为调查近年来浙江省猪流行性腹泻病毒流行毒株与疫苗株的S基因和ORF3基因遗传变异情况,2011-2016年,从浙江省各地区采集疑似猪流行性腹泻病料并提取其RNA,用3对特异性引物对S基因和ORF3基因进行RT-PCR扩增、克隆及测序,并对其序列变化、遗传进化情况和抗原位点进行分析.结果表明:与国内疫苗株CV777相比,12个样品株的S基因存在15个核苷酸插入和6个核苷酸缺失,导致氨基酸序列存在5个氨基酸插入(58QGVN61和l40N)和2个氨基酸缺失(163DI164),且在主要突变区S1区的2个中和表位(499-638和764-771 aa)有8处氨基酸突变;样品株的ORF3基因有7处氨基酸变异.遗传进化分析结果显示,12株分离株的S基因与亚洲主要疫苗株(国内疫苗株CV777和韩国弱毒疫苗株DR13)的同源性仅为93.3％～94.9％,而与2011-2016年中国流行株(BJ-2011和HuN)的同源性高达97.8％～99.9％,表明近年来猪流行性腹泻病毒呈现较大程度的变异,可为研制更加有效的疫苗奠定基础.%In order to investigate recent genetic variations of S and ORF3 genes in Porcine epidemic diarrhea virus(PEDV) epidemic and vaccine strains in Zhejiang Province, virus RNA was extracted from suspected PEDV samples which were collected from various areas in Zhejiang Province since 2011 to 2016, then RT-PCR, cloning and sequencing on S and ORF3 genes were performed using three pairs of specific primers to analyze the sequence variation, genetic evolution and antigen sites.The results showed that as compared to vaccine strain CV777, 15 nucleotides were inserted and 6 nucleotides were absent in the S genes of 12 epidemic strains, leading to the insertion of 5 amino acids (58QGVN61and140N) and deletion of 2 amino acids (163DI164) relatively.Moreover, in epidemic strains, totally 8 amino acid mutations were found in two neutralizing epitopes (499-638 and 764-771 aa) on the major mutation region S1;7 amino acids were variated in ORF3 gene.According to the analysis results of genetic evolution, these 12 epidemic strains shared 97.8％-99.9％ homology with China epidemic strains BJ-2011 and HuN in S gene, but only 93.3％-94.9％ homology with vaccine strains (CV777 and Korean attenuated strain DR13), indicating that huge variations and fast evolution had emerged in PEDV recently.This study could lay a foundation for development of effective vaccines and therapies.
展开▼
