Advancing Genomic Surveillance of Respiratory Syncytial Virus in Portugal Through an Adapted Amplicon-Based Whole-Genome Sequencing Workflow.

BACKGROUND:Respiratory syncytial virus (RSV) poses a significant global health burden, especially among young children. Whole-genome sequencing (WGS) is key for tracking RSV evolution and epidemiology, underscoring the importance of establishing efficient workflows for routine surveillance. OBJECTIVES:To optimize a streamlined, one-step, multiplex RT-PCR method for RSV WGS, enabling precise genetic characterization of circulating RSV viruses across Portugal. STUDY DESIGN:A one-step RT-PCR protocol was adapted from a published method and used to characterize RSV-positive samples collected by the National RSV Surveillance Network (VigiRSV) from 2021 to 2024. Of the 1167 samples received, those with real-time PCR cycle threshold values below 25 were considered eligible for sequencing. From this subset, 166 samples were randomly selected for assay development and validation, ensuring proportional geographic and seasonal representation. RESULTS:From the 166 samples tested, genome completeness averaged 92.69% (95% CI: 90.15%-95.23%), with an interquartile range of 6.60%, reflecting a generally consistent sequencing performance. This resulted in 134 near-complete genomes (≥30 × depth of coverage across ≥90% of the reference genome). Phylogenetic analysis revealed the circulation of 13 RSV A lineages and three RSV B lineages across Portugal over the study period. CONCLUSIONS:This adapted protocol achieved high overall horizontal coverage with consistent performance across seasons and regions, demonstrating its reliability for RSV whole-genome sequencing. The strong performance observed over multiple seasons and geographic regions indicates that the method is reproducible and suitable for integration into routine surveillance workflows. These characteristics make it a practical and scalable tool for enhancing RSV epidemiology and public health monitoring.