Preparation And Characterization Of North American Type â…¡ PRRSV Infectious Clone Expressing Green Fluorescent Protein

[Objective] Porcine reproductive and respiratory syndrome(PRRS), caused by PRRS virus(PRRSV), is one of the most serious infectious diseases in swine industry leading to huge economic loss worldwide. To investigate the interaction between virus and its host and to explore viral pathogenic mechanism at the molecular level, the objective of this study is to construct North American type II porcine reproductive and respiratory syndrome virus infectious clone containing the green fluorescent protein(GFP) gene and analyze its infectivity and replication capacity. [Method] Based on the genomic sequence of North American type II PRRSV(Gen Bank: AY545985.1) in p FL12 plasmid and gfp gene sequence in pc DNA-EF1-GFP plasmid, the gfp gene fused with PRRSV nonstructural protein 2(Nsp2) partial sequence at 5′and 3′terminus was amplified by overlap PCR from the two plasmids. The amplified gfp gene fused with Nsp2 fragment was inserted into Nsp2 gene of PRRSV infectious clone in p FL12 plasmid at Spe I and Xho I sites to generate recombinant plasmid p FL12-GFP containing gfp gene. After linearization with Acl I enzyme, the linearized p FL12-GFP was purified by protease K/SDS digestion, phenol/chloroform extraction and ethanol precipitation. The 5′-end capped GFP-PRRSV m RNA was transcribed by T7 RNA polymerase in vitro with m MESSAGE m MACHINE transcription kid using linearized plasmid as a template. The poly(A) tail was added at the 3′-end of the capped GFP-PRRSV m RNA by Yeast poly(A) polymerase to generate 5′-end capped and 3′-end tailed GFP-PRRSV m RNA. By using Transmessenger Transfection reagents, the GFP-PRRSV m RNA was transfected into baby hamster kidney-21( BHK-21) cells for GFP-PRRSV packaging and the rescued recombinant GFP-PRRSV virus was amplified in Marc-145 cells and identified by RT-PCR to amplify the specific fragment containing gfp and Nsp2 partial sequence and by fluorescence imaging at 48 h postinfection. The parent PRRSV and generated GFP-PRRSV were separately infected to Marc-145 cells and porcine alveolar macrophages(PAMs) to analyze their infectivity and replication capacity. [Results] By adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in PRRSV infectious clone plasmid p FL12 to construct PRRSV infectious clone plasmid containing gfp gene, p FL12-GFP. PRRSV m RNA containing gfp gene was synthesized in vitro by transcription with p FL12-GFP as the template, and then transfected into BHK-21 cells for virus packing and infected Marc-145 cells with the packed virus harvested from BHK-21 cells to obtain the recombinant GFP-PRRSV. Like its parental virus, the recombinant GFP-PRRSV maintains its infectivity and replication in Marc-145 cells and PAMs although its replication activity was not so strong as its parent. The replication speed of both PRRSV in Marc-145 cells were quicker than that in PAMs. [Conclusion] By using overlap PCR and ligation, the North American type II PRRSV infectious clone containing gfp gene inserted into and fused with Nsp2 gene was constructed. The rescued GFP-PRRSV virus, like its parental PRRSV, still maintains its infectivity and replication capability. This study provides favorable conditions for further investigation on PRRSV.