Whole-genome sequence characterization of respiratory syncytial virus in the Johns Hopkins Health System during the 2024-2025 respiratory season.

Respiratory syncytial virus (RSV) remains a leading cause of lower respiratory tract infections in young children, older adults, and immunocompromised individuals worldwide. In 2023, the first RSV vaccines and the widespread use of monoclonal antibodies were approved, underscoring the need for genomic surveillance to monitor their effectiveness and the emergence of antibody-resistant genotypes. This study details the whole-genome characterization of RSV strains circulating between September and December 2024. Whole RSV genome amplification was performed using overlapping amplicons, followed by Nanopore sequencing. Viral genomes were analyzed to determine subtypes, clade prevalence, and amino acid substitutions. Epidemiological and clinical data were collected to assess associations with specific viral variants. A total of 336 RSV-positive samples were collected for the study. RSV activity followed expected seasonal patterns, with a predominance of RSV-A (94.9%) and the regional evolution of clade A.D.1.6 (68.34%). Over half of the infections occurred in children aged 1-5 years (56.2%). Comorbidities, including immunosuppression, were significantly associated with severe clinical outcomes. Phylogenetic analyses revealed tight clustering and low intra-clade diversity. Our results highlight the ongoing genetic evolution of RSV-A following the coronavirus disease 2019 (COVID-19) pandemic. Amino acid substitutions were detected across surface and internal proteins, potentially affecting the effectiveness of vaccines, monoclonal antibodies, or antivirals. Predicted gains and losses of glycosylation sites may further influence antigenic presentation. These findings underscore the need for integrated RSV genomic and epidemiological surveillance.IMPORTANCEWith ongoing antiviral drug development, recent approvals of new vaccines, and the continued use of protective antibody therapies, it is crucial to monitor the genetic evolution of RSV. Here, we present a long-amplicon-based whole-genome sequencing protocol for RSV-A and RSV-B, enabling genomic surveillance and representing the first report of whole-genome analysis of RSV strains circulating in areas served by the Johns Hopkins Health System during the 2024-2025 respiratory viral season. Our findings demonstrate the value of whole-genome surveillance in identifying emerging clades and molecular variations, and highlight the continued genomic evolution of RSV-A in the post-COVID-19 era.