Vaccination and Antiviral Treatment against Avian Influenza H5Nx Viruses: A Harbinger of Virus Control or Evolution

Despite the panzootic nature of emergent highly pathogenic avian influenza H5Nx viruses in wild migratory birds and domestic poultry, only a limited number of human infections with H5Nx viruses have been identified since its emergence in 1996. Few countries with endemic avian influenza viruses (AIVs) have implemented vaccination as a control strategy, while most of the countries have adopted a culling strategy for the infected flocks. To date, China and Egypt are the two major sites where vaccination has been adopted to control avian influenza H5Nx infections, especially with the widespread circulation of clade 2.3.4.4b H5N1 viruses. This virus is currently circulating among birds and poultry, with occasional spillovers to mammals, including humans. Herein, we will discuss the history of AIVs in Egypt as one of the hotspots for infections and the improper implementation of prophylactic and therapeutic control strategies, leading to continuous flock outbreaks with remarkable virus evolution scenarios. Along with current pre-pandemic preparedness efforts, comprehensive surveillance of H5Nx viruses in wild birds, domestic poultry, and mammals, including humans, in endemic areas is critical to explore the public health risk of the newly emerging immune-evasive or drug-resistant H5Nx variants.


Introduction
Avian influenza viruses (AIVs) belong to the genus influenza A virus (IAV) within the Orthomyxoviridae family.The genome of IAV includes eight single-stranded ribonucleic acid (RNA) segments, encoding at least 10 fundamental viral proteins.The viral proteins of IAV can be categorized into (a) surface proteins (hemagglutinin (HA); neuraminidase (NA); and matrix protein 2 (M2); (b) internal structural proteins (polymerase basic 2 (PB2), polymerase basic 1 (PB1) and polymerase acidic (PA); nucleoprotein (NP); matrix protein 1 (M1); and nuclear export protein (NEP)); and (c) non-structural proteins (non-structural protein 1 (NS1)).Currently, 16 HA (H1-H16) and 9 NA (N1-N9) subtypes have been identified as causing AIV infections [1].In parallel, some bat-origin IAVs were detected in the New World "American continents" bat species (H17N10 and H18N11 influenza viruses) and in the Old World "Europe, Asia, and Africa" bat species (H9N2-like influenza viruses) [2,3].Most recently, the spectrum of the AIVs has been extended to include the newly characterized/distinct HA subtype in Common Pochard (Aythya ferina), nominally influenza viruses) [2,3].Most recently, the spectrum of the AIVs has been extended to include the newly characterized/distinct HA subtype in Common Pochard (Aythya ferina), nominally H19 [4].The natural reservoirs of all AIVs are waterfowl and shore birds from where they can be transmitted to new hosts [1,5] (Figure 1).Human, swine, marine animal, tiger, mink, horse, and domestic dog and cat AIVs are all assumed to have been transmitted from aquatic wild birds and emerged in avian reservoirs, and domestic poultry, to infect mammalian hosts.Unidirectional arrow refers to zoonotic potential of AIVs while bidirectional arrow refers to potential reverse zoonosis events "human-to-animal transmission" following zoonosis.
IAVs constantly evolve via two main genetic forces.The first is caused by the errorprone viral polymerase genetic mutations that constantly accumulate, leading to structural and functional changes, nominally genetic drift.On the other hand, co-infections of a cell/host by different viruses' subtypes can result in new virus variants that carry viral genome segments from two or more viruses and often demonstrate novel hybrid viruses of unidentified characteristics in a well-known process, nominally genetic reassortment or antigenic shift [1].
AIVs of all subtypes (H1-H16) exist in their natural reservoirs, wild waterfowl and shorebirds, in a low pathogenic (LP) form and can induce only mild illness in birds, if any, whereas the virulence of certain H5 and H7 AIV strains in birds varies widely, ranging from asymptomatic to highly lethal infections [6].The pathogenicity of AIVs for domestic poultry was confirmed to be associated with alteration in the proteolytic cleavage site (PCS) of the HA protein from a monobasic (e.g., PQIETR▼GLF) to a polybasic amino acid motif (e.g., PQRERRRKKR▼GLF).This alteration facilitates the HA cleavability of highly pathogenic (HP) AIVs by ubiquitous furin-like host proteases, not only in the respiratory and digestive tracts but also throughout the host vital systems, inducing a systemic infection.Besides the PCS, it is also important to note that the amino acid (aa) residues in its vicinity are also important as replication and virulence determinants in mammals [7].Moreover, virulence markers were documented in structural and non-structural influenza Human, swine, marine animal, tiger, mink, horse, and domestic dog and cat AIVs are all assumed to have been transmitted from aquatic wild birds and emerged in avian reservoirs, and domestic poultry, to infect mammalian hosts.Unidirectional arrow refers to zoonotic potential of AIVs while bidirectional arrow refers to potential reverse zoonosis events "human-to-animal transmission" following zoonosis.
IAVs constantly evolve via two main genetic forces.The first is caused by the errorprone viral polymerase genetic mutations that constantly accumulate, leading to structural and functional changes, nominally genetic drift.On the other hand, co-infections of a cell/host by different viruses' subtypes can result in new virus variants that carry viral genome segments from two or more viruses and often demonstrate novel hybrid viruses of unidentified characteristics in a well-known process, nominally genetic reassortment or antigenic shift [1].
AIVs of all subtypes (H1-H16) exist in their natural reservoirs, wild waterfowl and shorebirds, in a low pathogenic (LP) form and can induce only mild illness in birds, if any, whereas the virulence of certain H5 and H7 AIV strains in birds varies widely, ranging from asymptomatic to highly lethal infections [6].The pathogenicity of AIVs for domestic poultry was confirmed to be associated with alteration in the proteolytic cleavage site (PCS) of the HA protein from a monobasic (e.g., PQIETR GLF) to a polybasic amino acid motif (e.g., PQRERRRKKR GLF).This alteration facilitates the HA cleavability of highly pathogenic (HP) AIVs by ubiquitous furin-like host proteases, not only in the respiratory and digestive tracts but also throughout the host vital systems, inducing a systemic infection.Besides the PCS, it is also important to note that the amino acid (aa) residues in its vicinity are also important as replication and virulence determinants in mammals [7].Moreover, virulence markers were documented in structural and non-structural influenza proteins which extend beyond the HA [8][9][10][11].Thus, the virulence of AIVs is a multigenic trait, requiring a specific genome constellation for full virulence [12,13].
In the last two decades, zoonotic infections of humans caused by LP or high-pathogenic (HP) AIVs have dramatically increased [1,14].Transmission of the H5N1, H5N6, H7N3, H7N7, H7N9, H9N2, and H10N8 subtype AIVs from poultry to humans has been reported to the World Health Organization (WHO) [1].Since 2003, 457 out of 868 confirmed human laboratory cases died due to infections with H5N1 [15].Moreover, 515 out of 1568 cases died due to infections with H7N9 [1].For H9N2, a total of 89 cases of human infections, including two deaths, were reported from 2015 to 2023 by the WHO in the Western Pacific Region, mostly in China [16].The rate of infection due to subclinical or undetected cases is thought to be considerably more than currently documented cases.Consequently, the transmission of AIVs to humans poses a serious pandemic risk.

Introductions and Evolutionary Events of AIVs in Egypt
Egypt is a habitat for an impressive number (378) of bird species moving through 34 important bird areas (IBAs) [17] via two major migratory flyways: the Black Sea-Mediterranean and West Asia-East Africa flyways.Both flyways overlap in Egypt at the Nile Delta and the North Mediterranean Coast of Egypt, respectively, providing stopover areas for millions of migratory birds [18].Moreover, Egypt is particularly important for migratory soaring birds (MSBs), which depend on hot thermal uplifts to fly over and cross narrow water bodies without much effort, such as storks, ibises, pelicans, cranes, and many birds of prey.The Rift Valley/Red Sea Flyway is one of the main flyways for migratory soaring birds in the world.Egypt is of critical passage for these birds as it is located on the only land bridge between the Eurasian and African landmasses that links breeding grounds in Europe and Asia with wintering areas in Africa.The area of Southern Sinai and the Gulf of Suez are very critical to MSB migration as they include some bottlenecks of migration.On the bottlenecks, the flyway divides into two main corridors of migration along the river Nile and the Red Sea Cost (Figure 2).In 1996/1997, H5N1 AIVs with the potential to infect humans were first isolated in birds from live bird markets (LBMs) in Hong Kong.The parent H5N1 AIVs were isolated from geese in Guangdong province, China, (Gs/Gd) in 1996 and they acquired gene segments from H9N2, H6N2, and H5N1 AIVs.This Gs/Gd AIV killed 6 out of 18 human cases in Hong Kong [20].Effective control measures and closure of LBMs successfully eradicated the virus.However, the precursors circulating in the wild bird reservoir resulted in the re-emergence of the virus in 2002/2003.In 2005/2006, this virus was transmitted mainly by wild birds to about 60 countries in Asia, Europe, and Africa, causing severe socioeconomic losses in the poultry industry, particularly in developing countries.The virus became endemic in poultry in several countries, including China, Indonesia, Thailand, Vietnam, and Egypt.Phylogenetically, the H5-type HA was diversified into ten clades (clades 0 to 9) and several second-, third-, fourth-, or fifth-order clades (e.g., clades 2, 2.3, 2.3.4,2.3.4.4,2.3.4.4b) [21].
In Egypt, the HPAI H5N1 was documented in domestic poultry in early 2006 (Figure 3) shortly after its detection in wild migratory birds in Damietta Governorate in late 2005 [22].This virus continued to circulate, leading to accumulated amino acid substitutions in the surface immunogenic glycoproteins (HA and NA), and the virus was declared as endemic in Egypt in 2008 [17,23].From late 2009 to 2011, two vaccine-escape H5N1 mutant subclades, namely 2.2.1 and 2.2.1.1,co-circulated and were detected in poultry [17].Meanwhile, the H5N1 viruses of subclades 2.2.1 and 2.2.1.1 continued to change under improper vaccination circumstances to form new phylogenetic clusters, namely 2.2.1.2and 2.2.1.2a[17,24,25].In 2013/2014, H5N8 viruses of clade 2.3.4.4 spread from China to Korea and Japan, and then to Siberia, Europe, and Beringia and thereafter into the United States (US) via wild migratory birds [39,40].This is currently the most widespread H5-type AIV in recent history.The parent viruses from 2014/2015 were characterized by a high mean chicken infectious dose, lack of seroconversion in surviving chickens, low replication in different organs, a transmissibility indicating poor adaptation to poultry, and were clearly less virulent than the parent Gs/Gd virus [41].In ducks and geese, H5N8 viruses replicated and were excreted to a certain extent without inducing clinical signs or mortality, representing high adaptation [41,42].Moreover, a hallmark of the 2014/2015 outbreak in the US and elsewhere was the poor adaptation to mammals (mice, ferrets, dogs, cats), demonstrating mild or absent clinical signs and lack of transmission among experimental animals [43,44].Several studies showed that serial passages in mice increased virulence and viral replication in mammals, which was accompanied by mutations in the PB2, PB1, PA, HA, and/or NP segments [45].The virus had a lower affinity to human-type receptors, replicated at Synchronously, several clades of the LP influenza A/H9N2 viruses (e.g., G1, Y280, and Korean lineages) are well entrenched or endemic in several countries in Asia, Europe, and the Middle East, representing the most widespread AIV subtype in birds worldwide [26].H9N2 infections in poultry are mostly mild, with slight respiratory symptoms and a sudden drop in egg production (14-75% in breeder or layer flocks) [27].In 2011, H9N2 AIVs were first detected in Egypt in a quail farm [28].Although H9N2 viruses are LP in poultry, they pose a serious public health threat.The H9N2 AIVs of the G1-like lineage were isolated from humans in China, Hong Kong [29,30], and Egypt [31].In early 2015, three infections with H9N2 AIVs were documented among Egyptians that were recently exposed to infected poultry, confirming the high zoonotic potential of H9N2 viruses and ranking Egypt in the third position after China and Bangladesh in the cumulative numbers of H9N2 AIV human infections [32,33].Importantly, in China, H9N2 viruses donated internal-protein coding gene segments to other AIVs that then became fatal in humans, like the parent H5N1 Gs/Gd in 1996/1997 [34], H10N8 in 2013 [35,36], H7N9 (circulating since 2013) [34,37], and H5N6 in 2014 [38].
In 2013/2014, H5N8 viruses of clade 2.3.4.4 spread from China to Korea and Japan, and then to Siberia, Europe, and Beringia and thereafter into the United States (US) via wild migratory birds [39,40].This is currently the most widespread H5-type AIV in recent history.The parent viruses from 2014/2015 were characterized by a high mean chicken infectious dose, lack of seroconversion in surviving chickens, low replication in different organs, a transmissibility indicating poor adaptation to poultry, and were clearly less virulent than the parent Gs/Gd virus [41].In ducks and geese, H5N8 viruses replicated and were excreted to a certain extent without inducing clinical signs or mortality, representing high adaptation [41,42].Moreover, a hallmark of the 2014/2015 outbreak in the US and elsewhere was the poor adaptation to mammals (mice, ferrets, dogs, cats), demonstrating mild or absent clinical signs and lack of transmission among experimental animals [43,44].Several studies showed that serial passages in mice increased virulence and viral replication in mammals, which was accompanied by mutations in the PB2, PB1, PA, HA, and/or NP segments [45].The virus had a lower affinity to human-type receptors, replicated at lower levels than human H1N1 and H5N1 viruses in human cell cultures, and was highly susceptible to neuraminidase inhibitors (NAIs) (e.g., oseltamivir, zanamivir, peramivir) [43,46].

Imported and Locally Produced Avian Influenza Vaccines in Egypt
In an attempt to alleviate the risk of avian influenza (AI) infections in domestic poultry, vaccination has been adopted as a control strategy in South East Asia since 2003 and in Egypt since 2006 [56].In Egypt, several avian influenza vaccines engineered from both non-matching and matching strains were imported and subjected to evaluation and approval for field immunization (Table 1).The main criteria of an effective AI vaccination are: (1) vaccine composition that matches the circulating field strains; (2) reliable cold chain and logistics to target all poultry farms, including smallholder poultry farms; and (3) availability of sufficient financial and human resources to ensure the validity of the vaccination campaigns [57].An improper choice of vaccine strain or the incorrect implementation of vaccination campaigns can also contribute to the emergence of new field variants [58].
To approve the use of imported or locally generated AI vaccines in Egypt, an experimental assessment of the vaccine efficacy must be performed.The efficacy of a vaccine in controlling laboratory challenge infection with a highly pathogenic circulating virus in vaccinated poultry must exceed 80%, with a remarkable drop in virus shedding in vaccinated poultry when compared to the unvaccinated control group [59].Nevertheless, the effectiveness of an approved vaccine in the field remains unquantified and may differ from the experimental evaluation of "efficacy" due to many factors, including poor biosecurity, timing of vaccination, improper application, the circulation of different quasispecies or subclades of the same virus, and coinfections in poultry flocks that may impact the overall efficacy [57,60].Therefore, the adoption of laboratory vaccine efficacy as a sole criterion to deduce vaccine field effectiveness is sometimes deceptive.
As illustrated in Table 1, different H5-type viruses were used as vaccine seed strains, including non-circulating clades (e.g., clades 0, 1, and 2.3.2) and lineages (e.g., classical, Eurasian, and north American H5-lineage viruses).Those partially matching vaccine strains showed variable reactivity against earlier antigens and were approved for application, but reactivity declined over time with virus evolution due to antibody-mediated evolutionary selection pressure [61].

Improper Antiviral Drug Prescription to Control AIVs in Avian Species and Its Impact on the Evolution of Drug-Resistant Variants
In addition to the application of vaccines to control IAV infections, antivirals are considered a major line of defense with therapeutic activities against IAVs [64].Three classes of IAV antivirals were approved for the treatment of human influenza virus infections: M2 blocker adamantanes (amantadine and rimantadine), neuraminidase inhibitors (NAIs) (oseltamivir, zanamivir, and peramivir), and polymerase acidic (PA) protein inhibitor (Baloxavir marboxil) [65][66][67].Adamantanes work by blocking the matrix 2 (M2) ion channel protein of the influenza virus that is essential for the fusion of virus and host-cell membranes, thus preventing virus uncoating and the release of vRNPs into the host cell cytoplasm [68].These M2 blockers have historically been used as a symptomatic treatment for Parkinson's disease [69].Due to the extensive use of amantadine in treating influenza virus infections, the percentage of drug-resistant strains increased from 0.4% during 1994-1995 to 12.3% during 2003-2004 [70].In 2006, the percentage of amantadine-resistant strains continued to increase and 92% of human H3N2 influenza viruses were resistant to amantadine [70].Therefore, in 2007, amantadine was not recommended by the WHO for the treatment of human virus infections [71].
Although amantadine has not been approved for use in poultry, several reports revealed the widespread illegal use of adamantanes in treating AIV infection in poultry and/or as a nutrient supplement for different poultry species in different countries, including China and Egypt [72,73].The cheap price of amantadine and lack of strict veterinary supervision facilitated its wide application in the poultry production sector and the subsequent emergence of avian influenza adamantane-resistant strains.Also, the segmented nature of the influenza virus genome and lacking polymerase proofreading ability lead to a high mutation rate of the influenza virus and subsequently its escape from the antiviral effect.Indeed, a single amino acid substitution at positions 26, 27, 30, 31, and 34 in the transmembrane region of M2 is sufficient to confer virus resistance to amantadine [74].Hence, several studies reported the emergence of amantadine-resistant AIVs worldwide [75,76].In Egypt, AIV amantadine-resistant mutants emerged in 2007 among H5N1 isolates of different clades, harboring mainly the amino acid substitutions V27A and S31N, then continued to circulate until 2011 [77,78].Amantadine-resistant mutants were not detected before the re-emergence of H5N1 IAVs in 2015 [77,79].In addition to H5N1 AIVs, amantadine-resistant mutants were reported in Egypt among H9N2 AIVs strains [80], which are currently endemic in Egypt and co-circulating with H5N1 and other AIV subtypes in poultry species with zoonotic potential to induce human infections [28,81,82].
Due to the unavailability of rimantadine, the second M2 blocker, in most countries, there are no reports of using it in poultry.Nevertheless, H7N9 AIVs isolated in China, which induced several human infections, were resistant to both rimantadine and amantadine [83].
Oseltamivir is one of the most used NAIs and is stockpiled as a part of pandemic preparedness plans [84].Oseltamivir interacts with the virus's NA and hinders its function in mediating the cell release of new virions from infected cells [85].While oseltamivir is currently the drug of choice for the treatment of influenza infections, oseltamivir-resistant mutants have emerged among different influenza subtypes.However, due to the structural difference in the NA of different influenza subtypes, mutations responsible for conferring oseltamivir resistance are also different: amino acid substitutions H274Y (N2 numbering) for A/H1N1 viruses; E119V, R292K, and N294S for A/H3N2 viruses; and R150K and D197N for influenza B viruses [86].Fortunately, the administration of oseltamivir as prophylaxis in poultry flocks is costly, which renders its wide application difficult; nevertheless, oseltamivir-resistant H5N1 AIVs have been also reported [87][88][89][90].Experimentally, several studies revealed the development of oseltamivir-resistant mutants in aquatic birds (mallards) after exposure to oseltamivir treatment and the ability of these mutants to transmit to domestic chickens [91,92].Although oseltamivir-resistant IAVs are documented in humans [93,94], the drug is still effective and is considered the drug of choice in treating human influenza infections.However, the application of oseltamivir as a prophylactic or therapeutic against AIV infections in birds will accelerate the emergence and widespread of oseltamivir-resistant mutants that subsequently will limit the treatment options for human influenza infections.
Another NAI is zanamivir, which also works as a synthetic analog of sialic acid receptors to hinder influenza virus infection.While oseltamivir is the drug of choice in treating human influenza virus, zanamivir is often used when the effect of oseltamivir is limited.For influenza A/H1N1 viruses, the amino acid substitution H274Y is proved to confer oseltamivir resistance while I223R substitution could reduce the effectiveness of both oseltamivir and zanamivir [95].Most of the zanamivir resistance incidences were reported after a period of treatment in immunocompromised patients [96][97][98].
Baloxavir marboxil (BxM) was developed as a new anti-influenza drug via inhibiting the cap-dependent endonuclease activity of the polymerase acidic (PA) protein which is essential for the generation of capped RNA primers (mature mRNA) for viral transcription [99].BxM was first approved in Japan and the US in 2018, and since then it has been used in several other countries.Amino acid substitutions at position 38 of PA correlated with decreased effectiveness of BxM [100]; however, the degree of resistance varies according to the substituted amino acid.Briefly, leucine (L) confers a 10-fold and a 3-fold reduction in the susceptibility of A/H1N1 and A/H3N2 IAVs, respectively [99,101,102].Also, the amino acid substitutions threonine (T), methionine (M), or phenylalanine (F) confer a 10-> 50-fold reduction in the susceptibility to BxM, while valine (V) does not seem to have any effect [102].Virological surveillance conducted in Japan during the 2018-2019 influenza season revealed that reduced susceptibility to BxM has been reported at a low frequency, but did not spread widely during the 2019-2020 influenza season due to amino acid substitutions at position 38 of the PA (I38T and 38F) [103].
Overall, antivirals available for the treatment of human influenza virus infections are limited, particularly with the rapid emergence of antiviral-resistant variants.However, the application of any of these antivirals to control AIV infection in birds will undoubtedly increase the prevalence of antiviral-resistant variants, which will thereby result in reduced susceptibility or loss of activity of these antivirals in humans.

Adaptive Mammalian Mutation Markers in Egyptian AIVs
AIV circulation in animal reservoirs under subclinical control measures results in the emergence of genetic traits which permit the virus to cross the avian-to-human species barrier.These acquired genetic changes to improve the viral fitness of zoonotic AIVs can be gained either in avian hosts or the new mammalian host [1].However, we assume that the minimum essential genetic changes to initiate the zoonosis process are likely to occur in the original avian host before transmitting to mammals, including humans.
Despite the vaccination programs in poultry in Egypt to control AIV circulation, the majority of the reported human infections with AIV H5N1 worldwide in the last two decades (2003-2023) were documented in Egypt (41%, 359/868) with a considerable casefatality rate (26%, 120/457) [15].We assume that these high morbidity and mortality rates might be a potential consequence of prophylactic antiviral drug misuse for poultry and/or the application of non-matching/partially matching vaccine strains to control the circulating and emerging AIVs.
A diverse range of AIVs have infected various mammalian hosts during the past two decades and have posed a persistent threat to both human and animal health.This is an outcome of breaking down the interspecies barrier, which is primarily attributed to non-silent changes in the viral genome.The molecular basis for interspecies transmission of influenza viruses involves several factors that affect the transmissibility of these viruses between avian and mammalian host species.Several molecular characteristics are crucial for interspecies transmission, including (1) HA receptor binding specificity; (2) HA fusion stability; (3) the presence or absence of potential N-glycosylation sites in the HA; (4) the increased viral replication by the viral ribonucleoprotein (vRNP) complex in a new host; (5) the balance between receptor binding and fusion via HA protein and the receptorcleaving capacity of the NA protein in the new host; and (6) genetic reassortments that result in different gene constellations [104][105][106].The tripartite polymerase complex (PB2, PB1, and PA), together with viral HA, control the ability of influenza viruses to infect different species [107,108].As the binding preferences of HA in avian and human influenza viruses are α2,3-SA and α2,6-SA, respectively, it is believed that sialic acid (SA) receptors act as a host barrier in AIV zoonotic potential and interspecies transmission.For interspecies transmission, an alteration of the binding preference is needed so that the HA of AIV can bind the mammalian-or human-type SA receptor (α2,6-SA) [108][109][110].Furthermore, the viral polymerase complex has a crucial role in viral adaptation in the new hosts and is a major determinant of pathogenicity because it is considered the cornerstone of viral genome replication and gene transcription [111][112][113][114][115][116].Mainly, this adaptation to the new host is caused by single or multiple mutations in the genome of avian and human influenza viruses.A positive selection of certain amino acid substitutions, encoded by single or multiple mutations in the viral genome following virus adaptation to the new host, is considered an adaptive mutation marker.Interestingly, multiple sporadic human illnesses have been reported following adaptive mutations in the PB2 polymerase subunit [117][118][119][120]. PB2-E627K substitution is the most commonly occurring human adaptation polymerase mutation [121][122][123].The high virulence of H5N1 AIV in mice and the ability to replicate effectively in the mouse's upper respiratory tract (URT) were shown to be associated with the presence of E627K adaptive mutation in the PB2 gene [124][125][126].Moreover, the replication of AIV in mammalian cells was boosted by the human virus-like residue T271A in PB2 [127].Additionally, it has been shown that certain mutations in PB1, such as L473V and L598P, increase AIV polymerase activity in mammalian hosts [128,129].Researchers have also investigated the function of other viral proteins, including HA, NS1, NEP, and matrix proteins M1 and M2, in mammalian transmission [130][131][132][133][134][135][136].
Because of its distinct characteristics, the HPAI H5N1 currently imposes serious impacts on both animal and human health.Even though H5N1 viruses have been implicated in hundreds of human cases, this subtype lacks the potential for prolonged transmission in humans, which may be owing to the avian-type receptor-binding specificity in HA (α2,3-SA) [137,138].Molecular factors, particularly changes in HA that affect its receptorbinding specificity by either enhancing the binding to α2,6-SA or reducing the binding affinity to α2,3-SA, have been linked to multiple sporadic infections in humans [137,139].Numerous amino acid substitutions in HA, including Q226L, G228S, R193K, and E190D, have been found to improve the binding of HPAI H5N1 HA to α2,6-SA in both natural and experimental settings [137][138][139][140][141][142][143].Three other mutations were also identified as being crucial for the respiratory droplet infectiousness of H5N1 virus in ferrets, including H99Y in PB1 and H110Y and T160A in HA [113].It has been shown that an airborne-transmissible H5N1 might be produced by N224K and Q226L mutations together with the HA substitutions N158D and T318I, which change the receptor binding preference [144].As seen in H5N1 with the T160A amino acid change, the deletion of the glycosylation site close to the receptor-binding domain (RBD) at position 158-160 is favorable for human receptor specificity, increasing the binding to human-type receptors [113,144,145].Furthermore, it was shown that the amino acid substitutions L473V and L598P in PB1 increased the H5N1 virus's polymerase activity in human cells and enhanced the replication of the pandemic H1N1 virus [129].
Despite the fact that no human infections with H5N8 AIVs have been reported since its emergence in 2014, a genomic investigation of H5N8, which belongs to clade 2.3.4.4,revealed several human-like mutation markers, including PA-404S, PB2-613I, PB2-702R, HA-137A, HA-227R, and HA-A160T [145,146].The A160T amino acid substitution, which is an important glycosylation site in the HA protein, could potentially increase binding specificity for human-type receptors and increase the transmissibility of H5N8 AIVs from avian species to mammalian models [147].
Regarding H9N2 viruses, it was found that L226Q in HA improved the efficiency of direct transmissibility, and it was also shown that a higher incidence of the L226Q-containing H9N2 viruses in China increased the risk of human infection with this subtype [148].Swine H9N2 viral isolates were found to have distinctive amino acid alterations in the HA at residue 227 inside the receptor-binding pocket and residues 274, 279, and 286 outside the receptor-binding area [149].Moreover, the affinity for the α2,6-SA human receptor has also been found to be influenced by position 200, with valine (V) exerting the highest affinity, threonine (T) showing intermediate affinity, and alanine (A) demonstrating the lowest binding affinity [150].Moreover, H9N2 viruses were experimentally able to infect and disseminate in ferrets via respiratory droplets by promptly acquiring typical mammalian-adaptive markers including E627K, D701N, and Q591K mutations in PB2 with or without adaptation to mammalian hosts [151,152].
A cumulative summary of the distinctive molecular markers among currently circulating AIVs in Egyptian poultry is illustrated in Table 2. Interestingly, some of these adaptive mutations were acquired in poultry before transmission to mammals or humans, representing the minimum essential elements to recognize mammalian or human host cells and infect them.Following transmission to humans or mammals, the virus acquires several host-specific adaptive mutations due to differences between its original natural hosts and the mammalian hosts, including humans [1].Table 3 summarizes the impact of certain adaptive amino acid mutations/substitutions on the characteristics of AIVs in mammalian systems.

Conclusions and Perspectives
Despite the fact that the risk of H5Nx virus transmission to the public is still low, close monitoring of these AIVs and persons exposed to them is imperative [275].These AIVs are continuously evolving in endemic areas with improper control plans in place, and under inadequate immune and drug pressures.Taking into consideration the COVID-19 scenarios and the evolution of immunoescape variants in certain geographical areas, followed by the devastating spatiotemporal transmission of these SARS-CoV-2 variants of concern (VoCs) in a few days/months to all continents, we urge global health systems within the "One Health" approach to detect signals of potential variants of interest (VOIs) or variants of concern (VOCs) for the newly emerging avian influenza H5Nx viruses and rapidly assess their risk(s).Likewise, the application of evolution-driving control strategies, including vaccination, in certain geographical areas of the world must be subjected to assertive regulations, including safe farming practices and implementation of locally matching vaccine strains, because these viruses can affect the country of origin, neighboring countries, and may pave the way for a devastating pandemic if a virus acquires the minimum essential substitutions that support viral infection and person-to-person airborne transmission.Therefore, international collaboration to implement unified control strategies against AIVs must be urgently established and applied properly among the different sectors of the One Health approach.

Figure 1 .
Figure 1.Schematic representation of the disseminated IAV subtypes in different mammalian hosts.Human, swine, marine animal, tiger, mink, horse, and domestic dog and cat AIVs are all assumed to have been transmitted from aquatic wild birds and emerged in avian reservoirs, and domestic poultry, to infect mammalian hosts.Unidirectional arrow refers to zoonotic potential of AIVs while bidirectional arrow refers to potential reverse zoonosis events "human-to-animal transmission" following zoonosis.

Figure 1 .
Figure 1.Schematic representation of the disseminated IAV subtypes in different mammalian hosts.Human, swine, marine animal, tiger, mink, horse, and domestic dog and cat AIVs are all assumed to have been transmitted from aquatic wild birds and emerged in avian reservoirs, and domestic poultry, to infect mammalian hosts.Unidirectional arrow refers to zoonotic potential of AIVs while bidirectional arrow refers to potential reverse zoonosis events "human-to-animal transmission" following zoonosis.

Figure 2 .
Figure 2. The migration routes (flyways) of migratory birds all over the world.The eight flyways include one main flyway that passes by Egypt and connects west Asia with east Africa.Global flyway boundaries in this map were created according to Boere and Stroud, 2006 [19].

Figure 3 .
Figure 3. Timeline of different avian influenza viruses, including H5Nx viruses that emerged in the Egyptian poultry sector.From 2006 to 2017, the HPAI H5N1 virus of clade 2.2.1 and its subclades 2.2.1.1 and 2.2.1.2resulted in devastating economic losses in poultry and a remarkable public health hazard with zoonotic potential in humans.From 2016 to the present, the HPAI H5N8 virus of subclade 2.3.4.4 has been circulating, resulting mainly in losses in poultry.Recently, in 2022, HPAI H5N1 reassortants of clade 2.3.4.4b have been documented in migratory birds and domestic poultry in Egypt.

Figure 3 .
Figure 3. Timeline of different avian influenza viruses, including H5Nx viruses that emerged in the Egyptian poultry sector.From 2006 to 2017, the HPAI H5N1 virus of clade 2.2.1 and its subclades 2.2.1.1 and 2.2.1.2resulted in devastating economic losses in poultry and a remarkable public health hazard with zoonotic potential in humans.From 2016 to the present, the HPAI H5N8 virus of subclade 2.3.4.4 has been circulating, resulting mainly in losses in poultry.Recently, in 2022, HPAI H5N1 reassortants of clade 2.3.4.4b have been documented in migratory birds and domestic poultry in Egypt.

Table 2 .
Documented avian-to-mammalian adaptive mutations in AIVs in Egypt.

Table 3 .
Phenotype drift markers in AIVs and their altered characteristics.