Two residue mutations in VP1 of FMDV serotype A cause significant antigenic variation via reorientation of G-H loop revealed by lineage-specific neutralizing antibodies from the natural hosts

Foot-and-mouth disease virus (FMDV) serotype A exhibits extensive genetic and antigenic diversity, complicating vaccine strain selection. Here, we identified two distinct panels of host-derived neutralizing antibodies (nAbs) specific to the SEA97 lineage. The majority of nAbs (10/11) targeted antigenic site 2, while one bovine-derived antibody recognized residue 138 in the VP1 G-H loop. Compared with the ancestral A/AF72 strain of the A22 lineage, the T138A substitution and 140 N deletion on VP1 significantly enhanced the cross-neutralizing activity of SEA97 lineage-specific antibodies against the VP1 G-H loop and antigenic site 2, extending their coverage to the mutated A/AF72 strain. Neutralization assays with porcine immune sera further demonstrated that these mutations are key contributors to antigenic divergence between the A22 and SEA97 lineages. Structural simulations indicated that the T138A substitution and 140 N deletion in VP1 shifted the G-H loop conformation of A/AF72 strain toward that of SEA97 lineages, implicating them as molecular determinants of lineage-specific antigenicity. These findings suggest that substitution or deletion of upstream polar residues of the RGD motif reshaped G-H loop orientation, thereby contributing to antigenic variation in Asia topotype of FMDV serotype A.