Wastewater genomic surveillance of influenza A and B reveals seasonal and off-season circulation differences between municipal sites and an international airport, Germany, 2024

Wastewater surveillance has proved to be a valuable tool for monitoring human pathogens. Whole-genome sequencing of SARS-CoV-2 in wastewater has been extensively applied, allowing for early detection of variants and population-level surveillance of viral diversity and abundance; however, limited studies have applied this methodology to influenza. Here, we investigated influenza A and B diversity year-round in five metropolitan areas and one major international airport in Germany during 2024 using wastewater-based whole-genome sequencing. We applied amplicon-based sequencing covering all eight genome segments of both influenza A and B to 401 wastewater samples, yielding 339 influenza-positive samples. Bioinformatic analysis classified sequencing data, enabling subtype assignment and segment-level assessment of circulating influenza diversity. We recovered partial to near-complete genomes for influenza A and B for many influenza-positive samples. The results largely paralleled clinical subtype patterns across Germany, with winter peaks of H1N1 and small proportions of influenza B. In contrast, airport samples showed persistent off-season detection, a higher baseline of read counts across the entire year, and a greater proportion of H3N2 and influenza B reads. Segment-level characterization also revealed low-abundance reads assigned to several avian influenza subtypes, including H5N1, which were not clinically reported. These findings demonstrate the utility of wastewater whole-genome sequencing for population-level influenza surveillance. Distinct seasonal and off-season patterns highlight the value of year-round monitoring at international airports and seasonal monitoring in municipal systems, providing a framework to guide future influenza wastewater surveillance strategies.