Establishment of a DNA-launched infectious clone for a highly pneumovirulent strain of type 2 porcine reproductive and respiratory syndrome virus: Identification and in vitro and in vivo characterization of a large spontaneous deletion in the nsp2 region

A highly pneumovirulent strain of porcine reproductive and respiratory syndrome virus (PRRSV), ATCC VR2385, was isolated from a pig exhibiting typical PRRS in the early 90s. While passaging the virus in monkey kidney cells, we identified a large spontaneous deletion of a 435-bp in the nsp2 gene. To assess the biological significance of this spontaneous deletion, we first determined the full-length genomic sequence of this virus and established a DNA-launched infectious clone of the passage 14 virus containing the 435-bp nsp2 deletion (designated as pIR-VR2385-CA). The full-length viral genome engineered with two ribozyme elements at both ends was placed under the control of the eukaryotic CMV promoter. The infectious virus was successfully rescued from pIR-VR2385-CA DNA-transfected BHK-21 cells. To characterize the biological and pathological significance of this large nsp2 deletion, we subsequently constructed another DNA-launched infectious clone, pIR-VR2385-R, in which we restored the deleted 435-bp nsp2 sequence back to the pIR-VR2385-CA backbone. The growth characteristics of the two rescued viruses (VR2385-CA and VR2385-R) were compared, and the results showed that the VR2385-CA virus with the nsp2 deletion replicated more efficiently in vitro (1.0-1.5 log titer higher) than the VR2385-R virus with the restored nsp2 sequence but the VR2385-CA virus exhibited a significantly reduced serum viral RNA load in vivo. A comparative pathogenicity study in pigs (n = 10) revealed that the nsp2 deletion had no effect on virus virulence, and the restored nsp2 sequence in the VR2385-R virus remains stable during virus replication in pigs. The results from this study indicates that the spontaneous nsp2 deletion plays a role for enhanced PRRSV replication in vitro but has no effect on the pathogenicity of the virus.

► In this study, we identified a 435-bp spontaneous deletion in the nsp2 gene of PRRSV. ► The 435-bp nsp2 deletion enhanced PRRSV replication in vitro. ► The nsp2 deletion significantly reduced serum viral RNA loads in infected pigs. ► The nsp2 deletion has no effect on virus pathogenicity in vivo.