鼠肝炎病毒(mouse hepatitis virus，MHV)是研究冠状病毒的模式病毒，但传统的病原学研究方法局限于自然界分离的病毒，不利于研究的开展。反向遗传操作系统可快速获得研究所需的重组病毒，为研究MHV及其他冠状病毒的基因组功能开辟了新途径。本研究通过反转录聚合酶链式反应(reverse transcription-polymerase chain reaction，RT-PCR)将MHV的原始毒株A59全长基因组分成6个片段进行扩增，分别克隆到质粒载体，经酶切、体外连接和转录获得全长基因组RNA，然后电转细胞并进行病毒扩增。结果显示，重组病毒rA59与原始毒株的复制特性相似，其为研究MHV的生物学特性提供了实验工具。为方便检测病毒的复制水平，在病毒基因组开放阅读框4(open reading frame 4，ORF4)内分别插入绿色荧光蛋白ZsGreen(Zoanthus sp. green fluorescent protein)和荧光素酶Nluc(NanoLuc luciferase)报告基因，获得rA59-ZsGreen和rA59-Nluc重组报告病毒。结果显示，报告病毒所携带的报告基因并不影响病毒复制，可很好地反映病毒的复制特性。使用这两种病毒验证瑞德西韦(remdesivir)的抗病毒活性，分别检测药物处理后病毒的荧光亮度和荧光素酶活性。结果表明，rA59-ZsGreen和rA59-Nluc重组报告病毒适用于高通量药物筛选，具有重要的应用前景。综上所述，本研究成功建立了MHV A59毒株六质粒反向遗传操作系统，为研究MHV的生物学特性和测试抗病毒药物等提供了有力的工具。

Mouse hepatitis virus (MHV) is a model coronavirus, but the traditional etiological methods are limited to the viruses isolated from nature. The reverse genetic system can rapidly obtain the recombinant virus for research, creating a new method to study the genomic functions of MHV and other coronaviruses. In this study, whole genome of the original MHV A59 strain was divided into six fragments by reverse transcription-polymerase chain reaction (RT-PCR). All fragments were individually cloned into plasmid vectors, then the full-length genomic RNA was obtained by restriction enzyme digestion, in vitro ligation and in vitro transcription. After RNA was electroporated into cells, the recombinant virus was rescued. The results revealed that the replication characteristics of recombinant virus rA59 is similar to the original strain, providing an experimental tool for studying the biological characteristics of the virus. To detect the virus replication level, the recombinant reporter viruses rA59-ZsGreen and rA59-Nluc were obtained by inserting the green fluorescent protein ZsGreen (Zoanthus sp. green fluorescent protein) and luciferase Nluc (NanoLuc luciferase) into the open reading frame 4 (ORF4) genome, respectively. The results indicate that the reporter genes do not compromise viral replication and the reporter viruses can well reflect the viral replication property of their parental recombinant virus. These two viruses were used to verify the antiviral activity of remdesivir, by detecting the fluorescence intensity or luciferase activity after treatment. The results demonstrate that the recombinant reporter viruses rA59-ZsGreen and rA59-Nluc are suitable for high-throughput drug screening and have promising potential for application. In summary, the successful establishment of the 6-plasmid-based reverse genetic system for MHV strain A59 provides a powerful tool for studying the biological characteristics of MHV and testing antiviral drugs.