从PRRSV BJ-4株基因组全长cDNA获得感染性病毒

对已构建的覆盖猪繁殖与呼吸综合征病毒(PRRSV)BJ-4全长cDNA的6个重组质粒进行测序,并对部分点突变进行定点回复突变,将突变片段顺次连接,获得了全长cDNA克隆pWSK-DCBA。通过体外转录获得病毒基因组RNA,将RNA与脂质体混合后直接转染MARC-145细胞,获得拯救病毒(rV68)。rV68能在MARC-145细胞上稳定传代,并可引起PRRSV特征性的细胞病变(CPE)。增殖动态分析表明,rV68在MARC-145细胞上的生长有所迟滞,达到最高滴度的培养时间比亲本病毒延迟12h,但滴毒无显著差异(P>0.05)。结果表明,构建的BJ-4全长cDNApWSK-DCBA具有感染性,为研究中国PRRSV的分子致病与免疫机制、新型疫苗等奠定了基础。

Recovery of an infectious virus from the full-length cDNA of PRRSV BJ-4

Six recombinant plasmids covering cDNA of porcine reproductive and respiratory syndrome virus BJ-4 were sequenced,respectively,and 23 point mutations were reverted with site-directed mutagenesis kit.The full-length cDNA clone pWSK-DCBA was assembled and re-sequenced.The capped viral genomic RNA was transcribed in vitro,mixed with liposome and transfected into MARC-145 cells,and an infectious virus(designated rV68) was rescued.The rescued virus was able to induce CPE typical of PRRSV on MARC-145 and stably propagated in vitro.Growth kinetics curve of the rV68 exhibited a delayed replication in MARC-145 cell,namely its peak titer time was 12h later than that of parental virus.However,there was no significant difference between the peak titers of the rescued and parental virus(P>0.05).These results suggest that the full-length cDNA clone pWSK-DCBA of PRRSV BJ-4 is infectious, which provide a basis for further study on molecular pathogenicity and immunity,as well as developing novel vaccine of PRRSV.