Fluorescent antibody test for rapid detection of West Nile virus antigen in avian tissues

An indirect fluorescent antibody test (IFA) was developed to detect West Nile virus (WNV) antigens in tissues from avian species. The test samples used in the study consisted of 100 sets of tissues from dead crows that had been collected during the 2001 surveillance in Connecticut. The test tissues were punctured with a fine point Dacron cotton-tipped applicator and smeared in duplicate on 10-well diagnostic printed glass slides. Among several fixatives tested, 4% paraformaldehyde was the best. Reagent calibration for the IFA test was done in WNV-infected Vero cells and control uninfected Vero cells. Optimized antibody and fluorescent conjugate concentrations were then applied for the detection of WNV antigen on fixed tissue impression smears. Several tissues, including brain, heart, liver, kidney, and spleen were tested by the IFA test. The brain and heart seemed to be unsuitable for the test because of excessive background. Both virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) were used for validation, with the latter technique having a higher sensitivity. Therefore, IFA results were compared with RT-PCR results. The diagnostic sensitivity was 96.8% for liver, 96.4% for kidney, and 100% for spleen. The diagnostic specificity was 69% for liver, 95.3% for kidney and 95.8 for spleen. The IFA test performed best with spleen and kidney. The IFA test described here is a useful, practical, and rapid test for screening for WNV.