Functional implications of respiratory syncytial virus F sequence variability: a comparative analysis using contemporary RSV isolates

Respiratory syncytial virus (RSV) remains a major global health issue. Therapeutic options are limited, but new prophylactics, all targeting the fusion (F) glycoprotein, were recently licensed. Although F sequence variation is limited, it is unclear if and how this variability translates to phenotypical differences. We analyzed full F sequences of 105 contemporary RSV isolates, gathered from children presenting with acute lower respiratory tract infections. Based on F antigenic site variation, 20 isolates were selected for functional characterization. Although RSV F diversity is generally low, the overall mean pairwise distance between RSV-A strains is higher than RSV-B (0.014 vs 0.008, <i>P</i> < 0.001). Sequence variability in the known monoclonal antibody (mAb)-binding sites seems insufficient to explain the divergent and often subgroup-dependent sensitivity to mAb neutralization. Despite comparable growth rates, mean syncytium size was higher (mean 61.68 [SD 24.51] vs 31.72 [SD 11.64], <i>P</i> < 0.001), and mean syncytium frequency was lower (mean 25.37 [SD 9.20] vs 47.85 [11.52], <i>P</i> < 0.001) in RSV-A strains. For all isolates, higher temperatures were associated with increased inactivation, although some RSV-A isolates displayed manifestly higher stability. Genomic and phenotypic variability among our contemporary RSV isolates was limited, with noticeable exceptions. The availability of new prophylactics targeting RSV F corroborates the importance of continued RSV surveillance to identify changes in the F sequence, including mutations that reduce mAb efficacy or lead to escape mutants. Surveillance should include both sequencing data and an evaluation of sensitivity to prophylactic antibodies, using contemporary clinical isolates.IMPORTANCERespiratory syncytial virus (RSV) is a major cause of respiratory infections in young children worldwide. Recent progress has led to new ways to prevent serious RSV-associated disease. The virus's fusion (F) protein is a key focus for vaccine development because it helps the virus enter host cells and is well conserved across different virus strains. However, it is unclear if small differences in the F protein sequence could affect how the virus behaves <i>in vitro</i>. In this study, we, therefore, analyzed 105 RSV samples from children under two who presented with respiratory infections. We selected 20 samples (12 RSV-A and 8 RSV-B) for functional testing, based on their F protein sequences. Phenotypic differences between clinical isolates and reference strains, such as virus stability at 4°C and susceptibility to monoclonal antibody neutralization, highlight the importance of using viruses isolated from recent clinical samples. Although significant functional differences were observed in traits related to the F protein, both between the RSV subgroups and within, the underlying molecular mechanisms remain unclear. Ongoing monitoring of RSV is critical to ensure current and future vaccines remain effective.