PEDV流行株的分离鉴定及其遗传进化分析
发布时间：2018-10-11 16:01
【摘要】：为了解中国猪流行性腹泻病毒(Porine epidemic diarrhea virus,PEDV)的流行现状,本研究对上海和江苏地区猪场进行了PEDV流行株的分离鉴定。根据GenBank中PEDV的基因序列,对其M基因设计检测引物。检测临床采集的猪腹泻样品,2份临床样品能扩增出特异性目的条带,测序结果证实为PEDV M基因片段。将检测为PEDV阳性的样品接种Vero细胞,并添加不同浓度的胰酶进行病毒传代培养,在添加适量胰酶的Vero细胞上分离到两株病毒并能稳定传代、增殖。选择第22代病毒饲喂新生仔猪,进行回归试验,结果两株细胞毒均能使1日龄仔猪出现典型腹泻症状,进一步证实成功分离到PEDV,命名为JSLS/PEDV/1/2014和JS/PEDV/2/2014株。遗传进化分析,JS/PEDV/2/2014和JSLS/PEDV/1/2014分离株的M基因分别与中国毒株HLJ-2012、BJ-2012-1亲缘性最近;S基因与I群PEDV流行毒株亲缘性最近;两个毒株的ORF3基因均存在51个碱基缺失,与DR13处于同一大分支,与CV777亲缘性较远。
[Abstract]:In order to understand the epidemic status of porcine epidemic diarrhea virus (Porine epidemic diarrhea virus,PEDV) in China, PEDV epidemic strains were isolated and identified from pig farms in Shanghai and Jiangsu. According to the sequence of PEDV gene in GenBank, primers were designed to detect its M gene. The specific target bands were amplified from 2 clinical samples of porcine diarrhea, which were confirmed to be PEDV M gene fragments by sequencing. The PEDV positive samples were inoculated into Vero cells, and the virus was subcultured with different concentrations of trypsin. Two strains of viruses were isolated from Vero cells supplemented with proper amount of trypsin and could be subcultured and proliferated stably. The 22 ~ (th) generation virus was selected to feed newborn piglets. The results showed that both of the two strains could induce typical diarrhea symptoms in 1-day-old piglets. It was further confirmed that PEDV, was successfully isolated from piglets named JSLS/PEDV/1/2014 and JS/PEDV/2/2014 strains. Genetic evolution analysis showed that M gene of JS/PEDV/2/2014 and JSLS/PEDV/1/2014 isolate was most closely related to HLJ-2012,BJ-2012-1 of Chinese strain, S gene was most closely related to group I PEDV epidemic strain, and the ORF3 gene of both strains had 51 base deletion, which was at the same branch as DR13. The phylogenetic relationship with CV777 was far away.
【作者单位】： 上海市农业科学院畜牧兽医研究所;上海市农业遗传育种重点实验室;
【基金】：上海市科技兴农重点攻关项目(项目号:沪农科攻字(2013)第3-6号);上海市科技兴农重点攻关项目(项目号:沪农科攻字(2013)第5-5号) 上海市生猪产业技术体系建设项目 上海市扬帆计划(项目号:15YF1410300) 上海市农口青年成长计划项目(项目号:沪农青字(2016)第1-35号)
【分类号】：S852.65
[Abstract]:In order to understand the epidemic status of porcine epidemic diarrhea virus (Porine epidemic diarrhea virus,PEDV) in China, PEDV epidemic strains were isolated and identified from pig farms in Shanghai and Jiangsu. According to the sequence of PEDV gene in GenBank, primers were designed to detect its M gene. The specific target bands were amplified from 2 clinical samples of porcine diarrhea, which were confirmed to be PEDV M gene fragments by sequencing. The PEDV positive samples were inoculated into Vero cells, and the virus was subcultured with different concentrations of trypsin. Two strains of viruses were isolated from Vero cells supplemented with proper amount of trypsin and could be subcultured and proliferated stably. The 22 ~ (th) generation virus was selected to feed newborn piglets. The results showed that both of the two strains could induce typical diarrhea symptoms in 1-day-old piglets. It was further confirmed that PEDV, was successfully isolated from piglets named JSLS/PEDV/1/2014 and JS/PEDV/2/2014 strains. Genetic evolution analysis showed that M gene of JS/PEDV/2/2014 and JSLS/PEDV/1/2014 isolate was most closely related to HLJ-2012,BJ-2012-1 of Chinese strain, S gene was most closely related to group I PEDV epidemic strain, and the ORF3 gene of both strains had 51 base deletion, which was at the same branch as DR13. The phylogenetic relationship with CV777 was far away.
【作者单位】： 上海市农业科学院畜牧兽医研究所;上海市农业遗传育种重点实验室;
【基金】：上海市科技兴农重点攻关项目(项目号:沪农科攻字(2013)第3-6号);上海市科技兴农重点攻关项目(项目号:沪农科攻字(2013)第5-5号) 上海市生猪产业技术体系建设项目 上海市扬帆计划(项目号:15YF1410300) 上海市农口青年成长计划项目(项目号:沪农青字(2016)第1-35号)
【分类号】：S852.65
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