Adaptive VP1 sites 93 and 97 modulate antigenic evolution and receptor binding of Senecavirus A: Phylogenetic analysis and validation with recombinant viruses.

Abstract

Since 2014, Senecavirus A (SVA) has caused recurrent vesicular disease outbreaks in pigs and continues to circulate widely. However, functional evidence is limited as to whether mutations accumulated during long-distance spread alter receptor engagement or antigenicity, constraining sequence-based risk prioritization. We analyzed 263 quality-controlled, coding-complete SVA genomes after removing recombinant signals and integrated time-scaled phylogenetics with structural mapping and reverse genetics. Root-to-tip regression supported temporal signal. In time-scaled inference, the root location was assigned the highest posterior probability to the United States among sampled locations, and BSSVS supported inter-country connectivity links. Markov jump summaries, conditional on posterior ancestral-state reconstructions, were consistent with international transitions beginning around 2006. SkyGrid reconstruction suggested an increase in effective population size from about 2010-2020, while estimates near the ends of the time series may be sensitive to uneven sampling. Site-level analyses identified candidate selection signals mainly in capsid proteins and the RNA-dependent RNA polymerase, and structural mapping highlighted VP1 residues 62, 63, 93, and 97 near the ANTXR1 receptor-binding interface. We generated V93A, D97G, and double-mutant viruses and assessed replication, ANTXR1 binding, and cross-neutralization. Mutants displayed reduced replication in IB-RS-2 cells and porcine intestinal organoids, weaker ANTXR1 binding, and altered neutralization profiles relative to the parental strain. These results demonstrate that global SVA diversification is consistent with repeated connectivity and substantial drift under heterogeneous surveillance, while VP1 positions 93 and 97 exert clear biological effects and merit surveillance and antigenic follow-up.