Genomic characterization of circulating respiratory syncytial virus strains in Lombardy before and after the introduction of nirsevimab prophylaxis

Respiratory syncytial virus (RSV) significantly affects infants and vulnerable populations. Monoclonal antibodies (mAbs), together with vaccine strategies, mark progress in RSV prevention, necessitating ongoing monitoring of viral evolution and resistance. RSV-positive upper respiratory tract samples from patients who tested positive for RSV between October 2023 and March 2025 were collected. Whole genomes were obtained using amplicon protocol, sequenced on Illumina platforms, and assembled with vcf-consensus-builder. Maximum-likelihood trees (IQ-TREE; GTR + F+I+G6/G4) and Bayesian coalescent methods (GTR + F+I+G6/G4; constant population and strict molecular clock) defined clades and large phylogenetic clusters (LPCs: posterior probability = 1, sequences > 10). Amino acid variability in nucleoprotein (N) and fusion protein (F), targets of the small-molecule inhibitor zelicapavir and the mAbs, was investigated. Multivariable analysis compared 2023–2024 and 2024–2025 seasons. 302 RSV sequences (129 from 2023 to 2024, 173 from 2024 to 2025), mostly from infants (≤ 1 year, 56.0%) and males (57.3%), were retrieved; 72.7% of cases had lower-respiratory-tract infections, 67.2% were hospitalized, 15.0% were intensive-care-unit (ICU)-admitted, and 17 were nirsevimab breakthrough infections (N_BI). The most frequent clades were A.D.3 (26.8%) and B.D.E.1 (20.2%), followed by A.D.1.2 (17.5%) and A.D.5.2 (14.2%). Four LPCs were identified: two of 56 A.D.3 and 17 A.D.1.2 strains were detected exclusively in 2024–2025. mAb resistance-associated mutations were identified in two 2023–2024 and two 2024–2025 samples (palivizumab: K272E in A.D.5.3 and N262Y in A.D.3; nirsevimab: N208D in B.D.E.1 and K68I plus L204S in a B.D.E.1 N_BI). Among the 17 N_BI, the F antigenic site Ø S213R was also detected in an A.D.1.2 sample. No clesrovimab resistance was detected, though four mutations (three in 2024–2025 season) were found at its binding site (K433R in B.D.4.1.1; C439R in A.D.1.2; N437S in A.D.5.2; N444S in A.D.3). The N-I129M substitution associated with zelicapavir resistance was detected with an intrapatient relative-abundance of 0.05 in a 2024–2025 B.D.E.1 sample. By multivariable analysis, the 2024–2025 season was associated with an increased number of sequences in LPCs, increased F site Ø variability and Ct values, and fewer ICU entries ( P < 0.05). RSV genomic surveillance in Lombardy revealed ongoing viral diversification and F protein variability, underscoring the need for continuous monitoring to inform prophylaxis strategies.