Monoclonal antibodies directed against conserved epitopes on the nucleocapsid protein and the major envelope glycoprotein of equine arteritis virus

We recently developed a highly effective immunization procedure for the gen eration of monoclonal antibodies (MAbs) directed against the porcine reprod uctive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171-186, 1999), The same method was used to produce a panel of 16 MAbs specific for the eq uine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleoca psid (N) protein, and five MAbs recognized the major viral envelope glycopr otein (G(L)). Two of the EAV G(L)-specific MAbs and one antibody of unknown specificity neutralized virus infectivity, A comparison of the reactivitie s of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G (L) MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conse rved epitopes, In contrast, the two MAbs with the highest neutralization ti ters bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the fiel d isolates. Ten of the virus isolates reacted with only one of these two MA bs, indicating that they recognized different epitopes, The G(L)-specific M Abs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) we re used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c 1-specific NR escape mutant confirmed that these antibodies reacted with di stinct antigenic sites. Immunoelectron microscopy revealed for the first ti me that the antigenic determinants recognized by the anti-G, MAbs were loca lized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral en velope than the nonneutralizing anti-G(L) MAbs.