qRT-PCR and genotyping
hMPV
MINOR
Central laboratory testing of respiratory secretions was performed by Viracor-Eurofins (Lees Summit, MO, USA) using the 510(k) United States FDA-cleared and Conformité Européenne (European Conformity)-marked in vitro diagnostic Lyra® RSV + human metapneumovirus (hMPV) quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay (Quidel Corporation, San Diego, CA, USA). All RSV isolates were subtyped and evaluated for genotypic and phenotypic resistance to nirsevimab and palivizumab. To characterize isolates as RSV subtype A or B, the second hypervariable region of the attachment protein G gene was amplified and sequenced using Sanger sequencing; details of the primer sequences used can be found in Supplementary Table The full-length RSV F gene was amplified and sequenced using NGS assays for determination of consensus polymorphism(s); major variants were defined as having ≥25% MAF, while minor variant(s) were defined as RSV A ≥ 4% to <25% MAF or RSV B ≥ 5% to <25% MAF. NGS assays (Eurofins-Viracor, MO, USA) were validated by measurements of analytical sensitivity, accuracy (i.e., detection of mutation in a mixture of sequences), and precision. RSV A and RSV B F gene sequences in FASTA format were translated to amino acid sequences and aligned against year 2013 Netherlands RSV A/13-5275 or Netherlands RSV B/13-1273 reference sequences and amino acid substitutions in the full-length RSV F protein (AA 1-574), including the nirsevimab binding site (AA 62-69 and AA 196–212Prevalence of all RSV F protein sequence variations were reported relative to consensus sequences of RSV A (