Abstract
The emergence of highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b in U.S. dairy cattle highlights the urgent need to understand transmission dynamics within and among farms. House flies (Musca domestica) and blow flies (Calliphoridae), ubiquitous in agricultural settings, are suspected mechanical vectors of numerous pathogens, including viruses. We investigated the presence of H5N1 viral RNA in filth flies collected opportunistically from four H5N1-positive farms (three dairy and one poultry) in California during the 2024 outbreak. H5N1 RNA was detected via qRT-PCR in fly pools from all four locations, with the lowest Ct values (highest viral RNA) in house flies collected near milking parlors and dead animals. Whole-genome sequencing confirmed H5N1 viral genomes in flies from dairy farms, demonstrating high similarity (99.92–99.95%) to the B3.13 lineage circulating in the region and grouping closely with farm-associated viral sequences from milk. Although infectious virus was not detected in this study, our findings suggest that filth flies acquire HPAI H5N1 RNA from their environment, supporting their potential role as sentinels and/or mechanical vectors. These results underscore the critical importance of fly control, targeted surveillance, and integrated pest management strategies in agricultural settings to enhance biosecurity and potentially mitigate HPAI H5N1 transmission. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-29856-9.