Mutations and predicted glycosylation patterns in respiratory syncytial virus isolates correlate with disease severity.

Respiratory syncytial virus (RSV) remains an important cause of lower respiratory tract infections in young children, producing mild to life-threatening disease. Although rapid viral evolution through genetic drift is well established, the structural and functional impacts of specific pathoadaptive mutations linked to enhanced virulence are poorly defined. We investigated these relationships by isolating RSV from available nasal swabs of five hospitalized infants during the 2022-2023 winter season and conducting comparative viral genomic analysis. Severity of disease was evaluated using a validated clinical scoring system. Whole-genome sequencing followed by reference-guided assembly and structural modeling revealed distinct amino acid polymorphisms correlating with disease severity. Phylogenetic analysis placed all isolates within the RSV-A GA2.3.5 G clade. Isolates from mild moderate and severe cases clustered in A.D.1.5 and A.D.1.8 subclades. Nineteen amino acid differences were associated with clinical severity and isolates from moderate or severe cases replicated more rapidly in vitro than mild isolates. Computational glycosylation predictions indicated an increasing number of glycosylation sites in the G protein corresponding with greater disease severity. Together, these data suggest that specific pathoadaptive mutations may contribute to enhanced viral replication and severity, and are relevant for future surveillance efforts and the development of immune-based strategies targeting virulence-associated residues.