Background and aim: Highly pathogenic avian influenza virus (HPAI) H5N1 continues to threaten poultry biosecurity worldwide due to rapid antigenic drift and reassortment. Since late 2020, clade 2.3.4.4b strains have dominated outbreaks across multiple continents. This study genetically characterized H5N1 isolates circulating in Upper Egypt during 2023-2025, clarified their phylogenetic origin, and compared them with vaccine strains used nationally.

Materials and methods: A total of 100 samples from 25 broiler flocks showing respiratory and neurological symptoms across New Valley, Assiut, and El-Minya governorates were examined. Specimens were screened for avian influenza subtypes (H5N1, H9N2, H5N8, H6N2) and differential viral pathogens (Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, infectious bursal disease virus) using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Positive isolates were propagated in specific-pathogen-free embryonated chicken eggs and identified through hemagglutination and hemagglutination inhibition assays. Partial hemagglutinin gene sequencing and phylogenetic analyses were performed using Molecular Evolutionary Genetics Analysis version 7.0.

Results: HPAI-H5N1 was detected in 16% (4/25) of flocks, showing 25%-50% mortality. Five isolates displayed high hemagglutination titers (7-8 log2) and were confirmed as H5N1 subtype by RT-qPCR. Phylogenetic analysis classified New Valley-1-H5N1-2023 and New Valley-2-H5N1-2024 within clade 2.3.4.4b. These strains shared 96%-99% nucleotide and amino acid identity with recent Egyptian and Eurasian H5N1 isolates but only 72%-84% with currently used Egyptian vaccine seeds (e.g., MEFLUVAC [Kemin Industries, Inc., USA], EgyFlu [Nagy Awad Group, Cairo, Egypt]). Mutations R72S, A83D, and T140A were identified in receptor-binding and antigenic regions of hemagglutination, implying potential antigenic drift.

Conclusion: This is the first documentation of clade 2.3.4.4b HPAI-H5N1 circulation in broiler flocks of Upper Egypt. The low genetic relatedness to existing vaccine strains indicates probable vaccine mismatch and reduced protection. Continuous molecular surveillance, integration of full-genome sequencing, and periodic vaccine seed updates are essential for effective containment. Enhanced monitoring at the domestic-wild bird interface will help mitigate cross-species transmission and align with One Health strategies for zoonotic risk reduction.