肾综合征出血热汉坦病毒分型及序列特征的研究

目的 分析黑龙江省肾综合征出血热（HFRS）患者携带汉坦病毒的基因型别及序列特征.方法 采集临床诊断为肾综合征出血热病人的全血60例,其中通过金标法检测出血热特异性抗体阳性40例、阴性20例,血凝块提取汉坦病毒RNA,应用RT-Nest-PCR及核苷酸序列测定分型技术对黑龙江地区的汉坦病毒进行分型研究,设计汉坦病毒M片段通用引物（MOF,MOR）及HTNV和SEOV的M片段特异性引物（HTNMF、HTNMR,SEOMF、SEOMR）,应用Nest-PCR进行扩增,回收阳性扩增产物进行基因序列测定,将所测基因序列与国内外HV病毒株序列进行核苷酸同源性分析,构建M基因种系进化树.结果 出血热特异性抗体阳性的病例中扩增出HTN型19份,抗体阴性的病例中扩增出HTN型1份、SEO型l份.总体来看黑龙江省流行的汉坦病毒毒株主要为HTN型和SEO型,以HTN型为主.经核苷酸及氨基酸同源性和种系进化分析表明其黑龙江地区的病毒株同源性较高,其中HTN型与76 ～ 118株同源性较高,SEO型与Z37株的同源性较高.结论 黑龙江省的HV感染病例多为HTN型,与其他GenBank中的各地标准株的核苷酸及推导的氨基酸序列均有一定差异,在系统进化树中分析HTN型与76-118株较为接近.SEO型与国内Z37较为接近.结合临床症状发现其临床症状较为相似的在系统进化发生树中较为接近.

Study on the Genotypes and Sequence Characteristics of Hanta Virus

Objective To study the genotypes and sequence characteristics of hantavirus carried by patients of HFRS in Heilongjiang province. Methods Whole blood from 60 patients with the clinical diagnosis of HFRS was collected. All cases were tested with gold standard assay,40 cases of which were positive,20 cases negative. Then the Hantaviruses RNA from the blood clots were extracted. Genotyping Hantaviruses in Heilongjiang was Analyzed using RT - Nest - PCR and gene sequencing. The universal primers of hantavirus M gene （MOF,MOR） and the M genespecific primers of HTNV and SEOV （HTNMF, HTNMR; SEOMF, SEOMR）were designed and uesed in Nest - PCR. All products were collected and completed sequencing. A nucleotide homology analysis was done on the gene sequence and the other HV sequence, and phylogenetic tree of M gene was builf. Results Totally 19 cases of HFRS antibody positive and lcase of HFRS antibody negative were amplified by HTN genotype specific primes successfully. 1 case of HFRS antibody negative was amplified by SEO genotype specific primers. Generally, the hantavirus strains were mainly HTN and SEO genotype in Heilongjiang province, and HTN genotype mostly. The results of homology analysis of nucleotides and amino acid and the Phylogenetic tree indicated that the vi- rus strains in Heilongjiang province were highly homogenous. Compared to the strains in the GenBank of other area,the HTN and the strain 76 - 118 were high homology, and so were the SEO and Z37. Conclusion Most HV infections in Heilongjiang Province were caused byHTN genotype. The nucleotide and deduced amino acid sequences of HTN and the strains in the GenBank of other areas had some differences. The HTN and 76 - 118 were closer in the phylogenetic tree, and the SEO appeared to be closer to the Z37. Combined with clinical symptoms, we found that the clinical symptoms were more similar when the strains that caused them were closer in the phylogenetic tree.