Reverse genetics approach for arteriviruses using circular polymerase extension reaction.

Abstract

The circular polymerase extension reaction (CPER) has emerged as a high-fidelity, versatile and rapid tool for generating infectious cDNA clones of positive-sense, single-stranded RNA viruses. Here, we implemented this strategy to generate infectious cDNA clones of two closely related members of the family Arteriviridae: equine arteritis virus KY84 strain (EAV KY84) and porcine reproductive and respiratory syndrome virus VR2332 strain (PRRSV VR2332). Overlapping cDNA fragments spanning the entire viral genomes were generated and assembled with a linker sequence incorporating critical expression elements, yielding a circularized, full-length viral cDNA genome ready for direct transfection into permissive cells. Following transfection, infectious rEAV KY84 and rPRRSV VR2332 strains were recovered and characterized. Similarly, we generated reporter viruses by inserting the mCherry gene downstream of the nsp1/nsp2 cleavage junction in EAV KY84 ORF1a and the GFP gene downstream of ORF1b of PRRSV VR2332. All CPER-generated viruses showed high identity to their parental strains by next-generation sequencing. Growth kinetics of CPER-generated non-reporter and rEAV KY84-RFP mCherry viruses were comparable to their parental strains; however, the reporter rPRRSV VR2332-GFP showed a significant reduction in viral titres despite its stability for at least 15 passages. In contrast, mCherry expression from rEAV KY84-mCherry was negligible at passage 15 and associated with the deletion of the reporter gene. Taken together, the CPER-based method can be used as a rapid tool to generate infectious cDNA clones of arteriviruses, allowing the easy generation of mutants or reporter viruses that can facilitate the investigation of viral gene functions and their interactions with the host.