南京都市圈肾综合征出血热疑似病例血清学检测及基因亚型分析

Serological detection and genotype analysis of Hantavirus isolated from patients with suspected hemorrhagic fever with renal syndrome(HFRS)in the Nanjing metropolitan area

目的通过检测肾综合征出血热疑似患者血清标本中特异性抗体和病毒基因组RNA,了解其基因型别及序列特征。方法采用胶体金法、荧光定量PCR法、巢式PCR法进行样本检测,对巢式PCR扩增后的阳性产物进行双向测序获得M基因部分片段序列,与各亚型代表株对应序列进行同源性分析并构建系统进化树,确定其基因亚型。结果122份标本中,IgM阳性27份,阳性率22.1%,IgG阳性57份,阳性率46.7%;荧光定量PCR法检测汉坦病毒核酸阳性13份,阳性率10.7%;15份样本经巢式PCR扩增测序后获得目的片段,其中13株分别属于汉滩病毒H5、H6、H9亚型和汉城病毒S3亚型,另有两株病毒株已形成新的亚型。结论南京都市圈存在汉滩病毒和汉城病毒流行,其中汉滩病毒存在多种亚型。

To investigate the infection status of the population in Nanjing,a total of 122 serum samples from patients with suspected hemorrhagic fever with renal syndrome(HFRS)were collected.Through the detection of specific antibodies and viral genomic RNA in these samples,we assessed their genotype and sequence characteristics,and further performed population infection investigation.Samples were detected with the colloidal gold method,fluorescence quantitative PCR and nested PCR.Partial fragment sequences of the M gene were obtained through bidirectional sequencing of the positive products amplified by nested PCR.Homology analysis was conducted with the corresponding sequences of each subtype representative strain,and a phylogenetic tree was constructed to determine the genotypes.The IgM antibody and IgG antibody positivity rates were 22.1%and 46.7%respectively;13 samples of Hantavirus nucleic acids were analyzed by fluorescence quantitative PCR and showed a positivity rate of 10.7%.A total of 15 samples were amplified and sequenced by nested PCR to obtain the target fragments,among which 13 strains belonged to Hantaan virus subtypes H5,H6,H9 and Seoul virus subtype S3,and two other virus strains had formed new subtypes.Therefore,the Nanjing metropolitan area is a mixed epidemic area with multiple subtypes of circulating HV and the emergence of new subtypes.