title: A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit.
abstract: Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (PRRSV) carrying an enhanced green fluorescent protein (EGFP) reporter gene as a separate transcription unit. A copy of the transcription regulatory sequence for ORF6 (TRS6) was inserted between the N protein and 3'-UTR to drive the transcription of the EGFP gene and yield a general purpose expression vector. Successful recovery of PRRSV was obtained using an RNA polymerase II promoter to drive transcription of the full-length virus genome, which was assembled in a bacterial artificial chromosome (BAC). The recombinant virus showed growth replication characteristics similar to those of the wild-type virus in the infected cells. In addition, the recombinant virus stably expressed EGFP for at least 10 passages. EGFP expression was detected at approximately 10 h post infection by live-cell imaging to follow the virus spread in real time and the infection of neighbouring cells occurred predominantly through cell-to-cell-contact. Finally, the recombinant virus generated was found to be an excellent tool for neutralising antibodies and antiviral compound screening. The newly established reverse genetics system for PRRSV could be a useful tool not only to monitor virus spread and screen for neutralising antibodies and antiviral compounds, but also for fundamental research on the biology of the virus.
authors.forename: Chengbao
authors.initials: C
authors.lastname: Wang
authors.forename: Baicheng
authors.initials: B
authors.lastname: Huang
authors.forename: Ning
authors.initials: N
authors.lastname: Kong
authors.forename: Qiongyi
authors.initials: Q
authors.lastname: Li
authors.forename: Yuping
authors.initials: Y
authors.lastname: Ma
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Li
authors.forename: Jiming
authors.initials: J
authors.lastname: Gao
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.forename: Xiangpeng
authors.initials: X
authors.lastname: Wang
authors.forename: Chao
authors.initials: C
authors.lastname: Liang
authors.forename: Lu
authors.initials: L
authors.lastname: Dang
authors.forename: Shuqi
authors.initials: S
authors.lastname: Xiao
authors.forename: Yang
authors.initials: Y
authors.lastname: Mu
authors.forename: Qin
authors.initials: Q
authors.lastname: Zhao
authors.forename: Yani
authors.initials: Y
authors.lastname: Sun
authors.forename: Fernando
authors.initials: F
authors.lastname: Almazan
authors.forename: Luis
authors.initials: L
authors.lastname: Enjuanes
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, No, 22 Xinong Road, Yangling, Shaanxi 712100, China. zhouem@nwsuaf.edu.cn.
authors.forename: En-Min
authors.initials: EM
authors.lastname: Zhou
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000378:metabolism*; D000914:Antibodies, Viral / Q000378:metabolism*; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D000882:Haplorhini; D008969:Molecular Sequence Data; D019316:Porcine respiratory and reproductive syndrome virus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000662:veterinary; D014162:Transfection / Q000662:veterinary; D014779:Virus Replication
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000998:Antiviral Agents; D005819:Genetic Markers; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins