| Porcine reproductive and respiratory syndrome virus(PRRS)can be divided into type 1 and type 2,and both of them cause tremendous economic loss to the global swine industry.Although PRRSV2 isolates were still predominant in China,more and more PRRSV1 have been detected in different areas and caused reproductive failure in swine herbs.However,the permissive vaccines in China are all derived from PRRSV2 strains,which fail to provide enough heterologous protection.Thus,it is necessary to breed an effective vaccine against the PRRS disease caused by PRRSV1.Nevertheless,most PRRSV1 isolates failed to adapt to Marc-145 but can only breed in porcine alveolar macrophages(PAMs)in vitro.How to make the PRRSV1 isolates adapted to cell lines,such as Marc-145,can be a vital problem for the development of the PRRSV1 vaccine.Previous studies suggested that minor envelop proteins,coded by ORF2-4,decide the cell tropism of PRRSV.Moreover,when the 88/94/95 amino of GP2a were "F/I/L",the replication level of PRRSV1 isolates could be significantly raised.In this study,an infectious cDNA clone of a wild PRRSV1 isolate(rHLJB1)was generated and rescued from PAMs,which was insensitive to Marc-145.To obtain the Marc-145 adapted PRRSV1 virus,the“88/94/95"amino of GP2a were modified as "F/I/L" in rHLJB1.However,these mutations could not lead to rHLJB1 adapted Marc-145;Nest,the ORF2-4 genes from "Amervac",a Marc-145 adapted PRRSV1,were constructed into rHLJB1 and the rescued virus acquired the ability to infect Marc-145.To further investigate the key site of ORF2-4,the ORF2,ORF3,and ORF4 of the rHLJB1 were substituted by the sequence from "Amervac" respectively and the "F/I/L" model was also retained in these chimeric viruses.Finally,we found that on the premise of the "F/I/L/"model in GP2a,the Marc-145 tropism of rHLJB1 was decided by the overlapping sequence of ORF3 and ORF4.To sum up,this study successfully rescued the virus(rHLJB 1)from a cDNA clone of a Chinese avirulent PRRSV1 isolate.Based on this platform,the key sites for PRRSV1 adapting Marc145 were identified by testing the cell tropism of a series of chimeric viruses,which is beneficial to the prevention and control of PRRSV1 in China.This study provides a crucial platform to study the fundamental biology of the PRRSV1 virus and breed potential new PRRSV1 engineering vaccine with higher safety and efficiency. |
