One serotype-specific and three serotype-cross-reactive neutralizing monoclonal antibodies (N-mAbs) were utilized to study the neutralization epitopes involved in the formation of antigenic sites. Single, double, or triple neutralization-resistant mutants were selected by using the N- mAbs sequentially in vitro. Nucleotide and deduced amino acid (aa) substitutions found on the VP7 of such mutants showed that: (i) in addition to variable regions VR-5 (aa 87-100), VR-7 (aa 141-150), VR-8 (aa 208-224), and VR-9 (aa 235-242), aa 290 and 291 in the constant region of the VP7 are involved in neutralization, and (ii) VR-5, VR-7, VR-8, VR-9, and aa 290 and 291 are functionally related to one another. In order to further analyze neutralization sites on the VP7 and to study possible synergistic or antagonistic effects among the N-mAbs employed in this study, antigenic variants were generated in the presence of three N-mAbs. Nucleotide sequence analysis of the VP7-encoding gene of the mutants revealed no synergistic or antagonistic effects among the mAbs used in the selection of neutralization escape mutants.
