2017年河南省猪流行性腹泻病毒检测及基于S1基因片段的遗传变异分析

Detection and genetic characteristics of S1 fragments in spike gene of porcine epidemic diarrhoea virus in Henan in 2017

为了解河南省引起仔猪腹泻的猪肠道冠状病毒的感染和流行情况,我们对2017年度发生仔猪腹泻的河南省25家养殖场送检的94份仔猪肠道或粪便样品开展了相关检测。结果显示猪流行性腹泻病毒(porcine epidemic diarrhoea virus,PEDV)的样品检出率为68.1%(64/94),猪场阳性率为76.0%(19/25),但未检测到猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus,TGEV)、猪德尔塔冠状病毒(porcine deltacoronavirus,PDcoV)和猪急性腹泻综合征冠状病毒(swine acute diarrhoea syndrome coronavirus,SADS-CoV)。为进一步分析PEDV的遗传变异情况,利用RT-PCR和基因克隆测序技术,我们成功获得了8株PEDV的S1基因片段的核苷酸序列。分子进化树分析显示,我国流行毒株可以分为G1a(疫苗及其衍生株)、G1b(经典毒株)、G2a(2011-2013年变异株)、G2b(2016-2017年变异株)和G2c(重组毒株)5个进化分支,本研究获得的S1基因序列均处于G2b进化分支,且与2016-2017年间的流行毒株遗传距离更近。同源性分析显示,8株分离株彼此之间S1基因片段的核苷酸同源性为97.3%~99.5%,与变异毒株AJ1102的同源性为97.2%~98.0%,而与经典毒株CV777、CH/S的同源性仅为91.0%~92.3%。氨基酸序列比对分析显示,与CV777和CH/S等经典毒株相比,分离株S1基因片段除了多个氨基酸位点发生变异外,在58NQGV61、140N、157H这3个位置有氨基酸的插入,在163NI164 有2个氨基酸的缺失,这与之前关于PEDV变异株的研究报道相一致。此外,与其他变异株相比,本研究发现分离株HeNPDS/2017在491位有1个新的丝氨酸(Ser)缺失位点,其生物学意义有待进一步研究。本研究丰富了PEDV的分子流行病学数据,有助于了解河南省PEDV流行和遗传变异的最新情况,为该地区猪流行性腹泻病的防控提供一定的参考。

To investigate the epidemic status of porcine enteric coronavirus in Henan in 2017,a total of 94 clinical piglets diarrhoea samples and faeces were collected and conducted RT-PCR detection.The results showed that the porcine epidemic diarrhoea virus(PEDV) was the mainly causative agent for piglets viral diarrhoea.Porcine transmissible gastroenteritis virus(TGEV),porcine deltacoronavirus(PDcoV) and swine acute diarrhoea syndrome coronavirus(SADS-CoV) were not detected in this study.To further study the genetic characteristics of PEDV,we sequenced S1 fragments of spike gene of 8 isolates.The phylogenetic tree showed that all the epidemic strains were divided into five clusters including G1a(vaccine and derivatives),G1b(classical strains),G2a(variants during 2011-2013 years),G2b(variants during 2016-2017 years)and G2c(recombinant strains).The isolates were clustered into G2 bbranch and close to variants during 2016-2017 years.Homological analysis displayed that the isolates have a 97.3%-99.5% nucleotide identity with each other,97.2%-98.0%identity with variant strain AJ1102 and 91.0%-92.3%similarity with classical strain CV777/or CH/S.Besides,the sequence alignment showed that there are insertions at 58NQGV61,140N,157H sites and deletion at163NI164site in the S1 fragments of isolates.Compared with the other variants,one isolate HeNPDS/2017 has a new serine deletion at 491 th site.Our study enriched the genetic characteristics of PEDV and would be helpful to better understand the prevalent status and genetic variations of PEDV in Henan Province.