Establishing protocol for sequencing HA, NA and M segments Influenza A (H1N1 & H3N2) from wastewater.

Influenza A virus (IAV) poses significant public health challenges due to seasonal outbreaks and pandemics. Its rapid evolution requires continuous monitoring to inform vaccine and antiviral development. Wastewater-based surveillance (WBS) offers an unbiased and cost-effective approach to track infectious diseases, including dynamics and genomic evolution. We developed a novel primer panel for genomic WBS of IAV, targeting surface protein segments (HA, NA, M) of circulating H1N1 and H3N2 subtypes. Using this panel, we sequenced nucleic acid extracts from 59 wastewater samples from Switzerland during the 2022/2023 and 2023/2024 winter seasons. We found a significant correlation between clinical and wastewater-based abundance estimates of the dominant H1N1 clades in the 2023/2024 season. Additionally, we detected mutations in vaccine and drug target sites, aligning with clinical data. Our findings demonstrate that Influenza A evolution, including acquisition of mutations relevant to vaccine and antiviral strategies, can be effectively derived through wastewater-based surveillance.