Molecular Characterization, Pathogenicity, and Phylogenetic Analysis of H5N8 and H5N1 Avian Influenza Strains Isolated From Poultry in Egypt 2023

T HE widespread transmission of highly pathogenic avian influenza (HPAI) clade 2.3.4.4b has resulted in significant economic and public health concerns. This study analyzed four HPAI isolates-two H5N1 and two H5N8-collected from poultry and backyard birds across four Egyptian governorates. The isolates, obtained from chickens, turkeys, and ducks, exhibited high mortality rates. The misuse of avian influenza vaccines and weak biosecurity measures has contributed to viral evolution, forming distinct phylogenetic clusters that may compromise current vaccine effectiveness. Molecular characterization confirmed that all isolates belong to clade 2.3.4.4b. Phylogenetic analysis of the hemagglutinin (HA) gene revealed 97.1%-99.2% identity with Egyptian H5 viruses from 2016-2023 but only 84.8%-85.8% similarity with older H5N1 (clade 2.2.1.1) strains. HA gene comparison showed 85.8% identity with the rHVT-H5 vaccine strain and 76.6% with the H5N2 vaccine strain, raising concerns about vaccine efficacy. The presence of a polybasic cleavage site (PLREKRRKR/GLF) confirmed their high pathogenicity. Notable mutations, including N182, G221, Q222, G224, T140A, and N236D, indicate ongoing genetic adaptation. Pathogenicity studies in SPF chicks yielded IVPI scores of 2.55 (H5N1) and 2.48 (H5N8). Experimental infections showed species-specific mortality, with H5N1 causing 100% mortality in chickens and quails but 33% in ducks, while H5N8 caused 93% mortality in chickens and 40% in ducks. Prolonged viral shedding in Muscovy ducks underscores their role in viral transmission. These findings highlight the urgency of improving biosecurity and updating vaccine strategies to combat evolving HPAI strains in Egypt.