汉坦病毒M、S基因分型及种系进化分析

摘要

目的 探讨湖南省肾综合征出血热(HFRS)患者及宿主动物黑线姬鼠携带汉坦病毒的基因型别及序列特征.方法 应用免疫荧光试验(IFA)对鼠肺标本进行粗筛,提取IFA阳性鼠肺组织及HFRS患者血清总RNA,设计汉坦病毒M、S基因特异性引物,应用RT-PCR技术进行扩增,回收阳性扩增产物并克隆到pMD-18T载体进行序列测定,将所测序列与国内外HTNV及SEOV病毒株序列进行核苷酸同源性分析,应用MEGA5.0.4软件构建M、S基因种系进化树,结果 IFA阳性鼠肺标本荧光显微镜下可见做在的黄绿色针尖大小样颗粒;RT-PCR法扩增汉坦病毒M、S基因,HTNV通用引物可见490bp与593 bp的阳性条带;阳性产物进一步应用HTNV分型引物进行M、S基因检测,分别扩增出-约242 bp与276 bp大小的条带;SEO型特异性M、S基因片段内侧引物扩增反应均为阴性;编号为Hunan01、Hunan02、Hunan03的M、S基因序列与HTNV型84FLi株、SN7株的同源性最高,与SEOV型Gou3株的同源性最低;种系进化分析表明,Hunan01、Hunan02、Hu-nan03为汉坦病毒H5亚型.结论湖南省存在HTNV型感染病例与宿主动物,基因分型技术能进一步对病毒亚型进行鉴定.%To investigate the genotype and characteristic of hantavirus M and S hemorrhagic fever with renal syndrome (HFRS) , lung tissues of rats were screened by immunofluorescent antibody assay (IFA) , and the total RNA was extracted from the serum of HFRS patients and IFA positive lung tissues. RT PCR was applied to amplify the M and S gene of hantavir us after specific primers were designed. PCR positive product was recovered and purified before cloning to the pMD 18 T vector and the sequencing was carried by ABI3730 gene sequencer. The obtained sequencings were compared to the HTNV and SEOV sequences of hantavirus for nucleotide homology analysis and applied MEGA5. 0. 4 software to construct the phylogenetic tree. The yellow green needle like particles were scattered in the positive lung tissues under fluorescence microscope. The PCR prod ucts of M and S gene was about 490bp and 593bp with general primers and the further amplification with HTNV typing primers to the M and S gene was respectively 242bp and 276bp, while the amplified results of M and S gene were negative with the primers of SEOV. The sequences of HunanOl, HunanO2 and HunanO3 had a high nucleotide homology to the 84FLi and SN7 strains,which had a lower homology to the Gou3 strain. HunanOl, HunanO2 and HunanO3 were belonged to H5 subtype of HTNV. In conclusion, the technology of genotyping is appropriate for hantavirus diagnosis, and the HTNV patients and ani mal hosts are exist in Hunan Province.