Immunofluorescence Assay for Pestivirus Detection

The immunofluorescence assays were performed as previously described [8 (link)]. Briefly, the cells were fixed with 4% paraformaldehyde for 20 min at 4 °C, permeabilized with 1% (vol/vol) Triton-X 100 (Merck, Darmstadt, Germany) in PBS and stained with the mouse MAb 6A5 [35 (link)] and A18 [36 (link)]. The monoclonal antibody 6A5 was used to detect the E2 molecule of BVDV-1, BVDV-2, BVDV-3, BDV, BUNGO, giraffe pestivirus and LINDA infections. CSFV E2 was detected by MAb A18. Goat anti-mouse IgG conjugated with Cy3 (Dianova, Hamburg, Germany) was used as a secondary antibody. A porcine BUNGO antiserum (748-09.10-1) collected from a naturally infected sow, which had produced an abnormal litter, was kindly provided by the Elizabeth Macarthur Agricultural Institute.