猪流行性腹泻病毒YZ2016全基因组测序与稳定性分析

Complete genome sequencing and stability analysis of porcine epidemic diarrhea virus PEDV YZ2016

2016年我国江苏扬州某规模化养猪场暴发严重腹泻,本实验室通过RT-PCR确定其病原为猪流行性腹泻病毒(PEDV)。将阳性样品接种Vero细胞,盲传5代后出现典型病变,并通过RT-PCR及间接免疫荧光试验确定成功分离到PEDV毒株,命名为PEDVYZ2016。采用分段重叠法对分离株基因组扩增测序,获得其全长序列。遗传进化分析显示PEDVYZ2016与近年流行毒株亲缘关系较近,与经典毒株亲缘关系较远。PEDVYZ2016与BJ-2011-1、LZW全基因组核苷酸序列一致性可达99.2%,与BJ-2011-1、OKN-1/JPN/2013S基因推导氨基酸序列一致性可达98.5%。此外,分离株S基因发生多处点突变,导致其抗原表位及糖基化位点均发生部分改变。同时,序列比对分析显示不同代次的S、ORF3、N基因核苷酸突变率均极低。结果表明,本研究分离的PEDVYZ2016株属流行变异毒株,且在传代过程中遗传稳定性良好。

In 2016,severe diarrhea occurred on a large-scale pig farm in Yangzhou,Jiangsu Province,China.The pathogen was determined to be porcine epidemic diarrhea virus(PEDV)by RT-PCR.The Vero cells infected with the positive sample were passaged five times and then subjected to RT-PCR and indirect immunofluorescence assays(IFA).The results demonstrated that a new PEDV strain was successfully isolated and named PEDV YZ2016.Lately,the isolate was cloned and sequenced for the whole genome sequence.Phylogenetic analysis showed that PEDV YZ2016 was closely related to the epidemic strains in recent years,but far from the classical strains.The identity of whole-genome nucleotide sequences of PEDV YZ2016 with BJ-2011-1 and LZW was up to 99.2%,and the identity of S gene derived-amino acid sequences of PEDV YZ2016 with BJ-2011-1 and OKN-1/JPN/2013 was up to 98.5%.In addition,multiple point mutations of S gene resulted in important changes in antigen epitopes and glycosylation sites.Meanwhile,alignment analysis showed that the nucleotide mutation rates of S,ORF3 and N genes of different generations were extremely low.In conclusion,PEDV YZ2016 was an epidemic strain,and genetically stable during the passaging.