Efficient and robust reverse genetics system for bovine rotavirus generation and its application for antiviral screening

Song-Kang Qin; Kuan-Hao Li; Ben-Jin Liu; Cun Cao; De-Bin Yu; Zhi-Gang Jiang; Jun Wang; Yu-Xin Han; Fang Wang; Ying-Lin Qi; Chao Sun; Li Yu; Ji-Tao Chang; Xin Yin

doi:10.1016/j.virs.2024.09.010

December 2024

Citation: Song-Kang Qin, Kuan-Hao Li, Ben-Jin Liu, Cun Cao, De-Bin Yu, Zhi-Gang Jiang, Jun Wang, Yu-Xin Han, Fang Wang, Ying-Lin Qi, Chao Sun, Li Yu, Ji-Tao Chang, Xin Yin. Efficient and robust reverse genetics system for bovine rotavirus generation and its application for antiviral screening .VIROLOGICA SINICA, 2024, 39(6) : 917-928. http://dx.doi.org/10.1016/j.virs.2024.09.010

Efficient and robust reverse genetics system for bovine rotavirus generation and its application for antiviral screening

Song-Kang Qin ^a,b ,
Kuan-Hao Li ^a ,
Ben-Jin Liu ^a ,
Cun Cao ^a ,
De-Bin Yu ^a ,
Zhi-Gang Jiang ^a ,
Jun Wang ^a ,
Yu-Xin Han ^a ,
Fang Wang ^a ,
Ying-Lin Qi ^a ,
Chao Sun ^a ,
Li Yu ^a ,
Ji-Tao Chang ^{a,c
,,} ,
Xin Yin ^{a
,,}

cstr: 32224.14.j.virs.2024.09.010

a. State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China;
b. Laboratory of Molecular and Cellular Epigenetics, Grappe Interdisciplinaire de Génoprotéomique Appliquée, University of Liège, 4000 Liège, Belgium; Molecular Biology, Teaching and Research Center, 5030 Gembloux, Belgium;
c. Institute of Western Agriculture, The Chinese Academy of Agricultural Sciences, Changji, 831100, China

Corresponding author: Ji-Tao Chang, changjitao@caas.cn
Xin Yin, yinxin@caas.cn
Received Date: 17 May 2023
Accepted Date: 26 September 2024
Available online: 29 September 2024

Abstract

Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics (RG) system in the past. Since 2017, multiple plasmid-based RG systems for simian, human, and murine-like rotaviruses have been established. However, none of the described methods have supported the recovery of bovine rotaviruses (BRVs). Here, we established an optimized plasmid-based RG system for BRV culture-adapted strain (BRV G10P [15] BLR) and clinical isolates (BRV G6P [1] C73, G10P [11] HM26) based on a BHK-T7 cell clone stably expressing T7 polymerase. Furthermore, using this optimized RG system, we successfully rescued the reporter virus BRV rC73/Zs, rHM26/Zs and rBLR/Zs, harboring a genetically modified 1.8-kb segment 7 encoding full-length nonstructural protein 3 (NSP3) fused to ZsGreen, a 232-amino acid green fluorescent protein. Analysis of the stability of genomic insertions showed that the rC73/Zs and rBLR/Zs replicated efficiently and were genetically stable in seven rounds of serial passaging, while rHM26/Zs can be stabilized only up to the third generation, indicating that the BRV segment composition may influence the viral fitness. In addition, we adopted the recombinant reporter viruses for high-throughput screening application and discovered 12 candidates out of 1440 compounds with potential antiviral activities against rotavirus. In summary, this improved RG system of BRVs represents an important tool with great potential for understanding the molecular biology of BRV and facilitates the development of novel therapeutics and vaccines for BRV.
- Bovine rotavirus (BRV)
- , Optimized reverse genetics system
- , Reporter virus
- , High-throughput screen
- , Small chemical compound

Electronic Supplementary Material

10.1016j.virs.2024.09.010-ESM3.docx
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