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

. doi: 10.1016/j.virs.2024.09.010

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

高效稳定的牛轮状病毒反向遗传系统的建立及其在抗病毒药物筛选中的应用

秦松康 ^a,b ,
李宽昊 ^a ,
刘本金 ^a ,
曹存 ^a ,
于德斌 ^a ,
姜志刚 ^a ,
王俊 ^a ,
韩宇鑫 ^a ,
王芳 ^a ,
齐瑛琳 ^a ,
孙超 ^a ,
Li Yu ^a ,
常继涛 ^a,c,, ,
尹鑫 ^a,,

cstr: 32224.14.j.virs.2024.09.010

a. 中国农业科学院哈尔滨兽医研究所动物疾病控制与预防国家重点实验室, 哈尔滨 150000;
b. 比利时列日大学分子与细胞表观遗传学实验室, 比利时列日大学应用基因组学跨学科研究所;比利时列日大学分子生物学教研中心, 比利时列日市;
c. 中国农业科学院西部农业研究中心, 昌吉 831100

通讯作者： 常继涛, changjitao@caas.cn; 尹鑫, yinxin@caas.cn
收稿日期： 2023-05-17
录用日期： 2024-09-26

摘要

过去，由于缺乏反向遗传（RG）系统，人们对轮状病毒复制和致病机制的理解受阻。自2017年以来，基于质粒的猿猴、人类和小鼠轮状病毒的反向遗传系统已经建立，但尚无方法被报道用于拯救牛轮状病毒（BRVs）。基于此，我们通过筛选稳定表达T7 RNA聚合酶的BHK-T7单克隆细胞，建立了BRV细胞培养适应株（BRV G10P [15] BLR）和临床分离株（BRV G6P[1] C73，G10P[11] HM26）的反向遗传系统。此外，通过该反向遗传系统，我们成功拯救了非结构蛋白3（NSP3）与ZsGreen融合的报告病毒BRV rC73/Zs，rHM26/Zs和rBLR/Zs。对基因组插入稳定性的分析表明，rC73/Zs和rBLR/Zs在7轮连续传代中均能复制并且遗传稳定，而rHM26/Zs只能稳定到第三代，表明BRV的基因片段可能影响外源基因的稳定传代。此外，我们利用重组报告病毒对1440种化合物进行高通量筛选，从中筛选到12种抗轮状病毒候选药物。总之，我们优化后的牛轮状病毒反向遗传系统既为深入了解BRV分子生物学提供了重要工具，也为今后开发针对牛轮状病毒的新型治疗药物和疫苗提供了手段。
- 牛轮状病毒
- , 优化的反向遗传系统
- , 报告病毒
- , 高通量筛选
- , 小分子化合物

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,,

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

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

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