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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散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593bp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,分别扩增出一约242bp与276bp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。
Objective To investigate the genotypes and sequence characteristics of hantavirus carrying Apodemus agrarius in patients with hemorrhagic fever with renal syndrome (HFRS) and host animals in Hunan Province. Methods Immunofluorescence assay (IFA) was used to screen the rat lung specimens. Total RNA was extracted from IFA-positive lungs and HFRS patients. Specific primers for Hantavirus M and S gene were designed and amplified by RT-PCR. The positive amplification product was recovered and cloned into pMD-18T vector for sequence analysis. Nucleotide homology analysis was carried out with the sequences of HTNV and SEOV strains at home and abroad. MEGA5.0.4 software was used to construct M and S gene lines Evolutionary tree. Results IFA positive rat lung specimens were observed under the fluorescence microscope scattered yellow-green needle-like particle size; RT-PCR amplification of Hantaan virus M, S gene, HTNV universal primers 490bp and 593bp positive bands; positive products further application of HTNV Genotyping primers were used to detect the M and S genes, and a band of about 242bp and 276bp were amplified respectively. In the SEO-specific M and S gene fragments, the amplification reaction was negative in internal primers. The numbers were Hunan01, Hunan02 and Hunan03 The sequences of M and S genes showed the highest homology with HTNV 84FLi and SN7 strains and the lowest homology with SEOV Gou3 strains. Phylogenetic analysis showed that Hunan01, Hunan02 and Hu-nan03 were the most common Hantavirus H5 subtype . Conclusion There are cases of HTNV infection and host animals in Hunan Province. Genotyping can further identify the virus subtypes.