陕西省汉滩病毒分离株全基因组序列分析及生物学特性研究

Complete genome sequence analysis of the Hantaan viruses isolated from Shaanxi Province and its Characteristics

目的 从肾综合征出血热（HFRS）患者外周血单个核细胞（peripheral blood leukocytes,PBMC）中分离汉坦病毒,并对分离株全基因组序列测定及进化分析.方法 采集急性期HFRS患者外周血,分离PBMC后与Vero-E6细胞混合培养分离汉坦病毒,通过Real-time PCR和IFA法检测,扩增分离株全基因组片段进行序列测定及分析,并采用SX26分离株样本与HFRS患者恢复期血清和免疫接种者血清各50份进行微量中和试验.结果 成功分离到两株汉滩型毒株并获得全基因组序列,进化分析显示与西安分离株XAAa10091712同源性较高;微量中和试验结果显示50份HFRS患者恢复期血清分别中和分离株SX26与疫苗株Z10,中和抗体滴度差异有统计学意义,另50份免疫接种者血清样本针对SX26和Z10毒株产生的中和抗体阳转率分别为72％及88％,差异有统计学意义.结论 通过PBMC分离汉坦病毒可作为病毒分离的另一种途径;流行株与Z10疫苗株的抗原性存在差别,疫苗株产生的抗体中和流行株效果较弱,提示病毒进化过程中已发生部分变异,为疫苗免疫效果研究和肾综合征出血热的防治工作提供依据.

Objective To sequence the whole genome and to analyze the molecular and evolutionary of Hantavirus（HV） isolated from peripheral blood leukocytes （PBMC） of HFRS patients.Methods PBMC were isolated from peripheral blood of HFRS patients and cocultured with Vero-E6 cells.Quantitative realtime RT-PCR and IFA tests were used to detect virus titer.The genome segments were amplified by PCR with predesigned primers,whole sequence were obtained and further study on its evolution was analyzed.The differences between the isolate strains and Vaccine strain were tested by microneutralization test.Results Two Hantaan virus strains were isolated from PBMC of HFRS patients successfully.The whole genomic fragments were obtained.Molecular phylogenetic analysis result showed that both of the strains had the comparatively high similarity with XAAa10091712 strain.The seroneutralization titer of SX26 and Z10 had significant differences.Conclusion Hantaan virus could be successfully isolated from PBMC of HFRS patients,and it proved to be a feasible way of isolation of hantavirus from peripheral white blood cells;The microneutralization test proved that the antigenicity of two strains exist significant differences.It provided basis for the further study of hantavirus epidemic strains in this region.