Molecular Characterization of Highly Pathogenic Avian Influenza H5N1 Viruses Circulating in Bulgaria During 2024-2025: Evidence for Hidden Circulation and Zoonotic Risk Markers.

The highly pathogenic avian influenza virus HPAI A(H5N1) genotype AF was detected in southern Europe during the 2021/2022 season and spread widely. It emerged in Bulgaria in 2022/2023, mainly affecting mallard ducks. The DA genotype of the virus was detected in a diverse group of birds, including wild birds, zoo birds, and domestic poultry, across a wide area of eastern and southern Europe in 2023. In Bulgaria, following its introduction in 2023, the DA genotype became the predominant virus in laying hens. During 2024-2025, DA spread throughout the country, displacing AF from mallard flocks. The predominant subtype in Europe in 2025 was H5N1 genotype DI.2. This genotype became dominant after December 2024, accounting for over 90% of viruses within the EA-2024-DI genotype lineage, and has been detected in a wide range of bird species. In Bulgaria, DI.2 was identified in only one outbreak in a flock of laying hens in autumn 2024 and in a single case involving a western marsh harrier (Circus aeruginosus) in early 2025. These observations are consistent with a pattern of putative hidden circulation of avian influenza virus in duck farms in Bulgaria, potentially establishing a cycle of continuous circulation of the same viral subtype. In this study, we analysed viruses originating from Bulgaria, with a particular focus on EA-2024-DI genotype DI.2, and examined mutations related to host cell receptor binding, host specificity shifts, ligand binding, antibody recognition sites, viral oligomerization interfaces, and other functional regions. Some of these mutations have been associated with antigenic drift, immune escape, and virulence. Importantly, several are linked to changes in host specificity, a critical step in the potential transition of avian influenza viruses to humans. Consequently, such mutations represent key factors in the spread of highly pathogenic avian influenza and may pose a pandemic risk.