浙江省猪流行性腹泻病毒S1基因克隆与序列分析

本研究旨在分析浙江省猪流行性腹泻病毒（PEDV）的遗传变异情况，利用实时荧光定量RT-PCR方法对2015-2016年浙江省内收集的58份猪腹泻样品进行检测，设计2对特异性引物对16份来自浙江不同地区PEDV阳性样品的S1基因进行RT-PCR扩增、克隆及序列测定，并应用生物信息学软件对16株PEDV浙江毒株的S1基因进行分析。结果显示，48份样品为PEDV阳性。16个毒株之间S1基因片段核苷酸和氨基酸同源性分别为93.1%~99.8%和92.4%~99.7%，与疫苗株CV777的核苷酸同源性为92.3%~95.7%，氨基酸同源性为90.7%~95.7%。与疫苗株CV777相比，15个毒株在S1基因区域存在着15个核苷酸插入和6个核苷酸缺失。系统进化分析表明，大部分毒株与国内外流行的基因Ⅱ型PEDV毒株亲缘关系较近，15个毒株与2011-2016年中国流行的基因Ⅱ型PEDV毒株核苷酸和氨基酸同源性均在96.6%以上；与早期分离的CV777株、LZC株亲缘关系较远，核苷酸和氨基酸同源性均在93.4%以下；1个毒株（ZJ16NB6）与国内外流行的S-INDEL样毒株较近，核苷酸和氨基酸的同源性较高，均在98.4%~99.5%之间。本研究结果表明，2015-2016年浙江省仔猪腹泻主要是由PEDV感染引起的，浙江省流行的PEDV同时存在着基因Ⅱ型和S-INDEL样毒株，但以基因Ⅱ型毒株为主。

Cloning and Sequence Analysis of S1 Gene of Porcine Epidemic Diarrhea Virus from Zhejiang Province

To analyze the genetic variation of porcine epidemic diarrhea virus (PEDV),the piglet diarrhea samples from Zhejiang province during 2015 to 2016 were detected by Real-time RT-PCR.Two pairs of primers were designed and the S1 gene of 16 PEDV positive samples from different regions were amplified by RT-PCR,cloned and sequenced.The sequence of S1 gene was analyzed by biological information software.The results showed that 48 samples were positive for PEDV.Homologous analysis showed that the S1 gene of 16 PEDV strains shared 93.1% to 99.8% nucleotide homology and 92.4% to 99.7% amino acid homology.The S1 gene of 16 PEDV strains shared 92.3% to 95.7% nucleotide homology and 90.7% to 95.7% amino acid homology with vaccine strain CV777.Compared to vaccine strain CV777,15 nucleotides were inserted and 6 nucleotides were absent in the S1 gene of 15 epidemic strains.The phylogenetic tree based on sequences of S1 gene suggested that 15 PEDV strains had a close relationship with the genotype Ⅱ PEDV,nucleotide and amino acid homologous were all more than 96.6%.Compared with the early separation of CV777 and LZC strains,nucleotide and amino acid homologous were all less than 93.4%.However,strain ZJ16NB6 had a close relationship with the S-INDEL-like strain,nucleotide and amino acid homologous were all 98.4% to 99.5%.The results indicated that PEDV infection was the main pathogen of piglets diarrhea in Zhejiang province.The genotype Ⅱ and S-INDEL-like PEDV strain existed in Zhejiang province,but mainly prevailing the genotype Ⅱ PEDV strains.