Recombinant Pseudorabies Virus (PRV) Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds.
Recombinant Pseudorabies Virus (PRV) Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds.
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DOI：
10.3390/v8040090
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发表时间：
2016-03-29
期刊：
影响因子：
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通讯作者：
Cai XH
中科院分区：
文献类型：
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作者：
Tang YD;Liu JT;Fang QQ;Wang TY;Sun MX;An TQ;Tian ZJ;Cai XH
自2011年以来，中国出现了一种伪狂犬病病毒（PRV）变体，该变体不受商业疫苗的保护，并且尚未得到充分研究。PRV基因组很大，难以操作，但使用成簇的、规律间隔的短回文重复序列（CRISPR）/Cas9技术是可行的。然而，通过筛选鉴定单向导RNA（sgRNA）对于CRISPR/Cas9系统至关重要，并且传统上是时间和劳动密集型的，并且不适合快速和高通量筛选有效的PRV sgRNA。在这项研究中，我们开发了一种表达萤火虫荧光素酶和增强型绿色荧光蛋白（EGFP）的重组PRV毒株，作为PRV特异性sgRNA筛选和快速评估抗病毒化合物的报告病毒。荧光素酶活性是明显的，只要4小时后感染，并通过10代稳定表达。在原理筛选的证明中，我们能够鉴定几种PRV特异性sgRNA，并证实它们使用传统方法抑制PRV复制。使用报告病毒，我们还鉴定了缺乏US 3、US 2和US 9基因功能的PRV变体，并显示出对氯喹的抗PRV活性。我们的研究结果表明，报告PRV株将是一个有用的工具，为基础病毒学研究，并制定PRV的控制和预防措施。
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.