COMPARISON OF EQUINE ARTERITIS VIRUS ISOLATES USING NEUTRALIZING MONOCLONAL-ANTIBODIES AND IDENTIFICATION OF SEQUENCE CHANGES IN G(L) ASSOCIATED WITH NEUTRALIZATION RESISTANCE

Three murine monoclonal antibodies (MAbs) that neutralize equine arter itis virus (EAV) infectivity were identified and characterized. The an tibodies, 93B, 74D(B) and 38F, recognized the major envelope glycoprot ein (G(L)) encoded by open reading frame (ORF) 5 in immunoblots and by immunoprecipitation. All three MAbs were used to compare the Bucyrus isolate of EAV and MAb neutralization-resistant (NR) escape mutants wi th the vaccine virus and 19 independent field isolates of EAV by virus neutralization. The different abilities of the MAbs to neutralize vir us isolates indicated that they recognize non-identical epitopes. Susc eptibility to virus neutralization could not be used to distinguish vi ruses from acutely and persistently infected horses. Comparison of the ORF 5 nucleotide and deduced amino acid sequence from NR and neutrali zation-sensitive virus isolates revealed amino acid sequence changes a t positions 99 and 100 which correlate with the NR phenotype. Addition al unique changes in the amino acid sequence of MAb NR viruses at posi tions 96 and 113 may also contribute to neutralization resistance. The sequence data further showed that the Bucyrus-derived viruses contain one N-glycosylation site, whereas the held isolates DL8 and DL11 poss ess two sites, both of which are used. Most of the non-conservative am ino acid sequence changes were located within the second half of the N -terminal hydrophilic domain. Sequence changes within the first half o f the N-terminal ectodomain, the predicted transmembrane domain and th e C-terminal hydrophilic domain were mainly silent base substitutions or resulted in conservative amino acid substitutions, suggesting that these regions of the protein are functionally conserved.