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Increased Zoonotic Risk Posed by Emerging Haemagglutinin Variants of Clade 2.3.4.4b A(H5Nx) High Pathogenicity Avian Influenza Virus

This study identifies the novel HA E185K mutation in clade 2.3.4.4b H5Nx viruses as a key factor enhancing immune escape and binding to human-like receptors, particularly in cattle-derived strains, though it does not yet confer efficient transmission in ferrets, thereby highlighting an evolving zoonotic risk that warrants continued surveillance.

Original authors: Munir Iqbal, Jiayun Yang, Elizabeth Billington, William Harvey, Sofia Riccio, Simon Johnson, Valdez, Hannah Klim, Ksenia Sukhova, Jin Yu, Thusitha Karunarathna, Mehnaz Qureshi, Samuel Richardson, Toby

Published 2026-11-03

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Original authors: Munir Iqbal, Jiayun Yang, Elizabeth Billington, William Harvey, Sofia Riccio, Simon Johnson, Valdez, Hannah Klim, Ksenia Sukhova, Jin Yu, Thusitha Karunarathna, Mehnaz Qureshi, Samuel Richardson, Toby Carter, Jafar Hassan, Cecilia Genova, Georgina Ward, Kelly Roper, Jean-Remy Sadeyen, Dorothy Hawkins, Tereza Masonou, Maximillian Woodall, Claire Smith, Yan Liu, Marek Slomka, Ashley Banyard, Thomas Peacock, Ian Brown, Paul Digard, Samantha Lycett, Wendy Barclay, Joe James

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

Viruses that jump from animals to humans are a constant concern in the world of infectious diseases. Among them, avian influenza, often called bird flu, is a major player. These viruses usually live in wild waterfowl and are adapted to bind to specific receptors on the cells of birds. To infect a human, a virus must be able to latch onto different receptors found in the human respiratory tract. This ability to switch targets is a critical step in a virus becoming a threat to people. Furthermore, viruses are constantly changing. As they circulate, they accumulate mutations, some of which help them escape the immune systems of the animals they infect, while others might accidentally make them better at infecting new species. Scientists watch these changes closely, looking for the specific genetic tweaks that could turn a bird virus into a human one.

In a recent study, researchers investigated a specific group of bird flu viruses known as clade 2.3.4.4b. These viruses have been spreading globally and have recently been found infecting dairy cattle in the United States, raising fresh alarms about their ability to adapt to mammals. The team focused on a part of the virus called the hemagglutinin protein, which acts like a key that unlocks the door to a host cell. They wanted to see if natural changes in this key were allowing the virus to open doors it previously could not, specifically those found in humans. By analyzing thousands of virus sequences collected between 2020 and 2025, the scientists identified a single change in the genetic code that was becoming increasingly common, particularly in viruses found in cattle. This change, where one building block of the protein was swapped for another, appeared to be driven by the pressure of the immune system trying to fight the virus off.

The researchers took viruses carrying this specific change and tested them in the lab to see how they behaved. They found that the altered virus could bind to both the bird-type receptors and the human-type receptors, whereas the original virus could only bind to the bird ones. This dual ability is significant because it suggests the virus is becoming more flexible in the types of cells it can infect. The team also showed that this change helped the virus evade antibodies, the proteins the immune system uses to neutralize threats. In experiments where they grew the virus in the presence of antibodies from vaccinated chickens or infected ducks, the virus evolved to carry this specific change to survive. This confirmed that the mutation is a way for the virus to hide from the immune system while simultaneously gaining the ability to stick to human cells.

However, the story does not end with a virus that is ready to spread easily among people. The scientists took the viruses with this new mutation and tested them in ferrets, which are often used as a model for how flu might spread between humans. While the virus was still very dangerous to the ferrets that were directly infected, it did not spread to other ferrets that were in the same cage or in a nearby cage. The mutation did not make the virus stable enough to travel through the air effectively. The researchers also checked the physical properties of the virus's key protein and found that it did not become more stable at the temperatures required for airborne transmission. This means that while the virus has taken a step toward being able to infect humans, it has not yet taken the final step toward becoming a virus that could spread rapidly from person to person.

The findings highlight a complex evolutionary path. The mutation that allows the virus to escape immunity and bind to human cells is emerging naturally, especially in cattle populations. This suggests that as the virus circulates in new animal hosts, it is gathering the tools it needs to potentially infect people. Yet, the study also provides a reassuring boundary: the virus currently lacks the full package of traits needed for an outbreak in humans. The research underscores the importance of watching these animal populations closely. If the virus continues to evolve, it might eventually acquire the missing pieces, such as the ability to survive in the air. For now, the virus is a warning sign rather than an immediate pandemic threat, but its ability to change and adapt means that vigilance remains essential.

Technical Summary: Increased Zoonotic Risk Posed by Emerging Haemagglutinin Variants of Clade 2.3.4.4b A(H5Nx) High Pathogenicity Avian Influenza Virus

Problem Statement
The clade 2.3.4.4b H5Nx high pathogenicity avian influenza (HPAI) virus has evolved into a global panzootic, characterized by unprecedented outbreaks in wild and domestic birds and an expanding host range that now includes mammals such as sea mammals, cats, minks, and, notably, dairy cattle. While human infections remain sporadic, the surge in mammalian infections raises significant concerns regarding zoonotic spillover and pandemic potential. A critical determinant of host range is the viral haemagglutinin (HA) protein's binding specificity to sialic acid (SA) receptors: avian viruses typically bind $\alpha$ 2,3-linked SA, while human-adapted viruses prefer $\alpha$ 2,6-linked SA. Furthermore, the HA protein is the primary target of neutralizing antibodies, meaning its evolution is driven by immune pressure from natural infection or vaccination. The study addresses the urgent need to identify specific HA mutations in circulating clade 2.3.4.4b viruses that may simultaneously confer immune escape and altered receptor binding, thereby increasing zoonotic risk.

Methodology
The researchers employed a multi-faceted approach combining bioinformatics, reverse genetics (RG), and in vivo models:

Genomic Surveillance and Selection Analysis: The team analyzed HA sequences from clade 2.3.4.4b viruses (2020–2025) to identify non-conserved residues in the receptor-binding site (RBS). They used a "renaissance counting" approach on a posterior sample of time-resolved phylogenies to estimate codon-specific dN/dS ratios, identifying sites under positive selection.
Reverse Genetics and Phenotypic Characterization: A panel of 18 naturally occurring HA variants was generated using RG viruses with a monobasic cleavage site and PR8 internal genes. Receptor binding profiles were assessed using Bio-layer Interferometry (BLI) with synthetic avian-like (3SLN) and human-like (6SLN) receptor analogues.
Immune Escape Selection: To mimic antigenic drift, a dominant genotype strain (Ck/ENG/52/21) was serially passaged in embryonated chicken eggs under pressure from pooled chicken post-vaccination antiserum and duck post-infection antiserum.
Structural and Functional Validation: The study investigated the role of electrostatic charge at residue 185 by generating mutants (E185K, E185R, E185D, E185A, E185V). Pseudovirus entry assays were conducted in HEK293T cells engineered to mask $\alpha$ 2,3-linked SA to confirm receptor usage.
In Vivo Ferret Studies: Two RG HPAIVs representing cattle-derived lineages (one with wild-type E185 and one with the emerging K185 mutation) were tested for pathogenicity and airborne transmission in ferrets. Directly infected ferrets were co-housed with direct contacts and separated from indirect contacts to monitor transmission.

Key Contributions and Results

Identification of E185K as a Dual-Function Mutation: The study identified the HA mutation E185K (mature H5 numbering; E189K in H3 numbering) as a critical variant. Phylogenetic analysis revealed strong evidence of diversifying positive selection at position 185.
Enhanced Receptor Binding: Unlike the consensus virus which binds exclusively to avian-like 3SLN, the E185K mutation conferred increased binding avidity to both avian-like (3SLN) and human-like (6SLN) receptor analogues. This dual-binding capability was confirmed in cattle-derived H5N1 viruses carrying the K185 mutation, which showed enhanced entry into cells expressing $\alpha$ 2,6-linked SA.
Immune Escape Mechanism: The E185K mutation was identified as an immune escape variant. Serial passage under immune pressure selected for this mutation, which resulted in an 8-fold reduction in haemagglutination inhibition (HI) titres and a 3-fold reduction in virus neutralization (VNT) titres. The mechanism appears to involve enhanced receptor-binding avidity, allowing the virus to outcompete neutralizing antibodies.
Prevalence in Cattle: The E185K mutation is increasingly prevalent in H5N1 viruses isolated from dairy cattle in the USA, with phylogenetic analysis identifying two distinct lineages of cattle viruses harboring this mutation.
Replication and Stability: The E185K mutation did not significantly enhance viral replication kinetics in various cell lines or embryonated eggs compared to the wild-type. Furthermore, thermostability and fusion pH (pH 5.8) remained comparable to the wild-type, indicating the mutation did not confer the increased acid stability often associated with airborne transmission.
Transmission in Ferrets: Crucially, ferret studies demonstrated that while the cattle-derived viruses (both E185 and K185) were highly pathogenic (causing 100% mortality or euthanasia in directly infected ferrets), the E185K mutation did not confer efficient airborne transmission. No virus shedding was detected in indirect-contact ferrets, and transmission to direct-contact ferrets was limited (25% mortality in one group, none in the other).

Significance and Claims
The paper concludes that the HA E185K mutation represents a significant evolutionary step for clade 2.3.4.4b H5N1 viruses. It functions as a dual driver: facilitating immune escape from pre-existing antibodies and expanding receptor binding specificity to include human-like receptors. This expansion is particularly concerning given the mutation's emergence and prevalence in cattle populations, which serve as a potential bridge for mammalian adaptation.

However, the authors maintain a modest and cautious stance regarding immediate pandemic risk. They explicitly state that while E185K enhances receptor binding and immune evasion, it does not independently increase airborne transmission in the ferret model, likely due to the lack of increased HA acid stability. The significance of the findings lies in the identification of a "potential pathway for mammalian adaptation." The authors argue that the combination of immune escape and expanded receptor specificity creates a foundation upon which further adaptive mutations (such as those affecting pH stability) could build, potentially leading to increased zoonotic risk. Consequently, the paper underscores the critical need for continued surveillance and functional characterization of evolving H5Nx variants, particularly those circulating in mammalian hosts like cattle, to mitigate emerging threats.

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