Yang Fan, Tan Jinlong, Fang Yongxiang, Chen Guohua, Zhang Yongzhi, Hu Qianqian, Han Wuweiyi, Liu Yongsheng, Fu Baoquan, Jing Zhizhong, Li Weike
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Public Health of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
College of Animal Science, Anhui Science and Technology University, Fengyang, China.
Front Microbiol. 2022 Apr 4;13:846426. doi: 10.3389/fmicb.2022.846426. eCollection 2022.
Vesicular stomatitis virus (VSV) has a wide range of cell tropism, making it a prototype of studying the negative-strand RNA virus (NSRV), including virus-host interactions and vaccine development. Although VSV rescue systems have been progressively optimized throughout time, the T7-based expression system is the most commonly utilized to rescue VSV. However, it remains a significant barrier for many labs. In our study, we found that rescue VSV's efficiency is associated with the various multiplicities of infection (MOIs) of recombinant vaccinia virus expressing the T7 RNA polymerase (vTF-7.3). It works at maximum efficiency while the MOI of vTF-7.3 is 5, which is analyzed by quantitative PCR, Western blot, and flow cytometry, compared to the lowest rescue level with MOI of 1. Meanwhile, our data also suggest that purification of vTF-7.3 prior to transfection is a prerequisite for VSV rescue. Overall, our study reveals for the first time a precise correlation between vTF-7.3 and rescue efficiency, which may aid in resolving the uncertainties in the quest to build the VSV reverse genetic system.
水泡性口炎病毒（VSV）具有广泛的细胞嗜性，使其成为研究负链RNA病毒（NSRV）的原型，包括病毒与宿主的相互作用以及疫苗开发。尽管VSV拯救系统一直以来都在不断优化，但基于T7的表达系统是最常用于拯救VSV的系统。然而，这对许多实验室来说仍然是一个重大障碍。在我们的研究中，我们发现拯救VSV的效率与表达T7 RNA聚合酶的重组痘苗病毒（vTF-7.3）的不同感染复数（MOI）相关。通过定量PCR、蛋白质免疫印迹和流式细胞术分析，当vTF-7.3的MOI为5时，其工作效率最高，而MOI为1时拯救水平最低。同时，我们的数据还表明，转染前对vTF-7.3进行纯化是VSV拯救的先决条件。总体而言，我们的研究首次揭示了vTF-7.3与拯救效率之间的精确关联，这可能有助于解决构建VSV反向遗传系统过程中的不确定性。
