安徽省2011年新分离的19株狂犬病病毒的N基因序列分析

目的对安徽省2011年新分离的19株狂犬病病毒(Rabies virus,RABV)的N基因进行序列分析,并比较其与代表性RABV街毒株及疫苗株之间的差异。方法采集安徽省淮北地区狗肉市场的犬脑组织121份,伤人犬脑组织10份,采用直接免疫荧光法和双抗体夹心ELISA法检测RABV抗原,并通过颅内接种昆明乳鼠进一步鉴定毒株;RT-PCR扩增分离病毒的N基因,与pMD18-T载体连接测序后,与代表性RABV街毒株及疫苗株的N基因序列进行比对,并进行遗传学分析。结果在送检的131份犬脑组织样品中检出RABV阳性19株,其中伤人犬脑组织阳性率为90%,狗肉市场犬脑组织阳性率为8.26%。19株RABV N基因核苷酸序列同源性为97.5%～99.7%,推导的氨基酸序列同源性为98.7%～100%;与代表性人用和兽用RABV疫苗株相比,N基因核苷酸序列同源性为85.4%～89.9%,推导的氨基酸序列同源性为94.9%～99.1%,核苷酸序列的变异主要为同义突变。分离的19株RABV在系统进化树上高度聚集,与以往国内分离的代表性街毒株系统进化关系较近,在同一个分支(HN10除外)与我国疫苗株CTN-181株亲缘关系最近,处于同一个亚群。结论从安徽省伤人犬和狗肉市场的犬脑组织中成功分离并鉴定了19株RABV,这些病毒株均属于基因I型RABV,且具有地域性特征。本研究对特定区域RABV的流行及监测具有一定的意义。

Sequence analysis on N genes of 19 rabies virus strains isolated from Anhui Province, China in 2011

Objective To analyze the sequence of N genes of 19 rabies virus(RABV)strains isolated from Anhui Province,China in 2011 as well as its difference with other representative street strains and vaccine strains.Methods The brain tissues of 10 dogs biting people and 121 dogs from dog meat market in Huaibei District,Anhui Province were collected,and detected for RABV antigen by direct immunofluorescent assay,then further identified by intracranial inoculation to Kunming suckling mice.The N gene of RABV was amplified by RT-PCR and cloned into vector pMD18-T for sequencing and genetic analysis,and the results were compared with those of other representative street strains and vaccine strains.Results Of the 131 brain tissue samples of dogs,19 were positive for RABV.The positive rates of RABV in the dogs biting people and the dogs from dog meat market were 90% and 8.26% respectively.The homologies between nucleotide and deduced amino acid sequences of N gene of the 19 positive strains were 97.5% ~ 99.7% and 98.7% ~ 100% respectively.However,the homologies of nucleotide and deduced amino acid sequences of N gene of the 19 strains to those of representative strains for production of vaccines for human use and for animal use were 85.4% ~ 89.9% and 94.9% ~ 99.1% respectively.Most of the nucleotide variations were synonymous mutations.The 19 strains were highly aggregated on phylogenetic tree,which belonged to the same branch with the representative street strains isolated in China previously(except HN10)and to the same subgroup as that of CTN-181 strain.Conclusion Nineteen rabies virus strains were successfully isolated and identified from the brain tissues of dogs biting people and dogs from dog meat market in Anhui Province,which belonged to RABV genotype I and showed regional characteristic.The study is of a certain significance in monitoring the epidemic of RABV in specified regions.