A single amino acid change within antigenic domain II of the spike proteinof bovine coronavirus confers resistance to virus neutralization

The spike glycoprotein is a major neutralizing antigen of bovine coronaviru s (BCV). Conformational neutralizing epitopes of group A and group B monocl onal antibodies (MAbs) have previously been mapped to two domains at amino acids 351 to 403 (domain I) and amino acids 517 to 621 (domain II). To furt her map antigenic sites, neutralization escape mutants of BCV were selected with a group A MAb which has both in vitro and in vivo virus-neutralizing ability. The escape mutants were demonstrated to be neutralization resistan t to the selecting group A MAb and remained sensitive to neutralization by a group B MAb. In radioimmunoprecipitation assays, the spike proteins of ne utralization escape mutants were shown to have lost their reactivities with the selecting group A MAb. Sequence analysis of the spike protein genes of the escape mutants identified a single nucleotide substitution of C to T a t position 1583, resulting in the change of alanine to valine at amino acid position 528 (A528V). The mutation occurs in domain II and in a location w hich corresponds to the hypervariable region of the spike protein of the co ronavirus mouse hepatitis virus. Experimental introduction of the A528V mut ation into the wild-type spike protein resulted in the loss of MAb binding of the mutant protein, confirming that the single point mutation was respon sible for the escape of BCV from immunological selective pressure.