猪流行性腹泻病毒GD-A株的分离鉴定及S基因序列分析

Isolation and identification of GD-A strain of porcine epidemic diarrhea virus and phylogenetic tree analyses based on S gene

【目的】在广东省某疑似发生猪流行性腹泻的猪场中,采集病猪的粪便样品,经RT-PCR检测阳性的样品接种于Vero细胞,以分离猪流行性腹泻病毒.【方法】通过在细胞上的连续传代,直至能稳定增殖.并通过RT-PCR对细胞培养物进行特异性片段的检测,观察细胞病变(CPE)的发生情况,以及间接免疫荧光(IFA)检测猪流行性腹泻病毒的M蛋白,对病毒进行分离与鉴定.【结果和结论】病毒在盲传5代时能观察到典型病变,传至20代时能在Vero细胞上增殖稳定.RT-PCR、CPE与IFA的结果证明,所分离到的病毒为PEDV,并命名为GD-A株.

【Objective】The fecal samples were collected from a pig farm with similar clinical symptoms of porcine epidemic diarrhea in Guangdong Province. In order to isolate a virus strain,the samples with positive RT-PCR for porcine epidemic diarrhea virus（ PEDV） were inoculated in Vero cell lines.【Method】The isolated virus strain was serially passaged until it was proliferated stably in Vero cells.During the process,observation of the typical cytopathic effect（ CPE） at different time points,indirect fluorescent antibody（ IFA） assay detected for M protein of virus,and RT-PCR method applied for specific gene detection of PEDV were used to isolate and identify the PEDV proliferated in Vero cells. 【Result and conclusion】The typical CPE was observed at the 5th bind passage,and the isolated virus strains proliferated stably in Vero cells at the 20 th passage. According to the results of RT-PCR,CPE and IFA,the isolated virus strain is determined to be PEDV,which is named GD-A strain.