猪传染性胃肠炎病毒HB-FP株的分离鉴定及其S基因序列分析

Isolation and identification of porcine transmissible gastroenteritis virus HB-FP strain and phylogenetic analysis of S gene

为了解目前河北地区流行的猪传染性胃肠炎病毒(TGEV)的生物学特征及基因的遗传进化关系,从河北某地采集仔猪粪便或小肠内容物,病料处理后在PK-15细胞上进行盲传,通过RT-PCR、间接免疫荧光(IFA)验证,成功分离出1株猪传染性胃肠炎病毒,命名为HB-FP株。对HB-FP株S基因进行RT-PCR扩增,获得的分离株S全基因与国内外TGEV毒株进行序列比对和遗传进化分析:结果显示,HB-FP株S基因与其他参考毒株的核苷酸同源性为94.9%~98.8%,编码氨基酸同源性为65%~98%,与我国的TGEV JS2012(KT696544)株核苷酸同源性最高为98.8%,氨基酸同源性为98%。由进化树分析表明,HB-FP株除与英国TGEV株(AF104420)不在一个分支上外,与其他参考毒株都在同一分支上,中国的TGEV JS2012(KT696544)株、美国的TOY56-165(M94103)株、韩国的TGEV(AF298211)株与分离株HB-FP株亲缘关系最近。氨基酸变异情况分析表明,分离株与常用疫苗株相比在S蛋白的抗原表位区有7处点突变,这种抗原位点的突变对TGEV生物学特性的影响还有待进一步研究。本试验成功从河北省分离鉴定得到1株猪传染性胃肠炎病毒,为该地区TGEV防控及候选疫苗毒株的筛选提供了物质基础。

In order to further study the characteristics of the current transmissible gastroenteritis virus(TGEV) and its genetic evolutionary relationship in Hebei Province, the feces and small intestine contents of piglets were collected from Hebei Province,inoculated and cultured in the PK-15 cells. By RT-PCR and indirect immunofluorescence assay(IFA), a TGEV isolate was successfully identified and named as HB-FP strain. The S gene of HB-FP strain was amplified by RT-PCR and analyzed along with the sequence alignment and phylogenetic trees of TGEV S gene sequences from other domestic and international strains. The results showed that the S gene of HB-FP strain had 94.9%-98.8% nucleotide homology and 65%-98% amino acid homology with other reference strains. The highest homology with TGEV JS2012(KT696544) was 98.8% in nucleotide and 98% in amino acid. Phylogenetic tree analysis showed that the HB-FP strain was on the same branch with the other reference strains except the British TGEV strain(AF104420), and the genetic relationship of HB-FP strain was the closest to the TGEV JS2012(KT696544) strain of China, the TOY56-165(M94103) strain of United States, and the TGEV(AF298211) strain of Korea. The analysis of amino acid variation showed that there were 7 point mutations in the epitope of S protein,and the effect of mutation of these antigen sites on the biological characteristics of TGEV remains to be further studied. The results showed that a strain of transmissible gastroenteritis virus in Hebei Province was successfully isolated and identified in this experiment, which provided a material basis for the selection of transmissible gastroenteritis vaccine strains in this area.