犬瘟热病毒BJ16B2株的分离鉴定及H基因遗传进化分析

Isolation and identification of the canine distemper virus BJ16B2 strain and the genetic variability analysis of its H gene

采用Vero/SLAM细胞,从疑似犬瘟热病毒（CDV）感染犬的眼鼻分泌物拭子中分离到1株病毒,经细胞病变（CPE）观察、RT-PCR检测和间接免疫荧光试验鉴定,确定该分离株为CDV,命名为BJ16B2。对其H基因进行克隆和测序分析,BJ16B2株属于Asia-1型;潜在N-糖基化位点分析,共有9个位点,且在309~311位点,为强毒株。同源性比对分析发现,BJ16B2与参考毒株核苷酸和氨基酸同源性分别为88.7%~99.5%和86.5%~99.5%。其中与Asia-1型SY株同源性最高,核苷酸和氨基酸同源性均为99.5%,与疫苗株Snyder Hill、CDV3、Convac以及Onderstepoort亲缘关系较远,核苷酸和氨基酸同源性分别为90.6%~91.1%和90.3%~91.4%。强毒株BJ16B2的成功分离以及对其H基因序列分析,有利于进一步了解当前中国CDV流行株变异情况,为CDV毒力变化和宿主嗜性的研究提供参考依据。

This study was to isolate and identify the prevalent canine distemper virus （CDV） in domestic dogs and to analyze the evolution-ary genetic characteristics of its H gene. Swab samples collected from the dogs suspected with CDV infection was proliferated on Vero/SLAM cells and the isolated strains were identified by cytopathic effect observation, the reverse transcription polymerase chain reaction test and the indirect immunofluorescence test. The results showed that the isolate was CDV and named BJ16B2. Its H gene was cloned and sequenced. Phylogenetic analysis of the nueleotide of the H gene showed that BJ16B2 was clustered with the Asia-1 genotype, and it was a virulent strain since it possessed a potential N- glycosylation site on aa309-aa311 and a total of nine potential N-glycosylation sites in the H gene. Homol-ogy analysis showed that the nucleotide and amino acid homologies of BJ16B2 with the reference strains were 88. 7%-99. 5% and 86. 5% ~ 99. 5%, respectively. BJ16B2 exhibited a high homology with the SY strain, with both the nucleotide and amino acid homologies being at 99. 5%, but it was less homologous with the vaccine strains, Snyder Hill, CDV3, Convac and Onderstepoort, with the nucleotide and amino acid homologies being 90. 6% -91. 1% and 90. 3% -91. 4%, respectively. Here, the successful isolation of highly pathogenic and virulent strain BJ16B2 and complete sequence determination of its H gene provided a better understanding of the genetic variation of CDVs in China and might support research on virulence change and host tropism of CDV.