2014—2018年猪流行性腹泻病毒N基因遗传变化分析

Analysis of Genetic Variation of Porcine Epidemic Diarrhea Virus N Gene from 2014 to 2018

为了解猪流行性腹泻病毒(PEDV)N基因的分子流行病学情况,采用RT-PCR方法对2014—2018年采集到的河南、河北、山东、山西、甘肃、湖北、江西以及上海8省市共205份疑似PEDV阳性病料进行PEDV检测,使用RT-PCR方法扩增得到PEDV的N基因片段,回收PCR产物并与pMD20-T载体进行连接,挑选阳性克隆进行测序,分析研究PEDV遗传变异情况。结果显示,205份样品中,有191份样品为PEDV阳性,共得到19株PEDV的N基因序列,N基因全长均为1 326 bp,没有碱基的缺失和插入;将检测到的PEDV的N基因序列与国内外分离株进行比对发现,核苷酸的同源性为94.8%~99.5%,氨基酸的同源性为95.0%~99.3%。进化树分析发现,得到的19株N基因序列与CV777、CH-S等国内外毒株亲缘关系较远;其中,16株与MN、USA-Indiana-2013、USA-Iowa107-2013等近年分离株在一个分支上,为G2a亚群;CH-HENWZ-2015、CH-HENPY-2014、CH-HENZMDPY-2017这3株序列形成另一个分支,为G2b亚群。综上可知,我国PEDV流行广泛,病毒变异频繁。研究结果可为新型疫苗研发以及检测方法的建立奠定基础。

To investigate molecular epidemiology of porcine epidemic diarrhea virus(PEDV),a total of 205 samples were collected from 2014 to 2018 in Henan,Hebei,Shandong,Shanxi,Gansu,Hubei,Jiangxi provinces and Shanghai.All the samples were subjected to RT-PCR detection and the positive samples were sequenced to obtain the whole N gene,products of expected size were retrieved,ligated with pMD20-T vector and transformed to JM109 competent cell.Positive clones were clone to sequence commercially.The results showed that 191 samples were PEDV positive according to RT-PCR,and 19 N genes were acquired,the N gene was 1 326 bp in length.Sequence comparision showed that the nucleotide sequence homologies of N genes were up to 94.8%-99.5%,and the amino acid homologies were 95.0%-99.3%.Phylogenetic tree indicated that the 19 N sequences obtained in this study did not cluster with CV777,CH-S classic strains,among the 19 N sequences,16 N sequences clustered together with MN,USA-Indiana-2013,USA-Iowa107-2013,that was G2a,besides,CH-HENWZ-2015,CH-HENPY-2014,CH-HENZMDPY-2017 clustered together,that was G2b.To sum up,PEDV was prevalent widely in China and the virus mutation was frequent.This study result will lay a foundation for the development of new vaccines and the establishment of detection methods.