五株猪流行性腹泻病毒的分离鉴定及序列分析

为研究猪流行性腹泻病毒的流行情况并分析该病毒的培养特性,对甘肃、北京、河北、山西、浙江五个猪场送检的疑似猪流行性腹泻病猪小肠内容物,用RT-PCR方法进行检测,证明为猪流行性腹泻病毒核酸阳性。然后将阳性病料进行传代培养、病毒含量测定以及特异性检验,证明获得五株猪流行性腹泻病毒,分别命名为Ningxia9、Beijing5、Gaocheng8、Shanxi1、Zhejiang08。对分离的毒株进行S基因全长测序及氨基酸序列分析。结果表明:五株分离毒株在盲传19代开始出现明显的细胞病变,盲传到24代,出现稳定的细胞病变且五株分离毒株的24代病毒含量在104.5~5.0TCID50/m L之间;用S蛋白进行进化树分析表明,五株分离毒株与近年来国内流行毒株亲缘关系很近,在同一个进化分支上;分离的五株野毒株之间的氨基酸同源性高达99.2%~99.9%,而这五株野毒与Gen Bank上提交的2011年、2012年分离的野毒株的之间的氨基酸同源性为98.4%~99.9%;实验室分离毒株及国内近年流行毒株均与国内外经典毒株存在一定的共性,即特定位置氨基酸的插入、缺失或置换。

Isolation and identification of Porcine Epidemic Diarrhea Virus from different areas and the sequence analysis of their gene

Five Porcine epidemic diarrhea virus strains were isolated from the feces of piglets suspected to be suffering from a severe diarrhea in Farms from Gansu, Beijing, Hebei, Shanxi and Zhejiang. The viruses were detected by the RT-PCR amplification assay, virus content and specificity was assessed. Furthermore, sequencing and sequence analysis of the full long of S gene were carried out in the test. Result: The phylogenetic relationship between the five strains and China epidemic strains is very similar; The amino acid homology between 99.2% - 99.2% of five wild strains of Ningxia9, Beijing5, Gaocheng8, Shanxi1 and ZJ08, while the five strains of wild and the wild strains of Genbank submission for 2011 years to 2012 years amino acid homology was 99.1% - 99.9%; Isolate and domestic pandemic strain both at home and abroad in recent years with the classic strain has certain commonality that specific amino acid insertions and deletions or replacement position.