猪流行性腹泻病毒jiaozuo1012/2019株的分离鉴定及遗传进化分析

Isolation and characterization of a porcine epidemic diarrhea virus strain Jiaozuo1012/2019

为确定2019年河南焦作某猪场哺乳仔猪发生腹泻的病因,本研究采用RT-PCR方法对该发病猪场送检的5份仔猪肠内容物样品进行仔猪腹泻相关病毒检测,对检测的猪流行性腹泻病毒(PEDV)阳性样品进行病毒分离,对分离的PEDV S基因和ORF3基因进行PCR扩增,并分析这两个基因与GenBank中的PEDV参考株相应基因的同源性及遗传进化分析。结果显示,送检的5份猪肠道内容物样品PEDV检测结果均为阳性,而猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PoRV)的检测结果均为阴性;将随机选取的1份PEDV阳性样品接种Vero细胞并经传代培养,对培养5代的病毒经PCR检测表明分离到一株PEDV,并将其命名为jiaozuo1012/2019株;基于S基因同源性分析结果显示,该分离株与河南分离的PEDV CH/HNLH/2015株同源性最高,为99.3%,与近期河南分离株的同源性为96.4%~99.0%,与经典PEDV CV777株的同源性较低为93.4%。分离株S基因编码的氨基酸在aa59~aa62位存在4个氨基酸(59QGVN62)的插入,在aa140和aa163~aa164分别存在1个和2个氨基酸的缺失(140N和163NI164);ORF3基因的同源性分析结果显示,分离的PEDV与江苏分离的PEDV JSCZ1601株的同源性最高,为99.7%,与近年来河南分离株的同源性为98.5%~99.6%,与经典株CV777的同源性较低为96.6%;S基因与ORF3基因的进化树结果显示,该分离株与目前国内流行株遗传关系最近。上述结果表明,本研究分离的PEDV具有近年来新流行PEDV株的序列特征,与经典PEDV株具有相对较远的遗传进化关系。本研究为进一步探究PEDV在河南地区的变异规律提供了参考依据。

In order to determine the causative agent of diarrhea of suckling piglets in a pig farm in Jiaozuo city in 2019,RTPCR was used to detect the related viral pathogens from five clinical samples.The results showed that all five samples were positive for PEDV,but negative for TGEV and PoRV.A PEDV strain named as Jiaozuo1012/2019 was isolated from the positive samples by using Vero cells through detecting the fifth passaged virus by RT-PCR.The S gene and ORF3 gene of the isolated PEDV were amplified by RT-PCR,and the homology and phylogenetic tree of these two genes were analyzed by compared with PEDV reference strain in GenBank.Based on the homology analysis of S gene and ORF3 gene,S gene of this isolated PEDV had the highest homology(99.3%)with PEDV CH/HNLH/2015 strain isolated in Henan province.The homology of S gene of PEDV Jiaozuo1012/2019 strain with other strains isolated in Henan province was 96.4%-99.0%.The homology of S gene of this isolated virus with the classic PEDV CV777 strain was 93.4%,and there are 4 insertions(59QGVN62)and 3deletions(140N and 163NI164)in S protein of this isolated virus.The ORF3 gene shared 99.7%homology with PEDV JSCZ1601 strain isolated in Jiangsu province,among 96.4%-99.0%homology with other strains isolated in Henan province,and 96.6%homology with classical PEDV CV777 strain.The phylogenetic tree analysis of S gene and ORF3 gene showed that the isolated virus had the closest genetic relationship with the current domestically epidemic strains.This study provides reference data for further studying the variation of PEDV in Henan province.