Characterization and application of monoclonal antibodies to bovine viral diarrhea virus nonstructural protein 5A

Abstract Bovine viral diarrhea virus (BVDV) nonstruc-turalprotein5A(NS5A)isonetheleaststudiedoftheBVDVproteins. Therefore, to develop a tool for unraveling thefunctions performed by BVDV NS5A, monoclonal anti-bodies (MAbs) were generated by fusion of myeloma cellswith spleen cells from mice immunized with recombinantE.coli-expressedGST-NS5Aprotein.TwoMAbs(1H12and2F9) were established on the basis of immunofluorescenceand Western blot analysis. Both the MAbs were of IgG1subclass and recognized an epitope clustered within theN-terminal region of NS5A. Furthermore, the MAb 1H12was used successfully todetect NS5A protein inBVDV fieldisolates belonging to genotypes 1 and 2. Temporal expres-sion pattern studies during an infectious cycle revealed thatBVDV NS5A could be detected 12–60 h postinfection.Confocal microscopy studies showed a cytoplasmic stainingpattern and revealed that NS5A is localized on the endo-plasmic reticulum membrane in BVDV infected cells.IntroductionMembers of the genus Pestivirus such as Bovine viraldiarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2(BVDV-2), Classical swine fever virus, and Border diseasevirus are economically important animal pathogens affect-ing cattle, pigs, and sheep, respectively. These envelopedpositive-sense RNA viruses together with the generaFlavivirus and Hepacivirus constitute the family Flavivi-ridae. Pestivirus genome RNA is typically about 12.3 kband consists of 5