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目的了解江西省啮齿动物携带的肾综合征出血热汉坦病毒的基因型别。方法采用Vero-E6细胞对8份免疫荧光汉坦病毒抗原阳性鼠肺标本进行病毒分离,采用直接免疫荧光法和RT-PCR方法对培养物进行鉴定,对分离株部分M基因进行序列测定和分析。结果 1份标本培养后经直接免疫荧光试验检测到汉坦病毒特异性免疫荧光颗粒,培养物能在Vero-E6细胞中稳定传代,毒株命名为AYW89-15。毒株核酸经汉坦病毒M基因特异性分型引物进行RT-PCR检测,获得汉滩型汉坦病毒(Hantaan virus,HTNV)预期大小的目的片段;通过序列分析发现,分离株与HTNV参考株核苷酸同源性为83.1%~94.7%,氨基酸同源性为95.0%~99.0%,与汉城型等其他型别汉坦病毒核苷酸和氨基酸同源性均低于75%。进化树分析表明AYW89-15位于HTNV所在的枝系。结论新分离株AYW89-15为HTNV。
Objective To understand the genotypes of Hantavirus in rodent-bearing hemorrhagic fever with renal syndrome in Jiangxi Province. Methods Vero-E6 cells were used to isolate the virus from eight Hantavirus immunogen-positive lung samples. The immunofluorescence and RT-PCR methods were used to identify the culture. The M gene sequences of the isolated strains were sequenced and analyzed . Results One specimen was detected by direct immunofluorescence assay. The specific immunofluorescent particles of Hantavirus were detected and the culture was stable in Vero-E6 cells. The strain was named AYW89-15. The target nucleic acid of Hantaan virus (HTNV) was detected by reverse transcription-polymerase chain reaction (RT-PCR) using specific genotyping primers of Hantavirus M gene. By sequence analysis, it was found that the isolate and HTNV reference strain Nucleotide homology was 83.1% ~ 94.7%, amino acid homology was 95.0% ~ 99.0%, homology with Han type and other types of Hantavirus nucleotides and amino acids were less than 75%. Phylogenetic tree analysis showed that AYW89-15 is located in the branch where HTNV is located. Conclusion The new isolate AYW89-15 is HTNV.