Recent advances in avian influenza virus: Molecular pathogenesis, emerging strains, and next-generation therapeutics

Abstract

Avian influenza viruses (AIVs) represent an ongoing threat to global health due to their capacity for genetic evolution, zoonotic transmission, and pandemic emergence. This review highlights recent progress in understanding the molecular mechanisms underlying AIV infection, including viral immune evasion strategies and host-virus interactions. It discusses recent outbreaks involving reassortant strains such as H5N1 and H9N2, and examines their public health consequences. Advancements in antiviral therapy, including resistance patterns, and the development of next-generation vaccines such as messenger RNA and universal influenza vaccines are reviewed. The integration of genomic surveillance, artificial intelligence-driven prediction, and One Health approaches is emphasized as essential for pandemic preparedness. This comprehensive overview aims to provide researchers and policymakers with actionable insights for controlling the evolving threat of avian influenza.

Key Words: Avian influenza virus; Molecular pathogenesis; Surveillance; Vaccines; Antiviral therapy; Zoonotic transmission; Pandemic preparedness

Core Tip: Avian influenza viruses continue to evolve through re-assortment and mutation, posing persistent zoonotic and pandemic threats. This review synthesizes recent advances in molecular virology, highlights emerging high-risk strains, and evaluates next-generation vaccines and antiviral therapies. A deeper understanding of host–virus interactions, combined with advanced surveillance and innovative therapeutics, will be crucial for future pandemic prevention.

INTRODUCTION

Avian influenza viruses (AIVs) are a major global health concern due to their zoonotic potential and capacity to cause pandemics. These viruses, members of the Orthomyxoviridae family, possess a segmented negative-sense RNA genome that facilitates genetic re-assortment during co-infection, enabling the emergence of novel strains with unpredictable pathogenicity and transmissibility[1].

Influenza A viruses contain eight RNA segments, each encoding at least one essential viral protein. These include the polymerase complex proteins (PB1, PB2, PA), surface glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M), and non-structural proteins (NS). This segmented architecture promotes re-assortment when two distinct strains co-infect a host cell, potentially generating progeny with new genetic constellations. Such antigenic shift plays a pivotal role in viral evolution, host adaptation, and the emergence of pandemic strains[2].

Historical pandemics underscore the critical role of re-assortment in shaping influenza virus evolution. The 1957 H2N2 and 1968 H3N2 pandemics both arose from re-assortment events between human and AIVs, resulting in novel hemagglutinin and neuraminidase subtypes that escaped pre-existing immunity. These pandemics led to significant global morbidity and mortality, emphasizing the necessity of vigilant surveillance and early warning systems to monitor viral evolution and cross-species transmission events[3].

Pathogenesis involves coordinated action of viral proteins such as hemagglutinin, neuraminidase, and non-structural proteins that modulate host immune responses. Critical molecular events include suppression of type I interferon signaling through the non-structural protein 1 (NS1) and modulation of host mRNA processing, which aids in immune evasion[4]. The viral PB2 and PA subunits interact with host polymerase co-factors to facilitate replication in mammalian cells, particularly when carrying adaptive mutations like E627K or D701N[5]. Host pattern recognition receptors such as RIG-I and TLR7 detect viral RNA and activate NF-κB and IRF3/7 pathways, leading to pro-inflammatory cytokine production[6]. However, uncontrolled immune activation and high viral load may result in a cytokine storm, contributing to acute lung injury and severe disease.

Epidemiologically, the last decade has witnessed repeated emergence of novel reassortants, such as H7N9 in China (2013) and H5N1 clade 2.3.4.4b in Europe and the Americas (2021–2024). A 2023 meta-analysis reported a global case fatality rate of 35% among laboratory-confirmed H5N1 human infections, mostly linked to direct poultry contact[7]. Recent spillover events into cattle and associated human infections in the United States further amplify pandemic risks[8]. The H5N1 clade 2.3.4.4b virus has demonstrated remarkable cross-species adaptability. In 2023, widespread outbreaks were reported not only in wild birds and poultry but also in mammalian species including red foxes, seals, and farmed minks, raising concerns over mammalian transmission chains[9]. Notably, in early 2024, the virus was detected in dairy cattle across multiple U.S. states, marking the first documented cases of bovine infection with influenza A(H5N1)[10]. Subsequent zoonotic transmission was confirmed in human farm workers, although symptoms were mild. These events underscore the expanding host range of AIVs and highlight the critical need for enhanced surveillance at the animal-human interface.

The molecular processes that drive the evolution of AIVs; particularly genetic reassortment and adaptive mutations, can give rise to viruses with novel antigenic properties, enhanced host range, or resistance to existing antivirals. These changes may occur rapidly and unpredictably, as demonstrated by past pandemics and recent zoonotic spillovers. As such, a deep understanding of these mechanisms is essential for risk assessment, vaccine design, and the implementation of effective public health interventions. Strengthening global genomic surveillance and predictive modeling systems is vital to detect high-risk viral variants early and mitigate their spread.

This review provides an integrated overview of the molecular pathogenesis, emerging strains, and next-generation therapeutic strategies for avian influenza. By synthesizing recent advances in genomics, epidemiology, and biotechnology, it aims to inform future preparedness and response strategies.

METHODOLOGY

This review was conducted through a systematic literature search using PubMed, Scopus, and Web of Science databases covering studies published from January 2010 to April 2025. Search terms included “avian influenza virus”, “molecular pathogenesis”, “avian influenza outbreaks”, “antiviral resistance”, and “vaccine development”.

Articles were included if they provided original data or comprehensive reviews related to the molecular biology, epidemiology, and therapeutic interventions of AIVs. Exclusion criteria were non-English language papers, studies lacking molecular or clinical relevance, and reports not indexed in PubMed or major scientific databases.

Selected articles were critically appraised to synthesize the most relevant findings regarding viral evolution, immune evasion, antiviral resistance mechanisms, and advances in next-generation therapeutics.

MECHANISMS OF GENETIC REASSORTMENT IN AVIAN INFLUENZA VIRUSES

Genetic reassortment and mammalian host adaptation

AIVs possess an eight-segmented, negative-sense RNA genome, which facilitates genetic reassortment when two or more different influenza A strains co-infect the same host cell[11,12]. During co-infection, gene segments can be exchanged, leading to the emergence of novel reassortants.

However, not all segment combinations result in viable progeny. Segment compatibility is critical—successful reassortants require coordinated interactions among polymerase subunits (PB2, PB1, PA), nucleoproteins, and packaging signals to ensure efficient replication and assembly. Each RNA segment carries segment-specific packaging signals at both ends, which guide selective incorporation into new virions[13]. Incompatible or mismatched segments may produce defective or attenuated viruses incapable of propagation. Thus, reassortment success is governed not only by co-infection, but also by intrinsic segment-level constraints that act as evolutionary bottlenecks.

This process of genomic segment exchange is a major driver of viral evolution, enabling the emergence of novel genotypes with altered antigenicity, host range, and virulence[14,15]. Reassortment is particularly common in host species permissive to multiple influenza subtypes—such as wild birds, domestic poultry, and swine, which serve as mixing vessels[16]. Intermediate hosts such as swine play a pivotal role in the emergence of novel influenza genotypes. Swine possess both α2,3-linked (avian-like) and α2,6-linked (human-like) sialic acid receptors in their respiratory tract, allowing them to be infected by both avian and human influenza viruses[17]. This dual susceptibility creates a unique environment for co-infection and genetic reassortment, enabling the generation of progeny viruses with mixed avian-human gene constellations. Historical analyses have shown that the 2009 H1N1 pandemic virus emerged from such a complex reassortment process involving swine as a key intermediate[18]. The ecological proximity of swine to both domestic poultry and humans in certain agricultural settings further amplifies this risk.

The random packaging of eight RNA segments into progeny virions can yield viruses with gene combinations from both parental strains, resulting in antigenic shift. This phenomenon may enhance zoonotic transmission or pandemic potential[19,20]. Historical examples include the 1957 H2N2 and 1968 H3N2 pandemics, both caused by reassortment between avian and human influenza viruses, underscoring the importance of this mechanism in shaping influenza outbreaks[21,22].

Several key mutations in the PB2 and HA proteins have been implicated in the adaptation of AIVs to mammalian hosts. The PB2-E627K and D701N mutations enhance polymerase activity at the cooler temperatures of the mammalian upper respiratory tract, improving viral replication and virulence[23]. Similarly, changes in the HA receptor binding domain, such as Q226L and G228S, shift viral preference from avian-type α2,3-linked sialic acid receptors to human-type α2,6-linked sialic acid receptors[24]. These molecular adaptations have been identified in several zoonotic strains, including recent H5N1 and H7N9 outbreaks, and are considered important indicators of pandemic potential.

Structural biology has provided critical insights into how mutations in the hemagglutinin (HA) protein influence host receptor binding. High-resolution X-ray crystallography studies have demonstrated that specific amino acid substitutions—such as Q226L and G228S—alter the shape and charge distribution of the receptor-binding domain, enabling a shift in affinity from avian-type (α2,3-linked) to human-type (α2,6-linked) sialic acid receptors[25]. These conformational changes facilitate viral entry into human respiratory epithelial cells and represent key molecular determinants of zoonotic potential. Understanding these receptor-binding interactions at the structural level is essential for predicting viral transmissibility and informing vaccine design.

Host factors influencing reassortment

Reassortment efficiency is influenced by several host-related factors: Several key mutations in the PB2 and HA proteins have been implicated in the adaptation of AIVs to mammalian hosts. The PB2-E627K and PB2-D701N mutations enhance polymerase activity at the lower temperatures of the mammalian upper respiratory tract, thereby promoting viral replication and increased virulence[26]. Similarly, mutations in the HA receptor binding domain, such as Q226L and G228S, shift the virus’s binding preference from avian α2,3-linked sialic acid receptors to human α2,6-linked receptors, a critical step in enabling efficient human-to-human transmission[27]. These molecular adaptations have been observed in several zoonotic isolates, including recent H5N1 and H7N9 strains, and are considered important predictors of pandemic potential.

Receptor binding specificity: AIVs primarily bind to α-2,3-linked sialic acid receptors, while human influenza viruses preferentially recognize α-2,6-linked receptors[21,22]. However, certain reassortant strains have adapted to bind both receptor types, increasing their potential for human infection[28].

Intracellular compatibility of viral proteins: The polymerase complex, particularly the PB2 subunit, plays a crucial role in host adaptation and efficient replication in mammalian cells[29,30]. Mutations in PB2 (e.g., E627K) have been associated with increased replication efficiency in human airway cells[31,32].

Immune pressure and selective advantage: Viral reassortants that evade host immunity through changes in hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins are more likely to persist and spread[33,34]. The acquisition of mutations that confer resistance to existing vaccines or antiviral treatments further enhances their survival[35,36].

Immune evasion and host–virus interaction mechanisms

AIVs have evolved sophisticated mechanisms to evade host immune responses. A key viral factor is the NS1, which antagonizes host type I interferon (IFN) pathways by inhibiting both IFN production and downstream signaling, thereby impairing antiviral defense[37]. NS1 also binds to cleavage and polyadenylation specificity factor 30 (CPSF30), blocking host pre-mRNA processing and reducing the expression of critical immune genes[38].

Another immune evasion factor is the PA-X protein, expressed through a ribosomal frameshift from the PA gene. PA-X contributes to host shutoff by degrading cellular mRNAs, further dampening innate immune responses[39]. In addition, glycosylation of HA modulates antigenic masking, allowing the virus to escape recognition by neutralizing antibodies[40].

AIVs must also overcome host restriction factors such as MxA and interferon-induced transmembrane proteins. This is achieved, in part, through adaptive mutations in viral polymerase proteins. Notably, mutations such as PB2-E627K and PB2-D701N enhance polymerase activity and replication efficiency in mammalian cells, contributing to host adaptation and virulence[41]. These interactions illustrate the dynamic co-evolution between AIVs and host immune defenses.

The life cycle of AIVs and their immune evasion strategies are summarized schematically in Figure 1. Upon entry via hemagglutinin binding to sialic acid receptors, the virus replicates within the host nucleus. NS1 inhibits interferon signaling, while PA-X degrades host mRNA, collectively facilitating viral replication and immune evasion. The sequential stages of infection and host interference are depicted to provide a visual overview of these processes.

Figure 1 Life cycle of avian influenza virus and host immune evasion. HA: Hemagglutinin; NS1: Non-structural protein 1.

Recent evidence of reassortment in avian influenza outbreaks

Recent studies have documented reassortment events leading to the emergence of novel avian influenza strains:

H5N1 clade 2.3.4.4b (2021-Present): This clade has undergone multiple reassortment events, incorporating genes from low-pathogenicity avian influenza (LPAI) viruses, leading to enhanced transmissibility in mammals[42,43].

H7N9 evolution in China: Since its emergence in 2013, H7N9 has reassorted with local LPAI strains, resulting in highly pathogenic variants with increased zoonotic potential[44,45].

H9N2 as a genetic backbone: H9N2 viruses have repeatedly donated internal genes to emerging reassortants, including H5N6 and H10N8, facilitating cross-species transmission[46,47].

These findings underscore the role of reassortment in influenza virus evolution and highlight the need for continuous genomic surveillance.

EMERGING AVIAN INFLUENZA THREATS: RECENT OUTBREAKS AND ZOONOTIC POTENTIAL

AIVs continue to pose a significant public health concern, with multiple subtypes evolving through genetic reassortment and adaptation in avian and mammalian hosts[30]. The highly pathogenic avian influenza (HPAI) H5N1 and the LPAI H9N2 viruses have been particularly concerning due to their increasing zoonotic spillover potential[46,47].

Recent outbreaks and epidemiological patterns

The 2021-2022 outbreak of H5N1 in Europe marked one of the largest recorded HPAI outbreaks, with extensive transmission among wild birds and poultry[42]. This outbreak underscored the virus’s enhanced ability to persist in various ecological niches, increasing the risk of human exposure[42,48]. Concurrently, H9N2 viruses have undergone extensive reassortment, facilitating the emergence of novel zoonotic strains such as H7N9 and H10N8, both of which have caused sporadic human infections with severe outcomes[45-47].

In China, ongoing surveillance indicates that the H9N2 Lineage continues to act as a donor for internal gene segments in novel reassortant influenza strains, heightening concerns about its pandemic potential. Human infections with H9N2 are typically mild but serve as a warning sign of a virus that may adapt for efficient human-to-human transmission[46,47].

The evolution and global dissemination of major AIVs strains from 2010 to 2025 have been marked by significant outbreaks, including the emergence of H7N9 in China (2013), the intercontinental spread of H5N8 (2014-2015), and the recent detection of H5N1 infections in dairy cattle and humans in the United States (2024). A chronological overview of these events is presented in Figure 2.

Figure 2 Evolution and global spread of avian influenza virus strains (2010–2025).

Case study analysis: Zoonotic outbreaks and public health response

In 2024, the spillover of H5N1 clade 2.3.4.4b into dairy cattle in the United States marked a significant shift in host tropism, resulting in a cluster of human infections with respiratory symptoms and conjunctivitis. Genomic sequencing revealed PB2 mutations (E627K and T271A) associated with mammalian adaptation[48].

Since 2021, the H5N1 clade 2.3.4.4b has caused widespread outbreaks across Europe, Asia, Africa, and the Americas, with over 130 million poultry culled globally as of late 2024[49]. Although human infections remain rare, they are often severe; the World Health Organization (WHO) reports a 35%-50% case fatality rate among laboratory-confirmed cases, primarily following close contact with infected poultry. Meanwhile, H9N2 remains endemic in many Asian countries, where sporadic zoonotic infections and serological evidence of exposure in poultry workers have been documented[50].

These trends underscore the urgent need for enhanced genomic surveillance. The adoption of next-generation sequencing (NGS) has improved the detection and tracking of novel variants through real-time phylogenetic analysis. Additionally, artificial intelligence (AI)-based predictive models have shown promise in forecasting zoonotic spillover events—particularly for H7N9 and H5N8—by integrating genomic, migratory, and environmental data[51]. Expanding access to these technologies in resource-limited settings is essential for strengthening global early warning systems.

In parallel, China reported a surge in H5N6 human infections in Guangxi Province, many with direct poultry exposure, but a subset lacking known animal contact—raising concern over silent reservoirs or environmental contamination as potential transmission sources[52].

Collectively, these outbreaks highlight the urgent need to integrate veterinary and public health surveillance systems. The 2024 U.S. H5N1 outbreak exposed serious gaps in preparedness, including limited personal protective equipment (PPE) and inadequate training for frontline workers—despite the hard-won lessons of the coronavirus disease 2019 (COVID-19) pandemic[53].

Zoonotic potential and mammalian adaptation

Ecological and environmental factors significantly influence the emergence and spread of AIVs. Migratory birds, particularly waterfowl such as ducks and geese, serve as natural reservoirs and play a pivotal role in long-distance viral dissemination via major flyways, including the East Asian–Australasian and Central Asian routes[54]. Climate change is reshaping migratory patterns, altering breeding grounds, and affecting viral persistence in the environment, thereby modifying transmission dynamics[55]. Concurrently, human encroachment into wildlife habitats, intensification of poultry farming, and the proliferation of live animal markets are increasing avian–human interactions and facilitating spillover events[54]. Integrating ecological surveillance with virological monitoring is therefore critical for early detection and pandemic preparedness.

On the molecular level, studies on H5N1 and H9N2 viruses have shown that polymerase mutations, such as PB2-E627K, significantly enhance viral replication efficiency in mammalian cells. These mutations are considered key molecular determinants of cross-species transmission[56]. In ferret models, H5N1 viruses carrying mammalian-adaptive mutations have demonstrated increased airborne transmissibility, reinforcing their pandemic potential[57].

Furthermore, genetic reassortment between H9N2 and other influenza subtypes has led to the emergence of novel variants with increased affinity for human-like α2,6-linked sialic acid receptors, a hallmark of zoonotic adaptation[58]. The discovery of H10N8 as a reassortant containing internal genes derived from H9N2 further illustrates how ongoing genomic evolution can give rise to previously unrecognized pandemic threats[59].

Conclusions and future directions

The ongoing evolution of AIVs necessitates enhanced global surveillance to monitor genetic changes that may facilitate human adaptation. The integration of NGS and AI-driven predictive modeling could significantly improve early detection of high-risk strains[43,47]. Additionally, strengthening biosecurity measures in poultry farms and live bird markets remains a critical strategy for mitigating the risk of zoonotic spillovers[8].

ANTIVIRAL RESISTANCE AND VACCINE DEVELOPMENT IN EMERGING AVIAN INFLUENZA STRAINS

The increasing incidence of avian influenza infections in both birds and humans necessitates a comprehensive approach to antiviral strategies and vaccine development. The frequent genetic reassortment of AIVs poses challenges in antiviral efficacy and vaccine strain selection[60,61]. Recent studies have highlighted concerning trends in antiviral resistance and gaps in current vaccine coverage, necessitating novel intervention strategies[62,63].

Antiviral resistance in avian influenza viruses

Antiviral resistance among AIVs has emerged as a significant challenge to treatment and control efforts. Resistance mutations in the NA gene, such as the H275Y substitution, reduce the binding affinity of oseltamivir to the enzyme’s active site while preserving sialidase function—thus diminishing drug efficacy without compromising viral fitness[62,63]. This mutation has been observed in both H5N1 and H7N9 isolates.

The recently identified PA-I38T mutation confers resistance to baloxavirmarboxil, a cap-dependent endonuclease inhibitor. It alters the endonuclease binding domain of the PA protein, leading to reduced drug efficacy in circulating H5N6 and H9N2 strains. Clinical studies have correlated the presence of such mutations with prolonged viral shedding and worsened patient outcomes, highlighting the clinical relevance of resistance surveillance[64,65].

In contrast to seasonal influenza viruses, where resistance mutations typically emerge under direct therapeutic pressure, AIVs may acquire resistance through natural selection in animal hosts, especially in settings where antivirals are used to control poultry outbreaks. Dual resistance to adamantanes and neuraminidase inhibitors, observed in some H9N2 isolates, further complicates therapeutic options[66,67].

These developments underscore the necessity of monitoring resistance patterns via genotypic and phenotypic surveillance, especially as next-generation antivirals enter the pipeline. Recent global surveillance of highly pathogenic H5N1 strains has revealed emerging resistance to existing antivirals, highlighting the need for updated therapeutic strategies. Concurrently, advances in mRNA vaccine platforms offer a promising adjunct to traditional immunization and therapeutic approaches. Agents targeting host factors and viral proteins less prone to mutational escape are urgently needed. Promising candidates include combination therapies and monoclonal antibodies, which may help overcome resistance and improve clinical outcomes[68-70].

Next-generation therapeutic strategies

In response to the growing challenge of antiviral resistance, several next-generation therapeutic strategies are under investigation. Monoclonal antibodies (mAbs) targeting conserved epitopes of HA, such as the stem domain, have shown broad neutralizing activity against H5N1, H7N9, and H5N6 strains in preclinical models[71]. mAbs like VIS410 and CR8020 have demonstrated protective efficacy in both ferrets and mice, and early-phase clinical trials are ongoing[72].

Innovative approaches such as CRISPR-based antivirals, which use RNA-guided nucleases (e.g., Cas13) to degrade viral RNA selectively, are also being explored. These tools offer the potential for strain-specific targeting while sparing host transcripts, though delivery systems and off-target effects remain challenges[73].

Additionally, combination therapies—pairing neuraminidase inhibitors (e.g., oseltamivir) with endonuclease inhibitors (e.g., baloxavir) or host-targeted drugs (e.g., nitazoxanide)—have shown synergistic effects in vitro and in animal models[74]. These regimens may reduce the emergence of resistance and improve clinical outcomes. As such, expanding clinical trials and translational research into these modalities is a crucial component of pandemic preparedness. A comparative overview of current and emerging therapeutic strategies is summarized in Table 1, highlighting mechanisms of action, target stages, and clinical applicability.

Table 1 Comparative overview of current and emerging therapeutic strategies for avian influenza virus infection.

Therapeutic agent/strategy	Mechanism of action	Targeted strains	Development stage	Resistance issues
Oseltamivir (Tamiflu)	Neuraminidase inhibitor – blocks virus release	H5N1, H7N9, H9N2	Approved	H275Y mutation reduces efficacy[69]
BaloxavirMarboxil	Cap-dependent endonuclease inhibitor	H5N6, H9N2, H1N1	Approved (limited use in AIV)	PA-I38T mutation linked to resistance[64]
Amantadine	M2 ion channel blocker – inhibits viral uncoating	H1–H3 (historic use)	Obsolete (due to resistance)	Widespread resistance in H5/H9[67]
Nitazoxanide	Host-targeted antiviral; interferon modulator	Multiple AIV subtypes	Clinical trials	Minimal–host-directed mechanism[68]
Monoclonal antibodies	Neutralize HA or NA epitopes	H5N1, H7N9, H5N6	Preclinical/early clinical	Escape mutants possible[65]
mRNA vaccines	Encode HA/NA antigens for in vivo expression	Broad-spectrum (experimental)	Preclinical/early clinical	No direct resistance yet observed[70]
Universal vaccines	Target conserved HA stalk/M2e epitopes	Multisubtype AIVs	Experimental	Incomplete efficacy in human trials

AIVs: Avian influenza viruses; HA: Hemagglutinin; NA: Not available.

Advancements in avian influenza vaccines

The rapid antigenic drift and reassortment of AIVs complicate the development of effective vaccines[75]. Traditional inactivated vaccines, such as those used for H5 and H7 AIVs, have been employed extensively in poultry, but their effectiveness is compromised by genetic divergence among circulating strains[76,77]. The emergence of immune escape variants further underscores the need for updated vaccine formulations[78].

Several next-generation vaccine platforms are currently being explored

mRNA vaccines: Inspired by the success of mRNA COVID-19 vaccines, efforts are underway to develop mRNA-based influenza vaccines targeting conserved viral proteins[79,80]. These vaccines offer rapid adaptability to emerging variants and have shown promising immunogenicity in preclinical models[70]. Beyond humoral immunity, mRNA vaccines have demonstrated the ability to induce strong CD4+ and CD8+ T-cell responses, which may confer protection even in the presence of antigenic drift[75]. Lipid nanoparticle-formulated mRNA encoding HA has shown cross-protective efficacy in animal models, supporting its pandemic application[81].

Viral vector vaccines: Adenovirus-vectored vaccines expressing HA have demonstrated robust immune responses and cross-protection in animal models, particularly against H5N1 and H7N9 strains[82,83].

Universal influenza vaccines: Research is increasingly focused on vaccines targeting conserved viral epitopes, such as the stalk region of HA or the matrix protein 2 (M2), to confer broad-spectrum immunity against multiple influenza subtypes[84,85]. Recent developments include mosaic nanoparticle platforms, which co-display antigens from diverse influenza subtypes on a single scaffold to stimulate broadly neutralizing B cell responses[86]. Additionally, chimeric HA constructs and self-assembling protein scaffolds are being designed to focus immune responses on conserved domains, such as the HA stem, while minimizing reactivity to immunodominant variable regions. Early-phase clinical trials of the NIH’s universal vaccine candidate have demonstrated favorable safety and immunogenicity profiles[87].

The introduction of recombinant protein vaccines, including nanoparticle-based formulations, has also been explored as a strategy to improve immunogenicity while avoiding the risks associated with live-attenuated virus platforms[88,89]. However, the widespread implementation of these next-generation vaccines faces challenges related to regulatory approval, manufacturing scalability, and cost-effectiveness in resource-limited settings[90].

Conclusion and future directions

The continuous evolution of AIVs necessitates a proactive and integrated approach to antiviral drug development, vaccine innovation, and outbreak surveillance. Surveillance programs should incorporate real-time genomic sequencing, AI-driven predictive modeling, and global data-sharing frameworks to identify emerging resistance mutations and antigenic drift patterns rapidly[91,92]. Future vaccine strategies must prioritize adaptable platforms—such as mRNA and universal vaccine candidates—to offer broad and durable protection against zoonotic influenza strains[93].

Recent outbreaks, including H5N1 in cattle and H5N6 in humans, have exposed significant vulnerabilities in public health systems. These include inadequate PPE availability, limited frontline training, and insufficient cross-sectoral coordination during zoonotic spillovers. Addressing these gaps requires a renewed commitment to biosafety protocols, especially in high-risk agricultural settings[94].

A One Health approach—uniting human, animal, and environmental health domains—is essential for comprehensive pandemic preparedness. This should include interoperable surveillance platforms, strengthened biosecurity in live animal markets, stockpiling of protective equipment, and investment in cross-disciplinary response training. Finally, aligning scientific innovation with governance, equity, and policy enforcement will be key to preventing the next pandemic-scale influenza threat[94].

PUBLIC HEALTH PREPAREDNESS AND GLOBAL RESPONSE STRATEGIES FOR AVIAN INFLUENZA OUTBREAKS

Emerging AIVs continue to pose a significant threat to global public health. The increasing frequency of zoonotic spillover events underscores the need for robust surveillance systems, rapid containment measures, and coordinated international response strategies[95,96]. The WHO, the Food and Agriculture Organization (FAO), and the World Organisation for Animal Health (WOAH) have emphasized a One Health approach, integrating human, animal, and environmental health strategies to mitigate the risks associated with avian influenza[97,98].

Surveillance and early warning systems

Comprehensive surveillance is critical for early detection and containment of emerging avian influenza outbreaks. Recent advancements in genomic surveillance and AI-driven outbreak prediction have significantly improved the ability to monitor viral evolution in real time[99,100].

Genomic surveillance: Whole-genome sequencing of circulating AIV strains enables the identification of genetic reassortment events, virulence markers, and antiviral resistance mutations[64,101,102]. Countries such as China and the United States have established real-time genomic databases to facilitate rapid risk assessment and vaccine strain selection[103,104].

AI and predictive modeling: Machine learning algorithms trained on epidemiological and genomic data have shown promise in predicting avian influenza hotspots and cross-species transmission risk[105,106]. AI-driven risk assessment models have been successfully applied in avian influenza surveillance networks, enabling proactive intervention strategies[107,108].

Despite these advancements, gaps in global surveillance infrastructure remain a major challenge, particularly in resource-limited settings where underreporting and lack of laboratory capacity hinder outbreak detection[7,109]. Strengthening cross-border collaboration and data-sharing mechanisms is crucial for improving global preparedness[110].

Containment measures and public health interventions

Once an outbreak is detected, rapid containment measures are essential to prevent human-to-human transmission and minimize the risk of a pandemic[111,112].

Culling and biosecurity in poultry farms: Mass culling of infected poultry remains a primary control strategy, particularly in countries with intensive poultry farming industries[113,114]. However, concerns about economic losses and ethical considerations have led to the exploration of alternative containment strategies, such as avian influenza vaccination in poultry[78,115,116].

Personal protective measures: Frontline workers and individuals exposed to infected birds are advised to follow strict biosafety protocols, including the use of N95 respirators, gloves, and protective clothing to reduce zoonotic transmission risk[117,118].

Public health communication and risk awareness: Effective risk communication strategies are essential to mitigate misinformation and public panic during avian influenza outbreaks[119,120]. WHO guidelines emphasize the importance of transparent communication with the public and healthcare professionals to ensure timely response measures[121].

Global response and policy recommendations

The international response to avian influenza outbreaks has improved in recent years, but challenges remain in ensuring equitable access to vaccines and antiviral treatments, especially in low- and middle-income countries[122,123]. Key policy recommendations include:

Expanding international vaccine stockpiles: WHO’s global influenza vaccine action plan has called for increased investment in pre-pandemic influenza vaccines to ensure rapid deployment during future outbreaks[124,125].

Strengthening pandemic preparedness frameworks: Countries should regularly update their national influenza preparedness plans, incorporating lessons learned from the COVID-19 pandemic to enhance response efficiency[126,127].

Improving one health collaboration: Integrating human, animal, and environmental health surveillance systems is essential for early detection and containment of emerging zoonotic viruses[128,129].

FUTURE DIRECTIONS

Moving forward, the global scientific community must prioritize the development of broad-spectrum antiviral therapies and universal influenza vaccines to reduce the impact of future avian influenza pandemics[72,130]. Additionally, leveraging AI and big data analytics for real-time outbreak prediction and genomic surveillance will be instrumental in improving global influenza preparedness[131-133].

CONCLUSION

Concluding remarks and future directions

The ongoing evolution and adaptation of AIVs pose significant threats to both animal and human health. The rapid reassortment of viral genomes and the emergence of highly pathogenic strains highlight the necessity of continuous surveillance, genomic analysis, and the development of next-generation vaccines and therapeutics[133,134]. While antiviral agents such as baloxavir, marboxil and neuraminidase inhibitors have been instrumental in controlling influenza outbreaks, the increasing resistance to these drugs necessitates the exploration of novel treatment strategies, including monoclonal antibodies, host-targeted therapies, and RNA-based antivirals[65,72,135,136].

One of the major challenges in influenza research is the zoonotic transmission of AIVs, which requires enhanced surveillance and early detection mechanisms. Real-time genomic surveillance and artificial intelligence-driven predictive models are now being integrated into public health frameworks to detect and contain potential outbreaks before they escalate into pandemics[137,138]. Advances in CRISPR-based diagnostics and molecular epidemiology have further improved our ability to monitor viral mutations and track the spread of emerging variants[139,140].

The future of influenza research must also focus on the development of universal vaccines that provide broad-spectrum protection against multiple subtypes of influenza viruses. Current strategies, such as mosaic hemagglutinin vaccines and nanoparticle-based platforms, have shown promising results in preclinical and early-phase clinical trials[141,142]. However, large-scale validation and long-term immunogenicity studies are required before these vaccines can be widely implemented.

Research gaps and challenges

Despite substantial advances in AIVs surveillance and characterization, several critical research gaps persist. The precise molecular determinants that facilitate efficient interspecies transmission—particularly across less-studied hosts such as swine, cattle, and wild carnivores—remain incompletely understood. In addition, there is limited insight into host immune barriers, viral shedding dynamics in subclinical infections, and the role of environmental reservoirs in sustaining zoonotic potential. Future studies that integrate functional virology, comparative immunology, and ecological modeling are essential to close these gaps and improve our ability to predict and prevent pandemics.

Key challenges in AIV pathogenesis and control include

Mechanisms of host adaptation: The genetic and molecular determinants that enable AIVs to efficiently replicate and spread in mammalian hosts need further elucidation[27,143].

Long-term vaccine efficacy: The duration of immunity conferred by next-generation influenza vaccines remains uncertain, necessitating further longitudinal studies[144,145].

Resistance mechanisms: The molecular basis of drug resistance in influenza viruses, particularly in relation to newly developed antivirals, requires continuous monitoring[36,69,146,147].

Impact of climate change: The role of environmental factors in influencing the ecology and migration patterns of avian reservoirs is still not fully understood[148,149].

One health approach: Strengthening interdisciplinary collaborations between virologists, epidemiologists, veterinarians, and policymakers is crucial for effective pandemic preparedness and response[150-152].

Addressing these research gaps through multidisciplinary collaboration and international data sharing will be instrumental in mitigating the threat of future influenza pandemics. The strategic integration of genomics, artificial intelligence, and synthetic biology into influenza research holds transformative potential for developing next-generation diagnostics, vaccines, and therapeutics.

ACKNOWLEDGEMENTS

Authors are thankful to Prof. Anant Kale, Stratizen Research & Innovation Services Pvt. Ltd., Pune, India for revising this article for syntax and scientific language style.