Li Yanhua, Ren Cicheng, Li Chenxi, Xiao Yihong, Zhou Yanyang
College of Veterinary Medicine, Yangzhou University, Yangzhou, China.
Comparative Medicine Research Institute, Yangzhou University, Yangzhou, China.
Front Microbiol. 2022 May 13;13:907281. doi: 10.3389/fmicb.2022.907281. eCollection 2022.
The reverse genetics system is a valuable tool in the virological study of RNA viruses. With the availability of reverse genetics, the porcine reproductive and respiratory syndrome virus (PRRSV) has been utilized as a viral vector for the expression of foreign genes of interest. Here, we constructed a full-length cDNA clone of a highly pathogenic PRRSV (HP-PRRSV) TA-12 strain. Using this cDNA clone, we generated a reporter virus expressing a gaussia luciferase (Gluc) an additional subgenomic RNA between ORF7 and 3'UTR. This reporter virus exhibited similar growth kinetics to the wild-type (WT) virus and remained genetically stable for at least ten passages in MARC-145 cells. In cells infected with this reporter virus, the correlation between the expression levels of Gluc in culture media and the virus titers suggested that Gluc is a good indicator of the reporter virus infection. With this reporter virus, we further established the Gluc readout-based assays for antiviral drug screening and serum neutralizing antibody detection that exhibited comparable performance to the classical assays. Taken together, we established a reverse genetics system of HP-PRRSV and generated a novel reporter virus that could serve as a valuable tool for antiviral drug screening and serum neutralizing antibody detection.
反向遗传学系统是RNA病毒病毒学研究中的一种宝贵工具。随着反向遗传学的出现，猪繁殖与呼吸综合征病毒（PRRSV）已被用作表达感兴趣的外源基因的病毒载体。在此，我们构建了高致病性PRRSV（HP-PRRSV）TA-12株的全长cDNA克隆。利用该cDNA克隆，我们产生了一种表达高斯荧光素酶（Gluc）的报告病毒，在ORF7和3'UTR之间还有一个额外的亚基因组RNA。该报告病毒表现出与野生型（WT）病毒相似的生长动力学，并且在MARC-145细胞中至少传代十次后仍保持遗传稳定性。在感染该报告病毒的细胞中，培养基中Gluc的表达水平与病毒滴度之间的相关性表明，Gluc是报告病毒感染的良好指标。利用该报告病毒，我们进一步建立了基于Gluc读数的抗病毒药物筛选和血清中和抗体检测方法，其性能与经典方法相当。综上所述，我们建立了HP-PRRSV的反向遗传学系统，并产生了一种新型报告病毒，可作为抗病毒药物筛选和血清中和抗体检测的宝贵工具。
