Construction And Pathogenicity Evaluation Of HP-PRRSV HuN4Infectious Clone And Chimeric Viruses

The porcine reproductive and respiratory syndrome (PRRS) is one of the fatal infectious diseases infected by porcine reproductive and respiratory syndrome virus (PRRSV), which leads to the reproductive failure in sows, dysfunction in sperm among boars, respiratory disease in piglets. Although the relative research poses great insight in this area, the virulence genes, so far, still remains to be unclear and require the further investigation.In this study, based on the developed cDNA infectious clone(attenuated for171generations of CH-la)originated from the strain of CH-1R in our laboratory, we successfully constructed the infectious clone of HuN4named rHuN4, and identified the rescued virus and parental virus with a gene marker introduced by the site directed mutation. It has revealed that rHuN4has similar characterizations with HuN4via cell pathogenic effect(CPE)observation, RT-PCR, indirect immunofluorescence assay, growth kinetics methods and electron microscopy. It also suggested that there were no significant difference between rHuN4and HuN4through the vivo experimentation by detecting the clinical and pathological diagnosis, virus loads in tissue and serum samples and investigation of thymus atrophy. All this results show the infectious virus clone rHuN4of HP-PRRSV HuN4was recovered successfully.The chimeric viruses were developed in exchange of5’-UTR/ORF1and ORF2-7/3’-UTR between the rHuN4and rCH-1R, nominated as Cnsp-recHuN4and Hnsp-recCH-1R, respectively. In vitro experiments indicate that the structural proteins affect the type of CPE of the chimeric viruses. Thus, in comparison to Cnsp-recHuN4, Hnsp-recCH-1R remains continuingly to be1074TCID50/mL after84h infection and reflects a higher virulence during the late infection period. As well, comparing with rHuN4, it has revealed in vivo that Cnsp-recHuN4and Hnsp-recCH-1R held significant difference(p<0.05) or highly significant difference (p<0.01) in clinical and pathological diagnosis, virus loads in tissue and serum samples, and the investigation of thymus atrophy, nor does rCH-1R. Therefore,5’UTR/ORF1and ORF2-7/3’UTR serve to attenuate the virulence of virus stains. It implicate that the virulence of PRRSV is not full influenced by the non-structure protein or the structure proteins.