Construction And Rescue Of A Full-length Infectious CDNA Clone Of Highly Pathogenic Porcine Reproduction And Respiratory Syndrome Virus

PRRS(Porcine reproduction and respiratory syndrome,PRRS)broke out in the United States in the late 1980s.In 1991,The first time PRRSV(Porcine reproduction and respiratory syndrome virus,PRRSV)was isolated in the Netherlands and named PRRSV Lelystad strain(LV).there are still no effective measures to prevent and cure the disease,The global pig industry has caused huge economic losses.In prior studies,a strain GXNN1396 of HP-PRRSV((Highly pathogenic porcine reproductive and respiratory syndrome virus,HP-PRRSV)was isolated and identified from a pig lung infected with PRRSV.The strain was subcultured in MARC-145 cells to obtain a cell adapted strain of the GXNN1396-P96.This study is intended to build a full-length infectious cDNA clone of GXNN1396-P96.Based on the existing PRRSV infective clone pAJX25HB,the segment of GXNN1396-P96 was replaced with the gene fragment of pAJX25HB.Finally,a full-length cDNA clone of the GXNN1396-P96 strain was finished and named pGXAM.To distinguish the constructed clone from the parental virus,Mlu I was inserted at the pGXAM site by mutational PCR.pGXAM was transfected into BHK-21 cells and passaged on MARC-145 cells 48 hours later.Obviously cytopathic changes were observed after 3 days.The rescued virus was subjected by indirect immunofluorescence assay and the results showed that there was a specific PRRSV N protein expression in the virus infected cells.RT-PCR amplification and sequencing analysis also showed that the passaged rescue virus contained the "genetic marker" site Mlu I.These results indicate that we successfully rescue the full-length infectious cDNA clone of PRRSV and named rGXAM.The multistep growth curve results showed that the rescue virus rGXAM and the parent virus GXNN1396-P96 have similar growth curves,titer of virus,and size of plaques,indicating that the rescued virus has similar biological characteristics as the parent virus.The construction of infectious clones that cause attenuated strains lays the foundation for studying the pathogenesis of HP-PRRSV and developing new genetic engineering vaccines.