Avian Influenza (H5N1): Global Spread, Clinical Manifestations, and One Health Surveillance

Introduction: Virological and Epizootiological Context

Highly pathogenic avian influenza (HPAI) H5N1 is a subtype of influenza A virus (family Orthomyxoviridae, genus Alphainfluenzavirus) characterized by a multibasic cleavage site (MBCS) in the hemagglutinin (HA) glycoprotein that confers systemic tropism in gallinaceous poultry [3, 9]. The MBCS contains multiple basic amino acid residues (e.g., -RERRRKR-) which are cleavable by ubiquitous furin-like proteases, enabling systemic endothelial and parenchymal infection [9, 53]. Since its emergence in domestic geese in Guangdong Province, China, in 1996, H5N1 has undergone extensive evolutionary diversification into multiple clades, with clade 2.3.4.4b emerging as the dominant lineage during the ongoing panzootic that began in 2020 [3, 27, 53]. The global dissemination of clade 2.3.4.4b viruses has been unparalleled in scale, affecting wild birds, poultry, and a rapidly expanding range of mammalian species across six continents [27, 50, 64].

Global Spread and Evolutionary Dynamics

Phylogeographic Dissemination

The current panzootic is characterized by sustained intercontinental transmission via wild bird migratory flyways, particularly among Anseriformes (ducks, geese, swans) and Charadriiformes (gulls, terns) [32, 50, 55]. Phylogenetic analyses have documented repeated introductions of clade 2.3.4.4b viruses into Europe, Africa, Asia, and the Americas [6, 42, 65]. In North America, the emergence of D1.1 reassortant H5N1 viruses has been detected, indicating ongoing genomic reassortment between Eurasian and North American lineage viruses. Similarly, in South America, multiple introductions into Brazil in 2025 were followed by intra-epidemic reassortment events that generated novel genotypes. Phylogeographic modeling using continuous diffusion approaches has revealed shifting environmental drivers of H5 spread, with land surface temperature, precipitation, and wild bird density serving as key predictors of viral diffusion velocity in regions such as Italy.

Host Range Expansion and Spillover Events

H5N1 clade 2.3.4.4b viruses have demonstrated a remarkable ability to infect a phylogenetically broad range of avian and mammalian hosts [27, 49, 50]. Among wild birds, mortality events have been documented in common terns (Sterna hirundo) in Namibia, black-headed gulls (Chroicocephalus ridibundus) and common terns in Germany, and griffon vultures (Gyps fulvus) in southern Europe. The panzootic has also severely impacted procellariiform seabirds, including albatrosses and petrels, prompting the establishment of dedicated public databases to track mortality in these vulnerable populations. The incursion of H5N1 onto remote islands, such as Gough Island in the South Atlantic Ocean, underscores the capacity for long-distance maritime transport via infected seabirds. Environmental and ecological risk factors for spillover from wild birds to poultry operations have been systematically characterized, with factors such as proximity to wetlands, poultry density, and biosecurity compliance levels identified as significant predictors in studies conducted in British Columbia, Canada.

Mammalian spillover events have increased substantially since 2022 [18, 27, 49]. Infections have been confirmed in multiple pinniped species (seals, sea lions) in North and South America, with mortality events raising conservation concerns. Domestic cats, particularly those with outdoor access or fed raw poultry products, have been infected with H5N1, exhibiting severe neurological and respiratory disease. Fur seals (Callorhinus ursinus) in Russia were found to carry genotype A3 H5N1 viruses that demonstrated high virulence in mammalian models but lacked airborne transmission competence. In Japan, H5N1 was isolated from Ezo red foxes (Vulpes vulpes schrencki), and experimental mouse modeling confirmed neurotropism and systemic pathogenicity. Most notably, H5N1 clade 2.3.4.4b viruses have been detected in dairy cattle in the United States, marking the first sustained transmission of HPAI in Bovidae [63, 69]. Experimental infection of Jersey cows with a human isolate of H5N1 resulted in mammary gland tropism and viral shedding in milk. Genomic features associated with sustained mammalian transmission include adaptive mutations in the PB2 polymerase subunit (e.g., E627K, D701N) that enhance replication efficiency at lower temperatures typical of the mammalian respiratory tract [28, 60]. An emerging PB2-627 polymorphism has been shown to overcome ANP32 host restriction factors, further increasing the zoonotic risk of circulating strains.

Molecular Pathogenesis and Virulence Determinants

Hemagglutinin Structure and Receptor Binding

H5N1 HA preferentially binds to alpha-2,3-linked sialic acid receptors, which are abundant in the intestinal and respiratory tracts of avian species [9, 51]. Mammalian influenza A viruses typically bind alpha-2,6-linked sialic acids, which predominate in the human upper respiratory tract. However, recent studies using lectin histochemistry and glycan array analysis have demonstrated that HA proteins of both clade 1 and clade 2.3.4.4b H5N1 viruses exhibit comparable attachment patterns to avian and mammalian tissues, suggesting that mammalian adaptation does not require a complete switch in receptor specificity. The acquisition of specific amino acid substitutions in the receptor binding site (e.g., Q226L, G228S in H3 numbering) can enhance alpha-2,6 binding affinity and is considered a critical step for pandemic potential [28, 62].

Polymerase Complex and Replication Fitness

The viral RNA-dependent RNA polymerase (RdRp) complex, composed of PB1, PB2, and PA subunits, is a major determinant of host range and virulence [9, 60]. The PB2 E627K substitution, which is a hallmark of mammalian adaptation, increases polymerase activity at 33 degrees Celsius (the temperature of the mammalian upper airway) by facilitating interaction with host ANP32 family proteins [60, 63]. Studies comparing human and bovine H5N1 isolates from Texas identified specific amino acid residues in PB2 that confer differential replication and pathogenicity in mammalian cells. The PA subunit also contributes to virulence through cap-snatching activity and modulation of host innate immune responses.

Immune Evasion and Antigenic Drift

H5N1 viruses employ multiple strategies to evade host immune responses. The NS1 protein functions as a potent interferon antagonist by inhibiting RIG-I signaling and limiting the induction of type I interferons [9, 37]. Antigenic drift in the HA and neuraminidase (NA) glycoproteins has been documented, necessitating periodic updates of vaccine strains [10, 29]. Cross-reactivity of antibodies generated by prior infection or vaccination is shaped by immune history, with pre-existing immunity to seasonal influenza strains providing minimal protection against H5N1 due to antigenic distance between group 1 (H5) and group 2 hemagglutinins [5, 38]. Computational design and nanoparticle display platforms have been employed to engineer HA immunogens that elicit broadly cross-reactive antibodies against conserved epitopes in the HA stem domain [29, 81].

Clinical Manifestations in Avian Species

Poultry

In gallinaceous poultry (chickens, turkeys), HPAI H5N1 infection typically results in peracute to acute disease characterized by depression, decreased feed and water consumption, and sudden death [59, 64]. Clinical signs include cyanosis of the comb and wattles, edema of the head and neck, petechial hemorrhages on the shanks, and neurological deficits such as torticollis, ataxia, and opisthotonos [9, 59]. Respiratory signs (coughing, sneezing, rales) may be present but are often overshadowed by systemic involvement. In laying hens, egg production drops precipitously, and eggs may exhibit shell deformities or loss of pigmentation. Necropsy findings include hemorrhagic tracheitis, pulmonary congestion, hydropericardium, splenomegaly, and petechial hemorrhages on serosal surfaces [9, 59].

Domestic ducks and geese infected with HPAI H5N1 may exhibit a wider spectrum of clinical outcomes, ranging from asymptomatic infection to lethal neurological disease [27, 59]. In scavenging ducks in Bangladesh, clade 2.3.4.4b viruses caused neurological signs including head tremors, circling, and paralysis, with histopathological evidence of non-suppurative encephalitis and gliosis. The variability in clinical presentation among waterfowl complicates passive surveillance and underscores the importance of active virological monitoring [23, 27].

Wild Birds

Clinical manifestations in wild birds vary by species and viral genotype [50, 32]. Mortality events in colonial seabirds, such as terns and gulls, have been associated with acute death without premonitory signs [1, 12]. In contrast, broader passerine and raptor populations may exhibit neurological signs, lethargy, and respiratory distress [50, 70]. Subclinical infections are common in dabbling ducks, which serve as efficient reservoirs for viral dissemination along migratory routes [25, 33].

Clinical Manifestations in Mammalian Species

Pinnipeds and Cetaceans

Pinniped infections with H5N1 clade 2.3.4.4b have been documented in South America, North America, and Europe [18, 27]. Affected animals present with respiratory distress, neurological signs including seizures and disorientation, and high mortality rates in outbreak settings. Necropsy findings include interstitial pneumonia, meningoencephalitis, and myocarditis [18, 35].

Felidae and Canidae

Domestic cats and captive felids (e.g., tigers, leopards) are highly susceptible to HPAI H5N1, likely due to abundant alpha-2,3 sialic acid receptors in the feline respiratory tract. Clinical signs include pyrexia, depression, dyspnea, conjunctivitis, and rapidly progressive neurological dysfunction. Mortality rates in infected domestic cats are high, and horizontal transmission among cats in contact settings has been documented. Canids, including Ezo red foxes in Japan, have been found with H5N1 infection manifesting as neurological disease, with viral antigen detected in brain tissue.

Bovidae

The emergence of H5N1 in dairy cattle represents a novel host range expansion [63, 69]. Clinical signs in affected dairy cows include decreased feed intake, ruminal stasis, pyrexia, and a marked drop in milk production. Milk from infected cows exhibits high viral titers, and mammary gland tissue shows histopathological evidence of necrosis and inflammatory infiltration. The potential for bovine-to-bovine transmission via contaminated milking equipment has been identified as a plausible route of within-herd spread [63, 69].

One Health Surveillance Framework

The term "One Health" refers to the collaborative, multisectoral, and transdisciplinary approach to achieving optimal health outcomes across the human-animal-environment interface [15, 40]. For H5N1, a robust One Health surveillance framework integrates virological monitoring in wild birds, domestic poultry, and at-risk mammalian populations, coupled with real-time genomic data sharing and environmental risk assessment [15, 27, 40].

Surveillance Components

Wild bird surveillance relies on both passive reporting of mortality events and active sampling of apparently healthy birds, particularly along migratory flyways [2, 24, 36]. A taxonomically harmonized global dataset of wild bird hosts has been developed to standardize surveillance efforts and facilitate comparative analyses across regions. Spatiotemporal co-occurrence patterns between migratory birds and H5N1 outbreaks have been modeled to identify high-risk zones for targeted surveillance [25, 55]. Machine learning approaches have been applied to geospatial data to generate global risk maps predicting avian influenza outbreak probability based on environmental, climatic, and anthropogenic variables [11, 31].

Poultry surveillance includes both passive clinical monitoring and active virological testing at multiple points along the production chain, including live bird markets [23, 75]. Systematic reviews have identified risk factors for H5N1 incursion into commercial poultry operations, emphasizing the importance of biosecurity measures such as controlled farm access, boot and vehicle disinfection, and exclusion of wild birds.

Mammalian surveillance has become increasingly important following the detection of H5N1 in dairy cattle and multiple wildlife species [18, 27, 63]. Surveillance programs should include testing of clinical cases in domestic and wild mammals, with particular attention to species that may serve as bridging hosts for transmission to humans [27, 40].

Diagnostic Assays

Molecular diagnostics for H5N1 rely on real-time reverse transcription polymerase chain reaction (RT-PCR) targeting the matrix (M) gene for influenza A identification, followed by subtyping assays specific for H5 and N1 genes [9, 64]. The hemagglutinin cleavage site can be sequenced to distinguish low pathogenicity from high pathogenicity viruses. Whole-genome sequencing using high-throughput sequencers enables detailed phylogenetic and phylogeographic analyses, detection of reassortment events, and identification of molecular markers associated with mammalian adaptation [6, 14, 28, 42]. Serological assays for the detection of nucleoprotein (NP) or HA-specific antibodies may be used for retrospective surveillance in unvaccinated populations. The workflow below illustrates a molecular diagnostic algorithm for H5N1 detection and characterization.

flowchart TD
 A["Clinical sample: oropharyngeal/cloacal swab, tissue, milk"] --> B{RNA extraction}
 B --> C[RT-PCR targeting influenza A M gene]
 C --> D{Ct value < 40?}
 D -- No --> E[Report negative]
 D -- Yes --> F["Subtype RT-PCR: H5 and N1"]
 F --> G[H5 positive and N1 positive?]
 G -- Yes --> H[HA cleavage site sequencing for pathotyping]
 G -- No --> I[Further subtyping for other H/N combinations]
 H --> J{Multibasic cleavage site present?}
 J -- Yes --> K[Classify as HPAI H5N1]
 J -- No --> L[Classify as LPAI or report as untyped]
 K --> M[Whole genome sequencing for phylogenetics and reassortment analysis]
 M --> N[Data submission to public repositories for One Health surveillance]

Vaccine Technologies

Vaccination of poultry against H5N1 is employed in some endemic countries as a component of integrated control strategies [3, 7, 64]. Inactivated whole-virus vaccines and recombinant vector vaccines (e.g., fowlpox virus-vectored HA) are commercially available, though antigenic drift necessitates periodic strain updates [7, 79]. Novel vaccine platforms under development include mRNA vaccines, virus-like particle vaccines, and nanoparticle-based immunogens designed to elicit broadly protective antibody responses [29, 41, 52, 81]. Computationally designed HA proteins displayed on self-assembling nanocages have demonstrated the ability to induce pan-H5 cross-neutralizing antibodies in animal models. Adjuvanted subunit vaccines, such as MF59-adjuvanted A/Astrakhan strain vaccines, have been shown to induce cross-neutralizing antibodies against circulating clade 2.3.4.4b viruses in ferrets. However, the evaluation of vaccine efficacy against emerging genotypes must account for antigenic distance between vaccine strains and field isolates [10, 79]. Antiviral agents, including baloxavir marboxil and neuraminidase inhibitors, have been evaluated for treatment of H5N1 in endangered avian species and mammalian models, with variable efficacy [16, 48, 78].

Environmental and Biophysical Considerations

H5N1 virus survival in the environment is influenced by temperature, pH, humidity, and the presence of organic matter. The virus can remain infectious in water for extended periods at low temperatures and is inactivated by heat (above 60 degrees Celsius for 30 minutes), lipid solvents, and oxidizing agents [17, 30]. Electron beam irradiation has been evaluated as a method for inactivating H5N1 in duck meat, achieving a 4-log reduction at specific doses. Low-concentration gaseous chlorine dioxide has demonstrated virucidal efficacy against airborne H5N1 in experimental settings. Aqueous solutions containing quaternary ammonium cations and sulfobetaine have been reported as effective virucidal agents against HPAI. These environmental stability characteristics have direct implications for biosecurity protocols in poultry production and live bird markets [23, 67].

Data Integration and Computational Modeling

The integration of genomic, epidemiological, and environmental data into open-access databases is essential for real-time monitoring and response [2, 14, 36]. Public databases for tracking H5N1 impacts on albatrosses and petrels exemplify the value of species-specific surveillance platforms. Machine learning and deep learning methods have been applied to heterogeneous data sources, including web-based syndromic surveillance and social media analysis, for early warning of avian influenza outbreaks [31, 54, 76]. Continuous phylogeographic models reconstruct viral dispersal pathways and identify environmental covariates driving spread dynamics at high spatiotemporal resolution. Risk mapping using geographic information systems (GIS) and multi-criteria decision analysis has been implemented at national and regional scales to prioritize surveillance zones and allocate resources efficiently [21, 55].

Ethical and Governance Considerations

The panzootic nature of H5N1 has prompted discussions of zoonoethics, which extends traditional One Health governance frameworks to include co-responsibility among stakeholders across the human-animal-environment interface. Mass culling of infected and potentially exposed poultry remains a cornerstone of outbreak control, but raises ethical questions regarding animal welfare, economic impacts on producers, and environmental consequences of carcass disposal [15, 34]. Mathematical modeling studies have quantified the outbreak potential and the effectiveness of control measures (e.g., stamping out, movement restrictions, vaccination) in high-risk agricultural populations. Public perceptions of H5N1 vaccination and dietary changes have been assessed in urban and rural populations, revealing heterogeneities in acceptance and trust that must be addressed in risk communication strategies.

Conclusions

Avian influenza H5N1, particularly the clade 2.3.4.4b lineage, represents an ongoing panzootic threat with profound implications for poultry production, wildlife conservation, and mammalian health. The unprecedented geographic expansion, host range diversification, and emergence of sustained transmission in dairy cattle underscore the need for integrated, transdisciplinary surveillance systems grounded in molecular diagnostics and genomic epidemiology. A functional One Health framework must incorporate wild bird and mammal surveillance, rapid virological characterization, and transparent data sharing to enable early detection of incursions and informed deployment of control measures.

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