title: Recombinant Pseudorabies Virus (PRV) Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds.
abstract: 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.
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001, China. tangyandong2008@163.com.
authors.forename: Yan-Dong
authors.initials: YD
authors.lastname: Tang
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001, China. liujiting1989@163.com.|College of Animal Science and Technology, Jilin Agriculture University, Changchun 130018, China. liujiting1989@163.com.
authors.forename: Ji-Ting
authors.initials: JT
authors.lastname: Liu
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001, China. fang19922020@163.com.
authors.forename: Qiong-Qiong
authors.initials: QQ
authors.lastname: Fang
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001, China. sdndwty@163.com.
authors.forename: Tong-Yun
authors.initials: TY
authors.lastname: Wang
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001, China. qzsmx122@163.com.
authors.forename: Ming-Xia
authors.initials: MX
authors.lastname: Sun
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001, China. antongqing@hvri.ac.cn.
authors.forename: Tong-Qing
authors.initials: TQ
authors.lastname: An
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001, China. tzj@hvri.ac.cn.
authors.forename: Zhi-Jun
authors.initials: ZJ
authors.lastname: Tian
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001, China. cai139@hvri.ac.cn.
authors.forename: Xue-Hui
authors.initials: XH
authors.lastname: Cai
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D064113:CRISPR-Cas Systems; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015870:Gene Expression; D023061:Gene Order; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D011558:Herpesvirus 1, Suid / Q000187:drug effects* / Q000235:genetics; D049409:Luciferases, Firefly / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D000094704:RNA, Guide, CRISPR-Cas Systems; D049409:Luciferases, Firefly
keywords: CRISPR/Cas9; Cyclosporine A; chloroquine; firefly luciferase; pseudorabies virus