Naturally occurring mutations in Ross River virus E2 glycoprotein modulate antigenic surfaces without compromising antibody recognition

Ross River virus (RRV) is the most prevalent arthritogenic alphavirus in Australia, yet the structural consequences of envelope protein variation on antibody recognition remain poorly understood. Here, we analysed 85 complete RRV E2 sequences from Australia associated with documented infections and compared them with the earliest available wild type isolate to identify recurrent amino acid substitutions. Five top mutant variants (M1–M5) were selected for detailed investigation. In silico predictions revealed that Y5H and R238K may destabilize local structure, while other substitutions variably modulate protein folding and surface exposure. Structural modelling and molecular docking with the broadly neutralizing RRV-12 Fab antibody indicated that M1 and M3 maintained interactions with epitopes similar to WT, whereas M2, M4, and M5 exhibited redistributed antibody contacts, with M4 showing the most pronounced shift, although overall binding energetics remained largely conserved. Molecular dynamics simulations revealed structural plasticity in surface-exposed loops, suggesting that these mutations may modulate antigenic presentation while preserving attachment and entry functions. These results reveal that naturally occurring E2 mutations can reshape antigenic surfaces without necessarily abolishing antibody recognition. Importantly, the conserved residue engaged by RRV-12 highlights a potential target for rational vaccine design aimed at eliciting broad, cross-protective immunity against circulating RRV variants.