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目的 用RT PCR方法扩增肾综合征出血热病人血样品中的汉滩病毒RNA ,对扩增产物进行测序并分析汉滩病毒基因组的变异。方法 用引物PⅠ PⅡ对 2 2例血样品中的汉滩病毒RNA进行第一次PCR扩增 ,PⅠ PⅡ扩增样品再用PⅠ 1 PⅡ 2引物进行槽式RT PCR扩增 ,将扩增效果良好的PCR产物进行测序 ,对测序结果用生物化学软件进行同源性比较和进化树分析。其中HV2 2 6样品用引物P1 P2进行RT PCR扩增。结果 2 2例肾综合征病人血样品用PⅠ PⅡ和P1 P2引物扩增有 19例阳性 ,其中编号W6 13、W1141样品用PⅠ 1 PⅡ 2进行槽式扩增后对其扩增产物直接进行测序。HV2 2 6号样品用引物P1 P2进行扩增后 ,其扩增产物也直接进行测序。对该 3株序列测定结果进行比较分析发现 ,编号W6 13、W1141样品核苷酸序列与HV114株的同源性为 84% ,而与HTN76 118株的同源性为 99%。HV2 2 6号样品与HV114有 95 %的同源性 ,而与HTN76 118有 82 %的同源性。进化树分析表明 ,W6 13,W1141与HTN76 118株位于同一分枝 ,而HV2 2 6与HV114、A9株处于同一分枝。结论 湖北地区汉滩病毒至少存在 2个亚型 ,其中W6 13、W1141与HTN76 118的核苷酸序列高度同源 ,属于同一亚型。而HV2 2 6与HV114属于另一亚型。
Objective To amplify Hantaan virus RNA from blood samples of patients with hemorrhagic fever with renal syndrome by RT-PCR and to sequence the amplified products and analyze the variation of Hantaan virus genome. Methods The first PCR amplification of Hantaan virus RNA was carried out in 22 blood samples with primer PⅠPⅡ. The amplified PCR products were amplified by PCR using PⅠ 1 PⅡ 2 primer, and the amplification effect was good Of the PCR products were sequenced, sequencing results using biochemical software for homology comparison and phylogenetic tree analysis. The HV2 2 6 samples were amplified by RT PCR using primer P1 P2. Results The blood samples of 22 cases of patients with renal syndrome were amplified with PⅠPⅡ and P1P2 primers and 19 cases were positive. The samples of W113 and W1141 were directly amplified by PCR using PⅠ 1 PⅡ 2 . HV2 26 samples amplified with primers P1 P2, the amplification products were also directly sequenced. The comparison of the three sequencing results showed that the nucleotide sequences of W613 and W1141 had 84% homology with HV114 and 99% with HTN76 118. HV2 26 samples have 95% homology to HV114 and 82% homology to HTN76 118. Phylogenetic tree analysis showed that W6 13, W1141 and HTN76 118 were located in the same branch, while HV2 26 was in the same branch with HV114 and A9. Conclusion There are at least two subtypes of Hantaan virus in Hubei Province. The nucleotide sequences of W6 13, W1141 and HTN76 118 are highly homologous and belong to the same subtype. While HV2 2 6 and HV114 belong to another subtype.