Researcher Profile and Settings

Master

Affiliation (Master)

• Institute for Genetic Medicine　Laboratory of Animal Experiment

Affiliation (Master)

• Institute for Genetic Medicine　Laboratory of Animal Experiment

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Profile and Settings

Degree

• (BLANK)（Hokkaido University）
• (BLANK)（Hokkaido University）

Profile and Settings

• Name (Japanese)

  MORIMATSU

• Name (Kana)

  Kumiko

• Name

  200901099501828291

通称等の別名

Yoshimatsu Kumiko

Achievement

Research Interests

• bunyaviruses   腎症候性出血熱   HFRS   ワクチン   核蛋白   実験動物   診断法   マウス   抗原提示   診断抗原   カイコ   Chronic kidney disease   インテグリン   感染モデル   膜蛋白   抗原性   エピトープ   組換えバキュロウイルス   組み換え抗原   外皮蛋白   ペプチドワクチン   細胞融合   モノクローナル抗体   中和抗体   レセプター   精製法   感染防御   エンベロープ   血清学   齧歯類   人獣共通感染症   ハンタウイルス   serology   zoonosis   hantavirus   

Research Areas

• Life sciences / Hygiene and public health (non-laboratory)
• Life sciences / Hygiene and public health (laboratory)
• Life sciences / Healthcare management, medical sociology
• Life sciences / Virology
• Life sciences / Applied molecular and cellular biology
• Life sciences / Veterinary medicine
• Life sciences / Laboratory animal science

Research Experience

• 2019/07 - Today Institute for Genetic Medicine, Hokkaido University Laboratory of Animal Experimentation Associate Professor
• 2011/05 - 2019/06 Graduate School of Medicine, Hokkaido University Department of Microbiology and Immunology Associate Professor
• 1997/05 - 2011/05 Hokkaido Univesristy School of Medicine Institute for Animal Experimentation Assitant Professor
• 1989 - 1997 北海道大学免疫科学研究所 教務職員

Education

•        - 1989  Hokkaido University
•        - 1989  Hokkaido University  Graduate School, Division of Veterinary Medicine

Published Papers

• Development of an entirely cloned cDNA-based reverse genetics system for Tofla virus of orthonairovirus

  Shelly Wulandari, Samuel Nyampong, Sithumini M.W. Lokupathirage, Kumiko Yoshimatsu, Hiroshi Shimoda, Daisuke Hayasaka

  Virology 598 110170 - 110170 0042-6822 2024/10
• Virological characteristics of the SARS-CoV-2 Omicron XBB.1.5 variant

  Tomokazu Tamura, Takashi Irie, Sayaka Deguchi, Hisano Yajima, Masumi Tsuda, Hesham Nasser, Keita Mizuma, Arnon Plianchaisuk, Saori Suzuki, Keiya Uriu, Mst Monira Begum, Ryo Shimizu, Michael Jonathan, Rigel Suzuki, Takashi Kondo, Hayato Ito, Akifumi Kamiyama, Kumiko Yoshimatsu, Maya Shofa, Rina Hashimoto, Yuki Anraku, Kanako Terakado Kimura, Shunsuke Kita, Jiei Sasaki, Kaori Sasaki-Tabata, Katsumi Maenaka, Naganori Nao, Lei Wang, Yoshitaka Oda, Hirofumi Sawa, Ryoko Kawabata, Yukio Watanabe, Ayaka Sakamoto, Naoko Yasuhara, Tateki Suzuki, Yukari Nakajima, Zannatul Ferdous, Kenji Shishido, Yuka Mugita, Otowa Takahashi, Kimiko Ichihara, Yu Kaku, Naoko Misawa, Ziyi Guo, Alfredo Hinay, Yusuke Kosugi, Shigeru Fujita, Jarel M. Tolentino, Luo Chen, Lin Pan, Mai Suganami, Mika Chiba, Ryo Yoshimura, Kyoko Yasuda, Keiko Iida, Naomi Ohsumi, Adam P. Strange, Yuki Shibatani, Tomoko Nishiuchi, Shiho Tanaka, Olivia Putri, Gustav Joas, Yoonjin Kim, Daichi Yamasoba, Kazuhisa Yoshimura, Kenji Sadamasu, Mami Nagashima, Hiroyuki Asakura, Isao Yoshida, So Nakagawa, Akifumi Takaori-Kondo, Kotaro Shirakawa, Kayoko Nagata, Ryosuke Nomura, Yoshihito Horisawa, Yusuke Tashiro, Yugo Kawai, Takamasa Ueno, Chihiro Motozono, Mako Toyoda, Terumasa Ikeda, Akatsuki Saito, Keita Matsuno, Jumpei Ito, Shinya Tanaka, Kei Sato, Takao Hashiguchi, Kazuo Takayama, Takasuke Fukuhara

  Nature Communications 15 (1) 2024/02/08 

  Abstract Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the S486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determine the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. We provide the intrinsic pathogenicity of XBB.1 and XBB.1.5 in hamsters. Importantly, we find that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC suppression. In vivo experiments using recombinant viruses reveal that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, our study identifies the two viral functions defined the difference between XBB.1 and XBB.1.5.
• Akaluc bioluminescence offers superior sensitivity to trackin vivodynamics of SARS-CoV-2 infection

  Tomokazu Tamura, Hayato Ito, Shiho Torii, Lei Wang, Rigel Suzuki, Shuhei Tusjino, Akifumi Kamiyama, Yoshitaka Oda, Yuhei Morioka, Saori Suzuki, Kotaro Shirakawa, Kei Sato, Kumiko Yoshimatsu, Yoshiharu Matsuura, Satoshi Iwano, Shinya Tanaka, Takasuke Fukuhara

  2023/10/13 

  Summary Monitoringin vivoviral dynamics can improve our understanding of pathogenicity and tissue tropism. For positive-sense, single-stranded RNA viruses, several studies have attempted to monitor viral kineticsin vivousing reporter genomes. The application of such recombinant viruses can be limited by challenges in accommodating bioluminescent reporter genes in the viral genome. Conventional luminescence also exhibits relatively low tissue permeability and thus less sensitivity for visualizationin vivo. Here we show that unlike NanoLuc bioluminescence, the improved method, termed AkaBLI, allows visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Syrian hamsters. By successfully incorporating a codon-optimized Akaluc luciferase gene into the SARS-CoV-2 genome, we visualizedin vivoinfection, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of neutralizing antibodies and mRNA vaccination by monitoring changes in Akaluc signals. Overall, AkaBLI is an effective technology for monitoring viral dynamics in live animals.
• Multiple mutations of SARS-CoV-2 Omicron BA.2 variant orchestrate its virological characteristics.

  Izumi Kimura, Daichi Yamasoba, Hesham Nasser, Hayato Ito, Jiri Zahradnik, Jiaqi Wu, Shigeru Fujita, Keiya Uriu, Jiei Sasaki, Tomokazu Tamura, Rigel Suzuki, Sayaka Deguchi, Arnon Plianchaisuk, Kumiko Yoshimatsu, Yasuhiro Kazuma, Shuya Mitoma, Gideon Schreiber, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Akifumi Takaori-Kondo, Jumpei Ito, Kotaro Shirakawa, Kazuo Takayama, Takashi Irie, Takao Hashiguchi, So Nakagawa, Takasuke Fukuhara, Akatsuki Saito, Terumasa Ikeda, Kei Sato

  Journal of virology e0101123  2023/10/05 

  Previous studies on the Omicron BA.2 variant suggested that the virological characteristics of BA.2 are determined by the mutations in at least two different regions of the viral genome: in the BA.2 spike gene (enhancing viral fusogenicity and intrinsic pathogenicity) and the non-spike region of the BA.2 genome (leading to intrinsic pathogenicity attenuation). However, the mutations modulating the BA.2 virological properties remain elusive. In this study, we demonstrated that the L371F substitution in the BA.2 spike protein confers greater fusogenicity and intrinsic pathogenicity. Furthermore, we revealed that multiple mutations downstream of the spike gene in the BA.2 genome are responsible for attenuating intrinsic viral pathogenicity and replication capacity. As mutations in the SARS-CoV-2 variant spike proteins could modulate certain virological properties, such as immune evasion and infectivity, most studies have previously focused on spike protein mutations. Our results underpin the importance of non-spike protein-related mutations in SARS-CoV-2 variants. IMPORTANCE Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.
• Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5.

  Tomokazu Tamura, Daichi Yamasoba, Yoshitaka Oda, Jumpei Ito, Tomoko Kamasaki, Naganori Nao, Rina Hashimoto, Yoichiro Fujioka, Rigel Suzuki, Lei Wang, Hayato Ito, Yukie Kashima, Izumi Kimura, Mai Kishimoto, Masumi Tsuda, Hirofumi Sawa, Kumiko Yoshimatsu, Yuki Yamamoto, Tetsuharu Nagamoto, Jun Kanamune, Yutaka Suzuki, Yusuke Ohba, Isao Yokota, Keita Matsuno, Kazuo Takayama, Shinya Tanaka, Kei Sato, Takasuke Fukuhara

  Communications biology 6 (1) 772 - 772 2023/07/24 

  The unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates ongoing control measures. Given its rapid spread, the new Omicron subvariant BA.5 requires urgent characterization. Here, we comprehensively analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1. Although in vitro growth kinetics of BA.5 was comparable among the Omicron subvariants, BA.5 was much more fusogenic than BA.1 and BA.2. Airway-on-a-chip analysis showed that, among Omicron subvariants, BA.5 had enhanced ability to disrupt the respiratory epithelial and endothelial barriers. Furthermore, in our hamster model, in vivo pathogenicity of BA.5 was slightly higher than that of the other Omicron variants and less than that of ancestral B.1.1. Notably, BA.5 gains efficient virus spread compared with BA.1 and BA.2, leading to prompt immune responses. Our findings suggest that BA.5 has low pathogenicity compared with the ancestral strain but enhanced virus spread /inflammation compared with earlier Omicron subvariants.
• Insufficient serological evidence of the association between chronic kidney disease and leptospirosis in Badulla and Kandy districts, Sri Lanka

  Regina Amanda Fonseka, Pavani Senarathne, Devinda Shameera Muthusinghe, Nishantha Nanayakkara, Lishantha Gunaratne, Kumiko Yoshimatsu, Nobuo Koizumi, Chandika Damesh Gamage

  2023/06/01
• Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants.

  Tomokazu Tamura, Jumpei Ito, Keiya Uriu, Jiri Zahradnik, Izumi Kida, Yuki Anraku, Hesham Nasser, Maya Shofa, Yoshitaka Oda, Spyros Lytras, Naganori Nao, Yukari Itakura, Sayaka Deguchi, Rigel Suzuki, Lei Wang, Mst Monira Begum, Shunsuke Kita, Hisano Yajima, Jiei Sasaki, Kaori Sasaki-Tabata, Ryo Shimizu, Masumi Tsuda, Yusuke Kosugi, Shigeru Fujita, Lin Pan, Daniel Sauter, Kumiko Yoshimatsu, Saori Suzuki, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Yuki Yamamoto, Tetsuharu Nagamoto, Gideon Schreiber, Katsumi Maenaka, Takao Hashiguchi, Terumasa Ikeda, Takasuke Fukuhara, Akatsuki Saito, Shinya Tanaka, Keita Matsuno, Kazuo Takayama, Kei Sato

  Nature communications 14 (1) 2800 - 2800 2023/05/16 [Refereed]
   

  In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions.
• Convergent evolution of SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant.

  Jumpei Ito, Rigel Suzuki, Keiya Uriu, Yukari Itakura, Jiri Zahradnik, Kanako Terakado Kimura, Sayaka Deguchi, Lei Wang, Spyros Lytras, Tomokazu Tamura, Izumi Kida, Hesham Nasser, Maya Shofa, Mst Monira Begum, Masumi Tsuda, Yoshitaka Oda, Tateki Suzuki, Jiei Sasaki, Kaori Sasaki-Tabata, Shigeru Fujita, Kumiko Yoshimatsu, Hayato Ito, Naganori Nao, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Yuki Yamamoto, Tetsuharu Nagamoto, Jin Kuramochi, Gideon Schreiber, Akatsuki Saito, Keita Matsuno, Kazuo Takayama, Takao Hashiguchi, Shinya Tanaka, Takasuke Fukuhara, Terumasa Ikeda, Kei Sato

  Nature communications 14 (1) 2671 - 2671 2023/05/11 [Refereed]
   

  In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022.
• Effects of Diazinon Exposure on Urinary Shedding of Leptospira interrogans Serogroup Hebdomadis in Mice

  So Shinya, Devinda S. Muthusinghe, Nobuo Koizumi, Kumiko Yoshimatsu, Shouta M. M. Nakayama, Mayumi Ishizuka, Yoshinori Ikenaka

  Toxics 11 (4) 2023/04/11 

  Wild rodents are natural hosts of Leptospira spp. and are exposed to various pesticides, some of which are immunotoxic. Rodent urine is an important source of infection for humans and other animals. We evaluated the effects of pesticide exposure on Leptospira growth in mice. Diazinon, at doses of 0.2, 1, and 5 mg/kg/day, was orally administered continuously to mice infected with Leptospira interrogans serogroup Hebdomadis for 32 days. The numbers of L. interrogans in urine and kidney tissues were significantly lower in mice exposed to 5 mg/kg/day diazinon than in unexposed mice (p < 0.05). The urinary concentration of 2-isopropyl-6-methyl-4-pyrimidinol, the metabolite of diazinon, was comparable with the concentration at which viability of L. interrogans was decreased in in vitro assay, suggesting that it had toxic effects on L. interrogans in the proximal renal tubules. Diazinon exposure reinforced Leptospira-induced expression of inflammatory cytokine genes in kidney tissues, and an enhanced immune system might suppress Leptospira growth. These results suggest that diazinon exposure may not increase the risk of Leptospira transmission to humans through mice. This novel study evaluated the relationship between pesticide exposure and Leptospira infection in mice, and the results could be useful for risk assessment of leptospirosis.
• Hantavirus infection as a risk factor for chronic kidney disease of unknown aetiology (CKDu) and its prevalence in endemic areas of Sri Lanka since 2010 according to a retrospective serological analysis

  Chandika Damesh Gamage, Shanika Nanayakkara, Yomani D. Sarathkumara, Devinda S. Muthusinghe, Kenta Shimizu, Jiro Arikawa, Sithumini M. W. Lokupathirage, Nishantha Nanayakkara, Lishanthe Gunarathne, Rohana Chandrajith, Kouji H. Harada, Akio Koizumi, Kumiko Yoshimatsu

  Journal of Medical Microbiology 71 (12) 0022-2615 2022/12/16 [Refereed][Not invited]
   

  Background. Chronic kidney disease of unknown aetiology (CKDu) is a major public health problem in Sri Lanka, especially among agrarian communities. Although the cause of CKDu is still unknown, hantavirus infection has been proposed as a risk factor. Methods. This study was performed using serological samples collected from two CKDu-endemic areas, Anuradhapura (2010) and Badulla districts (2010 and 2016), and a non-endemic area, Matale (2016) district. The presence of anti-Thailand orthohantavirus IgG antibodies was investigated in serum samples. Hantavirus seroprevalence and demographic data were epidemiologically analysed. Results. Seroprevalence was higher in CKDu patients (40.6–60.0 %) and healthy individuals in CKDu-endemic areas (17.6–25.5 %) than in healthy individuals in non-endemic areas (3.0 %). Statistically significant odds ratios (ORs) for hantavirus infection in CKDu patients were detected in CKDu-endemic areas [ORs: 3.2 and 3.1; 95 % confidence interval (CI): 1.8–5.5 and 1.8–5.2 in Anuradhapura and Badulla districts in 2010; and OR: 4.4, 95 % CI: 2.3–8.5 in 2016 in Badulla district). Furthermore, the OR for hantavirus infection in Badulla district has increased in the last decade from 3.1 (95 % CI: 1.8–5.3) to 4.4 (95 % CI: 2.3–8.5). Conclusion. Hantavirus infection has been prevalent in two distant CKDu-endemic areas since 2010. The observed significant association of hantavirus seropositivity with CKDu indicates a possible role of hantavirus infection in CKDu pathogenesis.
• Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant.

  Akatsuki Saito, Tomokazu Tamura, Jiri Zahradnik, Sayaka Deguchi, Koshiro Tabata, Yuki Anraku, Izumi Kimura, Jumpei Ito, Daichi Yamasoba, Hesham Nasser, Mako Toyoda, Kayoko Nagata, Keiya Uriu, Yusuke Kosugi, Shigeru Fujita, Maya Shofa, Mst Monira Begum, Ryo Shimizu, Yoshitaka Oda, Rigel Suzuki, Hayato Ito, Naganori Nao, Lei Wang, Masumi Tsuda, Kumiko Yoshimatsu, Jin Kuramochi, Shunsuke Kita, Kaori Sasaki-Tabata, Hideo Fukuhara, Katsumi Maenaka, Yuki Yamamoto, Tetsuharu Nagamoto, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Takamasa Ueno, Gideon Schreiber, Akifumi Takaori-Kondo, Kotaro Shirakawa, Hirofumi Sawa, Takashi Irie, Takao Hashiguchi, Kazuo Takayama, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, Kei Sato

  Cell host & microbe 30 (11) 1540 - 1555 2022/11/09 

  The SARS-CoV-2 Omicron BA.2.75 variant emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically distinct from BA.5, the currently predominant BA.2 descendant. Here, we show that BA.2.75 has a greater effective reproduction number and different immunogenicity profile than BA.5. We determined the sensitivity of BA.2.75 to vaccinee and convalescent sera as well as a panel of clinically available antiviral drugs and antibodies. Antiviral drugs largely retained potency, but antibody sensitivity varied depending on several key BA.2.75-specific substitutions. The BA.2.75 spike exhibited a profoundly higher affinity for its human receptor, ACE2. Additionally, the fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were greater than those of BA.2. Our multilevel investigations suggest that BA.2.75 acquired virological properties independent of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.
• Virological characteristics of the SARS-CoV-2 Omicron BA.2 subvariants, including BA.4 and BA.5.

  Izumi Kimura, Daichi Yamasoba, Tomokazu Tamura, Naganori Nao, Tateki Suzuki, Yoshitaka Oda, Shuya Mitoma, Jumpei Ito, Hesham Nasser, Jiri Zahradnik, Keiya Uriu, Shigeru Fujita, Yusuke Kosugi, Lei Wang, Masumi Tsuda, Mai Kishimoto, Hayato Ito, Rigel Suzuki, Ryo Shimizu, Mst Monira Begum, Kumiko Yoshimatsu, Kanako Terakado Kimura, Jiei Sasaki, Kaori Sasaki-Tabata, Yuki Yamamoto, Tetsuharu Nagamoto, Jun Kanamune, Kouji Kobiyama, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Kotaro Shirakawa, Akifumi Takaori-Kondo, Jin Kuramochi, Gideon Schreiber, Ken J Ishii, Takao Hashiguchi, Terumasa Ikeda, Akatsuki Saito, Takasuke Fukuhara, Shinya Tanaka, Keita Matsuno, Kei Sato

  Cell 185 (21) 3992 - 4007 2022/10/13 [Refereed]
   

  After the global spread of the SARS-CoV-2 Omicron BA.2, some BA.2 subvariants, including BA.2.9.1, BA.2.11, BA.2.12.1, BA.4, and BA.5, emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these BA.2 subvariants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1/2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. We further provided the structure of the BA.4/5 spike receptor-binding domain that binds to human ACE2 and considered how the substitutions in the BA.4/5 spike play roles in ACE2 binding and immune evasion. Moreover, experiments using hamsters suggested that BA.4/5 is more pathogenic than BA.2. Our multiscale investigations suggest that the risk of BA.2 subvariants, particularly BA.4/5, to global health is greater than that of original BA.2.
• Pathological Studies on Hantaan Virus-Infected Mice Simulating Severe Hemorrhagic Fever with Renal Syndrome

  Zhouoxing Wei, Kenta Shimizu, Rakiiya S. Sarii, Devinda S. Muthusinghe, Sithumini M. W. Lokupathirage, Junko Nio-Kobayashi, Kumiko Yoshimatsu

  Viruses 14 (10) 2247 - 2247 2022/10/13 [Refereed]
   

  Hantaan virus is the causative agent of hemorrhagic fever with renal syndrome (HFRS). The Hantaan virus strain, Korean hemorrhagic fever virus clone-5 (KHF5), causes weight loss and renal hemorrhage in laboratory mice. Clone-4 (KHF4), which has a single E417K amino acid change in its glycoprotein, is an avirulent variant. In this study, KHF4 and KHF5 were compared to evaluate pathological differences in mice in vitro and in vivo. The characteristics of the two glycoproteins were not significantly different in vitro. However, the virulent KHF5 strain targeted the lungs and caused pneumonia and edema in vivo. Both strains induced high infectivity levels in the liver and caused hepatitis; however, petechial hemorrhage and glycogen storage reduction were observed in KHF5-infected mice alone. Renal hemorrhage was observed using viral antigens in the tubular region of KHF5-infected mice. In addition, an increase in white blood cell levels and neutrophilia were found in KHF5-infected mice. Microarray analysis of liver cells showed that CD8+ T cell activation, acute-phase protein production, and neutrophil activation was induced by KHF5 infection. KHF5 infectivity was significantly increased in vivo and the histological and clinicopathological findings were similar to those in patients with HFRS.
• Identification of cap-dependent endonuclease inhibitors with broad-spectrum activity against bunyaviruses.

  Shinsuke Toba, Akihiko Sato, Makoto Kawai, Yoshiyuki Taoda, Yuto Unoh, Shinji Kusakabe, Haruaki Nobori, Shota Uehara, Kentaro Uemura, Keiichi Taniguchi, Masanori Kobayashi, Takeshi Noshi, Ryu Yoshida, Akira Naito, Takao Shishido, Junki Maruyama, Slobodan Paessler, Michael J Carr, William W Hall, Kumiko Yoshimatsu, Jiro Arikawa, Keita Matsuno, Yoshihiro Sakoda, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Hiroshi Kida

  Proceedings of the National Academy of Sciences of the United States of America 119 (36) e2206104119  0027-8424 2022/09/06 [Refereed]
   

  Viral hemorrhagic fevers caused by members of the order Bunyavirales comprise endemic and emerging human infections that are significant public health concerns. Despite the disease severity, there are few therapeutic options available, and therefore effective antiviral drugs are urgently needed to reduce disease burdens. Bunyaviruses, like influenza viruses (IFVs), possess a cap-dependent endonuclease (CEN) that mediates the critical cap-snatching step of viral RNA transcription. We screened compounds from our CEN inhibitor (CENi) library and identified specific structural compounds that are 100 to 1,000 times more active in vitro than ribavirin against bunyaviruses, including Lassa virus, lymphocytic choriomeningitis virus (LCMV), and Junin virus. To investigate their inhibitory mechanism of action, drug-resistant viruses were selected in culture. Whole-genome sequencing revealed that amino acid substitutions in the CEN region of drug-resistant viruses were located in similar positions as those of the CEN α3-helix loop of IFVs derived under drug selection. Thus, our studies suggest that CENi compounds inhibit both bunyavirus and IFV replication in a mechanistically similar manner. Structural analysis revealed that the side chain of the carboxyl group at the seventh position of the main structure of the compound was essential for the high antiviral activity against bunyaviruses. In LCMV-infected mice, the compounds significantly decreased blood viral load, suppressed symptoms such as thrombocytopenia and hepatic dysfunction, and improved survival rates. These data suggest a potential broad-spectrum clinical utility of CENis for the treatment of both severe influenza and hemorrhagic diseases caused by bunyaviruses.
• Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike.

  Daichi Yamasoba, Izumi Kimura, Hesham Nasser, Yuhei Morioka, Naganori Nao, Jumpei Ito, Keiya Uriu, Masumi Tsuda, Jiri Zahradnik, Kotaro Shirakawa, Rigel Suzuki, Mai Kishimoto, Yusuke Kosugi, Kouji Kobiyama, Teppei Hara, Mako Toyoda, Yuri L Tanaka, Erika P Butlertanaka, Ryo Shimizu, Hayato Ito, Lei Wang, Yoshitaka Oda, Yasuko Orba, Michihito Sasaki, Kayoko Nagata, Kumiko Yoshimatsu, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Jin Kuramochi, Motoaki Seki, Ryoji Fujiki, Atsushi Kaneda, Tadanaga Shimada, Taka-Aki Nakada, Seiichiro Sakao, Takuji Suzuki, Takamasa Ueno, Akifumi Takaori-Kondo, Ken J Ishii, Gideon Schreiber, Hirofumi Sawa, Akatsuki Saito, Takashi Irie, Shinya Tanaka, Keita Matsuno, Takasuke Fukuhara, Terumasa Ikeda, Kei Sato

  Cell 185 (12) 2103 - 2115 2022/06/09 [Refereed]
   

  Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.
• Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant.

  Rigel Suzuki, Daichi Yamasoba, Izumi Kimura, Lei Wang, Mai Kishimoto, Jumpei Ito, Yuhei Morioka, Naganori Nao, Hesham Nasser, Keiya Uriu, Yusuke Kosugi, Masumi Tsuda, Yasuko Orba, Michihito Sasaki, Ryo Shimizu, Ryoko Kawabata, Kumiko Yoshimatsu, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Hirofumi Sawa, Terumasa Ikeda, Takashi Irie, Keita Matsuno, Shinya Tanaka, Takasuke Fukuhara, Kei Sato

  Nature 603 (7902) 700 - 705 2022/03 [Refereed]
   

  The emergence of the Omicron variant of SARS-CoV-2 is an urgent global health concern1. In this study, our statistical modelling suggests that Omicron has spread more rapidly than the Delta variant in several countries including South Africa. Cell culture experiments showed Omicron to be less fusogenic than Delta and than an ancestral strain of SARS-CoV-2. Although the spike (S) protein of Delta is efficiently cleaved into two subunits, which facilitates cell-cell fusion2,3, the Omicron S protein was less efficiently cleaved compared to the S proteins of Delta and ancestral SARS-CoV-2. Furthermore, in a hamster model, Omicron showed decreased lung infectivity and was less pathogenic compared to Delta and ancestral SARS-CoV-2. Our multiscale investigations reveal the virological characteristics of Omicron, including rapid growth in the human population, lower fusogenicity and attenuated pathogenicity.
• Subcellular localization of nucleocapsid protein of SFTSV and its assembly into the ribonucleoprotein complex with L protein and viral RNA.

  Sithumini M W Lokupathirage, Yoshimi Tsuda, Kodai Ikegame, Kisho Noda, Devinda S Muthusinghe, Fumiya Kozawa, Rashid Manzoor, Kenta Shimizu, Kumiko Yoshimatsu

  Scientific reports 11 (1) 22977 - 22977 2021/11/26 [Refereed]
   

  Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes novel zoonotic diseases in Asian countries including China, Japan, South Korea, and Vietnam. In phleboviruses, viral proteins play a critical role in viral particle formation inside the host cells. Viral glycoproteins (GPs) and RNA-dependent RNA polymerase (RdRp) are colocalized in the Golgi apparatus and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The nucleocapsid (N) protein was widely expressed in the cytoplasm, even in cells coexpressing GP. However, the role of SFTSV N protein remains unclear. The subcellular localization of SFTSV structural proteins was investigated using a confocal microscope. Subsequently, minigenome and immunoprecipitation assays were carried out. The N protein interacts with viral RNA (vRNA) and further shows translational activity with RdRp which is L protein and localized in the ERGIC and Golgi apparatus when co-expressed with GP. On the other hand, mutant N protein did not interact with vRNA either localized in the ERGIC or Golgi apparatus. The interaction between the N protein of SFTSV and vRNA is important for the localization of viral proteins and viral assembly. This study provides useful insights into the life cycle of SFTSV, which will lead to the detection of antiviral targets.
• Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation.

  Akatsuki Saito, Takashi Irie, Rigel Suzuki, Tadashi Maemura, Hesham Nasser, Keiya Uriu, Yusuke Kosugi, Kotaro Shirakawa, Kenji Sadamasu, Izumi Kimura, Jumpei Ito, Jiaqi Wu, Kiyoko Iwatsuki-Horimoto, Mutsumi Ito, Seiya Yamayoshi, Samantha Loeber, Masumi Tsuda, Lei Wang, Seiya Ozono, Erika P Butlertanaka, Yuri L Tanaka, Ryo Shimizu, Kenta Shimizu, Kumiko Yoshimatsu, Ryoko Kawabata, Takemasa Sakaguchi, Kenzo Tokunaga, Isao Yoshida, Hiroyuki Asakura, Mami Nagashima, Yasuhiro Kazuma, Ryosuke Nomura, Yoshihito Horisawa, Kazuhisa Yoshimura, Akifumi Takaori-Kondo, Masaki Imai, Shinya Tanaka, So Nakagawa, Terumasa Ikeda, Takasuke Fukuhara, Yoshihiro Kawaoka, Kei Sato

  Nature 602 (7896) 300 - 306 2021/11/25 [Refereed]
   

  During the current SARS-CoV-2 pandemic, a variety of mutations have accumulated in the viral genome, and currently, four variants of concern (VOCs) are considered potentially hazardous to human society1. The recently emerged B.1.617.2/Delta VOC is closely associated with the COVID-19 surge that occurred in India in the spring of 20212. However, its virological properties remain unclear. Here, we show that the B.1.617.2/Delta variant is highly fusogenic and notably more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates spike protein cleavage and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity than its parental virus. Our data suggest that the P681R mutation is a hallmark of the virological phenotype of the B.1.617.2/Delta variant and is associated with enhanced pathogenicity.
• Identification of Novel Rodent-Borne Orthohantaviruses in an Endemic Area of Chronic Kidney Disease of Unknown Etiology (CKDu) in Sri Lanka

  Devinda S. Muthusinghe, Kenta Shimizu, Sithumini M, W. Lokupathirage, Zhouoxing Wei, Yomani D. Sarathkumara, G. R. Am, a Fonseka, Pavani Senarathne, Nobuo Koizumi, Tomonori Kawakami, Akio Koizumi, Chaminda Wickramasinghe, Hideki Ebihara, Keita Matsuno, Yoshimi Tsuda, Jiro Arikawa, Chandika D. Gamage, Kumiko Yoshimatsu

  Viruses 13 (10) 1984 - 1984 1999-4915 2021/10/02 [Refereed]
   

  We reported the genetic evidence of circulating hantaviruses from small mammals captured in a chronic kidney disease of unknown etiology (CKDu) hotspot area of Sri Lanka. The high seroprevalence of anti-hantavirus antibodies against Thailand orthohantavirus (THAIV) has been reported among CKDu patients and rodents in Sri Lankan CKDu hotspots. We captured 116 small mammals from CKDu endemic regions in the Polonnaruwa District of Sri Lanka. Seven animals (five out of 11 Mus booduga and two out of 99 Rattus rattus) were PCR-positive for the hantavirus. A rat-borne sequence was grouped with a THAIV-like Anjozorobe virus. In contrast, Mus-borne sequences belonged to the THAIV lineage, suggesting a novel orthohantavirus species according to the phylogenetic analyses and whole-genome comparisons. Our genetic evidence indicates the presence of two THAIV-related viruses circulating in this CKDu endemic area, suggesting a basis for further investigations to identify the infectious virus in patients with CKDu and the CKDu induction mechanism of these viruses.
• Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Zambia.

  Masahiro Kajihara, Martin Simuunza, Ngonda Saasa, George Dautu, Akina Mori-Kajihara, Yongjin Qiu, Ryo Nakao, Yoshiki Eto, Hayato Furumoto, Bernard M Hang'ombe, Yasuko Orba, Hirofumi Sawa, Edgar Simulundu, Shuetsu Fukushi, Shigeru Morikawa, Masayuki Saijo, Jiro Arikawa, Swithine Kabilika, Mwaka Monze, Victor Mukonka, Aaron Mweene, Ayato Takada, Kumiko Yoshimatsu

  PLoS neglected tropical diseases 15 (6) e0009452  2021/06/01 [Refereed]
   

  Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis with a high case fatality rate in humans. Although the disease is widely found in Africa, Europe, and Asia, the distribution and genetic diversity of CCHF virus (CCHFV) are poorly understood in African countries. To assess the risks of CCHF in Zambia, where CCHF has never been reported, epidemiologic studies in cattle and ticks were conducted. Through an indirect immunofluorescence assay, CCHFV nucleoprotein-specific serum IgG was detected in 8.4% (88/1,047) of cattle. Among 290 Hyalomma ticks, the principal vector of CCHFV, the viral genome was detected in 11 ticks. Phylogenetic analyses of the CCHFV S and M genome segments revealed that one of the detected viruses was a genetic reassortant between African and Asian strains. This study provides compelling evidence for the presence of CCHFV in Zambia and its transmission to vertebrate hosts.
• Immunological Responses to Seoul Orthohantavirus in Experimentally and Naturally Infected Brown Rats (Rattus norvegicus).

  Shumpei P Yasuda, Kenta Shimizu, Takaaki Koma, Nguyen Thuy Hoa, Mai Quynh Le, Zhuoxing Wei, Devinda S Muthusinghe, Sithumini M W Lokupathirage, Futoshi Hasebe, Tetsu Yamashiro, Jiro Arikawa, Kumiko Yoshimatsu

  Viruses 13 (4) 2021/04/12 [Refereed]
   

  To clarify the mechanism of Seoul orthohantavirus (SEOV) persistence, we compared the humoral and cell-mediated immune responses to SEOV in experimentally and naturally infected brown rats. Rats that were experimentally infected by the intraperitoneal route showed transient immunoglobulin M (IgM) production, followed by an increased anti-SEOV immunoglobulin G (IgG) antibody response and maturation of IgG avidity. The level of SEOV-specific cytotoxic T lymphocytes (CTLs) peaked at 6 days after inoculation and the viral genome disappeared from serum. In contrast, naturally infected brown rats simultaneously had a high rate of SEOV-specific IgM and IgG antibodies (28/43). Most of the IgM-positive rats (24/27) had the SEOV genome in their lungs, suggesting that chronic SEOV infection was established in those rats. In female rats with IgG avidity maturation, the viral load in the lungs was decreased. On the other hand, there was no relationship between IgG avidity and viral load in the lungs in male rats. A CTL response was not detected in naturally infected rats. The difference between immune responses in the experimentally and naturally infected rats is associated with the establishment of chronic infection in natural hosts.
• U.S.-Japan cooperative medical sciences program: 22nd International Conference on Emerging Infectious Diseases in the Pacific Rim.

  Kristina T Lu, Takuya Yamamoto, David McDonald, Wei Li, Marissa Tan, Meng Ling Moi, Eun-Chung Park, Kumiko Yoshimatsu, Marie Ricciardone, Allan Hildesheim, Yukari Totsuka, Asuka Nanbo, Opass Putcharoen, Gompol Suwanpimolkul, Watsamon Jantarabenjakul, Leilani Paitoonpong, F Gray Handley, K Gayle Bernabe, Masahiko Noda, Miwa Sonoda, Patrick Brennan, Diane E Griffin, Ichiro Kurane

  Virology 555 71 - 77 2021/03 

  This review summarizes the presentations given at the 22nd International conference on Emerging Infectious Diseases in the Pacific Rim. The purpose of this annual meeting is to foster international collaborations and address important public health issues in the Asia-Pacific region. This meeting was held in Bangkok in February 2020 and focused on emerging virus infections. Unexpectedly, the SARS-CoV-2 pandemic was in the initial stages leading to a special session on COVID-19 in addition to talks on dengue, influenza, hepatitis, AIDS, Zika, chikungunya, rabies, cervical cancer and nasopharyngeal carcinoma.
• Serological methods for detection of infection with shrew-borne hantaviruses: Thottapalayam, Seewis, Altai, and Asama viruses.

  Zhouoxing Wei, Kenta Shimizu, Kumpei Nishigami, Yoshimi Tsuda, Yomani Sarathukumara, Devinda S Muthusinghe, Chandika D Gamage, Lishanta Granathne, Sithumini M W Lokupathirage, Nishanta Nanayakkara, Jiro Arikawa, Fuka Kikuchi, Keiko Tanaka-Taya, Motoi Suzuki, Shigeru Morikawa, Satoru Arai, Kumiko Yoshimatsu

  Archives of virology 166 (1) 275 - 280 2021/01 [Refereed]
   

  The infectivity of shrew-borne hantaviruses to humans is still unclear because of the lack of a serodiagnosis method for these viruses. In this study, we prepared recombinant nucleocapsid (rN) proteins of Seewis orthohantavirus, Altai orthohantavirus (ALTV), Thottapalayam thottimvirus (TPMV), and Asama orthohantavirus. Using monospecific rabbit sera, no antigenic cross-reactivity was observed. In a serosurvey of 104 samples from renal patients and 271 samples from heathy controls from Sri Lanka, one patient serum and two healthy control sera reacted with rN proteins of ALTV and TPMV, respectively. The novel assays should be applied to investigate potential infectivity of shrew-borne hantaviruses to humans.
• The Polarity of an Amino Acid at Position 1891 of Severe Fever with Thrombocytopenia Syndrome Virus L Protein Is Critical for the Polymerase Activity.

  Kisho Noda, Yoshimi Tsuda, Fumiya Kozawa, Manabu Igarashi, Kenta Shimizu, Jiro Arikawa, Kumiko Yoshimatsu

  Viruses 13 (1) 2020/12/27 [Refereed]
   

  Severe fever with thrombocytopenia syndrome virus subclone B7 shows strong plaque formation and cytopathic effect induction compared with other subclones and the parental strain YG1. Compared to YG1 and the other subclones, only B7 possesses a single substitution in the L protein at the amino acid position 1891, in which N is changed to K (N1891K). In this study, we evaluate the effects of this mutation on L protein activity via a cell-based minigenome assay. Substitutions of N with basic amino acids (K or R) enhanced polymerase activity, while substitutions with an acidic amino acid (E) decreased this activity. Mutation to other neutral amino acids showed no significant effect on activity. These results suggest that the characteristic of the amino acid at position 1891 of the L protein are critical for its function, especially with respect to the charge status. Our data indicate that this C-terminal domain of the L protein may be crucial to its functions in genome transcription and viral replication.
• Simultaneous serodetection of major rat infectious pathogens by a multiplex immunochromatographic assay.

  Noriko Tosa, Tomoko Ishida, Kumiko Yoshimatsu, Nobuhito Hayashimoto, Kanae Shiokawa, Akira Takakura, Jiro Arikawa

  Experimental animals 2020/11/12 [Refereed]
   

  Rapid and simple serologic tests that require only a small amount of blood without the euthanization of animals are valuable for microbial control in colonies of laboratory animals. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to Sendai virus (also known as hemagglutinating virus of Japan), hantavirus, and sialodacryoadenitis virus, which are causative agents of major infectious diseases in rats. For this assay, an ICA strip was placed into a microtube containing 150 μl PBS and either 0.75 μl of rat serum or 1.5 μl of whole blood. Binding antibodies were visualized by using anti-rat IgG antibody-conjugated colloidal gold. Under these conditions, the multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. Positive serum samples for each infectious disease were used to evaluate the sensitivity and specificity of the multiplex ICA. The sensitivities of the multiplex ICA for Sendai virus, hantavirus, and sialodacryoadenitis virus were 100%, 100%, and 81%, respectively. No nonspecific reactions were observed in any of the 52 positive sera against heterologous antigens. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid and simple serological testing of laboratory rats.
• 抗核蛋白質抗体を用いた各種ハンタウイルス抗原の新規検出法の確立

  三橋 健斗, 小林 進太郎, 好井 健太朗, 吉松 組子, 苅和 宏明

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 163回 253 - 253 1347-8621 2020/10
• Multilocus sequence typing reveals diverse known and novel genotypes of Leptospira spp. circulating in Sri Lanka.

  Lilani Karunanayake, Chandika D Gamage, Chandima P Gunasekara, Sajiv De Silva, Hidemasa Izumiya, Masatomo Morita, Devinda S Muthusinghe, Kumiko Yoshimatsu, Roshan Niloofa, Panduka Karunanayake, Wimalasiri Uluwattage, Makoto Ohnishi, Nobuo Koizumi

  PLoS neglected tropical diseases 14 (8) e0008573  2020/08 [Refereed][Not invited]
   

  BACKGROUND: Leptospirosis has gained much attention in Sri Lanka since its large outbreak in 2008. However, most of the cases were clinically diagnosed and information on Leptospira genotypes and serotypes currently prevailing in the country is lacking. METHODOLOGY/PRINCIPAL FINDINGS: We retrospectively analyzed 24 Leptospira strains from human patients as well as isolated and characterized three Leptospira strains from black rats using the microscopic agglutination test with antisera for 19 serovars and multilocus sequence typing. The isolates were identified as Leptospira borgpetersenii sequence types (STs) 143 and 144; L. interrogans STs 30, 34, 43, 44, 74, 75, 80, 308, 313, 314, 316, and 317; and L. kirschneri ST318. Six of the 15 STs were identified for the first time in this study. Five serogroups such as Autumnalis, Grippotyphosa, Hebdomadis, Javanica, and Pyrogenes were detected among the isolates. Contrary to previous studies, various genotypes including novel STs were isolated during an outbreak in Southern Province. L. borgpetersenii serogroup Javanica ST143 was isolated both from a human and black rat. CONCLUSIONS/SIGNIFICANCE: This study revealed that genetically diverse Leptospira strains currently circulate in Sri Lanka: some genotypes have been circulating and others have emerged recently, which may explain the recent surge of leptospirosis patients with varying clinical manifestations and frequent outbreaks of leptospirosis. Black rats were identified as the source of infection for humans, but reservoir animals for other genotypes remain unknown.
• Species and genetic diversity of Bandicota (Murinae, Rodentia) from Myanmar based on mitochondrial and nuclear gene sequences

  Satoko Mori, Thidalay Thwe, Wai Min Thu, Shumpei P. Yasuda, Saw Bawm, Kimiyuki Tsuchiya, Ken Katakura, Satoru Arai, Kumiko Yoshimatsu, Hitoshi Suzuki

  Mammal Research 65 (3) 493 - 502 2199-2401 2020/07 [Refereed][Not invited]
  
• [Hantavirus infection as a risk for chronic kidney disease of unknown etiology (CKDu) in Sri Lanka].

  Kumiko Yoshimatsu

  Uirusu 70 (2) 175 - 184 2020 [Not refereed]
   

  Chronic kidney disease of unknown etiology (CKDu) has emerged in endemic areas of Sri Lanka since the 1990s. The disease is a chronic but fatal disease. Until now, heavy metals and agrochemicals have been suspected as the cause of CKDu, but it has been still unknown. Recently, we have found a high seroprevalence to hantavirus in CKDu patients and reported that hantavirus infection is a risk of CKDu. Hantaviruses are rodent-borne zoonotic viruses. Here, I would like to introduce a story of the research from sero-epidemiology to the search for host animals.
• Reply to Comments by Yih et al. (Exposure to Hantavirus is a Risk Factor Associated with Kidney Diseases in Sri Lanka: A Cross-Sectional Study)

  Y D Sarathkumara, Chandika Damesh Gamage, Sithumini Lokupathirage, Devinda S. Muthusinghe, Nishantha Nanayakkara, Lishanthe Gunarathne, Kenta Shimizu, Yoshimi Tsuda, Jiro Arikawa, Yoshimatsu

  Viruses 11 (8) 2019/12/11 [Refereed][Not invited]
  
• Multiplex Immunochromatographic Assay for Serologic Diagnosis of Major Infectious Diseases in Laboratory Mice.

  Noriko Tosa, Tomoko Ishida, Kumiko Yoshimatsu, Nobuhito Hayashimoto, Kanae Shiokawa, Akira Takakura, Jiro Arikawa

  Journal of the American Association for Laboratory Animal Science : JAALAS 58 (6) 790 - 795 2019/11/01 [Refereed]
   

  Serologic monitoring of infectious diseases is important for microbial control in colonies of laboratory mice. Rapid and simple tests that do not require killing animals are valuable for this purpose. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to mouse hepatitis virus (MHV), Sendai virus (also known as hemagglutinating virus of Japan [HVJ]), and Clostridium piliforme (The pathogen that causes Tyzzer disease), which are major infectious diseases in mice. For this assay, an ICA strip was put into a microtube containing 150 μL PBS and either 0.75 μL mouse serum or 1.5 μL whole blood. Binding antibodies were visualized by using protein A-conjugated colloidal gold. Under these conditions, multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. To evaluate the sensitivity and specificity of multiplex ICA, positive serum samples for each infectious disease were used. Sensitivities of the multiplex ICA test for MHV, HVJ, and C. piliforme were 100%, 100%, and 90%, respectively. No nonspecific reaction was observed in any of the 30 positive sera. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA test. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid, simple, and safe serologic testing of laboratory mice.
• Serological Evidence of Thailand Orthohantavirus or Antigenically Related Virus Infection Among Rodents in a Chronic Kidney Disease of Unknown Etiology Endemic Area, Girandurukotte, Sri Lanka

  Lokupathirage, S.M.W., Muthusinghe, D.S., Shimizu, K., Nishigami, K., Noda, K., Tsuda, Y., Sarathkumara, Y.D., Gunawardana, S., Arikawa, J., Gamage, C.D., Yoshimatsu, K.

  Vector-Borne and Zoonotic Diseases 19 (11) 1530-3667 2019/07 [Refereed][Not invited]
  
• Development of a multiplex immunochromatographic test for serological diagnosis of major infectious diseases in laboratory mice

  Tosa N, Ishida T, Yoshimatsu K, Hayashimoto N, Kanae S, Takakura A, Arikawa J

  J Am Assoc Lab Anim Sci 2019/06 [Refereed][Not invited]
  
• Thailand orthohantavirus infection in patients with chronic kidney disease of unknown aetiology in Sri Lanka

  Yoshimatsu, K., Gamage, C.D., Sarathkumara, Y.D., Kulendiran, T., Muthusinghe, D.S., Nanayakkara, N., Gunarathne, L., Shimizu, K., Tsuda, Y., Arikawa, J.

  Archives of Virology 164 (1) 267 - 271 2019/01/04 [Refereed][Not invited]
  
• Comparison of immune response in mice sensitized to an animal allergen, Can f 1, and to a food allergen, ovalbumin

  Noriko Tosa, Kumiko Yoshimatsu, Motoko Takahashi, Jiro Arikawa

  Biomedical Research 40 (1) 9 - 15 2019 [Refereed][Not invited]
   

  Can f 1 belongs to the lipocalin superfamily and is considered to be an animal allergen. The immune response induced by Can f 1 in mice was compared with that induced by ovalbumin (OVA), a typical food allergen. Female BALB/c and C57BL/6 mice (6 weeks of age) were subcutaneously injected with Can f 1 or OVA with or without aluminum hydroxide (Alum) three times with intervals of two weeks. Serum levels of total IgE or antigen-specific IgE and production of IL13 and IFNγ from splenocytes were analyzed. Immunization with Can f 1 or OVA increased serum levels of both total IgE and antigen-specific IgE significantly irrespective of Alum. These results indicate that Can f 1 and OVA were able to induce allergic sensitization in mice. Splenocyte production of IL13 in mice immunized with Can f 1 or OVA with and without Alum were significantly increased after stimulation with each antigen. However, IL13 levels in the mice immunized with Can f 1 with Alum were significantly lower than those immunized without Alum. Increases in IFNγ levels after stimulation with Can f 1 or OVA were not remarkable. No influence of genetic backgrounds of BALB/c and C57BL/6 mice was found. Although Can f 1 induced Th2 type immune responses as was also the case for immunization with OVA, an inhibitory effect of Alum on induction of IL13 was observed only in mice immunized with Can f 1. These results suggest that the immune mechanism for allergic sensitization with Can f 1 is different from that with OVA.
• Development and evaluation of monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for the diagnosis of acute leptospirosis in humans

  Gamage, C., Sarathkumara, Y., Weerakkodi, V., Shiokawa, K., Yoshimatsu, K., Arikawa, J.

  Journal of Immunological Methods 463 134 - 136 0022-1759 2018/09 [Refereed][Not invited]
  
• Involvement of CD8+ T cells in the development of renal hemorrhage in a mouse model of hemorrhagic fever with renal syndrome

  Kenta Shimizu, Kumiko Yoshimatsu, Midori Taruishi, Yoshimi Tsuda, Jiro Arikawa

  Archives of Virology 163 (6) 1577 - 1584 0304-8608 2018/06/01 [Refereed][Not invited]
   

  Hemorrhagic fever with renal syndrome (HFRS) is caused by hantavirus infection. Although host immunity is thought to be involved in the pathogenesis of HFRS, the mechanism remains to be elucidated. A mouse model of HFRS, which showed renal hemorrhage similar to that seen in patients, has been developed previously. In this study, we aimed to clarify whether CD4+ and CD8+ T cells are involved in the development of renal hemorrhage in the mouse model. At 2 days before virus inoculation, CD4+ or CD8+ T cells in 6-week-old BALB/c mice were depleted by administration of antibodies. The CD4+ T cell-depleted mice developed signs of disease such as transient weight loss, ruffled fur and renal hemorrhage as in non-depleted mice. In contrast, the CD8+ T cell-depleted mice showed no signs of disease. After determination of CTL epitopes on the viral glycoprotein in BALB/c mice, the quantity of virus-specific CTLs was analyzed using an MHC tetramer. The quantity of virus-specific CTLs markedly increased in spleens and kidneys of virus-infected mice. However, the quantity in high-pathogenic clone-infected mice was comparable to that in low-pathogenic clone-infected mice. We previously reported that the high-pathogenic clone propagated more efficiently than the low-pathogenic clone in kidneys of mice during the course of infection. Therefore, there is a possibility that the balance between quantities of the target and effector is important for disease outcome. In conclusion, this study showed that CD8+ T cells are involved in the development of renal hemorrhage in a mouse model of HFRS.
• Expression of a Recombinant Nucleocapsid Protein of Rift Valley Fever Virus in Vero Cells as an Immunofluorescence Antigen and Its Use for Serosurveillance in Traditional Cattle Herds in Zambia

  Ngonda Saasa, Masahiro Kajihara, George Dautu, Akina Mori-Kajihara, Shuetsu Fukushi, Yona Sinkala, Shigeru Morikawa, Aaron Mweene, Ayato Takada, Kumiko Yoshimatsu, Jiro Arikawa

  Vector-Borne and Zoonotic Diseases 18 (5) 273 - 277 1557-7759 2018/05/01 [Refereed][Not invited]
   

  The open reading frame of the nucleocapsid protein (NP) of Rift Valley fever virus (RVFV) strain MP12 was cloned and expressed in Vero E6 cells. The recombinant NP (rNP)-expressing cells were used as antigens for an indirect immunofluorescent antibody assay (IFA). The rNP-based IFA and RVFV-infected Vero E6 cell (authentic antigen)-based IFA showed similar IFA profiles with immune rabbit serum, which was prepared by immunization with rNP expressed using a baculovirus vector. A total of 942 traditional cattle sera obtained in five districts in Central, Southern, and Western provinces of Zambia were screened for anti-RVFV antibodies by the authentic antigen-based and rNP-based IFAs. Significant agreement was obtained between the two IFAs. The findings show that the rNP-based IFA is a safe and useful diagnostic tool as an alternative to the authentic antigen-based IFA. The antibody titers given by the rNP-based IFA were higher than those by the authentic antigen-based IFA. Therefore, the rNP-based IFA might be useful for serosurveillance of RVFV infection among cattle. Antibody prevalence rates in the five districts were 1.3% to 13.5% in the authentic antigen-based IFA and 6.0% to 21.4% in the rNP-based IFA. The results indicated that despite no reports of active cases of RVF in these provinces of Zambia, the virus is circulating among cattle herds.
• Targeting of severe fever with thrombocytopenia syndrome virus structural proteins to the ERGIC (endoplasmic reticulum Golgi intermediate compartment) and Golgi complex

  Tapiwa LUNDU, Yoshimi TSUDA, Ryo ITO, Kenta SHIMIZU, Shintaro KOBAYASHI, Kentaro YOSHII, Kumiko YOSHIMATSU, Jiro ARIKAWA, Hiroaki KARIWA

  Biomedical Research 39 (1) 27 - 38 0388-6107 2018 [Refereed][Not invited]
  
• Establishment of Subclones of the Severe Fever with Thrombocytopenia Syndrome Virus YG1 Strain Selected Using Low pH-Dependent Cell Fusion Activity

  Sanae Nishio, Yoshimi Tsuda, Ryo Ito, Kenta Shimizu, Kumiko Yoshimatsu, Jiro Arikawa

  JAPANESE JOURNAL OF INFECTIOUS DISEASES 70 (4) 388 - 393 1344-6304 2017/07 [Refereed][Not invited]
   

  The first clinical case of the YG1 strain of the severe fever with thrombocytopenia syndrome virus (SFTSV) has been isolated in Japan. We found that only some of the cells underwent low pH-dependent cell fusion, although all of the cells were confirmed to have been infected with the virus. This suggested that the YG1 strain consists of a heterogeneous mixture of related viruses. Here, we established 3 subclones (termed E3, A4, and B7) from the YG1 strain, using the limiting dilution method with the pH-dependent cell fusion activity. Subclone E3 showed weak fusion activity and cytopathic effects (CPE) in Vero E6 cells. The amino acid sequence of E3 was identical to the published sequence for the YG1 strain, and it likely comprises a subpopulation of the YG1 strain. Subclone A4 displayed strong fusion activity under acidic conditions. In contrast, subclone B7 showed strong fusion activity and CPE under neutral and acidic conditions. Two amino acid differences shared between B7 and A4 were found in the envelope glycoproteins. In addition, an amino acid variant of the RNA-dependent RNA polymerase was found only in B7. These subclones will be valuable tools to elucidate cell fusion mechanisms of SFTSV and the relationship between viral proteins and their functions.
• Evidence of infection with Leptospira interrogans and spotted fever group rickettsiae among rodents in an urban area of Osaka City, Japan

  Kenta Shimizu, Rie Isozumi, Kazutoshi Takami, Isao Kimata, Kanae Shiokawa, Kumiko Yoshimatsu, Yoshimi Tsuda, Sanae Nishio, Jiro Arikawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 79 (7) 1261 - 1263 0916-7250 2017/07 [Refereed][Not invited]
   

  We examined 33 rodents captured in an urban area of Osaka City, Japan for IgG antibodies against Seoul virus, severe fever with thrombocytopenia syndrome virus, hepatitis E virus, Leptospira interrogans, Yersinia pestis, spotted fever, typhus and scrub typhus group rickettsiae. We found that 3 (9.1%) and 1 (3.0%) of the 33 rodents had antibodies against L. interrogans and spotted fever group rickettsiae, respectively. DNAs of leptospires were detected from 2 of the 3 seropositive rodents, but DNA of rickettsia was not detected. Phylogenetic analysis and multiple locus sequence typing revealed that the 2 leptospires were L. interrogans belonging to a novel sequence type. There is a potential risk for acquiring rodent-borne zoonotic pathogens even in cities in developed countries.
• Serological evidence of hantavirus infection in Girandurukotte, an area endemic for chronic kidney disease of unknown aetiology (CKDu) in Sri Lanka

  Chandika D. Gamage, Kumiko Yoshimatsu, Yomani D. Sarathkumara, Thiviyaaluxmi Kulendiran, Nishantha Nanayakkara, Jiro Arikawa

  INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES 57 77 - 78 1201-9712 2017/04 [Refereed][Not invited]
  
• Antibody detection from Middendorf's vole (Microtus middendorffii) against Tula virus captured in Mongolia

  Kumiko Yoshimatsu, Satoru Arai, Kenta Shimizu, Yoshimi Tsuda, Bazartseren Boldgiv, Bazartseren Boldbaatar, Erdenebaatar Sergelen, Dagvatseren Ariunzaya, Orsoo Enkhmandal, Sukhbaatar Tuvshintugs, Shigeru Morikawa, Jiro Arikawa

  JAPANESE JOURNAL OF VETERINARY RESEARCH 65 (1) 39 - 44 0047-1917 2017/02 [Refereed][Not invited]
   

  Seroepizootiological surveys among wild rodents were carried out on the east side of Lake Khovsgol in Mongolia in 2010 and 2011. A total of 76 voles belonging to the genera Myodes and Microtus were captured. Most of the voles that were seropositive to Tula virus antigen were Middendorf's voles (Microtus middendorffii (6/31)). Two of the 18 Myodes voles were also seropositive to Tula virus antigen. On the other hand, only one vole was seropositive to Puumala virus antigen. The results suggest that Tula virus was maintained in Middendorf's vole. This is the first report of detection of anti-Tula virus antibody in the central part of the Eurasia continent.
• Establishment of subclones of the severe fever with thrombocytopenia syndrome virus YG1 strain selected using low pH-dependent cell fusion activity

  Sanae Nishio, Yoshimi Tsuda, Ryo Ito, Kenta Shimizu, Kumiko Yoshimatsu, Jiro Arikawa

  Japanese Journal of Infectious Diseases 70 (4) 388 - 393 1884-2836 2017 [Refereed][Not invited]
   

  SUMMARY: The first clinical case of the YG1 strain of the severe fever with thrombocytopenia syndrome virus (SFTSV) has been isolated in Japan. We found that only some of the cells underwent low pH-dependent cell fusion, although all of the cells were confirmed to have been infected with the virus. This suggested that the YG1 strain consists of a heterogeneous mixture of related viruses. Here, we established 3 subclones (termed E3, A4, and B7) from the YG1 strain, using the limiting dilution method with the pH-dependent cell fusion activity. Subclone E3 showed weak fusion activity and cytopathic effects (CPE) in Vero E6 cells. The amino acid sequence of E3 was identical to the published sequence for the YG1 strain, and it likely comprises a subpopulation of the YG1 strain. Subclone A4 displayed strong fusion activity under acidic conditions. In contrast, subclone B7 showed strong fusion activity and CPE under neutral and acidic conditions. Two amino acid differences shared between B7 and A4 were found in the envelope glycoproteins. In addition, an amino acid variant of the RNA-dependent RNA polymerase was found only in B7. These subclones will be valuable tools to elucidate cell fusion mechanisms of SFTSV and the relationship between viral proteins and their functions.
• Epizootiological study of rodent-borne hepatitis E virus HEV-C1 in small mammals in Hanoi, Vietnam

  Satomu Obana, Kenta Shimizu, Kumiko Yoshimatsu, Futoshi Hasebe, Kozue Hotta, Rie Isozumi, Hoa Thuy Nguyen, Mai Quynh Le, Tetsu Yamashiro, Yoshimi Tsuda, Jiro Arikawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 79 (1) 76 - 81 0916-7250 2017/01 [Refereed][Not invited]
   

  There is concern about the zoonotic potential of rodent-borne hepatitis E virus, designated as HEV-C1. However, epizootiological information about HEV-C1 is limited. To address this issue, serum samples from 443 small mammals captured at 5 sites in Hanoi, Vietnam, were examined for anti-HEV-Cl IgG antibodies. In addition, livers of seropositive animals were examined for viral RNA. Anti-HEV-C1 antibodies were detected in 57 (12.9%) of the 443 serum samples. Seropositive animals were found in all of the sites (4.7% to 22.2%). Anti-HEV-C1 antibodies were detected from 48 (12.3%) of 389 Rattus norvegicus and 9 (19.6%) of 46 R. tanezumi, but were not detected from 8 Suncus murinus. Viral RNAs were detected from 13 (22.8%) of the 57 seropositive rodents. The detectioh rate of viral RNA in seropositive R. tanezumi (66.7%, 6/9) was significantly higher than that in seropositive R. norvegicus (14.6%, 7/48). The results suggest that R. tanezumi is more susceptible than R. norvegicus to HEV-Cl infection. Phylogenetic analysis revealed that Vietnamese strains were divided into 3 clusters in genetic group 2 of HEV-C1. Multiple clusters of viruses were detected at several sites without species specificity, suggesting that 3 clusters of HEV-C1 co-circulate in Hanoi, Vietnam.
• Appearance of renal hemorrhage in adult mice after inoculation of patient-derived hantavirus

  Kenta Shimizu, Takaaki Koma, Kumiko Yoshimatsu, Yoshimi Tsuda, Yuji Isegawa, Jiro Arikawa

  VIROLOGY JOURNAL 14 (1) 13  1743-422X 2017/01 [Refereed][Not invited]
   

  Background: Hemorrhagic fever with renal syndrome (HFRS) caused by hantavirus infection is characterized by fever, renal dysfunction and hemorrhage. An animal model mimicking symptoms of HFRS remains to be established. In this study, we evaluated the pathogenicity of an HFRS patient-derived Hantaan virus (HTNV) in adult mice. Methods: Five clones of HTNV strain KHF 83-61 BL (KHFV) that was derived from blood of an HFRS patient were obtained by plaque cloning. The pathogenicity of the virus clones was evaluated by using 6-week-old female BALB/c mice. Sequence analysis of the viral genome was performed by conventional methods. Results: All of the mice intravenously inoculated with KHFV clone (cl)-1, -2, -3 and -5 showed signs of disease such as transient body weight loss, ruffled fur, reduced activity and remarkably prominent hemorrhage in the renal medulla at 6 to 9 days post-inoculation (dpi) and then recovered. In contrast, mice intravenously inoculated with KHFV cl-4 did not show any signs of disease. We selected KHFV cl-5 and cl-4 as representative of high-pathogenic and low-pathogenic clones, respectively. Quantities of viral RNA in kidneys of KHFV cl-5-infected mice were larger than those in KHFV cl-4-infected mice at any time point examined (3, 6, 9 and 12 dpi). The quantities of viral RNA of KHFV cl-5 and cl-4 peaked at 3 dpi, which was before the onset of disease. Sequence analysis revealed that the amino acid at position 417 in the glycoprotein Gn was the sole difference in viral proteins between KHFV cl-5 and cl-4. The result suggests that amino acid at position 417 in Gn is related to the difference in pathogenicity between KHFV cl-5 and cl-4. When the inoculum of KHFV cl-5 was pretreated with a neutralizing antibody against HTNV strain 76-118, which belongs to the same serotype as KHFV clones, mice did not show any signs of disease, confirming that the disease was caused by KHFV infection. Conclusion: We found that an HFRS patient-derived HTNV caused renal hemorrhage in adult mice. We anticipate that this infection model will be a valuable tool for understanding the pathogenesis of HFRS.
• The amino acid at position 624 in the glycoprotein of SFTSV (severe fever with thrombocytopenia virus) plays a critical role in low-pH-dependent cell fusion activity

  Yoshimi Tsuda, Manabu Igarashi, Ryo Ito, Sanae Nishio, Kenta Shimizu, Kumiko Yoshimatsu, Jiro Arikawa

  BIOMEDICAL RESEARCH-TOKYO 38 (2) 89 - 97 0388-6107 2017 [Refereed][Not invited]
   

  Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus responsible for causing an emerging zoonotic disease. We previously established subclones from SFTSV strain YG1 based on differences in low-pH-dependent cell fusion activities and found two amino acid substitutions, Y328H and R624W, in the envelope glycoprotein (GP) of high fusion subclones. In this study, we show that transiently expressed GP with the R624W mutation, but not the Y328H mutation, induced cell fusion under acidic conditions. GP possessing either tryptophan, serine, glycine or aspartic acid at position 624 induced cell fusion, whereas GP possessing basic amino acids such as arginine or lysine did not induce cell fusion. These results indicated that the amino acid at position 624 has an important role for inducing low-pH-dependent cell fusion.
• Serological evidence of infection with rodent-borne hepatitis E virus HEV-C1 or antigenically related virus in humans

  Kenta Shimizu, Sugihiro Hamaguchi, Cuong Chi Ngo, Tian-Cheng Li, Shuji Ando, Kumiko Yoshimatsu, Shumpei P. Yasuda, Takaaki Koma, Rie Isozumi, Yoshimi Tsuda, Hiromi Fujita, Thuy Thanh Pham, Mai Quynh Le, Anh Duc Dang, Tuan Quang Nguyen, Lay-Myint Yoshida, Koya Ariyoshi, Jiro Arikawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 78 (11) 1677 - 1681 0916-7250 2016/11 [Refereed][Not invited]
   

  Zoonotic potential of a rat-derived hepatitis E virus (HEV), designated as HEV-Cl, remains unknown. To evaluate the risk for HEV-Cl infection in humans, paired sera of 208 hospitalized febrile patients collected from 2001 to 2003 in Hanoi, Vietnam, were examined for IgG antibodies to HEV-Cl and genotype 1 HEV (HEV-1), which is common in humans. IgG antibodies to virus-like particles (VLPs) of HEV-Cl and/or HEV-1 were detected from 99 of the 208 convalescent sera in enzyme-linked immunosorbent assay (ELISA). IgG antibody titers to HEV-Cl antigen in 3 of the 99 sera were more than 8-fold higher than those to HEV-1 antigen. IgM antibodies to HEV-Cl antigen were detected in acute sera from 2 of the 3 patients in ELISA and Western blotting. However, no HEV genome was detected. Clinical information was available for 1 of the 2 patients. Hepatic enzymes, aspartate aminotransferase and alanine aminotransferase, were mildly elevated (156 IU/l and 68 IU/l, respectively), and hepatomegaly was detected by ultrasonography. The patient recovered from the illness after 17 days. These results indicated that HEV-Cl or its variants infect humans in Vietnam and may cause acute febrile illness with mild liver dysfunction.
• Identification of causative Leishmania species in Giemsa-stained smears prepared from patients with cutaneous leishmaniasis in Peru using PCR-RFLP

  Yu Koarashi, Abraham G. Caceres, Florencia Margarita Zuniga Saca, Elsa Elvira Palacios Flores, Adela Celis Trujillo, Jose Luis Abanto Alvares, Kumiko Yoshimatsu, Jiro Arikawa, Ken Katakura, Yoshihisa Hashiguchi, Hirotomo Kato

  ACTA TROPICA 158 83 - 87 0001-706X 2016/06 [Refereed][Not invited]
   

  A PCR-Restriction Fragment Length Polymorphism (RFLP) targeting the mannose phosphate isomerase gene was established to differentiate Leishmania species distributed near the Department of Huanuco, Peru. The technique was applied to 267 DNA samples extracted from Giemsa-stained smears of cutaneous lesions taken from patients suspected for cutaneous leishmaniasis in the area, and the present status of causative Leishmania species was identified. Of 114 PCR-amplified samples, 22, 19, 24 and 49 samples were identified to be infected by Leishmania (Viannia) braziliensis, L. (V.) peruviana, L. (V.) guyanensis, and a hybrid of L. (V.) braziliensis/L. (V.) peruviana, respectively, and the validity of PCR-RFLP was confirmed by sequence analysis. Since PCR-RFLP is simple and rapid, the technique will be a useful tool for the epidemiological study of leishmaniasis. (C) 2016 Elsevier B.V. All rights reserved.
• Evaluation of truncated LipL32 expressed by Escherichia coli and Pichia pastoris for serodiagnosis of Leptospira infection in rodents

  Kanae Shiokawa, Chandika D. Gamage, Nobuo Koizumi, Yoshihiro Sakoda, Kenta Shimizu, Yoshimi Tsuda, Kumiko Yoshimatsu, Jiro Arikawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 78 (2) 221 - 230 0916-7250 2016/02 [Refereed][Not invited]
   

  The applicability of the recombinant LipL32 for serodiagnosis of leptospiral infection in field rodents was assessed in this study. An immunodominant region of LipL32 was determined by monoclonal antibodies, and then, truncated LipL32 (tLipL32) was designed to contain the region (87-188th amino acid). The tLipL32 was compared between two recombinant expression hosts Escherichia coli and Pichia pastoris in ELISA. With field rat sera, tLipL32 expressed by P. pastoris (tLipL32p) had high antigenicity without background reactions, while tLipL32 expressed by E. coli (tLipL32e) showed high background reactions, which were reduced by pre-adsorption of sera with E. coli. To evaluate tLipL32-ELISA, field rat sera were tentatively divided into a Leptospira infection positive (12 sera) and a negative group (12 sera) based on the results from flaB gene PCR of kidney samples and WB with whole Leptospira cell. Consequently, the sensitivity of tLipL32p-ELISA for field rat sera was 83%. A similar result was obtained from tLipL32e-ELISA with adsorbed sera, (92%). However, sensitivity of tLipL32e-ELISA using sera without an adsorption treatment was 50%. Regardless of the expression host, tLipL32-ELISA had 100% specificity and sensitivity in experimentally infected laboratory rats. These results suggest that recombinant LipL32 expressed by P pastoris is more applicable for serodiagnosis in field rats due to a lack of background reaction.
• Multiple-locus variable-number tandem repeat analysis of Leptospira interrogans and Leptospira borgpetersenii isolated from small feral and wild mammals in East Asia

  Nobuo Koizumi, Hidemasa Izumiya, Jung-Jung Mu, Zbigniew Arent, Shou Okano, Chie Nakajima, Yasuhiko Suzuki, Maki Mizutani Muto, Tsutomu Tanikawa, Kyle R. Taylor, Noriyuki Komatsu, Kumiko Yoshimatsu, Hoang Thi Thu Ha, Makoto Ohnishi

  INFECTION GENETICS AND EVOLUTION 36 434 - 440 1567-1348 2015/12 [Refereed][Not invited]
   

  Leptospira spp. are the causative agents of a worldwide zoonosis, leptospirosis, maintained by various mammals. Each Leptospira serovar is frequently associated with a particular maintenance host, and recently, Leptospira genotype-host association has also been suggested to limit serovars to restricted areas. We investigated the molecular characteristics of L. interrogans and L. borgpetersenii which were isolated from small feral and wild animals in four East Asian states using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA using 11 loci was performed on 110 L. interrogans serogroups from Japan (79 strains of 5 serogroups from 3 animal species), Philippines (21; 3; 2), Taiwan (7; 2; 3), and Vietnam (3; 1; 1). A MLVA method using 4 loci for L. borgpetersenii was established and performed on 52 isolates from Japan (26; 3; 7), Philippines (13; 1; 2), and Taiwan (13; 1; 3). In L. interrogans, serogroups Autumnalis and Hebdomadis appeared more genetically diverse than serogroups Bataviae, Grippotyphosa, Icterohaemorrhagiae, Pomona, or Pyrogenes. The former serogroup strains with the exception of one Hebdomadis strain were isolated from Apodemus speciosus while all the latter serogroup strains with the exception of Grippotyphosa were isolated from Rattus norvegicus. L. borgpetersenii was isolated from at least 11 animal species while L. interrogans was isolated from five species, which might suggest a wider host range for L. borgpetersenii. Broad host preference in a single genotype was also observed, which colonized not only different species of the same genera but also multiple animal genera. This study demonstrates that there may be variability in the range of genetic diversity among different Leptospira serogroups, which may be attributed to maintenance host animals and environmental factors. (C) 2015 The Authors. Published by Elsevier B.V.
• First evidence of Seoul hantavirus in the wild rat population in the Netherlands

  Jenny Verner-Carlsson, Mare Lõhmus, Karin Sundström, Tanja M. Strand, Monique Verkerk, Chantal Reusken, Kumiko Yoshimatsu, Jiro Arikawa, Frank van de Goot, Åke Lundkvist

  African Journal of Disability 5 (1) 27215  2226-7220 2015 [Refereed][Not invited]
   

  We report the first detection of Seoul hantavirus (SEOV)-specific antibodies in the wild brown rat population in the Netherlands. SEOV-reactive antibodies were found in three rats out of 16 in a repeated series of tests including immunofluorescence assay, immunoblot, and enzyme-linked immunosorbent assay. Focus reduction neutralization test confirmed the presence of SEOV-specific antibodies, and reverse-transcription polymerase chain reaction (RT-PCR) confirmed the presence of hantaviral RNA. This discovery follows the recent findings of SEOV infections in wild and pet brown rats and humans in England, Wales, France, Belgium, and Sweden, indicating an even higher importance of this hantavirus for public health in large areas of Europe.
• Distinct genetic characteristics of Sri Lankan Rattus and Bandicota (Murinae, Rodentia) inferred frommitochondrial and nuclear markers

  Shumpei P. Yasuda, Chandika D. Gamage, Nobuo Koizumi, Sanae Nishio, Rie Isozumi, Kenta Shimizu, Takaaki Koma, Takako Amada, Hitoshi Suzuki, Kumiko Yoshimatsu, Jiro Arikawa

  Genes and Genetic Systems 89 (2) 71 - 80 1880-5779 2014/09/01 [Refereed][Not invited]
   

  We examined genetic variation in black rats (the Rattus rattus complex) from Kandy District, Sri Lanka using mitochondrial cytochrome b (cytb, 1140 bp) and nuclear melanocortin 1 receptor (Mc1r, 954 bp) gene sequences together with database sequences. We confirmed the existence of two divergent mitochondrial lineages in Sri Lankan black rats, with genetic distance of 2.2% and estimated divergence time of 0.3 million years ago. Because one lineage is unique to the island and the other is closely related to R. rattus populations on the Indian subcontinent, two migration events of R. rattus from the subcontinent are inferred, one ancient and one recent. Mc1r analyses revealed 12 haplotypes among the Sri Lankan black rats. A median-joining network together with other availablesequences separated the 12 haplotypes into two groups, one unique to the island and the other related to previously reported R. rattus sequences. Notably, most individuals possessed various combinations of both haplotype groups which had no association with the cytb clades. These results imply that old and new R. rattuslineages are now intermingled as a result of hybridization in Sri Lanka. Specimensof the lesser bandicoot rat (Bandicota bengalensis) collected from Sri Lanka (n = 24) were shown to have no genetic variability in the cytb sequence. Our results indicate that the two most abundant groups of commensal rats in Sri Lanka, black rats and lesser bandicoot rats, are the product of contrasting evolutionary histories on different timescales.
• Antigenic Properties of N Protein of Hantavirus

  Kumiko Yoshimatsu, Jiro Arikawa

  VIRUSES-BASEL 6 (8) 3097 - 3109 1999-4915 2014/08 [Refereed][Not invited]
   

  Hantavirus causes two important rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in North and South America. Twenty-four species that represent sero-and genotypes have been registered within the genus Hantavirus by the International Committee on Taxonomy of Viruses (ICTV). Among the viral proteins, nucleocapsid (N) protein possesses an immunodominant antigen. The antigenicitiy of N protein is conserved compared with that of envelope glycoproteins. Therefore, N protein has been used for serological diagnoses and seroepidemiological studies. An understanding of the antigenic properties of N protein is important for the interpretation of results from serological tests using N antigen. N protein consists of about 430 amino acids and possesses various epitopes. The N-terminal quarter of N protein bears linear and immunodominant epitopes. However, a serotype-specific and multimerization-dependent antigenic site was found in the C-terminal half of N protein. In this paper, the structure, function, and antigenicity of N protein are reviewed.
• Serological diagnosis with recombinant N antigen for hantavirus infection

  Kumiko Yoshimatsu, Jiro Arikawa

  VIRUS RESEARCH 187 77 - 83 0168-1702 2014/07 [Refereed][Not invited]
   

  Hantaviruses are causative agents of two rodent-borne zoonoses, hemorrhagic fever with renal syndrome (HERS) and nephropathia epidemica (NE) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. Serological examinations to detect hantavirus antibodies have been most widely used for surveillance among humans and rodent reservoirs. Here, we will review antigenic structure of nucleocapsid (N) protein of hantaviruses and application of recombinant N protein as diagnostic antigen for screening and serotyping. (C) 2014 The Authors. Published by Elsevier B.V. All rights reserved.
• Neutrophil Depletion Suppresses Pulmonary Vascular Hyperpermeability and Occurrence of Pulmonary Edema Caused by Hantavirus Infection in C.B-17 SCID Mice

  Takaaki Koma, Kumiko Yoshimatsu, Noriyo Nagata, Yuko Sato, Kenta Shimizu, Shumpei P. Yasuda, Takako Amada, Sanae Nishio, Hideki Hasegawa, Jiro Arikawa

  JOURNAL OF VIROLOGY 88 (13) 7178 - 7188 0022-538X 2014/07 [Refereed][Not invited]
   

  Hantavirus infections are characterized by vascular hyperpermeability and neutrophilia. However, the pathogenesis of this disease is poorly understood. Here, we demonstrate for the first time that pulmonary vascular permeability is increased by Hantaan virus infection and results in the development of pulmonary edema in C. B-17 severe combined immunodeficiency ( SCID) mice lacking functional T cells and B cells. Increases in neutrophils in the lung and blood were observed when pulmonary edema began to be observed in the infected SCID mice. The occurrence of pulmonary edema was inhibited by neutrophil depletion. Moreover, the pulmonary vascular permeability was also significantly suppressed by neutrophil depletion in the infected mice. Taken together, the results suggest that neutrophils play an important role in pulmonary vascular hyperpermeability and the occurrence of pulmonary edema after hantavirus infection in SCID mice.
• Development of an immunochromatography strip test based on truncated nucleocapsid antigens of three representative hantaviruses

  Takako Amada, Kumiko Yoshimatsu, Takaaki Koma, Kenta Shimizu, Chandika D. Gamage, Kanae Shiokawa, Sanae Nishio, Clas Ahlm, Jiro Arikawa

  VIROLOGY JOURNAL 11 87  1743-422X 2014/05 [Refereed][Not invited]
   

  Background: Hantaviruses are causative agents of hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica (NE) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. There is a need for time-saving diagnostic methods. In the present study, recombinant N antigens were used as antigens in an immunochromatography strip (ICG) test to detect specific IgG antibodies. Methods: The N-terminal 103 amino acids (aa) of Hantaan virus (HTNV), Puumala virus (PUUV) and Andes virus (ANDV) nucleocapsid (N) protein were expressed in E. coli as representative antigens of three groups (HFRS, NE and HPS-causing viruses) of hantavirus. Five different types of ICG test strips, one antigen line on one strip for each of the three selected hantaviruses (HTNV, PUUV and ANDV), three antigen lines on one strip and a mixed antigen line on one strip, were developed and sensitivities were compared. Results: A total of 87 convalescent-phase patient sera, including sera from 35 HFRS patients, 36 NE patients and 16 HPS patients, and 25 sera from healthy seronegative people as negative controls were used to evaluate the ICG test. Sensitivities of the three-line strip and mixed-line strip were similar to those of the single antigen strip (97.2 to 100%). On the other hand, all of the ICG test strips showed high specificities to healthy donors. Conclusion: These results indicated that the ICG test with the three representative antigens is an effective serodiagnostic tool for screening and typing of hantavirus infection in humans.
• Role of nucleocapsid protein of hantaviruses in intracellular traffic of viral glycoproteins

  Kenta Shimizu, Kumiko Yoshimatsu, Takaaki Koma, Shumpei P. Yasuda, Jiro Arikawa

  Virus Research 178 (2) 349 - 356 0168-1702 2013/12/26 [Refereed][Not invited]
   

  To understand the role of nucleocapsid protein (NP) of hantaviruses in viral assembly, the effect of NP on intracellular traffic of viral glycoproteins Gn and Gc was investigated. Double staining of viral and host proteins in Hantaan virus (HTNV)-infected Vero E6 cells showed that Gn and Gc were localized to cis-Golgi, in which virus particles are thought to be formed. When HTNV Gn and Gc were expressed by a plasmid encoding glycoprotein precursor (GPC), which is posttranslationaly cleaved into Gn and Gc, Gn was localized to cis-Golgi, whereas Gc showed diffuse distribution in the cytoplasm in 32.9% of Gc-positive cells. The ratio of the diffused Gc-positive cells was significantly decreased to 15.0% by co-expression of HTNV NP. Co-expression of HTNV GPC with NPs of other hantaviruses, such as Seoul virus, Puumala virus and Sin Nombre virus, also reduced the ratios of diffused Gc-positive cells to 13.5%, 25.2%, and 11.6%, respectively. Among amino- and carboxyl-terminally truncated HTNV NPs, NP75-429, NP116-429, NP1-333, NP1-233, and NP1-155 possessed activity to reduce the ratio of diffused Gc-positive cells, while NP155-429 and NP1-116 did not. NP30-429 has partial activity. These results indicate that amino acid region 116-155 of NP is important for the activity, although amino acid region 1-30 is partially related. Truncation of the HTNV Gc cytoplasmic tail caused an increase in diffused Gc-positive cells. In addition, the effect of coexpression of HTNV NP was weakened. These results suggest that HTNV NP has a role to promote Golgi localization of Gc through a mechanism possibly mediated by the Gc cytoplasmic tail. © 2013 Elsevier B.V.
• Rapid, whole blood diagnostic test for detecting anti-hantavirus antibody in rats

  Takako Arnada, Kumiko Yoshimatsu, Shumpei P. Yasuda, Kenta Shimizu, Takaaki Koma, Nobuhito Hayashimoto, Chandika D. Gamage, Sanae Nishio, Akira Takakura, Jiro Arikawa

  JOURNAL OF VIROLOGICAL METHODS 193 (1) 42 - 49 0166-0934 2013/10 [Refereed][Not invited]
   

  Hantavirus is a causative agent of rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. Seoul virus (SEOV) is a causative agent of urban and laboratory rat-associated HFRS worldwide. Surveillance of rodents has been done mainly by serological detection of hantavirus-specific antibodies by enzyme linked immunosorbent assay (ELISA) and immunofluorescent antibody assay (IFA). An immunochromatographic (ICG) test was developed with the N-terminal 103 amino acids of nucleocapsid protein of Hantaan virus expressed by Escherichia coli as an antigen to detect IgG antibody specific to hantavirus in sera from Rattus sp. animals. Antibody-detecting sensitivity of the ICG test was the same as that of ELISA and about 100-times higher than that of IFA. Overall sensitivities and specificities of the ICG test in comparison to ELISA and IFA for sera from 192 urban rats and 123 laboratory rats were 99.3% and 100%, respectively. Diluted whole blood samples without separation could be used for the ICG test. The ICG test enabled detection of antibodies to SEOV, Hantaan, Dobrava/Belgrade, and Thailand viruses, which are causative agents of HFRS throughout Eurasia. The ICG test is a rapid, simple and safe method for diagnosis of SEOV infection in rats. (c) 2013 Elsevier B.V. All rights reserved.
• A survey of rodent-borne pathogens carried by wild Rattus spp. in Northern Vietnam

  T. Koma, K. Yoshimatsu, S. P. Yasuda, T. Li, T. Amada, K. Shimizu, R. Isozumi, L. T.Q. Mai, N. T. Hoa, V. Nguyen, T. Yamashiro, F. Hasebe, J. Arikawa

  Epidemiology and Infection 141 (9) 1876 - 1884 0950-2688 2013/09 [Refereed][Not invited]
   

  To examine the prevalence of human pathogens carried by rats in urban areas in Hanoi and Hai Phong, Vietnam, we live-trapped 100 rats in January 2011 and screened them for a panel of bacteria and viruses. Antibodies against Leptospira interrogans (22·0%), Seoul virus (14·0%) and rat hepatitis E virus (23·0%) were detected in rats, but antibodies against Yersinia pestis were not detected. Antibodies against L. interrogans and Seoul virus were found only in adult rats. In contrast, antibodies to rat hepatitis E virus were also found in juvenile and sub-adult rats, indicating that the transmission mode of rat hepatitis E virus is different from that of L. interrogans and Seoul virus. Moreover, phylogenetic analyses of the S and M segments of Seoul viruses found in Rattus norvegicus showed that Seoul viruses from Hai Phong and Hanoi formed different clades. Human exposure to these pathogens has become a significant public health concern. Copyright © Cambridge University Press 2012.
• A survey of rodent-borne pathogens carried by wild Rattus spp. in Northern Vietnam

  T. Koma, K. Yoshimatsu, S. P. Yasuda, T. Li, T. Amada, K. Shimizu, R. Isozumi, L. T.Q. Mai, N. T. Hoa, V. Nguyen, T. Yamashiro, F. Hasebe, J. Arikawa

  Epidemiology and Infection 141 (9) 1876 - 1884 0950-2688 2013/09 [Refereed][Not invited]
   

  To examine the prevalence of human pathogens carried by rats in urban areas in Hanoi and Hai Phong, Vietnam, we live-trapped 100 rats in January 2011 and screened them for a panel of bacteria and viruses. Antibodies against Leptospira interrogans (22·0%), Seoul virus (14·0%) and rat hepatitis E virus (23·0%) were detected in rats, but antibodies against Yersinia pestis were not detected. Antibodies against L. interrogans and Seoul virus were found only in adult rats. In contrast, antibodies to rat hepatitis E virus were also found in juvenile and sub-adult rats, indicating that the transmission mode of rat hepatitis E virus is different from that of L. interrogans and Seoul virus. Moreover, phylogenetic analyses of the S and M segments of Seoul viruses found in Rattus norvegicus showed that Seoul viruses from Hai Phong and Hanoi formed different clades. Human exposure to these pathogens has become a significant public health concern. Copyright © Cambridge University Press 2012.
• Epidemiology of Hantavirus Infection in Thousand Islands Regency of Jakarta, Indonesia

  Ima-Nurisa Ibrahim, Kenta Shimizu, Kumiko Yoshimatsu, Andre Yunianto, Ervi Salwati, Shumpei P. Yasuda, Takaaki Koma, Rika Endo, Jiro Arikawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 75 (8) 1003 - 1008 0916-7250 2013/08 [Refereed][Not invited]
   

  Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne zoonotic disease caused by hantavirus infection. Many HFRS cases have been reported in East Asia and North Europe, while the situation in Southeast Asia remains unclear. In this study, the prevalence of hantavirus infection in rodents and humans in Thousand Islands regency, which is close to the port of Jakarta, one of the largest historic ports in Indonesia, was investigated. A total of 170 rodents were captured in 2005, and 27 (15.9%) of the rodents were antibody-positive against Hantaan virus antigen in an immunofluorescence assay (IFA) and Western blotting. Despite the high prevalence in rodents, human sera collected from 31 patients with fever of -unknown origin and 20 healthy volunteers in the islands in 2009 did not show positive reaction to the antigen in IFA. To identify the virus in rodents genetically, a total of 59 rodents were captured in 2009. Sera from the rodents were screened for antibody by ELISA, and lung tissues were subjected to RT-PCR. 20 (33.9%) of the 59 rodents were antibody-positive, and 3 of those 20 rodents were positive for S and M genome segments of hantaviruses. Genetic analysis showed that the viruses belonged to Seoul virus and formed a cluster with those in Vietnam and Singapore. These results suggest that a unique group of Seoul viruses has spread widely in Southeast Asia.
• Cross-Reactivity of Secondary Antibodies against African Rodents and Application for Sero-Surveillance

  Ichiro Nakamura, Bernard Mudenda Hang'ombe, Hirofumi Sawa, Shintaro Kobayashi, Yasuko Orba, Akihiro Ishii, Yuka Thomas, Rie Isozumi, Kumiko Yoshimatsu, Aaron S. Mweene, Ayato Takada, Chihiro Sugimoto, Jiro Arikawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 75 (6) 819 - 825 0916-7250 2013/06 [Refereed][Not invited]
   

  A total of 466 rodents were captured in the Republic of Zambia from 2006 to 2010. Based on morphological observations and phylogenetic analyses of mitochondrial gene sequences, rodents were divided into 10 groups consisting of 39 Rattus rodents, 263 multimammate rats, 18 other Murinae rodents, 95 gerbils, 11 pouched mice, 1 giant-pouched rat, 38 fat mice and 1 dormouse. Rodent antibodies except that from Rattus were examined for their cross-reactivity to commercially available antibody detection reagents. Anti-mouse immunoglobulin G (IgG) was most cross-reactive to heterologous antibodies including multimammate rat, gerbil, pouched mouse and fat mouse. Thus, anti-mouse IgG would be a useful detection tool in serological examination of the Zambian rodent population. Preliminary sero-surveillance for plague, leptospirosis and hantavirus infection was performed by ELISA.
• Characterization of Full Genome of Rat Hepatitis E Virus Strain from Vietnam

  Tian-Cheng Li, Yasushi Ami, Yuriko Suzaki, Shumpei P. Yasuda, Kumiko Yoshimatsu, Jiro Arikawa, Naokazu Takeda, Wakita Takaji

  EMERGING INFECTIOUS DISEASES 19 (1) 115 - 118 1080-6040 2013/01 [Refereed][Not invited]
   

  We amplified the complete genome of the rat hepatitis E virus (HEV) Vietnam strain (V-105) and analyzed the nucleotide and amino acid sequences. The entire genome of V-105 shared only 76.8%-76.9% nucleotide sequence identities with rat HEV strains from Germany, which suggests that V-105 is a new genotype of rat HEV.
• Susceptibility of laboratory rats against genotypes 1, 3, 4, and rat hepatitis E viruses

  Tian-Cheng Li, Sayaka Yoshizaki, Yasushi Ami, Yuriko Suzaki, Shumpei P. Yasuda, Kumiko Yoshimatsu, Jiro Arikawa, Naokazu Takeda, Takaji Wakita

  Veterinary Microbiology 163 (1-2) 54 - 61 0378-1135 2013 [Refereed][Not invited]
   

  To determine whether or not rats are susceptible to hepatitis E virus (HEV) infection, each of group containing three laboratory rats (Wistar) were experimentally inoculated with genotypes 1, 3, 4 and rat HEV by intravenous injection. Serum and stool samples were collected and used to detect HEV RNA and anti-HEV antibodies by RT-PCR and ELISA, respectively. The virus infection was monitored up to 3 months after inoculation. None of the serum or stool samples collected from the rats inoculated with G1, G3, or G4 HEV indicated positive sign for virus replication. Although no alteration was observed in ALT level, rat HEV RNA was detected in stools from both of the rats inoculated with rat HEV, and both rats were positive for anti-rat HEV IgG and IgM from 3 weeks after inoculation. These results demonstrated that rats are susceptible to rat HEV but not to G1, G3, and G4 HEV. We also confirm that the nude rats were useful for obtaining a large amount of rat HEV and that the rat HEV was transmitted by the fecal-oral route. © 2012 Elsevier B.V.
• Introgressive hybridization of two major lineages of invasive Black Rats, Rattus rattus and R. tanezumi on the Japanese Islands inferred from Mc1r sequences

  Kambe Yoshikazu, Suzuki Sosuke, Yabe Tatsuo, Nakata Katsushi, Maezono Yasunori, Abe Shintaro, Ishida Ken, Tanikawa Tsutomu, Hashimoto Takuma, Takeda Mikako, Tsuchiya Kimiyuki, Yoshimatsu Kumiko, Suzuki Hitoshi

  Honyurui Kagaku (Mammalian Science) The Mammal Society of Japan 53 (2) 289 - 299 0385-437X 2013 

  Two invasive lineages of Black Rat (the Rattus rattus species complex) occur in Japan: the newly introduced Rattus rattus (2n=38) of Indian and European origin, and the anciently introduced R. tanezumi (2n=42) of East Asian origin. To assess the current distribution and invasion history of these lineages, we determined the nucleotide sequences of a coat color-related gene, melanocortin 1 receptor (Mc1r, 954 bp), in 36 rats from Japan and conducted phylogenetic analyses for a total of 133 rats mainly from Japan (17 localities). Mc1r haplotypes representing R. rattus were recovered from Otaru, the Ogasawara Islands, and Tokyo, and heterozygous individuals with the R. tanezumi type sequences were detected in these localities. These results imply that introgressive hybridization is currently going on in a variety of places in Japan such as urban and port areas, and on remote islands. In contrast, the R. rattus haplotypes were not detected in the natural forests of the Ryukyu Islands here represented by the islands of Amamioshima and Okinawa, implying the presence of some mechanism preventing the introduction of the invading lineage. We recovered some endemic haplotypes from the Ryukyu Islands, suggesting genetic differentiation between the Ryukyu Islands and other parts of Japan.
  
• 新世界ハンタウイルス感染のためのハンタウイルス組み換え核蛋白を用いた血清型鑑別ELISA法の開発

  駒 貴明, 吉松 組子, 垂石 みどり, 宮下 大輔, 遠藤 理香, 清水 健太, 安田 俊平, 天田 貴子, 瀬戸 隆, 村田 亮, 吉田 喜香, 苅和 宏明, 高島 郁夫, 有川 二郎

  北海道医学雑誌 88 (1) 35 - 35 2013/01 [Not refereed][Not invited]
  
• Variation in the bacterial conditions inside cages is correlated with intracage humidity and ammonia levels.

  Tosa N, Yoshimatsu K, Tadasuke Tsukiyama, Hatakeyama S, Arikawa J

  Lab Animal and Environ 21 (2) 87 - 98 2013 [Refereed][Not invited]
  
• Negative Strand RNA Virusのウイルス学 7.ブニヤウイルスとその生態

  吉松組子, 有川二郎

  ウイルス 日本ウイルス学会 62 (2) 239 - 250 0042-6857 2012/12 [Not refereed][Not invited]
   

  The family Bunyaviridae consists of over 300 virus species and strains that are divided into 5 genera: orthobunyavirus, hantavirus, nairovirus, phlebovirus, and tospovirus. All members of family Bunyaviridae possess a negative-sense, single stranded tripartite RNA genome, consisting of large (L), medium (M) and small (S) segments, which encode an RNA-dependent RNA polymerase, two envelope glyoproteins (Gn and Gc) and nucleocapsid (N) protein, respectively. Insects and arthropods serve as vectors of viruses in the Bunyaviridae, except for hantviruses, which instead are harbored by rodents. However, phylogenetically distinct soricomorph-associated hantaviruses have been discovered in widely separated geographical regions spanning four continents. This new finding strongly suggests that evolutionary record of hantaviruses is far more complex and ancient than originally expected. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease recently described in northeast and central China. The causative agent of SFTS is phylogenetically classified to genus phlebivirus, but unlike to other member in genus phlebovirus, SFTV transmit by ticks. This review provides a brief overview of hantavirus and hantavirus infection and describes about two newly appeared viruses in the family Bunyaviridae.
• Novel serological tools for detection of Thottapalayam virus, a Soricomorpha-borne hantavirus

  Mathias Schlegel, Erdenesaikhan Tegshduuren, Kumiko Yoshimatsu, Rasa Petraityte, Kestutis Sasnauskas, Baerbel Hammerschmidt, Robert Friedrich, Marc Mertens, Martin H. Groschup, Satoru Arai, Rika Endo, Kenta Shimizu, Takaaki Koma, Shumpei Yasuda, Chiaki Ishihara, Rainer G. Ulrich, Jiro Arikawa, Bernd Koellner

  ARCHIVES OF VIROLOGY 157 (11) 2179 - 2187 0304-8608 2012/11 [Refereed][Not invited]
   

  We developed serological tools for the detection of hantavirus-specific antibodies and hantavirus antigens in shrews. The work was focussed to generate Thottapalayam virus (TPMV)-specific monoclonal antibodies (mAbs) and anti-shrew immunoglobulin G (IgG) antibodies. The mAbs against TPMV nucleocapsid (N) protein were produced after immunization of BALB/c mice with recombinant TPMV N proteins expressed in Escherichia coli, baculovirus and Saccharomyces cerevisiae-mediated expression systems. In total, six TPMV N-protein-specific mAbs were generated that showed a characteristic fluorescent pattern in indirect immunofluorescence assay (IFA) using TPMV-infected Vero cells. Out of the six mAbs tested, five showed no cross-reaction to rodent-associated hantaviruses (Hantaan, Seoul, Puumala, Tula, Dobrava-Belgrade and Sin Nombre viruses) in IFA and enzyme-linked immunosorbent assay (ELISA), although one mAb reacted to Sin Nombre virus in IFA. None of the mAbs cross-reacted with an amino-terminal segment of the shrew-borne Asama virus N protein. Anti-shrew-IgG sera were prepared after immunization of rabbits and BALB/c-mice with protein-G-purified shrew IgG. TPMV-N-protein-specific sera were raised by immunisation of Asian house shrews (Suncus murinus) with purified yeast-expressed TPMV N protein. Using these tools, an indirect ELISA was developed to detect TPMV-N-protein-specific antibodies in the sera of shrews. Using an established serological assay, high TPMV N protein specific antibody titres were measured in the sera of TPMV-N-protein-immunized and experimentally TPMV-infected shrews, whereas no cross-reactivity to other hantavirus N proteins was found. Therefore, the generated mAbs and the established ELISA system represent useful serological tools to detect TPMV, TPMV-related virus antigens or hantavirus-specific antibodies in hantavirus-infected shrews.
• Development of a serotyping enzyme-linked immunosorbent assay system based on recombinant truncated hantavirus nucleocapsid proteins for New World hantavirus infection

  Takaaki Koma, Kumiko Yoshimatsu, Midori Taruishi, Daisuke Miyashita, Rika Endo, Kenta Shimizu, Shumpei P. Yasuda, Takako Amada, Takahiro Seto, Ryo Murata, Haruka Yoshida, Hiroaki Kariwa, Ikuo Takashima, Jiro Arikawa

  JOURNAL OF VIROLOGICAL METHODS 185 (1) 74 - 81 0166-0934 2012/10 [Refereed][Not invited]
   

  New World hantaviruses were divided into five groups based on the amino acid sequence variability of the internal variable region (around 230-302 amino acids) of hantavirus nucleocapsid protein (NP). Sin Nombre virus (SNV), Andes virus, Black Creek Canal virus (BCCV). Carrizal virus (CARV) and Cano Delgadito virus belong to groups 1, 2, 3,4 and 5, respectively. Patient and rodent sera were serotyped successfully by an enzyme-linked immunosorbent assay (ELISA) with recombinant truncated NP lacking 99 N-terminal amino acids (trNP100) of SNV, CARV and BCCV. The trNP100 of BCCV showed lower reactivity to heterologous sera. In contrast, whole recombinant NP antigens detected both homologous and heterologous antibodies equally. The results together with results of a previous study suggest that trNP100 can distinguish infections among viruses in groups 1, 2, 3 and 4 of New World hantaviruses. The serotyping ELISA with trNP100 is useful for epidemiological surveillance in humans and rodents. (C) 2012 Elsevier BM. All rights reserved.
• Isolation of Hokkaido virus, genus Hantavirus, using a newly established cell line derived from the kidney of the grey red-backed vole (Myodes rufocanus bedfordiae)

  Takahiro Sanada, Takahiro Seto, Yuka Ozaki, Ngonda Saasa, Kumiko Yoshimatsu, Jiro Arikawa, Kentaro Yoshii, Hiroaki Kariwa

  JOURNAL OF GENERAL VIROLOGY 93 (10) 2237 - 2246 0022-1317 2012/10 [Refereed][Not invited]
   

  Hantaviruses belong to the family Bunyaviridae and are maintained in wild rodents. Although Vero E6 cells, which originate from African green monkey kidney, are used widely in hantavirus research, isolation of hantaviruses from this cell line is difficult. To develop an efficient method of propagation and isolation of hantaviruses we established a novel cell line, MRK101, derived from the kidney of the grey red-backed vole (Myodes rufocanus bedfordiae), the natural host of Hokkaido virus (HOKV). The MRK101 cells showed a significantly higher susceptibility to Puumala virus (PUUV) hosted by Myodes glareolus than Vero E6 cells. Viral nucleocapsid protein in PUUV-infected MRK101 cells was detected earlier than in Vero E6 cells, and the viral titre in the culture fluid of MRK101 cells was higher than that of Vero E6 cells during the early phase of infection. In contrast, MRK101 cells showed no susceptibility to Hantaan virus. HOKV, which has not been isolated to date, was isolated successfully using MRK101 cells. Moreover, the newly isolated HOKV was successfully propagated in MRK101, but not Vero E6, cells. Phylogenic analyses of the S (small), M (medium) and L (large) segment sequences revealed that HOKV is related most closely to PUUV, but is distinct from other hantaviruses. These data suggest that the MRK101 cell line is a useful tool for the isolation and propagation of hantaviruses. Moreover, this is (to our knowledge) the first report of hantavirus isolation in a cell line that originated from the natural host.
• Development of a Diagnostic Method Applicable to Various Serotypes of Hantavirus Infection in Rodents

  Takahiro Sanada, Hiroaki Kariwa, Ngonda Saasa, Keisuke Yoshikawa, Takahiro Seto, Vyacheslav G. Morozov, Evgeniy A. Tkachenko, Leonid I. Ivanov, Kumiko Yoshimatsu, Jiro Arikawa, Kentaro Yoshii, Ikuo Takashima

  JOURNAL OF VETERINARY MEDICAL SCIENCE 74 (9) 1237 - 1242 0916-7250 2012/09 [Refereed][Not invited]
   

  Antigenic diversity among different hantaviruses requires a variety of reagents for diagnosis of hantavirus infection. To develop a diagnostic method applicable to various hantavirus infections with a single set of reagents, we developed an enzyme-linked immuno-sorbent assay (ELISA) using recombinant nucleocapsid proteins of three hantaviruses, Amur, Hokkaido, and Sin Nombre viruses. This novel cocktail antigen-based ELISA enabled detection of antibodies against Hantaan, Seoul, Amur, Puumala, and Sin Nombre viruses in immunized laboratory animals. In wild rodent species, including Apodemus, Ratios, and Myodes, our ELISA detected antibodies against hantaviruses with high sensitivity and specificity. These data suggest that our novel diagnostic ELISA is a useful tool for screening hantavirus infections and could be effectively utilized for serological surveillance and quarantine purposes.
• Studies on Hantavirus Infection in Small Mammals Captured in Southern and Central Highland Area of Vietnam

  Vu Dinh Luan, Kumiko Yoshimatsu, Rika Endo, Midori Taruishi, Vo Thi Huong, Dang Tuan Dat, Pham Cong Tien, Kenta Shimzu, Takaaki Koma, Shumpei P. Yasuda, Le Nhi, Vu Thi Que Huong, Jiro Arikawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 74 (9) 1155 - 1162 0916-7250 2012/09 [Refereed][Not invited]
   

  To investigate the distribution of hantaviruses among animals in Southern and Central Highland area of Vietnam, a total of 1311 serum samples were obtained from rats and Asian house shrews (Suncus murinus) captured at 11 locations between 2006 and 2009. A total of 1066 serum samples from rats were examined for IgG antibodies against Hantaan virus, and there were 30 antibody-positive serum samples from rats that had been captured mainly in a port area and urban area in Ho Chi Minh City (HCMC) (2.8%). All of the antibody-positive rats were Rattus norvegicus, and they had Seoul virus (SEOV) genome in their lungs. SEOV sequences detected from rats captured in Southern Vietnam belonged to the same lineage as those from rats captured at Haiphong Port and a market area in Hanoi City. SEOV strain CSG5 was isolated from a rat captured at Saigon Harbor. Strain CSG5 showed a cross-neutralization pattern almost the same as that of a representative strain of SEOV. A total of 245 Asian house shrews were captured in the Central Highland area and near HCMC. Sera were examined for IgG antibodies against Thottapalayam virus (TPMV), and 32 (13.1%) of the antibody-positive shrews were mainly from the Central Highland area and showed a neutralizing antibody against TPMV. These results indicated that SEOV is distributed among R. norvegicus inhabiting harbor and urban areas of Southern Vietnam and that TPMV or an antigenically related virus is distributed among Asian house shrews in Central Highland area.
• Ecology of hantaviruses in Mexico: Genetic identification of rodent host species and spillover infection

  Ngonda Saasa, Cornelio Sanchez-Hernandez, Maria de Lourdes Romero-Almaraz, Ezequiel Guerrero-Ibarra, Alberto Almazan-Catalan, Haruka Yoshida, Daisuke Miyashita, Mariko Ishizuka, Takahiro Sanada, Takahiro Seto, Kentaro Yoshii, Celso Ramos, Kumiko Yoshimatsu, Jiro Arikawa, Ikuo Takashima, Hiroaki Kariwa

  VIRUS RESEARCH 168 (1-2) 88 - 96 0168-1702 2012/09 [Refereed][Not invited]
   

  In our recent epidemiological survey conducted in Mexico for hantavirus infection, we identified three distinct viruses circulating in Mexican wild rodents, namely Montano virus (MTNV), Huitzilac virus (HUIV), and Carrizal virus (CARV). To gain a detailed understanding of hantavirus epidemiology and its associated hosts, 410 rodents were captured at eight collecting points in Morelos and Guerrero, Mexico, and examined for hantavirus seroprevalence, the presence of viral RNA, and rodent host species identification using cytochrome b gene sequences. Of the 32 species captured, seven species were positive for hantavirus: Peromyscus beatae (31/127; 24.4%), Reithrodontomys sumichrasti (6/15; 40%), Reithrodontomys megalotis (2/25; 8%), Peromyscus aztecus evides (1/1; 100%), Peromyscus megalops (1/41; 2.4%), Megadontomys thomasi (1/9; 11.1%), and Neotoma picta (1/6; 16.7%), with an overall prevalence of 10.5%; virus genome persisted in the majority of seropositive rodents. Nucleotide sequence and phylogenetic analysis showed that the viruses belonged mainly to the three lineages previously identified. The data showed that MTNV and CARV were primarily carried by P. beatae and R. sumichrasti, respectively. In addition, the data revealed an apparent complex interaction between hantaviruses and their hosts, suggesting active transmission and/or spillover infections within sympatric rodent species. (c) 2012 Elsevier B.V. All rights reserved.
• ベトナムのラットおよびヒトにおけるラットE型肝炎ウイルスの感染状況の調査

  清水 健太, 李 天成, 安田 俊平, 吉松 組子, 駒 貴明, 長谷部 太, 山城 哲, Hoa Nguyen Thuy, Mai Le Thi Quynh, 有川 二郎

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 154回 265 - 265 1347-8621 2012/08
• 新たに分離されたHokkaidoウイルスの性状解析

  真田 崇弘, 尾崎 由佳, 瀬戸 隆弘, 中尾 桃子, Saasa Ngonda, 吉松 組子, 有川 二郎, 好井 健太朗, 苅和 宏明

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 154回 265 - 265 1347-8621 2012/08 [Refereed][Not invited]
  
• bla(NDM-1)-positive Klebsiella pneumoniae from Environment, Vietnam

  Rie Isozumi, Kumiko Yoshimatsu, Tetsu Yamashiro, Futoshi Hasebe, Binh Minh Nguyen, Tuan Cuong Ngo, Shumpei P. Yasuda, Takaaki Koma, Kenta Shimizu, Jiro Arikawa

  EMERGING INFECTIOUS DISEASES 18 (8) 1383 - 1385 1080-6040 2012/08 [Refereed][Not invited]
  
• The N-terminus of the Montano virus nucleocapsid protein possesses broadly cross-reactive conformation-dependent epitopes conserved in rodent-borne hantaviruses

  Ngonda Saasa, Haruka Yoshida, Kenta Shimizu, Cornelio Sanchez-Hernandez, Maria de Lourdes Romero-Almaraz, Takaaki Koma, Takahiro Sanada, Takahiro Seto, Kentaro Yoshii, Celso Ramos, Kumiko Yoshimatsu, Jiro Arikawa, Ikuo Takashima, Hiroaki Kariwa

  VIROLOGY 428 (1) 48 - 57 0042-6822 2012/06 [Refereed][Not invited]
   

  The hantavirus nucleocapsid (N) protein is an important immunogen that stimulates a strong and cross-reactive immune response in humans and rodents. A large proportion of the response to N protein has been found to target its N-terminus. However, the exact nature of this bias towards the N-terminus is not yet fully understood. We characterized six monoclonal antibodies (mAbs) against the N protein of Montano virus (MTNV), a Mexican hantavirus. Five of these mAbs recognized eight American hantaviruses and six European and Asian hantaviruses, but not the Soricomorpha-borne Thottapalayam hantavirus. The N protein-reactive binding regions of the five mAbs were mapped to discontinuous epitopes within the N-terminal 13-51 amino acid residues, while a single serotype-specific mAb was mapped to residues 1-25 and 49-75. Our findings suggest that discontinuous epitopes at the N-terminus are conserved, at least in rodent-borne hantaviruses, and that they contribute considerably to N protein cross-reactivity. (C) 2012 Elsevier Inc. All rights reserved.
• 腎症候性出血熱の動物モデルの開発

  清水 健太, 吉松 組子, 駒 貴明, 伊勢川 裕二, 安田 俊平, 天田 貴子, 五十棲 理恵, 有川 二郎

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 153回 247 - 247 1347-8621 2012/03
• イムノクロマト法による抗ハンタウイルス抗体の迅速抗体検出法の開発

  天田 貴子, 吉松 組子, 安田 俊平, 駒 貴明, 清水 健太, 五十棲 理恵, 林元 展人, 高倉 彰, 有川 二郎

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 153回 273 - 273 1347-8621 2012/03
• Isolation and Characterization of Hantaviruses in Far East Russia and Etiology of Hemorrhagic Fever with Renal Syndrome in the Region

  Hiroaki Kariwa, Keisuke Yoshikawa, Yoichi Tanikawa, Takahiro Seto, Takahiro Sanada, Ngonda Saasa, Leonid I. Ivanov, Raisa Slonova, Tatyana A. Zakharycheva, Ichiro Nakamura, Kumiko Yoshimatsu, Jiro Arikawa, Kentaro Yoshii, Ikuo Takashima

  AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE 86 (3) 545 - 553 0002-9637 2012/03 [Refereed][Not invited]
   

  Hemorrhagic fever with renal syndrome (HFRS) is a serious public health issue in Far East Russia. Two different hantaviruses were isolated from rodents captured in the Khabarovsk region: Amur virus (AMRV; Khekhtsir/AP209/2005 strain from Apodemus peninsulae) and Hantaan virus (HTNV; Galkino/AA57/2002 strain from A. agrarius). Genetic analysis of the new isolates revealed that the M and L segments were apparently different between AMRV and HTNV, but S segments of the two viruses were closer. The antigenicities of AMRV, HTNV, and Seoul virus (SEOV) were differentiated by cross-neutralization. Serological differential diagnoses of 67 HFRS patients in the Prymorsky and Khabarovsk regions of Far East Russia were conducted using a neutralization test. The results revealed that the major cause of HFRS varied with location in Far East Russia: SEOV for Vladivostok city in the Prymorsky region, AMRV in rural areas of the Primorsky region, and probably HTNV for the Khabarovsk region.
• Application of Truncated Nucleocapsid Protein (N) for Serotyping ELISA of Murinae-Associated Hantavirus Infection in Rats

  Shumpei P. Yasuda, Kumiko Yoshimatsu, Takaaki Koma, Kenta Shimizu, Rika Endo, Rie Isozumi, Jiro Arikawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 74 (2) 215 - 219 0916-7250 2012/02 [Refereed][Not invited]
   

  Truncated recombinant nucleocapsid proteins (trNs) that lack N-terminally located cross-reactive epitopes of four Murinae rodent-associated hantaviruses, Seoul virus (SEOV). Thailand virus, Hantaan virus (HTNV) and Dobrava-Belgrade virus, were produced by using a baculovirus expression system. ELISA with the trNs as antigens enabled serotyping of immune sera from rats experimentally inoculated with the corresponding hantaviruses with cut-off OD values of 60% of those of whole N of HTNV. The trN-based ELISA could serotype 12 out of 13 sera obtained from wild rodents (Ratios norvegicus) naturally infected with SEOV using the 60% cut-off value. These results indicate that screening with whole N followed by serotyping with trNs using a cut-off OD value of 60% of that of whole N is a useful method for serological surveillance of Murinae-associated hantavirus infection among rodents.
• Genetic diversity of hantaviruses in Mexico: Identification of three novel hantaviruses from Neotominae rodents

  Hiroaki Kariwa, Haruka Yoshida, Cornelio Sanchez-Hernandez, Maria de Lourdes Romero-Almaraz, Jose Alberto Almazan-Catalan, Celso Ramos, Daisuke Miyashita, Takahiro Seto, Ayako Takano, Masashi Totani, Ryo Murata, Ngonda Saasa, Mariko Ishizuka, Takahiro Sanada, Kentaro Yoshii, Kumiko Yoshimatsu, Jiro Arikawa, Ikuo Takashima

  VIRUS RESEARCH 163 (2) 486 - 494 0168-1702 2012/02 [Refereed][Not invited]
   

  A variety of hantaviruses are harbored by rodents in North and South America, some of which can cause hantavirus pulmonary syndrome. To obtain greater evolutionary insight into hantaviruses in the Americas, a total of 211 rodents were captured in the Mexican states of Guerrero and Morelos in 2006. Anti-hantavirus antibodies were detected in 27 of 211 serum samples (12.8%) by ELISA. The distribution of seropositive rodents was: 17 Peromyscus beatae, 1 Megadontomys thomasi,1 Neotoma picta, 6 Reithrodontomys sumichrasti, and 2 Reithrodontomys megalotis. The hantavirus small (S), medium (M), and large (L) genome segments from P. beatae, R. sumichrasti, and R. megalotis were amplified and the sequences covering the open reading frames were determined. The hantaviruses from P. beatae, R. sumichrasti, and R. megalotis were provisionally designated Montano (MTN), Carrizal (CAR), and Huitzilac (HUI), respectively. The M segment amino acid identities among the Mexican hantaviruses were 80.8-93.0%. When these M segments were compared to those of known hantaviruses, MTN virus was most closely related to Limestone Canyon (LSC) virus (88.9% amino acid identity), while the CAR and HUI viruses were most closely related to El Moro Canyon (ELMC) virus (90-91% identity). Phylogenetic analysis revealed that the MTN, CAR, and HUI viruses occupy a monophyletic clade with the LSC, ELMC, and Rio Segundo viruses, which are harbored by Peromyscus boylii, R. megalotis, and Reithrodontomys mexicanus, respectively. The data obtained in this study provide important information for understanding the evolution of hantaviruses in the Americas. (C) 2011 Elsevier B.V. All rights reserved.
• Outbreak of Leptospirosis after Flood, the Philippines, 2009

  Al-shere T. Amilasan, Mugen Ujiie, Motoi Suzuki, Eumelia Salva, Maria Cecilia P. Belo, Nobuo Koizumi, Kumiko Yoshimatsu, Wolf-Peter Schmidt, Shane Marte, Efren M. Dimaano, Jose Benito Villarama, Koya Ariyoshi

  EMERGING INFECTIOUS DISEASES 18 (1) 91 - 94 1080-6040 2012/01 [Refereed][Not invited]
   

  After a typhoon in September 2009, an outbreak of leptospirosis occurred in Metro Manila, the Philippines; 471 patients were hospitalized and 51(10.8%) died. A hospital-based investigation found risk factors associated with fatal infection to be older age, hemoptysis, anuria, jaundice, and delayed treatment with antimicrobial drugs.
• [Bunyavirus and its ecology].

  Yoshimatsu, K., Arikawa, J.

  Uirusu 62 (2) 239 - 250 0042-6857 2012 [Refereed][Not invited]
  
• Characterization of self-assembled virus-like particles of rat hepatitis E virus generated by recombinant baculoviruses

  Tian-Cheng Li, Kumiko Yoshimatsu, Shumpei P. Yasuda, Jiro Arikawa, Takaaki Koma, Michiyo Kataoka, Yasushi Ami, Yuriko Suzaki, Le Thi Quynh Mai, Nguyen Thuy Hoa, Tetsu Yamashiro, Futoshi Hasebe, Naokazu Takeda, Takaji Wakita

  JOURNAL OF GENERAL VIROLOGY 92 (Pt 12) 2830 - 2837 0022-1317 2011/12 [Refereed][Not invited]
   

  Hepatitis E virus (HEV) is a causative agent of hepatitis E. Recently, a novel hepatitis E-like virus was isolated from Norway rats in Germany. However, the antigenicity, pathogenicity and epidemiology of this virus are unclear because of the lack of a cell-culture system in which to grow it. In this study, an N-terminally truncated ORF2 protein was expressed in insect Tn5 cells using a recombinant baculovirus expression system and a large amount of 53 kDa protein was expressed and efficiently released into the supernatant. Electron microscopic analyses of the purified 53 kDa protein revealed that the protein self-assembled into two types of empty HEV-like particles (rat HEVLPs). The smaller rat HEVLPs were estimated to be 24 nm in diameter, which is similar to the size of genotype G1, G3 and G4 HEVLPs. The larger rat HEVLPs were estimated to measure 35 nm in diameter, which is similar to the size of native rat HEV particles. An ELISA to detect antibodies was established using rat HEVLPs as the antigens, which demonstrated that rat HEVLPs were cross-reactive with G1, G3 and G4 HEVs. Detection of IgG and IgM antibodies was performed by examination of 139 serum samples from wild rats trapped in Vietnam, and it was found that 20.9 % (29/139) and 3.6 % (5/139) of the samples were positive for IgG and IgM, respectively. In addition, rat HEV RNA was detected in one rat serum sample that was positive for IgM. These results indicated that rat HEV is widespread and is transmitted among wild rats.
• Effect of environmental enrichment after the occurrence of wet bedding created by mice and abnormal fur in mice.

  Tosa N, Yoshimatsu K, Arikawa J

  J Am Assoc Lab Anim Sci 50 (5) 779 - 780 2011/09 [Refereed][Not invited]
  
• Puumala virus infection in Syrian hamsters (Mesocricetus auratus) resembling hantavirus infection in natural rodent hosts

  Takahiro Sanada, Hiroaki Kariwa, Noriyo Nagata, Yoichi Tanikawa, Takahiro Seto, Kumiko Yoshimatsu, Jiro Arikawa, Kentaro Yoshii, Ikuo Takashima

  VIRUS RESEARCH 160 (1-2) 108 - 119 0168-1702 2011/09 [Refereed][Not invited]
   

  The mechanism of hantavirus persistent infection in natural hosts is poorly understood due to a lack of laboratory animal models. Herein, we report that Syrian hamsters (Mesocricetus auratus) infected with Puumala virus (PUUV) at 4 weeks old show persistent infection without clinical symptoms for more than 2 months. IgG and IgM antibodies against the viral nucleocapsid protein and neutralizing antibody were first detectable at 14 days postinoculation (dpi) and maintained through 70 dpi. Viral RNA was first detected from 3 dpi in lungs and blood clots, and was detected in all tissues tested at 7 dpi. The viral RNA persisted for at least 70 days in the lungs, kidney, spleen, heart, and brain. The highest level of RNA copies was observed at 14 dpi in the lungs. Slight inflammatory reactions were observed in the lungs, adrenal glands, and brain. Immunohistochemical analysis revealed that PUUV antigen persisted until 56 dpi in the kidneys and adrenal glands. Infected hamsters showed no body weight loss or clinical signs. These results indicate that PUUV infection in hamsters is quite similar to the hantavirus infection of natural host rodents. (C) 2011 Elsevier B.V. All rights reserved.
• ハンタウイルス糖蛋白質の細胞内輸送に関与するウイルス蛋白質とその機能領域

  清水 健太, 吉松 組子, 駒 貴明, 安田 俊平, 有川 二郎

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 152回 236 - 236 1347-8621 2011/08
• 日本のげっ歯類のハンタウイルス感染状況に関する血清疫学調査とエゾヤチネズミが保有するHokkaidoウイルスの遺伝的多様性

  尾崎 由佳, 萩谷 友洋, 真田 崇弘, 瀬戸 隆弘, Kyle Taylor, 吉川 佳佑, Leonid Ivanov, 好井 健太朗, 坪田 敏男, 池中 良徳, 石塚 真由美, 吉松 組子, 有川 二郎, 苅和 宏明

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 152回 256 - 256 1347-8621 2011/08 [Refereed][Not invited]
  
• 日本のげっ歯類のハンタウイルス感染状況に関する血清疫学調査とエゾヤチネズミが保有するHokkaidoウイルスの遺伝的多様性

  尾崎由佳, 萩谷友洋, 真田崇弘, 瀬戸隆弘, TAYLOR Kyle, 吉川佳佑, IVANOV Leonid I, 好井健太朗, 坪田敏男, 池中良徳, 石塚真由美, 吉松組子, 有川二郎, 苅和宏明

  北海道獣医師会雑誌 (公社)北海道獣医師会 55 (8) 415 - 415 0018-3385 2011/08 [Not refereed][Not invited]
  
• Valine 1532 of human BRC repeat 4 plays an important role in the interaction between BRCA2 and RAD51

  Kazuhiko Ochiai, Yasunaga Yoshikawa, Kumiko Yoshimatsu, Toshina Oonuma, Yukiko Tomioka, Eichi Takeda, Jiro Arikawa, Katsumi Mominoki, Toshinori Omi, Kazuyoshi Hashizume, Masami Morimatsu

  FEBS LETTERS 585 (12) 1771 - 1777 0014-5793 2011/06 [Refereed][Not invited]
   

  The breast cancer susceptibility protein BRCA2 is essential for recombinational DNA repair. BRCA2 specifically binds to RAD51 via eight BRC repeat motifs and delivers RAD51 to double-stranded DNA breaks. In this study, a mammalian two-hybrid assay and competitive ELISA showed that the interaction between BRC repeat 4 (BRC4) and RAD51 was strengthened by the substitution of a single BRC4 amino acid from valine to isoleucine (V1532I). However, the cancer-associated V1532F mutant exhibited very weak interaction with RAD51. This study used a comparative analysis of BRC4 between animal species to identify V1532 as an important residue that interacts with RAD51. Structured summary of protein interactions: cRAD51 physically interacts with cRAD51 by two hybrid (View interaction) fBRC4 physically interacts with cRAD51 by two hybrid (View interaction) cBRC4 physically interacts with cRAD51 by two hybrid (View interaction) hBRC4 physically interacts with hBRC4 and hRAD51 by competition binding (View Interaction 1, 2) hBRC4 physically interacts with cRAD51 by two hybrid (View interaction) hBRC4 binds to hRAD51 by enzyme linked immunosorbent assay (View interaction) (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• An efficient in vivo method for the isolation of Puumala virus in Syrian hamsters and the characterization of the isolates from Russia

  Takahiro Seto, Evgeniy A. Tkachenko, Vyacheslav G. Morozov, Yoichi Tanikawa, Sergey I. Kolominov, Sergey N. Belov, Ichiro Nakamura, Nobuo Hashimoto, Yasuhiro Kon, Alexander E. Balakiev, Tamara K. Dzagurnova, Olga A. Medvedkina, Mina Nakauchi, Mariko Ishizuka, Kentaro Yoshii, Kumiko Yoshimatsu, Leonid V. Ivanov, Jiro Arikawa, Ikuo Takashima, Hiroaki Kariwa

  JOURNAL OF VIROLOGICAL METHODS 173 (1) 17 - 23 0166-0934 2011/04 [Refereed][Not invited]
   

  Puumala virus (PUUV) and other Arvicolinae-borne hantaviruses are difficult to cultivate in cell culture. To isolate these hantaviruses efficiently, hantavirus nucleocapsid protein (NP)-positive but seronegative wild rodents were selected by NP-detection ELISA. Three of 68 Myodes glareolus captured in Samara, Russia, were NP-positive and seronegative. Syrian hamsters were inoculated with lung homogenates from NP-positive rodents for virus propagation. Virus isolation in vitro was carried out by inoculation of lung homogenates of NP-positive hamsters to Vero E6 cell monolayers. Two PUUV strains (Samara49/CG/2005 and Samara94/CG/2005) from M. glareolus were isolated in Vero E6 cells. Nucleotide and amino acid sequence identities of the S segment of these isolates to those of PUUV F-s808 from a fatal HFRS patient in Samara region were 96.7-99.3% and 99.3-100.0%, respectively. Morphologic features of Vero E6 cells infected with PUUV strain Samara49/CG/2005 were quite similar to those of Hantaan virus-infected cells. Isolation of Hokkaido virus from Myodes rufocanus captured in Hokkaido, Japan, was also performed. Hokkaido virus NP and RNA were recovered and maintained in hamsters. These results suggest that inoculation of Syrian hamsters with rodent samples is an efficient method for the isolation and maintenance of PUUV and other Arvicolinae-borne hantaviruses. (C) 2011 Elsevier B.V. All rights reserved.
• Serological Evidence of Thailand Virus-Related Hantavirus Infection among Suspected Leptospirosis Patients in Kandy, Sri Lanka

  Chandika D. Gamage, Shumpei P. Yasuda, Sanae Nishio, Senanayake A. Kularatne, Kosala Weerakoon, Jayanthe Rajapakse, Chinyere Nwafor-Okoli, Romeo B. Lee, Yoshi Obayashi, Kumiko Yoshimatsu, Jiro Arikawa, Hiko Tamashiro

  JAPANESE JOURNAL OF INFECTIOUS DISEASES 64 (1) 72 - 75 1344-6304 2011/01 [Not refereed][Not invited]
   

  A cross-sectional study was undertaken to determine the current prevalence of leptospirosis and hantaviral infections, and the socio-demographic characteristics and risk factors of infected patients, in Kandy, Sri Lanka. This report discusses the serological evidence of hantavirus infections among 105 suspected leptospirosis patients, 8 of whom had hantavirus antibodies. Serotyping ELISA showed that these 8 patients had high optical density values for Thailand virus. Most of the sera showed that the focus reduction neutralization test titer against Thailand virus was higher than that against Seoul virus, thereby suggesting that the hantaviral antibodies found in Sri Lanka are different from Seoul virus but closely related to Thailand virus. These findings imply that the hantaviral infection found in Kandy, Sri Lanka appears to be due to a virus similar to Thailand virus. Epidemiological analysis revealed that the association between hantavirus infection and socio-demographic characteristics was not statistically significant.
• Serological Evidence of Thailand Virus-Related Hantavirus Infection among Suspected Leptospirosis Patients in Kandy, Sri Lanka

  Chandika D. Gamage, Shumpei P. Yasuda, Sanae Nishio, Senanayake A. Kularatne, Kosala Weerakoon, Jayanthe Rajapakse, Chinyere Nwafor-Okoli, Romeo B. Lee, Yoshi Obayashi, Kumiko Yoshimatsu, Jiro Arikawa, Hiko Tamashiro

  JAPANESE JOURNAL OF INFECTIOUS DISEASES 64 (1) 72 - 75 1344-6304 2011/01 [Refereed][Not invited]
   

  A cross-sectional study was undertaken to determine the current prevalence of leptospirosis and hantaviral infections, and the socio-demographic characteristics and risk factors of infected patients, in Kandy, Sri Lanka. This report discusses the serological evidence of hantavirus infections among 105 suspected leptospirosis patients, 8 of whom had hantavirus antibodies. Serotyping ELISA showed that these 8 patients had high optical density values for Thailand virus. Most of the sera showed that the focus reduction neutralization test titer against Thailand virus was higher than that against Seoul virus, thereby suggesting that the hantaviral antibodies found in Sri Lanka are different from Seoul virus but closely related to Thailand virus. These findings imply that the hantaviral infection found in Kandy, Sri Lanka appears to be due to a virus similar to Thailand virus. Epidemiological analysis revealed that the association between hantavirus infection and socio-demographic characteristics was not statistically significant.
• Different cross-reactivity of human and rodent sera to Tula virus and Puumala virus

  Erdenesaikhan Tegshduuren, Kumiko Yoshimatsu, Midori Taruishi, Rika Endo, Kenta Shimizu, Takaaki Koma, Shumpei P. Yasuda, Hiroaki Kariwa, Jiro Arikawa, Chiaki Ishihara

  COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 33 (6) E67 - E73 0147-9571 2010/12 [Refereed][Not invited]
   

  Tula virus (TULV) and Puumala virus (PUUV) are hantaviruses carried by the bank vole (Myodes glareolus) and European common vole (Microtus arvalis), respectively. PUUV is a causative agent of hemorrhagic fever with renal syndrome (HFRS), while TULV is thought to be apathogenic to humans. The N-terminal regions of the N proteins from TULV and PUUV were expressed and applied as enzyme-linked immunosorbent assay (ELISA) antigens. Colonized Japanese grass voles (Microtus montebelli) and BALB/c mice were used for experimental inoculation of the vole-borne hantaviruses TULV and PUUV. Voles and mice showed significant antibody production toward both viruses, but these antisera showed little cross-reactivity between TULV and PUUV in the immunofluorescence antibody assay and ELISA. In contrast, sera from patients with HFRS caused by PUUV exhibited high cross-reactivity against the TULV antigen, and sera from a natural rodent reservoir showed moderate cross-reactivity against the heterologous antigen, indicating that the antigenic cross-reactivity between TULV and PUUV differs in sera from rodents and humans. (C) 2010 Elsevier Ltd. All rights reserved.
• ハンタウイルスNucleocapsid proteinはGlycoprotein Gcのシスゴルジへの局在を促進する

  清水 健太, 吉松 組子, 駒 貴明, 遠藤 理香, 安田 俊平, Erdenesaikhan Tegshduuren, 有川 二郎

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 150回 232 - 232 1347-8621 2010/09
• メキシコ由来のハンタウイルスに対するモノクローナル抗体の作出と各種ハンタウイルスに対する反応性の検討

  吉田 喜香, 苅和 宏明, 真田 崇弘, Saasa Ngonda, 瀬戸 隆弘, 吉松 組子, 有川 二郎, 好井 健太朗, 高島 郁夫

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 150回 249 - 249 1347-8621 2010/09 [Refereed][Not invited]
  
• Puumalaウイルスを感染させたシリアンハムスター(Mesocricetus auratus)の感染動態の解析

  真田 崇弘, 苅和 宏明, 谷川 洋一, Nur Hardy Abu Daud, 瀬戸 隆弘, 永田 典代, 吉松 組子, 有川 二郎, 好井 健太朗, 高島 郁夫

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 150回 252 - 252 1347-8621 2010/09 [Refereed][Not invited]
  
• Truncated Hantavirus Nucleocapsid Proteins for Serotyping Sin Nombre, Andes, and Laguna Negra Hantavirus Infections in Humans and Rodents

  Takaaki Koma, Kumiko Yoshimatsu, Noemi Pini, David Safronetz, Midori Taruishi, Silvana Levis, Rika Endo, Kenta Shimizu, Shumpei P. Yasuda, Hideki Ebihara, Heinz Feldmann, Delia Enria, Jiro Arikawa

  JOURNAL OF CLINICAL MICROBIOLOGY 48 (5) 1635 - 1642 0095-1137 2010/05 [Refereed][Not invited]
   

  Sin Nombre virus (SNV), Andes virus (ANDV), and Laguna Negra virus (LANV) have been known as the dominant causative agents of hantavirus pulmonary syndrome (HPS). ANDV and LANV, with different patterns of pathogenicity, exist in a sympatric relationship. Moreover, there is documented evidence of person-to-person transmission of ANDV. Therefore, it is important in clinical medicine and epidemiology to know the serotype of a hantavirus causing infection. Truncated SNV, ANDV, and LANV recombinant nucleocapsid proteins (trNs) missing 99 N-terminal amino acids (trN100) were expressed using a baculovirus system, and their applicability for serotyping SNV, ANDV, and LANV infection by the use of enzyme-linked immunosorbent assays (ELISA) was examined. HPS patient sera and natural-reservoir rodent sera infected with SNV, ANDV, and LANV showed the highest optical density (OD) values for homologous trN100 antigens. Since even patient sera with lower IgM and IgG antibody titers were serotyped, the trN100s are therefore considered useful for serotyping with early-acute-phase sera. In contrast, assays testing whole recombinant nucleocapsid protein antigens of SNV, ANDV, and LANV expressed in Escherichia coli detected homologous and heterologous antibodies equally. These results indicated that a screening ELISA using an E. coli-expressed antigen followed by a serotyping ELISA using trN100s is useful for epidemiological surveillance in regions where two or more hantavirus species cocirculate.
• Hemorrhagic Fever with Renal Syndrome, Vietnam

  Vu Thi Que Huong, Kumiko Yoshimatsu, Vu Dinh Luan, Le Van Tuan, Le Nhi, Jiro Arikawa, Tran Minh Nhu Nguyen

  EMERGING INFECTIOUS DISEASES 16 (2) 363 - 365 1080-6040 2010/02 [Refereed][Not invited]
  
• Extensive Host Sharing of Central European Tula Virus

  Jonas Schmidt-Chanasit, Sandra Essbauer, Rasa Petraityte, Kumiko Yoshimatsu, Kirsten Tackmann, Franz J. Conraths, Kestutis Sasnauskas, Jiro Arikawa, Astrid Thomas, Martin Pfeffer, Jerrold J. Scharninghausen, Wolf Splettstoesser, Matthias Wenk, Gerald Heckel, Rainer G. Ulrich

  JOURNAL OF VIROLOGY 84 (1) 459 - 474 0022-538X 2010/01 [Refereed][Not invited]
   

  To examine the host association of Tula virus (TULV), a hantavirus present in large parts of Europe, we investigated a total of 791 rodents representing 469 Microtus arvalis and 322 Microtus agrestis animals from northeast, northwest, and southeast Germany, including geographical regions with sympatric occurrence of both vole species, for the presence of TULV infections. Based on serological investigation, reverse transcriptase PCR, and subsequent sequence analysis of partial small (S) and medium (M) segments, we herein show that TULV is carried not only by its commonly known host M. arvalis but also frequently by M. agrestis in different regions of Germany for a prolonged time period. At one trapping site, TULV was exclusively detected in M. agrestis, suggesting an isolated transmission cycle in this rodent reservoir separate from spillover infections of TULV-carrying M. arvalis. Phylogenetic analysis of the S and M segment sequences demonstrated geographical clustering of the TULV sequences irrespective of the host, M. arvalis or M. agrestis. The novel TULV lineages from northeast, northwest, and southeast Germany described here are clearly separated from each other and from other German, European, or Asian lineages, suggesting their stable geographical localization and fast sequence evolution. In conclusion, these results demonstrate that TULV represents a promiscuous hantavirus with a large panel of susceptible hosts. In addition, this may suggest an alternative evolution mode, other than a strict coevolution, for this virus in its Microtus hosts, which should be proven in further large-scale investigations on sympatric Microtus hosts.
• Epidemiological Study of Hantavirus Infection in the Samara Region of European Russia

  Hiroaki Kariwa, Evgeniy A. Tkachenko, Vyacheslav G. Morozov, Takahiro Seto, Yoichi Tanikawa, Sergey I. Kolominov, Sergey N. Belov, Ichiro Nakamura, Nobuo Hashimoto, Alexander E. Balakiev, Tamara K. Dzagurnova, Nur Hardy bin Abu Daud, Daisuke Miyashita, Olga A. Medvedkina, Mina Nakauchi, Mariko Ishizuka, Kentaro Yoshii, Kumiko Yoshimatsu, Jiro Arikawa, Ikuo Takashima

  JOURNAL OF VETERINARY MEDICAL SCIENCE 71 (12) 1569 - 1578 0916-7250 2009/12 [Refereed][Not invited]
   

  European Russia is a highly endemic area of hemorrhagic fever with renal syndrome (HFRS), a rodent-borne zoonotic disease, caused by hantaviruses. In total, 145 small mammals of four species (Myodes glareolus, Apodemus flavicollis, A. agrarius, and A. uralensis) were trapped in the Samara region of European Russia in August 2005 and examined for the presence of hantavirus (HV). Anti-HV antibodies were found in six of 68 (8.8%) M. glareolus and in one of 19 (5.3%) A. flavicollis by indirect immunofluorescent antibody assay (IFA). The Puumala virus (PUUV), which is one of the hantavirus species, was detected in the lungs of seven M. glareolus by RT-PCR. The virus S-segment was extremely similar (96.2% to 99.3%) to the sequence found in a fatal case of HFRS in the Samara region. Phylogenetic analyses of S and M segments showed that the Samara PUUVs form a cluster within the Russian Volga lineage and apparently differ from other European PUUVs. Anti-PUUV antibodies were found in blood sera from seven HFRS patients and from one undiagnosed patient from the Samara region, using IFA and an enzyme-linked immunosorbent assay (ELISA). These data Suggest that the bank vole M. glareolus is a primary natural reservoir and vector for PUUV, which is the main causative agent of HFRS in humans in the Samara region.
• Molecular Epidemiological and Serological Studies of Hantavirus Infection in Northern Vietnam

  Thua Thang Truong, Kumiko Yoshimatsu, Koichi Araki, Byoung-Hee Lee, Ichiro Nakamura, Rika Endo, Kenta Shimizu, Shumpei P. Yasuda, Takaaki Koma, Midori Taruishi, Megumi Okumura, Uyen Ninh Truong, Jiro Arikawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 71 (10) 1357 - 1363 0916-7250 2009/10 [Refereed][Not invited]
   

  The distribution of anti-hantavirus antibodies in humans and rodents in northern Vietnam was examined. In total, 837 seruml samples from healthy humans (617) and patients with fever (220), living in six different areas were screened for IgG antibodies against Hantaan or Seoul virus (SEOV) by ELISA, IFA, and Western blot analysis. Antibody-positive sera were identified ill 7/617 (1.1%) healthy donors, 51150 port workers in the port of Hai Phong, and 2/185 residents of Ha Nam Province. In comparison, positive sera were detected in 5/220 (2.3%) fever patients ill the provinces of Ha Nam (1/58) and Thanh Hoa (4/146). Antibody-positive Rattus norvegicus were found in the provinces of Ha Nam (7/52) and Thanh Hoa (1/67), in Haibatrung District (7/43) in Hanoi, and in Hai Phong" Port (21/62), while antibody-positive R. rattus (2/17) were found in Hai Phong Port. Part of the Gc region from the viral genome was amplified by RT-PCR using lung tissue samples front R. norvegicus in Haibatrung (2/7) and Hai Phong Port (7/9), bill not from R. rattus (0/2). Viral sequences were located in the SEOV clade and formed a single lineage with Indonesian SEOV. suggesting that Vietnamese SEOV is part of a distinct lineage among Asian SEOVs.
• Hantavirus species in India : A retrospective study

  S. Chandy, M. Okumura, K. Yoshimatsu, R. G. Ulrich, G. T. John, P. Abraham, J. Arikawa, G. Sridharan

  INDIAN JOURNAL OF MEDICAL MICROBIOLOGY 27 (4) 348 - 350 0255-0857 2009/10 [Not refereed][Not invited]
   

  Hantaviruses cause hemorrhagic fever with renal syndrome in Europe and Asia. There are about 20 documented hantavirus species and newer species are being described worldwide, especially in non-rodent reservoirs, i.e shrews. Focus reduction neutralization test is the classical serotyping technique for hantavirus. However, this study employs a previously established serotyping ELISA, to retrospectively analyze known hantavirus IgG reactive samples for infecting serotypes. The result suggests presence of Thailand virus- like and Hantaan virus -like strains in India.
• メキシコの野生げっ歯類が保有するハンタウイルスの遺伝子解析

  吉田 喜香, 苅和 宏明, 高野 絢子, 戸谷 理詩, 宮下 大輔, Ngonda Saasa, 瀬戸 隆弘, 真田 崇弘, 吉川 佳佑, 好井 健太朗, 吉松 組子, 有川 次郎, 高島 郁夫

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 148回 237 - 237 1347-8621 2009/09 [Refereed][Not invited]
  
• 極東ロシアの野鼠からのハンタウイルスの分離と人におけるウイルス感染状況の調査

  吉川 佳佑, 苅和 宏明, 瀬戸 隆弘, 真田 崇弘, 石塚 万里子, 吉松 組子, 有川 二郎, Ivanov Leonid, Slonova Raisa, 好井 健太朗, 高島 郁夫

  北海道獣医師会雑誌 (公社)北海道獣医師会 53 (8) 112 - 112 0018-3385 2009/08 [Refereed][Not invited]
  
• Seroepidemiological study in a Puumala virus outbreak area in South-East Germany

  Marc Mertens, Roman Woelfel, Katrin Ullrich, Kumiko Yoshimatsu, Jana Blumhardt, Ina Roemer, Jutta Esser, Jonas Schmidt-Chanasit, Martin H. Groschup, Gerhard Dobler, Sandra S. Essbauer, Rainer G. Ulrich

  MEDICAL MICROBIOLOGY AND IMMUNOLOGY 198 (2) 83 - 91 0300-8584 2009/05 [Refereed][Not invited]
   

  Puumala virus (PUUV) is the cause of the majority of haemorrhagic fever with renal syndrome cases in Germany. In 2004, a nephropathia epidemica outbreak was recorded in Lower Bavaria, South-East Germany. For a seroepidemiological study in this region including the resident population at four locations (n = 178) and soldiers from one location (n = 208) indirect immunoglobulin M (IgM) and immunoglobulin G (IgG) enzyme-linked immunosorbent assays (ELISAs) and immunoblot tests based on a yeast-expressed PUUV nucleocapsid protein were established. The validation using human serum panels originating from Germany revealed a diagnostic sensitivity and specificity of 98/100% for the IgM ELISA, 99/99% for the IgG ELISA, 99/100% for the IgM immunoblot test and 100/96% for the IgG immunoblot test. Using the novel IgG assays as well as a commercial IgG ELISA and an immunofluorescence assay for the resident population an average prevalence of 6.7% (12 of 178) with a range of 0% (0 of 21) to 11.9% (7 of 59) was observed. Positive serological results were equally distributed between males and females with an average age of 63 for males and 52 for females. The seroprevalence in the soldier group was found to be about 1% with one positive male of 203 (age 46 years) and one positive female of five (age 47 years). In conclusion, the PUUV seroprevalence in the residents of the outbreak region in Lower Bavaria was found to be up to fivefold higher than the average hantavirus seroprevalence of the German population.
• Acute febrile illness caused by hantavirus: serological and molecular evidence from India

  Sara Chandy, Kumiko Yoshimatsu, Hari Kishan Boorugu, Anugrah Chrispal, Kurien Thomas, Abraham Peedicayil, Priya Abraham, Jiro Arikawa, Gopalan Sridharan

  TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE 103 (4) 407 - 412 0035-9203 2009/04 [Refereed][Not invited]
   

  Study of hantavirus infections in India is in its early stages. As early symptoms of hantavirus disease can be non-specific and the diagnosis confirmed only by laboratory testing, use of appropriate diagnostic toots is important. To improve the diagnosis of hantavirus infections in India, commercial ELISA systems followed by indirect immunofluorescence assays were used to detect anti-hantavirus IgM and IgG in samples from patients with acute febrile illness. Of 347 patients tested, 5.2% showed serological evidence of hantavirus infection. Sequences obtained from patients showing molecular evidence of hantavirus infection were related to Hantaan virus. In the absence of mu-capture ELISA, we recommend the use of combination testing systems in areas non-endemic for hantavirus infections. In India there is an increased risk of rodent-borne infections and the differential diagnosis of undifferentiated febrile illness should include hantavirus infection. (C) 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
• 極東ロシアのげっ歯類からのハンタウイルスの分離とウイルス遺伝子の性状解析

  吉川 佳佑, 苅和 宏明, 瀬戸 隆弘, 真田 崇弘, 石塚 万里子, 吉松 組子, 有川 二郎, Ivanov Lenid, 好井 健太朗, 高島 郁夫

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 147回 260 - 260 1347-8621 2009/03 [Refereed][Not invited]
  
• Genetic and antigenic analyses of a Puumala virus isolate as a potential vaccine strain

  Nur Hardy Abu Daud, Hiroaki Kariwa, Evgeniy Tkachenko, Tamara Dzagurnova, Olga Medvedkina, Petr Tkachenko, Mariko Ishizuka, Takahiro Seto, Daisuke Miyashita, Takahiro Sanada, Mina Nakauchi, Kentaro Yoshii, Akihiko Maeda, Kumiko Yoshimatsu, Jiro Arikawa, Ikuo Takashima

  JAPANESE JOURNAL OF VETERINARY RESEARCH 56 (3) 151 - 165 0047-1917 2008/11 [Refereed][Not invited]
   

  Puumala virus (PUUV), a causative agent of hemorrhagic fever with renal syndrome (HFRS), is prevalent in Europe and European Russia. No vaccine has been developed for PUUV-associated HFRS, primarily because of the low viral yield in cultured cells. A PUUV strain known as DTK/Ufa-97 was isolated in Russia and adapted for growth in Vero E6 cells maintained in serum-free medium. The DTK/Ufa-97 strain produced a higher viral titer in serum-free medium, suggesting that it may prove useful in the development of an HFRS vaccine. When PUUV-infected Vero E6 cells were grown in serum-free medium, the DTK/Ufa-97 strain yielded more copies of intracellular viral RNA and a higher viral titer in the culture fluid than did the Sotkamo strain. Phylogenetic analysis revealed that PUUVs can be classified into multiple lineages according to geographical origin, and that the DTK/Ufa-97 strain is a member of the Bashkiria-Saratov lineage. The deduced amino acid sequences of the small, medium, and large segments of the DTK/Ufa-97 strain were 99.2% to 100%, 99.3% to 99.8%, and 99.8% identical, respectively, to those of the Bashkirian PUUV strains and 96.9%, 92.6%, and 97.4% identical, respectively, to those of the Sotkamo strain, indicating that the PUUVs are genetically diverse. However, DTK/Ufa-97 and other strains of PUUV exhibited similar patterns of binding to a panel of monoclonal antibodies against Hantaan virus. In addition, diluted antisera (i.e., ranging from 1:160 to 1:640) specific to three strains of PUUV neutralized both homologous and heterologous viruses. These results suggest that the DTK/Ufa-97 strain is capable of extensive growth and is antigenically similar to genetically distant strains of PUUV.
• 多種類のハンタウイルス血清型の検出が可能な抗原検出法の開発

  真田 崇弘, 苅和 宏明, 瀬戸 隆弘, 谷川 洋一, 宮下 大輔, 吉松 組子, 有川 二郎, 好井 健太朗, 高島 郁夫

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 146回 225 - 225 1347-8621 2008/09 [Refereed][Not invited]
  
• Lack of vertical transmission of Hantaan virus from persistently infected dam to progeny in laboratory mice

  Midori Taruishi, Kumiko Yoshimatsu, Rei Hatsuse, Megumi Okumura, Ichiro Nakamura, Jiro Arikawa

  ARCHIVES OF VIROLOGY 153 (8) 1605 - 1609 0304-8608 2008/08 [Refereed][Not invited]
   

  It is unclear how the hantaviruses are transferred from infected to uninfected rodents. We studied the status of persistently infected laboratory mice and examined the frequency of viral transmission to their offspring. Expression of Hantaan virus nucleocapsid protein was detected in the lungs of persistently infected dams. None of the progeny displayed viral antigen, although they were strongly positive for IgG antibodies against hantavirus. There was neither hantavirus RNA nor virus-specific IgM antibodies or virus-specific CD8(+) T cells in the progeny. These results did not show any indication for a vertical transmission of hantaviruses, at least in the laboratory mouse model studied.
• Development of a serotyping ELISA system for Thailand virus infection

  Ichiro Nakamura, Kumiko Yoshimatsu, Byoung-Hee Lee, Megumi Okumura, Midori Taruishi, Koichi Araki, Hiroaki Kariwa, Ikuo Takashima, Jiro Arikawa

  ARCHIVES OF VIROLOGY 153 (8) 1537 - 1542 0304-8608 2008/08 [Refereed][Not invited]
   

  To distinguish Thailand virus infection from infections with other hantaviruses, we established an ELISA serotyping system using a truncated nucleocapsid protein of Thailand virus lacking 49 amino acids at the N-terminus. In evaluations using patient and rodent sera, Thailand virus infection was readily distinguished from Hantaan and Seoul virus infections. Therefore, this ELISA system is an effective alternative to neutralization tests.
• Arch Virol

  Nakamura, I, Yoshimatsu, K, Lee, B. H, Okumura, M, Taruishi, M, Araki, K, Kariwa, H, Takashima, I, Arikawa, J

  Development of a serotyping ELISA system for Thailand virus infection. 153 (1537) 1542  2008 [Not refereed][Not invited]
  
• Seroepidemiological study on hantavirus infections in India

  Sara Chandy, Kumiko Yoshimatsu, Rainer G. Ulrich, Marc Mertens, Megumi Okumura, P. Rajendran, George T. John, Vinohar Balraj, Jayaprakash Muliyil, Joy Mammen, Priya Abraham, Jiro Arikawa, Gopalan Sridharan

  TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE 102 (1) 70 - 74 0035-9203 2008/01 [Refereed][Not invited]
   

  Hantaviruses are etiological agents of hemorrhagic fever with renal syndrome in many parts of Asia and Europe. There has been no documented case of hantavirus disease from India, although serological evidence exists. We investigated the prevalence of hantavirus in the Indian population and tried to identify potential risk groups for hantavirus infections. The presence of hantavirus-specific IgG antibodies was prospectively evaluated in 661 subjects belonging to different groups, i.e. patients with chronic renal disease, warehouse workers and tribal members engaged in rodent trapping. Healthy volunteer Wood donors were included as a control group. Thirty-eight seropositive samples were found using a combination of a commercial ELISA followed by an indirect immunofluorescence assay. Western blot using recombinant Hantaan virus nucleocapsid antigen confirmed the presence of anti-hantavirus IgG in 28 (74%) of the 38 sera tested. This study confirms the presence of hantaviruses in India and warrants increasing awareness of the problems of emerging pathogens and the threats they may pose to the public health system. (c) 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
• Analysis of the immune response of Hantaan virus nucleocapsid protein-specific CD8+ T cells in mice

  Midori Taruishi, Kumiko Yoshimatsu, Koichi Araki, Megumi Okumura, Ichiro Nakamura, Kilchl Kajino, Jiro Arikawa

  VIROLOGY 365 (2) 292 - 301 0042-6822 2007/09 [Refereed][Not invited]
   

  The major histocompatibility complex (MHC) class-I restricted epitope of Hantaan virus nucleocapsid protein (N) was identified using overlapping peptides and BALB/c mice. Using the MHC tetramer derived from the epitope, we found that the level of N-specific CD8(+) T cells increased to approximately 20% of all antigen-specific CD8(+) T cells in a mouse model of transient infection. However, N-specific CD8(+) T cells were undetectable in a mouse model of persistent infection, both in the persistently infected phase and in the convalescent phase. Levels of CD8(+) T cells producing interferon-gamma were weak in both the acute and convalescent phases in the persistently infected model. These results indicate that hantavirus strongly suppresses the production of N-specific CD8(+) T cells throughout the course of infection in persistently infected mice. Moreover, N-specific CD8(+) T cells were not effective in recovering persistently infected mice, despite the existence of abundant N antigen in vivo. (C) 2007 Elsevier Inc. All rights reserved.
• Hantavirus infection in East Asia

  Hiroaki Kariwa, Kumiko Yoshimatsu, Jiro Arikawa

  COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 30 (5-6) 341 - 356 0147-9571 2007/09 [Refereed][Not invited]
   

  Hantaviruses are enveloped RNA viruses that belong to the Hantavirus genus of the family Bunyaviridae. These viruses persistently infect their rodent reservoirs without causing disease. The virus is transmitted to humans via the inhalation of infectious aerosols generated from contaminated animal secretions or through the contaminated saliva of animal bites. Hantaviruses cause haemorrhagic fever with renal syndrome in Euro-Asia, and hantavirus pulmonary syndrome (HPS) in North and South America. Here, we review the epidemiology and epizootiology of hantavirus infection in Asian countries. (C) 2007 Elsevier Ltd. All rights reserved.
• Hantavirus Infection - typical rodent-borne viral zoonosis.

  Arikawa J, Yoshimatsu K, Truong UT, Troung UN

  Trop Med Health Japanese Society of Tropical Medicine 35 (2) 55 - 59 1348-8945 2007/09 [Refereed][Not invited]
  
• Prevalence of antibody to hepatitis E virus among wild sika deer, Cervus nippon, in Japan

  Y. Matsuura, M. Suzuki, K. Yoshimatsu, J. Arikawa, I. Takashima, M. Yokoyama, H. Igota, K. Yamauchi, S. Ishida, D. Fukui, G. Bando, M. Kosuge, H. Tsunemitsu, C. Koshimoto, K. Sakae, M. Chikahira, S. Ogawa, T. Miyamura, N. Takeda, T. C. Li

  ARCHIVES OF VIROLOGY 152 (7) 1375 - 1381 0304-8608 2007/07 [Not refereed][Not invited]
   

  We examined 976 sika deer serum samples, 159 liver tissue samples and 88 stool samples collected from 16 prefectures in Japan, and performed ELISA and RT-PCR assays to detect antibodies to HEV and HEV RNA, respectively. Although 25 (2.6%) of 976 samples were positive for anti-HEV IgG, the antibody titers were very low. The OD values ranged between 0.018 and 0.486, forming a single distribution rather than a bimodal distribution, suggesting that the antibody detected in this study was not induced by HEV infection, or that deer have low sensitivity to HEV. HEV RNA was not detected in these samples, also suggesting that deer may not play a role as an HEV reservoir.
• Development of serological assays for Thottapalayam virus, an insectivore-borne Hantavirus

  Megumi Okumura, Kumiko Yoshimatsu, Sanit Kumperasart, Ichiro Nakamura, Michiko Ogino, Midori Taruishi, Araya Sungdee, Sirima Pattamadilok, Ima Nurisa Ibrahim, Sri Erlina, Takashi Agui, Richard Yanagihara, Jiro Arikawa

  CLINICAL AND VACCINE IMMUNOLOGY 14 (2) 173 - 181 1556-6811 2007/02 [Refereed][Not invited]
   

  Thottapalayam virus (TPMV), a member of the genus Hantavirus in the family Bunyaviridae, was isolated from an insectivore, Suncus murinus (musk shrew), captured in southern India in 1964. While the isolation of TPMV predates the discovery of the prototype Hantaan virus, little is known about its genetics and biology. To date, preliminary evidence suggests that TPMV differs significantly, both antigenically and genetically, from all known rodent-borne hantaviruses. However, since detailed epizootiological studies have not been conducted, it is unclear if TPMV is naturally harbored by an insectivore host or if TPMV represents a "spillover" from its natural rodent reservoir host. Moreover, to what extent TPMV causes infection and/or disease in humans is not known. To address these issues, we first studied the antigenic profile of TPMV using monoclonal antibodies against Hantaan and Seoul viruses and pollyclonal immune sera against Puumala virus and TPMV. Armed with this newfound information, we developed an enzyme-linked immunosorbent assay system for the diagnosis of TPMV infections in shrews and humans, using a recombinant TPMV N antigen manipulated to have an E5/G6 epitope to be captured by monoclonal antibody clone E5/G6. Using this assay, we found anti-TPMV antibodies in sera from a patient with high fever of unknown etiology in Thailand and from two shrews captured in Indonesia. Seropositivity was verified by the indirect immunofluorescence antibody test, Western blotting analysis, and focus reduction neutralization test. Collectively, our data indicate that TPMV is harbored by Suncus murinus as its host in nature and is capable of infecting humans.
• A comparative epidemiological study of hantavirus infection in Japan and Far East Russia

  Hiroaki Kariwa, Kumari Lokugamage, Nandadeva Lokugamage, Hironobu Miyamoto, Kentaro Yoshii, Mina Nakauchi, Kumiko Yoshimatsu, Jiro Arikawa, Leonid I. Ivanov, Takuya Iwasaki, Ikuo Takashima

  JAPANESE JOURNAL OF VETERINARY RESEARCH 54 (4) 145 - 161 0047-1917 2007/02 [Refereed][Not invited]
   

  Hantaviruses are causative agents of some severe human illnesses, including hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The viruses are maintained by rodent hosts, and humans acquire infection by inhaling virus-contaminated excreta from infected animals. To examine the epidemiology of hantavirus infections in Japan and Far East Russia, we conducted epidemiological surveys in these regions. In Japan, anti-hantavirus antibodies were found in four rodent species, Clethrionomys rufocanus, Rattus norvegicus, R. rattus, and Apodemus speciosus. Although no new HFRS cases have been officially reported over the past 20 years in Japan, one member of the Japan Ground Self-Defense Force did test positive for hantavirus antibody. Repeated surveys in Far East Russia have revealed that two distinct hantavirus types cause severe HFRS in this region. Hantavirus sequences identified from A. peninsulae, fetal HFRS cases in Vladivostok, and Amur virus are highly similar to each other (> 92% identity), but they are less similar (similar to 84% identity) to the prototypical Hantaan virus, which is carried by A. agrarius. Phylogenetic analysis also indicates that Amur and A. peninsulae-associated viruses are distinct from Hantaan virus, suggesting that A. peninsulae is the reservoir animal for Amur virus, which causes severe HFRS. From HFRS patients in the Khabarovsk region, we identified viruses with nucleotide sequences that are more similar to Far East virus (> 96% identity) than to the Hantaan (88 - 89% identity) or Amur (81 - 83% identity) viruses. Phylogenetic analysis also indicates that the viruses from Khabarovsk HFRS patients are closely related to the Far East virus, and distinct from Amur virus.
• Mode of infection of Hokkaido virus (Genus hantavirus) among grey red-backed voles, Myodes rufocanus, in Hokkaido, Japan

  Nur Hardy Abu Daud, Hiroaki Kariwa, Yoich Tanikawa, Ichiro Nakamura, Takahiro Seto, Daisuke Miyashita, Kentaro Yoshii, Mina Nakauchi, Kumiko Yoshimatsu, Jiro Arikawa, Ikuo Takashima

  MICROBIOLOGY AND IMMUNOLOGY 51 (11) 1081 - 1090 0385-5600 2007 [Refereed][Not invited]
   

  Hokkaido virus (HOKV) is a member of the genus Hantavirus, in the family Bunyaviridae. To investigate HOKV infection in the host Myodes rufocanus, the grey red-backed vole, 199 animals were captured at Tobetsu (October 2004 and July 2005) and Nakagawa (October 2004) in Hokkaido, Japan, for detection of antibody, antigen, and viral RNA. In the surveys in Tobetsu (2004) and Nakagawa (2004), seropositive animals were detected at a frequency of 6.0% (5/84) and 10.4% (5/48), respectively. No seropositive animals were detected in Tobetsu in 2005. Seroprevalence in males in Tobetsu and Nakagawa in 2004 was 25 % (1/4) and 45.5 % (5111), respectively, which was higher than in females, at 5.0 % (4/80) and 0% (0/37), respectively (P<0.01). These results suggest that male animals play an important role in the maintenance of HOKV in M. rufocanus. Two females were seronegative but viral RNA-positive, indicating that these animals had acute infections before antibody was produced. Another five infected animals in Nakagawa were all male and had high levels of antibodies and viral RNA, suggesting that they had persistent infections. Viral RNA copies in organs of infected animals in Nakagawa were quantified by real-time polymerase chain reaction. Two acutely infected animals had >= 10 times the number of RNA copies in their lungs compared to those of persistently infected animals. In most cases, lungs or spleen had the highest RNA copy number, regardless of infection status.
• Geographical distribution of hantaviruses in Thailand and potential human health significance of Thailand virus

  Sirima Pattamadilok, Byoung-Hee Lee, Sanit Kumperasart, Kumiko Yoshimatsu, Megumi Okumura, Ichiro Nakamura, Koichi Araki, Yuvaluk Khoprasert, Prayadh Dangsupa, Pornpitak Panlar, Burkhard Jandrig, Detlev H. Krueger, Boris Klempa, Thomas Jaekel, Jonas Schmidt, Rainer Ulrich, Hiroaki Kariwa, Jiro Arikawa

  AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE 75 (5) 994 - 1002 0002-9637 2006/11 [Refereed][Not invited]
   

  Phylogenetic investigations, sequence comparisons, and antigenic cross-re activity studies confirmed the classification of Thailand virus (THAIV) as a distinct hantavirus species. The examination of sera from 402 rodents trapped in 19 provinces of Thailand revealed that five greater bandicoot rats (Bandicota indica) and one lesser bandicoot rat (B. savilei) from four provinces were focus reduction neutralization test (FRNT) antibody-positive for THAIV. One of 260 patients from Surin province in Thailand (initially suspected of having contracted leptospirosis, but found to be negative) showed symptoms compatible with hemorrhagic fever with renal syndrome (HFRS). The serum of this patient showed high titers of hantavirus-reactive IgM and IgG. FRNT investigations confirmed virus-neutralizing antibodies against THAIV. These observations suggest that THAIV or THAI-like viruses occur throughout Indochina and may represent an additional causative agent of HFRS.
• A pseudotype vesicular stomatitis virus containing Hantaan virus envelope glycoproteins G1 and G2 as an alternative to hantavirus vaccine in mice

  BH Lee, K Yoshimatsu, K Araki, M Okumura, Nakamura, I, J Arikawa

  VACCINE 24 (15) 2928 - 2934 0264-410X 2006/04 [Not refereed][Not invited]
   

  We examined whether a vesicular stomatitis virus (VSV) pseudotype bearing the hantavirus envelope glycoproteins (GPs) G1 and G2 (VSV Delta G*HTN) could be used as a safe and effective alternative to native hantavirus. Mice were immunized with purified particles of VSV Delta G*HTN. After the second immunization, all mice produced anti-GP antibody as detected in ELISA and a neutralization test. After the third immunization, the mice were challenged with Hantaan virus. Neither anti-NP antibody production nor Hantaan virus-specific CD8 T-cell reactions were detected in these mice. The present study demonstrated the potential of using a pseudotype VSV system as a tool for developing a hantavirus vaccine. (c) 2005 Elsevier Ltd. All rights reserved.
• A pseudotype vesicular stomatitis virus containing Hantaan virus envelope glycoproteins G1 and G2 as an alternative to hantavirus vaccine in mice

  BH Lee, K Yoshimatsu, K Araki, M Okumura, Nakamura, I, J Arikawa

  VACCINE 24 (15) 2928 - 2934 0264-410X 2006/04 [Refereed][Not invited]
   

  We examined whether a vesicular stomatitis virus (VSV) pseudotype bearing the hantavirus envelope glycoproteins (GPs) G1 and G2 (VSV Delta G*HTN) could be used as a safe and effective alternative to native hantavirus. Mice were immunized with purified particles of VSV Delta G*HTN. After the second immunization, all mice produced anti-GP antibody as detected in ELISA and a neutralization test. After the third immunization, the mice were challenged with Hantaan virus. Neither anti-NP antibody production nor Hantaan virus-specific CD8 T-cell reactions were detected in these mice. The present study demonstrated the potential of using a pseudotype VSV system as a tool for developing a hantavirus vaccine. (c) 2005 Elsevier Ltd. All rights reserved.
• Soochong virus: An antigenically and genetically distinct hantavirus isolated from Apodemus peninsulae in Korea

  LJ Baek, H Kariwa, K Lokugamage, K Yoshimatsu, J Arikawa, Takashima, I, JI Kang, SS Moon, SY Chung, EJ Kim, HJ Kang, KJ Song, TA Klein, R Yanagihara, JW Song

  JOURNAL OF MEDICAL VIROLOGY 78 (2) 290 - 297 0146-6615 2006/02 [Refereed][Not invited]
   

  Hantaan (HTN) virus, the etiologic agent of clinically severe hemorrhagic fever with renal syndrome (HFRS), was first isolated in 1976 from lung tissue of a striped-field mouse (Apodemus agrarius) captured in Songnae-ri, Gyeonggi Province, Republic of Korea. Found primarily in mountainous areas, the Korean field mouse (A. peninsulae) is the second-most dominant field rodent species found throughout Korea. A new hantavirus, designated Soochong (SOO), was isolated in Vero E6 cells from four A. peninsulae captured in August 1997 at Mt. Gyebang in Hongcheon-gun, Mt. Gachil, Inje-gun, Gangwon Province, and in September 1998 at Mt. Deogyu, Muju-gun, Jeollabuk Province. The entire S, M, and L genomic segments of SOO virus, amplified by RT-PCR from lung tissues of seropositive A. peninsulae and from virus-infected Vero E6 cells, diverged from HTN virus (strain 76-118) by 15.6%, 22.8%, and 21.7% at the nucleotide level and 3.5%, 9.5%, and 4.6% at the amino acid level, respectively. Phylogenetic analyses of the nucleotide and deduced amino acid sequences, using the maximum parsimony and neighbor-joining methods, indicated that SOO virus was distinct from A. agrarius-borne HTN virus. SOO virus shared a common ancestry with Amur virus from Far East Russia, as well as with H5 and B78 hantaviruses, previously isolated from HFRS patients in China. Cross-focus-reduction neutralizating antibody tests showed that SOO virus which is the first hantavirus isolated in cell culture from A. peninsulae, could be classified as a new hantavirus serotype.
• A pilot study for serological evidence of hantavirus infection in human population in south India

  S Chandy, S Mitra, N Sathish, TS Vijayakumar, OC Abraham, MV Jesudason, P Abraham, K Yoshimatsu, J Arikawa, G Sridharan

  INDIAN JOURNAL OF MEDICAL RESEARCH 122 (3) 211 - 215 0971-5916 2005/09 [Refereed][Not invited]
   

  Background & objectives: Hantaviruses are rodent-borne viruses of the family Bunyaviridae that have been identified as aetiological agents of two human diseases, haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). There are no reports of hantavirus infections in humans from India, hence this pilot study was undertaken to provide the serological evidence of hantavirus infections in humans in south India. Methods: Serum samples were obtained from individuals with acute febrile illness and from voluntary blood donors, majority of whom were from south India. Serum samples were tested for anti-hantavirus IgM using a commercial enzyme immunoassay (EIA). Samples found positive by the EIA were tested by an indirect immunofluorescence assay (IFA) using slides coated with Seoul virus (SEOV) infected cells as substrate. Results: Of the 152 serum samples from individuals with pyrexic illness, 23 (14.7%) were positive for anti-hantavirus IgM by EIA. In contrast, only 5.7 per cent of healthy blood donors were positive by this assay. Eighteen of the 22 (82%) EIA-positive samples from patients were positive by the IFA assay. In contrast, only 2 of the 5 (40%) blood donor EIA positive samples were positive in the IFA assay. Interpretation & conclusion: The finding of this study indicated the possible presence of hantavirus infections in the human population of India presenting both as asymptomatic and symptomatic infections. Further studies need to be done to confirm the findings on a larger sample using molecular techniques.
• 野生げっ歯類からのハンタウイルス抗原検出用ELISAの開発

  谷川 洋一, 苅和 宏明, 萩谷 友洋, Lokugamage Nandadeva, Lokugamage Kumari, 舘 敦史, 好井 健太朗, 吉松 組子, 有川 二郎, 高島 郁夫

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 139回 185 - 185 1347-8621 2005/03 [Refereed][Not invited]
  
• Nucleocapsid protein of cell culture-adapted Seoul virus strain 80-39: Analysis of its encoding sequence, expression in yeast and immuno-reactivity

  Jonas Schmidt, Burkhard Jandrig, Boris Klempa, Kumiko Yoshimatsu, Jiro Arikawa, Helga Meisel, Matthias Niedrig, Christian Pitra, Detlev H. Krüger, Rainer Ulrich

  Virus Genes 30 (1) 37 - 48 0920-8569 2005/01 [Not refereed][Not invited]
   

  Seoul virus (SEOV) is a hantavirus causing a mild to moderate form of hemorrhagic fever with renal syndrome that is distributed mainly in Asia. The nucleocapsid (N) protein-encoding sequence of SEOV (strain 80-39) was RT-PCR-amplified and cloned into a yeast expression vector containing a galactose-inducible promoter. A survey of the pattern of synonymous codon preferences for a total of 22 N protein-encoding hantavirus genes including 13 of SEOV strains revealed that there is minor variation in codon usage by the same gene in different viral genomes. Introduction of the expression plasmid into yeast Saccharomyces cerevisiae resulted in the high-level expression of a hexahistidine-tagged N protein derivative. The nickel-chelation chromatography purified, yeast-expressed SEOV N protein reacted in the immunoblot with a SEOV-specific monoclonal antibody and certain HTNV- and PUUV-cross-reactive monoclonal antibodies. The immunization of a rabbit with the recombinant N protein resulted in the induction of a high-titered antibody response. In ELISA studies, the N protein was able to detect antibodies in sera of experimentally infected laboratory rats and in human anti-hantavirus-positive sera or serum pools of patients from different geographical origin. The yeast-expressed SEOV N protein represents a promising antigen for development of diagnostic tools in serology, sero prevalence studies and vaccine development. © 2005 Springer Science+Business Media, Inc.
• Nucleocapsid protein of cell culture-adapted Seoul virus strain 80-39: Analysis of its encoding sequence, expression in yeast and immuno-reactivity

  J Schmidt, B Jandrig, B Klempa, K Yoshimatsu, J Arikawa, H Meisel, M Niedrig, C Pitra, DH Kruger, R Ulrich

  VIRUS GENES 30 (1) 37 - 48 0920-8569 2005/01 [Refereed][Not invited]
   

  Seoul virus (SEOV) is a hantavirus causing a mild to moderate form of hemorrhagic fever with renal syndrome that is distributed mainly in Asia. The nucleocapsid (N) protein-encoding sequence of SEOV (strain 80-39) was RT-PCR-amplified and cloned into a yeast expression vector containing a galactose-inducible promoter. A survey of the pattern of synonymous codon preferences for a total of 22 N protein-encoding hantavirus genes including 13 of SEOV strains revealed that there is minor variation in codon usage by the same gene in different viral genomes. Introduction of the expression plasmid into yeast Saccharomyces cerevisiae resulted in the high-level expression of a hexahistidine-tagged N protein derivative. The nickel-chelation chromatography purified, yeast-expressed SEOV N protein reacted in the immunoblot with a SEOV-specific monoclonal antibody and certain HTNV- and PUUV-cross-reactive monoclonal antibodies. The immunization of a rabbit with the recombinant N protein resulted in the induction of a high-titered antibody response. In ELISA studies, the N protein was able to detect antibodies in sera of experimentally infected laboratory rats and in human anti-hantavirus-positive sera or serum pools of patients from different geographical origin. The yeast-expressed SEOV N protein represents a promising antigen for development of diagnostic tools in serology, sero prevalence studies and vaccine development.
• A pilot study for serological evidence of hantavirus infection in human population in south India

  Chandy, S, Mitra, S, Sathish, N, Vijayakumar, T. S, Abraham, O. C, Jesudason, M. V, Abraham, P, Yoshimatsu, K, Arikawa, J, Sridharan, G

  Indian J Med Res 122 (211) 215  2005 [Not refereed][Not invited]
  
• Epitope analysis of monoclonal antibody E5/G6, which binds to a linear epitope in the nucleocapsid protein of hantaviruses

  M. Okumura, K. Yoshimatsu, K. Araki, B. H. Lee, A. Asano, T. Agui, J. Arikawa

  Archives of Virology 149 (12) 2427 - 2434 0304-8608 2004/12 [Not refereed][Not invited]
   

  Monoclonal antibody E5/G6 recognized a linear epitope common to hantavirus nucleocapsid proteins. Using synthetic peptides, we identified epitope E5/G6 as the 9 mer YEDVNGIRK (NP 165-173), in which D167, G170, I171, and R172 are indispensable. Furthermore, all the peptides synthesized using various hantavirus sequences bound MAb E5/G6 consistently, despite the existence of several amino acid variations in this region. These results indicate that MAb E5/G6 is a useful tool for detecting hantavirus antigen in rodent or patient tissues using Western blotting or other immunohistochemical assays. © Springer-Verlag 2004.
• Cell fusion activities of Hantaan virus envelope glycoproteins

  Michiko Ogino, Kumiko Yoshimatsu, Hideki Ebihara, Koichi Araki, Byoung-Hee Lee, Megumi Okumura, Jiro Arikawa

  Journal of Virology 78 (19) 10776 - 10782 0022-538X 2004/10 [Not refereed][Not invited]
   

  Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.
• Cell fusion activities of Hantaan virus envelope glycoproteins

  M Ogino, K Yoshimatsu, H Ebihara, K Araki, BH Lee, M Okumura, J Arikawa

  JOURNAL OF VIROLOGY 78 (19) 10776 - 10782 0022-538X 2004/10 [Refereed][Not invited]
   

  Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.
• 日本におけるハンタウイルス感染の動物伝染病学的及び疫学的研究(Epizootiological and epidemiological study of hantavirus infection in Japan)

  Lokugamage Nandadeva, 苅和 宏明, 好井 健太朗, 舘 敦史, 安藤 秀二, 福島 博, 土屋 公幸, 岩崎 琢也, 吉松 組子, 有川 二郎, 高島 郁夫

  日本獣医学会学術集会講演要旨集 138回 130 - 130 1347-8621 2004/08 [Refereed][Not invited]
  
• Amur型ハンタウイルス糖蛋白の哺乳類細胞での発現と抗原性解析

  舘 敦史, 苅和 宏明, Lokugamage Kumari, Lokugamage Nandadeva, 谷川 洋一, 好井 健太朗, 吉松 組子, 有川 二郎, 高島 郁夫

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 138回 131 - 131 1347-8621 2004/08 [Refereed][Not invited]
  
• Age-dependent hantavirus-specific CD8(+) T-cell responses in mice infected with Hantaan virus

  K Araki, K Yoshimatsu, BH Lee, M Okumura, H Kariwa, Takashima, I, J Arikawa

  ARCHIVES OF VIROLOGY 149 (7) 1373 - 1382 0304-8608 2004/07 [Not refereed][Not invited]
   

  To investigate age-dependent differences in hantavirus-specific CD8(+) T-cell responses, mice were inoculated with 0.1 50% newborn mouse lethal dose of Hantaan virus (HTNV) at 0, 3, 7, 14, or 35 days after birth. HTNV-specific CD8(+) T cells producing gamma interferon (IFN-gamma) were measured on day 30 after HTNV inoculation. Although no IFN-gamma-producing HTNV-specific CD8(+) T cells were detected in most of the mice inoculated with HTNV on day 0 after birth, most mice inoculated at 3, 7, 14, or 35 days had HTNV-specific CD8(+) T cells. The production of tumor necrosis factor alpha (TNF-alpha) by IFN-gamma-producing CD8(+) T cells and the cytotoxic activity against HTNV-infected target cells were similar in immature and adult mice. However, the number of IFN-gamma-producing HTNV-specific CD8(+) T cells was significantly less in mice inoculated with HTNV at 3 days than in older mice. In addition, a strong correlation between HTNV persistence and a lack of HTNV-specific CD8(+) T cells was observed. These results suggest that mice over 7 days old have the ability to induce functional HTNV-specific CD8(+) T-cell responses that are indistinguishable from the responses of adult mice, and that HTNV-specific CD8(+) T cells are important for clearance of HTNV.
• Genetic and antigenic characterization of the Amur virus associated with hemorrhagic fever with renal syndrome

  K Lokugamage, H Kariwa, N Lokugamage, H Miyamoto, M Iwasa, T Hagiya, K Araki, A Tachi, T Mizutani, K Yoshimatsu, J Arikawa, Takashima, I

  VIRUS RESEARCH 101 (2) 127 - 134 0168-1702 2004/05 [Refereed][Not invited]
   

  The genetic and antigenic characteristics of the Amur (AMR) and Far East (FE) virus lineages, which are both within the genus Hantavirus, were studied. Representative viruses, H5 and B78 for AMR and Bao, 14 for FE, were used. The entire small (S) and medium (M) segments, except for the 3'- and 5'-ends, were sequenced. The deduced amino acid sequences of AMR had 96.7 and 92.0-92.2% identities with the Hantaan (HTN) virus in the S and M segments, respectively. The amino acid sequences of FE had 99.1 and 97.9% identities in the S and M segments, respectively. The three viral strains and HTN virus had similar binding patterns to a panel of monoclonal antibodies (MAbs), except that one MAb did not bind AMR. However, sera from Apodemus peninsulae, naturally infected with AMR virus, neutralized homologous viruses at 1: 160 to 1:320 dilutions and HTN at 1:20 to 1:40 dilutions. The anti-AMR serum neutralized homologous viruses at a 1:80 dilution and HTN at a 1:40 dilution. The anti-HTN serum did not neutralize AMR (< 1:40 dilution), although it had a high neutralizing titer (1:320) against the homologous virus. Therefore, we suggest that AMR virus may constitute a distinct serotype within the genus Hantavirus. (C) 2004 Elsevier B.V. All rights reserved.
• A new model of Hantaan virus persistence in mice: the balance between HTNV infection and CD8(+) T-cell responses

  K Araki, K Yoshimatsu, BH Lee, H Kariwa, Takashima, I, J Arikawa

  VIROLOGY 322 (2) 318 - 327 0042-6822 2004/05 [Refereed][Not invited]
   

  We established a viral persistence model that involves the adoptive transfer of spleen cells from immunocompetent mice (H-2(d)) into Hantaan virus (HTNV)-infected severe combined immunodeficient (SCID, H-2(d)) mice. The infection is maintained despite the presence of neutralizing antibodies, without apparent signs of disease, and there is a correlation between HTNV persistence and the lack of HTNV-specific CD8(+) T cells. In addition, disseminated HTNV infection before the initiation of immune responses appears to be important for virus persistence. The suppression of HTNV-specific CD8(+) T cells in the present model appears to occur at the periphery. The present study also demonstrates that CD8(+) T cells contribute to the clearance of HTNV Thus, it seems that HTNV-specific CD8(+) T cells play a key role in HTNV persistence in mice. This model of viral persistence is useful for studies of immune responses and immunocytotherapy against viral infection. (C) 2004 Elsevier Inc. All rights reserved.
• Comparison of virulence of various hantaviruses related to hemorrhagic fever with renal syndrome in newborn mouse model.

  K Lokugamage, H Kariwa, N Lokugamage, M Iwasa, T Hagiya, K Araki, A Tachi, T Mizutani, K Yoshimatsu, J Arikawa, T Iwasaki, Takashima, I

  JAPANESE JOURNAL OF VETERINARY RESEARCH 51 (3-4) 143 - 149 0047-1917 2004/02 [Refereed][Not invited]
   

  The virulence of hantaviruses that are antigenically related but have different genetic characteristics from the prototype of hantavirus, Hantaan (HTN) virus, was examined in newborn mice. The H5 and B78 strains of the Amur (AMR) genotype, the Bao14 strain of the Far East (FE) genotype, and the 76 - 118 strain of HTN virus were inoculated subcutaneously ( 1 focus-forming unit; FFU) into newborn mice. All of the AMR and FE genotype viruses inoculated mice were died by 16 days post-infection (dpi) and 21 dpi, respectively, while 50% of the HTN virus inoculated mice survived until 30 dpi. The AMR and FE genotype viruses inoculated mice had high viral titers in the lung (1.3x10(6) to 1.3x10(8) FFU/gram [g] tissue), brain (2.1x10(7) to 1.2x10(9) FFU/g tissue), and kidney(2.5x10(5) to 1.6x10(7) FFU/g tissue), and showed a detectable level of antibodies (titers 1 : 16 -1 : 32) at 14 dpi. In contrast, the HTN virus infected mice had viruses only in the lungs at low titers (1.1-5.3x10(5) FFU/g tissue). Observations of body-weight changes revealed that the AMR and FE genotype viruses inoculated mice had lower growth rates than the HTN virus inoculated mice. These data suggest that the AMR and FE genotype viruses are more virulent than the HTN virus in newborn mice.
• A new model of Hantaan virus persistence in mice: The balance between HTNV infection and CD8⁺ T-cell responses

  Araki, K., Yoshimatsu, K., Lee, B.-H., Kariwa, H., Takashima, I., Arikawa, J.

  Virology 322 (2) 2004 [Not refereed][Not invited]
  
• Epizootiological and epidemiological study of hantavirus infection in Japan

  N Lokugamage, H Kariwa, K Lokugamage, MA Iwasa, T Hagiya, K Yoshii, A Tachi, S Ando, H Fukushima, K Tsuchiya, T Iwasaki, K Araki, K Yoshimatsu, J Arikawa, T Mizutani, K Osawa, H Sato, K Takashima

  MICROBIOLOGY AND IMMUNOLOGY 48 (11) 843 - 851 0385-5600 2004 [Refereed][Not invited]
   

  Epizootiological surveys on hantavirus infections in rodents were carried out in various areas of Japan, including the four major islands of Hokkaido, Honshu, Shikoku, and Kyushu from 2000 to 2003. A total of 1,221 rodents and insectivores were captured. Seropositive animals were found in Apodemus (A.) speciosus (5/482, 1.0%), Rattus (R.) norvegicus (4/364, 1.1%), R. rattus (3/45, 6.7%), and Clethrionomys (C.) rufocanus (7/197, 3.6%). The partial S segment was amplified from one seropositive R. rattus captured at Hakodate. The nucleotide sequence showed 96% identity with the Seoul virus (SEOV) prototype strain SR-11. In addition, we conducted an epidemiological survey on human hantavirus infection in a high-risk population, the personnel of the Japan Ground Self-defense Force on Hokkaido. One out of 207 human blood samples was positive for anti-hantavirus antibody by IFA, ELISA, and WB analysis. The result of the serotype specific ELISA indicates that this individual acquired SEOV infection. This study indicates that A. speciosus, R. norvegicus, R. rattus, and C. rufocanus carry hantaviruses as the reservoir animals in Japan. Infected R. rattus and R. norvegicus in port areas could be the sources of human SEOV infection and a threat to travelers and individuals working in seaports.
• Association of the nucleocapsid protein of the Seoul and Hantaan hantaviruses with small ubiquitin-like modifier-1-related molecules

  BH Lee, K Yoshimatsu, A Maeda, K Ochiai, M Morimatsu, K Araki, M Ogino, S Morikawa, J Arikawa

  VIRUS RESEARCH 98 (1) 83 - 91 0168-1702 2003/12 [Not refereed][Not invited]
   

  We performed yeast two-hybrid screening of a human kidney cell cDNA library to study the biological role of the hantavirus nucleocapsid protein (NP). We found that Seoul virus (SEOV) and Hantaan virus (HTNV) NPs were associated with small ubiquitin-like modifier (SUMO)-1-interacting proteins PIAS 1, PIASxbeta, HIPK2, CHD3, and TTRAP, which interacted with the SUMO-1 conjugating enzyme (Ubc-9) and SUMO-1 in the yeast two-hybrid assay. Interactions between the HIPK2, CHD3, and TTRAP proteins and SEOV NP were also shown in a mammalian two-hybrid assay. However, there was no interaction between PIAS proteins and NP, which was probably due to the inhibitory effect of PIAS on transcription in the mammalian two-hybrid assay. Nevertheless, a co-expression experiment suggested the existence of a PIAS-NP interaction in the cytoplasm. The region spanning amino acids 100-125 of SEOV NP, which represents a critical region for NP-NP polymerization. was found to be responsible for the interaction with SUMO-1-related molecules in both the yeast and mammalian two-hybrid assays. These results add to the information on interactions of hantavirus NP and host cellular proteins. (C) 2003 Elsevier B.V. All rights reserved.
• Association of the nucleocapsid protein of the Seoul and Hantaan hantaviruses with small ubiquitin-like modifier-1-related molecules

  BH Lee, K Yoshimatsu, A Maeda, K Ochiai, M Morimatsu, K Araki, M Ogino, S Morikawa, J Arikawa

  VIRUS RESEARCH 98 (1) 83 - 91 0168-1702 2003/12 [Refereed][Not invited]
   

  We performed yeast two-hybrid screening of a human kidney cell cDNA library to study the biological role of the hantavirus nucleocapsid protein (NP). We found that Seoul virus (SEOV) and Hantaan virus (HTNV) NPs were associated with small ubiquitin-like modifier (SUMO)-1-interacting proteins PIAS 1, PIASxbeta, HIPK2, CHD3, and TTRAP, which interacted with the SUMO-1 conjugating enzyme (Ubc-9) and SUMO-1 in the yeast two-hybrid assay. Interactions between the HIPK2, CHD3, and TTRAP proteins and SEOV NP were also shown in a mammalian two-hybrid assay. However, there was no interaction between PIAS proteins and NP, which was probably due to the inhibitory effect of PIAS on transcription in the mammalian two-hybrid assay. Nevertheless, a co-expression experiment suggested the existence of a PIAS-NP interaction in the cytoplasm. The region spanning amino acids 100-125 of SEOV NP, which represents a critical region for NP-NP polymerization. was found to be responsible for the interaction with SUMO-1-related molecules in both the yeast and mammalian two-hybrid assays. These results add to the information on interactions of hantavirus NP and host cellular proteins. (C) 2003 Elsevier B.V. All rights reserved.
• Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of mongolian gerbils

  N Lokugamage, H Kariwa, K Lokugamage, T Hagiya, H Miyamoto, MA Iwasa, K Araki, K Yoshimatsu, J Arikawa, T Mizutani, Takashima, I

  JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (11) 1189 - 1194 0916-7250 2003/11 [Not refereed][Not invited]
   

  Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10(5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.
• Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of mongolian gerbils

  N Lokugamage, H Kariwa, K Lokugamage, T Hagiya, H Miyamoto, MA Iwasa, K Araki, K Yoshimatsu, J Arikawa, T Mizutani, Takashima, I

  JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (11) 1189 - 1194 0916-7250 2003/11 [Refereed][Not invited]
   

  Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10(5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.
• Detection of antibody for the serodiagnosis of hantavirus infection in different rodent species

  BH Lee, K Yoshimatsu, K Araki, M Ogino, M Okumura, K Tsuchiya, H Kariwa, J Arikawa

  ARCHIVES OF VIROLOGY 148 (10) 1885 - 1897 0304-8608 2003/10 [Not refereed][Not invited]
   

  Peroxidase-labeled staphylococcal protein A, streptococcal protein G, and antibodies directed against Mus musculus (mouse), Rattus norvegicus (rat), Mesocretus auratus (hamster), and Peromyscus leucopus were examined for their reactivity with immunoglobulin G (IgG) from various rodent species. The purpose of this study was to identify the optimal secondary antibodies or reagents for specific serodiagnosis of hantavirus infection in various rodent species. Using ELISA, a total of 65 sera from 29 rodent species of the family Muridae and one serum sample from family Octodontidae were compared for IgG reactivity with the six different reagents. The results demonstrate that the reactivities of the secondary antibodies and reagents to the sera varied, even among sera from rodents of the same genus. Hantavirus-specific antibody ELISA revealed that hantavirus-infected rodent sera obtained from M. musculus, R. norvegicus, Apodemus agrarius, A. peninsulae, and Bandicota indica bound to the six different conjugates in a similar pattern as that detected in IgG ELISA. These results indicate that the applicability of secondary antibodies and protein A and G should be carefully evaluated before use for serodiagnosis in different rodent species.
• Synthesis of Seoul virus RNA and structural proteins in cultured cells

  H Kariwa, H Tanabe, T Mizutani, Y Kon, K Lokugamage, N Lokugamage, MA Iwasa, T Hagiya, K Araki, K Yoshimatsu, J Arikawa, Takashima, I

  ARCHIVES OF VIROLOGY 148 (9) 1671 - 1685 0304-8608 2003/09 [Not refereed][Not invited]
   

  Seoul virus is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). The virion has a tripartite (S, M, and L) negative-stranded RNA genome, which is characteristic of the family Bunyaviridae. However, the molecular basis of virus replication is not well known. We established a Northern blot hybridization (NB) procedure using digoxygenin-labeled RNA probes, to quantitate the hantaviral plus- and minus-strand RNAs separately. Virus RNA replication was analyzed in infected Vero E6 cells. When the Vero E6 cells were infected with Seoul virus strain KI-83-262 (KI) at m.o.i.=0.25, the plus-strand RNA was detected within 1 h post-infection (hpi), and the minus-strand RNA was detected subsequently. Using laser confocal microscopy, the nucleocapsid protein (NP) was detected within 2 hpi, and accumulated as scattered granules in the cytoplasm until 24 hpi. In contrast, the G2 protein first appeared at 8 hpi, was immediately transported to the Golgi, and accumulated in the Golgi until 24 hpi. Infectious virus particles were released into the medium at 24 h hpi. These findings indicate that hantavirus RNA replication starts with the appearance of NP at 2 hpi, glycoproteins then accumulate gradually in the Golgi, and virion formation is initiated once the viral RNAs and proteins have accumulated.
• Serological analysis of hemorrhagic fever with renal syndrome (HFRS) patients in Far Eastern Russia and identification of the causative hantavirus genotype

  H Miyamoto, H Kariwa, K Araki, K Lokugamage, D Hayasaka, BZ Cui, N Lokugamage, LI Ivanov, T Mizutani, MA Iwasa, K Yoshimatsu, J Arikawa, Takashima, I

  ARCHIVES OF VIROLOGY 148 (8) 1543 - 1556 0304-8608 2003/08 [Not refereed][Not invited]
   

  Hemorrhagic fever with renal syndrome (HFRS) is endemic in East Asia and Europe. The disease is caused by several viruses belonging to the genus Hantavirus, including the Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava Belgrade virus (DOBV), and Puumala virus (PUUV). Recently, HTNV-related viruses, Amur (AMR) and Far East (FE) genotypes were identified as causative agents of HFRS in Far Eastern Russia. To investigate the epidemiology of HFRS and virus transmission, we collected sera from 17 acute and 32 convalescent patients who were clinically diagnosed with HFRS in the Khabarovsk region of Far Eastern Russia, and detected anti-hantavirus antibodies using an ELISA that can differentiate the infected virus serotype using truncated hantavirus nucleocapsid protein antigen. Sixteen of the 17 acute phase patients had antibodies to hantavirus, and all the positive sera had higher optical densities for HTNV-specific antigen than for SEOV-, DOBV-, or PUUV-specific antigens. The partial M segment of the viral genome was amplified from blood clots from three acute patients by PCR. The nucleotide sequences had closer identities to the FE genotype (> 96%) than to the prototype HTNV (88 to 89%) or AMR genotype (81 to 83%). A phylogenetic analysis found that the virus sequences from the patients clustered with the FE type, and were distinct from the AMR type. Thirty-one of 32 convalescent patient sera had antibodies to HTNV-specific antigen. These data suggest ELISA system can detect HTNV-specific antibodies to the FE type, which may be responsible for most of the HFRS in Khabarovsk.
• Hantavirus-specific CD8(+)-T-cell responses in newborn mice persistently infected with Hantaan virus

  K Araki, K Yoshimatsu, BH Lee, H Kariwa, Takashima, I, J Arikawa

  JOURNAL OF VIROLOGY 77 (15) 8408 - 8417 0022-538X 2003/08 [Refereed][Not invited]
   

  The relationship between virus-specific CD8(+)-T-cell responses and viral persistence was studied in mice by using Hantaan virus (HTNA). We first established a simple method for measuring levels of virus-specific CD8(+) T cells by How cytometry. Next, to produce a mouse model of persistent HTNV infection, newborn mice were inoculated subcutaneously within 24 h of birth with 1 or 0.150% newborn mouse lethal dose of HTNV. All mice that escaped lethal infection were persistently infected with HTNV until at least 30 days after virus inoculation and had no virus-specific CD8(+) T cells producing gamma interferon (IFN-gamma). Subsequently, the virus was eliminated from some of the mice, depending on the appearance of functional virus-specific CD8(+) T cells, which have the ability to produce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) and have cytotoxic activity. Neutralizing antibodies were detected in all mice, regardless of the presence or absence of virus. In the acute phase, which occurs within 30 days of infection, IFN-gamma-producing HTNV-specific CD8(+) T cells were detected on day 15 after virus inoculation. However, TNF-alpha production and the cytotoxic activity of these specific CD8(+) T cells were impaired and HTNV was not removed. Almost all of these specific CD8(+) T cells disappeared by day 18. These results suggest that functional HTNV-specific CD8(+) T cells are important for clearance of HTNV.
• Enzyme-linked immunosorbent assay using recombinant antigens expressed in mammalian cells for serodiagnosis of tick-borne encephalitis

  K Yoshii, D Hayasaka, A Goto, M Obara, K Araki, K Yoshimatsu, J Arikawa, L Ivanov, T Mizutani, H Kariwa, Takashima, I

  JOURNAL OF VIROLOGICAL METHODS 108 (2) 171 - 179 0166-0934 2003/03 [Refereed][Not invited]
   

  A recombinant plasmid that expresses the tick-borne encephalitis (TBE) virus premembrane (prM) and envelope (E) proteins in mammalian cells was constructed. Recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins, and transfected cells released virus-like particles (VLPs), which were 1.13-1.14 g/ml in density and 20-30 nm in diameter, into the culture medium. Recombinant E proteins were used for the development of an enzyme-linked immunosorbent assay (ELISA) to detect TBE virus-specific IgM and IgG antibodies in serum. The results of this ELISA correlated well with the results of commercial ELISA, when tested with 95 serum samples from clinically TBE-suspected patients. In addition, ELISA using recombinant antigens showed no cross-reactivity against serum from Japanese encephalitis (JE) patients, despite the cross-reactivity shown by commercial ELISA systems. These observations indicated that this newly developed ELISA system could distinguish tick-borne encephalitis from Japanese encephalitis infection, and that it constitutes a useful and safe alternative to conventional ELISA systems. (C) 2002 Published by Elsevier Science B.V.
• Hantavirus-specific CD8⁺-T-cell responses in newborn mice persistently infected with Hantaan virus

  Araki, K., Yoshimatsu, K., Lee, B.-H., Kariwa, H., Takashima, I., Arikawa, J.

  Journal of Virology 77 (15) 2003 [Not refereed][Not invited]
  
• Enzyme-linked immunosorbent assay using recombinant antigens expressed in mammalian cells for serodiagnosis of tick-borne encephalitis

  Kentarou Yoshii, Daisuke Hayasaka, Akiko Goto, Mayumi Obara, Koichi Araki, Kumiko Yoshimatsu, Jiro Arikawa, Leonoid Ivanov, Tetsuya Mizutani, Hiroaki Kariwa, Ikuo Takashima

  Journal of Virological Methods 108 (2) 171 - 179 0166-0934 2003 [Not refereed][Not invited]
   

  A recombinant plasmid that expresses the tick-borne encephalitis (TBE) virus premembrane (prM) and envelope (E) proteins in mammalian cells was constructed. Recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins, and transfected cells released virus-like particles (VLPs), which were 1.13-1.14 g/ml in density and 20-30 nm in diameter, into the culture medium. Recombinant E proteins were used for the development of an enzyme-linked immunosorbent assay (ELISA) to detect TBE virus-specific IgM and IgG antibodies in serum. The results of this ELISA correlated well with the results of commercial ELISA, when tested with 95 serum samples from clinically TBE-suspected patients. In addition, ELISA using recombinant antigens showed no cross-reactivity against serum from Japanese encephalitis (JE) patients, despite the cross-reactivity shown by commercial ELISA systems. These observations indicated that this newly developed ELISA system could distinguish tick-borne encephalitis from Japanese encephalitis infection, and that it constitutes a useful and safe alternative to conventional ELISA systems. © 2002 Published by Elsevier Science B.V.
• Establishment of an Enzyme-Linked Immunosorbent Assay for Detection of Hantavirus Antibody of Rats Using a Recombinant of Nucleocapsid Protein Expressed in Escherichia coli.（共著）

  Exp. Anim. 52(1),25-30 2003 [Not refereed][Not invited]
  
• Use of vesicular stomatitis virus pseudotypes bearing hantaan or Seoul virus envelope proteins in a rapid and safe neutralization test

  M Ogino, H Ebihara, BH Lee, K Araki, A Lundkvist, Y Kawaoka, K Yoshimatsu, J Arikawa

  CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY 10 (1) 154 - 160 1071-412X 2003/01 [Not refereed][Not invited]
   

  A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVDeltaG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVDeltaG*G. Pseudotype VSV with the Hantaan (VSVDeltaG*-HTN) or Seoul (VSVDeltaG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 10(5) to 10(6)/ml. The infectivity of VSVDeltaG*-HTN and VSVDeltaG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVDeltaG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVDeltaG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4degreesC for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.
• The Multimerization of Hantavirus nucleocapsid protein depends on type-specific epitopes.(共著)

  Journal of Virology J. Virol. 77(2),943-952 2003 [Not refereed][Not invited]
  
• The intracellular association of the nucleocapsid protein (NP) of hantaan virus (HTNV) with small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9)

  A Maeda, BH Lee, K Yoshimatsu, M Saijo, Kurane, I, J Arikawa, S Morikawa

  VIROLOGY 305 (2) 288 - 297 0042-6822 2003/01 [Not refereed][Not invited]
   

  Small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9) conjugates SUMO-1 to target proteins and modulates cellular processes such as signal transduction, transcription regulation, and cell growth regulation. We demonstrated here that the nucleocapsid protein (NP) of Hantaan virus (HTNV) was associated with Ubc9 and SUMO-1 in vivo. Analysis of the interaction between the truncated NPs and Ubc9 revealed that the amino acid residues at the positions between 101 and 238 in the NP were responsible for the interaction. Furthermore, a consensus binding motif of Ubc9 and SUMO-1, MKAE, within this region, especially the second amino acid of the motif, K residue, was crucial for the interaction, and the interaction was essential for the NP to be localized in the perinuclear region. These results indicate that the assembly of the HTNV-NP is regulated by the interaction between the NP and Ubc9. This is the first report to demonstrate the interaction of Ubc9 with a structural protein of negative-strand RNA viruses. (C) 2003 Elsevier Science (USA).
• The multimerization of hantavirus nucleocapsid protein depends on type-specific epitopes

  K Yoshimatsu, BH Lee, K Araki, M Morimatsu, M Ogino, H Ebihara, J Arikawa

  JOURNAL OF VIROLOGY 77 (2) 943 - 952 0022-538X 2003/01 [Refereed][Not invited]
   

  Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.
• Use of vesicular stomatitis virus pseudotypes bearing hantaan or Seoul virus envelope proteins in a rapid and safe neutralization test

  M Ogino, H Ebihara, BH Lee, K Araki, A Lundkvist, Y Kawaoka, K Yoshimatsu, J Arikawa

  CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY 10 (1) 154 - 160 1071-412X 2003/01 [Refereed][Not invited]
   

  A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVDeltaG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVDeltaG*G. Pseudotype VSV with the Hantaan (VSVDeltaG*-HTN) or Seoul (VSVDeltaG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 10(5) to 10(6)/ml. The infectivity of VSVDeltaG*-HTN and VSVDeltaG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVDeltaG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVDeltaG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4degreesC for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.
• The intracellular association of the nucleocapsid protein (NP) of hantaan virus (HTNV) with small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9)

  A Maeda, BH Lee, K Yoshimatsu, M Saijo, Kurane, I, J Arikawa, S Morikawa

  VIROLOGY 305 (2) 288 - 297 0042-6822 2003/01 [Refereed][Not invited]
   

  Small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9) conjugates SUMO-1 to target proteins and modulates cellular processes such as signal transduction, transcription regulation, and cell growth regulation. We demonstrated here that the nucleocapsid protein (NP) of Hantaan virus (HTNV) was associated with Ubc9 and SUMO-1 in vivo. Analysis of the interaction between the truncated NPs and Ubc9 revealed that the amino acid residues at the positions between 101 and 238 in the NP were responsible for the interaction. Furthermore, a consensus binding motif of Ubc9 and SUMO-1, MKAE, within this region, especially the second amino acid of the motif, K residue, was crucial for the interaction, and the interaction was essential for the NP to be localized in the perinuclear region. These results indicate that the assembly of the HTNV-NP is regulated by the interaction between the NP and Ubc9. This is the first report to demonstrate the interaction of Ubc9 with a structural protein of negative-strand RNA viruses. (C) 2003 Elsevier Science (USA).
• Establishment of an enzyme-linked immunosorbent assay for detection of Hantavirus antibody of rats using a recombinant of nucleocapsid protein expressed in Escherichia coli

  A Takakura, K Goto, T Itoh, K Yoshimatsu, Takashima, I, J Arikawa

  EXPERIMENTAL ANIMALS 52 (1) 25 - 30 1341-1357 2003/01 [Refereed][Not invited]
   

  A recombinant nucleocapsid protein of Hantaan virus (HTN) 76-118 strain expressed in E. coli was applied as a serodiagnostic antigen in an enzyme-linked immunosorbent assay (rHTN-ELISA) for detection of hantavirus antibody in rat sera. The sensitivity and specificity of the rHTN-ELISA were compared with those of the indirect immunofluoresent assay (IFA) using virus-infected cells. The sensitivity of rHTN-ELISA was similar to that of the IFA both in experimentally SR-11 infected rat and naturally infected rat sera. Sera showing a low antibody titer in IFA and suspected to be negative by other methods were also found to be negative in rHTN-ELISA. These results indicate that rHTN-ELISA is effective as a screening method for serodiagnosis of hantaviruses, because of its high sensitivity, specificity, safety and suitability for processing large number of samples.
• The multimerization of Hantavirus nucleocapsid protein depends on type-specific epitopes

  Kumiko Yoshimatsu, Byoung-Hee Lee, Koichi Araki, Masami Morimatsu, Michiko Ogino, Hideki Ebihara, Jiro Arikawa

  Journal of Virology 77 (2) 943 - 952 0022-538X 2003/01 [Refereed][Not invited]
   

  Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.
• Genetic characterization of hantaviruses transmitted by the Korean field mouse (Apodemus peninsulae), Far East Russia

  K Lokugamage, H Kariwa, D Hayasaka, BZ Cui, T Iwasaki, N Lokugamage, LI Ivanov, Volkov, VI, VA Demenev, R Slonova, G Kompanets, T Kushnaryova, T Kurata, K Maeda, K Araki, T Mizutani, K Yoshimatsu, J Arikawa, Takashima, I

  EMERGING INFECTIOUS DISEASES 8 (8) 768 - 776 1080-6040 2002/08 [Refereed][Not invited]
   

  In an epizootiologic survey of 122 rodents captured in Vladivostok, Russia, antibodies positive for hantavirus were found in Apodemus peninsulae (4/70), A. agrarius (1/39), and Clethrionomys rufocanus (1/8). The hantavirus sequences identified in two seropositive A. peninsulae and two patients with hemorrhagic fever with renal syndrome (HFRS) from the Primorye region of Far East Russia were designated as Solovey and Primorye, respectively. The nucleotide sequences of the Solovey, Primorye, and Amur (obtained through GenBank) sequences were closely related (>92% identity). Solovey and Primorye sequences shared 84% nucleotide identity with the prototype Hantaan 76-118. Phylogenetic analysis also indicated a close relationship between Solovey, Primorye, Amur, and other viruses identified in Russia, China, and Korea. Our findings suggest that the Korean field mouse (A. peninsulae) is the reservoir for a hantavirus that causes HFRS over a vast area of east Asia, including Far East Russia.
• Genetic characterization of hantaviruses transmitted by the Korean field mouse (Apodemus peninsulae), Far East Russia

  Kumari Lokugamage, Hiroaki Kariwa, Daisuke Hayasaka, Bai Zhong Cui, Takuya Iwasaki, Nandadeva Lokugamage, Leonid I. Ivanov, Vladimir I. Volkov, Vladimir A. Demenev, Raisa Slonova, Galina Kompanets, Tatyana Kushnaryova, Takeshi Kurata, Kenji Maeda, Koichi Araki, Tetsuya Mizutani, Kumiko Yoshimatsu, Jiro Arikawa, Ikuo Takashima

  Emerging Infectious Diseases 8 (8) 768 - 776 1080-6040 2002 [Not refereed][Not invited]
   

  In an epizootiologic survey of 122 rodents captured in Vladivostok, Russia, antibodies positive for hantavirus were found in Apodemus peninsulae (4/70), A. agrarius (1/39), and Clethrionomys rufocanus (1/8). The hantavirus sequences identified in two seropositive A. peninsulae and two patients with hemorrhagic fever with renal syndrome (HFRS) from the Primorye region of Far East Russia were designated as Solovey and Primorye, respectively. The nucleotide sequences of the Solovey, Primorye, and Amur (obtained through GenBank) sequences were closely related (> 92% identity). Solovey and Primorye sequences shared 84% nucleotide identity with the prototype Hantaan 76-118. Phylogenetic analysis also indicated a close relationship between Solovey, Primorye, Amur, and other viruses identified in Russia, China, and Korea. Our findings suggest that the Korean field mouse (A. peninsulae) is the reservoir for a hantavirus that causes HFRS over a vast area of east Asia, including Far East Russia.
• Epizootiological survey of hantavirus among rodent species in Ningxia Hui Autonomous Province, China

  H Kariwa, CB Zhong, K Araki, K Yoshimatsu, K Lokugamage, N Lokugamage, ME Murphy, T Mizutani, J Arikawa, H Fukushima, H Xiong, JHC Chen, Takashima, I

  JAPANESE JOURNAL OF VETERINARY RESEARCH 49 (2) 105 - 114 0047-1917 2001/08 [Not refereed][Not invited]
   

  Hantaviral antibodies were detected in the sera from Apodemus (A.) agrarius and A. peninsulae captured in Ningxia province, China by several different serological diagnostic methods. A total of 409 sera from rodent and insectivore species were collected in 1999 and examined by indirect immunofluorescent antibody assay (IFA). Among them, 19 of 191 (9.9%) sera of A. agrarius and 1 of 13 (7.7%) sera of A. peninsulae were positive for hantaviral antibodies. The other species (Rattus norvegicus, Mus musculus, Cricetulus triton, and Sorex cylindricauda) were negative. The reaction pattern of positive serum was characterized as scattered and granular virus antigens in the cytoplasm of hantavirus infected Vero E6 cells. Some of the A. agrarius sera positive for hantavirus were further examined by Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and the focus reduction neutralization test (FRNT). By WB, positive sera showed the same specific reaction pattern of baculovirus-expressed recombinant hantaviral nucleocapsid protein, as shown in hantavirus-immune serum. By ELISA, IFA-positive sera showed significantly higher optical densities (around 1.0) than the negative A. agrarius sera. Hantaan type hantavirus was neutralized with the positive sera. These results suggest that A. agrarius have hantavirus infection and may play a role as a reservoir animal for hantavirus in Ningxia Hui Autonomous Province, China.
• Characterization of in vitro and in vivo antiviral activity of lactoferrin and ribavirin upon hantavirus

  ME Murphy, H Kariwa, T Mizutani, H Tanabe, K Yoshimatsu, J Arikawa, Takashima, I

  JOURNAL OF VETERINARY MEDICAL SCIENCE 63 (6) 637 - 645 0916-7250 2001/06 [Not refereed][Not invited]
   

  Mechanisms of anti-hantaviral activities of bovine lactoferrin (LF) and ribavirin (Rbv) were investigated. Hantavirus focus formation at 48 hr was 1546 of the control in cells treated with 400 mug/ml LF for 1 hr at 37 degreesC prior to viral infection. Post infection treatment with 100 mug/ml Rbv also inhibited the focus formation to 2.5% of the control. Combined LF pre- and Rbv post-infection treatment completely inhibited focus formation. Viral glycoprotein (G2) and nucleocapsid protein (NP) syntheses were delayed in LF pre treated cells up to 24 hr post infection (hpi) but became comparable to the control by 48 hpi. Further, LF inhibited viral shedding at 24 hpi but did not inhibit shedding after 48 hpi. However, Rbv was able to inhibit synthesis of viral proteins, (+) and (-) strand RNAs also inhibited viral shedding after 24 hr. These results suggest that LF inhibits viral adsorption to cells, while Rbv inhibits viral RNA synthesis. For in vivo trials of LF and Rbv, LF pre- and Rbv post-treatment were evaluated in suckling mice infected with hantavirus, of which 7% survived. LF concentrations of 40 and 160 mg/cg administered prior to viral challenge improved survival rates to 15% and 70%, respectively for single administration and 85% and 94%. respectively, for double administration. Rbv concentrations of 25 and 50 mg/kg gave survival rates of 68% and 81%, respectively. This suggests that both LF and Rbv are efficacious in hantavirus infection in vivo.
• Truncated hantavirus nucleocapsid proteins for serotyping Hantaan, Seoul, and Dobrava hantavirus infections

  K. Araki, K. Yoshimatsu, M. Ogino, H. Ebihara, A. Lundkvist, H. Kariwa, I. Takashima, J. Arikawa

  Journal of Clinical Microbiology 39 (7) 2397 - 2404 0095-1137 2001 [Not refereed][Not invited]
   

  Truncated recombinant nucleocapsid proteins (rNPs) of Hantaan virus (HTNV), Seoul virus (SEOV), and Dobrava virus (DOBV) were expressed by a baculovirus system. The truncated rNPs, which lacked 49 (rNP50) or 154 (rNP155) N-terminal amino acids of the NPs of HTNV, SEOV, and DOBV, were able to differentiate HTNV-, SEOV-, and DOBV-specific immune sera. Recombinant NP50s retained higher reactivities than rNP155s and were proven useful for enzyme-linked immunosorbent assay (ELISA). The ELISAs based on the rNP50s of HTNV, SEOV, and DOBV successfully differentiated three groups of patient sera, previously defined by neutralization tests: 17 with HTNV infection, 12 with SEOV infection, and 20 with DOBV infection. The entire rNP of Puumala virus (PUUV) distinguished PUUV infection from the other types of hantavirus infection. Serotyping with these rNP50s can be recommended as a rapid and efficient system for hantavirus diagnosis.
• Mouse Mx2 protein inhibits hantavirus but not influenza virus replication

  Jin, H.K., Yoshimatsu, K., Takada, A., Ogino, M., Asano, A., Arikawa, J., Watanabe, T.

  Archives of Virology 146 (1) 2001 [Not refereed][Not invited]
  
• Pathogenicity of Hantaan virus in newborn mice: Genetic reassortant study demonstrating that a single amino acid change in glycoprotein G1 is related to virulence

  H Ebihara, K Yoshimatsu, M Ogino, K Araki, Y Ami, H Kariwa, Takashima, I, DX Li, J Arikawa

  JOURNAL OF VIROLOGY 74 (19) 9245 - 9255 0022-538X 2000/10 [Not refereed][Not invited]
   

  Two Hantaan virus strains, clone 1 (cl-1), which is virulent in newborn mice, and its attenuated mutant (mul1E10), were used to examine the pathogenesis of Hantaan virus infection in a mouse model and identify virus factors relating to virulence. After subcutaneous inoculation of newborn BALB/c mice, cl-1 caused fatal disease with high viral multiplication in peripheral organs, but mul1E10 produced nonfatal infection with a low level of virus multiplication. Intracerebral inoculation of either strain caused fatal disease. Histopathological changes in the dead animals were prominent in the brain, indicating that the brain is the target organ and produces the fatal outcome. These results indicate that mul1E10 has a generally less virulent phenotype, and because of decreased multiplication in peripheral tissues, neuroinvasiveness is also decreased. An experiment with genetic reassortant viruses showed that in newborn mice the M segment is the most related to virulence and the L segment is partly related. Sequence comparison detected a single deduced amino acid change (cl-l Ile to mul1E10 Thr) at amino acid number 515 in glycoprotein G1. One nucleotide change, but no amino acid substitution, was observed in the noncoding region of the L segment. In mouse brain microvascular endothelial cells in vitro, viruses possessing a cl-1-derived M segment grew more rapidly than viruses containing a mul1E10 derived M segment. These results suggest that the single amino acid change in the glycoprotein alters peripheral growth, which affects invasion of the central nervous system in mice.
• In vitro antiviral activity of lactoferrin and ribavirin upon hantavirus

  ME Murphy, H Kariwa, T Mizutani, K Yoshimatsu, J Arikawa, Takashima, I

  ARCHIVES OF VIROLOGY 145 (8) 1571 - 1582 0304-8608 2000 [Not refereed][Not invited]
   

  Bovine lactoferrin (LF) and ribavirin (Rbv) were tested as antiviral agents against Seoul type hantavirus (SR-11 strain) in vitro. Hantaviral foci number in Vero E6 cells infected with SR-11 was reduced with LF treatment by 5 days post infection to obtain a 50% effective dose (ED50) Of 2500 mu g/ml, while pretreatment with LF was highly efficacious having an ED50 of 39 mu g/ml. Conversely, 1 h pretreatment with Rbv revealed no inhibition of viral focus formation but could significantly reduce the number of viral foci (ED50: 10 mu g/ml) when used from the time of viral infection. One hour pre-treatment of the cell monolayer with LF and subsequent addition of Rbv revealed a synergistic anti-hantaviral effect against SR-11, <20 FFU/ml as compared to 10(5) foci/ml in the control. One hour treatment of SR-11 with LF prior to cell inoculation gave an ED50 of 312.5 mu g/ml. Whereas, washing the LF-pretreated cell monolayer with PBS demonstrated minimal focus reduction, suggesting LF lightly adheres to cells. These results indicate that LF has anti-hantaviral activity in vitro and inhibition of virus adsorption to cells which play an important role in revealing the anti-hantaviral activity of LF. This paper reports for the first time the anti-hantaviral effect of LF.
• Genetic diversity of hantaviruses isolated in China and characterization of novel hantaviruses isolated from Niviventer confucianus and Rattus rattus

  Hua Wang, Kumiko Yoshimatsu, Hideki Ebihara, Michiko Ogino, Koichi Araki, Hiroaki Kariwa, Zhaoxiao Wang, Zhaozhuang Luo, Dexin Li, Changshou Hang, Jiro Arikawa

  Virology 278 (2) 332 - 345 0042-6822 2000 [Not refereed][Not invited]
   

  The antigenic and genetic properties of 46 hantaviruses from China, 13 from patients, 23 from rodents, and 10 from unknown hosts, were compared with those of other hantaviruses. The viruses were classified as either Hantaan (HTN) or Seoul (SEO) viruses. A phylogenetic analysis of the partial M (300 bp) and S (around 485 bp) genomes of HTN viruses identified nine distinct genetic subtypes, one consisting of isolates from Korea. The SEO viruses were divided into five genetic subtypes, although they had less variability than the HTN subtypes. There was a correlation between the subtype and province of origin for four subtypes of HTN viruses, confirming geographical clustering. Hantaan virus NC167 isolated from Niviventer confucianus and SEO virus Gou3 isolated from Rattus rattus were the basal clades in each virus. The phylogenetic trees constructed from the entire S and M segments suggested that NC167 was introduced to N. confucianus in a host-switching event. The reactivity of a panel of 35 monoclonal antibodies was almost exactly the same in NC167 and a representative HTN virus and in Gou3 and a representative SEO virus. However, there was a one-way cross-neutralization between them. These results confirm the varied nature of Murinae-associated hantaviruses in China. © 2000 Academic Press.
• Detection of hantaviral antibodies among patients with hepatitis of unknown etiology in Japan

  H Kariwa, K Yoshimatsu, K Araki, K Chayama, H Kumada, M Ogino, H Ebihara, ME Murphy, T Mizutani, Takashima, I, J Arikawa

  MICROBIOLOGY AND IMMUNOLOGY 44 (5) 357 - 362 0385-5600 2000 [Not refereed][Not invited]
   

  Hantaviral antibodies were detected in the sera from patients with hepatic disease of unknown etiology in Japan by several different serological diagnostic methods. A total of 105 sera from diseased patients which were negative to A-G hepatitis virus infections in the Tokyo area were tested. Among them, 3 out of 73 sera from patients with chronic hepatic disease were positive to hantaviral antibody by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent antibody assay (IFA) and Western blot analysis (WB), Neutralizing antibody titers of the 3 sera to Seoul virus (SEO) were 4 to 8 times higher than those to Hantaan virus (HTN), However, all of the 32 sera from patients with acute hepatitis were negative for hantaviral antibody, Among the 60 patients with chronic hepatitis in Hokkaido which were serologically negative to B and C hepatitis virus infection, one was positive for hantaviral antibody by ELISA and WE. In contrast, the sera from healthy adults in Japan, 550 from the Honshu and Kyushu regions, and 1,000 from the Hokkaido region, were negative for hantavirus antibody. These results show that hantaviral antibodies are more frequently detected in patients with hepatic disease than in healthy adults. However, the observation that no positive sera were detected from patients with acute hepatitis implies that hantavirus might not be directly related to hepatitis.
• 26.マウスのインターフェロン誘導性抗ウイルス遺伝子Mxに関する研究

  陳 喜慶, 高田 礼人, 森松 組子, 荻野 倫子, 有川 二郎, 渡辺 智正

  日本疾患モデル学会記録 公益社団法人 日本実験動物学会 16 33 - 33 0918-8991 2000
• A serosurvey of Borna disease virus infection in wild rats by a capture ELISA

  K Tsujimura, T Mizutani, H Kariwa, K Yoshimatsu, M Ogino, Y Morii, H Inagaki, J Arikawa, Takashima, I

  JOURNAL OF VETERINARY MEDICAL SCIENCE 61 (2) 113 - 117 0916-7250 1999/02 [Not refereed][Not invited]
   

  For a serological diagnostic test for Borna disease (BD), we developed a capture ELISA with specificity and sensitivity based on detection of antibodies against BD virus (BDV) p40 protein. Using our capture ELISA system, the antibody response of rats inoculated intracerebrally with BDV at 4 weeks after birth showed a sharp increase from 1 to 4 weeks postinoculation (p.i.) and a steady level after 5 weeks p.i. To investigate prevalence of BDV infection among wild rats, we examined sera of Rattus norvegicus in Kami-iso town, Oshima district, Hokkaido, suggesting that rats in this area had not been infected by BDV.
• Genetic diversities of hantaviruses among rodents in Hokkaido, Japan and Far East Russia

  H Kariwa, K Yoshimatsu, J Sawabe, E Yokota, J Arikawa, Takashima, I, H Fukushima, A Lundkvist, FN Shubin, LM Isachkova, RA Slonova, GN Leonova, N Hashimoto

  VIRUS RESEARCH 59 (2) 219 - 228 0168-1702 1999/02 [Not refereed][Not invited]
   

  Seroepizootiologic surveys among wild rodents were carried out in Japan and Far East Russia in 1995 and 1996. Seropositive animals were only identified in Clethrionomys rufocanus (23/134) in Hokkaido, Japan. On the other hand, seropositives were identified in C. rufocanus (1/8), Apodemus argrarius (2/66), Apodemus spp. (2/26) and Microtus fortis (3/22) in Vladivostok, Far East Russia. Total RNA was isolated from lungs of seropositive animals and the S genome segments were amplified by PCR, cloned and sequenced. The S and M genomes of hantavirus, derived from Japanese C. rufocanus (Tobetsu genotype), were most closely related with Puumala viruses (76-79% nucleotide and 95% amino acid identities for S genome, 70-78% nucleotide and 87-92% amino acid identities for M genome). The recombinant nucleocapsid protein of Tobetsu genotype was antigenically quite similar with that of Sotkamo. These suggest that the virus endemic in Japanese C. rufocanus belongs to Puumala virus. Phylogenetic analysis indicates that the genotype forms a distinct lineage within Puumala viruses. Partial S segment (1-1251 nt), derived from seropositive M. fortis in Vladivostok, was sequenced and analyzed. The S genome segment, which was designated Vladivostok genotype, was most closely related with Khabarovsk virus (79% nucleotide and 90% amino acid identities) which was isolated from M. fortis. (C) 1999 Elsevier Science B.V. All rights reserved.
• A Serosurvey of Borna Disease Virus Infection in Wild Rats by a Capture ELISA

  Koji Tsujimura, Tetsuya Mizutani, Hiroaki Kariwa, Kumiko Yoshimatsu, Michiko Ogino, Yuko Morii, Hisae Inagaki, Jiro Arikawa, Ikuo Takashima

  Journal of Veterinary Medical Science 61 (2) 113 - 117 1782-155X 1999/02 [Refereed][Not invited]
   

  For a serological diagnostic test for Borna disease (BD), we developed a capture ELISA with specificity and sensitivity based on detection of antibodies against BD virus (BDV) p40 protein. Using our capture ELISA system, the antibody response of rats inoculated intracerebrally with BDV at 4 weeks after birth showed a sharp increase from 1 to 4 weeks postinoculation (p.i.) and a steady level after 5 weeks p.i. To investigate prevalence of BDV infection among wild rats, we examined sera of Rattus norvegicus in Kami-iso town, Oshima district, Hokkaido, suggesting that rats in this area had not been infected by BDV.
• Hantavirus infection of SD rats reared in domestic laboratory animal facilities.（共著）

  Korean Journal of the laboratory Animal Science 15(1),49-52 1999 [Not refereed][Not invited]
  
• N-acetylgalactosamine (GalNAc)-specific lectins mediate enhancement of Hantaan virus infection

  M Ogino, K Yoshimatsu, H Ebihara, J Arikawa

  ARCHIVES OF VIROLOGY 144 (9) 1765 - 1777 0304-8608 1999 [Not refereed][Not invited]
   

  N-acetylgalactosamine (GalNAc)-specific lectins, Dolichos biflorus agglutinin (DBA), and soybean agglutinin (SBA), enhanced Hantaan (HTN) virus infections in Vero E6 and P388D1 cells. Treatment of Vero E6 cells with the lectins either before or during, but not after, virus inoculation resulted in lectin-mediated enhancement of infection (LME), indicating that GalNAc-specific lectin affects an early stage of the infection. Lectin blot and FAGS analysis showed that the ability of HTN virus envelope glycoproteins and cell surface molecules to bind DBA and SEA was essential for LME. GalNAc clearly inhibited LME, indicating that the lectins bind with their specific carbohydrate-binding site. These results suggest that a lectin cross-link between the virus and the cell surface is the most plausible mechanism for inducing infection enhancement.
• Antigenic characterization of Hantaan and Seoul virus nucleocapsid proteins expressed by recombinant baculovirus: Application of a truncated protein, lacking an antigenic region common to the two viruses, as a serotyping antigen

  M Morii, K Yoshimatsu, J Arikawa, GZ Zhou, H Kariwa, Takashima, I

  JOURNAL OF CLINICAL MICROBIOLOGY 36 (9) 2514 - 2521 0095-1137 1998/09 [Not refereed][Not invited]
   

  Hantaan virus (HTN) and Seoul virus (SEO) are members of the genus Hantavirus in the family Bunyaviridae and are causative agents of hemorrhagic fever with renal syndrome. The complete and truncated nucleocapsid proteins (NP) of HTN and SEO were expressed by a recombinant baculovirus system, Antigenic characterization of the NP using monoclonal antibodies (Mabs)indicated that the binding sites For the serotype-specific MAbs were located between amino acids (aa) 155 and 429, a Western blot assay indicated that the serotype-specific epitopes were conformation dependent. An indirect immunofluorescence antibody (IFA) assay with the truncated NP (aa 155 to 429) was able to distinguish convalescent-phase sera from HTN and SEO patients. However, the antibody liters with the truncated NP were lower than those with the whole NP, The truncated NP of SEO (aa 155 to 429) could be used as an enzyme-linked immunosorbent assay (ELISA) antigen, but the truncated NP from HTN lost its reactivity when used for ELISA. The LFA assay using baculovirus-expressed truncated NP as an antigen is a rapid, simple, and safe test for distinguishing between HTN and SEO infections by serotype.
• Evaluation of serological diagnosis of Borna disease virus infection using recombinant proteins in experimentally infected rats

  M Ogino, K Yoshimatsu, K Tsujimura, T Mizutani, J Arikawa, Takashima, I

  JOURNAL OF VETERINARY MEDICAL SCIENCE 60 (4) 531 - 534 0916-7250 1998/04 [Not refereed][Not invited]
   

  We produced two recombinant Borna disease virus (BDV) proteins, p40 and p24, by using a baculovirus vector as a diagnostic antigen; Antigenicities of these recombinant proteins were evaluated by immune rabbit sera; Recombinant p40 was a more sensitive antigen than p24 for the detection of antibodies in infected rats. Rats inoculated with BDV within 24 hi after birth showed higher detection rates of viral RNA and viral proteins from the brain than rats inoculated at 4 weeks-old. Depending on the age of infection and the time postinfection, the detection of BDV RNA, protein, or anti-BDV antibody did not always correlate in individuals, We suggest both serological and molecular biological methods are needed in the diagnosis of BDV infection.
• Adjuvant activity of muramyl dipeptide derivatives to enhance immunogenicity of a hantavirus-inactivated vaccine

  YC Yoo, K Yoshimatsu, Y Koike, R Hatsuse, K Yamanishi, O Tanishita, J Arikawa, Azuma, I

  VACCINE 16 (2-3) 216 - 224 0264-410X 1998/01 [Not refereed][Not invited]
   

  The adjuvant effect of two lipophilic derivatives of muramyl dipeptide (MDP), B30-MDP and MDP-Lys(L18), on the ability of an inactivated vaccine of B-I virus (B-l vaccine) to induce immune response against Hantavirus causing hemorrhagic fever with renal syndrome (HFRS) was examined, When mice were immunized subcutaneously (s.c.) twice at 2-week intervals with B-I vaccine admixed with or without 100 mu g mouse(-1) of B30-MDP (B-1/B30-MDP) or MDP-Lys(L18) [B-1/MDP-Lys(L18)], mice immunized with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed Significantly higher indirect fluorescent antibody (IFA) titers against HFRS virus than mice immunized with B-I vaccine alone, Both mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) also exhibited significantly higher neutralizing antibody titers against HFRS virus than mice immunized with B-I ,vaccine alone during 3-9 weeks after-the primary immunization. The evaluation of antibody-producing cells by enzyme-linked immunospot (ELISPOT) assay on week 4 revealed that both MDP derivatives,es enhanced the number of HFRS virus-specific IgG1 and IgM antibody-producing cells. Furthermore, mice treated with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed a higher level of Th-2 type cytokines, IL-4 and IL-6, in sera than mice treated with B-l alone. In an in-vitro analysis of T lymphocyte proliferation to baculovirus-expressed recombinant nucleocapsid pi protein (rNP) of Hantaan 76-118 sa strain, the splenocytes of mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) on week 4 showed a Significantly higher proliferating activity than those treated with B-1 vaccine alone, In addition when mice were immunized once with B-l vaccine admixed with or without B30-MDP and MDP-Lys(L18) and followed by intrafootpad (i.f.) injection of B-I vaccine on day 7, mice immunized with B-1/B30-MDP and B-1/MDP-Lys(LIS) induced a higher delayed-type hypersensitivity (DTH) reaction than mice immunized with B-I vaccine alone, These results suggest that B30-MDP and MDP-Lys(L18) are useful immunoadjuvants to enhance the ability of inactivated B-l vaccine to induce a humoral and cellular response to HFRS virus, (C) 1997 Elsevier Science Ltd, All rights reserved.
• Characterization of neutralizing monoclonal antibody escape mutants of Hantaan virus 76118

  M Kikuchi, K Yoshimatsu, J Arikawa, R Yoshida, YC Yoo, Y Isegawa, K Yamanishi, S Tono-oka, Azuma, I

  ARCHIVES OF VIROLOGY 143 (1) 73 - 83 0304-8608 1998 [Not refereed][Not invited]
   

  Neutralizing monoclonal antibody (MAb) escape mutants of Hantaan virus were generated using MAbs to envelope protein G1 (16D2) and G2 (11E10). The mutant viruses (mu16D2 and mu11E10), lacked reactivity only to the selecting MAb, or a MAb belonging to the same antigenic site. Both mutants had a single amino acid (a.a.) substitution. The a.a. substitution, found in mu16D2, was different from that found in another mutant selected with the same MAb (16D2). Although MAb 11E10 immunoprecipitated G2 protein, a deduced a.a. substitution was located in the G1 region. These results suggest that antigenic sites defined by neutralizing MAbs are composed of discontinuous epitopes over the G1 and G2 proteins. Mutant 11E10 showed a significant decrease in virulence in suckling mice. A virulence revertant of mu11E10, selected through passages in suckling mice brain, showed exactly the same deduced a.a. sequence as mu11E10 and still was not neutralized by MAb 11E10. Since mutant 16D2 was virulent for suckling mice, neutralization related epitopes found with MAbs 11E10 and 16D2 were independent of pathogenicity in BALB/c mice.
• Urine-associated horizontal transmission of Seoul virus among rats

  H Kariwa, M Fujiki, K Yoshimatsu, J Arikawa, Takashima, I, N Hashimoto

  ARCHIVES OF VIROLOGY 143 (1) 15 - 24 0304-8608 1998 [Not refereed][Not invited]
   

  To understand the mode of transmission of Seoul type hantavirus in Wistar rats, we examined the shedding of the virus and antibody production in infected rats. When 1-day-old rats were inoculated with the KI-83-262 strain of Seoul virus, S segment of the viral genome was detected in lungs, clots, urine, saliva, submaxillary glands, rectums, and kidneys by nested reverse transcriptase PCR. On the other hand, when 8-week-old rats were infected with the virus, viral genome was detected only in the lungs and rectum. In newborn rats intranasally administered urine from infected newborn rats, four of six rats shed the virus into their urine. In addition, three of eight rats kept in the same cage with infected animals also shed the virus into urine. Moreover, the virus genome was detected in the urine of urban rats (Rattus norvegicus) in an enzootic focus. These findings suggest that the urine containing virus from infected rats is an actual source of the Seoul virus infection.
• Urine-associated horizontal transmission of Seoul virus among rats

  H Kariwa, M Fujiki, K Yoshimatsu, J Arikawa, Takashima, I, N Hashimoto

  ARCHIVES OF VIROLOGY 143 (2) 365 - 374 0304-8608 1998 [Refereed][Not invited]
   

  To understand the mode of transmission of Seoul type hantavirus in Wistar rats, we examined the shedding of the virus and antibody production in infected rats. When 1-day-old rats were inoculated with KI-83-262 strain of Seoul virus, the S segment of the viral genome was detected in lungs, clots, urine, saliva, submaxillary glands, rectums, and-kidneys by nested reverse transcriptase PCR. On the other hand, when 8-week-old rats were infected with the virus, viral genome was detected only in the lungs and rectum. In uninfected newborn rats intranasally administered urine from infected newborn rats, four of six rats shed the virus into their urine. In addition, three of eight rats kept in a same cage with infected animals also shed the virus into urine. Moreover, the virus genome was detected in the urine of urban rats (Rattus norvegicus) in an enzootic focus. These findings suggest that the urine containing virus from infected rats is an actual source of Seoul virus infection.
• Hantavirus infection in SCID mice

  K Yoshimatsu, J Arikawa, S Ohbora, C Itakura

  JOURNAL OF VETERINARY MEDICAL SCIENCE 59 (10) 863 - 868 0916-7250 1997/10 [Not refereed][Not invited]
   

  Severe combined immunodeficiency (SCID) mice were inoculated with Hantaan virus strain 76-118 (HTN) or Seoul virus strain SR-11 (SR) of hantaviruses. Susceptibility of SCID mice was compared with those of immunocompetent adult mice, newborn mice and nude mice. SCID mice inoculated with HTN or SR died 32 to 35 days after infection. Unlike newborn mice which also died of hantavirus infection, SCID mice survived longer than newborn mice and showed typical wasting symptoms rather than nervous symptoms. Immunohistochemical staining and virus isolation indicated that both HTN and SR inoculated SCID and SR inoculated nude mice showed systemic infection, but nude mice inoculated with SR survived for longer than 8 weeks after inoculation. Passive transfer of spleen cells from immunocompetent BALB/c mice conferred protection on SCID mice within 2 weeks of HTN infection. Immune mediated pathologic mechanism was examined by transferring the spleen cells to SCID mice inoculated with HTN virus 3 weeks before the cell transfer. The recipient SCID mice showed an increase of serum BUN level coinciding with the appearance of serum antibody to HTN virus, suggesting the immune mediated pathogenicity.
• A case of tick-borne encephalitis in Japan and isolation of the virus

  Takashima, I, K Morita, M Chiba, D Hayasaka, T Sato, C Takezawa, A Igarashi, H Kariwa, K Yoshimatsu, J Arikawa, N Hashimoto

  JOURNAL OF CLINICAL MICROBIOLOGY 35 (8) 1943 - 1947 0095-1137 1997/08 [Not refereed][Not invited]
   

  A case of tick-borne encephalitis (TEE) has not been reported for many years in Japan, although a serological survey of sera from domestic animals suggested the presence of TEE foci in Hokkaido, the northern island of Japan. Studies were conducted to prove the presence of an endemic focus of TEE virus in Japan by means of serology and virus isolation. In October 1993 in Hokkaido, a severe case of encephalitis in a dairy farmer's nife was diagnosed as TEE. Serological examination of paired serum specimens showed a rise in the neutralization antibody titer to Russian spring summer encephalitis virus. A seroepizootiological survey of dogs showed that the TEE-related virus was prevalent in the area. Three virus isolates were obtained from the blood of sentinel dogs, and antigenic analysis grouped the isolates into TEE-related viruses. Sequence analysis of the envelope protein gene identified one of the isolates as being of the same subtype as the Russian spring summer encephalitis (Far Eastern TEE) virus. The results provide evidence that TEE is endemic in a certain area of Japan.
• Protective effect of mucosal administration of recombinant human macrophage colony stimulating factor (rhM-CSF) on mucosal infection of Sendai virus in mice

  K Matsuzawa, YC Yoo, A Fukushima, K Yoshimatsu, J Arikawa, Azuma, I

  VACCINE 15 (1) 85 - 89 0264-410X 1997/01 [Not refereed][Not invited]
   

  We investigated the protection confered by the mucosal administration of recombinant human macrophage colony-stimulating factor (rhM-CSF) against mucosal infection of Sendai virus in mice. In an experimental infection model using Sendai virus, an intranasal (i.n.) administration of rhM-CSF (20 mu g per mouse) 2 days before infection induced significant protection against a lethal infection of this virus. Also, its antiviral activity was dependent upon the dose of rhM-CSF. However; a subcutaneous (s.c.) administration of rhM-CSF with an effective close (20 mu g per mouse) i.n. did not confer protection. In a time course analysis of virus growth in the lungs, mice given rhM-CSF in. significantly inhibited the early period of infection, compared with the untreated mice. Moreover, the level of interferon-gamma (IFN-gamma) in lung wash fluids from the rhM-CSF-treated mice was higher than that of the untreated mice. These results suggested that the mucosal (i.n.), but not the systemic (s.c.) administration of rhM-CSF augments host resistance against mucosal infection with Sendai virus, and that its prophylactic activity is related to growth inhibition of the virus and enhanced IFN-gamma secretion in the lungs. Copyright (C) 1997 Elsevier Science Ltd.
• Effect of MDP-Lys(L18) as a mucosal immunoadjuvant on protection of mucosal infections by Sendai virus and rotavirus

  A Fukushima, YC Yoo, K Yoshimatsu, K Matsuzawa, M Tamura, S Tonooka, K Taniguchi, S Urasawa, J Arikawa, Azuma, I

  VACCINE 14 (6) 485 - 491 0264-410X 1996/04 [Not refereed][Not invited]
   

  To examine the effect of MDP-Lys(L18), a derivative of muramyl dipeptide (MDP), as a mucosal immunoadjuvant, we investigated its activity to augment host resistance against mucosal infections by Sendai virus and rotavirus in mice. lit an experimental infection model that suckling mice (10-day-old) were inoculated perorally (p.o.) with 1.5 x 10(6) p.f.u. mouse(-1) of rotavirus strain SA11, intrarectal (i.r.) as well as p.o., administration of MDP-Lys(L18) (50 mu g mouse(-1)) prior to virus infection markedly reduced rotavirus-induced diarrhea. Furthermore, when MDP-Lys(L18) was administered p.o. (1 mg mouse(-1)), i.r. (300 mu g mouse(-1)) or intranasally (i.n., 100 mu g mouse(-1)) various days before Sendai virus infection (2.6 x 10(4) HAD mouse(-1)), all the mucosal administration of MDP-Lys(L18) significantly protected a lethal infection of Sendai virus, showing a dose-dependent manner. However, the efficacy of MDP-Lys(L18) to induce the prophylactic activity against the viruses somewhat varied according to the administration route and timing. In time course analysis of virus isolation in vivo, the mice administered with MDP-Lys(L18) exhibited a significant reduction of both viruses in the lungs for Sendai virus and in the bowels for rotavirus. These results suggest that MDP-Lys(L18) is a potent mucosal immunoadjuvant to enhance nonspecific host resistance against two mucosal infectious viruses, Sendai virus and rotavirus. Copyright (C) 1996 Elsevier Science Ltd.
• Characterization of the nucleocapsid protein of Hantaan virus strain 76-118 using monoclonal antibodies

  K Yoshimatsu, J Arikawa, M Tamura, R Yoshida, A Lundkvist, B Niklasson, H Kariwa, Azuma, I

  JOURNAL OF GENERAL VIROLOGY 77 695 - 704 0022-1317 1996/04 [Not refereed][Not invited]
   

  We characterized the antigenic sites on the nucleocapsid protein (NP) of Hantaan virus (HTN) using 10 monoclonal antibodies (MAbs). At least seven antigenic sites were revealed by a competitive binding assay and divided into three partially overlapping antigenic regions (I, II and III). Regions I [amino acids (aa) 1-103], II (aa 104-204) and III (aa 205-402) were mapped on NP by examinincr the reactivity of truncated gene products. Those that corresponded to region I reacted with immune mouse serum, indicating that the region contained major linear epitopes as reported with Four corners virus (FCV) and Puumala virus (PUU) NP. At least one MAb to each region inhibited viral growth when they were introduced into cells by scrape-loading. In addition, they conferred protection from a lethal HTN challenge to newborn mice. A PEPSCAN assay localized the epitope of MAb E5/G6 between aa 166-175. Since E5/G6, which had the highest inhibitory effect both in cells and in mice, showed no virus neutralization activity by ordinary neutralization test, this region is suggested to be important for the virus growth after entry into the cells.
• Modes of Seoul virus infections: Persistency in newborn rats and transiency in adult rats

  H Kariwa, M Kimura, S Yoshizumi, J Arikawa, K Yoshimatsu, Takashima, I, N Hashimoto

  ARCHIVES OF VIROLOGY 141 (12) 2327 - 2338 0304-8608 1996 [Not refereed][Not invited]
   

  To understand the mode of persistent infection of Seoul virus in rodents, we examined the distribution of the virus genome and antibody production in infected rats. When 1-day-old rats were inoculated with the KI-83-262 strain, the S segment of viral genome was detected in sera, clots, lungs and kidneys from 3 to 184 days post inoculation (d.p.i.) by nested reverse transcriptase PCR. On the other hand, when 7-week-old rats were infected with this virus, viral genome was detected only in the lungs from 3 to 50 d.p.i. The neutralizing antibody titers of rats inoculated at 1-day of age were higher than those of rats inoculated at 7 weeks of age. In both a ge groups, however, the IgG avidity of antibody increased along with the course of infection. We found that urban rats (Rattus norvegicus) infected early in life harbored the virus for more than 6 months.
• Western blotting using recombinant hantaan virus nucleocapsid protein expressed in silkworm as a serological confirmation of hantavirus infection in human sera

  K Yoshimatsu, J Arikawa, H Li, H Kariwa, N Hashimoto

  JOURNAL OF VETERINARY MEDICAL SCIENCE 58 (1) 71 - 74 0916-7250 1996/01 [Not refereed][Not invited]
   

  Recombinant Hantaan virus nucleocapsid protein expressed in silkworm larvae was applied as a serological diagnostic antigen in Western blots (WE) of human sera. The sensitivity of this method was similar to that of the IFA test. Hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica diagnosed by their cross-reactivity in WE. The specificity of this method was higher than that of IFA test because the background was low. Sera that exhibited high background staining in the IFA test were readily diagnosed with this method. We recommended WE using recombinant Hantaan virus nucleocapsid antigen as a confirmatory procedure for the serodiagnosis of hantavirus.
• Production of recombinant hantavirus nucleocapsid protein expressed in silkworm larvae and its use as a diagnostic antigen in detecting antibodies in serum from infected rats

  K Yoshimatsu, J Arikawa, R Yoshida, H Li, YC Yoo, H Kariwa

  LABORATORY ANIMAL SCIENCE 45 (6) 641 - 646 0023-6764 1995/12 [Not refereed][Not invited]
   

  The recombinant nucleocapsid protein (rNP) of Hantaan virus was expressed by a baculovirus vector in silkworm hemolymph and was used as an antigen in western blotting (WB). The rNP is expressed in insoluble form in hemolymph; therefore simple washing of the insoluble fraction with phosphate-buffered saline by low-speed centrifugation allowed preparation of purified antigen for WB. The rNP had strain-specific and hantavirus-common epitopes similar to the authentic NP antigen of hantavirus and was stable after transfer to membrane. For detection of antibody in serially obtained sera from experimentally infected rats, WB enabled detection of IgM antibodies 3 days after infection, which was at least 2 days earlier than detection by the indirect immunofluorescent antibody test (IFA). Thus WB had a higher sensitivity than the IFA for detection of hantavirus antibody in the serum of experimentally infected rats. The WB-determined IgG antibody titer was about 10 times higher than that determined by the IFA. No background staining was observed by WB even at a 1:10 dilution of serum. The selected rat sera with strong background staining or confusing staining patterns by LFA, but not focus reduction neutralization test titers, could be interpreted as test-negative because they did not have a specific reaction to virus antigen by WB. Thus the specificity of WB was higher than that of the IFA. Moreover, WB can distinguish specific from nonspecific reactions by the detection of the specific antigen on the WB membrane. Therefore the IFA or enzyme-linked immunosorbent assay followed by WB is recommended for serologic confirmation of hantavirus infection.
• EFFECT OF MDP-LYS(L18), A DERIVATIVE OF MDP, ON ENHANCING HOST-RESISTANCE AGAINST HANTAAN VIRUS-INFECTION IN NEWBORN MICE

  YC YOO, K YOSHIMATSU, R HATSUSE, M TAMURA, R YOSHIDA, S TONOOKA, J ARIKAWA, AZUMA, I

  VACCINE 13 (14) 1300 - 1305 0264-410X 1995/10 [Not refereed][Not invited]
   

  We examined the effect of MDP-Lys(L18), a lipophilic derivative of muramyl dipeptide, on the enhancement of host resistance against virus infection in newborn mice Newborn mice were inoculated with 4 LD(50)/mouse of Hantaan virus strain 76-118 (HTN) one day after birth. Mice given 100 mu g/mouse of MDP-Lys(L18) before infection exhibited significantly higher survival rates than those of non-treated mice. The effect of MDP-Lys(L18) was also restorative when given to the mice 4 or 7 days after infection. The titers of virus isolated from the lungs and spleens 12 days after infection, were about 30-times lower in MDP-Lys(L18)-treated (lung: 1.0 x 10(3) FFU; spleen: 6.8 x 10(1) FFU/mouse), than those of non-treated mice (lung: 3.4 x 10(4) FFU; spleen: 1.9 x 10(3) FFU/mouse). Furthermore the virus was undetectable in the brains of MDP-Lys(L18)-treated mice, whereas viruses were isolated from 3 of 6 non-treated mice MDP-Lys(L18) augmented the number of peripheral leukocytes and splenocytes, as well as mitogenic responses of the cells from bone marrow and spleen of newborn mice. These results suggest that MDP-Lys(L18) enhanced the resistance of newborn mice against HTN virus in a systemic infection model, and that this mechanism is involved in the enhancement of hematopoiesis and responsiveness of immune-related cells to mitogens.
• EVIDENCE FOR THE EXISTENCE OF PUUMULA-RELATED VIRUS AMONG CLETHRIONOMYS RUFOCANUS IN HOKKAIDO, JAPAN

  H KARIWA, S YOSHIZUMI, J ARIKAWA, K YOSHIMATSU, K TAKAHASHI, TAKASHIMA, I, N HASHIMOTO

  AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE 53 (3) 222 - 227 0002-9637 1995/09 [Not refereed][Not invited]
   

  We conducted field surveys of indigenous rodent species in Hokkaido, Japan from 1980 to 1993. Serum samples were collected from 663 rodents, including Clethrionomys rufocanus, Apodemus speciosus, A. argenteus, and C. rutilus. Antibody to hantavirus was determined by the protein G antibody assay. Positive C. rufocanus were detected in seven of eight collection sites, but no antibody was detected in the remaining rodent species. To reveal the serotype of the circulating virus in C. rufocanus, antibody titers to Hantaan, Seoul, Puumala, and Prospect Hill viruses were compared by means of the focus reduction neutralization test. The titers in positive sera were extremely high to the Sotkamo strain of Puumala virus. Results were confirmed by the reverse transcriptase-polymerase chain reaction, and suggested that Puumala-related viruses are in circulation among C. rufocanus populations in Hokkaido.
• EFFECTS OF MURAMYL DIPEPTIDE DERIVATIVES AS ADJUVANTS ON THE INDUCTION OF ANTIBODY-RESPONSE TO RECOMBINANT HEPATITIS-B SURFACE-ANTIGEN

  M TAMURA, YC YOO, K YOSHIMATSU, R YOSHIDA, T OKA, K OHKUMA, J ARIKAWA, AZUMA, I

  VACCINE 13 (1) 77 - 82 0264-410X 1995/01 [Not refereed][Not invited]
   

  The ability of two muramyl dipeptide (MDP) derivatives, B30-MDP and MDP-Lys (L18), to enhance the immunogenicity of recombinant hepatitis B surface antigen (rHBsAg) was examined. When mice were immunized intraperitoneally with rHBsAg together with each MDP derivative, the antibody titres were higher than those in mice immunized with alum-adsorbed rHBsAg, which is a commercially available hepatitis B vaccine. When mice were given a subcutaneous or intramuscular injection of rHBsAg and either MDP derivative, the antibody titres were the same as those in mice given alum-adsorbed rHBsAg. These results indicate the usefulness of MDP derivatives as immunoadjuvants for a new-generation vaccine.
• CHARACTERIZATION OF THE MODE OF HANTAAN VIRUS-INFECTION IN ADULT MICE USING A NESTED REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION - TRANSIENT VIRUS-REPLICATION IN ADULT MICE

  H KARIWA, M KAMIMURA, J ARIKAWA, K YOSHIMATSU, TAKASHIMA, I, N HASHIMOTO

  MICROBIOLOGY AND IMMUNOLOGY 39 (1) 35 - 41 0385-5600 1995 [Not refereed][Not invited]
   

  A polymerase chain reaction (PCR) for the detection of hantavirus genome was established and applied to analyze the mode of infection of Hantaan virus in adult ICR mice. The cDNA for the S genome segment of Hantaan virus was reverse-transcribed from the total RNA of organs of the infected mice. The sequence in the S genome segment of Hantaan virus was successfully amplified by reverse transcriptase (RT)-PCR followed by nested PCR. In 5-week-old ICR mice inoculated intraperitoneally with Hantaan virus, strain 76-118 (1.3 x 10(5) FFU/mouse), the virus was detected in clots and lungs from 3 to 10 days post-inoculation (p.i.) by nested PCR and virus-isolation techniques. No virus was detected in any specimens collected on 1 day and after 28 days p.i., and in spleens and brains through the observation period by both methods, The antibody which was measured by indirect immunofluorescence antibody assay (IFA) appeared at 7 days p.i. and the geometric mean titer was elevated to its maximum level of 1 : 203 at 10 days p.i., maintaining the same level until 35 days p.i. These results suggest that adult mice are transiently infected with Hantaan virus.
• EFFECT OF THE SYNTHETIC LIPID A-RELATED COMPOUND, DT-5461, ON RESISTANCE TO SENDAI VIRUS-INFECTION IN MICE

  R YOSHIDA, K SATO, YC YOO, K YOSHIMATSU, M TAMURA, C ISHIHARA, J ARIKAWA, AZUMA, I

  IMMUNOPHARMACOLOGY 28 (2) 153 - 161 0162-3109 1994/09 [Not refereed][Not invited]
   

  DT-5461 enhanced host resistance to Sendai virus infection in mice. Intranasal (i.n.) administration of 200 mu g of DT-5461 per mouse 3 days before infection was the most effective administration route, dose and timing. DT-5461 enhanced the cytotoxicity of murine natural killer (NK) cells. In addition, DT-5461 activated murine peritoneal macrophages, resulted in augmented of cytotoxicity and the induction of tumor necrosis factor-alpha (TNF-alpha). Therefore, these immunomodulating activities presented by DT-5461 caused protection against Sendai virus infection.
• PROTECTIVE ACTIVITY OF MDP-LYS(L18) ON HANTAVIRUS INFECTION IN NEWBORN MICE AND POTENTIATION OF ANTIGENICITY BY B30-MDP AND MDP-LYS(L18) OF INACTIVATED HANTAVIRUS STRAIN B-1 VACCINE AND RECOMBINANT HEPATITIS-B VIRUS SURFACE-ANTIGEN

  AZUMA, I, YC YOO, M TAMURA, R YOSHIDA, K YOSHIMATSU, J ARIKAWA, K YAMANISHI

  IMMUNOTHERAPY OF INFECTIONS 15 191 - 203 1994 [Refereed][Not invited]
  
• APPLICATION OF A RECOMBINANT BACULOVIRUS EXPRESSING HANTAVIRUS NUCLEOCAPSID PROTEIN AS A DIAGNOSTIC ANTIGEN IN IFA TEST - CROSS-REACTIVITIES AMONG 3 SEROTYPES OF HANTAVIRUS WHICH CAUSES HEMORRHAGIC-FEVER RENAL SYNDROME (HFRS)

  K YOSHIMATSU, J ARIKAWA, H KARIWA

  JOURNAL OF VETERINARY MEDICAL SCIENCE 55 (6) 1047 - 1050 0916-7250 1993/12 [Not refereed][Not invited]
   

  Recombinant baculovirus-infected insect cells which expressed recombinant protein, analogous to the nucleocapsid protein (NP) of Seoul type hantavirus, strain SR-11 (rNP-SR-Sf9) were applied to the indirect immunofluorescent antibody (LFA) test. The rNP-SR-Sf9 reacted with anti NP MAb clones which recognized strain specific or hantavirus common epitopes in the IFA test. The recombinant antigen was detected by antibodies to 3 serotypes of hantaviruses (Hantaan 76-118, SR-11, Puumala). Antibody titers of a group of experimentally infected mouse and urban rat sera using rNP-SR-Sf9 and strain SR-11-infected Vero cell antigens were mutual correlative. These results indicated that the rNP-SR-Sf9 were an effective and safety substitute for Vero E6 cells in the IFA test for serosurveys of hantavirus infection.
• POSTNATAL CHANGE OF PIG INTESTINAL GANGLIOSIDE BOUND BY ESCHERICHIA-COLI WITH K99 FIMBRIAE

  Y YUYAMA, K YOSHIMATSU, E ONO, M SAITO, M NAIKI

  JOURNAL OF BIOCHEMISTRY 113 (4) 488 - 492 0021-924X 1993/04 [Not refereed][Not invited]
   

  Enterotoxigenic Escherichia coli possessing K99 fimbriae (E. coli K99) causes diarrhea in piglets of less than 1 week old. The first stage of the bacterial infection is adhesion by the fimbriae on the small intestinal mucosa and the adhesion is followed by colony formation. K99 fimbriae bind specifically to N-glycolylneuraminyl-lactosyl-ceramide, GM3(NeuGc) [Ono, E. et al. (1989) Infect. Immun. 57, 907-911]. We examined the postnatal change of the content and the molecular species of GM3(NeuGc) in the small intestinal mucosa of 0- to 14-day-old piglets and adult pigs. GM3(NeuGc) was a major ganglioside of piglet intestinal mucosa. GM3(NeuGc) content was maximal at birth and gradually decreased to 1/16 in adult animals (5 months old). The ceramide moiety of piglet intestinal GM3(NeuGc) was characterized by the presence of 2-hydroxylated palmitic acid. I-125-labeled bacteria strongly bound to GM3(NeuGc) containing 2-hydroxylated palmitic acid and phytosphingosine compared with GM3(NeuGc) containing any other ceramide moiety. The time when this particular GM3(NeuGc) appears coincides with the time that the infection occurs, and it may explain the susceptibility of newborn piglets to E. coli K99 infection.
• ANTIVIRAL ACTIVITY OF SULFATED CHITIN DERIVATIVES AGAINST FRIEND MURINE LEUKEMIA AND HERPES-SIMPLEX TYPE-1 VIRUSES

  C ISHIHARA, K YOSHIMATSU, M TSUJI, J ARIKAWA, SAIKI, I, S TOKURA, AZUMA, I

  VACCINE 11 (6) 670 - 674 0264-410X 1993/04 [Not refereed][Not invited]
   

  Sulfated chitin derivatives, selected for their low toxicity and high inhibitory activity of melanoma metastasis, were examined for anti-viral activity against Friend murine leukaemia, herpes simplex type-1 (HSV) and Sendai viruses. Carboxymethyl chitin with a 7.66% degree of sulfation (SCM-chitin III) showed a significant inhibition of Friend murine leukaemia helper virus (F-MuLV) and HSV, but not of Sendai virus growth in vitro. Sulfated N-deacetylated chitin had a significant but weak activity against F-MuLV and HSV infections. Carboxymethyl chitin showed no effect on these infections in vitro. SCM-chitin III also exhibited anti-viral activity in vivo by suppressing the splenomegaly which was caused by prior infection of mice with FV, a complex of F-MuLV and spleen focus-forming virus.
• PROTECTIVE IMMUNITY OF HANTAAN VIRUS NUCLEOCAPSID AND ENVELOPE PROTEIN STUDIED USING BACULOVIRUS-EXPRESSED PROTEINS

  K YOSHIMATSU, YC YOO, R YOSHIDA, C ISHIHARA, AZUMA, I, J ARIKAWA

  ARCHIVES OF VIROLOGY 130 (3-4) 365 - 376 0304-8608 1993 [Not refereed][Not invited]
   

  Recombinant Hantaan virus nucleocapsid protein (rNP) and recombinant envelope (rEnv) proteins were prepared using a baculovirus expression system to examine the role of Hantaan virus structural proteins in protective immunity. Passive transfer of spleen cells from mice immunized with rNP conferred partial protection or prolongation of time to death from fatal Hantaan virus infection in suckling mice which were challenged with Hantaan virus at 40 LD50 (survival rate: 43%) or 4 LD50 (survival rate: 43%). The T cell-enriched fraction protected one mouse from lethal infection but the B cell-enriched fraction had no such effect on fatal HTN infection. The protective effects of the antibody against HTN challenge were examined by passive immunization. The monoclonal antibody ECO 2 directed to NP also conferred partial survival and significant difference in time to death. Although rEnv antigen failed to induce neutralizing antibody, both immune spleen cells and immune serum to rEnv conferred partial protection upon suckling mice. These results indicate that both nucleocapsid and envelope proteins of Hantaan virus were responsible for induction of cell mediated protective immunity. Vero E 6 cells infected with Hantaan virus expressed envelope protein on the surface, as determined by flow cytometry. However, there was only negligible expression of nucleocapsid protein.
• COMPARISON OF VIRULENCE BETWEEN SEOUL VIRUS-STRAIN SR-11 AND HANTAAN VIRUS-STRAIN 76-118 OF HANTAVIRUSES IN NEWBORN MICE

  YC YOO, K YOSHIMATSU, R YOSHIDA, M TAMURA, AZUMA, I, J ARIKAWA

  MICROBIOLOGY AND IMMUNOLOGY 37 (7) 557 - 562 0385-5600 1993 [Not refereed][Not invited]
   

  Virulence of hantavirus strain of SR-11 Seoul virus and Hantaan 76-118 (HTN) of Hantaan virus were compared. Infections of both strains were lethal in newborn mice. However, inoculum required to cause lethal infection was about 4,000 times higher for strain HTN (1.65 x 10(3) PFU/mouse/LD50) than for strain SR-11 (0.36 PFU). Thus, both strains were considered pathogenic to newborn mice but they possessed different levels of virulence. The assay system used for these strains in newborn mice proved to be useful in the study of hantavirus vilurence. Growth curves of the two strains in CV-7 cell cultures were compared. Strain SR-11 was shown to have higher activity of virus replication and virus release into the culture fluids than strain HTN. The possibility of a relationship between replication activity and high levels of virulence in mice was suggested.
• EFFECT OF NEUTRALIZING MONOCLONAL-ANTIBODIES ON HANTAAN VIRUS-INFECTION OF THE MACROPHAGE P388D1-CELL LINE

  JS YAO, J ARIKAWA, H KARIWA, K YOSHIMATSU, TAKASHIMA, I, N HASHIMOTO

  JAPANESE JOURNAL OF VETERINARY RESEARCH 40 (2-3) 87 - 97 0047-1917 1992/09 [Refereed][Not invited]
   

  The effect of neutralizing monoclonal antibodies (N MAbs) on Hantaan virus infection of macrophages was investigated using P388D1 cells, a murine macrophage cell line. MAbs to the G1 protein (G1b) and the G2 protein (G2a and G2c) neutralized viral infectivity in P388D1 cells. N MAbs to G1b showed much higher neutralizing potency than those to G2a and G2c. With each N MAbs, two distinct effects were observed: neutralization of viral infectivity occurring at high concentrations and enhancement of that at low concentrations. Non-neutralizing MAbs, on the other hand, showed only enhancement of viral infectivity even at high concentrations without any inhibitory effects. The Fab fragments of N MAbs showed neither neutralizing nor enhancing activities. However, when the virus-Fab complexes were reacted with the anti-Fab antibodies, both neutralization and enhancement of viral infectivity were restored depending on the dose of Fab fragments. These results indicate that Hantaan virus infection of P388D1 cells is mediated by the Fc portion of the antibodies and neutralization is dependent on the concentration of N antibodies bound bivalently to the neutralization site on the virion.
• ISOLATION OF C-REACTIVE PROTEIN FROM CAT SERUM

  A WATANABE, M MORIMATSU, K YOSHIMATSU, S YAMAMOTO, A TERAO, K TSUKAZAKI, M SAITO, M NAIKI

  JOURNAL OF SMALL ANIMAL PRACTICE 33 (2) 71 - 77 0022-4510 1992/02 [Refereed][Not invited]
   

  C-reactive protein (CRP) was isolated from sera from healthy cats by calcium-dependent affinity chromatography on phosphorylcholine derivatives of bovine albumin-coupled Toyopearl, followed by an anion-exchange chromatography using DEAE cellulose. It was identified as CRP by its immunochemical cross reactivity with human CRP. The molecular weight of cat CRP was approximately 100 kilodaltons (kDa) and composed of two glycosylated subunits (23 kDa) and three non-glycosylated subunits (20 kDa) with non-covalent association. Under electron microscopic examination, cat CRP had a pentameric disc-like configuration which is characteristic of CRP. Immunoelectrophoresis and isoelectric focusing showed that cat CRP is an acidic alpha-1-globulin (pI 4.1 to 4.3). Serum concentrations of CRP in cats and kittens were measured by single radial immunodiffusion. In 12 healthy cats from various sources, values ranged from 38 to 168-mu-g/ml. In kittens, serum CRP levels also showed a wide distribution, 81 per cent of them were less than 40-mu-g/ml.
• Isolation and characterization of conglutinin as an influenza A virus inhibitor

  Wakamiya, N., Okuno, Y., Sasao, F., Ueda, S., Yoshimatsu, K., Naiki, M., Kurimura, T.

  Biochemical and Biophysical Research Communications 187 (3) 1992
• STIMULATION OF HOST-DEFENSE MECHANISM WITH SYNTHETIC ADJUVANTS AND RECOMBINANT CYTOKINES AGAINST VIRAL-INFECTION IN MICE

  AZUMA, I, C ISHIHARA, J IIDA, YC YOO, K YOSHIMATSU, J ARIKAWA

  MICROBIAL INFECTIONS 319 253 - 263 1992 [Refereed][Not invited]
  
• ANTIBODY-DEPENDENT ENHANCEMENT OF HANTAVIRUS INFECTION IN MACROPHAGE CELL-LINES

  JS YAO, H KARIWA, TAKASHIMA, I, K YOSHIMATSU, J ARIKAWA, N HASHIMOTO

  ARCHIVES OF VIROLOGY 122 (1-2) 107 - 118 0304-8608 1992 [Refereed][Not invited]
   

  Antibody-dependent enhancement (ADE) of hantavirus infections (strains Hantaan 76-118 and SR-11) was studied using macrophage-like cell lines (J774.1, P388D 1, and U937). Significantly higher virus titers (1,000 to 4,000 FFU/ml) were obtained by pretreatment of the virus with immune serum as compared to normal serum (< 20 FFU/ml). Monoclonal antibodies (MAbs) to strain Hantaan 76-118 were employed to determine the antigenic determinants responsible for the ADE activity. ADE of the infection occurred with MAbs to both G1 and G2 envelope glycoproteins, but not with MAbs to nucleocapsid protein. Antigenic determinants related to haemagglutination or virus neutralization were found to cause ADE of the infection.
• PROTECTIVE ROLE OF ANTIGENIC SITES ON THE ENVELOPE PROTEIN OF HANTAAN VIRUS DEFINED BY MONOCLONAL-ANTIBODIES

  J ARIKAWA, JS YAO, K YOSHIMATSU, TAKASHIMA, I, N HASHIMOTO

  ARCHIVES OF VIROLOGY 126 (1-4) 271 - 281 0304-8608 1992 [Refereed][Not invited]
   

  To investigate the role of Hantaan virus envelope glycoprotein in infection, a panel of monoclonal antibodies (MAbs) was examined in vitro with several serological tests and in vivo by passive transfer experiments in mice. An antigenic site, specific for the inhibition of infected cell focus was detected with the focus inhibition neutralization test (FINT), in addition to the neutralization related antigenic sites, which were revealed by the ordinary focus reduction neutralization test (FRNT). Suckling mice were given the MAbs by passive transfer followed by lethal Hantaan virus challenge. All neutralizing MAbs detected by either FRNT or FINT protected all mice from lethal infection, confirming the importance of the antigenic sites as a protective antigen. Mice given non-neutralizing MAbs by passive transfer, however, began to die earlier than the control group; mean time to death (18.2 +/- 2.1 to 21.5 +/- 2.8 days) being significantly shorter than that of the control group (25.8 +/- 1.8, p < 0.01, Mann-Whitney, U probability test). Virus titers in brains of mice which died early, were about 10 times higher than those of control mice. These results indicated the early death phenomenon of mice which was mediated by the anti-virus antibody.
• ELEVATION OF BOVINE SERUM C-REACTIVE PROTEIN AND SERUM AMYLOID-P COMPONENT LEVELS BY LACTATION

  M MORIMATSU, A WATANABE, K YOSHIMATSU, T FUJINAGA, M OKUBO, M NAIKI

  JOURNAL OF DAIRY RESEARCH 58 (3) 257 - 261 0022-0299 1991/08 [Refereed][Not invited]
   

  C-reactive protein (CRP) and serum amyloid P component (SAP), which are known to increase in sera from humans and many other animals with acute inflammation caused by infection, toxic drug administration or injury, were previously purified from bovine serum. These serum levels were determined by enzyme-linked immunosorbent assay (ELISA) using specific antiserum to bovine CRP or SAP which was prepared by immunizing rabbits and goats with each purified protein. Among 68 healthy Holstein cows, 45 non-lactating cows had levels of CRP and SAP of 20.6 +/- 1.4 and 27.6 +/- 1.3-mu-g/ml respectively; 23 lactating cows had higher levels of CRP and SAP (76.0 +/- 13.6 and 38.3 +/- 5.5-mu-g/ml respectively). In the latter group, there was a high correlation between milk yield and serum CRP levels (P < 0.001). From these observations, it was assumed that lactation might stimulate CRP synthesis rather than SAP synthesis in bovine liver as an acute phase reaction, and that CRP might be called a lactation-associated protein.
• ISOLATION AND CHARACTERIZATION OF C-REACTIVE PROTEIN AND SERUM AMYLOID-P COMPONENT FROM BOVINE SERUM

  M MORIMATSU, H SAKAI, K YOSHIMATSU, O MINOWA, S YAMAMOTO, K YATOMI, T FUJINAGA, M NAIKI

  JAPANESE JOURNAL OF VETERINARY SCIENCE 51 (4) 723 - 732 0021-5295 1989/08 [Refereed][Not invited]
  

MISC

• Broad-spectrum search for influenza virus CEN (cap endonuclease) inhibitors

  佐藤彰彦, 佐藤彰彦, 佐藤彰彦, 鳥羽晋輔, 鳥羽晋輔, 垰田善之, 日下部伸治, 日下部伸治, 小西慧, 小西慧, 松野啓太, 松野啓太, 吉松組子, 有川二郎, 佐々木道仁, 佐々木道仁, 大場靖子, 大場靖子, 大場靖子, 澤洋文, 澤洋文, 澤洋文  日本ウイルス学会学術集会プログラム・予稿集(Web)  70th-  2023
• ザンビアにおけるクリミア・コンゴ出血熱の疫学調査

  梶原将大, SIMUUNZA Martin, SAASA Ngonda, DAUTU George, 森亜紀奈, 邱永晋, 中尾亮, HANG’OMBE Bernard, HANG’OMBE Bernard, 澤洋文, 澤洋文, SIMULUNDU Edgar, 福士秀悦, 森川茂, 西條政幸, 有川二郎, 高田礼人, 高田礼人, 吉松組子  日本ウイルス学会学術集会プログラム・予稿集(Web)  69th-  2022
• Reply to comments by Yih et al. (Exposure to hantavirus is a risk factor associated with kidney diseases in Sri Lanka: A cross-sectional study)

  Yomani D. Sarathkumara, Chandika D. Gamage, Sithumini Lokupathirage, Devinda S. Muthusinghe, Nishantha Nanayakkara, Lishanthe Gunarathne, Kenta Shimizu, Yoshimi Tsuda, Jiro Arikawa, Kumiko Yoshimatsu  Viruses  11-  (12)  2019/12/11
• 東アジアの小型哺乳動物から分離されたレプトスピラの反復配列多型解析法による分子型別

  小泉信夫, 泉谷秀昌, 慕蓉蓉, 岡野祥, 中島千絵, 鈴木定彦, 谷川力, 小松謙之, 吉松組子, 武藤(水谷)麻紀, 武藤(水谷)麻紀, 大西真  レプトスピラ・シンポジウム  53rd-  2016
• イムノクロマトグラフィー法によるハンタウイルスの迅速診断法の開発

  小山 芽以, 吉松 組子, 好井 健太朗, 有川 二郎, 苅和 宏明  日本獣医学会学術集会講演要旨集  158回-  373  -373  2015/08  [Not refereed][Not invited]
  
• Hantavirus Pulmonary Syndrome

  吉松 組子, 有川 二郎  医学のあゆみ  253-  (1)  63  -67  2015/04/04  [Not refereed][Not invited]
  
• マウスを用いた腎症候性出血熱の疾患モデルの開発

  清水健太, 吉松組子, 駒貴明, 有川二郎  日本実験動物学会総会講演要旨集  61st-  2014
• 【人と動物の共通感染症最前線10】 日本のげっ歯類におけるハンタウイルス感染の血清疫学調査とエゾヤチネズミが保有するHokkaidoウイルスの分離

  苅和 宏明, 尾崎 由佳, 真田 崇弘, 池中 良徳, 石塚 真由美, 坪田 敏男, 好井 健太朗, 吉松 組子, 有川 二郎, 高島 郁夫  獣医畜産新報  66-  (4)  262  -264  2013/04  [Not refereed][Not invited]
   

  ハンタウイルスはげっ歯類媒介性の人獣共通感染症の病原体で、腎症候性出血熱(HFRS)またはハンタウイルス肺症候群(HPS)を引き起こす。近年の日本におけるげっ歯類のハンタウイルス感染状況を明らかにするために、1994年から2010年にかけて国内の様々な地域で捕獲された1658匹のげっ歯類の血清について、抗ハンタウイルス抗体の検出を行った。840例のRattus属げっ歯類(ドブネズミとクマネズミ)は全て抗体陰性であった。北海道以外の地域で捕獲された野生げっ歯類113例はいずれも抗体陰性であったのに対し、北海道で捕獲された705例の野性げっ歯類のうち、エゾヤチネズミの7.4%(26/352)とアカネズミの1.2%(2/168)が抗体を保有していた。(著者抄録)
• 新世界ハンタウイルス感染症の血清型鑑別診断のための組換え核蛋白を用いた新規ELISA法の開発

  駒 貴明, 吉松 組子, 垂石 みどり, 宮下 大輔, 遠藤 理香, 清水 健太, 安田 俊平, 天田 貴子, 瀬戸 隆弘, 村田 亮, 吉田 喜香, 苅和 宏明, 高島 郁夫, 有川 二郎  北海道醫學雜誌 = Acta medica Hokkaidonensia  88-  (1)  35  -35  2013/01/01  [Not refereed][Not invited]
  
• Environmental enrichment after the occurrence of wet bedding and the appearance of abnormal fur in mice is effective for solving those problems

  TOSA Noriko, YOSHIMATSU Kumiko, ARIKAWA Jiro  実験動物と環境 = Laboratory animal and environment  20-  (1)  1  -8  2012/04/01
• 腎症候性出血熱の動物モデルの開発

  清水健太, 吉松組子, 駒貴明, 伊勢川裕二, 安田俊平, 天田貴子, 五十棲理恵, 有川二郎  日本獣医学会学術集会講演要旨集  153rd-  2012
• ベトナムのラットおよびヒトにおけるラットE型肝炎ウイルスの感染状況の調査

  清水健太, 李天成, 安田俊平, 吉松組子, 駒貴明, 長谷部太, 山城哲, HOA Nguyen Thuy, MAI Le Thi Quynh, 有川二郎  日本獣医学会学術集会講演要旨集  154th-  2012
• 肺水腫を呈すハンタウイルス感染SCIDマウスに於ける好中球の役割

  駒貴明, 永田典代, 吉松組子, 佐藤由子, 清水健太, 天田貴子, 西尾佐奈恵, 長谷川秀樹, 有川二郎  日本ウイルス学会学術集会プログラム・抄録集  60th-  2012
• イムノクロマト法による抗ハンタウイルス抗体の迅速抗体検出法の開発

  天田貴子, 吉松組子, 安田俊平, 清水健太, 駒貴明, 林元展人, 高倉彰, 有川二郎  日本ウイルス学会学術集会プログラム・抄録集  60th-  2012
• イムノクロマト法による抗ハンタウイルス抗体の迅速抗体検出法の開発

  天田貴子, 吉松組子, 安田俊平, 駒貴明, 清水健太, 五十棲理恵, 林元展人, 高倉彰, 有川二郎  日本獣医学会学術集会講演要旨集  153rd-  2012
• 腎症候性出血熱の動物モデルの開発と病態発現機構の解析

  清水健太, 吉松組子, 駒貴明, 天田貴子, 西尾佐奈恵, 有川二郎  日本ウイルス学会学術集会プログラム・抄録集  60th-  2012
• イムノクロマト法によるラットとヒト血清のハンタウイルス抗体迅速検出法の開発

  有川二郎, 天田貴子, 林元展人, 吉松組子, 安田俊平, 清水健太, 駒貴明, 五十棲理恵, 高倉彰  日本実験動物学会総会講演要旨集  59th-  2012
• エゾヤチネズミ(Myodes rufocanus bedfordiae)の腎臓由来細胞株の樹立とHokkaidoウイルス分離への応用

  真田崇弘, 瀬戸隆弘, 尾崎由佳, NGONDA Saasa, 吉松組子, 有川二郎, 好井健太朗, 苅和宏明  日本ウイルス学会北海道支部夏季シンポジウム講演抄録  46th-  2012
• ハンタウイルス糖蛋白質の細胞内輸送に関与するウイルス蛋白質とその機能領域

  清水健太, 吉松組子, 駒貴明, 安田俊平, 有川二郎  日本獣医学会学術集会講演要旨集  152nd-  2011
• 免疫不全マウスを用いたハンタウイルス感染症病態モデルの検討

  駒貴明, 吉松組子, 永田典代, 清水健太, 安田俊平, 有川二郎  日本ウイルス学会学術集会プログラム・抄録集  58th-  2010
• ハンタウイルス持続感染メカニズム解明のための実験感染ラットを用いた細胞性免疫測定系の確立

  安田俊平, 吉松組子, 遠藤理香, 清水健太, 駒貴明, 有川二郎  日本ウイルス学会学術集会プログラム・抄録集  58th-  2010
• ハンタウイルスGlycoproteinの細胞内動態に及ぼすNucleocapsid proteinの影響

  清水健太, 吉松組子, 駒貴明, 安田俊平, 有川二郎  日本ウイルス学会学術集会プログラム・抄録集  58th-  2010
• ハンタウイルスNucleocapsid proteinはGlycoprotein Gcのシスゴルジへの局在を促進する

  清水健太, 吉松組子, 駒貴明, 遠藤理香, 安田俊平, ERDENESAIKHAN Tegshduuren, 有川二郎  日本獣医学会学術集会講演要旨集  150th-  2010
• メキシコの野生げっ歯類が保有するハンタウイルスの抗原性解析

  吉田喜香, 苅和宏明, 真田崇弘, NGONDA Saasa, 瀬戸隆弘, 吉松組子, 有川二郎, 好井健太朗, 高島郁夫  日本ウイルス学会学術集会プログラム・抄録集  58th-  2010
• インドネシアのげっ歯類におけるハンタウイルス感染症の疫学

  清水健太, イブラヒムイマヌリサ, 吉松組子, 遠藤理香, 安田俊平, 駒貴明, エルテネサイハンテグシドーレン, 有川二郎  日本ウイルス学会学術集会プログラム・抄録集  57th-  2009
• トガリネズミ目(旧食虫目)由来ハンタウイルスThottapalayamウイルス(TPMV)核蛋白の単クローン抗体を用いた抗原領域の解析

  エルテネサイハンテグシドーレン, 清水健太, 吉松組子, 遠藤理香, 駒貴明, 安田俊平, 有川二郎, 石原智明  日本ウイルス学会学術集会プログラム・抄録集  57th-  2009
• ハンタウイルス自然感染ラットと実験感染ラットにおける病態の比較

  安田俊平, 吉松組子, 遠藤理香, 清水健太, 駒貴明, TEGSHDUUREN Erdenesaikhan, 垂石みどり, 有川二郎  日本ウイルス学会学術集会プログラム・抄録集  57th-  2009
• 汎用PCRプライマーを用いたハンタウイルス遺伝子検出スクリーニング法の確立

  遠藤理香, 吉松組子, 駒貴明, 清水健太, 安田俊平, TEGSHDUUREN Erdenesaihan, 垂石みどり, 海老原秀喜, 宮下大輔, 瀬戸隆弘, HERNANDEZ Cornelio S., ALMARAZ Maria L.R., RAMOS Celso, 苅和宏明, 高島郁夫, 有川二郎  日本ウイルス学会学術集会プログラム・抄録集  57th-  2009
• 新世界ハンタウイルス感染の血清型鑑別ELISA法の確立

  駒貴明, 吉松組子, 垂石みどり, 遠藤理香, 清水健太, 安田俊平, エルテネサイハンテグシドーレン, 海老原秀喜, HERNANDEZ Cornelio S., ALMARAZ Maria L.R., RAMOS Celso, 宮下大輔, 瀬戸隆弘, 苅和宏明, 高島郁夫, ENRIA Delia, 有川二郎  日本ウイルス学会学術集会プログラム・抄録集  57th-  2009
• 新世界ハンタウイルス感染の血清型鑑別ELISA法の確立

  駒貴明, 吉松組子, 垂石みどり, 遠藤理香, 清水健太, 安田俊平, テグシドーレン エルデネサイハン, 海老原秀喜, HERNANDEZ Cornelio S., ALMARAZ Maria L.R., RAMOS Celso, 宮下大輔, 瀬戸隆弘, 苅和宏明, 高島郁夫, ENRIA Delia, 有川二郎  日本ウイルス学会北海道支部夏季シンポジウム講演抄録  43rd-  2009
• 複数のハンタウイルス血清型に対応可能な診断法の開発

  真田崇弘, 苅和宏明, 瀬戸隆弘, 谷川洋一, 宮下大輔, 吉松組子, 有川二郎, 好井健太朗, 高島郁夫  日本ウイルス学会北海道支部夏季シンポジウム講演抄録  43rd-  2009
• Evidence of Hantavirus Infection Among Rodents in South India

  S. Chandy, K. Yoshimatsu, R. G. Ulrich, M. Schlegel, D. J. Prakash, V. Balraj, P. Abraham, J. Arikawa, G. Sridharan  INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES  12-  E131  -E131  2008/12  [Not refereed][Not invited]
  
• 新世界ハンタウイルス感染の血清型鑑別診断法の確立

  駒貴明, 吉松組子, 垂石みどり, 遠藤理香, 海老原秀喜, 有川二郎  日本ウイルス学会学術集会プログラム・抄録集  56th-  2008
• ロシアのボルガ川流域におけるハンタウイルス感染症の疫学的研究

  瀬戸隆弘, 苅和宏明, 谷川洋一, 吉松組子, 中村一郎, 宮下大輔, 中内美名, 好井健太朗, 有川二郎, 高島郁夫  日本ウイルス学会学術集会プログラム・抄録集  55th-  2007
• メキシコの野生げっ歯類におけるハンタウイルス感染症の疫学調査

  宮下大輔, 苅和宏明, 瀬戸隆弘, 好井健太郎, 吉松組子, 有川二郎, 高島郁夫  日本ウイルス学会学術集会プログラム・抄録集  55th-  2007
• 東アジアの人とげっ歯類のおけるハンタウイルス感染の疫学的研究 (特集 人と動物の共通感染症最前線(2))

  有川 二郎, 吉松 組子, Kumperasart Sanit  獣医畜産新報  58-  (4)  326  -328  2005/04
• Nucleocapsid protein of cell culture-adapted Seoul virus strain 80-39: Analysis of its encoding sequence, expression in yeast and immuno-reactivity

  Jonas Schmidt, Burkhard Jandrig, Boris Klempa, Kumiko Yoshimatsu, Jiro Arikawa, Helga Meisel, Matthias Niedrig, Christian Pitra, Detlev H. Krüger, Rainer Ulrich  Virus Genes  30-  (1)  37  -48  2005/01  [Not refereed][Not invited]
   

  Seoul virus (SEOV) is a hantavirus causing a mild to moderate form of hemorrhagic fever with renal syndrome that is distributed mainly in Asia. The nucleocapsid (N) protein-encoding sequence of SEOV (strain 80-39) was RT-PCR-amplified and cloned into a yeast expression vector containing a galactose-inducible promoter. A survey of the pattern of synonymous codon preferences for a total of 22 N protein-encoding hantavirus genes including 13 of SEOV strains revealed that there is minor variation in codon usage by the same gene in different viral genomes. Introduction of the expression plasmid into yeast Saccharomyces cerevisiae resulted in the high-level expression of a hexahistidine-tagged N protein derivative. The nickel-chelation chromatography purified, yeast-expressed SEOV N protein reacted in the immunoblot with a SEOV-specific monoclonal antibody and certain HTNV- and PUUV-cross-reactive monoclonal antibodies. The immunization of a rabbit with the recombinant N protein resulted in the induction of a high-titered antibody response. In ELISA studies, the N protein was able to detect antibodies in sera of experimentally infected laboratory rats and in human anti-hantavirus-positive sera or serum pools of patients from different geographical origin. The yeast-expressed SEOV N protein represents a promising antigen for development of diagnostic tools in serology, sero prevalence studies and vaccine development. © 2005 Springer Science+Business Media, Inc.
• Epitope analysis of monoclonal antibody E5/G6, which binds to a linear epitope in the nucleocapsid protein of hantaviruses

  M Okumura, K Yoshimatsu, K Araki, BH Lee, A Asano, T Agui, J Arikawa  ARCHIVES OF VIROLOGY  149-  (12)  2427  -2434  2004/12  [Not refereed][Not invited]
   

  Monoclonal antibody E5/G6 recognized a linear epitope common to hantavirus nucleocapsid proteins. Using synthetic peptides, we identified epitope E5/G6 as the 9 merYEDVNGIRK (NP 165 - 173), in which D167, G170, I171, and R172 are indispensable. Furthermore, all the peptides synthesized using various hantavirus sequences bound MAb E5/G6 consistently, despite the existence of several amino acid variations in this region. These results indicate that MAb E5/G6 is a useful tool for detecting hantavirus antigen in rodent or patient tissues using Western blotting or other immunohistochemical assays.
• Cell fusion activities of Hantaan virus envelope glycoproteins

  Michiko Ogino, Kumiko Yoshimatsu, Hideki Ebihara, Koichi Araki, Byoung-Hee Lee, Megumi Okumura, Jiro Arikawa  Journal of Virology  78-  (19)  10776  -10782  2004/10  [Not refereed][Not invited]
   

  Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.
• Age-dependent hantavirus-specific CD8(+) T-cell responses in mice infected with Hantaan virus

  K Araki, K Yoshimatsu, BH Lee, M Okumura, H Kariwa, Takashima, I, J Arikawa  ARCHIVES OF VIROLOGY  149-  (7)  1373  -1382  2004/07  [Not refereed][Not invited]
   

  To investigate age-dependent differences in hantavirus-specific CD8(+) T-cell responses, mice were inoculated with 0.1 50% newborn mouse lethal dose of Hantaan virus (HTNV) at 0, 3, 7, 14, or 35 days after birth. HTNV-specific CD8(+) T cells producing gamma interferon (IFN-gamma) were measured on day 30 after HTNV inoculation. Although no IFN-gamma-producing HTNV-specific CD8(+) T cells were detected in most of the mice inoculated with HTNV on day 0 after birth, most mice inoculated at 3, 7, 14, or 35 days had HTNV-specific CD8(+) T cells. The production of tumor necrosis factor alpha (TNF-alpha) by IFN-gamma-producing CD8(+) T cells and the cytotoxic activity against HTNV-infected target cells were similar in immature and adult mice. However, the number of IFN-gamma-producing HTNV-specific CD8(+) T cells was significantly less in mice inoculated with HTNV at 3 days than in older mice. In addition, a strong correlation between HTNV persistence and a lack of HTNV-specific CD8(+) T cells was observed. These results suggest that mice over 7 days old have the ability to induce functional HTNV-specific CD8(+) T-cell responses that are indistinguishable from the responses of adult mice, and that HTNV-specific CD8(+) T cells are important for clearance of HTNV.
• Antigenic and genetic diversity of hantaviruses isolated in far eastern Asian region

  J Arikawa, K Yoshimatsu, H Wang, H Kariwa, K Lokugamage, N Lokugamage, Takashima, I  JOURNAL OF CLINICAL VIROLOGY  28-  S28  -S28  2003/12  [Not refereed][Not invited]
  
• Association of the nucleocapsid protein of the Seoul and Hantaan hantaviruses with small ubiquitin-like modifier-1-related molecules

  BH Lee, K Yoshimatsu, A Maeda, K Ochiai, M Morimatsu, K Araki, M Ogino, S Morikawa, J Arikawa  VIRUS RESEARCH  98-  (1)  83  -91  2003/12  [Not refereed][Not invited]
   

  We performed yeast two-hybrid screening of a human kidney cell cDNA library to study the biological role of the hantavirus nucleocapsid protein (NP). We found that Seoul virus (SEOV) and Hantaan virus (HTNV) NPs were associated with small ubiquitin-like modifier (SUMO)-1-interacting proteins PIAS 1, PIASxbeta, HIPK2, CHD3, and TTRAP, which interacted with the SUMO-1 conjugating enzyme (Ubc-9) and SUMO-1 in the yeast two-hybrid assay. Interactions between the HIPK2, CHD3, and TTRAP proteins and SEOV NP were also shown in a mammalian two-hybrid assay. However, there was no interaction between PIAS proteins and NP, which was probably due to the inhibitory effect of PIAS on transcription in the mammalian two-hybrid assay. Nevertheless, a co-expression experiment suggested the existence of a PIAS-NP interaction in the cytoplasm. The region spanning amino acids 100-125 of SEOV NP, which represents a critical region for NP-NP polymerization. was found to be responsible for the interaction with SUMO-1-related molecules in both the yeast and mammalian two-hybrid assays. These results add to the information on interactions of hantavirus NP and host cellular proteins. (C) 2003 Elsevier B.V. All rights reserved.
• Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of mongolian gerbils

  N Lokugamage, H Kariwa, K Lokugamage, T Hagiya, H Miyamoto, MA Iwasa, K Araki, K Yoshimatsu, J Arikawa, T Mizutani, Takashima, I  JOURNAL OF VETERINARY MEDICAL SCIENCE  65-  (11)  1189  -1194  2003/11  [Not refereed][Not invited]
   

  Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10(5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.
• Detection of antibody for the serodiagnosis of hantavirus infection in different rodent species

  BH Lee, K Yoshimatsu, K Araki, M Ogino, M Okumura, K Tsuchiya, H Kariwa, J Arikawa  ARCHIVES OF VIROLOGY  148-  (10)  1885  -1897  2003/10  [Not refereed][Not invited]
   

  Peroxidase-labeled staphylococcal protein A, streptococcal protein G, and antibodies directed against Mus musculus (mouse), Rattus norvegicus (rat), Mesocretus auratus (hamster), and Peromyscus leucopus were examined for their reactivity with immunoglobulin G (IgG) from various rodent species. The purpose of this study was to identify the optimal secondary antibodies or reagents for specific serodiagnosis of hantavirus infection in various rodent species. Using ELISA, a total of 65 sera from 29 rodent species of the family Muridae and one serum sample from family Octodontidae were compared for IgG reactivity with the six different reagents. The results demonstrate that the reactivities of the secondary antibodies and reagents to the sera varied, even among sera from rodents of the same genus. Hantavirus-specific antibody ELISA revealed that hantavirus-infected rodent sera obtained from M. musculus, R. norvegicus, Apodemus agrarius, A. peninsulae, and Bandicota indica bound to the six different conjugates in a similar pattern as that detected in IgG ELISA. These results indicate that the applicability of secondary antibodies and protein A and G should be carefully evaluated before use for serodiagnosis in different rodent species.
• Synthesis of Seoul virus RNA and structural proteins in cultured cells

  H Kariwa, H Tanabe, T Mizutani, Y Kon, K Lokugamage, N Lokugamage, MA Iwasa, T Hagiya, K Araki, K Yoshimatsu, J Arikawa, Takashima, I  ARCHIVES OF VIROLOGY  148-  (9)  1671  -1685  2003/09  [Not refereed][Not invited]
   

  Seoul virus is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). The virion has a tripartite (S, M, and L) negative-stranded RNA genome, which is characteristic of the family Bunyaviridae. However, the molecular basis of virus replication is not well known. We established a Northern blot hybridization (NB) procedure using digoxygenin-labeled RNA probes, to quantitate the hantaviral plus- and minus-strand RNAs separately. Virus RNA replication was analyzed in infected Vero E6 cells. When the Vero E6 cells were infected with Seoul virus strain KI-83-262 (KI) at m.o.i.=0.25, the plus-strand RNA was detected within 1 h post-infection (hpi), and the minus-strand RNA was detected subsequently. Using laser confocal microscopy, the nucleocapsid protein (NP) was detected within 2 hpi, and accumulated as scattered granules in the cytoplasm until 24 hpi. In contrast, the G2 protein first appeared at 8 hpi, was immediately transported to the Golgi, and accumulated in the Golgi until 24 hpi. Infectious virus particles were released into the medium at 24 h hpi. These findings indicate that hantavirus RNA replication starts with the appearance of NP at 2 hpi, glycoproteins then accumulate gradually in the Golgi, and virion formation is initiated once the viral RNAs and proteins have accumulated.
• Serological analysis of hemorrhagic fever with renal syndrome (HFRS) patients in Far Eastern Russia and identification of the causative hantavirus genotype

  H Miyamoto, H Kariwa, K Araki, K Lokugamage, D Hayasaka, BZ Cui, N Lokugamage, LI Ivanov, T Mizutani, MA Iwasa, K Yoshimatsu, J Arikawa, Takashima, I  ARCHIVES OF VIROLOGY  148-  (8)  1543  -1556  2003/08  [Not refereed][Not invited]
   

  Hemorrhagic fever with renal syndrome (HFRS) is endemic in East Asia and Europe. The disease is caused by several viruses belonging to the genus Hantavirus, including the Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava Belgrade virus (DOBV), and Puumala virus (PUUV). Recently, HTNV-related viruses, Amur (AMR) and Far East (FE) genotypes were identified as causative agents of HFRS in Far Eastern Russia. To investigate the epidemiology of HFRS and virus transmission, we collected sera from 17 acute and 32 convalescent patients who were clinically diagnosed with HFRS in the Khabarovsk region of Far Eastern Russia, and detected anti-hantavirus antibodies using an ELISA that can differentiate the infected virus serotype using truncated hantavirus nucleocapsid protein antigen. Sixteen of the 17 acute phase patients had antibodies to hantavirus, and all the positive sera had higher optical densities for HTNV-specific antigen than for SEOV-, DOBV-, or PUUV-specific antigens. The partial M segment of the viral genome was amplified from blood clots from three acute patients by PCR. The nucleotide sequences had closer identities to the FE genotype (> 96%) than to the prototype HTNV (88 to 89%) or AMR genotype (81 to 83%). A phylogenetic analysis found that the virus sequences from the patients clustered with the FE type, and were distinct from the AMR type. Thirty-one of 32 convalescent patient sera had antibodies to HTNV-specific antigen. These data suggest ELISA system can detect HTNV-specific antibodies to the FE type, which may be responsible for most of the HFRS in Khabarovsk.
• Hantavirus-specific CD8(+)-T-cell responses in newborn mice persistently infected with Hantaan virus

  K Araki, K Yoshimatsu, BH Lee, H Kariwa, Takashima, I, J Arikawa  JOURNAL OF VIROLOGY  77-  (15)  8408  -8417  2003/08  [Not refereed][Not invited]
   

  The relationship between virus-specific CD8(+)-T-cell responses and viral persistence was studied in mice by using Hantaan virus (HTNA). We first established a simple method for measuring levels of virus-specific CD8(+) T cells by How cytometry. Next, to produce a mouse model of persistent HTNV infection, newborn mice were inoculated subcutaneously within 24 h of birth with 1 or 0.150% newborn mouse lethal dose of HTNV. All mice that escaped lethal infection were persistently infected with HTNV until at least 30 days after virus inoculation and had no virus-specific CD8(+) T cells producing gamma interferon (IFN-gamma). Subsequently, the virus was eliminated from some of the mice, depending on the appearance of functional virus-specific CD8(+) T cells, which have the ability to produce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) and have cytotoxic activity. Neutralizing antibodies were detected in all mice, regardless of the presence or absence of virus. In the acute phase, which occurs within 30 days of infection, IFN-gamma-producing HTNV-specific CD8(+) T cells were detected on day 15 after virus inoculation. However, TNF-alpha production and the cytotoxic activity of these specific CD8(+) T cells were impaired and HTNV was not removed. Almost all of these specific CD8(+) T cells disappeared by day 18. These results suggest that functional HTNV-specific CD8(+) T cells are important for clearance of HTNV.
• Enzyme-linked immunosorbent assay using recombinant antigens expressed in mammalian cells for serodiagnosis of tick-borne encephalitis

  K Yoshii, D Hayasaka, A Goto, M Obara, K Araki, K Yoshimatsu, J Arikawa, L Ivanov, T Mizutani, H Kariwa, Takashima, I  JOURNAL OF VIROLOGICAL METHODS  108-  (2)  171  -179  2003/03  [Not refereed][Not invited]
   

  A recombinant plasmid that expresses the tick-borne encephalitis (TBE) virus premembrane (prM) and envelope (E) proteins in mammalian cells was constructed. Recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins, and transfected cells released virus-like particles (VLPs), which were 1.13-1.14 g/ml in density and 20-30 nm in diameter, into the culture medium. Recombinant E proteins were used for the development of an enzyme-linked immunosorbent assay (ELISA) to detect TBE virus-specific IgM and IgG antibodies in serum. The results of this ELISA correlated well with the results of commercial ELISA, when tested with 95 serum samples from clinically TBE-suspected patients. In addition, ELISA using recombinant antigens showed no cross-reactivity against serum from Japanese encephalitis (JE) patients, despite the cross-reactivity shown by commercial ELISA systems. These observations indicated that this newly developed ELISA system could distinguish tick-borne encephalitis from Japanese encephalitis infection, and that it constitutes a useful and safe alternative to conventional ELISA systems. (C) 2002 Published by Elsevier Science B.V.
• Establishment of an enzyme-linked immunosorbent assay for detection of Hantavirus antibody of rats using a recombinant of nucleocapsid protein expressed in Escherichia coli

  A Takakura, K Goto, T Itoh, K Yoshimatsu, Takashima, I, J Arikawa  EXPERIMENTAL ANIMALS  52-  (1)  25  -30  2003/01  [Not refereed][Not invited]
   

  A recombinant nucleocapsid protein of Hantaan virus (HTN) 76-118 strain expressed in E. coli was applied as a serodiagnostic antigen in an enzyme-linked immunosorbent assay (rHTN-ELISA) for detection of hantavirus antibody in rat sera. The sensitivity and specificity of the rHTN-ELISA were compared with those of the indirect immunofluoresent assay (IFA) using virus-infected cells. The sensitivity of rHTN-ELISA was similar to that of the IFA both in experimentally SR-11 infected rat and naturally infected rat sera. Sera showing a low antibody titer in IFA and suspected to be negative by other methods were also found to be negative in rHTN-ELISA. These results indicate that rHTN-ELISA is effective as a screening method for serodiagnosis of hantaviruses, because of its high sensitivity, specificity, safety and suitability for processing large number of samples.
• Use of vesicular stomatitis virus pseudotypes bearing hantaan or Seoul virus envelope proteins in a rapid and safe neutralization test

  M Ogino, H Ebihara, BH Lee, K Araki, A Lundkvist, Y Kawaoka, K Yoshimatsu, J Arikawa  CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY  10-  (1)  154  -160  2003/01  [Not refereed][Not invited]
   

  A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVDeltaG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVDeltaG*G. Pseudotype VSV with the Hantaan (VSVDeltaG*-HTN) or Seoul (VSVDeltaG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 10(5) to 10(6)/ml. The infectivity of VSVDeltaG*-HTN and VSVDeltaG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVDeltaG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVDeltaG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4degreesC for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.
• The multimerization of Hantavirus nucleocapsid protein depends on type-specific epitopes

  Kumiko Yoshimatsu, Byoung-Hee Lee, Koichi Araki, Masami Morimatsu, Michiko Ogino, Hideki Ebihara, Jiro Arikawa  Journal of Virology  77-  (2)  943  -952  2003/01  [Not refereed][Not invited]
   

  Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.
• Genetic characterization of hantaviruses transmitted by the Korean field mouse (Apodemus peninsulae), Far East Russia

  K Lokugamage, H Kariwa, D Hayasaka, BZ Cui, T Iwasaki, N Lokugamage, LI Ivanov, Volkov, VI, VA Demenev, R Slonova, G Kompanets, T Kushnaryova, T Kurata, K Maeda, K Araki, T Mizutani, K Yoshimatsu, J Arikawa, Takashima, I  EMERGING INFECTIOUS DISEASES  8-  (8)  768  -776  2002/08  [Not refereed][Not invited]
   

  In an epizootiologic survey of 122 rodents captured in Vladivostok, Russia, antibodies positive for hantavirus were found in Apodemus peninsulae (4/70), A. agrarius (1/39), and Clethrionomys rufocanus (1/8). The hantavirus sequences identified in two seropositive A. peninsulae and two patients with hemorrhagic fever with renal syndrome (HFRS) from the Primorye region of Far East Russia were designated as Solovey and Primorye, respectively. The nucleotide sequences of the Solovey, Primorye, and Amur (obtained through GenBank) sequences were closely related (>92% identity). Solovey and Primorye sequences shared 84% nucleotide identity with the prototype Hantaan 76-118. Phylogenetic analysis also indicated a close relationship between Solovey, Primorye, Amur, and other viruses identified in Russia, China, and Korea. Our findings suggest that the Korean field mouse (A. peninsulae) is the reservoir for a hantavirus that causes HFRS over a vast area of east Asia, including Far East Russia.
• Epizootiological survey of hantavirus among rodent species in Ningxia Hui Autonomous Province, China

  H Kariwa, CB Zhong, K Araki, K Yoshimatsu, K Lokugamage, N Lokugamage, ME Murphy, T Mizutani, J Arikawa, H Fukushima, H Xiong, JHC Chen, Takashima, I  JAPANESE JOURNAL OF VETERINARY RESEARCH  49-  (2)  105  -114  2001/08  [Not refereed][Not invited]
   

  Hantaviral antibodies were detected in the sera from Apodemus (A.) agrarius and A. peninsulae captured in Ningxia province, China by several different serological diagnostic methods. A total of 409 sera from rodent and insectivore species were collected in 1999 and examined by indirect immunofluorescent antibody assay (IFA). Among them, 19 of 191 (9.9%) sera of A. agrarius and 1 of 13 (7.7%) sera of A. peninsulae were positive for hantaviral antibodies. The other species (Rattus norvegicus, Mus musculus, Cricetulus triton, and Sorex cylindricauda) were negative. The reaction pattern of positive serum was characterized as scattered and granular virus antigens in the cytoplasm of hantavirus infected Vero E6 cells. Some of the A. agrarius sera positive for hantavirus were further examined by Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and the focus reduction neutralization test (FRNT). By WB, positive sera showed the same specific reaction pattern of baculovirus-expressed recombinant hantaviral nucleocapsid protein, as shown in hantavirus-immune serum. By ELISA, IFA-positive sera showed significantly higher optical densities (around 1.0) than the negative A. agrarius sera. Hantaan type hantavirus was neutralized with the positive sera. These results suggest that A. agrarius have hantavirus infection and may play a role as a reservoir animal for hantavirus in Ningxia Hui Autonomous Province, China.
• Truncated hantavirus nucleocapsid proteins for serotyping Hantaan, Seoul, and Dobrava hantavirus infections

  K Araki, K Yoshimatsu, M Ogino, H Ebihara, A Lundkvist, H Kariwa, Takashima, I, J Arikawa  JOURNAL OF CLINICAL MICROBIOLOGY  39-  (7)  2397  -2404  2001/07  [Not refereed][Not invited]
   

  Truncated recombinant nucleocapsid proteins (rNPs) of Hantaan virus (HTNV), Seoul virus (SEOV), and Dobrava virus (DOBV) were expressed by a baculovirus system. The truncated rNPs, which lacked 49 (rNP50) or 154 (rNP155) N-terminal amino acids of the NPs of HTNV, SEOV, and DOBV, were able to differentiate HTNV-, SEOV-, and DOBV-specific immune sera. Recombinant NP50s retained higher reactivities than rNP155s and were proven useful for enzyme-linked immunosorbent assay (ELISA). The ELISAs based on the rNP50s of HTNV, SEOV, and DOBV successfully differentiated three groups of patient sera, previously defined by neutralization tests: 17 with HTNV infection, 12 with SEOV infection, and 20 with DOBV infection. The entire rNP of Puumala virus (PUUV) distinguished PUUV infection from the other types of hantavirus infection. Serotyping with these rNP50s can be recommended as a rapid and efficient system for hantavirus diagnosis.
• Characterization of in vitro and in vivo antiviral activity of lactoferrin and ribavirin upon hantavirus

  ME Murphy, H Kariwa, T Mizutani, H Tanabe, K Yoshimatsu, J Arikawa, Takashima, I  JOURNAL OF VETERINARY MEDICAL SCIENCE  63-  (6)  637  -645  2001/06  [Not refereed][Not invited]
   

  Mechanisms of anti-hantaviral activities of bovine lactoferrin (LF) and ribavirin (Rbv) were investigated. Hantavirus focus formation at 48 hr was 1546 of the control in cells treated with 400 mug/ml LF for 1 hr at 37 degreesC prior to viral infection. Post infection treatment with 100 mug/ml Rbv also inhibited the focus formation to 2.5% of the control. Combined LF pre- and Rbv post-infection treatment completely inhibited focus formation. Viral glycoprotein (G2) and nucleocapsid protein (NP) syntheses were delayed in LF pre treated cells up to 24 hr post infection (hpi) but became comparable to the control by 48 hpi. Further, LF inhibited viral shedding at 24 hpi but did not inhibit shedding after 48 hpi. However, Rbv was able to inhibit synthesis of viral proteins, (+) and (-) strand RNAs also inhibited viral shedding after 24 hr. These results suggest that LF inhibits viral adsorption to cells, while Rbv inhibits viral RNA synthesis. For in vivo trials of LF and Rbv, LF pre- and Rbv post-treatment were evaluated in suckling mice infected with hantavirus, of which 7% survived. LF concentrations of 40 and 160 mg/cg administered prior to viral challenge improved survival rates to 15% and 70%, respectively for single administration and 85% and 94%. respectively, for double administration. Rbv concentrations of 25 and 50 mg/kg gave survival rates of 68% and 81%, respectively. This suggests that both LF and Rbv are efficacious in hantavirus infection in vivo.
• Epidemiology and epizootiology of hantavirus infection in Japan

  J Arikawa, K Yoshimatsu, H Kariwa  JAPANESE JOURNAL OF INFECTIOUS DISEASES  54-  (3)  95  -102  2001/06  [Not refereed][Not invited]
   

  Hantaviruses cause two severe human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Various rodent species act as animal reservoirs for hantavirus. In Japan, urban rat- (Rattus norvegicus) and laboratory rat-derived human infections were reported during the 1960s and 1970s-1984, respectively. Although no human cases of infection have been reported since 1984, infected urban rats have been found throughout Japan, and infected grey red-backed voles (Clethrionomys rufocanus) have been identified in Hokkaido. These carriers can be considered to be potential sources of human infection. This review examines the epidemiology and epizootiology of this important zoonosis in Japan.
• Mouse Mx2 protein inhibits hantavirus but not influenza virus replication

  HK Jin, K Yoshimatsu, A Takada, M Ogino, A Asano, J Arikawa, T Watanabe  ARCHIVES OF VIROLOGY  146-  (1)  41  -49  2001  [Not refereed][Not invited]
   

  The antiviral potential of Mx2 protein remains unknown, because the Mx2 gene in commonly used strains of laboratory mice is nonfunctional. Our previous study showed that functional Mx2 protein in some feral-origin strains was induced upon interferon treatment, was localized in the cytoplasm, and inhibited vesicular stomatitis virus replication. In the present study, we have demonstrated chat the embryonic fibroblastic cells from a feral-origin strain (SPR) expressed 74 kDa Mx2 protein, which prevented the accumulation of viral transcripts and proteins of hantaviruses when the Mx2 gene was constitutively expressed in transfected Vero cells. Furthermore, the cells showed significantly lower titers of the virus than control cells. In contrast, influenza virus replication was not affected by the expression of Mx2 protein in the Vero cells.
• Genetic diversity of hantaviruses isolated in China and characterization of novel hantaviruses isolated from Niviventer confucianus and Rattus rattus

  H Wang, K Yoshimatsu, H Ebihara, M Ogino, K Araki, H Kariwa, ZX Wang, ZZ Luo, DX Li, CS Hang, J Arikawa  VIROLOGY  278-  (2)  332  -345  2000/12  [Not refereed][Not invited]
   

  The antigenic and genetic properties of 46 hantaviruses from China, 13 from patients, 23 from rodents, and 10 from unknown hosts, were compared with those of other hantaviruses. The viruses were classified as either Hantaan (HTN) or Seoul (SEO) viruses. A phylogenetic analysis of the partial M (300 bp) and S (around 485 bp) genomes of HTN viruses identified nine distinct genetic subtypes, one consisting of isolates from Korea. The SEC viruses were divided into five genetic subtypes, although they had less variability than the HTN subtypes. There was a correlation between the subtype and province of origin for four subtypes of HTN viruses, confirming geographical clustering. Hantaan virus NC167 isolated from Niviventer confucianus and SEO virus Gou3 isolated from Rattus rattus were the basal clades in each virus. The phylogenetic trees constructed from the entire S and M segments suggested that NC167 was introduced to N. confucianus in a host-switching event. The reactivity of a panel of 35 monoclonal antibodies was almost exactly the same in NC167 and a representative HTN virus and in Gou3 and a representative SEC virus. However. there was a one-way cross-neutralization between them. These results confirm the varied nature of Murinae-associated hantaviruses in China. (C) 2000 Academic Press.
• Pathogenicity of Hantaan virus in newborn mice: Genetic reassortant study demonstrating that a single amino acid change in glycoprotein G1 is related to virulence

  H Ebihara, K Yoshimatsu, M Ogino, K Araki, Y Ami, H Kariwa, Takashima, I, DX Li, J Arikawa  JOURNAL OF VIROLOGY  74-  (19)  9245  -9255  2000/10  [Not refereed][Not invited]
   

  Two Hantaan virus strains, clone 1 (cl-1), which is virulent in newborn mice, and its attenuated mutant (mul1E10), were used to examine the pathogenesis of Hantaan virus infection in a mouse model and identify virus factors relating to virulence. After subcutaneous inoculation of newborn BALB/c mice, cl-1 caused fatal disease with high viral multiplication in peripheral organs, but mul1E10 produced nonfatal infection with a low level of virus multiplication. Intracerebral inoculation of either strain caused fatal disease. Histopathological changes in the dead animals were prominent in the brain, indicating that the brain is the target organ and produces the fatal outcome. These results indicate that mul1E10 has a generally less virulent phenotype, and because of decreased multiplication in peripheral tissues, neuroinvasiveness is also decreased. An experiment with genetic reassortant viruses showed that in newborn mice the M segment is the most related to virulence and the L segment is partly related. Sequence comparison detected a single deduced amino acid change (cl-l Ile to mul1E10 Thr) at amino acid number 515 in glycoprotein G1. One nucleotide change, but no amino acid substitution, was observed in the noncoding region of the L segment. In mouse brain microvascular endothelial cells in vitro, viruses possessing a cl-1-derived M segment grew more rapidly than viruses containing a mul1E10 derived M segment. These results suggest that the single amino acid change in the glycoprotein alters peripheral growth, which affects invasion of the central nervous system in mice.
• In vitro antiviral activity of lactoferrin and ribavirin upon hantavirus

  ME Murphy, H Kariwa, T Mizutani, K Yoshimatsu, J Arikawa, Takashima, I  ARCHIVES OF VIROLOGY  145-  (8)  1571  -1582  2000  [Not refereed][Not invited]
   

  Bovine lactoferrin (LF) and ribavirin (Rbv) were tested as antiviral agents against Seoul type hantavirus (SR-11 strain) in vitro. Hantaviral foci number in Vero E6 cells infected with SR-11 was reduced with LF treatment by 5 days post infection to obtain a 50% effective dose (ED50) Of 2500 mu g/ml, while pretreatment with LF was highly efficacious having an ED50 of 39 mu g/ml. Conversely, 1 h pretreatment with Rbv revealed no inhibition of viral focus formation but could significantly reduce the number of viral foci (ED50: 10 mu g/ml) when used from the time of viral infection. One hour pre-treatment of the cell monolayer with LF and subsequent addition of Rbv revealed a synergistic anti-hantaviral effect against SR-11, <20 FFU/ml as compared to 10(5) foci/ml in the control. One hour treatment of SR-11 with LF prior to cell inoculation gave an ED50 of 312.5 mu g/ml. Whereas, washing the LF-pretreated cell monolayer with PBS demonstrated minimal focus reduction, suggesting LF lightly adheres to cells. These results indicate that LF has anti-hantaviral activity in vitro and inhibition of virus adsorption to cells which play an important role in revealing the anti-hantaviral activity of LF. This paper reports for the first time the anti-hantaviral effect of LF.
• Detection of hantaviral antibodies among patients with hepatitis of unknown etiology in Japan

  H Kariwa, K Yoshimatsu, K Araki, K Chayama, H Kumada, M Ogino, H Ebihara, ME Murphy, T Mizutani, Takashima, I, J Arikawa  MICROBIOLOGY AND IMMUNOLOGY  44-  (5)  357  -362  2000  [Not refereed][Not invited]
   

  Hantaviral antibodies were detected in the sera from patients with hepatic disease of unknown etiology in Japan by several different serological diagnostic methods. A total of 105 sera from diseased patients which were negative to A-G hepatitis virus infections in the Tokyo area were tested. Among them, 3 out of 73 sera from patients with chronic hepatic disease were positive to hantaviral antibody by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent antibody assay (IFA) and Western blot analysis (WB), Neutralizing antibody titers of the 3 sera to Seoul virus (SEO) were 4 to 8 times higher than those to Hantaan virus (HTN), However, all of the 32 sera from patients with acute hepatitis were negative for hantaviral antibody, Among the 60 patients with chronic hepatitis in Hokkaido which were serologically negative to B and C hepatitis virus infection, one was positive for hantaviral antibody by ELISA and WE. In contrast, the sera from healthy adults in Japan, 550 from the Honshu and Kyushu regions, and 1,000 from the Hokkaido region, were negative for hantavirus antibody. These results show that hantaviral antibodies are more frequently detected in patients with hepatic disease than in healthy adults. However, the observation that no positive sera were detected from patients with acute hepatitis implies that hantavirus might not be directly related to hepatitis.
• A serosurvey of Borna disease virus infection in wild rats by a capture ELISA

  K Tsujimura, T Mizutani, H Kariwa, K Yoshimatsu, M Ogino, Y Morii, H Inagaki, J Arikawa, Takashima, I  JOURNAL OF VETERINARY MEDICAL SCIENCE  61-  (2)  113  -117  1999/02  [Not refereed][Not invited]
   

  For a serological diagnostic test for Borna disease (BD), we developed a capture ELISA with specificity and sensitivity based on detection of antibodies against BD virus (BDV) p40 protein. Using our capture ELISA system, the antibody response of rats inoculated intracerebrally with BDV at 4 weeks after birth showed a sharp increase from 1 to 4 weeks postinoculation (p.i.) and a steady level after 5 weeks p.i. To investigate prevalence of BDV infection among wild rats, we examined sera of Rattus norvegicus in Kami-iso town, Oshima district, Hokkaido, suggesting that rats in this area had not been infected by BDV.
• Genetic diversities of hantaviruses among rodents in Hokkaido, Japan and Far East Russia

  H Kariwa, K Yoshimatsu, J Sawabe, E Yokota, J Arikawa, Takashima, I, H Fukushima, A Lundkvist, FN Shubin, LM Isachkova, RA Slonova, GN Leonova, N Hashimoto  VIRUS RESEARCH  59-  (2)  219  -228  1999/02  [Not refereed][Not invited]
   

  Seroepizootiologic surveys among wild rodents were carried out in Japan and Far East Russia in 1995 and 1996. Seropositive animals were only identified in Clethrionomys rufocanus (23/134) in Hokkaido, Japan. On the other hand, seropositives were identified in C. rufocanus (1/8), Apodemus argrarius (2/66), Apodemus spp. (2/26) and Microtus fortis (3/22) in Vladivostok, Far East Russia. Total RNA was isolated from lungs of seropositive animals and the S genome segments were amplified by PCR, cloned and sequenced. The S and M genomes of hantavirus, derived from Japanese C. rufocanus (Tobetsu genotype), were most closely related with Puumala viruses (76-79% nucleotide and 95% amino acid identities for S genome, 70-78% nucleotide and 87-92% amino acid identities for M genome). The recombinant nucleocapsid protein of Tobetsu genotype was antigenically quite similar with that of Sotkamo. These suggest that the virus endemic in Japanese C. rufocanus belongs to Puumala virus. Phylogenetic analysis indicates that the genotype forms a distinct lineage within Puumala viruses. Partial S segment (1-1251 nt), derived from seropositive M. fortis in Vladivostok, was sequenced and analyzed. The S genome segment, which was designated Vladivostok genotype, was most closely related with Khabarovsk virus (79% nucleotide and 90% amino acid identities) which was isolated from M. fortis. (C) 1999 Elsevier Science B.V. All rights reserved.
• N-acetylgalactosamine (GalNAc)-specific lectins mediate enhancement of Hantaan virus infection

  M Ogino, K Yoshimatsu, H Ebihara, J Arikawa  ARCHIVES OF VIROLOGY  144-  (9)  1765  -1777  1999  [Not refereed][Not invited]
   

  N-acetylgalactosamine (GalNAc)-specific lectins, Dolichos biflorus agglutinin (DBA), and soybean agglutinin (SBA), enhanced Hantaan (HTN) virus infections in Vero E6 and P388D1 cells. Treatment of Vero E6 cells with the lectins either before or during, but not after, virus inoculation resulted in lectin-mediated enhancement of infection (LME), indicating that GalNAc-specific lectin affects an early stage of the infection. Lectin blot and FAGS analysis showed that the ability of HTN virus envelope glycoproteins and cell surface molecules to bind DBA and SEA was essential for LME. GalNAc clearly inhibited LME, indicating that the lectins bind with their specific carbohydrate-binding site. These results suggest that a lectin cross-link between the virus and the cell surface is the most plausible mechanism for inducing infection enhancement.
• Evaluation of serological diagnosis of Borna disease virus infection using recombinant proteins in experimentally infected rats

  M Ogino, K Yoshimatsu, K Tsujimura, T Mizutani, J Arikawa, Takashima, I  JOURNAL OF VETERINARY MEDICAL SCIENCE  60-  (4)  531  -534  1998/04  [Not refereed][Not invited]
   

  We produced two recombinant Borna disease virus (BDV) proteins, p40 and p24, by using a baculovirus vector as a diagnostic antigen; Antigenicities of these recombinant proteins were evaluated by immune rabbit sera; Recombinant p40 was a more sensitive antigen than p24 for the detection of antibodies in infected rats. Rats inoculated with BDV within 24 hi after birth showed higher detection rates of viral RNA and viral proteins from the brain than rats inoculated at 4 weeks-old. Depending on the age of infection and the time postinfection, the detection of BDV RNA, protein, or anti-BDV antibody did not always correlate in individuals, We suggest both serological and molecular biological methods are needed in the diagnosis of BDV infection.
• Antigenic characterization of Hantaan and Seoul virus nucleocapsid proteins expressed by recombinant baculovirus. application of a truncated protein, lacking an antigenic region common to the two virus."jointly worked"

  Journal of clinical Microbiology  36(9 ),2514-2521-  1998  [Not refereed][Not invited]
  
• Adjuvant activity of muramyl dipeptide derivatives to enhance immunogenicity of a hantavirus-inactivated vaccine

  YC Yoo, K Yoshimatsu, Y Koike, R Hatsuse, K Yamanishi, O Tanishita, J Arikawa, Azuma, I  VACCINE  16-  (2-3)  216  -224  1998/01  [Not refereed][Not invited]
   

  The adjuvant effect of two lipophilic derivatives of muramyl dipeptide (MDP), B30-MDP and MDP-Lys(L18), on the ability of an inactivated vaccine of B-I virus (B-l vaccine) to induce immune response against Hantavirus causing hemorrhagic fever with renal syndrome (HFRS) was examined, When mice were immunized subcutaneously (s.c.) twice at 2-week intervals with B-I vaccine admixed with or without 100 mu g mouse(-1) of B30-MDP (B-1/B30-MDP) or MDP-Lys(L18) [B-1/MDP-Lys(L18)], mice immunized with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed Significantly higher indirect fluorescent antibody (IFA) titers against HFRS virus than mice immunized with B-I vaccine alone, Both mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) also exhibited significantly higher neutralizing antibody titers against HFRS virus than mice immunized with B-I ,vaccine alone during 3-9 weeks after-the primary immunization. The evaluation of antibody-producing cells by enzyme-linked immunospot (ELISPOT) assay on week 4 revealed that both MDP derivatives,es enhanced the number of HFRS virus-specific IgG1 and IgM antibody-producing cells. Furthermore, mice treated with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed a higher level of Th-2 type cytokines, IL-4 and IL-6, in sera than mice treated with B-l alone. In an in-vitro analysis of T lymphocyte proliferation to baculovirus-expressed recombinant nucleocapsid pi protein (rNP) of Hantaan 76-118 sa strain, the splenocytes of mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) on week 4 showed a Significantly higher proliferating activity than those treated with B-1 vaccine alone, In addition when mice were immunized once with B-l vaccine admixed with or without B30-MDP and MDP-Lys(L18) and followed by intrafootpad (i.f.) injection of B-I vaccine on day 7, mice immunized with B-1/B30-MDP and B-1/MDP-Lys(LIS) induced a higher delayed-type hypersensitivity (DTH) reaction than mice immunized with B-I vaccine alone, These results suggest that B30-MDP and MDP-Lys(L18) are useful immunoadjuvants to enhance the ability of inactivated B-l vaccine to induce a humoral and cellular response to HFRS virus, (C) 1997 Elsevier Science Ltd, All rights reserved.
• Characterization of neutralizing monoclonal antibody escape mutants of Hantaan virus 76118

  M Kikuchi, K Yoshimatsu, J Arikawa, R Yoshida, YC Yoo, Y Isegawa, K Yamanishi, S Tono-oka, Azuma, I  ARCHIVES OF VIROLOGY  143-  (1)  73  -83  1998  [Not refereed][Not invited]
   

  Neutralizing monoclonal antibody (MAb) escape mutants of Hantaan virus were generated using MAbs to envelope protein G1 (16D2) and G2 (11E10). The mutant viruses (mu16D2 and mu11E10), lacked reactivity only to the selecting MAb, or a MAb belonging to the same antigenic site. Both mutants had a single amino acid (a.a.) substitution. The a.a. substitution, found in mu16D2, was different from that found in another mutant selected with the same MAb (16D2). Although MAb 11E10 immunoprecipitated G2 protein, a deduced a.a. substitution was located in the G1 region. These results suggest that antigenic sites defined by neutralizing MAbs are composed of discontinuous epitopes over the G1 and G2 proteins. Mutant 11E10 showed a significant decrease in virulence in suckling mice. A virulence revertant of mu11E10, selected through passages in suckling mice brain, showed exactly the same deduced a.a. sequence as mu11E10 and still was not neutralized by MAb 11E10. Since mutant 16D2 was virulent for suckling mice, neutralization related epitopes found with MAbs 11E10 and 16D2 were independent of pathogenicity in BALB/c mice.
• Urine-associated horizontal transmission of Seoul virus among rats

  H Kariwa, M Fujiki, K Yoshimatsu, J Arikawa, Takashima, I, N Hashimoto  ARCHIVES OF VIROLOGY  143-  (1)  15  -24  1998  [Not refereed][Not invited]
   

  To understand the mode of transmission of Seoul type hantavirus in Wistar rats, we examined the shedding of the virus and antibody production in infected rats. When 1-day-old rats were inoculated with the KI-83-262 strain of Seoul virus, S segment of the viral genome was detected in lungs, clots, urine, saliva, submaxillary glands, rectums, and kidneys by nested reverse transcriptase PCR. On the other hand, when 8-week-old rats were infected with the virus, viral genome was detected only in the lungs and rectum. In newborn rats intranasally administered urine from infected newborn rats, four of six rats shed the virus into their urine. In addition, three of eight rats kept in the same cage with infected animals also shed the virus into urine. Moreover, the virus genome was detected in the urine of urban rats (Rattus norvegicus) in an enzootic focus. These findings suggest that the urine containing virus from infected rats is an actual source of the Seoul virus infection.
• Hantavirus infection in SCID mice

  K Yoshimatsu, J Arikawa, S Ohbora, C Itakura  JOURNAL OF VETERINARY MEDICAL SCIENCE  59-  (10)  863  -868  1997/10  [Not refereed][Not invited]
   

  Severe combined immunodeficiency (SCID) mice were inoculated with Hantaan virus strain 76-118 (HTN) or Seoul virus strain SR-11 (SR) of hantaviruses. Susceptibility of SCID mice was compared with those of immunocompetent adult mice, newborn mice and nude mice. SCID mice inoculated with HTN or SR died 32 to 35 days after infection. Unlike newborn mice which also died of hantavirus infection, SCID mice survived longer than newborn mice and showed typical wasting symptoms rather than nervous symptoms. Immunohistochemical staining and virus isolation indicated that both HTN and SR inoculated SCID and SR inoculated nude mice showed systemic infection, but nude mice inoculated with SR survived for longer than 8 weeks after inoculation. Passive transfer of spleen cells from immunocompetent BALB/c mice conferred protection on SCID mice within 2 weeks of HTN infection. Immune mediated pathologic mechanism was examined by transferring the spleen cells to SCID mice inoculated with HTN virus 3 weeks before the cell transfer. The recipient SCID mice showed an increase of serum BUN level coinciding with the appearance of serum antibody to HTN virus, suggesting the immune mediated pathogenicity.
• A case of tick-borne encephalitis in Japan and isolation of the virus

  Takashima, I, K Morita, M Chiba, D Hayasaka, T Sato, C Takezawa, A Igarashi, H Kariwa, K Yoshimatsu, J Arikawa, N Hashimoto  JOURNAL OF CLINICAL MICROBIOLOGY  35-  (8)  1943  -1947  1997/08  [Not refereed][Not invited]
   

  A case of tick-borne encephalitis (TEE) has not been reported for many years in Japan, although a serological survey of sera from domestic animals suggested the presence of TEE foci in Hokkaido, the northern island of Japan. Studies were conducted to prove the presence of an endemic focus of TEE virus in Japan by means of serology and virus isolation. In October 1993 in Hokkaido, a severe case of encephalitis in a dairy farmer's nife was diagnosed as TEE. Serological examination of paired serum specimens showed a rise in the neutralization antibody titer to Russian spring summer encephalitis virus. A seroepizootiological survey of dogs showed that the TEE-related virus was prevalent in the area. Three virus isolates were obtained from the blood of sentinel dogs, and antigenic analysis grouped the isolates into TEE-related viruses. Sequence analysis of the envelope protein gene identified one of the isolates as being of the same subtype as the Russian spring summer encephalitis (Far Eastern TEE) virus. The results provide evidence that TEE is endemic in a certain area of Japan.
• Protective effect of mucosal administration of recombinant human macrophage colony-stimulating factor (rhM-CSF) on mucosal infection of Sendai virus in mice.

  Kaori Matsuzawa, Yung Choon Yoo, Akihisa Fukushima, Kumiko Yoshimatsu, Jiro Arikawa, Ichiro Azuma.  Collected papers from the Institute of Immunological Science Hokkaido University  20-  171  -175  1997
• Protective effect of mucosal administration of recombinant human macrophage colony stimulating factor (rhM-CSF) on mucosal infection of Sendai virus in mice

  K Matsuzawa, YC Yoo, A Fukushima, K Yoshimatsu, J Arikawa, Azuma, I  VACCINE  15-  (1)  85  -89  1997/01  [Not refereed][Not invited]
   

  We investigated the protection confered by the mucosal administration of recombinant human macrophage colony-stimulating factor (rhM-CSF) against mucosal infection of Sendai virus in mice. In an experimental infection model using Sendai virus, an intranasal (i.n.) administration of rhM-CSF (20 mu g per mouse) 2 days before infection induced significant protection against a lethal infection of this virus. Also, its antiviral activity was dependent upon the dose of rhM-CSF. However; a subcutaneous (s.c.) administration of rhM-CSF with an effective close (20 mu g per mouse) i.n. did not confer protection. In a time course analysis of virus growth in the lungs, mice given rhM-CSF in. significantly inhibited the early period of infection, compared with the untreated mice. Moreover, the level of interferon-gamma (IFN-gamma) in lung wash fluids from the rhM-CSF-treated mice was higher than that of the untreated mice. These results suggested that the mucosal (i.n.), but not the systemic (s.c.) administration of rhM-CSF augments host resistance against mucosal infection with Sendai virus, and that its prophylactic activity is related to growth inhibition of the virus and enhanced IFN-gamma secretion in the lungs. Copyright (C) 1997 Elsevier Science Ltd.
• Effect of MDP-Lys(L18) as a mucosal immunoadjuvant on protection of mucosal infections by Sendai virus and rotavirus

  A Fukushima, YC Yoo, K Yoshimatsu, K Matsuzawa, M Tamura, S Tonooka, K Taniguchi, S Urasawa, J Arikawa, Azuma, I  VACCINE  14-  (6)  485  -491  1996/04  [Not refereed][Not invited]
   

  To examine the effect of MDP-Lys(L18), a derivative of muramyl dipeptide (MDP), as a mucosal immunoadjuvant, we investigated its activity to augment host resistance against mucosal infections by Sendai virus and rotavirus in mice. lit an experimental infection model that suckling mice (10-day-old) were inoculated perorally (p.o.) with 1.5 x 10(6) p.f.u. mouse(-1) of rotavirus strain SA11, intrarectal (i.r.) as well as p.o., administration of MDP-Lys(L18) (50 mu g mouse(-1)) prior to virus infection markedly reduced rotavirus-induced diarrhea. Furthermore, when MDP-Lys(L18) was administered p.o. (1 mg mouse(-1)), i.r. (300 mu g mouse(-1)) or intranasally (i.n., 100 mu g mouse(-1)) various days before Sendai virus infection (2.6 x 10(4) HAD mouse(-1)), all the mucosal administration of MDP-Lys(L18) significantly protected a lethal infection of Sendai virus, showing a dose-dependent manner. However, the efficacy of MDP-Lys(L18) to induce the prophylactic activity against the viruses somewhat varied according to the administration route and timing. In time course analysis of virus isolation in vivo, the mice administered with MDP-Lys(L18) exhibited a significant reduction of both viruses in the lungs for Sendai virus and in the bowels for rotavirus. These results suggest that MDP-Lys(L18) is a potent mucosal immunoadjuvant to enhance nonspecific host resistance against two mucosal infectious viruses, Sendai virus and rotavirus. Copyright (C) 1996 Elsevier Science Ltd.
• Characterization of the nucleocapsid protein of Hantaan virus strain 76-118 using monoclonal antibodies

  K Yoshimatsu, J Arikawa, M Tamura, R Yoshida, A Lundkvist, B Niklasson, H Kariwa, Azuma, I  JOURNAL OF GENERAL VIROLOGY  77-  695  -704  1996/04  [Not refereed][Not invited]
   

  We characterized the antigenic sites on the nucleocapsid protein (NP) of Hantaan virus (HTN) using 10 monoclonal antibodies (MAbs). At least seven antigenic sites were revealed by a competitive binding assay and divided into three partially overlapping antigenic regions (I, II and III). Regions I [amino acids (aa) 1-103], II (aa 104-204) and III (aa 205-402) were mapped on NP by examinincr the reactivity of truncated gene products. Those that corresponded to region I reacted with immune mouse serum, indicating that the region contained major linear epitopes as reported with Four corners virus (FCV) and Puumala virus (PUU) NP. At least one MAb to each region inhibited viral growth when they were introduced into cells by scrape-loading. In addition, they conferred protection from a lethal HTN challenge to newborn mice. A PEPSCAN assay localized the epitope of MAb E5/G6 between aa 166-175. Since E5/G6, which had the highest inhibitory effect both in cells and in mice, showed no virus neutralization activity by ordinary neutralization test, this region is suggested to be important for the virus growth after entry into the cells.
• Effect of MDP-Lys(L18) as a mucosal immunoadjuvant on protection of mucosal infections by Sendai virus and rotavirus.

  Akihisa Fukushima, Yung Choon Yoo, Kumiko Yoshimatsu, Kaori Matsuzawa, Mizuho Tamura, Shuichi Tono-oka, Koki Taniguchi, Shozo Urasawa, Jiro Arikawa, Ichiro Azuma.  Collected papers from the Institute of Immunological Science Hokkaido University  19-  153  -159  1996
• Characterization of the nucleocapsid protein of Hantaan virus strain 76-118 using monoclonal antibodies.

  Kumiko Yoshimatsu, Jiro Arikawa, Mizuho Tamura, Ryu Yoshida, Lundkvist, B. Niklasson, H. Kariwa, Ichiro Azuma.  Collected papers from the Institute of Immunological Science Hokkaido University  19-  143  -152  1996
• Modes of Seoul virus infections: Persistency in newborn rats and transiency in adult rats

  H Kariwa, M Kimura, S Yoshizumi, J Arikawa, K Yoshimatsu, Takashima, I, N Hashimoto  ARCHIVES OF VIROLOGY  141-  (12)  2327  -2338  1996  [Not refereed][Not invited]
   

  To understand the mode of persistent infection of Seoul virus in rodents, we examined the distribution of the virus genome and antibody production in infected rats. When 1-day-old rats were inoculated with the KI-83-262 strain, the S segment of viral genome was detected in sera, clots, lungs and kidneys from 3 to 184 days post inoculation (d.p.i.) by nested reverse transcriptase PCR. On the other hand, when 7-week-old rats were infected with this virus, viral genome was detected only in the lungs from 3 to 50 d.p.i. The neutralizing antibody titers of rats inoculated at 1-day of age were higher than those of rats inoculated at 7 weeks of age. In both a ge groups, however, the IgG avidity of antibody increased along with the course of infection. We found that urban rats (Rattus norvegicus) infected early in life harbored the virus for more than 6 months.
• Western blotting using recombinant hantaan virus nucleocapsid protein expressed in silkworm as a serological confirmation of hantavirus infection in human sera

  K Yoshimatsu, J Arikawa, H Li, H Kariwa, N Hashimoto  JOURNAL OF VETERINARY MEDICAL SCIENCE  58-  (1)  71  -74  1996/01  [Not refereed][Not invited]
   

  Recombinant Hantaan virus nucleocapsid protein expressed in silkworm larvae was applied as a serological diagnostic antigen in Western blots (WE) of human sera. The sensitivity of this method was similar to that of the IFA test. Hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica diagnosed by their cross-reactivity in WE. The specificity of this method was higher than that of IFA test because the background was low. Sera that exhibited high background staining in the IFA test were readily diagnosed with this method. We recommended WE using recombinant Hantaan virus nucleocapsid antigen as a confirmatory procedure for the serodiagnosis of hantavirus.
• Production of recombinant hantavirus nucleocapsid protein expressed in silkworm larvae and its use as a diagnostic antigen in detecting antibodies in serum from infected rats

  K Yoshimatsu, J Arikawa, R Yoshida, H Li, YC Yoo, H Kariwa  LABORATORY ANIMAL SCIENCE  45-  (6)  641  -646  1995/12  [Not refereed][Not invited]
   

  The recombinant nucleocapsid protein (rNP) of Hantaan virus was expressed by a baculovirus vector in silkworm hemolymph and was used as an antigen in western blotting (WB). The rNP is expressed in insoluble form in hemolymph; therefore simple washing of the insoluble fraction with phosphate-buffered saline by low-speed centrifugation allowed preparation of purified antigen for WB. The rNP had strain-specific and hantavirus-common epitopes similar to the authentic NP antigen of hantavirus and was stable after transfer to membrane. For detection of antibody in serially obtained sera from experimentally infected rats, WB enabled detection of IgM antibodies 3 days after infection, which was at least 2 days earlier than detection by the indirect immunofluorescent antibody test (IFA). Thus WB had a higher sensitivity than the IFA for detection of hantavirus antibody in the serum of experimentally infected rats. The WB-determined IgG antibody titer was about 10 times higher than that determined by the IFA. No background staining was observed by WB even at a 1:10 dilution of serum. The selected rat sera with strong background staining or confusing staining patterns by LFA, but not focus reduction neutralization test titers, could be interpreted as test-negative because they did not have a specific reaction to virus antigen by WB. Thus the specificity of WB was higher than that of the IFA. Moreover, WB can distinguish specific from nonspecific reactions by the detection of the specific antigen on the WB membrane. Therefore the IFA or enzyme-linked immunosorbent assay followed by WB is recommended for serologic confirmation of hantavirus infection.
• EFFECT OF MDP-LYS(L18), A DERIVATIVE OF MDP, ON ENHANCING HOST-RESISTANCE AGAINST HANTAAN VIRUS-INFECTION IN NEWBORN MICE

  YC YOO, K YOSHIMATSU, R HATSUSE, M TAMURA, R YOSHIDA, S TONOOKA, J ARIKAWA, AZUMA, I  VACCINE  13-  (14)  1300  -1305  1995/10  [Not refereed][Not invited]
   

  We examined the effect of MDP-Lys(L18), a lipophilic derivative of muramyl dipeptide, on the enhancement of host resistance against virus infection in newborn mice Newborn mice were inoculated with 4 LD(50)/mouse of Hantaan virus strain 76-118 (HTN) one day after birth. Mice given 100 mu g/mouse of MDP-Lys(L18) before infection exhibited significantly higher survival rates than those of non-treated mice. The effect of MDP-Lys(L18) was also restorative when given to the mice 4 or 7 days after infection. The titers of virus isolated from the lungs and spleens 12 days after infection, were about 30-times lower in MDP-Lys(L18)-treated (lung: 1.0 x 10(3) FFU; spleen: 6.8 x 10(1) FFU/mouse), than those of non-treated mice (lung: 3.4 x 10(4) FFU; spleen: 1.9 x 10(3) FFU/mouse). Furthermore the virus was undetectable in the brains of MDP-Lys(L18)-treated mice, whereas viruses were isolated from 3 of 6 non-treated mice MDP-Lys(L18) augmented the number of peripheral leukocytes and splenocytes, as well as mitogenic responses of the cells from bone marrow and spleen of newborn mice. These results suggest that MDP-Lys(L18) enhanced the resistance of newborn mice against HTN virus in a systemic infection model, and that this mechanism is involved in the enhancement of hematopoiesis and responsiveness of immune-related cells to mitogens.
• EVIDENCE FOR THE EXISTENCE OF PUUMULA-RELATED VIRUS AMONG CLETHRIONOMYS RUFOCANUS IN HOKKAIDO, JAPAN

  H KARIWA, S YOSHIZUMI, J ARIKAWA, K YOSHIMATSU, K TAKAHASHI, TAKASHIMA, I, N HASHIMOTO  AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE  53-  (3)  222  -227  1995/09  [Not refereed][Not invited]
   

  We conducted field surveys of indigenous rodent species in Hokkaido, Japan from 1980 to 1993. Serum samples were collected from 663 rodents, including Clethrionomys rufocanus, Apodemus speciosus, A. argenteus, and C. rutilus. Antibody to hantavirus was determined by the protein G antibody assay. Positive C. rufocanus were detected in seven of eight collection sites, but no antibody was detected in the remaining rodent species. To reveal the serotype of the circulating virus in C. rufocanus, antibody titers to Hantaan, Seoul, Puumala, and Prospect Hill viruses were compared by means of the focus reduction neutralization test. The titers in positive sera were extremely high to the Sotkamo strain of Puumala virus. Results were confirmed by the reverse transcriptase-polymerase chain reaction, and suggested that Puumala-related viruses are in circulation among C. rufocanus populations in Hokkaido.
• Effects of muramyl dipeptide derivatives as adjuvants on the induction of antibody response to recombinant hepatitis B surface antigen.

  Mizuho Tamura, Yung Choon Yoo, Kumiko Yoshimatsu, Ryu Yoshida, Tetsuya Oka, Kunio Ohkuma, Jiro arikawa, Ichiro Azuma  Collected papers from the Institute of Immunological Science Hokkaido University  18-  155  -160  1995
• Effect of MDP-Lys(L18), a derivative of MDP, on enhancing host resistance against Hantaan virus infection in newborn mice.

  Yung Choon Yoo, Kumiko Yoshimatsu, Rei Hatsuse, Mizuho Tamura, Ryu Yoshida, Shuichi Tono-oka, Jiro Arikawa, Ichiro Azuma.  Collected papers from the Institute of Immunological Science Hokkaido University  18-  203  -208  1995
• EFFECTS OF MURAMYL DIPEPTIDE DERIVATIVES AS ADJUVANTS ON THE INDUCTION OF ANTIBODY-RESPONSE TO RECOMBINANT HEPATITIS-B SURFACE-ANTIGEN

  M TAMURA, YC YOO, K YOSHIMATSU, R YOSHIDA, T OKA, K OHKUMA, J ARIKAWA, AZUMA, I  VACCINE  13-  (1)  77  -82  1995/01  [Not refereed][Not invited]
   

  The ability of two muramyl dipeptide (MDP) derivatives, B30-MDP and MDP-Lys (L18), to enhance the immunogenicity of recombinant hepatitis B surface antigen (rHBsAg) was examined. When mice were immunized intraperitoneally with rHBsAg together with each MDP derivative, the antibody titres were higher than those in mice immunized with alum-adsorbed rHBsAg, which is a commercially available hepatitis B vaccine. When mice were given a subcutaneous or intramuscular injection of rHBsAg and either MDP derivative, the antibody titres were the same as those in mice given alum-adsorbed rHBsAg. These results indicate the usefulness of MDP derivatives as immunoadjuvants for a new-generation vaccine.
• CHARACTERIZATION OF THE MODE OF HANTAAN VIRUS-INFECTION IN ADULT MICE USING A NESTED REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION - TRANSIENT VIRUS-REPLICATION IN ADULT MICE

  H KARIWA, M KAMIMURA, J ARIKAWA, K YOSHIMATSU, TAKASHIMA, I, N HASHIMOTO  MICROBIOLOGY AND IMMUNOLOGY  39-  (1)  35  -41  1995  [Not refereed][Not invited]
   

  A polymerase chain reaction (PCR) for the detection of hantavirus genome was established and applied to analyze the mode of infection of Hantaan virus in adult ICR mice. The cDNA for the S genome segment of Hantaan virus was reverse-transcribed from the total RNA of organs of the infected mice. The sequence in the S genome segment of Hantaan virus was successfully amplified by reverse transcriptase (RT)-PCR followed by nested PCR. In 5-week-old ICR mice inoculated intraperitoneally with Hantaan virus, strain 76-118 (1.3 x 10(5) FFU/mouse), the virus was detected in clots and lungs from 3 to 10 days post-inoculation (p.i.) by nested PCR and virus-isolation techniques. No virus was detected in any specimens collected on 1 day and after 28 days p.i., and in spleens and brains through the observation period by both methods, The antibody which was measured by indirect immunofluorescence antibody assay (IFA) appeared at 7 days p.i. and the geometric mean titer was elevated to its maximum level of 1 : 203 at 10 days p.i., maintaining the same level until 35 days p.i. These results suggest that adult mice are transiently infected with Hantaan virus.
• EFFECT OF THE SYNTHETIC LIPID A-RELATED COMPOUND, DT-5461, ON RESISTANCE TO SENDAI VIRUS-INFECTION IN MICE

  R YOSHIDA, K SATO, YC YOO, K YOSHIMATSU, M TAMURA, C ISHIHARA, J ARIKAWA, AZUMA, I  IMMUNOPHARMACOLOGY  28-  (2)  153  -161  1994/09  [Not refereed][Not invited]
   

  DT-5461 enhanced host resistance to Sendai virus infection in mice. Intranasal (i.n.) administration of 200 mu g of DT-5461 per mouse 3 days before infection was the most effective administration route, dose and timing. DT-5461 enhanced the cytotoxicity of murine natural killer (NK) cells. In addition, DT-5461 activated murine peritoneal macrophages, resulted in augmented of cytotoxicity and the induction of tumor necrosis factor-alpha (TNF-alpha). Therefore, these immunomodulating activities presented by DT-5461 caused protection against Sendai virus infection.
• POSTNATAL CHANGE OF PIG INTESTINAL GANGLIOSIDE BOUND BY ESCHERICHIA-COLI WITH K99 FIMBRIAE

  Y YUYAMA, K YOSHIMATSU, E ONO, M SAITO, M NAIKI  JOURNAL OF BIOCHEMISTRY  113-  (4)  488  -492  1993/04  [Not refereed][Not invited]
   

  Enterotoxigenic Escherichia coli possessing K99 fimbriae (E. coli K99) causes diarrhea in piglets of less than 1 week old. The first stage of the bacterial infection is adhesion by the fimbriae on the small intestinal mucosa and the adhesion is followed by colony formation. K99 fimbriae bind specifically to N-glycolylneuraminyl-lactosyl-ceramide, GM3(NeuGc) [Ono, E. et al. (1989) Infect. Immun. 57, 907-911]. We examined the postnatal change of the content and the molecular species of GM3(NeuGc) in the small intestinal mucosa of 0- to 14-day-old piglets and adult pigs. GM3(NeuGc) was a major ganglioside of piglet intestinal mucosa. GM3(NeuGc) content was maximal at birth and gradually decreased to 1/16 in adult animals (5 months old). The ceramide moiety of piglet intestinal GM3(NeuGc) was characterized by the presence of 2-hydroxylated palmitic acid. I-125-labeled bacteria strongly bound to GM3(NeuGc) containing 2-hydroxylated palmitic acid and phytosphingosine compared with GM3(NeuGc) containing any other ceramide moiety. The time when this particular GM3(NeuGc) appears coincides with the time that the infection occurs, and it may explain the susceptibility of newborn piglets to E. coli K99 infection.
• ANTIVIRAL ACTIVITY OF SULFATED CHITIN DERIVATIVES AGAINST FRIEND MURINE LEUKEMIA AND HERPES-SIMPLEX TYPE-1 VIRUSES

  C ISHIHARA, K YOSHIMATSU, M TSUJI, J ARIKAWA, SAIKI, I, S TOKURA, AZUMA, I  VACCINE  11-  (6)  670  -674  1993/04  [Not refereed][Not invited]
   

  Sulfated chitin derivatives, selected for their low toxicity and high inhibitory activity of melanoma metastasis, were examined for anti-viral activity against Friend murine leukaemia, herpes simplex type-1 (HSV) and Sendai viruses. Carboxymethyl chitin with a 7.66% degree of sulfation (SCM-chitin III) showed a significant inhibition of Friend murine leukaemia helper virus (F-MuLV) and HSV, but not of Sendai virus growth in vitro. Sulfated N-deacetylated chitin had a significant but weak activity against F-MuLV and HSV infections. Carboxymethyl chitin showed no effect on these infections in vitro. SCM-chitin III also exhibited anti-viral activity in vivo by suppressing the splenomegaly which was caused by prior infection of mice with FV, a complex of F-MuLV and spleen focus-forming virus.
• Application of a recombinant baculovirus expressing hantavirus nucleocapsid protein as a diagnostic autgenin IFA test : cross reactivates among 3 serotypes of hantavirus which causes hemmorrhagic fever with renal syndrome(HFRS)."jointly worked"

  Journal of Veterinary Medical Science  55(6),1047-1050-  1993  [Not refereed][Not invited]
  
• COMPARISON OF VIRULENCE BETWEEN SEOUL VIRUS-STRAIN SR-11 AND HANTAAN VIRUS-STRAIN 76-118 OF HANTAVIRUSES IN NEWBORN MICE

  YC YOO, K YOSHIMATSU, R YOSHIDA, M TAMURA, AZUMA, I, J ARIKAWA  MICROBIOLOGY AND IMMUNOLOGY  37-  (7)  557  -562  1993  [Not refereed][Not invited]
   

  Virulence of hantavirus strain of SR-11 Seoul virus and Hantaan 76-118 (HTN) of Hantaan virus were compared. Infections of both strains were lethal in newborn mice. However, inoculum required to cause lethal infection was about 4,000 times higher for strain HTN (1.65 x 10(3) PFU/mouse/LD50) than for strain SR-11 (0.36 PFU). Thus, both strains were considered pathogenic to newborn mice but they possessed different levels of virulence. The assay system used for these strains in newborn mice proved to be useful in the study of hantavirus vilurence. Growth curves of the two strains in CV-7 cell cultures were compared. Strain SR-11 was shown to have higher activity of virus replication and virus release into the culture fluids than strain HTN. The possibility of a relationship between replication activity and high levels of virulence in mice was suggested.
• PROTECTIVE IMMUNITY OF HANTAAN VIRUS NUCLEOCAPSID AND ENVELOPE PROTEIN STUDIED USING BACULOVIRUS-EXPRESSED PROTEINS

  K YOSHIMATSU, YC YOO, R YOSHIDA, C ISHIHARA, AZUMA, I, J ARIKAWA  ARCHIVES OF VIROLOGY  130-  (3-4)  365  -376  1993  [Not refereed][Not invited]
   

  Recombinant Hantaan virus nucleocapsid protein (rNP) and recombinant envelope (rEnv) proteins were prepared using a baculovirus expression system to examine the role of Hantaan virus structural proteins in protective immunity. Passive transfer of spleen cells from mice immunized with rNP conferred partial protection or prolongation of time to death from fatal Hantaan virus infection in suckling mice which were challenged with Hantaan virus at 40 LD50 (survival rate: 43%) or 4 LD50 (survival rate: 43%). The T cell-enriched fraction protected one mouse from lethal infection but the B cell-enriched fraction had no such effect on fatal HTN infection. The protective effects of the antibody against HTN challenge were examined by passive immunization. The monoclonal antibody ECO 2 directed to NP also conferred partial survival and significant difference in time to death. Although rEnv antigen failed to induce neutralizing antibody, both immune spleen cells and immune serum to rEnv conferred partial protection upon suckling mice. These results indicate that both nucleocapsid and envelope proteins of Hantaan virus were responsible for induction of cell mediated protective immunity. Vero E 6 cells infected with Hantaan virus expressed envelope protein on the surface, as determined by flow cytometry. However, there was only negligible expression of nucleocapsid protein.
• NEW METHODS FOR ISOLATION OF K99 FIMBRIAE FROM ENTEROTOXIGENIC ESCHERICHIA-COLI

  K YOSHIMATSU, Y YUYAMA, E ONO, B SYUTO, M NAIKI  JOURNAL OF VETERINARY MEDICAL SCIENCE  53-  (6)  1119  -1121  1991/12  [Not refereed][Not invited]
  

Books etc

• ハンタウイルスエンベロープ蛋白の標的細胞への吸着・侵入における挙動の解析

  森松, 組子 
  [北海道大学大学院医学研究科] 2007/05 1冊
• 組換え可溶性外被蛋白を用いたハンタウイルスワクチンおよび迅速診断キットの開発

  森松, 組子 
  [北海道大学] 2004/03 61p

Association Memberships

• 日本獣医学会   日本実験動物学会   日本ウイルス学会   

Works

• スリランカにおける原因不明慢性腎臓病(CKDu)とハンタウイルス感染症の関連に関する研究

  森松(吉松) 組子 2016 - Today
• 腎症候性出血熱とハンタウイルス肺症候群の中和代替試験法による鑑別診断法の開発

  1996 -2008
• 組換え可溶性外皮蛋白を用いたハンタウイルスワクチンおよび迅速診断キットの開発

  2001 -2003
• 自然免疫情報伝達系による生体防御と胎児発生の制御～新規NFкB抑制タンパク質ファミリー分子欠損マウスの解析を中心に

  2003
• モノクローナル抗体および細胞内抗体導入（スクレイプローディング）法を用いたハンタウイルス核蛋白の機能の解析

  1995 -1996
• 蚕バキュロウイルス発現ハンタウイルス核蛋白の精製法の確立と診断法への応用

  1993 -1993
• 免疫不全マウス(SCID、ヌード)での腎症候性出血熱ウイルスによる腎症候性出血熱障害の解析

  1992 -1992
• 実験マウスにおける組換え腎症候性出血熱ウイルス蛋白を用いた免疫応答の解析－パキュロウイルスベクター発現系の新しい応用

  1991 -1991

Research Projects

• Studies on rodent-borne zoonotic diseases in Sri Lanka

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

  Date (from‐to) : 2023/04 -2026/03 

  Author : 森松 組子, 清水 健太, 浦田 秀造
• Development of rapid and simple individeual diagnostic method for major infectious diseases in laboratory animals : a multiplex immunochromatographic test

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2020/04 -2023/03 

  Author : 土佐 紀子, 林元 展人, 石田 智子, 森松 組子

   

  学術・科学技術の研究に供する動物の微生物モニタリングは、研究結果の信頼性の担保、および動物福祉の観点から必要不可欠である。近年、多系統少数個体維持の増加や個別換気型飼育機の普及等により、従来の微生物モニタリング方法、すなわち「おとり動物」の感染症の検出を指標とする間接的な方法では感染症を摘発することが困難となっている。この問題を解決するためには、動物を安楽死させることなく微量全血を用い、マウス、ラットの主要感染症を同時に且つ迅速・簡便に検出できる個体別血清診断法の確立が必要である。その方法として有望と考えられる「多項目イムノクロマト(ICG)法」の開発について申請者らは取り組んで来た。 本研究ではこれまでの成果を基に、１）多項目ICG法の検出条件が確定したマウス・ラットの感染症（マウス：マウス肝炎ウイルス感染症、センダイウイルス感染症、ティザー病。ラット：唾液腺涙腺炎ウイルス感染症、センダイウイルス感染症、腎症候性出血熱）の検出における本法の実用性を検証し、２）上記感染症に加え、主要感染症であるマイコプラズマ感染症（マウス、ラット）とティザー病（ラット）のICG法を確立することを目的としている。２年度（令和３年度）においては、以下の研究を実施した。 １．多項目ICGスティックの最適な保管条件・最長保管期間を明らかにする実験において、保管期間６ヶ月および１２ヶ月の多項目ICGスティックの各保管条件における検出・感度特異性への影響の結果を得た。 ２．乾燥血液を用いた時のICG法の検出・感度特異性を明らかにするため、乾燥血液を作製するための条件を検討した。 ３．多項目ICGスティックの輸送条件を確定するための実験設備の準備を行った。 ４．Myco（マウス、ラット）とTyzzer（ラット）のICG法を確立するため、抗原の準備を行い、ICGメンブレンに塗布する抗原処理方法の実験に着手した。
• エボラ出血熱の発症の分子基盤に関する研究

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  Date (from‐to) : 2019/04 -2022/03 

  Author : 津田 祥美, 森松 組子

   

  本課題ではエボラウイルスの主要標的細胞とされているマクロファージや肝細胞でのウイルス増殖がエボラウイルス感染症で観察される病態にどのように関与しているのか、ウイルスの組織指向性や主要標的細胞での増殖能力と致死的病態の関連性を明らかにすることを目的とする。これまでの解析によりマクロファージや樹状細胞で主に発現しているmicroRNAのターゲット配列をウイルス遺伝子に組込むことでマクロファージや樹状細胞でのウイルス増殖を抑制した組換えエボラウイルス作出可能であることを示した。また作出したウイルスは著しく病原性が減弱したことから、感染初期におけるマクロファージや樹状細胞でのウイルス増殖の重要性を示した。本研究ではクロファージや樹状細胞で増殖したウイルスが次にターゲットとし、重要な宿主応答に関与していると考えられる肝細胞でのウイルス増殖を抑制したウイルスの作成を試みた。これまでのC型肝炎ウイルスなどの研究により、肝臓で発現するいくつかのmicroRNAの重要性が報告されている。そこで本研究ではmicroRNA122をまずターゲットとして選び、microRNA122に対応する遺伝子配列を組み込んだ組換えウイルスの作出を試みた。これまでの研究と同様にマウスを用いた病原性解析を目的として、親株であるエボラウイルスとマウス馴化株に遺伝子組換えを行ったところ、ともにウイルスの作出を確認した。今後は作出したウイルスの細胞での増殖、マウスモデルを用いた病原性の解析を行う予定である。
• 熱帯性慢性腎臓病に関連する新規ハンタウイルス、ランカウイルスの解析

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  Date (from‐to) : 2019/04 -2022/03 

  Author : 森松 組子, 清水 健太, 津田 祥美

   

  慢性腎臓病(CKD)は2002年に米国腎臓財団によって提唱された概念である。先進国では成人人口の13％を占めるとされ、そのほとんどが糖尿病や高血圧等の生活習慣病を原疾患とする。一方で、このような原疾患を持たない原因不明のCKDがスリランカ、インドの一部、中南米から報告されており、CKDuと呼ばれている。特にスリランカでは重症型のCKDuが健康な労働年齢の男性に頻発し、国家的な問題となっている。近年申請者らは、CKDu患者の約半数が抗ハンタウイルス抗体陽性であることを見いだし、ハンタウイルス感染症が慢性腎臓病の発症に関与していることを疫学的解析により示した。さらに、CKDuの流行地のげっ歯類からハンタウイルスを検出し、このウイルスが感染源となっている可能性をみいだした。本研究では、スリランカ固有の新規ハンタウイルスを解析し、この情報を基礎に血清診断法、分子生物学的診断法を開発する。さらにこれらの診断法を用いて、ヒトおよびげっ歯類におけるハンタウイルスの感染状況およびCKDu発症機序の解明を試みることを最終的な目的とする。 今年度は特に、スリランカ固有のハンタウイルスの特定と遺伝子解析を目標としていた。結果的に、存在がすでに確認されていたランカウイルスに加えてもう一つのタイランド型ハンタウイルスであるアンゾロベウイルスを見いだし、それぞれの宿主がインドハツカネズミおよびクマネズミであることを明らかにした。さらにそれぞれのウイルスの遺伝子配列を決定し、ランカウイルスは最も進化的に離れたタイランド型ハンタウイルスであること、クマネズミ由来のウイルスはマダガスカルに棲息するクマネズミ由来のアンゾロベウイルスに極めて近縁であることが明らかとなった。また、遺伝子配列を基に血清診断法および遺伝子診断法の開発を行った。
• Molecular biological analysis of hantavirus of hantavirus relating to chronic kidney disease by unknown etiology (CKDu) in Sri Lanka

  日本医療研究開発機構：U.S.-Japan Cooperative Medical Science Program Collaborative Award 2018

  Date (from‐to) : 2018/04 -2019/03 

  Author : MORIMATSU-Yoshimatsu Kumiko
• 西條 政幸

  厚生労働省：感染症実用化研究事業

  Date (from‐to) : 2016/04 -2019/03 

  Author : Masayuki Saijo
• Studies on host animals for leptospirosis and hantavirus infection in Sri Lanka

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2015/04 -2018/03 

  Author : Yoshimatsu Kumiko, Gamage Chandika D

   

  To clarify the mode and host of Leptospira and hantavirus infection, rat-trapping in Sri Lanka was carried out. Pathogenic strains of Leptospires were isolated from rats and shrews. And high seroprevalence (19%) of anti-hantavirus antibody among black rats was found in rural area, Girandrukotte. In Sri Lankan endemic strain of rat was considered to be a host for Thailand virus-related hantavirus. Around 50% of chronic kidney disease by unknown etiology (CKDu) patients were seropositive to hantavirus, while 5% for healthy people in CKDu non-endemic area. In this study, in Sri Lanka, it was shown that Leptospira and hantavirus infection occurred frequently from endemic rats as the infection source, and furthermore, hantavirus infection could be the risk of progression of CKDu.
• スリランカにおけるレプトスピラ症とハンタウイルス感 染症の媒介げっ歯類に関する研究

  文部科学省：科学研究費補助金(基盤研究(C))

  Date (from‐to) : 2015 -2018/03 

  Author : 森松(吉松) 組子
• Development of diagnostic methods for viral zoonoses derived from wild animals and birds and the comparative epidemiological study of the viral zoonones

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2013/04 -2017/03 

  Author : KARIWA Hiroaki

   

  In this study, simple diagnostic methods were developed for zoonoses, such as hantavirus infections, tick-borne encephalitis, and West Nile fever, which are severe and fatal diseases, and are considered as major problems in many countries. ELISA and immunochromatography were developed to detect anti-hantavirus antibodies in various rodents. ELISA systems using viral-like particles (SPs) of tick-borne encephalitis virus conjunctive with IgG and Strep-tag were established. A stable reverse genetics system for West Nile virus was developed by using homologous recombination.
• Studies on fever with unknown origin (FUO0 in South and South East Asia

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2011 -2013 

  Author : MORIMATSU Kumiko, ARIKAWA Jiro, SHIMIZU Kenta

   

  The investigation of hantavirus-associated infectious diseases in Southeast Asia and South Asia had been conducted. Throughout the investigation, many examples of fever of unknown origin (FUO) were found from febrile diseases. In this report, it aimed to clarify FUO in this region. Febrile patient sera (not dengue fever and a flu victim) and wild rodent sera as a career of infectious agents conducted investigation of causative agent of febrile illness in Vietnam and Sri Lanka. Finally, it was suggested that seropositive cases against pathogenic organs, such as hepatitis E virus (HEV), leptospira, and hantaviruses are found from a part of FUO patient. Furthermore, it was shown that rodents in both countries also possessed these agents. Therefore rodents were able to be sources of these zoonotic agents.
• Study for preventing epidemic of viral zoonoses in Japan and theAmerican Continents

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2009 -2012 

  Author : KARIWA Hiroaki, ARIKAWA Jiro, YOSHII Kentaro, MORIMATSU Kumiko

   

  Three novel hantaviruses were detected in Mexican rodents. The antibody detection method for various hantavirus infections was developed. Five of six monoclonal antibodies to nucleocapsid protein (NP) of Mexican hantavirus were broadly reacted with NPs of rodent-borne hantaviruses. Glycosylation of West Nile virus E protein made great influence in the pathogenesis in chicks infected with West Nile virus.
• Studies on persistent hantavirus infection in rodents; throughoutanalysis of function of immune cells

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2006 -2008 

  Author : ARIKAWA Jiro, MORIMATSU Kumiko, ENDO Rika, KARIWA Hiroaki

   

  ハンタウイルスは持続感染齧歯類が感染源となる人獣共通感染症でヒトに腎症候性出血熱やハンタウイルス肺症候群という重篤な疾病を引き起こす。齧歯類は血中に高い中和抗体を保有しつつ不顕性にウイルスを排泄し続けるというきわめて特徴的な持続感染を成立させている。本研究では齧歯類におけるウイルス特異的CTL抑制を中心とする免疫抑制を介したハンタウイルス持続感染成立機構の詳細を明らかにすることを目的とする。
• Epidemiological study on zoonotic diseases endemic in North America which are potentially introduced into Japan

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2004 -2007 

  Author : Hiroaki KARIWA, 高島 郁夫, 有川 二郎, 吉松 組子

   

  In this study, new diagnostic methods were developed and the epidemiological surveys were carried out on West Nile fever and hantavirus infections which are potentially introduced into Japan.1) Development of diagnostic methods for West Nile fever: To establish the surveillance system to West Nile virus introduction to Japan, serological diagnostic methods for anti-West Nile virus antibodies were developed. The newly developed methods were the micro-neutralization test and inhibition ELISA.2) Epidemiological study of West Nile fever in wild birds in Far East Russia: Total RNA was extracted ...
• Studies on hantavirus nucleocapsid protein and envelope glycoproteins

  Grant-in-Aid for Scientific Research

  Date (from‐to) : 2001 -2007
• Studies on the behavior of hantavirus envelope glycoprotein in adsorption and invasion to target cells.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2005 -2006 

  Author : MORIMATSU Kumiko

   

  Hantaviruses are causative agents for rodent-borne viral zoonosis, HFRS and HPS. Envelope glycoproteins G1 and G2 induce neutralizing antibodies. Monoclonal antibodies possessing neutralizing activities showed fusion inhibition activities. This means vaccine target epitope for hantavirus is a fusion responsible region in envelope protein. I studied the role of envelope glycoproteins in establishment of infection in cells. At the first, we found highly sensitive cell lines (A549, BHK and VeroE6 cells) and non-permissive cell lines (MDBK) by using pseudotype VSV screening. AlphaVbeta3 integrin was reported as candidate of the cellular receptor for hantavirus entry. Therefore, we tried to induce alphaVbeta3 into MDBK. I could not detect change of infectivity in that cells. However, transfection efficiency of plasmid constract to MDBK cells were not enough to evaluate the effect of molecules. Otherwise, anti-beta3 integrin antibodies were not inhibit cellular entry of hantavirus in permissive cells found in this study. So that this molecules was considered not to be major receptor molecules in these cells. Next, I screened bindings of glycolipids from permissive cell line Vero E6 cells against hantavirus glycoprotein. Any significant binding was not detected between glycolipids and envelope proteins. These results suggested that the cellular receptor of hantavirus must be protein molecules except alphaVbeta3 integrin. Finally, a clone 5 of MDBK cells was established as relative high DNA induction and non-permissive line. And cDNA libraly from permissive cell line A5449 was produced. This study established the basis for analysis of interaction between hantavirus envelope glycoproteins and cellular molecules.
• アジアのヒトおよび動物におけるハンタウイルスの血清疫学的および分子疫学的研究

  科学技術振興調整費による中核的研究拠点（ＣＯＥ）育成

  Date (from‐to) : 2006 

  アジアにおける疫学的解析を通して、ハンタウイルス感染の浸淫状況を明らかにし、新規ハンタウイルスを検索する。
• Serological and molecular biological stdies on hantavirus infection among human and animals in Asia

  SCF System for Establishment and Support of Center's of Excellence

  Date (from‐to) : 2006 

  Serological surveylance of human and animal sera and hantavirus genome analysis by using molecular biological methods
• Regulation of host defense and development by innate-immunity signal transduction system

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2003 -2005 

  Author : Masami MORIMATSU, 吉松 組子, 山本 欣郎

   

  In innate immune system, specific pattern molecules such as LPS bind to Toll-like receptors and activate signal transduction pathway involving NF-kappaB. Recently we identified a novel nuclear IkB molecule, MAIL. In this study we focused on MAIL and other NF-kappaB related molecules.1. Analysis of MAIL deficient miceEmbryonic lethality was observed for about 90% of MAIL deficient mice. Hypoplasia of the liver was suggested to be a possible cause for this embryonic lethality. Remaining 10% of MAIL deficient mice were born alive. These mice developed severe atopic dermatitis-like disease.2. A...
• ハンタウイルス核蛋白およびエンベロープ蛋白の構造と機能に関する研究

  文部科学省：科学研究費補助金

  Date (from‐to) : 2001/04 -2004/03 

  Author : 森松(吉松)組子

   

  組換え蛋白を用いて粒子形成、出芽、細胞への侵入の感染サイクルにおける構造蛋白の役割を解析する
• Development of hantavirus vaccine and rapid diagnosis kit by using soluble recombinant envelope glycoproteins

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2001 -2003 

  Author : MORIMATSU Kumiko, ONO Eriko, MORIMATSU Masami, KARIWA Hiroaki, ARIKAWA Jiro

   

  1. It was clarified that recombinant Hantavirus envelope glycoproteins G1 and G2 (GPs) had the cell-fusion activity by using CAG promotor-expression system. Fusion-responsible epitope on GPs was found to locate in the neutralization (FRNT)-relating epitope. Therefore, the recombinant GPs was considered to possess neutralization-and fusion-relating epitopes. In addition, hantavirus nucleocapsid (N) protein was also expressed in mammalian cells by using same vector. As the result the N protein may suppress that expression and transportation of the envelope protein. It was also shown that the virus like particle was not formed by GPs and N protein alone. 2. By using recombinant GPs, pseudotype vesicular stomatitis virus (VSV) enveloped with hantavirus GPs altered to VSV G protein was produced (VSVΔG*HTN). Similar pseudotype VSV was produced with recombinant GPs of Seoul virus (VSVΔG*SEO). These pseudotypes were applied to rapid neutralization assay as safety alternatives of authentic viruses. These pseudotype virus particles were also applied to vaccination as alternatives to authentic virion. Mice were immunized with soluble recombinant GPs or pseudotype virion. In mice immunized with soluble recombinant GPs, FRNT antibody was not detected. Contrary, in mice immunized with VSVΔG*HTN virion, FRNT antibody was detected. Challenge administration of hantavirus was carried out by s.c. inoculation of 4 FFU of hantavirus. These mice did not show the elevation of anti-N antibody and hantavirus specific CD8 T cell response, indicating that the FRNT antibody could protect mice from hantavirus infection. On the other hand, all control mice immunized with VSVΔG*G or PBS could not escape from hantavirus infection. These results indicated that packaged recombinant GPs on VSV particle were possible to induce protective FRNT antibody. This study shows that novel approach for the development of vaccination of viruses that have difficulties in preparation of authentic virus particles.
• Study for the development of new gene targeting method utilizing epitope tags

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 1999 -2001 

  Author : Masami MORIMATSU, 吉松 組子, 小川 和重, 首藤 文榮, 吉村 佳典, 三好 一郎

   

  To establish the bases for developing new methods of gene targeting, we investigated the efficiency of gene knock-in and the use of specific markers called epitope-tags. By using this approach, gene of interest is to be analyzed by antibodies against epitope-tags. We selected some genes for targeting experiments. Detailed characterization of candidates for epitope-tags was also carried out.1. Investigation of a novel endotoxin-inducible gene, MAIL, as a target.MAIL is suitable for targeting experiment because it is known that transcription of the gene is markedly up-regulated by endotoxin s...
• Studies on the development of diagnosis for infectious diseases among laboratory rodents

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 1999 -2001 

  Author : ARIKAWA Jiro, MORIMATSU Kumiko, KARIWA Hiroaki, SATO Hiroshi, TAKAKURA Akira

   

  1. Nucleocapsid (N) proteins of Hantaan, Seoul and Dobrava viruses were expressed by E. coli and baculovirus systems. The recombinant proteins by both systems produced enough amount of recombinant proteins and retained the anti genicity similar to those of authentic viruses. They are considered to be a ble to apply for ELISA antigen. 2. The truncated N proteins which lacked 50 amino acids at N-terminal reduced the cross reactivity to antibody to heterologous serotypes. Therefore, the truncated antigens were applied as serotyping antigen.The ELISA with the truncated antigen able to serotype of patient and rodent sera from China and Far Eastern part of Russia as determined by ordinary neutralization test. 3. Mouse hepatitis virus N protein gene was expressed by yeast system of which maximum expression was observed at 72 hours after culture. However, the antigenicity was too low to apply for ELISA antigen. 4. cDNA of N protein of lymphocytic choriomeningitis (LCM) virus were cloned from Japanese isolate (strain OQ28) and prototype strain (strain WE). The cDNAs of full length, C-terminal region and central region were independently transfected to COS cells. Although they could express recombinant proteins, amount of antigen expressed was too small to apply to ELISA. 5. Recombinant baculovirus expressing LCM virus N protein was provided from National Institute of Infectious Diseases Japan. Both the recombinant baculovirus infected SF-9 cells and Tm5 cells were found to be applicable for IFA antigen and ELISA antigen, respectively. A total of 9,840 mouse sera from 1,117 animal facility in Japan were tested for LCM virus antibody by IFA and ELISA. From these results, screening with ELISA followed by confirmation by IFA was recommended.
• 実験動物での賢症候性出血熱とハンタウイルス肺症候群の鑑別診断法の確立に関する研究

  日本学術振興会：科学研究費助成事業 奨励研究(A)

  Date (from‐to) : 1999 -2000 

  Author : 森松 組子

   

  本研究はハンタウイルスの侵入を阻止するために齧歯類における診断法、特にHPS感染を摘発する方法を確立することを目的とする。ハンタウイルスの抗原性は大きく3つに分けられる.ネズミ亜科(Murinae)、ハタネズミ亜科(Microtinae/Arvicolinae)、アメリカネズミ亜科(Sigmodontinae)それぞれに属する齧歯類を宿主とするウイルスである.本年度はさらに応用範囲を拡大し、さらに鑑別診断系の評価を行った。 1.平成11年度に引き続き野鼠血清の収集を行った。本年度はHPSのreservoir動物となるアメリカネズミ亜科のげっ歯類の血清および抗血清を入手することができた。さらにより多くのネズミ亜科、ハタネズミ亜科のげっ歯類血清合計31種について適切な抗体検出系を決定することができた。この結果からより広範な抗体摘発体制を取ることが可能となった。 2.平成11年度に引き続き、Hantaan,Puumala,SinNombreハンタウイルスに対する標準抗血清の作成を行った。 3.3種類のハンタウイルス、Hantaan,Puumala,SinNombreハンタウイルス核蛋白の抗原性の交差は少ないことが明らかとなった。 4.ネズミ亜科のウイルス3種類(Hantaan,Seoul,Dobrava)の核蛋白をコードするcDNAから主要共通抗原領域をコードする部分を削除し、バキュロウイルスベクターを用いて発現させることで調整した抗原を用いた鑑別診断法の感度は約95%、特異性は100%であり、十分な感度と特異性を示した。 以上の結果から、ハンタウイルスの侵入を阻止するために、広範な野鼠の抗体検出法、適切な抗原を供給することが可能となった。さらにネズミ亜科のハンタウイルスについては詳細なウイルス型別も可能な診断系を提供できることが明らかとなった。
• Molecular biologic characterization of hatavirus pathogenicity

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B).

  Date (from‐to) : 1999 -2000 

  Author : ARIKAWA Jiro, MORIMATSU Kumiko, KARIWA Hiroaki, TAKASHIMA Ikuo, MORIKAWA Shigeru

   

  1. Epidemiologic conditions and status of vaccine development in China were investigated. Information regarding to nucleotide sequence of Chinese isolates and diagnostic procedures for serodiagnosis were exchanged. 2. Technical information for genetic characterization of hantavirus was obtained from Slovak scientist. 3. Technical information for characterization of hantavirus receptor and genetic reassortant virus was obtained from US scientist. 4. Techniques for construction of artificial hantavirus was obtained from scientist of Wisconsin University School of Veterinary Medicine. 5. Information regarding to Nephropathia epidemica virus pathogenicity was obtained from Dr.Antti Vaheri of Helsinki University. 6. Information of epidemiologic and epizootiologic conditions in South East Asia was obtained from scientist in Thai. 7. Infection enhancement by the GalNac specific lectin, DBA and SBA was confirmed. Involvement of cellular factor on the virus cell membrane fusion was elucidated. 8. Pathogenicity to mice was related to growth rated at peripheral cite. The difference was considered to caused by the point mutation at enveloped protein. 9. Viral S and M genome was cloned and expressed in the mammalian cells. Several kinds of mini-genomes were constructed for the examination of role for transcription and translation. For the next step, relationship between the minigenome expression and the proteins expressed by S and M genomes will be required.
• Studies on a mechanism of hantavirus persistent infection in central nervous system and immune system

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A).

  Date (from‐to) : 1998 -2000 

  Author : ARIKAWA Jiro, MORIKAWA Shigeru, KARIWA Hiroaki, MORIMATSU Kumiko

   

  1. Hantavirus infection was enhanced about 10 times by addition of lectins (DBA and SBA) which specifically bind to N-acetylgalactosamine. This enhancement was considered to caused by the cross linking between receptor to virion by the lectins. 2. Two Vero E6 cell subclones, one induce low pH dependent cell fusion after hantavirus infection and the other resistant to cause cell fusion, were established. By using the two cell clones, the possible role for the cellular factor for responsible for induce infected cell fusion was considered. 3. After peripheral infection of hantavirus to mice, virulent virus reached to brain 4 to 5 days earlier than that of avirulent virus. This difference was considered as a mechanism which define the virulence of hantavirus in the mouse model. 4. Virulent hantavirus showed higher growth rate in the brain micro vascular cells and peritoneal macrophage, suggesting that virulence relates to the growth ability in the target organs. 5. Genetic reassortant virus between virulent and avirulent viruses were established. The comparison of the virulence of the reassortant viruses indicated that one amino acid difference at enveloped protein and nucleotide difference in the polymerase gene are related to the virulence. 6. Virulent hantavirus infected SCID mice were passively transferred with spleen cells of immunized BALB/c mice 3 weeks after infection. The recipient SCID mice represented apparent weight loss 4 days after the transfer, indicating the immune mediated pathogenicity. 7. Hantaan virus S and M genome segment which encoding nucleocapsid protein and enveloped glycoprotein, respectively were cloned and expressed in the mammalian cells. 8. Entire L genome segment which encodes polymerase was cloned. The expression of the L clone is under going.
• Comparative ecological study of hantavirus in rodents in Japan and Far East Russia

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 1998 -1999 

  Author : Hiroaki KARIWA, 吉松 組子, 水谷 哲也, 有川 二郎, 高島 郁夫

   

  Epidemiological study was conducted in Japan and Far East Russia and the followings were revealed.1. Hantavirus was identified in the urine from infected rats. Since newborn rats intranasally inoculated with the urine containing virus had the virus in their organs and urine, the virus may be maintained by the transmission with virus-contaminated urine by intranasal route in nature.2. Enzyme linked immunosorbent assay (ELISA) using baculovirus-expressed nucleocapsid proteins of different hantaviruses as antigens was established. This assay made it possible to detect hantaviral antibody and a...
• 免疫カセットセオリーに基づくハンタウイルス核蛋白高度保存領域での感染防御免疫誘導

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 1997 -1998 

  Author : 森松 組子

   

  ハンタウイルスは腎症候性出血熱の原因ウイルスで、ブニヤウイルス科に属するRNAウイルスである。この研究ではハンタウイルス内部蛋白の特定のエピトープを標的抗原とする新しいタイプのペプチドワクチンの開発を目的として、マウス実験感染モデルを用いて単クローン抗体E5/G6エピトープペプチドの誘導する免疫応答とその感染防御活性について検討している。平成10年度は、カセット配列(主要組織適合抗原結合領域)を付加した,E5/G6エピトープのペプチド抗原による免疫応答の解析を明らかにするために、ペプチドを合成しマウスに免疫し免疫応答を測定することを試みた。1. ペプチドデザインPeptide 1 YEGF-EDVNGIRKP-KAKGITPeptide 2 YEGFS-EDVNGIR---KAKGITをMAP及び通常のペプチドとして合成した。これはH-2bのカセット配列に、E5G6エピトープ配列EDVNGIRKPKを挿入したものである。2. 免疫MAPのペプチド抗原を100ug/mouse complete adjuvantとともにC57BL6マウスに免疫し、経時的に採血し、抗体価の上昇を調べた。3. T細胞応答の測定系の確立牌臓細胞をX線照射によって不活化し、抗原提示細胞として用いることを試みた。昨年度の研究結果から非RI測定系を確立するため代替法として、市販のテトラゾリウム塩類、酵素を比...
• Studies on the control of hantavirus infection and rodent

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(国際学術研究)

  Date (from‐to) : 1996 -1997 

  Author : Jiro ARIKAWA, 宋 干, 李 徳新, Antti Vaheri, Bo Niklasson, 網 康至, 伊勢川 裕二, 五十嵐 章, Clarence Pet, 杉山 和良, 高島 郁夫, 森松 組子, 王 華, Clarence Peters

   

  Japanese investigator, Dr. Sugiyama carried out a epizootiologicstudy of wild rodents at Beijing and Xian city. A total of 100 rodents were captured from the two regions and were examined for their antibody and antigen distribution.Chinese investigator, Dr. Li, stayed at Japanese research institution for examining genetic comparison among Chinese hantaviruses obtained from above materials. He examined partial nucleotide sequence of two Chinese isolates and showed the higher relationship to Korean isolates than to Japanese one.Finish investigator, Dr. Vaheri, visited at Japanese investigator...
• Establishment of serodiagnostic method and surveylance system for hemorrhagic fever with renal syndrome virus infection in experimental animals

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

  Date (from‐to) : 1994 -1996 

  Author : ARIKAWA Jiro, TAKAKURA Akira, SUGIYAMA Kazuyoshi, YOSHIMATSU Kumiko, KARIWA Hiroaki

   

  1. Recombinant baculovirus expressing hantavirus nucleocapsid protein (Bac.HTN-NP) was applied for the antigen of ELISA.Specificity of the ELISA was the same to that of the IFA which uses infected Vero E6 cells as antigen. However, the ELISA had a sensitivity problem because of the relatively high background coloring. 2. cDNA which encoded hantavirus nucleocapsid protein was expressed by using E.coli system and established capture ELISA system. This ELISA was shown to have same specificity and sensitivity to that of the ordinally IFA,and therefore, considered as a useful screening method. 3. Bac.HTN-NP was successfully applied for the antigen for Western blotting (WB). The WB method readily distinguished specific reaction from nonspecific reaction from the WB pattern. Therefore, the WB was considered as an effective method for the serologic confirmation of hantavirus infection.
• 細胞内抗体導入(スクレイプロ-ディング)法でのハンタウイルス核蛋白の機能の解析

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 1995 -1995 

  Author : 吉松 組子

   

  ハンタウイルスは腎症候性出血熱の原因ウイルスで、ブニヤウイルス科に属するRNAウイルスである。この研究ではハンタウイルス核蛋白の誘導する液性抗体の感染防御について明らかにすることを目標として感染防御に有効なエピトープ領域の決定を行なった。1次構造上の抗原領域の決定; 大腸菌を使ってハンタウイルスHantaan 76-118株の核蛋白の様々な部分を組換え蛋白として発現させ、単クローン抗体との反応性を調べ、単クローン抗体の認識部位を明らかにした。リニアエピトープを認識する単クローン抗体E5/G6についてはcDNAから予想されるアミノ酸配列を元にペプチドを合成し反応性を確認し、エピトープの特定を行なったところ,アミノ酸166-175の配列,EDVNGIRKPKであることが明らかとなった。またこの実験に付随して,ハンタウイルスの核蛋白のアミノ酸1から103がimunodominant領域であることが分かった.すなわち,人及び動物がハンタウイルスに感染したとき,核蛋白に対する抗体が強く誘導されるが,そのほとんどのリニアエピトープはこの領域に集まっていることが明らかとなり,診断抗原として有用な領域であることが分かった。また、立体構造依存性のエピトープについては,バキュロウイルスでの同様の実験から,この1-103の領域と200-300の領域にもにも存在することが示唆された。スクレープロ-デ...
• Studies on development of HFRS vaccine and therapeutics

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(国際学術研究)

  Date (from‐to) : 1993 -1995 

  Author : Jiro ARIKAWA, Bo Niklasson, 宋 干, 杉山 和良, 橋本 信夫, Niklasson Bo, 周 桂珍, 森松 組子, Ake Lundkvis, Bo Niklasson

   

  1.In 1993 and 1995, head investigator (Dr. Arikawa), and Japanese investigators (Drs. Hashimoto, Morimatsu, Kariwa) visited Swedish Institute for Infectious Disease control, and Helsinki University Institute for Virology and made information exchange and discussions along with this research project.2.In 1993, Chinese investigators (Dr. Song and Hun), in 1995, Swedish investigators (Drs. Niklasson and Lundkvist) visited institutions of Japanese investigator such as Hokkaido University, National Institute Health of Japan and Osaka University to do information exchange and discussions along wi...
• 組換えハンタウイルス核蛋白を用いたRNPトランスフェクション系の確立

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 1994 -1994 

  Author : 吉松 組子

   

  これまでに、私は蚕のバキュロウイルス系を用いて、ハンタウイルスの核蛋白を大量に発現するシステムを開発してきた.今回の研究では、この組換え蛋白を用いて、RNPトランスフェクション系を確立する事を目的とした.ハンタウイルス核蛋白を蚕のヘモリンフ中に発現させた場合、不溶性となる.この不溶性蛋白は、尿素及びグアニジン塩酸塩で可溶化され、特に尿素存在下で陰イオン交換クロマトグラフィーを行った場合、可溶性の蛋白として精製が可能である.しかしながら、この蛋白は消化を受け、フラグメント化されている場合が多い.これをさらに精製し、全長の核蛋白のみを精製するため、核蛋白に対するモノクローナル抗体を使ったAffinity精製を試みた.結果は収率は著しく低いものの、全長を含む核蛋白も精製が可能であった.しかしながら、これらの核蛋白に対するモノクローナル抗体を用いて精製した場合、それぞれの抗体の認識部位によって精製されるフラグメントが異なる.このため、これらの抗体の一次構造上の認識部位を決定しておく必要が生じた.また、Derai et alは大腸菌による発現組換え蛋白がRNA結合能を保持していたことを報告している.このため、大腸菌の発現系を利用し、抗原部位の決定を試みた.結論は、比較的効率よく全長の組換え蛋白を精製できるモノクローナル抗体は、立体構造認識の一部の単クローン抗体と、N末端を認識する抗体で...
• 蚕バキュロウイルス発現組換えハンタウイルス核蛋白の精製法の確立と診断法への応用

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 1993 -1993 

  Author : 森松 組子, 吉松 組子

   

  1.感染蚕ヘモリンフ中の不溶性画分を遠心洗浄することによって、組換え蛋白を部分精製、濃縮を行った。この不溶性画分は、弱アルカリ性(pH8.0)、還元条件下で、4M Guanidin Hydrochrolide, 6M Urea,あるいは1%SDS等によって可溶化された。ここから可溶化のための物質を取り除くと再び不溶性の沈殿となった。尿素存在下の陰イオン交換クロマトグラフィーを行なったところ、0.15-0.2MのNaClで核蛋白は溶出され、この画分は50mM Tris-HCl pH8.0、または水に透析後も再沈殿することはなかった。しかしながらこの時、リン酸を含む緩衝液に透析すると沈殿を生じることが分かったので、以下Tris系の緩衝液を用いることとした。この可溶化核蛋白を含む画分は室温で取り扱うと断片化が生じ、これはセリンプロテアーゼ阻害剤であるPMSFを加えて4℃で取り扱うことによって妨げることが明かとなった。2.可溶化され、濃縮された組換え核蛋白は単クローン抗体を結合させたアフィニティーカラムを用いてさらに精製することが可能であった。この抗原をHigh-density Particleに結合させ凝集抗原の作製を試みた。しかしながら抗原を感作させるだけでParticleは凝集した。塩析では10%飽和硫安で沈殿し、しばしば不溶化した。これらの結果は、アミノ酸配列からも予想されて...
• Genetical and biochemical characterizations of hemorrhagic fever with renal syndrome virus infection in mice.

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(試験研究(B))

  Date (from‐to) : 1991 -1993 

  Author : Jiro ARIKAWA, 伊勢川 裕二, 石原 智明, 吉松 組子, 板倉 智敏, 橋本 信夫

   

  1. SCID mice inoculated i.p. with Hantaan virus and Seoul virus developed systemic infection and died after 32 to 38 days post infection.2. Viral antigen was detected from various organs including kidney. Although no hemorrhagic manifestations was seen in kidney, the SCID mice showed increased level of BUN after infection.3. RT-PCR followed by Nested-PCR method was established for detecting Hantaan virus genome in various organs in infected mice.4. SCID mice (21 days post infection) were passively administered with spleen cells which were prepared from normal BALB/c mice. Seven days after t...
• STUDIES ON THE DEVELOPMENT OF HEMORRHAGIC FEVER WITH RENAL SYNDROME VACCINE USING RECOMBINANT BACULOVIRUS EXPRESSED PROTEIN.

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(一般研究(C))

  Date (from‐to) : 1990 -1992 

  Author : Jiro ARIKAWA, 吉松 組子, 高島 郁夫, 橋本 信夫

   

  1) All the neutralizing monoclonal antibodies to Hantaan virus envelope proteins examined so far caused antibody-dependent enhancement of infection to macrophage cell lines with subneutralizing amount of the antibodies. Thus, it seemed to be difficult to separate neutralization specific antigens for preparation of vaccine.2) Recombinant Hantaan virus envelope proteins were successfully expressed by the baculovirus expression system to prepare the viral antigens without any biological hazard from the Hantaan virus infection. The antigenic properties of the expressed and authentic viral antig...
• 腎症候性出血熱ウイルス(ハンタウイルス)の病原性に関する研究

  科学研究費補助金
• Study on Virulency and pathogenicity of hantavirus, which causes hemorrhagic fever with renal syndrom

  Grant-in-Aid for Scientific Research

Industrial Property Rights

• 特許第3809502号:ハンタウィルス抗原蛋白質およびモノクローナル抗体  2006/06/02

  吉松 組子, 有川 二郎, 橋本 信夫, 三谷 勝男  有川 二郎, 橋本 信夫, 株式会社エイアンドティー  200903028180319252