Hemagglutinin is the linchpin of influenza A infectivity. It recognizes sialosides, sugar chains terminating in sialic acid, on the surface of host cells, and after the virus has been taken up by endocytosis it triggers the fusion of viral and endosomal membranes. Avian influenza viruses typically favor sialic acids linked through an alpha-2,3 configuration, which are found deeper in the human respiratory tract, whereas human-adapted viruses prefer alpha-2,6-linked sialosides enriched in the upper airway. Both linkage types exist in human airways, and their distribution is thought to shape where a given virus can replicate. Whether E190D or Q226H might switch H5 receptor preference has been a pressing question, because position 190 has been implicated in receptor switching in H1 viruses and leucine at position 226 in H5 hemagglutinin has been shown to confer alpha-2,6 binding, although histidine at that position had not shown the same effect.

To find out, a team led by Sriram Subramaniam at the University of British Columbia used cryogenic electron microscopy to solve the structures of three H5 hemagglutinin ectodomains expressed and purified under identical conditions: the BC24 protein carrying both mutations of interest, the clade 1 protein from the fatal 2004 Vietnam case A/Vietnam/1203/2004 (VN04), and the clade 2.3.4.4b protein from A/Michigan/90/2024 (MI24), a recent strain that caused only mild human disease and is closely related to BC24. Globally, the three proteins are structurally very similar, with root mean square deviations of 0.441 angstroms between BC24 and VN04 and 1.095 angstroms between BC24 and MI24. The mutations do not substantially alter the fold of the protein near or far from the receptor-binding site. The dramatic difference lay elsewhere: in the electron density maps.

Previous structural work on H5 hemagglutinins had revealed that an N-linked glycan attached to asparagine 169 folds back on itself and occupies the virus’s own receptor-binding pocket, acting as an auto-glycan that mimics an alpha-2,3-linked sialoside. In the new structures, clear density for this auto-glycan is visible in the receptor-binding sites of both VN04 and MI24 hemagglutinin, but it is entirely absent from BC24 hemagglutinin. The researchers ruled out the possibility that the glycan simply was not attached in their preparation, since the same expression system, comparable glycosylation at other sites, and the close sequence relationship between MI24 and BC24 all argue that N169 glycosylation should occur similarly in both. The most plausible explanation is that the BC24 receptor-binding site has lost the affinity needed to hold the glycan in place, which in turn implies greatly reduced affinity for alpha-2,3-linked sialosides on host cells.

The structural basis for this loss is geometrically precise. In the VN04 and MI24 structures, the glutamate at position 190 positions its carboxylate for hydrogen bonding 2.9 and 2.8 angstroms, respectively, from the C9 hydroxyl of the terminal sialic acid, while the glutamine at 226 places its amide 3.3 and 3.2 angstroms from the C4 hydroxyl of the second sugar, galactose. In BC24, the aspartate introduced by the E190D mutation sits one methylene shorter and ends up 4.9 angstroms from where the bound sialic acid would align, while the histidine of Q226H pivots away from the galactose hydroxyl. The residues that are ideally positioned to grip an alpha-2,3-sialylated glycan in other strains are precisely the ones mutated in the patient’s virus, offering a clean structural explanation for the empty binding pocket.

Biochemical assays confirmed and extended the structural finding. On N-linked and O-linked glycan microarrays, VN04 and MI24 hemagglutinin bound exclusively to alpha-2,3-linked sialosides, with MI24 showing the broader breadth toward fucosylated glycans such as sialyl Lewis X that has been reported for clade 2.3.4.4 proteins. BC24 hemagglutinin, by contrast, showed no detectable binding to any glycan on the arrays, neither alpha-2,3 nor alpha-2,6 linked. Dynamic light scattering confirmed that all protein preparations were comparably stable, indicating that the differences reflect genuine receptor selectivity rather than artifacts of sample quality. Enzyme-linked immunosorbent assays probing four representative biantennary sialosides told the same story: neither BC24 nor any mutant carrying aspartate 190 or histidine 226, on either the MI24 or BC24 backbone, bound significantly to either linkage type at concentrations up to 50 micrograms per milliliter, while restoring glutamate 190 and glutamine 226 on the BC24 background reverted binding to alpha-2,3-linked sialosides.

Crucially, the mutagenesis work addressed the mixed viral population observed in the patient. Because E190D and Q226H each appeared in roughly a third of sequence reads, and it is unknown how often both mutations reside on the same hemagglutinin allele, the team generated single and double mutants in both forward and reverse directions. Every combination carrying either single mutation reproduced the severely reduced binding phenotype, meaning that multiple distinct minority variants circulating in the patient independently shared the weak receptor-binding trait. The findings also rule out the hypothesis that these mutations switched receptor specificity from alpha-2,3 to alpha-2,6 sialosides; instead, they simply crippled binding to both.

Yet the virus was clearly functional. In cell-based fusion assays using A549 human alveolar carcinoma cells, which display both sialoside linkage types, full-length BC24 hemagglutinin drove membrane fusion and syncytium formation, albeit at substantially reduced levels, yielding about 0.32-fold the luminescent signal of VN04 hemagglutinin while MI24 reached 1.3-fold. The authors suggest that multivalent avidity effects, in which many weak interactions collectively support attachment, or binding to alternative receptors not represented on the arrays, such as sulfated glycans, may compensate for the low biochemical affinity. Notably, BC24 hemagglutinin mediated fusion at levels statistically significantly above those of the severely impaired Y98F mutant, a variant known to permit viral proliferation depending on cell type, suggesting the weakly binding variants remain biologically competent. The broadly neutralizing antibody MEDI8852, which targets the conserved stem region, potently inhibited BC24 hemagglutinin-driven fusion with estimated IC50 values comparable to VN04 and MI24, and the patient’s neuraminidase carried no known resistance mutations, leaving conventional antiviral options intact.

The immunological data add a further layer. Serum from the patient, collected at four time points, showed strong reactivity against both BC24 and MI24 hemagglutinin and cross-reacted robustly with H1, H2, and H3 hemagglutinins, indicating a vigorous humoral response that nonetheless failed to prevent severe disease. Healthy pediatric controls displayed modest cross-reactivity against the clade 2.3.4.4b proteins, likely from seasonal vaccination or prior influenza exposure, supporting growing evidence that the population may not be entirely naive to H5N1. The authors propose that weakened receptor binding may have helped these variants evade upper-airway receptors and enrich in the distal airways, where the patient’s viral loads were highest, a hypothesis consistent with the historical association between lower respiratory tract infection and H5N1 severity. No human-to-human transmission followed the case, consistent with the absence of alpha-2,6 binding, but the study demonstrates that severe human disease can occur without classical human-receptor adaptation, and that weakly binding hemagglutinin variants can be fit in a severe infection context, adding an important dimension to surveillance and pandemic preparedness efforts.

Subject of Research: Structural and functional analysis of H5N1 hemagglutinin receptor-binding mutations from a severe human influenza case

Article Title: Diminished sialoside binding in novel H5N1 influenza hemagglutinin variants identified in a human patient

Article References: Diminished sialoside binding in novel H5N1 influenza hemagglutinin variants identified in a human patient. (n.d.). https://doi.org/10.1038/s41467-026-77829-x

Image Credits: AI Generated

DOI: 10.1038/s41467-026-77829-x

Keywords: H5N1, influenza, hemagglutinin, sialoside binding, cryo-EM, receptor-binding site, E190D, Q226H, avian influenza, clade 2.3.4.4b, glycan microarray, zoonosis

Hemagglutinin, or HA, is the major surface glycoprotein of influenza A virus. Each of its roughly 500-amino-acid monomers is cleaved into two subunits: HA1, which forms the globular head that docks onto sialic acid receptors on host cells, and HA2, which forms the stem anchoring the trimer to the viral envelope. In highly pathogenic avian viruses, a polybasic cleavage site allows activation by the protease furin throughout the body, contributing to systemic infection in birds. After a virus particle is taken up by endocytosis, the acidification of the endosome triggers an irreversible conformational change in HA that exposes the hydrophobic fusion peptide at the N-terminus of HA2, enabling the viral and endosomal membranes to merge and release the segmented viral genome into the cell.

The prefusion form of the HA trimer is metastable by design. It must be stable enough to survive the journey between hosts, yet primed to refold when the right pH is reached. If the trimer is too labile, it may fire prematurely in transmission droplets or early endosomes, destroying infectivity; if it is too stable, fusion may fail altogether. This balance differs markedly between host species. In wild aquatic birds, the natural reservoir of avian influenza viruses, greater acid stability helps virions persist in water. The jump to poultry is typically accompanied by acquisition of the polybasic cleavage site and reduced acid stability, while adaptation to mammals generally demands the opposite: a lower fusion-triggering pH, shifting from roughly 5.7 to 6.0 in avian strains to about 5.0 to 5.5 in human-adapted viruses. That extra stability helps the virus survive the mildly acidic environment of the human respiratory tract and of airborne droplets, a prerequisite for efficient airborne transmission.

Clade 2.3.4.4b, which likely emerged around 2020 through reassortment between a highly pathogenic H5N8 strain and a low-pathogenic avian virus in Central Asia or Europe, still binds preferentially to avian-type α2,3-linked sialic acid receptors and shows only weak affinity for the human-type α2,6-linked receptors. Experimental work has shown that a small number of HA mutations, such as Gln226Leu, Asn224Lys and Gly228Ser in the receptor-binding pocket, can shift receptor specificity, and that increased acid stability is essential for airborne transmission in ferrets. Yet these receptor-shifting substitutions remain rare in circulating strains, suggesting that efficient human adaptation will require multiple coordinated changes across at least three viral functions: polymerase activity, receptor-binding specificity and HA stability.

The new analysis, led by Daniel Christian Lauster, Christian Sieben, Matthias Ballauff and Andreas Herrmann of Freie Universität Berlin and the Helmholtz Centre for Infection Research, does not generate new experimental data. Instead, it builds on the deep mutational scanning study by Dadonaite and colleagues published in 2024, which used a lentivirus-based pseudovirus platform to measure how nearly every possible amino acid substitution in the HA of A/American Wigeon/South Carolina/2021, a clade 2.3.4.4b strain, affected receptor specificity, acid stability and antibody escape. The Berlin team took the stabilising mutations identified in that screen and asked a structural question: what, mechanically, do these substitutions actually do to the protein?

Because no three-dimensional structure exists for the exact strain used in the mutational scan, the researchers used the crystal structure of the HA ectodomain from A/Texas/37/2024, the reference strain of the dairy cattle outbreaks, which shares 99.4 percent sequence identity with the scanned virus, differing at only three positions. Using PyMOL to map polar interactions and the Adaptive Poisson-Boltzmann Solver to compute electrostatic surface potentials at both neutral pH 7.4 and acidic pH 5.3, they dissected each stabilising substitution in molecular detail. The electrostatic calculations, performed at physiological salt concentrations, revealed a recurring theme: many stabilising mutations act by taming a patch of dense positive charge in the lower HA1 head domain that becomes more repulsive as acidic residues become protonated at low pH.

In the HA1 subunit, the team found that substitutions such as Lys57Glu, Lys62Glu, Arg81Gly, Arg90Glu, Lys262Ile and Lys269Glu all reduce the strongly positive local surface potential of the lower head domain, diminishing electrostatic repulsion that would otherwise destabilise the prefusion trimer under acidic conditions. Other mutations work through different physics. Glu106Asp preserves the existing contact with Asn71 of HA2 while forging an additional interaction with Arg76 of a neighbouring monomer, thereby reinforcing the HA1-HA2 interface both within and between monomers. Thr318Ile, located near the fusion peptide pocket, enhances hydrophobic packing with surrounding residues, consistent with earlier work by Imai and colleagues showing that this substitution lowers the fusion pH by about 0.3 units. Histidine substitutions at positions 18 and 110 also stabilise the protein, likely because replacing these protonatable pH-sensing residues with non-charged amino acids removes a destabilising positive charge that appears at acidic pH.

In the HA2 stem, where most mutations are destabilising because the region is so finely tuned, the researchers identified a distinct set of permissive sites concentrated in the long helix and its connecting loop. Here, hydrophobic substitutions dominate: Ile77Leu, Val91Met, Asp95Met and Leu99Met all introduce methionine or leucine side chains oriented toward the centre of the three-helix bundle, creating new networks of hydrophobic interactions between the long helices of the three HA2 subunits. Asp90Glu, by contrast, strengthens a salt bridge with Lys307 of the adjacent HA1 subunit, shortening it from 3.5 to 2.9 angstroms while preserving the surrounding polar network. Arg106Glu reduces repulsion between positively charged side chains projecting into the stem centre. The short helix, notably, tolerated no stabilising mutations at all, underscoring its critical role in maintaining the metastable prefusion architecture.

The authors are careful to frame their conclusions as computationally derived hypotheses. The analysis treats each mutation in isolation and cannot capture epistasis, the phenomenon whereby combinations of mutations produce effects not predictable from their individual contributions. Recent work by Dosey and colleagues has shown that simultaneous mutations in the HA stem of clade 2.3.4.4b can stabilise the protein in ways not observed when the same mutations are studied one at a time. The study also deliberately excludes the wider viral context: neuraminidase activity must remain balanced with HA binding for efficient mucus penetration and viral release, and polymerase mutations in the circulating genotype B3.13 already point toward mammalian adaptation. Effects on acid stability, moreover, do not necessarily correlate with effects on cell entry, meaning the two phenotypes must be evaluated separately.

Nevertheless, the structural framework has immediate practical value for pandemic preparedness. By identifying which positions in the circulating H5N1 hemagglutinin can accept acid-stabilising substitutions, and by explaining the mechanisms involved, the study gives genomic surveillance teams a prioritised watch list of mutations whose appearance in sequencing data would signal progressive adaptation toward mammalian hosts. The finding that HA retains substantial structural plasticity, with multiple independent routes to enhanced acid stability spanning electrostatic tuning, hydrogen-bond reinforcement and hydrophobic packing, is a sobering one: it suggests the virus does not need a single improbable mutation to clear this barrier, but could assemble the required stability incrementally. The authors emphasise that direct experimental validation with purified mutant proteins, biophysical stability measurements and mutant HA structures will be essential, and that HA-neuraminidase co-adaptation must be studied in parallel. As clade 2.3.4.4b continues its global expansion, this kind of structure-guided surveillance may prove to be one of the most sensitive early-warning tools available for detecting the molecular steps that could bring a bird virus closer to human pandemic capability.

Subject of Research: Computational structural analysis of acid-stabilising hemagglutinin mutations in highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b

Article Title: In silico analysis of pH stabilising mutations of hemagglutinin of influenza A virus H5N1 clade 2.3.4.4b

Article References: Lauster, D. C., Sieben, C., Ballauff, M., & Herrmann, A. (2026). In silico analysis of pH stabilising mutations of hemagglutinin of influenza A virus H5N1 clade 2.3.4.4b. npj Viruses, 4(1), Article 44. https://doi.org/10.1038/s44298-026-00236-y

Image Credits: AI Generated

DOI: 10.1038/s44298-026-00236-y

Keywords: H5N1, influenza A virus, hemagglutinin, acid stability, clade 2.3.4.4b, deep mutational scanning, pandemic preparedness, zoonosis, membrane fusion, electrostatics, host adaptation, structural biology

The model, which the authors call an interface-resilience framework, rests on three pillars. The first is spillover pressure, the sum of forces that push pathogens across the species boundary and into human populations. The second is microbial community stability, the resilience of the vast microbial ecosystems that inhabit humans, animals, plants, soil and water. The third is governance connectivity, the capacity of institutions, sectors and nations to share data, coordinate surveillance and act in concert. What makes the model distinctive is its explicit claim about how these three dimensions interact: systemic health risk is expected to be greatest when spillover pressure is high, microbial resilience is low and governance systems are poorly connected. Risk, in other words, is not a property of any single factor but of their configuration.

This framing represents a subtle but important departure from how outbreak preparedness is usually conceived. Conventional approaches tend to focus on detecting pathogens once they have already crossed into humans, then containing them through case isolation, contact tracing and, where available, vaccination. The interface-resilience model shifts the analytical focus upstream, to the conditions that determine whether a pathogen encounter becomes a spillover event, whether a spillover becomes an outbreak, and whether an outbreak becomes a pandemic. By quantifying pressure, resilience and connectivity as distinct but coupled variables, the framework gives public health authorities a set of indicators that can be tracked continuously, long before a pathogen has a name.

The review grounds this argument in a sweeping historical arc. One Health, as a formal term, is relatively recent, but the insight that human, animal and environmental health are entangled is old. The authors revisit the lessons of plague and rabies control, two of the oldest sustained efforts in disease management, both of which required intervening in animal reservoirs and environmental conditions rather than simply treating human patients. Plague control demanded an understanding of rodent populations and flea vectors; rabies control depended on managing dog populations and, eventually, wildlife reservoirs. These campaigns, the review argues, were One Health in practice long before the term existed, and they demonstrate that integrated approaches can succeed when the ecological dimensions of disease are taken seriously.

From that historical foundation, the review moves through the modern catalogue of threats that have made One Health urgent. SARS and COVID-19 demonstrated how rapidly a zoonotic virus can exploit global travel networks. H5N1 avian influenza continues to circulate across wild bird and poultry populations, repeatedly raising the specter of adaptation to efficient human transmission. Mosquito-borne diseases are expanding their geographic range as climate change alters vector habitats. Antimicrobial resistance, which the review treats as a quintessential One Health problem, accumulates at the interface of human medicine, veterinary practice and agricultural environments, where antibiotic use in one sector selects for resistance that ultimately threatens all of them. Food- and pet-associated zoonoses add further pathways by which pathogens move between species in everyday life.

One of the more technically interesting contributions of the review is its treatment of microbial communities as a variable in their own right. The human microbiome, the gut flora of livestock, the soil and water microbial ecosystems of agricultural landscapes: these communities are not passive backdrops to disease but active modulators of it. A stable, diverse microbial community can suppress pathogen overgrowth, compete for resources and buffer hosts against invasion. Conversely, disruption of these communities, whether through antibiotic overuse, land degradation, chemical contamination or dietary homogenization, can lower the barrier to pathogen establishment. By placing microbial community stability alongside spillover pressure in the model, the authors make the case that microbiome degradation is a form of systemic risk accumulation, one that unfolds gradually and largely invisibly until it compounds a pathogen threat.

The third pillar, governance connectivity, addresses a failure mode that became painfully visible during the COVID-19 pandemic: the fragmentation of information and authority across sectors and borders. Human health ministries, veterinary services, environmental agencies and agricultural departments often operate in silos, with separate data systems, separate mandates and separate budgets. When a pathogen moves across the human-animal-environment interface, it moves faster than the institutional response can reorganize. Governance connectivity, in the model’s terms, measures the ability of these sectors to share surveillance data, coordinate risk assessment and execute joint action. The review suggests that poor connectivity is not merely an inefficiency but a multiplier of risk, because it delays detection and response precisely when speed matters most.

The review does not remain at the level of theory. It examines practical experience from China, a country whose scale and density of human-animal-environment interactions make it a critical testing ground for integrated approaches. The authors document experience in integrated disease management, in the control of antimicrobial resistance, in sustainable vector control that reduces reliance on chemical insecticides, in food-safety governance and in ecological management. These case studies serve a dual purpose: they demonstrate that cross-sector coordination is operationally feasible, and they illustrate the kinds of institutional arrangements, data flows and regulatory instruments that the governance-connectivity pillar of the model requires. The Chinese experience, the review suggests, offers transferable lessons for other regions confronting similar interface pressures.

The practical payoff of the interface-resilience model, if adopted, would be a shift toward preventive, data-driven health governance. Instead of waiting for outbreaks to trigger emergency responses, health systems would continuously monitor the three dimensions of the model: tracking land-use change, wildlife-livestock contact rates, climate-driven vector expansion and trade flows as indicators of spillover pressure; monitoring microbial community composition and diversity across humans, animals and environments as indicators of resilience; and auditing the interoperability of surveillance systems as an indicator of governance connectivity. Where indicators trend toward the high-risk configuration, interventions could be deployed preemptively, whether that means regulating wildlife trade, restoring degraded habitats, stewarding antibiotic use or building cross-sector data platforms.

The review arrives at a moment when the costs of fragmented thinking are difficult to ignore. Pandemics, antimicrobial resistance, vector-borne disease expansion and microbiome disruption are usually studied by separate communities of researchers, funded by separate programs and managed by separate institutions. The interface-resilience model offers a common analytical language for all of them, one that makes explicit how biological pressure, ecological resilience and institutional capacity combine to determine systemic risk. The authors, affiliated with Wenzhou Medical University’s Institute of One Health, position the framework as a step toward an integrated global health architecture spanning humans, animals and the environment. Whether the model’s indicators can be operationalized at scale remains a question for future work, but the conceptual contribution is clear: the health of humanity is not a standalone variable, and the systems that protect it must be designed with the same interconnectedness that characterizes the threats themselves.

Subject of Research: One Health framework integrating pathogen spillover, microbiome stability and governance for global health risk assessment

Article Title: One Health model links spillover, microbiomes and governance to global risk

Article References: One Health model links spillover, microbiomes and governance to global risk. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: One Health, spillover, microbiome, zoonoses, governance, surveillance, antimicrobial resistance, H5N1, COVID-19, vector-borne disease, public health, global health

The urgency behind the new assays is difficult to overstate. Since its first detection in January 2022, highly pathogenic avian influenza H5 has infected more than 13,000 wild birds and roughly 173 million poultry, with economic losses estimated between 14 and 164 billion dollars. In March 2024, an unprecedented spillover of clade 2.3.4.4b A(H5N1) into dairy cattle was reported in Texas, and the virus has since spread across 17 US states, infecting more than 1,000 cattle herds with sustained mammalian transmission and 70 human cases reported as of June 3, 2025. Historically, HPAI outbreaks such as H5N1 in Hong Kong in 1997 and H7N9 in China in 2013 have produced case fatality rates of at least 30 percent in humans, underscoring the pandemic potential that hangs over every new host adaptation.

Current diagnostic approaches each carry trade-offs that limit their usefulness in the field. Virus isolation remains the gold standard but is confined to biosafety level-3 laboratories staffed by highly trained personnel. Rapid immunoassays deliver results in about 15 minutes but suffer from reduced sensitivity and cannot discriminate between viral clades. Reverse transcription PCR offers high sensitivity and adaptability yet depends on thermocycling equipment and skilled operators, while next-generation sequencing provides nucleotide-level resolution at the cost of high expense and long turnaround times. The result, the authors argue, is a diagnostic landscape in which the tools that respond fastest to outbreaks are either too insensitive or too infrastructure-dependent to support surveillance at farms, markets, and rural clinics where spillovers first emerge.

To close this gap, the team turned to SHINE, short for Streamlined Highlighting of Infections to Navigate Epidemics, a one-pot CRISPR diagnostic platform previously developed for SARS-CoV-2 and influenza detection. In the avian influenza version of the workflow, viral RNA undergoes reverse transcription, recombinase polymerase amplification, and T7 transcription within a single tube, generating RNA amplicons that activate the collateral cleavage activity of the Cas13a enzyme when they base-pair with a complementary CRISPR RNA guide. Activated Cas13a then cuts RNA reporters, producing either a fluorescent signal read out on a plate reader or a colorimetric band on a paper lateral flow strip, the latter requiring nothing more sophisticated than a smartphone camera to document.

Assay design leaned heavily on machine learning. The researchers used ADAPT, a software platform that predicts sensitive and specific Cas13 guide sequences, to select primer and crRNA sets targeting the hemagglutinin gene segment that uniquely defines H5 viruses. Two candidate designs achieved predicted coverage of 98.72 and 96.18 percent of aligned H5N1 sequences, and the top-performing guide, targeting nucleotides 847 to 874 of the H5 consensus, was carried forward. A series of optimization experiments followed: the team tuned the ratio of T7-attached to non-T7-attached forward primers, settled on a 1:3 ratio that reduces competition between amplification and detection, increased RPA primer concentrations, and adjusted reporter, RNase H, and magnesium levels. The resulting optimized assay, SHINE-H5, reliably detected synthetic RNA targets across all tested concentrations within 60 minutes, a substantial improvement over the prototype.

Analytical characterization of SHINE-H5 was rigorous. Testing against six vaccine-derived viral seedstocks from the CDC showed high fluorescence for the intended target and negligible signal for non-target viruses. Clinical specificity was evaluated using 64 nasopharyngeal swab specimens from Massachusetts General Hospital that tested positive for seasonal influenza A or B but negative for avian H5 by qPCR; SHINE-H5 correctly returned negative results for all 64 samples, covering infections with H1N1, H3N2, and influenza B, including co-infections. The limit of detection, established with serial dilutions of H5N1 viral seedstock in viral transport media and a logistic regression model requiring at least 95 percent detection, was 121.7 copies per microliter, with a 95 percent confidence interval of 63.14 to 234.55 copies per microliter. In a head-to-head comparison, a validated RT-qPCR assay detected samples down to 10 copies per microliter while SHINE-H5 detected down to 50 copies per microliter, a sensitivity gap the authors acknowledge but one that still significantly outperforms existing H5 immunoassays.

For field deployment, the researchers adapted SHINE-H5 to a lateral flow readout, replacing the fluorescent quenched reporter with a FAM-biotin reporter whose cleavage products migrate on a paper strip. Using a larger 40-microliter reaction volume, the lateral flow version detected H5N1 seedstock down to 25 copies per microliter while retaining strong specificity against a subset of 14 seasonal influenza-positive patient samples. The team also demonstrated that the assay can detect synthetic RNA targets spiked into milk, a proof of concept for surveillance in the dairy herds now at the center of the North American outbreak.

Beyond broad H5 detection, the study introduces SHINE-H5-CS, a clade-specific assay targeting 2.3.4.4b A(H5N1), the lineage that has dominated global H5 phylogeny since 2021 and is driving the US cattle outbreak. Guide design for this assay employed BADGERS, a successor to ADAPT that integrates advanced search algorithms to explore the fitness landscape of candidate crRNAs and achieve single-nucleotide discrimination. Of three candidates, crRNA3, targeting a region of the H5 segment starting at nucleotide position 880, showed the highest on-target activity with minimal off-target signal. The researchers also shifted the forward primer four nucleotides upstream to introduce an additional mismatch that sharpened discrimination. SHINE-H5-CS consistently detected clade 2.3.4.4b RNA at concentrations above 100 copies per microliter while showing minimal cross-reactivity with non-2.3.4.4b H5 sequences and seasonal influenza seedstocks, enabling lineage identification without sequencing.

The team extended the platform to the H7 subtype, the other hemagglutinin group with well-documented potential to evolve into highly pathogenic strains. Phylogenetic analysis of 25 representative H7 strains revealed clean separation into Eurasian and North American lineages that mirror migratory bird flyways, and the 2013 Eurasian H7N9 epidemic, which caused more than 1,500 human infections with a fatality rate near 40 percent, demonstrated the public health stakes of this lineage. The resulting SHINE-H7-Eurasian assay reliably detected Eurasian H7 RNA across a range of concentrations while discriminating against North American H7 strains and a panel of unrelated seasonal influenza viruses, and it too was adapted to a lateral flow format that preserved lineage specificity.

The authors are candid about the limitations of the work. Confirmed human H5 and Eurasian H7 cases remain rare, so the assays could not be evaluated on true positive human clinical specimens, and compatibility with specimen types beyond mock milk samples, such as poultry or environmental samples, has not yet been assessed. Even so, the combination of isothermal operation at 37 degrees Celsius, turnaround times of 60 to 120 minutes, minimal instrumentation, machine-learning-accelerated assay design, and compatibility with paper-based readouts positions the SHINE family of assays as a practical bridge between laboratory-grade accuracy and point-of-need accessibility. As avian influenza continues its advance through wild birds, poultry, cattle, and occasionally people, tools that can detect not just the virus but its specific clades, close to where it emerges, may prove decisive in blunting the next spillover before it becomes the next pandemic.

Subject of Research: Development of streamlined one-pot CRISPR-Cas13 diagnostic assays for detection and genetic subtyping of H5 and H7 avian influenza viruses

Article Title: Streamlined CRISPR-based assays for detection and subtyping of H5 and H7 avian influenza

Article References: Huang, Y., Guo, A., Adams, G., Lemieux, J. E., & Myhrvold, C. (2026). Streamlined CRISPR-based assays for detection and subtyping of H5 and H7 avian influenza. iScience, 29(10), Article 117556. https://doi.org/10.1016/j.isci.2026.117556

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117556

Keywords: avian influenza, CRISPR diagnostics, SHINE assay, Cas13, H5N1, H7 avian influenza, clade 2.3.4.4b, lateral flow assay, recombinase polymerase amplification, point-of-need testing, dairy cattle outbreak, pandemic preparedness