First confirmation of new bunyavirus-infected patients in Zhejiang province and molecular identification of the isolated virus
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. 
 
Key words: 
New bunyavirus;  Virus isolation;  Molecular identification;  Phylogenetic analysis