific enough for the rapid diagnosis of infections of influenza A virus in chickens and pigs.
  • JH Liu, K Okazaki, A Mweene, WM Shi, QM Wu, JL Su, GZ Zhang, GR Bai, H Kida
    VIRUS GENES 29 (3) 329 - 334 0920-8569 2004/12 [Refereed][Not invited]
    The hemagglutinin (HA) genes of 12 H9N2 influenza virus strains isolated from chickens in Mainland China during the period 1995 - 2002 were genetically analyzed. All the isolates possessed the same amino acid motif - R-S-S-R/G-L- at the cleavage site of HA. Except for the conserved amino acids, as is the case in the other avian influenza viruses, located in the receptor binding site, all of the 12 isolates possessed N at amino acid position 183; A, T, or V at position 190; K at position 137, whereas the representative strains of the other lineage ( except Dk/HK/Y280/97-like lineage) virus of H9N2 viruses had H, E, and R at these positions respectively. These could be considered as the partial molecular markers of the H9 viruses isolated from chickens in Mainland China. Phylogenetic analyses showed HA genes of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. No A/quail/Hong Kong/Gl/97-like virus was found in chicken, population since the outbreak of H9N2 influenza in Mainland China in 1992. The available evidence indicates that HA genes of H9 influenza virus circulating in Mainland China during the past years were well conserved.
  • JH Liu, K Okazaki, GR Bai, WM Shi, A Mweene, H Kida
    VIRUS GENES 29 (1) 81 - 86 0920-8569 2004/08 [Refereed][Not invited]
    H2 influenza virus caused a pandemic in 1957 and has the possibility to cause outbreaks in the future. To assess the evolutionary characteristics of H2 influenza viruses isolated from migratory ducks that congregate in Hokkaido, Japan, on their flyway of migration from Siberia in 2001, we investigated the phylogenetic relationships among these viruses and avian and human viruses described previously. Phylogenetic analysis showed that the PB2 gene of Dk/Hokkaido/107/01 (H2N3) and the PA gene of Dk/Hokkaido/95/01 (H2N2) belonged to the American lineage of avian virus and that the other genes of the isolates belonged to the Eurasian lineage. These results indicate that the internal protein genes might be transmitted from American to Eurasian avian host. Thus, it is further confirmed that interregional transmission of influenza viruses occurred between the North American and Eurasian birds. The fact that reassortants could be generated in the migratory ducks between North American and Eurasian avian virus lineage further stresses the importance of global surveillance among the migratory ducks.
  • K Okazaki, H Kida
    JOURNAL OF GENERAL VIROLOGY 85 (8) 2131 - 2137 0022-1317 2004/08 [Refereed][Not invited]
    Glycoprotein B (gB) is the most conserved glycoprotein of herpesviruses and plays important roles in virus infectivity. Two intervening heptad repeat (HR) sequences were found in the C-terminal half of all herpesvirus gBs analysed. A synthetic peptide derived from the HR region (aa 477-510) of bovine herpesvirus type 1 (BoHV-1) gB was studied for its ability to inhibit virus replication. The peptide interfered with cell-to-cell spread and consistently inhibited replication of BoHV-1, with a 50% effective concentration value (EC50) of 5 muM. Inhibition of replication was obtained not only with herpesviruses including pseudorabies virus and herpes simplex virus type 1 but also partly with Newcastle disease virus. Possible mechanisms of membrane fusion inhibition by the peptide are discussed.
  • Kida H
    Uirusu 1 54 93 - 96 0042-6857 2004/06 [Refereed][Not invited]
  • Nagai Y, Otsuki K, Abe N, Kawaoka Y, Kida H, Kurata T, Sata T, Tashiro M, Yamaguchi N, Yoshikura H
    Uirusu 1 54 123 - 141 0042-6857 2004/06 [Refereed][Not invited]
  • S Yazawa, M Okada, M Ono, S Fujii, Y Okuda, Shibata, I, H Kida
    VETERINARY MICROBIOLOGY 98 (3-4) 221 - 228 0378-1135 2004/03 [Refereed][Not invited]
    Dual infection of pigs with swine influenza virus (SIV) and Mycoplasma hyopneumoniae was carried out to compare the clinical and pathological effects of dual infection in caesarian derived and colostrums deprived (CDCD) pigs, with that of a single infection with M. hyopneumoniae. In Experiment 1, 40-day-old CDCD pigs were inoculated only with SIV (A/Sw/Hok/2/81, H1N1). The virus was isolated from nasal swabs for 5-6 days. None of these pigs showed clinical signs of infection throughout the experimental period. These results suggested that this strain can infect pigs but is only slightly pathogenic when it is inoculated singly to a CDCD pig. In Experiment 2, 60-day-old CDCD pigs were inoculated with M. hyopneumoniae and then were inoculated with SIV (A/Sw/Hok/2/8 1) at 1 week (MHYO-7d-SIV-7d group) or 3 weeks (MHYO-21 d-SIV-7d group) after M. hyopneumoniae inoculation. Macroscopically, dark red-to-purple lung lesions were observed in all of pigs at 14 or 28 days post-inoculation. Percentages of dark red-to-purple lung lesions in dual infection groups (MHYO-7d-SIV-7d group: 18.7 +/- 4.2%, MHYO-21 d-SIV-7d group: 23.0 +/- 8.0%) were significantly (P < 0.05) increased compared to those of each control group in which pigs were inoculated only with M. hyopneumoniae (MHYO-14d group: 4.7 +/- 2.9%, MHYO-28 group: 3.3 +/- 2.4%). Microscopically, bronchial epithelial lesions (epithelial disruption, degeneration, hyperplasia and formation of microabscess) were frequently observed in dark red-to-purple lung lesions of only the dual infection groups. These results demonstrate that the lung lesion of pigs inoculated with M. hyopneumoniae and SIV is more severe than that of pigs inoculated only with M. hyopneumoniae. (C) 2003 Elsevier B.V. All rights reserved.
  • K Ohishi, N Kishida, A Ninomiya, H Kida, Y Takada, N Miyazaki, AN Boltunov, T Maruyama
    MICROBIOLOGY AND IMMUNOLOGY 48 (11) 905 - 909 0385-5600 2004 [Refereed][Not invited]
    Antibodies to influenza A virus were detected using enzyme-linked immunosorbent assay (ELISA) in the sera from two of seven Baikal seals (Phoca sibrica) and from five of six ringed seals (Phoca hispida) in Russia. In a hemagglutination-inhibition test using H1-H15 reference influenza A viruses, ELISA-positive sera from one Baikal seal and four ringed seals reacted to A/Aichi/2/68 (H3N2) and A/Bangkok/1/79 (H3N2) strains. One ringed seal serum sample reacted to A/seal/Massachusetts/1/80 (H7N7). The present results suggested that human-related H3 viruses were prevalent in Baikal seals and ringed seals inhabiting the central Russian Arctic.
  • JH Liu, K Okazaki, WM Shi, H Kida
    VIRUS GENES 27 (3) 291 - 296 0920-8569 2003/12 [Refereed][Not invited]
    Genetic analysis indicated that the pandemic influenza strains derived from wild aquatic birds harbor viruses of 15 hemagglutinin (HA) and 9 neuraminidase (NA) antigenic subtypes. Surveillance studies have shown that H9N2 subtype viruses are worldwide in domestic poultry and could infect mammalian species, including humans. Here, we genetically analyzed the HA and NA genes of five H9N2 viruses isolated from the migratory ducks in Hokkaido, Japan, the flyway of migration from Siberia during 1997-2000. The results showed that HA and NA genes of these viruses belong to the same lineages, respectively. Compared with those of A/quail/Hong Kong/G1/97-like and A/duck/Hong KongIY280/97-like viruses, HA and NA of the migratory duck isolates had a close relationship with those of H9N2 viruses isolated from the chicken in Korea, indicating that the Korea H9N2 viruses might be derived from the migratory ducks. The NA genes of the five isolates were located in the same cluster as those of N2 viruses, which had caused a human pandemic in 1968, indicating that the NA genes of the previous pandemic strains are still circulating in waterfowl reservoirs. The present results further emphasize the importance of carrying out molecular epidemiological surveillance of H9N2 viruses in wild ducks to obtain more information for the future human influenza pandemics preparedness.
  • Kida H
    Nihon rinsho. Japanese journal of clinical medicine 11 61 1865 - 1871 0047-1852 2003/11 [Refereed][Not invited]
  • Sunden Y, Park CH, Matsuda K, Anagawa A, Kimura T, Ochiai K, Kida H, Umemura T
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 11 65 (11) 1185 - 1188 0916-7250 2003/11 [Refereed][Not invited]
  • JH Liu, K Okazaki, WM Shi, QM Wu, AS Mweene, H Kida
    VIRUS GENES 27 (2) 197 - 202 0920-8569 2003/10 [Refereed][Not invited]
    The neuraminidase (NA) genes of 12 H9N2 influenza virus strains isolated from diseased chickens in different farms in mainland China during 1995-2002 were amplified and sequenced. Amino acids at hemadsorbing (HB) site of these isolates are different from those of A/quail/Hong Kong/G1/97-like viruses and A/chicken/Korea/96-like viruses. Neuraminidases of the 12 strains had a deletion of 3 amino acid residues at positions 63-65 as compared to that of A/turkey/Wisconsin/189/66, while those of Korea and Pakistan H9N2 isolates had no deletion. Phylogenetic analyses showed NA gene of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. NA gene of the H9N2 viruses isolated in Korea and Pakistan belonged to lineage different from those of the 12 isolates. The present results indicate that the NA of H9N2 strains isolated in mainland China during the past 8 years were well preserved and the geographical distribution play a significant role in the evolution of the H9N2 influenza viruses.
  • JH Liu, K Okazaki, H Ozaki, Y Sakoda, QM Wu, FY Chen, H Kida
    AVIAN PATHOLOGY 32 (5) 551 - 560 0307-9457 2003/10 [Refereed][Not invited]
    Ten H9N2 influenza virus strains isolated from diseased chickens in different farms in China during 1995 to 1999 were antigenically and genetically characterized. The haemagglutinins of the isolates were not related to those of A/quail/ Hong Kong/G1/97 ( H9N2) (Qa/HK/G1/97), but were closely related to that of A/chicken/ Hong Kong/G9/97 ( H9N2) (Ck/HK/G9/97). The neuraminidase of these isolates had a deletion of three amino acid residues at positions 63 to 65 as compared with those of Ck/HK/G9/97, while that of Qa/HK/G1/97 lacked two amino acids at positions 38 and 39. The PB2 genes of the isolates were not related to those of Qa/HK/G1/ 97 or Ck/HK/G9/97, but showed some relationship to that of A/duck/Hong Kong/Y439/97 ( H9N2) (Dk/HK/Y439/ 97). The PB1 genes of the isolates were not related to those of the three representative strains. The PA, NP, M, and NS genes of the isolates belonged to the same lineage as those of Ck/HK/G9/97, and were distinct from those of Qa/HK/G1/ 97 and Dk/HK/Y439/97. The present results indicate that H9N2 influenza viruses prevalent in chicken populations in China belong genetically to one lineage and are distinct from Qa/HK/G1/ 97, presumed to be the donor of the internal protein genes of the highly pathogenic H5N1 influenza virus in Hong Kong in 1997.
  • H Tanaka, CH Park, A Ninomiya, H Ozaki, A Takada, T Umemura, H Kida
    VETERINARY MICROBIOLOGY 95 (1-2) 1 - 13 0378-1135 2003/08 [Refereed][Not invited]
    The direct transmission of H5N1 influenza A viruses from chickens to humans in Hong Kong in 1997 emphasized the need to have information on the pathogenesis of avian influenza virus infection in mammals. H5N1 influenza viruses isolated from patients during the incident killed experimentally infected mice. The principal lesions of the mice were broncho-interstitial pneumonia and nonsuppurative encephalitis. Infectious viruses and/or viral antigens were detected in the brain as well as in the trigeminal and vagal ganglia but not in the blood of the mice. These findings suggest that the virus reached the brain through the vagus and/or trigeminal nerves following replication in the respiratory mucosa. The results imply that neurotropism of the H5N1 virus in mice is a novel characteristic in the pathogenesis of infection by human influenza virus isolates. (C) 2003 Elsevier B.V. All rights reserved.
  • A Takada, S Matsushita, A Ninomiya, Y Kawaoka, H Kida
    VACCINE 21 (23) 3212 - 3218 0264-410X 2003/07 [Refereed][Not invited]
    It has been known that influenza A virus infection induces a cross-protective immunity against infection by viruses with different subtypes of viral envelope proteins, hemagglutinin (HA) and neuraminidase (NA). This heterosubtypic immunity is generally mediated by cytotoxic T lymphocytes (CTL) reactive to specific epitopes in the viral internal proteins, such as nucleoprotein and matrix protein. By contrast, immunization with inactivated virus antigens has been thought to be unable to generate heterosubtypic immunity, since inactivated antigens do not usually induce CTL responses. However, we show that intranasal immunization with formalin-inactivated intact virus, but not ether-split vaccines, induced a broad spectrum of heterosubtypic protective immunity in mice. The protection may be mediated by the mucosal immune response, most likely secretory IgA antibodies to the viral proteins. This approach may overcome limitations in the efficacy of inactivated influenza vaccines and confer potent immunity to humans against viruses with new pandemic potential. (C) 2003 Elsevier Science Ltd. All rights reserved.
  • Kida H
    Uirusu 1 53 (1) 71 - 74 0042-6857 2003/06 [Refereed][Not invited]
  • Kida H
    Uirusu 1 53 93 - 94 0042-6857 2003/06 [Refereed][Not invited]
  • NV Makarova, H Ozaki, H Kida, RG Webster, DR Perez
    VIROLOGY 310 (1) 8 - 15 0042-6822 2003/05 [Refereed][Not invited]
    Quail have emerged as a potential intermediate host in the spread of avian influenza A viruses in poultry in Hong Kong. To better understand this possible role, we tested the replication and transmission in quail of influenza A viruses of all 15 HA subtypes. Quail supported the replication of at least 14 subtypes. Influenza A viruses replicated predominantly in the respiratory tract. Transmission experiments suggested that perpetuation of avian influenza viruses in quail requires adaptation. Swine influenza viruses were isolated from the respiratory tract of quail at low levels. There was no evidence of human influenza A or B virus replication. Interestingly, a human-avian recombinant containing the surface glycoprotein genes of a quail virus and the internal genes of a human virus replicated and transmitted readily in quail; therefore, quail could function as amplifiers of influenza virus reassortants that have the potential to infect humans and/or other mammalian species. (C) 2003 Elsevier Science (USA). All rights reserved.
  • M Nagai, Y Sakoda, M Mori, M Hayashi, H Kida, H Akashi
    JOURNAL OF GENERAL VIROLOGY 84 (2) 447 - 452 0022-1317 2003/02 [Refereed][Not invited]
    The cytopathogenic bovine viral diarrhoea virus (cp BVDV) strain KS86-1 cp was isolated from a calf persistently infected with the noncytopathogenic (ncp) strain KS86-1 ncp after it was exposed to cp BVDV strain Nose and developed mucosal disease (MD). Molecular analysis revealed that an insertion of a cellular gene and a duplication of the viral RNA encoding the nucleocapsid protein C and part of N-pro had occurred in the C coding region of the Nose and KS86-1 cp genomes. The inserted cellular gene was closely related to the cINS sequence. Remarkably, the 5' upstream region from the insertion of KS86-1 cp had high sequence identity to that of Nose, but differed from that of KS86-1 ncp. In contrast, the region downstream from the insertion of KS86-1 cp showed high identity to KS86-1 ncp, but not to Nose. These data reveal that KS86-1 cp is a chimeric virus generated by homologous RNA recombination in a calf with MD.
  • C Sugimoto, M Hasegawa, HY Zheng, Demenev, V, Y Sekino, K Kojima, T Honjo, H Kida, T Hovi, T Vesikari, JA Schalken, K Tomita, Y Mitsunobu, H Ikegaya, N Kobayashi, T Kitamura, Y Yogo
    JOURNAL OF MOLECULAR EVOLUTION 55 (3) 322 - 335 0022-2844 2002/09 [Refereed][Not invited]
    Human polyomavirus JC virus (JCV) isolates around the world are classified into more than 10 geographically distinct genotypes (designated as subtypes). Evolutionary relationships among JCV subtypes were recently examined, and the following pattern of JCV evolution was indicated. The ancestral JCV first divided into three superclusters, designated Types A, B, and C. A split in Type A generated two subtypes, EU-a and -b, containing mainly European and Mediterranean isolates. The split in Type B generated Af 2 (the major African subtype), B1-c (a minor European subtype), and various Asian subtypes. Type C generated a single subtype (Afl), consisting of isolates derived from western Africa. In this study, JCV isolates prevalent among northeastern Siberians and Canadian Inuits were evaluated in the context of the above-described pattern of JCV evolution. The Siberian/Arctic JCV isolates were classified as belonging mainly to Type A, based on the result of a preliminary phylogenetic analysis. We then examined, using the whole-genome approach, the phylogenetic relationships among worldwide Type A isolates. In neighbor-joining and maximum-likelihood analyses, Type A JCVs worldwide consistently diverged into three subtypes, EU-a, -b, and -c, with high bootstrap probabilities. EU-c was constructed only by northeastern Siberian isolates, derived mainly from Nanais living in the lower Amur River region, and was shown to have been generated by the first split in Type A. Most Siberian/Arctic isolates derived from Chukchis, Koryaks, and Canadian Inuits formed a distinct cluster within the EU-a subtype, with a high bootstrap probability. Based on the present findings, we discuss ancient human migrations, accompanied by Type A JCVs, across Asia and to Arctic areas of North America.
  • Y Shengqing, N Kishida, H Ito, H Kida, K Otsuki, Y Kawaoka, T Ito
    VIROLOGY 301 (2) 206 - 211 0042-6822 2002/09 [Refereed][Not invited]
    A benign Newcastle disease virus (NDV) recently became highly virulent during replication in domestic chickens. It is still unclear whether NDVs circulating among wild waterfowl also have the potential to become highly pathogenic (velogenic) in chickens. To demonstrate experimentally the generation of velogenic NDV from a nonpathogenic waterfowl isolate, we passaged an avirulent goose isolate in chickens. After nine consecutive passages by air-sac inoculation, followed by five passages in chick brain, the virus became highly virulent in chickens, producing a 100% mortality rate, and demonstrating typical velogenic properties in pathogenicity tests. Sequence analysis at the fusion protein cleavage site showed that the original isolate contained the typical avirulent type sequence, E-R-Q-E-R/L, which progressed incrementally to a typical virulent type, K-R-Q-K-R/F, during repeated passage in chickens. These results demonstrate that avirulent viruses, maintained in wild waterfowl in nature and bearing the consensus avirulent type sequence, have the potential to become velogenic after transmission to and circulation in chicken populations. The results also suggest that chickens provide a mechanism for the selection of virulent viruses from an avirulent background. (C) 2002 Elsevier Science (USA).
  • A Ninomiya, A Takada, K Okazaki, KF Shortridge, H Kida
    VETERINARY MICROBIOLOGY 88 (2) 107 - 114 0378-1135 2002/08 [Refereed][Not invited]
    Pig serum samples collected in southeastern China were examined for antibodies to influenza A viruses. Since the hemagglutination inhibition (HI) test does not accurately detect antibodies to the hemagglutinins (HAs) of "avian" influenza viruses, we utilized the neutralization (NT) test to detect subtype-specific antibodies to the HA of avian viruses in pig sera. Neutralizing antibodies to H1, H3, H4, and H5 influenza viruses were detected in the serum samples collected in 1977-1982 and 1998, suggesting that pigs in China have been sporadically infected with avian H4 and H5 viruses in addition to swine and human HI and H3 viruses. Antibodies to H9 virus, on the other hand, were found only in the sera collected in 1998, not in those collected in 1977-1982, correlating with the recent spread in poultry and subsequent isolation of H9N2 viruses from pigs and humans in 1998. The present results indicate that avian influenza viruses have been transmitted to pig populations in southeastern China. (C) 2002 Elsevier Science B.V. All rights reserved.
  • A Ninomiya, K Ogasawara, K Kajino, A Takada, H Kida
    VACCINE 20 (25-26) 3123 - 3129 0264-410X 2002/08 [Refereed][Not invited]
    Mucosal immunity is critical for protection from viral infections. We attempted to activate mucosal cytotoxic T lymphocytes (CTLs) specific for influenza A virus nucleoprotem (NP) which play an important role in protective immunity. It has been shown that dendritic cells (DCs) activated by signaling via CD40-CD40 ligand (CD40L) interaction are required for the differentiation of naive CD8(+) T cells into antigen-specific CTLs in a non-mucosal environment. We herein inoculated mice intranasally with an anti-CD40 monoclonal antibody (anti-CD40 mAb) and NP366-374 peptide, corresponding to a CTL epitope on NP, encapsulated in liposome (liposomal NP366-374) to induce protective CTL responses against influenza A virus. Intranasal but not subcutaneous immunization with liposomal NP366-374 effectively induced mucosal immunity to reduce virus replication in the lung, suggesting that anti-CD40 mAb also functioned as a mucosal adjuvant. Interestingly, neither MHC class I- nor class II-deficient mice immunized intranasally with these materials were resistant to the infection. Since anti-CD40 mAb was considered to help replace CD4(+) T cells, another help of CD4(+) T cells are presumably required for the induction of CTL activity in the lung. This approach may prove promising for developing vaccines to induce mucosal CTL responses, and seems to highlight differences between mucosal and non-mucosal immunity. (C) 2002 Elsevier Science Ltd. All rights reserved.
  • T Watanabe, S Watanabe, H Kida, Y Kawaoka
    VIROLOGY 299 (2) 266 - 270 0042-6822 2002/08 [Refereed][Not invited]
    We propose a rational approach to the design of live virus vaccines against influenza infection by alteration of the influenza A virus M2 protein, which is responsible for ion channel activity. Previously we demonstrated that a mutant A/WSN/33 (H1N1) influenza virus with defective M2 ion channel activity did not show appreciable growth defects in cell culture, although its growth was attenuated in mice (T Watanabe, S. Watanabe, H. Ito, H. Kida, and Y Kawaoka, 2001, J. Virol. 75, 5656-5662). Here, we show that this M2 ion channel defective mutant virus, the M2del29-31, protected mice against challenge with lethal doses of influenza virus, indicating the potential of incorporating this M2 alteration in a live influenza vaccine as one of the attenuating mutations. (C) 2002 Elsevier Science (USA).
  • Miyoshi M, Okazaki K, Takiguchi M, Kida H, Hashimoto A
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 7 64 (7) 627 - 631 0916-7250 2002/07 [Refereed][Not invited]
  • T Noda, H Sagara, E Suzuki, A Takada, H Kida, Y Kawaoka
    JOURNAL OF VIROLOGY 76 (10) 4855 - 4865 0022-538X 2002/05 [Refereed][Not invited]
    Using biochemical assays, it has been demonstrated that expression of Ebola virus VP40 alone in mammalian cells induced production of particles with a density similar to that of virions. To determine the morphological properties of these particles, cells expressing VP40 and the particles released from the cells were examined by electron microscopy. VP40 induced budding from the plasma membrane of filamentous particles, which differed in length but had uniform diameters of approximately 65 nm. When the Ebola virus glycoprotein (GP) responsible for receptor binding and membrane fusion was expressed in cells, we found pleomorphic particles budding from the plasma membrane. By contrast, when GP was coexpressed with VP40, GP was found on the filamentous particles induced by VP40. These results demonstrated the central role of VP40 in formation of the filamentous structure of Ebola virions and may suggest an interaction between VP40 and GP in morphogenesis.
  • T Watanabe, S Watanabe, G Neumann, H Kida, Y Kawaoka
    JOURNAL OF VIROLOGY 76 (2) 767 - 773 0022-538X 2002/01 [Refereed][Not invited]
    Despite the success of influenza virus vaccines in reducing severe illness, their efficacy is suboptimal. We describe here the immunogenicity and protective capacity of replication-incompetent influenza virus-like particles (VLPs) which were generated entirely from cDNAs and lacked either the entire NS gene (encoding both the NS1 and NS2 protein) or only the NS2 gene. In mammalian cells infected with NS gene-deficient VLPs, the nucleoprotein, but not other viral proteins including hemagglutinin (RA) and neuraminidase (NA), was detected. In contrast, cells infected with VLPs expressing NS1 but not NS2 (NS2 knockout) expressed multiple viral proteins, including RA and NA. When challenged with lethal doses of an antigenically homologous mouse-adapted influenza virus, 94% of mice-vaccinated with the NS2-knockout VLPs survived, compared with less than 10% of those given the NS-deficient VLPs. These results demonstrate the potential of replication-incompetent NS2-knockout VLPs as novel influenza vaccines and perhaps also as vectors to express genes from entirely unrelated pathogens.
  • K Ohishi, A Ninomiya, H Kida, CH Park, T Maruyama, T Arai, E Katsumata, T Tobayama, AN Boltunov, LS Khuraskin, N Miyazaki
    MICROBIOLOGY AND IMMUNOLOGY 46 (9) 639 - 644 0385-5600 2002 [Refereed][Not invited]
    Seroepidemiological surveillance of influenza in Caspian seals (Phoca caspica) was conducted. Antibodies to influenza A virus were detected in 54% (7/13), 57% (4/7), 40% (6/15) and 26% (11/42) of the serum samples collected in 1993, 1997, 1998 and 2000 by enzyme-linked immunosorbent assay (ELISA). In an hemagglutination-inhibition (HI) test using H1-H15 reference influenza A viruses as antigens, more than half of the examined ELISA-positive sera reacted with an H3N2 prototype strain A/Aichi/2/68. These sera were then examined by HI test with a series of naturally occurring antigenic variants of human H3N2 virus, and H3 viruses of swine, duck, and equine origin. The sera reacted strongly with the A/Bangkok/1/79 (H3N2) strain, which was prevalent in humans in 1979-1981. The present results indicate that human A/Bangkok/1/79-like virus was transmitted to Caspian seals probably in the early 1980s, and was circulated in the population. Antibodies to influenza B virus were detected by ELISA in 14% (1/7) and 10% (4/42) serum samples collected from Caspian seals in 1997 and 2000, respectively. Our findings indicate that seal might be a reservoir of both influenza A and B viruses originated from humans.

Books etc


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Awards & Honors

  • 2017/12 タマサート大学 名誉公衆衛生学博士号
    受賞者: 喜田 宏
  • 2017/11 文化功労者
    受賞者: 喜田 宏
  • 2017/05 春の叙勲 瑞宝重光章
    受賞者: 喜田 宏
  • 2016/10 北海道功労賞
    受賞者: 喜田 宏
  • 2011/11 All Japan Agriculture Decoration
     for Achievement of Control of Avian Influenza 
    受賞者: Hiroshi KIDA
  • 2009/02 Animal Science Prize
     Clarification of Ecology of Influenza Viruses and Establishment of Influenza VIrus Library and its Application to the Diagnosisand Prevention of Highly Pathogenic Avian Influenza 
    受賞者: 喜田宏
  • 2005/06 The 95th Japan Academy Prize
     Studies for the Control of Influenza - Mechanism of Emergence of Pandemic Influenza VIrus Strains in Polutry, Domestic Animals and Humans, and Molecular Basis of the Neutralization of Viral Infectivity with Antibodies- 
    受賞者: 喜田宏
  • 2005/04 The Japan Prize of Agricultural Science and the Yomiuri Agriculture Prize
     Ecology of Influenza Viruses 
    受賞者: 喜田宏
  • 2004/11 The 58th Hokkaido Shimbun Cultural Prize
     Ecology of Influenza Viruses in Birds, Animals and Humans 
    受賞者: 喜田宏
  • 2002/03 Hokkaido Science and Tachnology Award
     Studies on Mechanisms of Emergence of Pandemic Influenza A Viruses and their Control Measures 
    受賞者: 喜田宏
  • 1982/04 The Japanese Society of Veterinary Science Award
     Studies on the Classification of Avian Paramyxoviruses 
    受賞者: 喜田宏

Research Grants & Projects

  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2007 -2009 
    Author : Takashi UMEMURA, Chon-ho PARK, Hiroshi KIDA
    Specific antibodies detected in cerebrospinal fluid by intrathecal vaccination (vaccination by an inoculating antigen into subarachnoidal space) were produced de novo in the brain. Intrathecal vaccination completely prevented clinical signs and death of mice inoculated with rabies or pseudorabies viruses. Eighty percent of the mice intrathecally vaccinated and inoculated rabies virus directly into the brain survived, and this result suggested that the intrathecal vaccination was applicable for the treatment of rabid animals and humans. Our novel murine model of influenzal encephalopathy of ...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(S))
    Date (from‐to) : 2003 -2006 
    Author : Hiroshi KIDA, 岡崎 克則, 河岡 義裕, 迫田 義博, 梅村 孝司, 伊藤 壽啓, 小笠原 一誠
    H5N1 highly pathogenic avian influenza (HPAI) virus has spread in poultry from Southeast Asia to the north, Europe, and Africa. We have been conducting global surveillance of avian influenza and the assessment of pathogenicity of the H5 virus isolates in birds and mammals. In 2006, 55 avirulent avian influenza viruses were isolated from fecal sample of water birds. Thus, avian influenza viruses of 133 combinations of HA and NA subtypes have been stocked for use in vaccine and diagnosis.It was revealed that the H5N1 isolates in Mongolia in 2006 were closely related to each other and classifi...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 2004 -2005 
    Author : Katsunori OKAZAKI, 喜田 宏
    Glycoprotein B (gB) is the most conserved glycoprotein of herpesviruses and plays important roles in virus infectivity. The glycoprotein of most herpesviruses, including psuedorabies virus (PrV), is cleaved by a cellular protease into two disulfide-linked subunits. In the present study, we demonstrated that the glycoprotein generated in human colon carcinoma Lo Vo cells, which lack the ubiquitous protease furin, remained the uncleaved form and that the virus replicated in Lo Vo cells without cell fusion. The uncleaved gB was converted into the subunits after furin digestion. The virus also ...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(A))
    Date (from‐to) : 2000 -2002 
    Author : Hiroshi KIDA, 高田 礼人, 河岡 義裕, 岡崎 克則, 伊藤 壽啓, 迫田 義博
    To provide information on the precursor genes of the next pandemic influenza, epidemiological surveillance of animal and avian influenza viruses was performed. In total, 3,987 fecal samples of migratory waterfowls were collected in Russia, Mongolia, China and Holdcaido, Japan. From these samples, 212 influenza A viruses were isolated H1N2 swine influenza viruses were isolated from pigs with respiratory syndrome in Miyagi Prefecture, Japan. Twenty-five strains of H9N2 viruses were exchanged between 5 centers of global animal influenza network and stated analyzing these viruses.Genetic analys...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(A))
    Date (from‐to) : 2000 -2002 
    Author : Yoshihiro KAWAOKA, 堀本 泰介, 喜田 宏
    Influenza is an example of a disease for which the viral pathogen has emerged into the human population many times over past centuries, sometimes with devastating consequencesIn 1997, an H5N1 influenza A virus was transmitted from birds to humans in Hong Kong, killing 6 out of the 18 people infected. When mice were infected with human isolates, two virulence groups became apparent. Using reverse genetics, we showed that a mutation at position 627 in the PB2 protein influenced the outcome of infection in mice. Moreover, high cleavability of the hemagglutinin glycoprotein was essential requir...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(A))
    Date (from‐to) : 1999 -2001 
    Author : Toshihiro ITO, 河岡 義裕, 堀本 泰介, 喜田 宏, 大槻 公一, 伊藤 啓史
    An H5N1 avian influenza A virus was transmitted to humans in Hong Kong in 1997. Although the virus causes systemic infection and is highly lethal in chickens because of the susceptibility of the hemagglutinin to furin and PC6 proteases, their virulence in mice revealed two distinct groups of viruses : group 1, for which the dose lethal for 50 % of mice (MLD_<50>) was between 0.3 and 11 PFU, and group 2, for which the MLD_<50> was more than 10^3 PFU. The failure of some H5N1 viruses to produce systemic infection in our model indicates that multiple, factors contribute to the severity of H5N1...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(国際学術研究, 基盤研究(A))
    Date (from‐to) : 1998 -1999 
    Author : Hiroshi KIDA, 河岡 義裕, 伊藤 壽啓, 高田 礼人, 岡崎 克則
    Influenza A viruses of different subtypes were isolated from fecal samples of ducks in their nesting areas in Siberia. Phylogenetic analysis of the NP genes of the isolates in Siberia and those in Hokkaido, Japan on their flyway of migration from Siberia to the south in autumn revealed that they belong to the Eurasian lineage of avian influenza viruses. It is noted that the genes of the isolates in Siberia are closely related to those of H5N1 influenza virus strains isolated from chickens and humans in Hong Kong in 1997 as well as to those of isolates from domestic birds in southern China. ...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 1998 -1999 
    Author : Kazumasa OGASAWARA, 喜田 宏, 小野江 和則, 早瀬 ヨネ子
    1. According to the cassette theory, we prepared a synthetic peptide vaccine against H5 subtype influenza viruses. The peptide, however, could not induce neutralizing antibodies specific for H5 subtype influenza viruses in mice. This result suggests that all antibodies produced by immunizing with the peptides prepared according to the cassette theory could not recognize their original proteins. Thus, we are preparing a new peptide which can mimic the original protein. Namely, it contains a circular peptide formed with a disulfied bond and an I-AィイD1bィエD1 binding component (46F/54A).2. After...
  • 文部科学省:科学研究費補助金(萌芽的研究)
    Date (from‐to) : 1998 -1999 
    Author : 岡崎 克則, 高田 礼人, 喜田 宏
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(A))
    Date (from‐to) : 1996 -1998 
    Author : Hiroshi KIDA, 中里 幸和, 板倉 智敏, 伊藤 壽啓, 岡崎 克則, 前出 吉光, 藤田 正一, 高田 礼人, 梅村 孝司, 中里 幸和
    Intravenous injection into chickens with the virus-free extracts from the allantoic membrane of chicken embryos infected with influenza viruses resulted in sudden death due to blood coagulation in the vessels. The chickens injected intravenously with heparin prior to the injection with the extract survived without any signs of disease. The results suggest that some coagulation factors exist in the tissues where virus replicated and that they showed toxiciry to the chickens.To provide information on the pathogenically of fowl plague virus, virological and histopathological comparison was car...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 1996 -1997 
    Author : Katsunori OKAZAKI, 喜田 宏, 高田 礼人
    An escape mutant of bovine herpesvirus 1 (BHV1) was obtained in the presence of monoclonal antibody against glycoprotein B (gB) , which required complement for virus neutralization. The mutant was also resistant to other 3 monoclonal antibodies against gB,which exhibited complement-independent neutralizing activity. Amino acid substitution of gB of the mutant is now being analyzed. The cytotoxic activity of peripheral blood lymphocytes from BHV1-infected cattle is also being investigated using the mutant-infected primary culture as a target to study whether the amino acid substitution affec...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(国際学術研究)
    Date (from‐to) : 1995 -1997 
    Author : Hiroshi KIDA, 伊藤 壽啓, 河岡 義裕, 岡崎 克則, デメネフ V, ヤムニコバ S, ルボフ D.K, Svetrana Yam, 高田 礼人, Yamnikova Sv
    In each summer of 1995-1997,3,000 fecal materials of waterfowls and lake-water samples were collected in Kamchatka, Khabarovsk, and Saha Republic. In 1996, out of 900 fecal samples collected in Kobyaysky area of the basin of the River Lena, 60゚30'N,19 H4N6,1 H4N9,1 H11N1,2 H11N6, and 8 H11N9 influenza viruses were isolated. Although no virus was isolated from the samples collected in "40 islands" area of the River Lena (65゚00'-64゚34'N) , 1 H4N6 and 5 H3N8 viruses were isolated from 120 samples collected in Kobyaysky area and 72 samples in Yakutsk (62゚05'N) , respectively, in 1997. No influe...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 1996 -1996 
    Author : 伊藤 寿啓, 喜田 宏
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(一般研究(B), 基盤研究(B))
    Date (from‐to) : 1994 -1996 
    Author : Jiro ARIKAWA, 橋本 信夫, 谷口 孝喜, 喜田 宏, 東 市郎, 苅和 宏明
    1. Animal models for studying respiratory infection, enteric infection and systemic infections were established by using mice and Sendai virus, rotavirus and hantavirus, respectively.2. MDP-Lys (L18) was selected as a immunoadjuvant and examined its effect on the enhancement of protection from infection after its administration by oral, intranasal, intrarectal and subcutaneous routes.3. Fatal Sendai virus infection was significantly reduced by administration of MDP-Lys (L18) through intranasal, ora and even intra rectal route.4. Rota virus diarrhea was significantly reduced by administratio...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(一般研究(C))
    Date (from‐to) : 1994 -1995 
    Author : Toshihiro ITO, 喜田 宏
    The receptor specificity of the HA differs amongsinfluenza viruses : most avian influenza viruses preferentially bind the sialic acid-alpha-2,3-galactose (SAalpha2-3Gal) linkage, while human influenza viruses preferentially bind the SAalpha2,6Gal linkage on cell surface sialyloligosaccharides. To understand the molecular basis of host range restriction, we examined the distribution of sialyloligosaccharides at the replication site of influenza virus in nonhuman animals. Using linkage-specific lectins, we found that epitherial cells lining duck intestine and horse trachea contain SAalpha2,3G...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(試験研究(A))
    Date (from‐to) : 1993 -1995 
    Author : 清水 悠紀臣, Hiroshi KIDA, 大塚 治城, 喜田 宏, 関川 賢二, 見上 彪, 小野 悦郎, 岡部 達二, 笠井 憲雪
    Intracellular immunization using dominant-negative mutants of viral proteins is prorposed as a new approach to antiviral therapy in humans and to germ-line transformation in animals to confer resistance to viral infections. To obtain effective repressors of Aujeszky's disease virus (ADV) immediate-early (IE) gene expression, mutant genes encoding dominant-negative mutants of ADV IE protein (IE180) and early protein 0 (EPO), and a chimeric gene encoding a fusion protein consisting of the DNA-binding domain of IE180 and a tail-trucated herpes simplex virus 1 VP16 lacking the transcription act...
  • 文部科学省:科学研究費補助金(国際学術研究)
    Date (from‐to) : 1994 -1994 
    Author : 板倉 智敏, 余 鋭萍, 落合 謙爾, 喜田 宏
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(国際学術研究)
    Date (from‐to) : 1993 -1994 
    Author : Hiroshi KIDA, R.G.Webster, 河岡 義裕, 岡崎 克則, 伊藤 寿啓, Webster R.G
    To provide information how influenza A viruses are maintained among waterfowl reservoirs at their nesting areas in the northern territory and on the mechanism of perpetuation of the viruses in nature, virological surveillance of avian influenza was continued in Alaska during their breeding season in summer from 1993 to 1994. Phylogenetic analyzes of the genes of the isolates were then carried out.Influenza viruses were isolated mainly from fecal samples of dabbling ducks in their nesting places in central Alaska. From 541 fecal samples of ducks, 33 influenza viruses and 15 paramyxoviruses w...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(総合研究(A))
    Date (from‐to) : 1992 -1993 
    Author : Takeshi MIKAMI, 喜田 宏, 生田 和良, 小沼 操, 速水 正憲, 長谷川 篤彦
    The purpose of the present studies is to compare the molecular biological nature of lentiviruese isolated from various animals. In these studies, we tried to find the genetic properities commonly observed among lentiviruese and the mechanisms for expression of pathogenesis of the viruses in vivo at a molecular level. We report here about feline immunodeficiency virus as a representative.1) When compared the nucleotide sequences of LTR of FIV isolated from Japan, Australia and U.S., Japanese isolates were considerable genetic devergence from both Australian and U.S.idolates. These results sh...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(一般研究(B))
    Date (from‐to) : 1992 -1993 
    Author : Chitoshi ITAKURA, 御領 政信, 落合 謙爾, 喜田 宏
    A rabbit hemorrhageic disease (RHD) virus was isolated from Korean rabbits. The purified virus particles were 35 to 40 nm in diameter and had hollow depressions on their surface. The structural protein of the virus was composed of a single major polypeptide of 63 kDa.This virus had the high virulence and caused acute deaths in rabbits. Histopathological lesions caused by RHD virus were acute necrotizing hepatitis consisting of focal or solitary necrosis of hepatocytes.Immunohistochemically, RHD virus antigens were detected in degenrated and necrotic hepatocytes in formalin fixed tissues fro...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(国際学術研究)
    Date (from‐to) : 1991 -1992 
    Author : Hiroshi KIDA, 河岡 義裕, 岡崎 克則, 伊藤 寿啓, 小野 悦郎, 清水 悠紀臣
    On the basis of the results of epidemiological studies of influenza in birds, animals, and humans, we have proposed the route of introduction of the hemagglutinin(HA) gene of the A/Hong Kong/68(H3N2) strain, i.e., from a H3influenza virus circulating in migratory ducks to domestic ducks in Southern China, then to pigs where genetic reassortment occurred with the H2N2 Asian influenza virus to produce the new pandemic strain, A/Hong Kong/68(H3N2).To provide information on the distribution of influenza A viruses in waterfowls at their nesting points in the northern territory, virological surve...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(一般研究(A))
    Date (from‐to) : 1989 -1992 
    Author : Shigeo NAMIOKA, 林 正信, 笠井 憲雪, 田口 文広, 喜田 宏, 橋本 信夫
    It has been shown that antisense RNAs and oligonucleotides against viral genes can be used as tools for inhibiting viral gene expression and viral replication in vitro. These results gave rise to the concept of unnatural intracellular antiviral immunity driven by specific antisense RNA. Recently, we reported that in the transfected DBT cell lines which expressed antisense RNA against the nucleocapsid (N) protein gene of mouse hepatitis virus(MHV), viral transcription and multiplication were inhibited. In order to provide further information concerning the ability of antisense RNA to inhibit...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(総合研究(A))
    Date (from‐to) : 1990 -1991 
    Author : Takeshi MIKAMI, 小沼 操, 松浦 善治, 川喜田 正夫, 喜田 宏, 永井 美之, 児玉 洋
    The purpose of the present study is to evaluate the ecology and pathogenicity of Newcastle disease virus (NDV). The hemagglutinin-neuraminidase (HN) and fusion (F) proteins of NDV were known to play key roles in the course of NDV infection. We examined biological and immunological properties of the expressed HN or F protein after inserting these genes into vaccinia virus (VN), fowlpox virus (FPV) or baculovius (BV). The following results were obtained.1) The role of immune responses to HN protein in protection against NDV infection was investigated using a recombinant vaccinia virus (rVV-HN...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(一般研究(A))
    Date (from‐to) : 1990 -1991 
    Author : Yukio SHIMIZU, 小野 悦郎, 首藤 文栄, 桑原 幹典, 喜田 宏, 伊藤 寿啓
    Newcastle disease virus (NDV) isolates from domestic and feral birds were antigenically analyzed and their virulence for chicken embryos was determined. The results indicate that virulent strains are still prevailing in domestic fowl populations including those in Japan, that heterogeneous NDV strains of different antigenicity and virulence are maintained by water fowls and cocirculating in nature, and that virulent NDV strains antigenically different from vaccine strains may be introduced into domestic fowls from migratory birds.In order to provide information on the factors responsible fo...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(一般研究(B))
    Date (from‐to) : 1989 -1990 
    Author : Hiroshi KIDA
    The H3 hemagglutinins (HA) of influenza virus isolates from domestic ducks in southern China were analyzed antigenically and genetically. The results provided molecular evidence of a role of these birds to introduce avian H3 influenza viruses to pigs as "mixing vessels" where A/Hong Kong/68 (H3N2) strain was produced.Experimental infection of pigs with H1-13 influenza A virus strains isolated from different host species provided the follwing informations ;The cells lining the upper respiratory tract of pig were susceptible not only to the HA of H3 influenza viruses of porcine and human orig...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(一般研究(A))
    Date (from‐to) : 1987 -1990 
    Author : 佐藤 文昭, 斉藤 文昭, Fumiaki SATO, 見上 彪, 久保 周一郎, 遠藤 大二, 林 正信, 桑原 幹典, 喜田 宏, 児玉 洋, 小沼 操, 見上 彪
    The aim of this research project is to get basic data to develop recombinant vaccines for veterinarians. Recombinant vaccines consisted of two components, vector virus and antigen genes. In this study, we used two viruses which can be used for vector of recombinant vaccines. One is herpesvirus of turkeys which is used as a vaccine of Marek's disease virus and the other is fowl pox virus. We decided thymidine kinase gene as a marker for genetic recombination between the vector virus gnome and antigen gene. And hemagglutinin-neuraminidase gene of Newcastle disease virus was cloned into thymid...

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