Integrated molecular, immunoinformatic, and structural analysis reveals emerging antigenic divergence of Foot-and-mouth disease virus serotype O during the 2022–2023 outbreaks in Indonesia

Background and Aim: After more than three decades of freedom from foot-and-mouth disease (FMD), Indonesia experienced widespread outbreaks in 2022–2023, raising major concerns regarding viral evolution and vaccine effectiveness. Foot-and-mouth disease virus (FMDV) serotype O remains the predominant circulating serotype in the region. However, the immunological and structural consequences of recent genetic variation have not been comprehensively evaluated. This study aimed to integrate molecular, immunoinformatic, and structural analyses to characterize FMDV serotype O isolates from West Java and South Sumatra and to assess their implications for antigenicity, immune recognition, and vaccine matching. Materials and Methods: Clinical epithelial samples were collected from naturally infected cattle during outbreaks in West Java and South Sumatra. Viral RNA was extracted, and the capsid genes VP1, VP2, and VP3 were amplified and sequenced. Phylogenetic relationships were inferred using VP1 nucleotide and amino acid sequences. Immunoinformatic analyses were conducted to predict VP1-derived T-cell (BoLA-restricted) and B-cell epitopes, followed by in silico evaluation of antigenicity, allergenicity, and toxicity. Structural analyses included prediction of VP1 ligand-binding pockets and molecular docking between VP1 and Toll-like receptor 7 (TLR7) to explore innate immune recognition. Results: Sequence comparison revealed reduced identity of VP1 (98.26–99.05%) and VP3 (as low as 98.48%) relative to the 2022 Indonesian reference strain. Phylogenetic analysis identified three nucleotide-based clusters and two amino acid–based clusters, indicating intra-country diversification and the emergence of potential micro-lineages. Several amino acid substitutions occurred near known immunogenic regions of VP1, resulting in altered T- and B-cell epitope binding profiles in selected isolates. Predicted epitopes were predominantly antigenic and non-toxic, although some showed potential allergenicity. Structural modeling demonstrated variability in VP1 binding-pocket composition among isolates. Docking analysis revealed favorable VP1–TLR7 interactions, particularly in selected South Sumatra isolates, suggesting strong innate immune engagement. Conclusion: This integrated molecular–immunoinformatic–structural analysis demonstrates that newly circulating Indonesian FMDV serotype O isolates exhibit genetic, antigenic, and structural divergence that may reduce current vaccine matching. Continuous molecular surveillance and regionally adapted vaccine design are therefore essential to maintain effective FMD control in Indonesia. Keywords: foot-and-mouth disease virus, immunoinformatics, Indonesia, molecular docking, phylogenetic analysis, serotype O, TLR7 interaction, VP1 gene.