Recombinant Pseudorabies Virus (PRV) Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds

文献类型： 外文期刊

第一作者： Tang, Yan-Dong

作者： Tang, Yan-Dong;Liu, Ji-Ting;Fang, Qiong-Qiong;Wang, Tong-Yun;Sun, Ming-Xia;An, Tong-Qing;Tian, Zhi-Jun;Cai, Xue-Hui;Liu, Ji-Ting

作者机构：

关键词： pseudorabies virus;firefly luciferase;CRISPR/Cas9;chloroquine;Cyclosporine A

期刊名称：VIRUSES-BASEL （ 影响因子：5.048； 五年影响因子：5.127 ）

ISSN： 1999-4915

年卷期： 2016 年 8 卷 4 期

页码：

收录情况： SCI

摘要： A Pseudorabies virus (PRV) variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. However, identification of single guide RNA (sgRNA) through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP) as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures.

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