[en] 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.
Disciplines :
Veterinary medicine & animal health
Author, co-author :
Qin, Songkang ✱; 列日大学 - 大学 > TERRA 研究中心 ; 列日大学 - 大学 > TERRA 研究中心 ; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Li, Kuan-Hao ✱; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Liu, Ben-Jin ✱; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Cao, Cun; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Yu, De-Bin; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Jiang, Zhi-Gang; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Wang, Jun; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Han, Yu-Xin; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Wang, Fang; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Qi, Ying-Lin; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Sun, Chao; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Yu, Li; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China
Chang, Ji-Tao ; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China, Institute of Western Agriculture, The Chinese Academy of Agricultural Sciences, Changji, 831100, China. Electronic address: changjitao@caas.cn
Yin, Xin ; State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150000, China. Electronic address: yinxin@caas.cn
The authors would like to thank the members from Yin Laboratory for their critical suggestions. This study was supported by the Heilongjiang Provincial Natural Science Foundation of China (grant no. LH2033C107), the National Key Research and Development Program of China (2023YFD1801302) and the Central Public-interest Scientific Institution Basal Research Fund (grant no. 1610302022010).
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