浙江省首次发现新型布尼亚病毒感染病例及分离病毒分子鉴定

First confirmation of new bunyavirus-infected patients in Zhejiang province and molecular identification of the isolated virus

目的 了解浙江省是否存在新型布尼亚病毒潜在自然疫源地,分离疑似病例血清中新型布尼亚病毒并进行鉴定.方法 免疫荧光法检测浙江省台州地区野生啮齿动物不同组织标本中新型布尼亚病毒抗原.实时荧光定量RT-PCR检测疑似病人血清中新型布尼亚病毒核酸,扩增产物进行序列测定.Vero细胞分离疑似病人血清中新型布尼亚病毒,以新型布尼亚病毒株核衣壳蛋白编码基因为靶基因,采用RT-PCR及扩增产物测序对分离的疑似新型布尼亚病毒株进行鉴定,另对该序列进行同源性分析和比较.结果 70只野生啮齿动物中,免疫荧光法检测阳性率为5.71%.实时荧光定量RT-PCR检测结果显示,4例疑似病人血清中有两例检测结果阳性.1例阳性血清样本中分离出1株疑似新型布尼亚病毒,RT-PCR和测序结果证实该病毒确为新型布尼亚病毒,其核衣壳蛋白编码基因序列与湖北省新型布尼亚病毒分离株相似性高达92.2%,但与国内其他地区新型布尼亚病毒分离株序列差异较大.结论 首次证实浙江省存在新型布尼亚病毒自然疫源地及感染病人,不同群新型布尼亚病毒分布可能存在一定的地理差异.

Objective To determine the potential natural foci of new bunyavirus,and isolate and identify the new bunyavirus strain in sera from suspected new bunyavirus-infected patients.Methods Immunofluorescence assay was used to detect the antigens of new bunyavirus in different tissue specimens of wild rodent animals in Tiantai area of Zhejiang province.Fluorescence quantitative real-time RT-PCR was applied to detect the viral nucleic acid in sera of suspected new bunyavirus-infected patients and the amplification products were analyzed by sequencing.The new bunyavirus in the pateints＇sera was isolated using Vero cells.Using nucleocapsid protein encoding gene of new bunyavirus as the target gene,the isolated suspected new bunyavirus strain was identified by RT-PCR and sequencing of the amplification product.Moreover,sequence identity of the amplification product of nucleocapsid protein encoding gene of new bunyavirus was analyzed and compared.Results Of the 70 wild rodent animals,5.71% were positive in the immunofluorescence assay.The fluorescence quantitative real-time RT-PCR confirmed that two of the four detected patients＇serum specimens were positive.One suspected strain of new bunyavirus was isolated from one pf the two positive patients＇serum specimens.The results of RT-PCR and sequencing confirmed that the viral strain exactly belongs to new bunyavirus with 92.2% sequence identity to that of the new bunyavirus isolates in Hubei province but distinct with the new bunyavirus isolates from other areas in China.Conclusion The presence of natural foci of new bunyavirus and new bunyavirus-infected patients in Zhejiang province are firstly confirmed by this study.There is a geographical diversity of the distribution of new bunyavirus in different groups.