青海省玉树州不同宿主间狂犬病病毒感染的分子流行病学分析

A molecular epidemiological analysis of rabies viruses infecting different hosts in Yushu,Qinghai

目的分析3株经野狼-家犬-牦牛传播的狂犬病病毒N基因部分核酸序列,以确定其传染源和传染途径。方法分离发病动物的RNA进行DFA检测和RT-PCR检测,阳性者进行N基因部分编码区核苷酸测序检测,采用ATCG ver4软件进行序列拼接,用DNAMAN将所测序列N基因进行核苷酸序列同源性比对,用ClustalX软件Bootstrap N-J Tree法构建系统发生树。结果发病宿主动物脑组织标本和脊髓经DFA检测到内基氏小体和病毒颗粒,RT-PCR扩增狂犬病毒核酸阳性,扩增产物大小为256bp,与目标片段大小一致。RT-PCR扩增产物经纯化后测序,N基因核苷酸序列3毒株间的同源性为99.28%~99.73%,推导的氨基酸序列同源性为99.46%~100.00%,牦牛狂犬病毒核苷酸序列与咬伤犬同源性99.28%,在自毙狼和病犬同源性为100%,牦牛狂犬病毒核苷酸序列与本室2012年测序的玛多犬N基因核苷酸同源性99.7%,与2012年内蒙古野生狸neimeng102B（EU652445.1）的同源性为95.28%,与2008年USA994-dog（EF611868.1）的同源性为95.94%,与2009BeijingHu1（EU700031.1）的同源性为85.9%。3株狂犬病毒株间N基因变异较小,稳定性较高,病毒核苷酸序列均为基因China IV,具有一定的地域性,进化分析显示均属于与野生动物最为相关的北极群。结论狂犬病可经野狼-家犬-家养牦牛传播,本起疫情中野狼为第一传染源。

Objective To analyze part of the nucleic acid sequence of the N gene of three strains of the rabies virus transmitted by wolves,dogs,and yaks in order to determine the source of infection and route of transmission. Methods RNA was isolated from infected animals for use in DFA and RT-PCR.Samples that tested positive were examined to determine the nucleotide sequence of the coding region of the N gene.ATCC software ver4 was to assemble sequences,DNAMAN was used to analyze the homology of the nucleotide sequence of the N gene,and the Bootstrap N-J Tree option in ClustalX software was used to construct a phylogenetic tree. Results DFA detected Negri bodies and virus particles in brain tissue and spinal cord specimens from host animals.Rabies virus RNA was amplified with RT-PCR,yielding a product that was positive for rabies virus RNA.This product was 256 bp in size,which coincided with the expected size of the target fragment.After amplification,the product was purified and sequenced.Among the 3strains of the virus,the similarity of the nucleotide sequence of the N gene was 99.28%-99.73% and the similarity of the amino acid sequence was 99.46%-100.00%.The rabies strain from yaks was 99.28%similar to the rabies strain from dogs.The rabies strain from wolves was 100%similar to the rabies strain from dogs.The nucleotide sequence of the N gene of the rabies strain from yaks was 99.7% similar to that of the rabies strain from mad dogs tested at this laboratory in 2012,95.28% similar to that of the neimeng102 Bstrain isolated from wild raccoon dogs（EU652445.1）in Inner Mongolia in 2012,95.94% similar to that of the USA994-dog strain isolated from dogs（EF611868.1）in the US in 2008,and 85.9% similar to that of the 2009BeijingHu1 strain isolated from humans（EU700031.1）.There was little variation in the N gene of the 3strains of rabies virus.The strains were highly stable,and sequencing indicated that the strains are of the China IV lineage,which has regional features to some extent.Evolutionary analysis indicated that the strains are most closely related to the Arctic clade. Conclusion Rabies can be transmitted by wolves,dogs,and domesticated yaks.Wolves are thefirst source of outbreaks.