The molecular epidemiology of human respiratory syncytial virus in a tertiary paediatric hospital between 2020 and 2024

Background: Respiratory syncytial virus (RSV) is a leading cause of respiratory illness in young children, contributing significantly to morbidity and mortality and placing a substantial burden on resource-limited healthcare systems. Molecular epidemiological data from sub-Saharan Africa remain limited. This study aimed to investigate the genetic diversity and evolution of RSV in a paediatric population in Cape Town, South Africa, from September 2020 to June 2024. Methods: Respiratory syncytial virus-positive respiratory samples with a cycle threshold (Ct) < 30 from patients at Red Cross War Memorial Children’s Hospital were sequenced and analysed. Phylogenetic analysis of the partial RSV genome (F and G genes) was conducted using reference sequences derived from a global sequence database. Mutational profiles were assessed for relevance to monoclonal antibody and vaccine effectiveness. Results: Of the 100 selected samples, 42 were sequenced, and 40 had sufficient coverage for analysis. Sequencing failure was associated with higher Ct values, older sample age and suboptimal storage at −20°C. Respiratory syncytial virus-A predominated (78.6%), with A.D.5.1 as the dominant lineage; RSV-B strains (16.7%) clustered within B.D.E.1 and B.D.4.1. Genotypes GA2.3.5 (RSV-A) and GB5.0.5a (RSV-B) were observed, consistent with post-pandemic global trends. G-gene analysis revealed multiple substitutions (G224E, I134K, K262E) suggestive of immune pressure. The F-gene remained more conserved; substitutions such as T122A, S211N and S389P were detected but were not linked to reduced susceptibility to monoclonals. Conclusion: This study demonstrated the co-circulation of genetically diverse RSV-A and RSV-B lineages in children between 2020 and 2024. No established resistance-associated substitutions to currently licensed RSV monoclonal antibodies were identified. Continued genomic surveillance is warranted to monitor RSV evolution. Contribution: Continued genomic surveillance is essential to strengthen molecular epidemiology and guide public health preparedness.