Culture, isolation and identification of new bunyavirus in African green monkey kidney(Vero) cells

Abstract

To culture, isolate and identify new bunyavirus in Vero cell line. Samples of 164 new bunyavirus positive by real time RT-PCR detection and well preserved serum specimens were selected from cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) in Xinyang, Henan province in 2009 - 2011. These sera were cultured in Vero cell line and new bunyavirus were detected by observing cytopathic effect (CPE), Real-time RT-PCR, indirect immunofluorescence assay (IFA) and thin-section electron microscopy observation. A total of 10 positive PCR products were selected randomly for sequencing and the results were compared with sequence in Genbank. Among 164 FTLS serum specimens cultured in Vero cell line, no special CPE were observed and 67 strains (40.85%) were positive detected by Real-time RT-PCR. Nucleic acid similarity of 10 specimens were 97.8% - 100% and there's also a high similarity (> 99%) between specimens and new bunyavirus isolates (Accession No. HQ141600.1). Among 67 positive strains, 58 of them showed specific fluorescence particles by IFA. The viral particles were observed to be spheres with a diameter of 80 - 100 nm by electron microscopy. Vero cell line is suitable for culture, isolation and identification of new bunyavirus.