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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1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。
Objective To analyze the genotypes and sequence characteristics of Hantaan virus in patients with hemorrhagic fever with renal syndrome (HFRS) in Heilongjiang Province. Methods Sixty patients with hemorrhagic fever with renal syndrome were enrolled in this study. Forty-four cases of hemorrhagic fever-specific antibodies were detected by gold standard method, 20 cases were negative. Hantavirus RNA was extracted from blood clots by RT-Nest- PCR and nucleotide sequencing were used to genotype the Hantaan virus in Heilongjiang Province. The Hantaan virus M fragment universal primer (MOF, MOR) and HTNV and SEOV M fragment specific primers (HTNMF, HTNMR, SEOMF and SEOMR). Nest-PCR was used to amplify and recover the positive amplification products for gene sequence analysis. Nucleotide homology analysis was performed between the tested gene sequences and HV strain sequences at home and abroad to construct the M gene germline evolution tree. Results 19 cases of HTN were amplified in cases of hemorrhagic fever-specific antibody. One case of HTN-type and one case of SEO-type were amplified in case of antibody negative. In general, the prevalence of Hantavirus strains in Heilongjiang Province is mainly HTN type and SEO type, mainly HTN type. Nucleotide and amino acid homology and phylogenetic analysis showed that the homology of the virus strains in Heilongjiang region was high. The homology between HTN strains and 76-118 strains was high, and the homology between SEO type strains and Z37 strains Higher. Conclusion The majority of HV infection cases in Heilongjiang Province are HTN-type. There are some differences between nucleotide and deduced amino acid sequence of the standard strains in other GenBanks. The phylogenetic tree analysis of HTN-type and HT-76-118 strains are more similar. SEO type and domestic Z37 closer. Combined with clinical symptoms found that the clinical symptoms are similar in the phylogenetic tree is more close.