猪流行性腹泻病毒JS2017株的分离鉴定及S基因序列分析

为了解江苏省某规模化猪场猪流行性腹泻病毒(PEDV)遗传变异情况,本试验采集腹泻仔猪小肠组织及肠道内容物,通过RT-PCR进行PEDV鉴定。将检测为阳性的病料组织经20μg/mL胰酶处理后,接种到Vero细胞中进行培养,将细胞培养物进行反复冻融后,收获病毒液,进行RT-PCR鉴定。采用分段重叠策略对其S基因进行全基因序列扩增,并在此基础上分析该毒株S基因的核苷酸同源性及遗传进化关系。结果显示,经RT-PCR鉴定,病料组织呈PEDV阳性,阳性病料接种Vero细胞盲传4代后,出现明显的细胞病变,表现为细胞合胞体病变,空泡化,最终裂解脱落。收获的病毒液经RT-PCR鉴定后,确认分离到1株PEDV,命名为JS2017株。核苷酸序列分析表明,JS2017株S基因序列与美国变异株OH1414、加拿大变异株ON-007、中国变异株PEDV-LYG和CH/PDS/2015位于同一进化分支,其核苷酸同源性高达99.0%以上;其中与中国变异株YC2014亲缘关系最近,核苷酸同源性为100.0%;与经典株CV777、DR13株同源性较低,亲缘关系较远。结果表明,本研究分离获得的JS2017毒株是一株PEDV地方流行变异毒株,与2014年中国江苏分离株(YC2014株)亲缘关系最密切,与当前猪流行性腹泻疫苗株亲缘关系较远。

Isolation and Identification of Porcine Epidemic Diarrhea Virus Strain JS2017 and Its S Gene Sequence Analysis

In order to understand the genetic variation of porcine epidemic diarrhea virus (PEDV) in a large-scale pig farm in Jiangsu,the intestinal content of piglets with watery diarrhea were collected and detected for PEDV by RT-PCR.The diseased tissue detected as positive was treated with 20 μg/mL trypsin,and then inoculated into Vero cells for culture.After the cell culture was repeatedly frozen and thawed,the virus solution was harvested and identified by RT-PCR.The S gene was amplified by a segmentation overlap strategy,and the nucleotide homology and genetic evolution of S gene was analyzed.The results showed that the pathological tissue was positive for PEDV by RT-PCR.After 4 generations of inoculation with Vero cells,the positive pathogens showed obvious cytopathic changes,which showed cell syncytosis,vacuolization and finally lysis.After the harvested virus solution was identified by RT-PCR,it was confirmed that one PEDV was isolated and designated as JS2017 strain.Sequence analysis showed that the S gene sequence of JS2017 strain was in the same evolutionary branch as the American variant strain OH1414,Canadian variant strain ON-007,Chinese variant strain PEDV-LYG and CH/PDS/2015,and its nucleotide homology was as high as 99.0%.Among them,the Chinese mutant strain YC2014 had the closest relationship,and its nucleotide homology was 100.0%;JS2017 strain had low homology with the classical strains CV777 and DR13,and had a distant relationship.The results showed that the JS2017 strain isolated in this study was a PEDV local epidemic strain,which was closely related to the 2014 Jiangsu isolate (YC2014 strain), and had a far-reaching relationship with the current swine epidemic diarrhea vaccine strain.