The molecular epidemiology of respiratory syncytial virus in Ontario, Canada from 2022-2024 using a custom whole genome sequencing assay and analytics package.

Background: Respiratory Syncytial Virus (RSV) infections are a cause of significant morbidity and mortality in children and the elderly. Despite the clinical burden of disease, very little is known about the inter- and intra-seasonal genomic variability of RSV. Furthermore, the recent approval of vaccines and monoclonal antibody therapies will likely lead to higher selective pressure on RSV. Genomic surveillance will be essential to monitor viral changes and inform future therapeutic developments and public health responses. Here, we describe the development of an amplicon-based whole-genome sequencing assay for RSV to enable genomic surveillance.

Methods: A 750-bp overlapping amplicon design was developed to co-amplify RSV-A/-B directly from patient samples collected during two respiratory illness seasons (2022/23, 2023/24) for whole-genome sequencing. RSV subtype, clade, and F-protein antigenic site sequences were determined with a custom analytical pipeline.

Results: Of the 429 specimens included in the study 410 (95.6 %) samples met acceptability. Our data demonstrated co-circulation of both RSV subtypes, with increasing predominance of RSV-A since 2022. There were seven genomic clades of RSV-A, while >95 % of RSV-B belonged to a single clade. 1.5 % of samples had amino acid changes within the binding sites of the current RSV therapeutics Palivizumab or Nirsevimab.

Conclusions: Continuous monitoring of RSV genotypes and mutations will be critical for understanding the impact of new therapeutics and vaccines on RSV epidemiology and detecting emergence of vaccine-escape and/or antiviral resistant mutations.