湖南省2008-2009年狂犬病病原学监测及病毒基因特征分析

摘要

目的 分析2008-2009年湖南省狂犬病病原学监测结果及新分离病毒的N基因分子特征.方法 采用直接免疫荧光法(DFA)、巢式PCR检测狂犬病监测标本,阳性标本应用ABI3730测序仪对N基因进行全序列测序;运用生物信息学方法分析N基因特征及序列同源性,构建系统进化树,分析新分离病毒株遗传特征并与以往分离株比较.结果 1451份监测犬脑组织标本中DFA初筛阳性31份,阳性率为2.14％;31份DFA阳性标本经巢式PCR复核,17份阳性,阳性率为1.17％;巢式PCR检测疑似狂犬病病例唾液、脑脊液、血清及尿液标本56份,3份阳性,阳性率为5.36％;新分离的阳性株与巴斯德株进行N基因序列比较,核苷酸和氨基酸同源性均在87.2％～ 87.9％之间;成功构建系统进化树,新分离的20株病毒全部属于基因Ⅰ型.新分离病毒株与湖南省内、邻省和国际株比较存在不同的亲缘关系.结论 湖南省狂犬病病例及犬携带病毒的情况较为稳定,流行株仍为基因Ⅰ型,未发生变异.%Objective To analyze the etiology of rabies in Hunan province and the genetic characteristics of rabies N gene isolated from 2008 to 2009.Methods Direct immunofluorescence assay (DFA) and nested PCR were employed to detect the monitoring samples including brain tissues of dogs and saliva,serum or urine which were collected in 2008 to 2009,from the rabies patients.Positive samples were sequenced by ABI3730 gene analyzer for the full length of the N gene target.The homology and hpylogeography of the rabies virus were analyzed after the phylogenetic tree was constructed by Blast,Clustal W and Mega 4.0 software.Results Of the 1451 tissue samples from the dogs' brain,31 were positive under DFA and the positive rate was 2.14％.The DFA positive samples were redeteeted by RT-PCR and the positive rate was 1.17％.56 samples of saliva,serum and urine samples were detected by RT-PCR from the rabies patients,with 3 positives and the positive rate was 5.36％.The length of nest PCR products were 255 bp.The rates of homology to the nucleotide and the amino acid of rabies N gene were 87.2％-87.9％ after compared to the pasture strain.The phylogenetic tree was successfully built and 20 strains isolated lately belonged to the rabies gene type Ⅰ.Conclusion The epidemic situation of human and dogs rabies in Human were relatively stable,with all the isolated rabies virus belonging to genotype Ⅰ,without any variation.