Genomic evolution and minority variant dynamics of RSV during the first season of nirsevimab implementation in Italy (2024-5)

Respiratory syncytial virus (RSV) remains a leading cause of hospitalization despite the recent implementation of infant immunoprophylaxis programs. The inter- and intrahost levels of genetic variation across the genome have not been extensively investigated. Hence, the aim of this study was to characterize the whole genome of RSV strains circulating within two Italian areas during the 2024-5 RSV season, corresponding to the first season after implementation of the national nirsevimab prophylaxis program. Whole genome sequencing (WGS) was performed on 88 respiratory samples that tested positive for RSV. Clinical records showed that none of the sequenced patients had received nirsevimab prophylaxis. The WGS strains obtained in the present study were correlated with the RSV strains circulating in Europe using phylogenetic inference. A newly designed bioinformatic pipeline was developed to evaluate the extent of genetic variation within hosts and reveal the distribution of minority mutations among RSV lineages. RSV-A (n = 54) showed significantly higher levels of nucleotide diversity than RSV-B (n = 34). Among the RSV-A strains, lineage A.D.3 was the most prevalent (35/54, 64%), followed by A.D.1 (18/54, 33%). The A.D.5 lineage was identified in only one strain, while all 34 RSV-B strains were found to be lineage B.D.E. Analysis of variants across the major lineages (A.D.1, A.D.3, and B.D.E) revealed heterogeneous mutation patterns. The G, L, and F genes showed the highest variability. Minority viral variants (0.02-0.05 frequency) followed the same gene-specific dynamics as the broader quasispecies population. A > G transitions consistent with ADAR activity were detected in all samples, but their frequency was similar to the other nucleotide substitutions across the genomes. The analysis of minor variants also targeted mutations known to reduce nirsevimab efficacy, and none were detected. Overall, RSV-A showed substantially greater intragenotypic and intrahost diversity than RSV-B. The elevated diversity in RSV-A resulted from a wider distribution of mutations across the viral genome, including both high-frequency substitutions that shaped the consensus sequence and low-frequency variants contributing to the underlying quasispecies complexity. Although no nirsevimab-treated patients were included in this study, the observed intrahost diversity highlights the importance of continued genomic surveillance to detect potential escape variants as prophylactic programs expand.