Emergence, migration and spreading of the high pathogenicity avian influenza virus H5NX of the Gs/Gd lineage into America

Published: December 30, 2025

Source : Alejandro J. Aranda 1; Gabriela Aguilar-Tipacamú 1,2; Daniel R. Perez 3; Bernardo Bañuelos-Hernandez 4; George Girgis 5; Xochitl Hernandez-Velasco 6; Socorro M. Escorcia-Martinez 6; Inkar Castellanos-Huerta 7; and Victor M. Petrone-Garcia 8,*.

Summary

Author details:

1 Maestría en Salud y Producción Animal Sustentable, Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Querétaro, Mexico; 2 Licenciatura en Medicina Veterinaria y Zootecnia, Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Querétaro, México; 3 Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA; 4 Facultad de Veterinaria, Universidad De La Salle Bajío, Avenida Universidad 602, Lomas del Campestre, León, México; 5 Nevysta Laboratory, Iowa State University Research Park, Ames, Lowa, USA; 6 Departamento de Medicina y Zootecnia de Aves, Facultad de Medicina Veterinaria y Zootecnia (FMVZ), Universidad Nacional Autónoma de México (UNAM), Cd. de México, México; 7 Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas, USA; 8 Departamento de Ciencias Pecuarias, Facultad de Estudios Superiores de Cuautitlán (FESC), Universidad Nacional Autónoma de México (UNAM), Cuautitlán, Mexico.

The high pathogenicity avian influenza virus H5N1, which first emerged in the winter of 2021, has resulted in multiple outbreaks across the American continent through the summer of 2023 and they continue based on early 2025 records, presenting significant challenges for global health and food security. The viruses causing the outbreaks belong to clade 2.3.4.4b, which are descendants of the lineage A/Goose/Guangdong/1/1996 (Gs/Gd) through genetic reassortments with several low pathogenicity avian influenza viruses present in populations of Anseriformes and Charadriiformes orders. This review addresses these issues by thoroughly analysing available epidemiological databases and specialized literature reviews. This project explores the mechanisms behind the resurgence of the H5N1 virus. It provides a comprehensive overview of the origin, timeline and factors contributing to its prevalence among wild bird populations on the American continent.

INTRODUCTION

Influenza is a highly contagious viral disease that affects avian species, giving rise to the term avian influenza (AI) and several species of mammals [1, 2] due to factors associated with viral replication, the capacity for mutation and infectivity [3, 4]. The avian influenza virus (AIV), part of the Orthomyxoviridae family, is the causative agent of AI [2, 5]. This viral family is constituted of seven genera: Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus, Deltainfluenzavirus, Quaranjavirus, Thogotovirus and Isavirus [5–7]. The first four genera consist of a single species: influenza A virus (IAV), influenza B virus (IBV), influenza C virus (ICV) and influenza D virus (IDV), respectively [5, 8–10]. Instead, the genus Quaranjavirus is composed of three species (Quaranfil virus, Johnston Atoll virus and Lake Chad virus) and the genus Thogotovirus of two species (Dhori virus and Thogoto virus), while the genus Isavirus is composed of a single species (infectious salmon anaemia virus) [5, 11]. However, IAV, IBV, ICV and IDV are regarded as the most significant concerning human and animal health because they are classified as responsible agents in respiratory disease outbreaks across numerous species [5, 7].

While waterfowl are considered the primary host for the ecological survival of AIV in the environment [12], different avian and mammal species also significantly contribute to acquiring and maintaining mutations to broaden the host range [13]. This panorama became evident when the A/Goose/Guangdong/1/1996 (Gs/Gd) lineage viruses emerged in 1996 [14, 15]; these viruses were known to cause disease in wildlife avian species, a characteristic not observed since the outbreaks in South Africa in 1961 [16, 17]. Furthermore, they began to cause outbreaks mainly in carnivorous mammals, such as mustelids, felids and canids, since the early 21st century [18]. Since 2021, structural changes in different viral proteins have led to cases appearing in other species, including canids, ursids, felids, mustelids, cetaceans, pinnipeds and bovids [18–20]. This review outlines the ecoepidemiological dynamics of significant epidemic events caused by Gs/Gd lineage viruses and highlights the essential molecular markers contributing to their adaptation to mammals.

GENOMIC AND PROTEIN FUNCTION OF ORTHOMYXOVIRUS

Generally, the Orthomyxoviruses present a segmented genome of single-stranded, negative-sense RNA (ssRNA(-)) [2, 21, 22]; however, there are differences among genera (e.g. IAV and IBV with ICV and IDV). The genome comprises eight segments for IAV and IBV, while ICV and IDV only have seven [3, 22–24]. Depending on the virus strain and genus, these segments present variations in the number of encoded proteins, from 9 to 17 [22, 25–27]. These differences directly influence the distribution and impact on ecosystems, e.g. ICV encodes six structural and three non-structural proteins [22], including polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acid (PA), haemagglutinin-esterase-fusion (HEF), nucleoprotein (NP), matrix protein (M1), CM2 protein (CM2), non-structural protein 1 (NS1) and nuclear export protein (NEP/NS2) [28]; given their closeness to IDV, it was determined that they could encode identical proteins [29]. These viruses are characteristically present in species such as humans, swines and canids [4, 28]; in the case of IDV in swines, equids, camelids and bovids [29], both viruses are generally related, with clinical presentations of type acute [30]. In contrast, IBV encodes nine structural, two non-structural proteins and two accessory proteins: PB2, PB1, PA, haemagglutinin (HA), NP, neuraminidase (NA), NB protein (NB), M1, BM2 protein (BM2); NS1, NS2 [22]; PB1-F2 and PB-N40 [31]. This viral genus is one of the leading representatives of clinical presentations associated with the respiratory tract in species such as humans, swines and marine mammals [4, 32], with clinical presentations ranging from acute to fatal cases [32].

In the specific context of the AIV, in addition to the ORFs coding previously proteins referred (PB2, PB1, PA, HA, NP, NA, NB, M1, M2, NS1 and NS2) [22], alternative ORFs for specific proteins are reported in segment 2 (87 amino acids peptide and PB1-N40 protein) [22, 33, 34], in segment 3 (PA-X, PA-N155 and PA-N182 proteins) [35, 36], in segment 7 (M42 protein) [37] and finally in segment 8 [non-structural protein 3 (NS3)] [38], showing a vast repertoire of strategies during infection and viral replication. These features in protein expression are related to crucial replication and genomic characteristics of AIV, like the segmented genome of ssRNA(−), the RNA-dependent RNA polymerase complex capable of inducing deletions and insertions into the genome [39–41], evolutionary plasticity [42] and the absence of correction and repair activity during RNA synthesis from cRNA [43–45], resulting in a high mutation rate of 10−3–10−6 nt per cell infection cycle [39, 43, 46].

The specific role and efficiency in the activity of each protein during an infection serve as a reference for understanding the effects of disease on an organism and its impact on the ecosystem [25, 26]. Concerning the viral proteins on the infectious capacity of viruses, it is necessary to consider the surface glycoproteins of AIV at the beginning of this description, HA and NA, referred to as HxNy [47, 48]. The HxNy profile determines the virus subtype according to the type of HA (1–19) and NA (1–11) (Fig. 1) [9, 42, 49–53], along with the regulation of the range of interaction with the host, affecting the virus transmissibility to new hosts and binding and releasing specific receptors [26, 42, 54, 55]. The role of the HA protein is to specifically bind to sialic acid (SA) in cellular receptors [25, 50, 51, 56], specifically 5-N-acetylneuraminic acid (Neu5Ac) and recently demonstrated binding to 5-N-glycolylneuraminic acid (Neu5Gc) [51, 57–60]. For Neu5Ac receptors, it has been named galactose-linked SA receptors (SA α−2,6 Gal) as mammalian receptors and SA α−2,3 Gal as avian receptors [61]. In addition, there is an adaptation, mainly referring to avian species: AIV adapted to chickens have an affinity to the SA α−2,3 Gal receptor with a β1,4 linkage to N-acetyl galactosamine (GalNac), and the particular case of AIV associated with waterfowl (ducks) has a preference for the SA α−2,3 Gal receptor with a β1,3 linkage to N-acetylglucosamine (GlcNac) [62]. In addition to its participation during cell binding, the amino acid sequence of the HA, in particular, the cleavage site (CS) sequence [48, 63], is decisive for the pathotypes [64] classified as either low pathogenicity avian influenza virus (LPAIV) or high pathogenicity avian influenza virus (HPAIV) [48, 63, 64], based on the capacity of CS of furin-like proteases, increasing the virus’s tissue tropism [65]. In the case of NA, its sialidase enzymatic activity promotes the degradation of the avian cellular receptor or mammalian cellular receptor [56, 58, 66]. Finally, the surface structural protein conforming the viral capsomer (M1) with ion channels (M2) [25, 67, 68]. Internal proteins play a role in viral replication and interacting with host cells, including forming the enzymatic polymerase complex vRNP (viral ribonucleoprotein; PA, PB1 and PB2) [25, 67, 69–72], packaging the genomic segments and transporting them into the nucleus for replication (NP) [25, 67, 69, 70, 72–74]. Finally, the exportation of M1 and the vRNP complex from the nucleus and cellular immune response type I IFN inhibition (NS1 and NS2) [25, 45, 68, 75]. Together, these proteins are associated with the stability, affinity, adaptability and pathogenicity of AIV [44, 56, 76].

ECOLOGY AND DYNAMIC OF TRANSMISSION OF AIV

Over 10 000 avian species have adapted to a wide range of ecological niches [73, 77, 78]. At least 100 species belonging to 12 of the 50 orders [79, 80] are crucial in disseminating and prevalences of AIV in their habitats, with special emphasis on migratory birds (Anseriformes and Charadriiformes) [79, 81–83]. These waterfowl species act as primary reservoirs for subtypes LPAIV (H1–H16, H19 and N1–N9) [52, 53, 79, 80, 84–89], with 120HA/NA combinations identified in different avian species [90–92].

Fig. 1. Reported influenza virus subtypes in various hosts. Dates marked in red indicate sporadic transmission of avian-origin AIV, while green dates signify direct transmission of AIV by other species. (1) The H16N3 subtype is documented in dolphins; (2) The H1N1 virus was reported in captive felines and seals; (3) Notification of HPAI H5N1 in captive felines; (4) The H7N7 subtype was last reported in 1970; (5) H1N1, H1N2 and H3N2 subtypes are endemic in pigs; (6) The H2 subtype has yet to be reported since 1957; (7) Avian H19 subtype was isolated from a common pochard (A. ferina) in Kazakhstan. It could be a descendant from the H9 bat lineage and the H9 avian lineage, as this subtype presents an affinity to bind to the major histocompatibility complex class II, like AIV circulating in bats. (8) H5N1 subtype has been reported in dairy cows and goats in several states of the USA since early 2024 (Created with BioRender.com).

These LPAIV often lead to asymptomatic infections [4, 13, 77]; however, symptomatic infections, especially in young birds, are reported in 0.7% to up to 30% of cases [77, 79, 93–95]. Consequently, these animal populations can readily transmit it to other species or help introduce them into different habitats or geographical areas because of their migratory patterns involving intraand intercontinental movements (Fig. 2) [93, 96–98]. An example of this can be the case of the Antarctic [2], considered free of AIV until the 80s [99, 100], when antibodies against AIV were detected in populations of Adélie penguins (Pygoscelis adeliae), chinstrap penguins (Pygoscelis antarcticus), gentoo penguins (Pygoscelis papua), south polar skuas (Catharacta maccormicki) and brown skuas (Catharacta antarctica lonnbergi) [101]. Consequently, in 2014, the isolation of the H11N2 virus in Adélie penguins, along with other AIV, demonstrated the current presence of these diseases [102, 103].

In addition to waterfowl species, other species are susceptible to disease and play a role in transmission between species, referring to these as intermediate and bridge hosts [13, 104, 105]. The intermediate hosts are considered those where coinfection is present, promoting the acquisition of adaptations to new hosts [13]. For instance, swine, felines and mustelids are considered significant candidates [106–108]. On the other hand, bridge hosts promote transmission from natural hosts to receptive populations, acting as a link in the transmission of the virus from wildlife to domestic avian and mammal species. Examples of a bridge host are the Passeriformes order, which is recognized as the primary candidate for such a mechanism [104], and species of Ratitae also participate in the retention of mutations acquired in mammalian infections before returning to waterbirds [83].

Fig. 2. Migratory patterns of migratory birds. Visualizing the interrelationship between different flyways worldwide (Created with BioRender.com).

According to the interspecies transmission model, it is important to mention the appearance of the spillover of subtypes H5 and H7 in the poultry industry [65, 109] mainly due to the acquisition of multibasic amino acid sequences in the CS, which allowed the emergence of HPAIV [65, 110]. However, this model becomes altered when examining the transmission of HPAIV in wildlife avian species [84, 111], which occurs less frequently, as exemplified by the H5N3 virus in common terns (Sterna hirundo), reported in the Cape Province coast between Elizabeth Harbour and Lamberts Bay in 1961 [14, 16, 17, 112]. It was not until the emergence of HPAIV H5NX of Gs/Gd lineage that the virus was first isolated in domestic goose in China in 1996, with the continuous outbreak in wildlife animals caused by HPAIV [14, 112]. These epidemiological events are called spill-back because transmission occurs from domestic species to wildlife counterparts [88].

EMERGENCE OF GS/GD LINEAGE VIRUSES

The evolution of LPAIV into lineage Gs/Gd viruses is closely linked to genomic reassortments that lead to new combinations of genomes [46, 113, 114]. This viral evolutionary process requires the matching and compatibility between the segments of the parental virus vRNA (viral RNA) with other viral proteins of different types of viruses during co-infections [70, 115–117]. The emergence of the Gs/Gd virus in 1996 [14] is believed to originate from LPAIV circulating in bird populations [88, 112], where viruses such as H3N2, H3N8, H6N1, H6N6 and H9N2 played an active role as genomic segment sources for their emergence [117–119]. This lineage marked a shift in the evolutionary dynamics of AIV, not only by diversification of its HA gene, resulting in the emergence of ten phylogenetic clades main (Fig. 3), but also by generating the first case of direct transmission in humans from an avian-origin virus in Hong Kong in 1997 [80, 112, 120–123]. Until December 2024, more than 900 human cases have been reported [124–126], including outbreaks caused by clade 2.3.4.4b viruses genotype B3.13 (Fig. 4) and D1.1 (Fig. 5) circulating in bovines, birds and poultry [19, 127–129].

Genetically, the viruses responsible for outbreaks between 2005 and 2010 were closely related to clades 1, 2 and 3, previously isolated from birds and humans between 1997 and 2003 [130]. However, since 2008, clade 0 (China and Hong Kong), clade 2.1.1 (Indonesia), clade 2.1.2 (Indonesia), clade 2.3.1 (Hunan and Guangdong provinces), clade 2.3.3 (Hunan and Guiyang provinces), clade 2.4 (Yunnan and Guangxi provinces), clade 2.5 (China, Korea and Japan), clade 3 (Hong Kong, China and Vietnam), clade 4 (Hong Kong and China), clade 5 (China and Vietnam), clade 6 (China), clade 8 (Hong Kong and China) and clade 9 (China) appear to have been replaced by new clades or subclades and are now considered inactive or extinct [22, 120, 122, 131]. In contrast, the following clades have continued to be detected, albeit with limited presence between 2008 and 2016: clade 1, including third-order subclades, reported in China, Malaysia, Cambodia, Vietnam, Hong Kong, Thailand and Laos; clade 2.1.3 in Indonesia; clade 2.2.1 in Egypt and Israel; clade 2.2.2 in India, Bangladesh and Bhutan; clade 2.3.2 in China, Hong Kong, Myanmar and Vietnam; clade 2.3.2.1, including fifth-order subclades, in Russia, Bulgaria, Romania and North-Southeast Asia; clade 7, including second-order subclades, in China and Vietnam [122, 131–135].

Fig. 3. Formation of clades of HPAI H5NX of Gs/Gd lineage. The phylogenetic tree of HA sequences from Gs/Gd lineage viruses was generated using the Markov Chain Monte Carlo method which was run for 100 million iterations and sampled every 1000 steps to allow all parameters to converge and GTR model with a proportion of invariant sites (I) and gamma-distributed rates (G) in BEAST v1.10.4, and FigTree v1.4.4 was used for the display of the phylogenetic tree obtained by Bayesian analysis. The dark blue line indicates the emergence of clade 2.3.4.4c in North America, and letter (a) indicates the emergence of clade 2.3.4.4b in the Americas region (Created with iTOL version 5).

Fig. 4. Dispersion of the 2.3.4.4b clade in the Americas region. Description of the start of the first epizootic event in 2014/17 and the second event in 2020/25, where the main genotypes currently circulating in the region of the Americas are identified. The green-underlined strains are examples of strains belonging to genotype B3.2 in South America. EA lineage (Eurasian), NA (North America) and SA (South America) (Created with iTOL version 5).

Fig. 5. Origin of the genotypes from H5N1 viruses 2.3.4.4b clade into America. The strains in which the different genotypes emerged from A/Eurasian wigeon/Netherlands/1/2020 (H5N1) virus and from which 2.3.4.4b clade viruses circulating in America descend; the pink line indicates that the genotype emerged in Europe, orange in Asia, blue in Africa, green in North America and red in South America. Cui et al. initially classified the genotypes that emerged from A/Eurasian wigeon/Netherlands/1/2020 (H5N1) virus into 16 genotypes (G1–G16) [265]; however, the nomenclature from 2.3.4.4b virus clade based in the European Union determined that during the 2020/21 outbreaks, 19 genotypes emerged which were identified by letters (i.e. a, b, c) and in the 2021/22 outbreaks, 31 genotypes emerged which were named with two letters (i.e. AA, AB, AC) and these genotypes have determined that genotype EA-2020-C corresponding to genotype A [266]. Genotypes named BB, AB (H5N1 A/duck/Saratov/29-02/2021-like), CH (H5N1 A/Mallard/Netherlands/18/2022-like), I (H5N5 A/whooper_swan/Romania/10123_21VIR849-1/2021-like), DA (H5N1 A/mute_swan/Slovenia/ PER1486-23TA_23VIR10323-22/2023-like), DB (H5N1 A/herring_gull/Germany-NI/2023AI08764/2023-like), DC (H5N1 A/Common_Buzzard/ Netherlands/23023642-002/2023-like), DD (H5N1 A/Pheasant/England/113705/2023-like), DE (H5N1-A/Chicken/Scotland/114176/2023-like), DF (H5N1-A/Sparrowhawk/Scotland/131359/2023-like) and DG (H5N1 A/chicken/Germany-NI/2023AI08838/2023-like) are the genotypes currently circulating in Europe [244]. Genotype B3.2 emerged in South America on 13 May 2023 and arrived in Brazil in June 2023 [261] (Created with BioRender. com).

Despite the diversity of phylogenetic clades, many have been reported in specific regions or countries. However, clade 2 has been the most important because, according to the last recorded isolations until 2024 (shown in Figs 3, 4 and 5) [22, 120, 121, 131, 133], it has expanded into fifth-order clades and caused the three main events of the inter- and intracontinental spread of the H5NX viruses [136].

ORIGIN OF HPAIV H5NX CLADE 2.3.4.4

Lineage Gs/Gd viruses caused outbreaks between 1997 and 2003 in Thailand, Vietnam, Cambodia, Indonesia and China [118, 137]. However, this epidemiological situation changed in 2005 when an unprecedented outbreak occurred among wildlife avian species at Qinghai Lake in China, a crucial area for migrating over 200 000 birds and a convergence site for flyways from Eurasia and East Asia–Australasia [118, 118, 138–140, 140, 141]. This outbreak, attributed to the H5N1 virus clade 2.2 [136, 142], resulted in the deaths of over 6184 waterfowl between 4 May and 29 June. The hardest impact was in bar-headed geese (Anser indicus), brown-headed gulls (Larus brunnicephalus), black-headed gulls (Larus ichthyaetus), ruddy shelducks (Tadorna ferruginea), great cormorants (Phalacrocorax carbo), hooper swans (Cygnus cygnus), black-headed cranes (Grus nigricollis) and pochards (Aythya ferina) [14, 118, 141]. The viruses descending from this outbreak, known as Qinghai Lake-like viruses [118], were related to the A/chicken/Jiangxi/25/04 and A/peregrine falcon/HK/D0028/04 isolations in China [118, 141]. From December 2003 to October 2004, H5N1 lineage Gs/Gd outbreaks in Suphanburi Province, Thailand, affecting mammal species, such as tigers (Panthera tigris), leopards (Panthera pardus) [143] and domestic dogs (Canis familiaris) [144]. Additionally, from December 2003 to January 2004, some cases occurred in lions (Panthera leo), tigers (P. tigris), Asiatic golden cats (Catopuma temminckii), leopards (P. pardus) and a clouded leopard (Neofelis nebulosa) in Cambodia [145]. However, clade 2.2 was reported in 2006 on Rügen Island, Germany, exclusively in a domestic cat (Felis catus) [146] and a stone marten (Martes foina) [147]. Due to the seasonal migratory patterns of wildlife avian species (Fig. 6), the geographic distribution of clade 2.2 was reported from Northern and Southern Europe (October 2005–2006) to the Middle East and Northern and Western Africa [112, 136, 148, 149], including 38 countries across Africa, Asia and Europe by 2009 [81, 112, 136, 148]. After a decline in the detection of clade 2.2 in Europe in 2009 and in West Africa until 2008 [148], Egypt became endemic in both domestic and wild bird populations [81] and clade 2.2 was reported in South Asia (2011) [148]. From 2003 to 2006, clade 2.3 was reported in China, Hong Kong, Vietnam, Thailand, Laos and Malaysia, leading to clade 2.3.4 (Fujian-like lineage) in China in 2005 [122, 150]. From 2005 to 2008, clade 2.3.4 became the dominant virus in various regions of Southern China, Laos, Malaysia, Thailand and Northern Vietnam [122, 151]. Evolving from clade 2.3.4 (H5N1, H5N2, H5N5 and H5N8) circulating [135, 152–154], fourth-order clades emerged between 2006 and 2014 [133, 135, 155] in the following order: clade 2.3.4.2 during 2006/07 in Yunnan province and Vietnam, clade 2.3.4.3 in 2007 in Vietnam, clade 2.3.4.1 during 2009/10 in Hynan and Guizhou provinces, Vietnam and Laos, clade 2.3.4.4 in 2010 in China [132, 133, 155] and clade 2.3.4.6 during 2013/14 in China, South Korea, Japan, Laos and Vietnam, currently named clade 2.3.4.4 [135, 155–158].

Fig. 6. Spread of clade 2.2 during the 2005 outbreak in Qinghai Lake. Potential migratory routes in the virus spread and countries (identified by numbers) where initial outbreaks of the first intercontinental wave were reported. Birds from the south/southeast arrive to breed at the Qinghai Lake (black arrows), and the virus would move to Mongolia (16), Kazakhstan (15) and Russia (14) (red arrows) when the spring migration begins. Birds present in breeding areas in Siberia would reach Romania (6) and Turkey (7) during the 2005 winter migration from the Black Sea/Mediterranean and Eurasian (green arrows). In early 2006, movement occurred towards France (1), Germany (2), Switzerland (3), Italy (4) and Sweden (5) (pink arrow); after its introduction to Europe, some populations moved along the Atlantic and Black Sea/Mediterranean flyways to Egypt (8), Sudan (9), Cameroon (10), Nigeria (11), Burkina Faso (12) and Ivory Coast (13) (purple arrow). Finally, birds wintering in Africa and Europe returned to Siberia (yellow dashed arrows), triggering a new wave of cases in 2007 in Europe and the Middle East. Upon returning to Russia from the intersection zone of the Eurasian and Australasia flyway, they reached China–Mongolia, Southeast Asia [Thailand (23), Laos (22), Vietnam (21) and Indonesia (24)] (black dashed arrows) and emerged in the winter of 2007 in Korea (25) and Japan (26) (blue dashed arrow) (Created with BioRender.com).

Clade 2.3.4.4 traces its origin to one of the first isolations, subtype H5N8 (A/duck/Jiangsu/k1203/2010), from domestic ducks in Jiangsu province, China, in January 2010 [159–162]. This clade emerged from reassortments with the H5N1 clade 2.3.4 Asian lineage and circulating LPAIV in Eurasia [135, 155, 159, 161, 163]. In November 2013, in Zhejiang province, China, domestic poultry witnessed the re-emergence of clade 2.3.4.4 [135, 160], marking the onset of the second epizootic event that began in the 2014/15 period, spreading to several countries (Fig. 7) [88, 132, 164–167].

GENETIC GROUPS AND GENOTYPES OF HPAIV H5NX CLADE 2.3.4.4

Clade 2.3.4.4 viruses have their genetic origin in genotype V (Fujian-like lineage) [168], which emerged from the internal genes of genotype Z except the PA gene, which came from circulating LPAIV in waterfowl [169, 170]. However, due to the wide variety of NA subtypes, high incidence in wild birds, as well as a wide geographical spread [133, 171], clade 2.3.4.4 was organized into four genetic groups: A, B, C and D [164, 172], which had key genotypic characteristics: group A, their internal genes came from LPAV circulating in eastern China gave rise to the D3 genotype [155, 158, 173], and when they presented their incursion into America gave rise to the genotypes AmN2 and AmN1 [155]; additionally, these viruses were considered high pathogenicity in poultry because they retained key residues to promote affinity SA α−2,3 Gal receptor [173]; group B, with five internal genes of Eurasian LPAIV gave rise to genotypes G1 and G3 [155, 164, 174], similarly present markers that led to infections in domestic birds with a slight variation in the CS sequence of HA [173] and finally the groups C and D, six internal genes came from Asian LPAIV which caused the genotype G1 (included genotypes of the second and third orders) to emerge for group C and the genotype G2 (included only genotypes of the second order) for group D [164, 174]. However, a key feature that determined its classification into groups was its wide range of evolutionary rate: group A for the HA gene was 12.14×10⁻³ substitutions site⁻¹ year⁻¹ and the NA gene, 9.10×10⁻³ substitutions site⁻¹ year⁻¹ [175, 176]; group B for HA gene from 4.81 to 8.58×10⁻³ substitutions site⁻¹ year⁻¹ and NA gene, from 4.95 to 7.32×10⁻³ substitutions site⁻¹ year⁻¹ [177]; group C for HA, 5.635×10⁻³ substitutions site⁻¹ year⁻¹ and NA 6.407×10⁻³ substitutions site⁻¹ year⁻¹; finally group D, HA 5.459×10⁻³ substitutions site⁻¹ year⁻¹ and NA 4.839×10⁻³ substitutions site⁻¹ year⁻¹ [174].

Fig. 7. Spread of clade 2.3.4.4 during 2014/17. The viruses of clade 2.3.4.4 descend from subtypes H5N2, H5N5 and H5N8 of clade 2.3.4 circulating in domestic populations in China since 2008. Through various recombination events, clade 2.3.4.4 emerged during 2012/13 and was divided into four genetic groups. Group A (H5N8) was isolated in early 2014 in the Republic of Korea from where it migrated to different Asian countries causing outbreaks before arriving in the breeding area of Siberia in the summer of 2014 and emerging in Western Europe and North America by the end of 2014 in response to winter migration. Group B (H5N8) emerged in China in May 2016 and was detected in Russia and Mongolia in June 2016, from where it moved to the Middle East, Africa and Europe during the fall migration of 2016. However, in Europe, between 2016/17, this group underwent recombination with LPAIV, giving rise to HPAI H5N5 virus. Group C (H5N6) emerged in China, spread to Laos and Vietnam in 2014, Hong Kong in 2015 and at the end of 2016 in the Republic of Korea and Japan. Group D (H5N6) migrated from China to Vietnam in 2013/14 (Created with BioRender.com).

Group A (A/broiler duck/Korea/Buan2/2014-like), clade 2.3.4.4A (2.3.4.4a), also known as subgroup A (like-Buan virus) [148, 178–181], emerged on the Korean Peninsula during the winter of 2013/14 [148]. It originated from a reassortment of A/duck/Zhejiang/W24/2013 (H5N8) (2010-descendant virus) and A/duck/Jiangsu/k1203/2010 (H5N8) [40, 159]. This group was reported in the Russian Federation in September 2014 [148, 182], reaching Northern Europe during the winter migration of late 2014 and finally reaching the Eastern part of the continent in February 2015 [148], returning to Asia (South Korea–Japan) during the spring–summer period [178, 180]. It marked the first intercontinental spread from Asia to America during the winter of 2014 [136, 148, 180]. The avian migratory pattern possibly contributed to the distribution from Japan to San Lorenzo Island in Alaska, culminating in their return to the Siberian tundra along the North American Pacific flyway through the Beringia region to the mainland [132, 149, 183, 184] in avian breeding areas in Siberia. These locations represent key geographic locations for intra- and intercontinental dissemination [155], allowing dissemination through the various avian migratory routes connecting East Asia–Australasia, Eurasia, East Asia–East Africa and Northern American Pacific flyways [185].

Due to these circumstances, the HPAIV H5N8 virus was introduced in North America [164]. The circulating virus of Eurasian lineage in South Korea and genomic reassortments with LPAIV subtypes from North American lineage gave rise to the H5N2 subtype, causing the first outbreaks of clade 2.3.4.4 in British Columbia, Canada, between late November and early December [14, 186, 187]. Subsequently, the viruses spread along the Pacific Americas flyway to the USA, causing the first outbreaks: H5N8 and H5N2 subtypes in Washington from 6 December to 11 December, H5N8 subtype in Oregon from 16 December to 22 December and Idaho on 22 December 2024 [135, 188–193]. Through genomic reassortments, H5N1, H5N2 and H5N8 viruses spread along the Central Americas and Mississippi flyway from late December 2014 to June 2015, affecting 12 states in the USA and re-emerging in Canada (Ontario) [97, 175, 190]. However, by mid-2015, due to eradication strategies implemented by the governments of the USA and Canada, outbreaks of 2.3.4.4 clade viruses were decreasing [190]. The viruses re-emerged in mid2016 in Alaska, isolating the strain A/mallard/Alaska/AH0008887/2016 (H5N2) [194].

According to the significant divergence and spread, group A was classified into six subgroups: C0, primitive H5N8 viruses, the first viruses circulating outside China (January to July 2014); C1, H5N8 viruses circulating only in South Korea (September 2014 to November 2015); C5, H5N8 reported in South Korea (May 2014 to April 2016); C2, intercontinental group A (icA) 3, composed of H5N8 viruses from Japan (December 2014) and South Korea (January 2015); C4 (icA1), a group composed of H5N8 viruses from Europe, Russia, Taiwan, Japan and South Korea (2014 to mid-2015); C3 (icA2), composed of H5N8 as well as H5N2 and H5N1 viruses (originated in North America) from North America and Japan (2014 to mid-2016) [135, 194–196].

Group B (A/breeder duck/Korea/Gochang1/2014-like), clade 2.3.4.4B (2.3.4.4b) or subgroup B (similar-Gochang virus) [148, 178–181], emerged in May and June 2016 [148], causing outbreaks in populations of brown-headed gulls (L. brunnicephalus), barred-headed geese (A. indicus) and great black-headed gulls (L. ichthyaetus) on Egg Island, lake Qinghai, China [163], as well as in the territory of the Tuva Republic, on the border between Russia and Mongolia, in populations of great crested grebes (Podiceps cristatus), black-headed gulls (Larus ridibundus), grey herons (Ardea cinerea), great cormorants (P. carbo) and common terns (S. hirundo) present in Uvs-Nuur Lake [148, 182, 197]. This subgroup was originated from a reassortment of A/ duck/Jiangsu/k1203/2010 (H5N8), A/duck/Hunan/8-19/2009 (H4N2) and A/environment/Jiangxi/28/2009 (H11N9) viruses [159]. However, after emergence in Russia/Mongolia, subgroup B crossed with circulating LPAIV in the Central Asian migratory route, giving rise to genotype 1 of Russia–Mongolia H5N8 viruses [159, 198]. The viruses then reached South Asia [148, 178] and Europe, rearranging with LPAIV, and finally, in mid-October 2016, genotype 2 of European H5N8 viruses resulted [159, 178]. Once established in Europe, the H5N8 virus rearranged with circulating LPAIV, giving rise to the H5N6 virus in 2017, causing outbreaks in the Netherlands, Switzerland, Greece and the United Kingdom [197] and the H5N5 virus during 2016/17 in the Netherlands, Germany, Poland, Italy, Croatia and Hungary [199]. Subsequently, this viral type was detected in North Africa in November 2016 [148, 200]. In December 2016, at Lake Victoria in the Lutembe Bay in Wakiso and Masaka districts [201], due to the migratory flow of Afro-Eurasian birds along the Black Sea/Mediterranean and Asia–East Africa migratory routes [200], the viral circulation began in the African continent (Nigeria, Uganda, Zimbabwe, Democratic Republic of the Congo and South Africa) of these viruses [148].

Group C (H5N6) viruses emerged in China (2013) from a reassortment with H5N6, H6NX and H7N9/H9N2 viruses circulating in this country [202]. Later reports from this group were recorded in Laos, Vietnam (2014), Hong Kong (2015), South Korea and Japan (2016). Finally, group D (H5N6) only circulated from China to Vietnam in 2013/14 [164]. The viruses from this clade that caused outbreaks during these periods were collectively referred to as H5NX viruses [121], including subtypes H5N1, H5N2 (2008), H5N3 (2004), H5N5 (2006), H5N6 (2013) and H5N8 (2010) [164, 203]. The HPAIV H5N8 was the most representative, as it improved its ability to infect wildlife avian species, causing infections with high virus shedding [136, 171, 183], a critical characteristic for transmission.

The extensive diversification and intercontinental spread of clade 2.3.4.4 resulted in eight groups (a–h) of fifth-order clades [204–206]. However, due to changes in the nomenclature established by World Health Organization [184, 207], the subgroups were reassigned as follows: 2.3.4.4a (2013), responsible for H5N6 virus outbreaks in Asia [205]; 2.3.4.4b (2016–2017), spread along Eurasia and Africa [164, 205], responsible for the third inter- and intracontinental spread [136]; 2.3.4.4c (2014–2016), formerly 2.3.4.4a [208]; 2.3.4.4e (2016–2017), leading H5N6 outbreaks in Japan and South Korea; 2.3.4.4 (2016), groups D, F, G and H predominated in China and East Asia [205].

HPAIV H5NX CLADE 2.3.4.4B

In viruses of the 2.3.4.4b clade, H5N6 and H5N8 were the primary circulating subtypes observed in avian wildlife and poultry during 2016–2017 and 2020–2021 [112, 208–210]. However, during the same period, subtypes H5N1, H5N2, H5N3, H5N4 and H5N5 without any epizootic impact were also reported in Europe and Asia [136, 209–211]. Due to the ongoing circulation of H5 viruses, specifically clade 2.3.4.4b, through the Eastern Atlantic, Eurasia and the Mediterranean–Black Sea flyways [184, 210], the invasion of subtype H5N1 into Northern Europe commenced [112, 136], marking the third intercontinental epizootic event. Subtype H5N1 A/Eurasian wigeon/Netherlands/1/2020 (H5N1) [135, 136] replaced subtype H5N8 as the predominant during 2021–2022 [208, 210], establishing itself from reassortments of LPAIV with H5N8 virus through the Mediterranean—Black Sea flyway [112, 136, 171]. This H5N1 subtype resulted from reassortments of the H5N8 virus segment 4 (HA) and segment 7 (M); H3N8 segment 1 (PB2) and 5 (NP) [A/gadwall/Chany/893/2018 (H3N8)-like virus]; H3N8 segment 2 (PB1) [A/duck/ Mongolia/217/2018 (H3N8)-like virus]; H1N1 segment 3 (PA) and 8 (NS) [A/Anas platyrhynchos/Belgium/10402-H195386/2017 (H1N1)-like virus]; H1N1 segment 6 (NA) [A/A. platyrhynchos/Belgium/9594-H191810/2016 (H1N1)-like virus] [136].

The presence of the HPAIV H5N1 virus clade 2.3.4.4b in migratory species from Northern or Northwestern Europe resulted in its detection in the American High Arctic in 2021. It initially emerged in a great black-backed gull (Larus marinus) between 4 November and 26 November 2021 and was subsequently reported in domestic birds on a farm in St. John’s from 9 December to 21 December 2021; both outbreaks occurred in the province of Newfoundland and Labrador on the Atlantic coast of Canada [135, 165, 212]. The virus was introduced along the East Atlantic flyway via three principal migration routes: migration from Northern Europe to Iceland or Svalbard, concluding in Greenland or the American High Arctic [165, 167, 213]; migration from North-West Europe to the High Arctic and/or North-West Greenland, where some birds migrate towards Baffin Bay, interacting with Arctic seabirds that head to the Canadian Atlantic coast in autumn; pelagic migration from Northern Europe to the American Atlantic coast [165]. After the initial cases, some waterbirds migrated southward along the Atlantic flyway, leading to the first outbreaks in the USA, which were initially recorded on 30 December 2021 in populations of American wigeon (Mareca americana) in South Carolina (A/American wigeon/South Carolina/AH0195145/2021) and on 8 January 2022 in North Carolina (A/ American wigeon/North Carolina/AH0182517/2022) [135]. After the initial outbreaks, the virus spread across North America via four flyways: Atlantic, Mississippi, Central and Pacific [97, 165, 166].

According to a study of wildlife bird population movements along flyways up until spring 2022, ~64.7% of these movements occurred on the Atlantic migratory flyway, 33.6% occurred between the Atlantic and Mississippi flyways and only 1.7% took place at the confluence of the Atlantic and Central flyways [97, 98]. However, in February 2022, in British Columbia, a white-tailed eagle (Haliaeetus albicilla) was diagnosed with an HPAIV H5N1 virus-like strain that had previously been reported in the Americas and was genetically related to viruses identified in Hokkaido, Japan, genotype G7, in early 2022 [136, 214]. This suggests that the mobilization to the continent could occur through the confluence of the flyways from East Asia–Australasia and the Pacific.

The spread of the HPAIV H5N1 virus through the Pacific flyway occurred outside the USA, leading to the first case on 13 October 2022 in a gyrfalcon in the State of Mexico, Mexico [215]. Subsequently, spreading to South America via the Atlantic flyway, with an initial outbreak reported in Colombia (A/duck/Choco/ICA-3501/2022) (9 October 2022), Peru, Venezuela and Ecuador (November 2022), Chile (December 2022), Bolivia (January 2023) and Argentina and Uruguay (February 2023). By May 2023, it reached Paraguay and Brazil [121, 135, 216]. The genotypes associated with these outbreaks included B1.1, B1.2 and B3.2 [103, 184, 217, 218]. Notably, genotype B3.2 marked the virus’s first incursion into Antarctica, leading to an outbreak in the sub-arctic on 30 October 2023 (A/Southern_fulmar/Falkland_Islands/133789/2023) and another in the Arctic (Bird Island, South Georgia) on 8 October 2023, affecting brown skua (Stercorarius antarcticus) populations. By the end of 2023, several outbreaks were also reported among kelp gulls (Larus dominicanus), Antarctic fur seals (Arctocephalus gazella), Southern elephant seals (Mirounga leonina) and Southern fulmars (Fulmarus glacialoides) in South Georgia and the South Sandwich Islands [103, 135].

EVOLUTIONARY ADAPTATIONS OF HPAIV H5NX CLADE 2.3.4.4B

Evidence of the first evolutionary changes lies in the isolations of H5N1 viruses that caused outbreaks in humans and wild cats (1997), continuing through to cases linked to descendants of clade 2 [108, 219]. These viruses showed a glutamine–lysine substitution at position 627 in the PB2 protein, related to enhanced replication in mammals [107, 141, 143, 219]. H5N1 avian and mammalian viruses showed changes in the HA sequence. Both contained a polybasic amino acid sequence (SPQRERKRKKR) in the HA1 CS, altering their pathotype to HPAIV [65]. However, several mutations have been key to increasing their pathogenicity in mammals, their ecological dispersion, as well as genotypic drift [220]. Other mutations have been present in all genotypes (except in genotypes B, W, Z+ and X0 –X3 ), including the deletion in the NA and NS1 proteins, associated with the adaptation of influenza viruses [219–221]. Due to the wide variety of isolations since 1997, the deletion in NA has been classified into four groups based on location and length of the deletion: (1) long NA stalk, (2) deletion of 20 amino acids from positions 49–68, (3) deletion of 20 amino acids from positions 55–74 and (4) deletion of 19 amino acids from positions 55–73 [221]. But since 2001, the majority of viruses have presented a deletion from positions 49–68 [220–222]; this deletion in the stalk NA generates various repercussions on the virulence of the virus, as it has been associated with: (1) adaptation of waterfowl viruses to domestic birds and (2) to promote viral replication by promoting the retention of the virion in the plasma membrane to improve the binding of HA to the cellular receptor (SA α−2,6 Gal or SA α−2,3 Gal) [223]. In relation to the deletion in NS1, it was initially reported in the amino acids of positions 88–92 (AIASS/V) [143, 219]; however, since 2003, it has been presented at positions 80–84 of the N-terminal end [221, 222, 224]. This mutation promotes NS1 to act as an antagonist in the response mediated by IFN-α or IFN-β [45] because it competes with retinoic acid-inducible gene I in the induction of IFN [221, 225]. On the other hand, all isolations prior to the 2005 outbreaks still retained the amino acids glutamine in position 226 (Q226) and glycine in position 228 (G228) in HA, which are associated with increased affinity to the SA α−2,6 Gal [108, 143].

Since then, several mutations have been reported in clade 2 virus descendants, which resulted in the adaptation of the virus to lower temperatures, thus promoting its replication in the respiratory tract and having an airborne transmission [226]. Since then, it has been proposed that these mutations may have been selected through the involvement of intermediate hosts, especially wildlife felids susceptible to viruses of this clade [107, 108, 143, 145, 226] or some bird species, such as Ratitae [83] before the virus returned to domestic or aquatic birds. Similar to the clade that caused the first epizootic event, clade 2.3.4.4 viruses had various mutations, e.g. (1) modifications in the receptor-binding domain of the HA protein, resulting in adaptive changes to affect new hosts [91, 164]; (2) dual affinity for both SA receptors because they had proline in position 128 (128P), arginine in position 137 (137A) and arginine in position 160 (160A) in HA; (3) all viruses contained a polybasic amino acid sequence (PLREKRRKR/ GLF) in the HA1 CS [153, 227]; (4) continued to express the substitution E627K and D701D mutation in PB2 [228, 229]; (5) some viruses showed a truncation of 11 amino acids in the PB1-F2 protein, which is associated with increased pathogenicity and adaptability in mammals [227, 230].

On the other hand, the descendants of the 2.3.4.4b clade have developed the peculiarity of being able to constantly infect mammals, where so far it has caused outbreaks in more than 30 species of mammals [231], and that in the case of terrestrial mammals (canids, mustelids, procyonids, ursids and mephitids) and marine (cetaceans and pinnipeds) has been considered a neurotropic agent due to the affinity towards this tissue during its viral pathogenesis [213, 232–234]. Due to this characteristic, several mutations associated with adaptability to non-avian hosts have been reported, e.g. (1) E627K mutation, as well as I192V (PB2) and N66S (PB1-F2), associated with adaptation and increases in mammalian virulence, (2) substitution of S137A and T160A in HA, which increases the affinity to SA α−2,6 Gal receptor and (3) deletion in the stalk of NA [213, 232, 235]. However, they were also responsible for the first outbreaks in cattle caused by IAV (H5N1) [135], an epidemiological event that was not thought possible because ruminants were only hosts of IDVs [29, 236] and express mainly the Neu5Gc receptor (instead of Neu5Ac) [237]. Several scenarios have been proposed for the occurrence of outbreaks: (1) a direct transmission from waterfowl, as these species of the viruses are able to bind to both receptor types (Neu5Gc and Neu5Ac), a quality that is lost when the virus begins to circulate in poultry [59]; (2) replication of the virus in tissues expressing its receptors (SA α−2,6 Gal and SA α−2,3 Gal), as in bovines, they are poorly expressed in the respiratory tract [238], but predominate in the mammary gland [237, 239]; (3) adaptability of the virus, as (i) mutation Y161A changes the affinity of the union of the virus from Neu5Ac to Neu5Gc (not yet reported in bovines) [240] or (ii) mutation M631L (PB2) [239, 241].

INTERCONTINENTAL SPREADING AND ENZOOTIC EVENTS OF HPAIV H5NX VIRUSES

The worldwide distribution of HPAIV H5NX Eurasian AIV, originating from Gs/Gd viruses, has led to outbreaks classified into several enzootic events and three instances of intercontinental spread [112, 136, 149, 171]. The first intercontinental spread of HPAIV H5N8, clade 2.3.4.4, was responsible for epidemic records in North America during 2014/16 [132, 164, 183, 188], attributed to migratory wildlife avian species from Asia to Alaska or through the Pacific Americas flyway [132, 149, 183, 184]. Subsequently, it came across the continent via the Central and Mississippi flyways [97]. During 2020, the lineage Gs/Gd re-emerged as HPAIV H5N1 [112, 136, 171], causing the second intercontinental spread to America at the end of 2021 [136]. But in this case, geese and swans were potentially involved in spreading the HPAIV H5N1 virus due to annual migratory patterns from Ireland and North America.

Additionally, seagulls are considered another potential species based on their extensive pelagic movements and breeding and wintering areas spanning from north and central North America to South America [242]. Their broad annual migratory patterns could be related to the spreading of the virus, into the Antarctic, and a possible third intercontinental dispersal (Oceania).

IMPACT AND PREVALENCE OF HPAIV H5NX IN AMERICA

According to records from the United States Department of Agriculture and the Canadian Food Inspection Agency, the first AI outbreak that significantly impacted North America was caused by H5NX viruses of the 2.3.4.4c clade during 2014/16 [187, 189]. This caused the death of 250 000 birds in Canada (Ontario and British Columbia) [187]. At the same time, in the USA, it affected 21 states, resulting in the death of 7.4million turkeys and 43million chickens and an economic impact of ~$3.3 billion for the country [192]. However, it was not until the third epizootic event emerged that all regions of the Americas were impacted.

The Pan American Health Organization reported that by the end of 2024, animal health and agricultural organizations across Argentina, Bolivia, Brazil, Canada, Chile, Colombia, Costa Rica, Cuba, Ecuador, the Falkland Islands, Guatemala, Greenland, Honduras, Mexico, Panama, Paraguay, Peru, the USA, Uruguay and Venezuela have identified 3648 outbreaks of HPAIV H5NX in poultry, wild birds and both terrestrial and marine mammals [19, 243]. Government sources describe the impact of the virus in America as significant because bird and mammal populations have never been exposed to this agent, causing substantial reductions in the population numbers of various wildlife species [19].

The current situation in North America shows that this region presents the incursion of H5N1, H5N5 and H5N6 subtypes [135, 165, 184, 213, 244]. H5N1 viruses in Canada since their incursion (2021–2025) have caused outbreaks in poultry in the provinces of Alberta, British Columbia, Manitoba, New Brunswick, Newfoundland and Labrador, Nova Scotia, Ontario, Quebec and Saskatchewan, affecting more than 15million poultry [245]. Moreover, 2229 cases of 90 bird species have been reported throughout the Canadian territory [244]. In contrast, in the 10 Canadian provinces, around 150 cases were reported in mammals, 11% of which were marine mammals (Quebec province) and the rest terrestrial mammals [244].

Starting in 2023, the initial outbreaks of the H5N5 virus were documented on the continent. So far, there have been two reported cases in Greenland, leading to the first notification of outbreaks involving Gs/Gd lineage HPAIV [20, 135] and 90 cases in Canada, specifically in New Brunswick, Newfoundland and Labrador, Nova Scotia, Nunavut, Ontario, Prince Edward Island and Quebec. Of these cases, 82% involved wild birds, 17% were found in terrestrial mammals and 1% in marine mammals [245]. Only two cases of the H5N6 subtype have been registered: one in Manitoba involving a blue-winged teal (Spatula discors) on 21 November 2022 and another in Minnesota concerning a bald eagle (Haliaeetus leucocephalus) on 8 May 2023 [135, 245].

The U.S. situation has evolved, with an economic impact initially estimated at $2.5–$3 billion due to poultry outbreaks up to 2023 [80]. However, by early 2025, the virus had spread to 50 states, affecting over 133 million birds [246]. Various estimates project a price increase of 50% for eggs, 45% for turkey, 44% for chicken, 103% for organic eggs, 60% for turkey breast and 82% for organic chicken since 2022. Price increases have soared to 125% for eggs, 85% for turkey and 47% for chicken [247]. Additionally, the virus has led to 10 948 cases in wild birds across the USA and 449 cases in mammals in 38 states, with only 6% reported in marine mammals located in Washington, Florida, Wisconsin and Maine [246, 248]. Furthermore, outbreaks in 924 dairy herds across 16 states have profoundly impacted the dairy industry, causing changes in milk’s organoleptic characteristics, a 35% decrease in rumination, a 22% reduction in production with a recovery period of ~2 months and nearly 2% mortality [249, 250].

By early 2023 in Mexico, reports indicated that 5 981 105 birds were affected, including ~300 685 wild birds. However, only 361 cases were documented in wild birds. In contrast, the poultry sector saw the euthanization of 5 311 815 birds to control outbreaks, leading to an economic loss of 2633 million pesos for the industry. Although the Ministry of Agriculture and Rural Development (SADER) declared the country free of H5N1 in June 2023, the virus re-emerged in October 2023 in the states of Veracruz, Jalisco, Sonora and Guanajuato. Since then and continuing into early 2025, there have been recurrent outbreaks among wildlife avian species and domestic birds across various regions of Mexico [215, 251–254].

While the virus has impacted the poultry sector in several countries in South America, wildlife populations of birds and marine mammals have been most affected, mainly in Chile and Peru. According to statistics from both countries, the virus caused the death of more than 40% of the pelican population [255]. In Peru, the virus caused the mass death of over 22 000 wildlife birds in 4weeks in 2022, with the most affected species being the Peruvian pelicans (Pelecanus thagus) and Peruvian boobies (Sula variegata) [256], and on 13 November 2022, it had caused the death of more than 50 000 wildlife birds and 3108 South American sea lions (Otaria flavescens) [257]. This resulted in 83% of the cases in marine mammals [216]. However, in Chile (September 2023), the virus caused the death of more than 29 000 seabirds, mainly the Peruvian booby (S. variegata), guanay cormorant (Phalacrocorax bougainvillii), kelp gull (L. dominicanus), Peruvian pelican (P. thagus), neotropic cormorant (Phalacrocorax brasilianus), grey gull (Larus modestus), Humboldt penguin (Spheniscus humboldti) and Magellanic penguin (Spheniscus magellanicus) [258–260]. On the other hand, 19 967 deaths were reported in marine mammals, of which 98% were recorded in sea lions (O. flavescens) and the rest in marine otter (Lontra felina) (n=40), Chilean dolphin (Cephalorhynchus eutropia) (n=17), Southern river otter (Lontra provocax) (n=1), Fernández fur seal (Arctocephalus philippii) (n=42) and Burmeister’s porpoise (Phocoena spinipinnis) (n=35) [260]. However, Argentina, Brazil, Falkland Islands and Uruguay have also reported cases in their populations of marine mammals, mainly the two-hair fur seal (Arctocephalus australis), the Southern elephant seal (M. leonina) and the South American sea lion (O. flavescens) [19, 103, 135]. Despite the genotypic diversification of viruses in South America [217, 261], all viruses recorded have retained the parental segments HA, NA and M from European lineages, and the remaining segments for NP, NS, PA, PB1 and PB2 have undergone reassortments with LPAIV circulating in wildlife bird populations on the continent [121].

Another impact that the virus has also had in the Americas region is that it has caused several cases in humans, e.g. (1) Canada reported its first case on 14 November 2024 in British Columbia caused by genotype D1.1 [19, 135]; (2) USA has reported 66 cases caused by genotypes B13.3 and D1.1 [135, 246]; (3) Chile reported one case on 29 March 2023; (4) Ecuador also reported one case on 9 January 2023 [19], genotype B3.2 caused both outbreaks [135]. Due to the morbidity and mortality that the virus has had in wildlife populations, animal health authorities on the continent have expressed concern about the population stability of endangered species where outbreaks have occurred, such as Humboldt penguins (S. humboldti) and marine otters (L. felina) [258], as well as those species with potential risk because of their feeding habits, such as the Andean condor (Vultur gryphus), California condor (Gymnogyps californianus) [258, 259] and several mammal species (mainly carnivores) that have contracted the disease mainly by oral route through consumption of infected material [18, 147]. In the face of this situation, it was the U.S. government which first authorized the implementation of vaccination for endangered species to safeguard populations of these species [262].

CONCLUSION

A wide range of factors promote the environmental permanence of AIV, considering natural reservoirs, as well as seasonal migration patterns intra- and intercontinental, facilitating the introduction and emergence of the virus in various geographical regions. The breeding area in Siberia plays a key role in the spread of the main epidemiological events caused by H5NX viruses descended from the Gs/Gd lineage since not only the main migratory routes from all continents are interconnected, but it also is a region where 200 000–400 000 wildlife birds converge during the breeding season [263]. However, factors like globalization also contributed to the increased activity of the poultry industry, which led to the emergence of HPAIV and the local spread of AIV worldwide. The leap between different species has been an observable phenomenon in Gs/Gd lineage viruses by acquiring mutations involved with pathogenicity, transmissibility and adaptability due to the participation of potential intermediary or bridge hosts that allow the permanence of these mutations.

Based on the eco-epidemiological review of the outbreaks caused by the Gs/Gd virus, it is of interest to evaluate whether the acquisition or permanence of the mutations involved in adaptation to a new host. As well, evaluate the implementation of biological tools such as vaccination to safeguard wildlife populations that are suffering from a never-before-seen imbalance and the potential risk presented for Antarctic and Greenland wildlife which have reported cases of H5NX virus of the Gs/ Gd lineage for the first time, as would be the spread of the virus to Oceania through pelagic species, which is increasingly becoming a target continent in the spread of the virus, since in March 2024 the first case of H5N1 clade 2.3.2.1a was reported in a human [264], opening the window of a possible third intercontinental incursion of Gs/Gd lineage viruses, mediated by ecological and human activity factors.

This article was originally published in Journal of General Virology 2025;106:002081. DOI 10.1099/jgv.0.002081. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.

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