Abstract
Respiratory syncytial virus (RSV) is a globally circulating virus, causing severe respiratory infections in infants and the elderly. Two RSV vaccines were recently approved, and passive immunization is now recommended in several countries for all newborns, therefore careful surveillance of RSV variants will be important in the future. We therefore develop an integrated whole genome RSV amplification, sequencing and bioinformatics analysis method ("RSVTyper"; https://anaconda.org/bioconda/rsv-typer ) that is suitable for patient samples as well as wastewater. 243 RSV isolates from 2008 to 2025 and wastewater samples from 2023/2024 were amplified in a multiplex tiling PCR with specific primers, generating 39 amplicons ~ 550 bp in length, and sequenced with Oxford Nanopore Technologies. Sequencing reads of patient isolates were analyzed with the RSVTyper pipeline, a tailored analysis pipeline including automatic reference selection, consensus sequence generation and clade assignment via Nextclade. Amplification and sequencing were successful for 213/243 samples. Whole genomes (> 90% coverage) were obtained from 98% of samples with > 10,000 copies/mL, from 3/14 samples with 1,000-10,000 copies/mL, and from none with < 1,000 copies/mL. Average genome-wide mean depth for successfully sequenced samples was 31,800x with an average mean depth of 41,400x in the F gene. Phylogenetic analysis showed seasonal clade and subtype shifting, with a good representation of clade frequencies from patients in wastewater sequences. No variants with known escape mutations from prophylactic monoclonal antibodies were detected. In conclusion, we developed RSVTyper, a cost-effective and scalable RSV sequencing pipeline by integrating sequencing and bioinformatic analysis. It is suitable for both resource-limited settings and high-throughput applications. It will facilitate enhanced RSV surveillance, allowing for further characterization of the RSV genome and rapid detection of potential escape mutations.