1. This study characterised the H5N8 highly pathogenic avian influenza (HPAI) viruses, clade 2.3.4.4b from backyard waterfowl selected from Menoufia governorate during 2022. Genetic and antigenic analysis of H5N8 isolates was performed and the genetic markers for waterfowl brain invasion were analysed by whole genome sequencing.

2. Molecular diagnosis of field samples showed that, out of 30 tested waterfowl flocks showing respiratory and nervous symptoms of the disease, 13 duck flocks and 11 geese flocks were confirmed positive for H5N8 by RT-qPCR.

3. The whole genome sequencing for four H5N8 strains were performed from duck and geese flocks. Phylogenetic analysis of the hemagglutinin (HA) gene revealed that the 2022 isolates clustered with Russian and European-like strains. All strains had a polybasic cleavage site (PLREKRRKRGLF), indicating high pathogenicity. Mutations, such as A140T, G268E, R82K, and I162M, were identified and linked to antigenic drift and adaptation.

4. Brain-derived isolates showed specific HA mutations (e.g. F244L, I248S, K234R) compared to tracheal isolates, which suggested possible roles in neurotropism. Internal genes revealed mammalian adaptation markers such as 504?V (PB2), 127?V and 672?L (PA), and 133?G (PB1), with additional unique mutations observed H5N8 strains isolated from geese in 2022. Reassortment analysis for the 8 gene segments showed that H5N8 duck strains isolated in 2018 belonged to genotype G1, while the 2022 strains belonged to genotype G4. This confirmed changes after 2018 and reflecting continuous viral evolution through segment reassortment.

5. Antigenic analysis revealed that the newly introduced H5N1 virus clade 2.3.4.4b is antigenically similar with H5N8 viruses from the same clade, while different from viruses in other clades.