Avian influenza viruses of the subtype H6Nx are being detected globally with increasing frequency. Some H6Nx lineages are becoming enzootic in Asian poultry and sporadic incursions into European poultry are occurring more frequently. H6Nx viruses that contain mammalian adaptation motifs pose a zoonotic threat and have caused human cases. Although currently understudied globally, H6Nx avian influenza viruses pose a substantial threat to both poultry and human health. In this review we examine the current state of knowledge of H6Nx viruses including their global distribution, tropism, transmission routes and human health risk.

Newcastle Disease Virus (NDV) genotype VII is a highly pathogenic Orthoavulavirus that has caused multiple outbreaks among poultry in Egypt since 2011. This study aimed to observe the prevalence and genetic diversity of NDV prevailing in domestic and wild birds in Egyptian governorates. A total of 37 oropharyngeal swabs from wild birds and 101 swabs from domestic bird flocks including chickens, ducks, turkeys, and pelicans, were collected from different geographic regions within 13 governorates during 2019–2020. Virus isolation and propagation via embryonated eggs revealed 91 swab samples produced allantoic fluid containing haemagglutination activity, suggestive of virus presence. The use of RT-PCR targeted to the F gene successfully detected NDV in 85 samples. The geographical prevalence of NDV was isolated in 12 governorates in domestic birds, migratory, and non-migratory wild birds. Following whole genome sequencing, we assembled six NDV genome sequences (70–99% of genome coverage), including five full F gene sequences. All NDV strains carried high virulence, with phylogenetic analysis revealing that the strains belonged to class II within genotype VII.1.1. The genetically similar yet geographically distinct virulent NDV isolates in poultry and a wild bird may allude to an external role contributing to the dissemination of NDV in poultry populations across Egypt. One such contribution may be the migratory behaviour of wild birds; however further investigation must be implemented to support the findings of this study. Additionally, continued genomic surveillance in both wild birds and poultry would be necessary for monitoring NDV dissemination and genetic diversification across Egypt, with the aim of controlling the disease and protecting poultry production.

Pigeon paramyxovirus serotype 1 (PPMV-1), an antigenic and host variant of avian paramyxovirus Newcastle disease virus (NDV), primarily originating from racing pigeons, has become a global panzootic. Egypt uses both inactivated PPMV-1 and conventional NDV vaccines to protect pigeons from disease and mortality. However, the impact of prevalent strains and the effectiveness of available vaccines in pigeons in Egypt are unclear. This study investigates the virulence of PPMV-1 (Pigeon/Egypt/Sharkia-19/2015/KX580988) and evaluates available paramyxovirus vaccines in protecting pigeons against a PPMV-1 challenge. Ten-day-old specific-pathogen-free (SPF) embryonated chicken eggs infected with this strain exhibited a mean death time (MDT) of 86.4 ± 5.88 h. The intracerebral pathogenicity index (ICPI) in day-old chickens was 0.8, while pigeons experienced an ICPI of 0.96 and an intravenous pathogenicity index (IVPI) of 2.11. These findings classify the strain as virulent and velogenic. Experimental infection of pigeons with this PPMV-1 strain at 10⁶ EID₅₀/0.1 mL resulted in a 62.5% mortality rate, displaying nervous and enteric distress. The virus caused extensive lesions in visceral organs, with strong immunohistochemistry signals in all examined organs, indicating the systemic spread of the virus concurrent to its neurotropic and viscerotropic tropism. Furthermore, vaccination using an inactivated PPMV-1 and live NDV LaSota vaccine regimen protected 100% of pigeons against mortality, while with a single NDV LaSota vaccine, it was 62.5%. The PPMV alone or combined with NDV LaSota induced protective levels of haemagglutination inhibition (HI) antibody titres and reduced virus shedding from buccal and cloacal cavities. Based on generalised linear gamma model analysis, both PPMV-1 and NDV LaSota are antigenically comparable by HI. These findings suggest that using both inactivated PPMV-1 (G-VI) and live attenuated NDV (LaSota) vaccines is an effective prophylactic regimen for preventing and controlling PPMV-1 and NDV in pigeons, thereby reducing the risk of interspecies transmission.

Identification of a universal influenza vaccine candidate has remained a global challenge for both humans and animals. This study describes an approach that uses consensus sequence building to generate chimeric HAs (cHAs): two resultant H1 HA-based chimeras comprising of conserved sequences (within several areas spanning the head and stalk regions) of H1 and H5 or H9 HAs. These cHAs expressed in Drosophila cells (S2) were used to immunize mice. All immunized mice were protected from an infectious H1 virus challenge. Seroconverted mice sera to the H1 cHAs inhibited both the challenge virus and an H5 virus isolate by haemagglutination inhibition (HI) assay. These findings further emphasize that cHAs induce cross-reactive antibodies against conserved areas of both head and stalk regions of the seasonal influenza A (H1N1) pdm09 virus’ HA and holds potential for further development of a universal influenza vaccine.

Newcastle disease virus (NDV) genotype VII is a highly pathogenic Orthoavulavirus that has caused multiple outbreaks among poultry in Egypt since 2011. This study aimed to investigate the genetic diversity of NDV prevailing in domestic and wild birds in Egyptian governorates. A total of 37 oropharyngeal swabs from wild birds and 101 swabs from domestic bird flocks including chickens, ducks, turkeys, and swans were collected from different geographic regions within 13 governorates during 2019-2020. Virus isolation and propagation via embryonated eggs revealed 91 swab samples produced allantoic fluid containing hemagglutination activity, suggestive of virus presence. The use of RT-PCR targeted to F gene successfully detected NDV in 85 samples. The geographical prevalence of NDV spread to 12 governorates in domestic birds, migratory and non-migratory wild birds. Following whole genome sequencing, we assembled six NDV genome sequences (70 - 99% of genome coverage), including five full F gene sequences. All NDV strains carried high virulence, based on the presence of polybasic amino acids (RRQRF) at the F gene cleavage site. Phylogenetic analysis revealed that the NDV strains belonged to class II within genotype VII.1.1. The presence of genetically similar virulent NDV in wild birds further highlights their role in the dissemination of NDV in poultry populations across Egypt. Continued genomic surveillance in both wild birds and poultry would be necessary for monitoring NDV incursions and genetic diversification.

Since its emergence in 1996, the H5 avian influenza virus (AIV) A/Goose/Guangdong/1/1996 (Gs/GD) hemagglutinin (HA) has evolved into over 30 genetically and antigenically distinct clades, including the widespread clade 2.3.4.4b. Vaccination is widely used in endemic regions to reduce poultry losses and zoonotic risk. However, the evolving antigenic diversity and global co-circulation of multiple clades challenge the protective efficacy of poultry vaccines with poor antigenic matching to field strains, resulting in immune escape and vaccine failure. This study aimed to improve vaccine seed selection by identifying HA epitopes contributing to inter-clade antigenic differences. Recombinant clade-representative viruses were generated using HA genes from circulating H5 AIVs via reverse genetics with A/Puerto Rico/8/1934 (PR8) internal and neuraminidase genes. Antigenic relationships were assessed using hemagglutination inhibition (HI) assays with homologous and heterologous chicken antisera. Antigenic cartography revealed a clear distinction of clade 2.3.4.4 from others and notable intra-clade diversity. Pairwise antigenic and genetic comparisons identified 48 putative antigenic residues. These were individually introduced into a candidate HA by site-directed mutagenesis, and antigenic influence was assessed by HI using sera raised against the non-mutated HA. Four residues R82K, A83T, T204I, and F229Y had significant antigenic effects, with three (R82K, T204I, and F229Y [H5 numbering]) being novel. These findings demonstrate that combining serology and in silico residue analysis can identify key antigenic determinants. This work highlights the need for precise antigenic matching in vaccine design and highlights the value of combining molecular and immunological tools to optimize vaccine seed selection against diverse and evolving H5 strains. IMPORTANCE: The continued evolution of H5 avian influenza viruses (AIVs), particularly the Gs/GD lineage, poses major challenges for poultry disease control and zoonotic risk mitigation. Vaccine effectiveness is undermined by antigenic drift and the co-circulation of diverse clades, often leading to mismatches between vaccine and field strains. This study addresses the critical need to improve vaccine strain selection by identifying hemagglutinin (HA) residues driving antigenic variation across H5 clades. Using recombinant viruses, antigenic cartography, hemagglutination inhibition assays, and mutagenesis, we pinpointed 48 key residues, with four R82K, A83T, T204I, and F229Y having major antigenic effects, including three novel markers. These findings advance our understanding of H5 antigenic evolution and provide a framework for predicting vaccine performance. By integrating molecular and serological data, our work informs rational vaccine seed strain selection, contributing to more broadly protective vaccines and improved control of H5 AIV in poultry, while reducing the risk of zoonotic transmission.