Isolation of Genetically Diverse H5N8 Avian Influenza Viruses in Poultry in Egypt, 2019-2021

Abstract

The global spread of avian influenza virus (AIV) of clade 2.3.4.4b since 2016 has caused severe losses in wild birds and poultry and has posed a risk for the infection of mammals including humans. The vaccination of poultry has been used to limit the spread of the virus and mitigate its socioeconomic impact. Here, we describe H5N8 epidemics in chickens, turkeys and ducks from different localities in Egypt from 2019 to 2021. About 41.7% (n = 88/211) flocks were tested positive by RT-qPCR for H5N8 viruses with prevalence rates of 45.1% (n = 65/144) and 34.3% (n = 23/67) in vaccinated and non-vaccinated flocks, respectively. A sequence analysis of the hemagglutinin and neuraminidase genes indicated not only the multiple introduction events of H5N8 viruses in Egypt but also the establishment of endemic viruses in commercial poultry in 2020/2021. The recent H5N8 viruses in poultry in Egypt are genetically distinct from the majority of licensed vaccines used in the field. Together, our findings indicate that poultry in Egypt is an endemic center for clade 2.3.4.4b in the Middle East. The efficiency of current vaccines should be regularly evaluated and updated to fully protect poultry flocks in Egypt against H5N8 viruses.

Keywords: Egypt; H5N8; avian influenza virus; clade 2.3.4.4b; poultry; vaccination failure.

Figure 1

Phylogenetic relatedness of the HA of Egyptian H5N8 to Eurasian viruses. Nucleotide sequences of all-full or near-full HA (n = 2700) genes of H5N8 viruses from Asia, Africa, the Middle East and Europe were retrieved on 21 January 2022 and aligned using MAFFT, and the tree was generated by IQTree. The tree shows clade 2.3.4.4a mainly circulated in wild birds and rarely in poultry from 2013 to 2015, and clade 2.3.4.4B viruses from 2016 to 2021 (A). The phylogenetic tree of the Egyptian H5N8 viruses in clade 2.3.4.4B, including new sequences generated in this study (marked in black triangles), and selected sequences from other countries was generated by MrBayes implemented in Topali v.2 using the GTR + G model. Trees were generated after selecting 4 chains of 10,000,000 replicates and 25% buried-in parameters. Posterior probability values are shown on the main nodes (B). Egyptian H5N8 sequences are shown in red, H5N8 sequences from the Middle East are shown in orange, sequences from Europe are shown in blue and Asian viruses are in black. Branches depicted in cyan in panel A refer to sequences from other African countries.

Figure 2

Phylogenetic relatedness of the NA of Egyptian H5N8 to Eurasian viruses. Nucleotide sequences of all-full or near-full NA (n = 1994) genes of H5N8 viruses from Asia, Africa, the Middle East and Europe were retrieved on 21 January 2022 and aligned using MAFFT and the tree was generated by IQTree (A). The phylogenetic tree of the Egyptian H5N8 viruses, including new sequences generated in this study (marked in black triangles), and selected sequences from other countries was generated by MrBayes implemented in Topali v.2 using the GTR + G model. Trees were generated after selecting 4 chains of 10,000,000 replicates and 25% buried-in parameters. Posterior probability values are shown on the main nodes (B). Egyptian H5N8 sequences are shown in red, H5N8 sequences from the Middle East are shown in orange, sequences from Europe are shown in blue and Asian viruses are in black. Branches depicted in cyan in panel A refer to sequences from other African countries.

Figure 3

Amino acid differences of Egyptian H5N8 from 2017 to 2021 and licensed H5 vaccine strains in poultry in Egypt. Amino acid sequences of Egyptian H5N8 from 2017 to 2021 were retrieved from GISAID. All sequences including those generated in this study were aligned against different vaccine strains. Number of amino acid differences compared to the vaccine strain. Number of amino acid (aa) differences are shown in the y-axis. Each dot represents one HA sequence and sequences are arranged from 2017 to 2021. The figure was generated by GraphPad Prism 9.0.0 and was further edited using Inkscape 0.92. For the abbreviations of the vaccine strains, refer to Table 2.

Figure 4

Number of amino acid differences of Egyptian H5N8 from 2017 to 2021 compared to selected vaccine strains. HA sequences of viruses isolated from 2017 (blue), 2018 (green), 2019 (orange), 2020 (pink) and 2021 (red) were compared to vaccine strains. Clade 2.3.4.4 EGY16/H5N8 (upper left) and Re8 (upper right) are commonly used in poultry in Egypt, while historic H5N2 virus-based vaccines (lower panels) are less used. Although clade 2.3.4.4 viruses are closely related to the Egyptian viruses, there is an increasing number of aa differences in the Egyptian H5N8 viruses particularly from 2020/2021 compared to the H5N2 vaccines. For the abbreviations of the vaccine strains, refer to Table 2.