绵羊伪狂犬病病毒的分离鉴定及其gD基因序列分析

ISOLATION AND IDENTIFICATION OF SHEEP PRV AND SEQUENCE ANALYSIS OF ITS GD GENE

本研究以1例疑似绵羊伪狂犬病病例为研究对象,进行了病毒分离鉴定及其g D基因分析。将病料接种BHK-21细胞,细胞出现病变,经间接免疫荧光和荧光定量PCR检测结果证实,所分离的病毒为伪狂犬病病毒,将其命名为SH1311毒株。在电镜下观察病毒,病毒粒子呈球形,囊膜表面有大量纤突。对分离株g D基因进行PCR扩增、测序和序列分析,并与国内外伪狂犬病病毒株的同源性进行比较发现,该分离株与2012年以来上海市分离的猪PRV毒株g D基因的同源性最高,为99.8%~100%,而与2010年上海市流行毒株相比,同源性只有80.5%。构建的g D基因进化树显示,SH1311分离株与上海市2012~2015年间分离的7株猪伪狂犬病病毒以及国内JS-2012、HNB、HN1201、HLJ8、BJ/RD、ZJ01毒株属于同一个进化分支,亲缘关系最近,与欧美以及韩国的流行毒株都处在不同的分支上。

Pseudorabies virus(PRV)strain SH1311 was isolated from a suspected sheep case.BHK-21 cells were inoculated with tissue samples and developed CPE.The presence of PRV in inoculated BHK-21 cells was confirmed by indirect immunofluorescence and fluorescence PCR.The morphology of the SH1311 strain was examined under electron microscope.The virus particles were round in shape and a lot of fibers were present on the surfaces of the capsules.In this study,gD gene of the SH1311 strain was amplified by PCR for sequencing.The sequence analysis of gD gene showed that the SH1311 strain shared 99.8%-100%nucleotide homology with 7 PRV isolates obtained since 2012 from Shanghai but had low nucleotide homology(80.5%)with those isolated in 2010 from this area.Furthermore,the SH1311 strain was classified in the same evolutionary branch with other PRV isolates from Shanghai during 2012-2015 and domestic epidemic stains JS-2012、HNB、HN1201、HLJ8、BJ/RD and ZJ01,but different from those from Europe,America and South Korea.