浙江省丽水地区2株汉坦病毒分离株的型别鉴定和基因差异研究

目的 从浙江省丽水地区肾综合征出血热(HFRS)疫区阳性鼠肺标本中分离汉坦病毒(HV),并对丽水分离株进行分子生物学鉴定,分析M和S片段基因序列以确定毒株的型别和基因差异程度.方法 收集宿主动物标本,采用直接免疫荧光检测鼠肺标本HV抗原.将HV抗原阳性的鼠肺标本接种Vero-E6细胞,分离HV.提取病毒总RNA,应用反转录聚合酶链反应(RT-PCR)扩增病毒M和S基因全片段,克隆入质粒载体,纯化后测定序列,并同HV其他病毒株进行比较.结果 成功分离到2株HV,扩增出2株病毒的M和S全片段并测定了序列,经对核苷酸序列分析表明,2株病毒与现有的HTNV有最高的同源性,均为HTNV,但与国内外其他的HTNV核苷酸差异高达13.4%～20.7%和10.3%～16.1%.用M和S片段核苷酸序列所构建的系统进化树显示,2毒株分在HTNV发生群,与HTNV的Z10、A9、Z5病毒株亲缘关系最近,但独自构成一进化支.结论 浙江丽水分离株可以定型为HTN型病毒,与国内外其他的HTN型病毒基因差异较大.

Study on the difference of genes and the type identification of hantavirus from Lishui,Zhejiang province

Objective To isolate hantavirus from Lishui county-one of the epidemic regions for hemorrhagic fever with renal syndrome(HFRS),in Zhejiang province,and to identify the serotype and molecular/biological characteristics of a new HTN subtype hantavirus(HV)strains,hopefully to provide evidence for HFRS prevention and therapy.Methods Data on the host animals was collected from Lishui,Zhejiang province in 2007.Direct immunofluorscece assay was adopted to determine HFRS antigens and the lung tissues from HV infeeted Vero-E6 cells for HV isolation,then total RNA was extracted from Hantavirus Lishui strains and amplified by RT-PCR M,S segments of strains genome were also clened and sequenced and compared with those of other strains of HV Results 2 strains virus(ZLS6-11 and ZLS-12)Were successfully isolated from 7 positive lung samples of mice and were identified as HTNV by anti-McAb and phylogenetic analysis.With sequence compation.We found that 2 strains with complete M and S segment had higher homology with HTN-type strains than with other types of HV,but 13.4%-20.7%and 10.3%-16.1%of the genes were found which were difierent from HTNV.The phylogenetic trees constructed by complete S and M segment showed that ZLS6-11 and ZLS-12 strains were located in HTNV group,and structured independent embranchment.Conclusion ZLS6-11 and ZLS-12 Strains were believed to belong to HTN-type and phylogenetically different from the HTNV.