近年我国部分省份PEDV流行毒株S基因及编码蛋白变异分析

Analysis of variation of sprike gene and encoded protein in PEDV epidemic strains in some provinces of China in recent years

为了解当前猪流行性腹泻（porcine epidemic diarrhea，PED）在我国部分省份的发病情况以及猪流行性腹泻病毒（porcine epidemic diarrheavirus，PEDV）的分子特征和遗传变异情况，本研究从山西、湖北、辽宁、江西、广东5个省份的部分猪场采集疑似PED症状的样品60份，利用RT-PCR方法对其进行PEDV检测。筛选其中的PEDV阳性毒株，利用分段克隆测序的方法获得其PEDVS基因全序列，并利用生物信息学方法对其进行分析。结果表明，PEDV检出率为61.7％；筛选的13株毒株s基因核苷酸同源性为96．6％～99．8％，与2010年以前我国分离株的同源性为92．6％～95．5％，与2010年以后我国分离株的同源性为93．8％～99．4％，与关韩毒株的一同源性为93．8％～99．0％，与疫苗株的同源性为92．1％～98．1％；13株S蛋白氨基酸序列与疫苗株Cv777氨基酸序列相比存在75个相同突变位点和12个差异突变位点，其中在s1区（1～789aa）占比71．26％，中和表位区域有2个相同突变位点，线性表位区域有9个相同突变位点，2个受体结合域有25个相同突变位点。其s蛋白均具有信号肽（1～20aa），剪切位点为20～21aa。S蛋白的二级结构主要是a螺旋32．97％、卢折叠27．78％、β-转角8．59％和无规卷曲30．66％。遗传进化分析表明，13株PEDV均属于G2b类群，而我国现今常用的疫苗株CV777归属于Gla类群。说明当前PED在我国的流行趋势依然严峻，本次分离的13株PEDV与2010年前的野毒株以及现行疫苗株相比发生了较大的变异，因此需要引起相关猪场及检疫部门的高度重视，并及早采取措施以防止该病的大规模暴发。

In order to investigate the current status of PED and the molecular characteristics and ge- netic variation of PEDV in some provinces in China, 60 samples collected from pig farms in Shanxi, Hubei, Liaoning,Jiangxi and Guangdong provinces were detected by method of RT-PCR. The PEDV-positive strains were randomly selected to amplify the S gene and sequence alignment by subcloning and sequencing. Then bioinformatics methods were used to analyze the sequences. The results showed that the positive rate of PEDV in this study was 61.7%. The nucleotide homology between 13 PEDV S genes was 96.6% to 99. 8%, and they respectively shared 92. 6%- 95.5 %, 93.8 %-99.4 %, 93.8 %-99 %,92.1%-98. 1 % identity with Chinese strains isolated before 2010 ,Chinese strains isolated after 2010, strains isolated from The United States and South Korea, and vaccine strain. Analysis of amino acid variation suggested that there were 75 identical mutation sites and 12 differential mutation sites in 13 PEDV S protein sequences compared with the CV777, which accounted for 71.26% of the S1 region （1-789 aa）. There were 2 same mutations sites in theneutralizing epitope regions and 9 same mutations sites in the linear epitope area,at the same time 25 mutations were found in the 2 receptor domains. Signal peptide analysis showed that all 13 S proteins had signal peptide （1-20 aa） ,and the shear site was between 20 aa and 21 aa. The seconda ry structure of S protein was composed of 32. 97% alpha-helix,27.78% beta folding,8.59% beta- Angle,and 30.66%o random curled. Genetic evolution analysis showed that 13 strains of PEDV belonged to G2b group,while vaccine strains such as CV777 belonged to Gla group. All this explains that the PED spreading in China is still severe, and apparent variation occurred between the 13 PEDV and the wild PEDV 2011 years ago and the current vaccine strains. Therefore more attention should be paid and early measures should be taken to prevent the large-scale outbreak of PED in above areas.