吉林省汉坦病毒宿主动物携带病毒的遗传进化分析

Genetics and evolution of viruses carried by animal hosts of Hantaviruses in Jilin Province,China

目的对吉林省汉坦病毒（Hantavirus，Hv）宿主动物携带病毒进行遗传进化分析。方法采集吉林省不同地区鼠肺样品，应用间接免疫荧光法（indirect immunofluorescence assay，IFA）鉴定出HV感染的阳性样本，利用Trizol试剂提取病毒RNA，RT-PCR法扩增阳性样本中M和S片段基因，并进行测序，应用DNASTAR软件对M和S片段基因的核苷酸同源性进行分析，并与汉城病毒（Seoulvirus，SEOV）参考株进行比较；利用MegAligh软件进行蛋白序列中氨基酸差异性分析；利用MEGA5.0软件进行系统发生分析。结果共捕获719只啮齿动物，其中褐家鼠（R.norvegicus）555只，黑线姬鼠（A.agrarius）74只，小家鼠（胍m12sclzllzs）90只，HV抗原阳性率为1.39％（10／719）；10份阳性样本中，7份来自褐家鼠，2份来自小家鼠，1份来自黑线姬鼠。用全长s引物扩增得到的10株病毒全长s基因片段，经同源性分析比较发现，各序列间的核苷酸同源性在92.8％～95.1％之间，与其他SEOV核苷酸同源性在90.2％～94.7％之间；用M引物扩增得到的部分基因片段，经同源性分析比较发现，各序列间的核苷酸同源性在96.5％～99.5％之间，而与其他SEOV核苷酸同源性在91.5％～97.7％之间；10株病毒全部属于SEOV。黑线姬鼠G蛋白365～769位点问有5个氨基酸差异位点（V494L、C546S、Y626C、M630L和V637I），小家鼠G蛋白365～769位点问主要有5个氨基酸差异位点（C378G／R、F420L、Q436P／R、E535G／A和C589G／W），但同种间在378、436、535和589位点也有差异；黑线姬鼠N蛋白1～430位点间存在P298L变异；小家鼠N蛋白变异位点较多，主要在M24K／N、A37D、1140M、M173K、D192E、L219V、P229A、D237E、H265Y、V284L、T333S、A389G和V425E变异。分离得到的10株病毒根据不同的来源地可分为两个不同的进化分支。结论研究表明吉林省宿主动物携带的HV仍以SEOV为主，其遗传进化呈现多样性。

Objective To analyze the genetics and evolution of viruses carried by animal hosts of Hantaviruses （HV） in Jilin Province, China. Methods Rat lung samples were collected in various regions of Jilin Province, from which the HV-positive ones were identified by indirect IFA. Viral RNA was extracted with Trizol reagent. The M and S gene fragments in positive samples were amplified by RT-PCR and sequenced. The homology of nucleotides of M and S genes was analyzed by using DNASTAR software, and compared with the reference strain of Seoul virus （SEOV）. The amino acid diversity in protein sequence was analyzed by MegAlign software. Phylogenetic analysis was performed by using MEGAS. 0 software. Results A total of 719 rodents, including 555 （77. 2%） R. norvegicus, 74 （10. 3%） A. agrarius and 90 （12. 5%） M. musculus were captured in Jilin Province, of which 10 （1.39%） were positive for HV antigen, including 7 from R. norvegicus, 2 from musculus and 1 from A. agraritts. The homology of nucleotide sequences of amplified full-length S genes from 10 strains was 92. 8% - 95. 1%, while that to other SEOV nucleotide sequence was 91. 5% ~ 97. 7%. All the 10 strains were SEOV. Five amino acid variation sites were observed at sites 365 - 769 of G protein of A. agrarius, i.e. V494L, C546S, Y626C, M630L and V637I. Similarly, five amino acid variation sites were also observed at sites 365 ～ 769 of G protein ofM. musculus, i.e. C37G/R, F420L, Q436P/R, E535G/A and C589G/W. However, the variations were also observed at sites 378, 436, 535 and 589 of the same species. The variation of P298L was observed at sites 1 ～ 430 of N protein of A. agrarius. Variations were observed in several sites of N protein of M. musculus, including M24K / N, A37D, I140M, M173K, D192E, L219V, P229A, D237E, H265Y, V284L, T333S, A389G and V425E. The isolated 10 strains belonged to two branches on phylogenetic tree. Conclusion Most of Hantaviruses carried by animal hosts in Jilin Province were SEOV, of which the genetics showed a diversity.