我国部分地区猪流行性腹泻病毒的分离与进化分析

发布时间：2018-11-09 13:14

【摘要】：2013年冬及2014年春,吉林省、陕西省猪场疑似发生流行性腹泻病毒感染。为确定病原、了解其与国内外其他毒株的基因差异,分别采集腹泻粪便样品和小肠组织,进行病毒分离培养并对关键基因进行测序和分析,获得如下结果:1.利用Vero细胞连续传代后,分离到猪流行性腹泻病毒株SP株(吉林),YL9099、YL9140、YL9234株(陕西)。2.用RT-PCR方法对分离的到的4株猪流行性腹泻病毒S基因及SP株的M、ORF3基因进行进行扩增、克隆、测序,用DNAStar、MEGA6.0、Net NGlyc 1.0 Server与Gene Bank中登录的猪流行性腹泻病毒株S、M、ORF3序列进行序列变化、遗传进化关系、抗原位点,糖基化位点的分析。(1)本实验的4株病毒与经典毒株株CV777、DR13相比,核苷酸同源性在94.0%-99.4%之间。其推导的氨基酸与疫苗株相比有79-84处突变,其中发生在S1区的突变较多,占整体突变的70%以上,这也表明其S1区为该病毒的高变区。糖基化位点与抗原性分析,该4株病毒与疫苗株对比在已报道的抗原表位COE、2C10、S1D6均发生了几处突变,在S1D5区未发现突变。(2)与国内外68株毒株的核苷酸对比分析后发现,本实验分离4株病毒与流行毒株AJ1102、CH/JLCC、CH18,同源性较高,在99.5%-100%之间,与经典毒株同源性低。(3)SP株ORF3基因核苷酸序列分析表明:SP株与中国毒株JS-HZ2012 China(杭州)、GD-B China(广东)的核苷酸同源性最高,达99.7%,与中国株LZC China(兰州)的同源性最低,为95.6%。SP株M基因核苷酸序列分析表明:SP株M基因与国内外13-019349 American、USA Colorado 2013 American、BJ-2011-3 China、GD-B China、HB-2012-1 China、HBMC2012 China、JS-HZ2012 China6株的同源性最高,均达100%。与中国毒株LZC China的同源性最低,为97.2%。分析结果提示现阶段PEDV呈现快速变异和进化的趋势,其地域性和时间性愈来愈明显,目前急需选择新的疫苗株来控制PEDV的爆发。由于本研究的SP、YL9099、YL9140、YL9234株具有近年来PEDV变异株流行的普遍分子特征,因而可为新的疫苗毒株筛选提供参考。
[Abstract]:In the winter of 2013 and spring of 2014, swine farms in Jilin Province and Shaanxi Province were suspected to have epidemic diarrhea virus infection. In order to determine the pathogen and understand the gene difference between the pathogen and other strains at home and abroad, the fecal samples and small intestine tissues of diarrhea were collected, the virus was isolated and cultured, and the key genes were sequenced and analyzed. The results were as follows: 1. The porcine epidemic diarrhea virus strains SP (Jilin) and YL9099,YL9140,YL9234 (Shaanxi) were isolated after continuous passage of Vero cells. The S gene of 4 strains of porcine epidemic diarrhea virus and the MNORF3 gene of SP strain were amplified, cloned, sequenced and sequenced by RT-PCR. The sequence changes, genetic evolution, antigenic sites and glycosylation sites of the swine epidemic diarrhoea virus strain Schim MN ORF3, which were registered in Net NGlyc 1.0 Server and Gene Bank, were analyzed. (1) the four strains of the virus and the classical strain of CV777, in this study were selected for the analysis of the sequence changes, genetic evolution, antigenic sites and glycosylation sites. The nucleotide homology of DR13 was 94. 0-99. 4%. Compared with the vaccine strain, the deduced amino acid had 79-84 mutations, among which more mutations occurred in the S1 region, which accounted for more than 70% of the total mutation, which also indicated that the S1 region was the high variable region of the virus. The glycosylation sites and antigenicity of the four strains were compared with those of the vaccine strains. Several mutations were found in the reported epitope COE,2C10,S1D6. No mutation was found in the S1D5 region. (2) the nucleotide analysis of 68 strains of viruses at home and abroad showed that the homology of AJ1102,CH/JLCC,CH18, between the four viruses isolated in this experiment and the prevalent strains was between 99.5- 100%. (3) the nucleotide sequence analysis of ORF3 gene of SP strain showed that SP strain had the highest nucleotide homology with Chinese strains JS-HZ2012 China (Hangzhou) and GD-B China (Guangdong), and the nucleotide sequence of SP strain was 99.7%. The nucleotide sequence analysis of M gene of 95.6%.SP strain showed that M gene of SP strain had the lowest homology with that of Chinese strain LZC China (Lanzhou. It was found that M gene of SP strain had the highest homology with 13-019349 American,USA Colorado 2013 American,BJ-2011-3 China,GD-B China,HB-2012-1 China, at home and abroad. The homology of HBMC2012 China,JS-HZ2012 China6 strain was the highest, which was 100%. The homology with the Chinese strain LZC China was the lowest (97.2%). The results suggest that PEDV presents a trend of rapid variation and evolution, and its regional and temporal characteristics are becoming more and more obvious. It is urgent to select new vaccine strains to control the outbreak of PEDV. Because the SP,YL9099,YL9140,YL9234 strain in this study has the prevalent molecular characteristics of PEDV variant in recent years, it can be used as a reference for the screening of new vaccine strains.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

【相似文献】