[Establishment of indirect immunofluorescence assay (IFA) for detection of IgG antibody against new bunyavirus].

OBJECTIVE: To develop an indirect immunofluorescence assay (IFA) for detection of IgG antibodies against new bunyavirus. METHODS: The antigen slides were prepared with 5 new bunyavirus strains isolated using Africa green monkey kidney (Vero) cells. Specificity and sensitivity evaluation of IFA were carried out by optimizing working conditions of IFA. Using established IFA, serum samples from both acute and recovery phases were tested for 126 cases with fever thrombocytopenia and leukopenia syndrome in Xinyang, Henan province in 2007 - 2011. The results were compared with detections by RT-PCR. RESULTS: The new bunyavirus stable immunofluorescence specific WZ69 strain was selected to prepare antigen slides of IFA. The optimum conditions of IFA were: optimum dilution for primary antibody (serum) and secondary antibody (isosulfocyanic acid fluorescence marked goat anti-human IgG antibody) was 1:40 and 1:150 respectively. The optimum dilution for Evans blue in secondary antibody was 1:20 000. Among the 126 patients, 96 paired serum specimens were tested positive to the new bunyavirus and 30 patients were tested negative to the virus. The positive rate of antibodies was 76.19%. There was no significant difference in results between IFA and RT-PCR (72.22% (91/126)) (P > 0.05). CONCLUSION: The IFA has high sensitivity and specificity with easy operation. It can be used in detecting the new bunyavirus infection in patients with fever, thrombocytopenia and leukopenia syndrome.

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

OBJECTIVE: To culture, isolate and identify new bunyavirus in Vero cell line. METHODS: 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. RESULTS: 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. CONCLUSION: Vero cell line is suitable for culture, isolation and identification of new bunyavirus.