甘肃NADC30-like PRRSV的分离及其基因组特性分析

Isolation and genetic characterization of NADC30-like PRRSV in Gansu

为了解甘肃省部分PRRSV流行株的分子生物学特征和遗传演化规律,对来自甘肃武威一猪场的疑似PRRS感染病料通过细胞培养进行PRRSV分离。该病料细胞培养物可以引起Marc-145细胞发生明显的细胞病变,PRRSV特异性引物RT-PCR扩增结果显示该病料组织为PRRSV阳性,IFA实验显示接种分离病毒株的Marc-145细胞可以与PRRSV N蛋白抗体反应,出现特异性荧光,将其命名为GSWW2018。设计引物对该毒株全基因组进行分段扩增,克隆测序后拼接得到全长序列。采用DNAstar软件分析其与48个参考毒株核苷酸及氨基酸的同源性,使用MEGA7.0软件绘制系统遗传进化树,并利用Simplot软件分析其重组事件。结果显示GSWW2018全长14 997 bp,其Nsp2蛋白存在与NADC30相似的"111+1+19"的131个氨基酸的不连续的缺失。Nsp2基因和ORF5基因系统进化树分析结果表明其属于类NADC30亚群。重组结果表明GSWW2018可能由HP-PRRSV与NADC30重组而来。本研究表明甘肃省已出现类NADC30毒株,需要进一步加强监测。

In order to understand the molecular characteristics and genetic evolution rules of some PRRSV strains in Gansu Province,the suspected PRRS infected materials from a pig farm in Wuwei,Gansu Province,were isolated by cell culture.The cell culture of this disease material can cause significant cytopathic effect on Marc-145 cells.The RT-PCR amplification results of PRRSV-specific primers indicated that the tissue of this disease material was PRRSV positive.The immunofluorescence assay showed that Marc-145 cells infected with isolated virus could react with PRRSV N protein antibody and generated specific fluorescence,so the isolated strain was named as GSWW2018.Primers were designed to amplify the whole genome of this strain.DNAstar software was used to analyze the homology of nucleotides and amino acids of the isolated strain and the 48 reference strains,MEGA7.0 software was used to draw the phylogenetic tree,and Simplot software was used to analyze the recombination events.The results showed that the total length of GSWW2018 genome was 14 997 bp,and the Nsp2 protein had a discontinuous deletion of 131 amino acids of "111+1+19"similar to NADC30.The phylogenetic tree analysis of Nsp2 and ORF5 genes indicated that GSWW2018 strain belonged to the NADC30 subgroup.The results showed that GSWW2018 could be recombined product of HP-PRRSV and NADC30.This study indicated that the NADC30-like strain has appeared in Gansu Province,which needs to be further monitored.