猪流行性腹泻病毒河南株ORF3基因分子流行病学分析

Molecular epidemiological analysis of ORF3 genes of PEDV endemic strains in Henan province

为了解猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)河南株的起源和遗传变异,本试验对2014年8月—2015年5月,来自河南省16个规模猪场仔猪表现为严重腹泻、食欲下降和脱水等临床特征的22份病料样品,利用RT-PCR方法,扩增PEDVORF3基因,回收PCR产物克隆至pMD18-T载体,并进行测序分析。结果显示,22份病料样品均能扩增出PEDVORF3基因,其序列长度均为849bp,包含一个完整阅读框(675bp),编码224个氨基酸残基。22个河南株之间核苷酸同源性为95.9%~100%,与欧洲经典毒株CV777同源性介于97.1%~97.9%,存在8个位点发生相同的氨基酸置换,与Truncated CV777株同源性介于94.8%~95.4%。基因遗传进化树分析表明,PEDV毒株可分为2大群,河南株属于第Ⅱ群,整体独立成支,与河南往年流行株、国内分离株、日本、美国及韩国毒株亲缘关系较近,而与CV777株、Truncated CV777株亲缘关系较远。结果表明PEDV河南株ORF3基因存在变异,为PEDVORF3基因的蛋白功能研究和河南省PED的防控提供技术支持。

To understand the genetic origin and variation of porcine epidemic diarrhea virus （PEDV） prevailing in Henan province,a total of 22 PEDV positive samples from suckling piglets with severe diarrhea were collected from 16 pig farms and amplified by RT-PCR. ORF3 genes were then cloned into pMD18-T vector and sequenced. The result of sequencing showed that the length of ORF3 genes all were 675 bp which encoded 224 amino acids. The strains in this study had eight amino acid substitutions when compared with CV777 strain. The nucleotide homologies of the 22 strains were 95. 9%-100% and amino acid homologies were 96.4%-100%. By comparing with those of European strain CV777 and vaccine strain truncated CV777, the nucleotide homologies were 97.1%-97.9% and 94.8%-95.4%, respectively. Based on the phylogenetic relationship of ORF3 genes, the PEDV field strains and PEDV reference strains could be divided into two groups,and all the field strains identified in this study belong to group 2. It suggested that preva- lent PEDV strains in this study seem to be closely related to Henan isolates in previous years,do- mestic strains,Janpanese strains,American strains as well as Korean strains but differ genetically from European strains or vaccine strains used in China. This experiment analyzed the ORF3 gene sequence features in prevailing of Henan region in recent years, which provides a new support of epidemiology study and protein function research of PEDV ORF3 gene.