Genetic Diversity of Avian Influenza Viruses Detected in Waterbirds in Northeast Italy Using Two Different Sampling Strategies

Simple Summary Avian influenza viruses (AIVs) significantly threaten the global poultry industry, and some strains are capable of infecting mammals, even humans. Wild aquatic migratory birds serve as the natural reservoir for low-pathogenic AIVs and, through migratory flyways, contribute to the global spread and genetic diversification of the virus. It is therefore essential to monitor AIVs circulating in wild hosts. In this study, the collection of cloacal swabs from waterbirds and avian faecal droppings in selected wetlands allowed for the molecular detection and characterization of different low-pathogenic AIV strains circulating in wild birds during the 2021-2022 avian influenza epidemic in the Italian poultry industry.Abstract Avian influenza viruses (AIVs), which circulate endemically in wild aquatic birds, pose a significant threat to poultry and raise concerns for their zoonotic potential. From August 2021 to April 2022, a multi-site cross-sectional study involving active AIV epidemiological monitoring was conducted in wetlands of the Emilia-Romagna region, northern Italy, adjacent to densely populated poultry areas. A total of 129 cloacal swab samples (CSs) and 407 avian faecal droppings samples (FDs) were collected, with 7 CSs (5.4%) and 4 FDs (1%) testing positive for the AIV matrix gene through rRT-PCR. A COI-barcoding protocol was applied to recognize the species of origin of AIV-positive FDs. Multiple low-pathogenic AIV subtypes were identified, and five of these were isolated, including an H5N3, an H1N1, and three H9N2 in wild ducks. Following whole-genome sequencing, phylogenetic analyses of the hereby obtained strains showed close genetic relationships with AIVs detected in countries along the Black Sea/Mediterranean migratory flyway. Notably, none of the analyzed gene segments were genetically related to HPAI H5N1 viruses of clade 2.3.4.4b isolated from Italian poultry during the concurrent 2021-2022 epidemic. Overall, the detected AIV genetic diversity emphasizes the necessity for ongoing monitoring in wild hosts using diverse sampling strategies and whole-genome sequencing.