Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b in Wild Birds and Live Bird Markets, Egypt

Clade 2.3.4.4 H5Nx influenza viruses have further diversified into several subclades. Sub-clade 2.3.4.4b H5N1 viruses have been widely circulating in wild birds and detected in Europe, Africa, Asia, and North America since October 2020. In this study, we report the first detection of highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses in wild birds and domestic ducks from live bird markets in Egypt. Phylogenetic analysis revealed that the Egyptian H5N1 virus retained the genomic composition of Eurasian strains. Mutations in the viral proteins associated with zoonotic potential and pathogenicity were detected in Egyptian isolates. Egypt is considered a hot spot for the evolution of the influenza virus, so active surveillance of avian influenza viruses in Egypt is warranted.


Introduction
Infectious diseases and pandemics in humans are often caused by pathogens transmitted from non-human animal reservoirs.Influenza A viruses (IAVs) spread among a variety of different hosts and cross species barriers to create new viral strains.Waterfowl serves as the primary reservoir and can perpetuate many avian influenza virus (AIV) subtypes via asymptomatic shedding, which plays an important role in the reassortment and transmission of influenza subtypes to domestic poultry [1].In 1996, a highly pathogenic (HP) AIV (H5N1) of Goose/Guangdong/1/96 (Gs/GD) lineage emerged in Chinese poultry and has been able to cross the species barrier and infect humans, which eventually spread to Europe, Africa, and the North American continent via migratory birds [2][3][4].Due to the accumulation of genetic mutations and reassortment with multiple influenza subtypes, Gs/Gd lineage viruses evolved into nine clades and multiple subclades.
Highly pathogenic AIV (H5N1) was initially introduced into Egypt in 2005 and became endemic in poultry in 2008.Since then, many outbreaks have been reported in domestic poultry farms.Multiple clades of H5N1 Gs/Gd lineage viruses (Clades 2.2, 2.2.1, 2.2.1.1,2.2.1.1a,and 2.2.1.2) were identified [7].The 2.3.4.4bH5N8 virus was first detected in Egypt in wild birds in 2016 [8].Since then, several cases of H5N8 were recorded among domestic poultry in live bird markets, backyard flocks, and commercial farms in several governorates in Egypt.Although all Egyptian H5N8 isolates belong to the clade 2.3.4.4,several independent introductions of the virus have been detected [9].The 2.3.4.4bH5N8 replaced the clade 2.2.1 H5N1 viruses that subsequently disappeared.H9N2 AIVs were widespread in poultry globally and endemic in poultry in many Middle Eastern countries including Egypt, where H9N2 G1-like lineages were introduced in 2010 [10][11][12].Extensive surveillance of the H9N2 virus has indicated that the virus was endemic in Egyptian domestic poultry in different geographical regions across the country and reassortant H9N2 viruses were detected [13].Co-circulation of H5Nx and H9N2 viruses increases the probability of genetic reassortment which might enhance the zoonotic potential.
To monitor the influenza viruses with pandemic potential at the human-animal interface, in this study, we identify the genetic and antigenic characteristics of HPAI H5N1 viruses that were introduced into Egypt through active surveillance of AIVs in live bird markets (LBMs) and migratory wild birds.

Sample Collection
Active surveillance of avian influenza viruses has been conducted in Egypt through collaborative efforts of the Center of Scientific Excellence for Influenza viruses, National Research Centre, Egypt, and Center of Excellence for Influenza Research and Surveillance at St. Jude (Memphis, TN, USA) since 2009.In April 2021, we sampled poultry and wild birds sold in LBMs in Egypt.We collected cloacal and oropharyngeal swab samples, which were kept chilled in virus transport medium until they reached the laboratory.

Sample Screening and Virus Isolation
Samples were thoroughly vortexed prior to viral nucleic acid extraction from 200 µL of viral transport media using either the automated MagNA Pure 96 platform, KingFisher Flex instrument (Thermo Fisher Scientific, Rocklin, CA, USA) or the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions.Nucleic acid extracts were screened by real-time RT-PCR (rRT-PCR) for the presence of the AIVs (universal M-gene) [14], and samples were classified as positive with a Ct value ≤ 36.All positive samples were individually injected into the allantoic cavity of 10-day-old specific pathogen-free embryonated hens' eggs, incubated for 48 h post-injection at 37 • C, and then chilled at 4 • C for 4 h or overnight.Allantoic fluids were then collected and analyzed by the hemagglutination assay (HA) using 0.5% chicken red blood cells (RBCs).Hemagglutination assays (HA) of the allantoic fluids from the inoculated eggs were performed to screen for IAV according to the World Health Organization (WHO) and the World Organization for Animal Health (OIE) protocols.The positive samples were aliquoted and stored at −80 • C.

Sequencing and Sequence Analysis
Viral RNA extracted from allantoic fluid was subjected to reverse transcription to synthesize the first cDNA strand using a SuperScript IV first-strand synthesis kit (Invitrogen, Waltham, MA, USA) and the Uni12 influenza primer.Then, Phusion high-fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) and Uni12/13 primers were used for multiplex PCR of all eight gene segments, and PCR products were purified.Sequencing library preparation was performed by using Illumina's Nextera XT DNA Sample Preparation Kit according to the manufacturer's protocol.Amplicons were sequenced on Illumina's MiSeq platform (Illumina, San Diego, CA, USA) by using the paired-end approach.The eight full segments of each H5N8 virus were assembled using CLC Genomics Workbench, version 21 (CLC Bio, Qiagen, Hilden, Germany).

Nucleotide Sequence Accession Numbers
The nucleotide sequences of the H5N1 AIVs described in this study were deposited in the GenBank database with the accession numbers shown in Table S1.

Results and Discussion
Through surveillance, we isolated H5N1 viruses from one wild pintail duck and three domestic Pekin ducks, A/pintail/Egypt/RA19853OP/2021 in late 2021 and A/duck/Egypt/ BA20360C/2022, A/duck/Egypt/BA20360OP/2022, and A/duck/Egypt/BA20361OP/2022 isolates in early 2022.The analysis of the complete HA gene segment showed that the HPAI H5N1 viruses belonged to phylogenetic clade 2.3.4.4b.The nucleotide sequence identities across all eight segments of the four viruses were 99.5-100%.As a representative virus, A/pintail/Egypt/RA19853OP/2021 (H5N1) had a high nucleotide identity (99-100%) to the HPAI A(H5N1) viruses of clade 2.3.4.4 from Europe and the Middle East (Table 1).These isolates were identified as HPAI viruses that harbored multiple basic amino acids (PLRERRRKR/G) within the cleavage site of the HA gene, which is characteristic of high pathogenicity in chickens.
We combined genome sequences generated in this study with all sequences of H5Nx viruses available in GenBank and the GISAID database (11).Phylogenetic analysis confirmed that the Egyptian A(H5N1) isolates are of clade 2.3.4.4b and clustered with the recent HPAIV A(H5N1) isolates from Europe, Africa, and the Middle East (Figure 1).The clade 2.3.4.4bHA genes of H5 viruses have evolved from a sub-linage under clade 2.3.4.4 which includes several subtypes of H5N1, H5N6, and H5N8 viruses.Our isolates detected in this study clustered with the HA genes of H5N1 viruses contemporarily detected in Europe and the Middle East.The time to the most recent common ancestor (tMRCA) was calculated to explain the emergence of the H5N1 viruses.Taking the intersection of the 95% highest posterior density (HPD) intervals of the tMRCA (Figure 2) suggests that the viruses from Europe, Africa, and the Middle East share a common ancestor of unknown origin that emerged around July 2020 (95%HPD: April 2020-October 2020).We did not find an amino acid deletion at position 133 in the HA protein (H3 numbering) in all our isolates, a feature common with clade 2.3.4.4 isolated from humans (Table 2), and associated with the alteration of the H5 HA receptor binding pocket [22].The analysis of the NA gene of H5N1 viruses revealed that none of these viruses displayed oseltamivir resistance markers E119, H275, R293, and N295 (N1 numbering) (Table 2).Deletions were also present in both neuraminidase (NA) (an 11-aa deletion in the stalk region) and nonstructural protein 1 (NS1) (deletion from residues 80-84; Table 2), which are associated with high pathogenicity in avian hosts [23].These analyses suggest that the newly detected H5N1 viruses in Egypt may be able to infect and cause disease in mammals.The receptor binding sites in the viral HA gene of the four viruses possess the conservative amino acid residues (including 190E, 220R, 225G, 226Q, and 228G; H3 numbering), which indicated that these viruses would preferentially bind to the α-2,3-sialic acid linkage, the avian-like receptors.

Conclusions
Several introductions of clade 2.3.4.4b viruses have been seen in Egypt.Those introductions are typically through wild migratory birds but eventually spill over to poultry.Some live bird markets in Northern Egypt sell both poultry and trapped wild birds for human consumption.Such an interface provides ample opportunity for cross-species virus spill-over.The viruses we detected were from such markets where the initial virus was detected in a migratory bird and then in domestic poultry.No human cases of clade 2.3.4.4bH5Nx infections were reported in Egypt but the mutations detected in analyzed viruses suggest that human infections can occur.A vigilant surveillance system at the human-wild bird-poultry interface is necessary.

Figure 1 .
Figure 1.Phylogenetic tree of H5N1 viruses sequenced in this study, in addition to other publicly available H5Nx clade 2.3.4.4 from GenBank and GISAID.Red dots represent the H5N1 viruses sequenced in this study.Topological support values (SH-like support) of selected nodes are displayed.To the right, a schematic representation of viral clustering of each gene segment (from left to right: PB2, PB1, polymerase acidic, haemagglutinin, nucleoprotein, neuraminidase, matrix, and non-structural) is shown.Segment colors indicate origin of the segment.Within each cluster, a unified color pattern indicates homogeneity and a different color pattern indicates reassortment.

Figure 2 .
Figure 2. Time to the most recent common ancestor of H5N1 viruses sequenced in this study; maximum clade credibility temporal phylogeny of the hemagglutinin (HA) gene.The H5N1 viruses from Egypt are represented by red dots.Posterior clade probabilities are indicated by the sizes of the internal node circles.Shaded bars represent the 95% highest probability distribution for the age of each node with posterior clade probability > 0.3.

Author Contributions:
Conceptualization, R.E.-S., R.J.W., G.K. and M.A.A..; methodology and investigation, R.E.-S., Y.M., S.H.M., Y.S., A.E.T., M.G., M.N.K. and M.E.S.; software, R.E.-S.and Y.S.; writing-original draft preparation, R.E.-S.and A.K.; writing-review and editing, R.E.-S., A.K., T.T.Y.L., G.K. and M.A.A.; supervision, R.E.-S., G.K. and M.A.A.; project administration, P.P.M. and G.K.; funding acquisition, T.T.Y.L., R.J.W., G.K. and M.A.A.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, US Department of Health and Human Services (under contract HHSN272201400006C), and the National Natural Science Foundation of China's Excellent Young Scientists Fund (Hong Kong and Macau) (31922087) and the InnoHK funding from Innovation and Technology Commission of HKSAR.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 1 .
Comparison of nucleotide sequence identities of the eight influenza A virus (IAV) gene sequences for the virus isolated in this study (A/pintail/Egypt/RA19853OP/2021 (H5N1)) and nearest virus homologs.

Table 2 .
Assessment of molecular amino acid markers for zoonotic potential of the influenza A(H5N1) virus detected in Egypt.

Table 3 .
Antigenic analysis of H5N1 influenza A viruses from Egypt by hemagglutination inhibition assay.
* Polyclonal antibodies were produced in ferrets.Homologous titers are bold and underlined.