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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(Seoul virus,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MEGA 5.0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(M.musculus)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位点间有5个氨基酸差异位点(V494L、C546S、Y626C、M630L和V637I),小家鼠G蛋白365~769位点间主要有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、I140M、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 genetic evolution of Hantavirus (HV) host virus in Jilin Province. Methods The murine lung samples were collected from different areas of Jilin Province. Positive samples were identified by indirect immunofluorescence assay (IFA). The viral RNA was extracted by Trizol reagent. The M and S fragments were amplified by RT-PCR The nucleotide and amino acid sequences of M and S fragments were analyzed by DNASTAR software and compared with Seoul virus (SEOV) reference strain. The amino acid differences of amino acid sequences were analyzed by using MegAligh software Analysis; Phylogenetic analysis using MEGA 5.0 software. Results A total of 719 rodents were captured, including 555 R. norvegicus, 74 A. agrarius and 90 M. musculus. The positive rate of HV antigen was 1.39% (10/719). Of the 10 positive samples, seven were from Rattus norvegicus, two from Mus musculus and one from Apodemus agrarius. The full-length S primers were used to amplify the full-length S gene of the 10 strains of viruses. By homology analysis, nucleotide homology between the sequences was between 92.8% and 95.1% The acid homology was between 90.2% and 94.7%. The partial DNA fragments amplified by M primer showed homology between 96.5% -99.5% by homology analysis , While the nucleotide homologies with other SEOVs ranged from 91.5% to 97.7%. All 10 viruses belonged to SEOV. There are five amino acid difference sites (V494L, C546S, Y626C, M630L and V637I) between the 365th and the 769th sites of G-protein of Apodemus agrarius. There are five amino acid difference sites (C378G / R, F420L, Q436P / R, E535G / A and C589G / W), but there were also differences at 378, 436, 535 and 589 in the same species. P298L variation. The results showed that there are many variations of N protein in Mus musculus, mainly in M24K / N, A37D, I140M, M173K, D192E, L219V, P229A, D237E, H265Y, V284L, T333S, A389G and V425E. The 10 viruses isolated can be divided into two different evolutionary branches according to different origins. Conclusions The results show that HV in host animals in Jilin Province is still dominated by SEOV, and its genetic evolution shows diversity.