Detection of Hemagglutinin H5 Influenza A Virus Sequence in Municipal Wastewater Solids at Wastewater Treatment Plants with Increases in Influenza A in Spring, 2024

Prospective influenza A (IAV) RNA monitoring at 190 wastewater treatment plants (WWTPs) across the US identified increases in IAV RNA concentrations at 59 plants in spring 2024, after the typical seasonal influenza period, coincident with the identification of highly pathogenic avian influenza (subtype H5N1) circulating in dairy cattle in the US. We developed and validated a hydrolysis-probe RT-PCR assay for quantification of the H5 hemagglutinin gene. We applied it retrospectively to samples from four WWTPs where springtime increases were identified and one WWTP where they were not. The H5 marker was detected at all four WWTPs coinciding with the increases and not detected in the WWTP without an increase. Positive WWTPs are located in states with confirmed outbreaks of highly pathogenic avian influenza, H5N1 clade 2.3.4.4b, in dairy cattle. Concentrations of the H5 gene approached overall influenza A virus gene concentrations, suggesting a large fraction of influenza virus inputs were H5 subtypes. At all four H5 positive WWTPs, industrial discharges containing animal waste, including milk byproducts, were permitted to discharge into sewers. Our findings demonstrate that wastewater monitoring can detect animal-associated influenza contributions and highlight the need to consider industrial and agricultural inputs into wastewater. This work illustrates wastewater monitoring's value for comprehensive influenza surveillance, including for influenzas that currently are thought to be primarily found in animals with important implications for animal and human health.