1株猪传染性胃肠炎病毒的分离鉴定及其生物学特性研究

摘要

The faeces samples were collected from a pig with severe diarrhoea,transmissible gastroenteritis virus(TGEV) nucleotide was amplified by reverse transcription polymerase chain reaction (RT-PCR) and serological methods to isolate the new TGEV strain.The filtered samples were inoculated into ST cells to isolate viruses,then the characterization of the viruses propagated in ST cells were determined by immunoperoxidase monolayer assay(IPMA) and RT-PCR.TGEV S1 gene was amplified by RT-PCR and sequenced,analyzed by solftware DNAStar 7.0 and Mega 5.0.Results showed that significant cytopathic effect(CPE) of ST cells inoculated the sample was observed;RT-PCR detection results showed that the culture samples were TGEV nucleotide positive,as well as serologic test results showed that the culture samples were TGEV antigenic positive.The isolated virus strain was named TGEV-NY.TGEV could be detected in the ST cells 12 hours post infection(hpi) by IPMA,as well as TGEV nueleotide could be detected by RT-PCR in the culture supernant.Nucleotide sequences showed that the open reading frame of S1 gene was 2 220 bp encoding 740 aa.Phylogenetic analysis showed that TGEV-NY strain belonged to the gene group Ⅰ,shared close genetic relationship with TGEV strain SC-Y which was isolated in 2006 from China.%采集1例疑似感染猪传染性胃肠炎病毒(TGEV)仔猪的粪便,运用RT-PCR方法和血清学方法对病料进行TGEV检测,并应用猪睾丸细胞(ST)进行病毒分离培养,分别采用免疫过氧化物酶单层细胞染色法(IPMA)和RT-PCR方法检测病毒在ST细胞中的增殖动态,应用RT-PCR方法扩增分离TGEV S1基因,经序列测定后,采用DNAStar 7.0和Mega 5.0软件对TGEV进行生物信息学分析.结果表明,病料接种到ST细胞培养后,出现明显细胞病变效应(CPE);RT-PCR检测结果显示,TGEV核酸阳性;血清学鉴定TGEV抗原阳性.将分离毒株命名为TGEV-NY.感染12h后,IPMA检测结果阳性,且可从细胞培养上清中检出TGEV核酸.序列测定分析表明,分离毒株S1基因开放阅读框(ORF)为2 220 bp,编码740个氨基酸.进化分析显示,分离毒株与我国2006年分离的SC-Y毒株亲缘关系最近,分离毒株属于基因Ⅰ群.