Establishment of mouse model for bovine coronavirus infection to accelerate development of live vaccines

• Project/Area Number: 20K15661
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 42020:Veterinary medical science-related
• Research Institution: Gifu University
• Principal Investigator: Nakagawa Keisuke 岐阜大学, 応用生物科学部, 准教授 (90853250)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2022: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2021: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2020: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 牛コロナウイルス / マウスモデル / 生ワクチン開発 / 下痢症

Outline of Research at the Start

現行の不活化ワクチン接種より優れた牛コロナウイルスの予防法として、有効性の高い弱毒生ワクチン接種が考えられる。しかしながら、牛を用いた感染実験の汎用性の低さ、ならびに経済的・技術的障礙から、その開発は進んでいない。本研究では、コロナウイルスの中でも宿主域が比較的広い牛コロナウイルスに着目し、汎用性の高いコンベンショナルマウスを用いて牛コロナウイルス病態再現マウスモデルの確立を試みる。また、確立されたマウスモデルを利用して、培養細胞にて長期継代した牛コロナウイルス生ワクチン候補株の有用性を評価する。

Outline of Final Research Achievements

Bovine coronavirus (BCoV) causes respiratory and diarrheal symptoms in cattle. Since there is no small animal models that reproduces the pathogenesis, it is difficult to understand the mechanism of the pathogenicity and establish candidates of attenuated live vaccine. In this study, we aimed to 1) establish a mouse model of the BCoV infection and 2) develop and evaluate attenuated vaccine candidate strain. By using the isolate of BCoV strain, we orally administered BCoV to two-day-old suckling mice. As a result, we found that two out of 10 mice showed diarrhea. We then attempted to detect infectious virus and viral genes from the diarrhea of the infected suckling mice. However, no infectious virus or viral genes were detected from the diarrhea. Furthermore, we could not find neutralizing antibodies in the suckling mice. We obtained a 20th passage of BCoV in HRT-18 cells, and are currently determining the growth property and complete genome sequence of the passage strain.

Academic Significance and Societal Importance of the Research Achievements

牛コロナウイルス（BCoV）は牛に呼吸器症と下痢症を引き起こす。牛以外に本感染症の病態を再現する動物モデルが存在しないため、病態解析や弱毒生ワクチン株の樹立が進んでいない。また、マウスのゲノム編集技術を用いて病態再現マウスを樹立するのではなく、日本で販売されているコンベンショナルマウスを用いてBCoV病態再現マウスを確立できたのなら、汎用性と経済性の面で大きな利点が期待される。残念ながら本研究では、BCoV病態再現マウスの樹立は叶わなかったものの、培養細胞にて長期継代したBCoVを得ることができた。今後、継代株の増殖性を解析することで、将来的なBCoVワクチン開発に貢献できる可能性がある。

Research Products

• [Journal Article] A comprehensive list of the Bunyavirales replication promoters reveals a unique promoter structure in Nairoviridae differing from other virus families (2022)
  • Author(s): Yutaro Neriya, Shohei Kojima, Arata Sakiyama, Mai Kishimoto, Takao Iketani, Tadashi Watanabe, Yuichi Abe, Hiroshi Shimoda, Keisuke Nakagawa, Takaaki Koma, Yusuke Matsumoto
  • Journal Title: Scientific Reports Volume: 12 Issue: 1 Pages: 13560-13560
  • DOI: 10.1038/s41598-022-17758-z
  • Peer Reviewed / Open Access
• [Journal Article] Molecular survey of infectious bronchitis virus on poultry farms in Gifu Prefecture, Japan from 2021 to 2022 by RT-PCR with an enhanced level of detection sensitivity for the S1 gene (2022)
  • Author(s): SAITO Harunobu、NAKAGAWA Keisuke、KITAMURA Yuko、KUWATA Keisuke、TANAKA Eiji
  • Journal Title: Journal of Veterinary Medical Science Volume: 84 Issue: 9 Pages: 1157-1163
  • DOI: 10.1292/jvms.22-0206
  • ISSN: 0916-7250, 1347-7439
  • Peer Reviewed / Open Access
• [Journal Article] Complete Genome Sequence of Bovine Coronavirus in Blood Diarrhea from Adult Cattle That Died from Winter Dysentery in Japan (2021)
  • Author(s): Nakagawa Keisuke、Kumano Hitomi、Kitamura Yuko、Kuwata Keisuke、Tanaka Eiji、Fukushi Hideto
  • Journal Title: Microbiology Resource Announcements Volume: 10 Issue: 42
  • DOI: 10.1128/mra.00807-21
  • Peer Reviewed / Open Access
• [Presentation] 冬季に死亡した牛の気道および腸管から検出された牛コロナウイルスのS遺伝子解析 (2020)
  • Author(s): 中川敬介
  • Organizer: 第１６３回日本獣医学会学術集会