Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenical ly distinct. from the standard North American isolates, were selected to im munize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV m onoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidin e-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and re ference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions u p to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to reco gnize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggest ing they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cell s transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural pr otein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our know ledge, these are the first mAbs produced against South American BVDV isolat es and will certainly be useful for research and diagnostic purposes.