4株猪流行性腹泻病毒S蛋白序列特征和遗传进化分析

Sequence Characteristics and Genetic Analysis of S Protein of 4 Porcine Epidemic Diarrhea Strains

关键词:  猪流行性腹泻病毒  结构基因S  序列特征  遗传进化分析

Key words:  Porcine epidemic diarrhea virus  S structural gene  Sequence characteristics  Genetic evolution analysis

摘要:

为了解目前国内猪流行性腹泻病毒流行毒株与疫苗毒株的S蛋白差异情况，为该病防控提供理论参考，本研究将2013年采自北京、河南、陕西、广东四个省市的疑似PED病料，抽提RNA，用设计的针对结构基因S的特异性引物进行RT-PCR扩增、克隆及测序，并对其序列变化、遗传进化情况、抗原位点进行分析。结果显示：4个样品株的S基因（4161bp）与国内疫苗株CV777（4152bp）相比，存在15个核苷酸的插入和6个核苷酸的缺失，导致推导的氨基酸序列存在5个氨基酸的插入（59QGVN62 and 140N）和2 个氨基酸的缺失（163NI164），且在主要突变区S1区的2个中和表位（499~638aa和764~771aa）有7处氨基酸突变。遗传进化分析结果显示，4个样品株与主要疫苗株同源性较低（93.8%~94.7%），而与2007~2009年韩国毒株，2011年日本毒株以及中国近年流行毒株同源性高（96.0%~99.6%），表明近年来国内PEDV呈现较大变异，可能需要研制更有效的疫苗用于PED的防控。

Abstract:

In order to investigate the differences of S protein between PEDV epidemic strains and main vaccine strains, and provide theoretical reference for prevention of PED，in this study we collected tissues and feces samples infected with PEDV from Beijing, Henan, Shanxi, Guangdong. By exacting RNA from the samples, we got the whole S genes by RT-PCR. After cloning and sequencing, we analyze sequence variation, genetic evolution and antigen sites. The results show that compared with national vaccine strain CV777（4152bp）, these four epidemic strains’ S genes（4161bp）have 15 nucleotides insertions, 6 nucleotides deletions. As a result, there are five amino acids insertion(59QGVN62 and 140N) and two amino acids deletion（163NI164） in the corresponding protein S. According to the results of amino acid sequence alignment of S protein, amino acid mutations are mainly concentrated in S1 domain (1 ~ 789 aa). There are totally 7 amino acid mutations of two neutralizing epitopes which belong to S1 domain. Genetic evolution analysis results show that 4 epidemic strains have low sequence homology （93.8%~94.7%）with main vaccine strains, while they share high sequence homology （96.0%~99.6%） with South Korean strain in the year of 2007~2009, Japanese strain of the year 2011 and China strains of recent years. The results reflect that PEDV has fast variation and evolution trends, which suggests that it is necessary for us to further understand PEDV variation and evolution trend and provide theoretical basis for prevention and control of swine diarrhea outbreak.