牦牛源牛冠状病毒部分基因的扩增、序列分析及病毒分离鉴定

Amplification,Sequence Analysis of Bovine Coronavirus Genes and Isolation of the Viruses from Yak

本试验的目的是对青藏高原地区牦牛进行牛冠状病毒(bovine coronavirus,BCoV)的分子流行病学调查,并分离牦牛源BCoV。采用RT-PCR方法检测西藏、青海、四川、云南的犊牦牛腹泻粪便中BCoV,并扩增其S、HE及N基因片段;选取阳性样本进行BCoV分离。结果显示:从336份犊牦牛腹泻粪便中检出232份BCoV阳性,检出率为69.05%。序列分析和系统发育分析显示,本试验克隆的32个BCoV阳性样本中的S1亚基序列、HE基因片段和N基因片段均有独特的遗传进化趋势;首次成功分离到1株牦牛源BCoV,蚀斑纯化后病毒TCID50为10-7.17·0.1mL-1,鉴定结果显示该毒株S基因发生了重组事件。本试验结果表明青藏高原牦牛源BCoV感染率很高,且有独特的遗传进化趋势。

The aim of this study was to investigate molecular prevalence of bovine coronavirus(BCoV)in yak in Qinghai-Tibetan Plateau and to isolate BCoV from clinical samples.RT-PCR assay was used to detect BCoV from 336 diarrhea feces samples of yak from Tibet,Qinghai,Sichuan and Yunnan,and the S,HE,and N gene fragments were further amplified,moreover,positive samples were used to isolate BCoV strain.Results were as follows:Approximate 69.05%(232/336)diarrheic samples were detected BCoV positive by RT-PCR.Sequence and phylogenetic analysis showed that BCoV S1 subunit sequence,HE gene fragment and N gene fragment from 32 clinical samples in this study had unique evolutionary features.Notably,a BCoV strain of yak was successfully isolated firstly,the TCID50 of this strain was 10^-7.17·0.1 mL^-1,and a recombinant event was predicted in S gene of this strain.These results indicate that BCoV widely circulates in yak in the Qinghai-Tibetan Plateau of China and exhibit unique evolution.