猪流行性腹泻病毒的分离鉴定及N基因序列分析

Isolation and identification of porcine epidemic diarrhea virus and its sequence analysis of N gene

【目的】了解引起仔猪腹泻的猪流行性腹泻病毒是否为变异株。【方法】从疑似猪流行性腹泻病毒感染的猪场采集死亡仔猪的肠黏膜及其内容物,将病料常规处理后接种VERO细胞,采用维持培养液加10μg/mL的胰酶的方法分离培养。用RT-PCR、间接免疫荧光、动物回归实验以及部分序列分析等方法对分离株进行鉴定。【结果】病料在接入细胞第一代即出现病变,细胞肿胀变圆,形成合胞体,逐渐脱落,细胞病变明显。随着病毒传代次数的增加,细胞病变逐渐稳定,进行RT-PCR检测、间接免疫荧光检测以及动物回归实验,最终确定分离株符合PEDV特征,并命名为BJ2015,测定毒株的TCID50为10-6.4/0.1mL。基因测序结果显示,N基因长1 329bp,编码442个氨基酸。与参考株N基因核苷酸序列比对及遗传进化分析表明,BJ2015与传统株CV777亲缘关系较远;与流行变异株BJ-2011-1N基因在进化树的同一个分支上,二者的核苷酸和氨基酸同源性分别为99.6%、99.5%,表明BJ2015与BJ-2011-1亲缘关系较近。【结论】结果表明BJ2015为PEDV流行性变异毒株。

【Objective】In this study,it was aimed to dig out whether the porcine epidemic diarrhea virus is a variant strain,which caused the swine epidemic diarrhea.【Methods】The intestinal mucosa and contents from dead diarrheal piglets in pig herds,were collected from those being suspected to be infected by porcine epidemic diarrhea virus（PEDV）.These samples were tansfected into VERO cells after being treated with general methods,VERO cells were cultured in DMEM supplemented with10% trypsin.The isolated strains were identified by reverse transcription-polymerase chain reaction（RT-PCR）,indirect immunofluorescence staining,animal regression and partial gene sequence analysis.【Results】After the first generation,VERO cells became tumescence and round,gathered together the package body and then gradually fall off.It is doubtful for CPE of PEDV.With the great increase of virus passages,CPE became more and more regular after being detected by RT-PCR,indirect immunofluorescence staining and animal regression to identify the virus.Ultimately,the isolate was characterized as PEDV and named as BJ2015,and the TCID50 was 10-6.4/0.1mL.Gene sequencing results showed that the length of the N gene is 1 329 bp,encoding 442 amino acids.Compared with the reference strains,the N gene nucleotide sequence and phylogenetic analysis showed that the genetic distance between BJ2015 strain and the traditional strain CV777 was very far.N genes of BJ2015 andpopular variant BJ-2011-1were in a same branch of the evolutionary tree,each of them had 99.6%,and 99.5% of nucleotide and amino acid homology,respectively.it also showed that the genetic distance between BJ2015 strain and the BJ-2011-1strain was not far away.【Conclusion】BJ2015strain was a popular PEDV variant.