Biology subjects

Boons, G.-J.

Publications and source records attributed to Boons, G.-J..

At least 19 recordsLinked to original sources

bioRxiv2026

Five decades of human H1N1 evolution reveal conserved glycan receptor specificity

H1N1 influenza viruses have circulated continuously among human populations for five decades. During evolution, the hemagglutinin has accumulated amino acid substitutions in and around the receptor-binding site (RBS), mainly due to antigenic pressure, potentially altering receptor recognition and binding profiles. Here, we investigated the glycan-binding properties of H1N1 strains collected from 1977 through 2024 using ELISA, flow cytometry, and tissue histochemistry to characterize changes in receptor-binding specificity over time. Across the strains examined, we observe a recurring preference for binding 2,6-sialylated di-LacNAc N-glycans. Binding to 2,3-sialylated glycans was generally undetectable, although A/Hongkong/117/1977 and A/New Caledonia/20/1999 bind chicken tracheal tissues. Interestingly, post-pandemic H1N1 viruses fluctuate in their relative binding avidities. The responsible amino acids, in turn, have different effects in pre-pandemic HA proteins. We also report a classical egg-adapted H1N1 mutant that shows null binding across platforms. These findings highlight the contribution of RBS mutations during initial adaptation and evolution of human H1N1 influenza viruses to glycan-binding properties, which, in contrast to H3N2 viruses, never fail to bind glycans that correlate with avian erythrocyte hemagglutination.

microbiology↗

bioRxiv2026

Enhanced Pathogenicity and Contact Transmissibility of Human-origin Avian Influenza H5N1 Clade 2.3.4.4b Genotype B3.13 Compared to D1.1 in Ferrets

Since its emergence in 2020, multiple genotypes of the H5N1 clade 2.3.4.4b have been identified, with B3.13 and D1.1 emerging in the USA as two major and concerning genotypes. However, their relative pathogenicity and transmissibility in mammals have not been fully elucidated. We compared the pathogenicity and transmissibility of the first two human H5N1 clade 2.3.4.4b cases caused by B3.13 in Texas (A/Texas/37/2024; HPhTX B3.13) and D1.1 in Louisiana (A/Louisiana/12/2024; HPhLA D1.1) in a ferret model of infection and transmission. HPhTX B3.13 infection resulted in more severe clinical disease and enhanced viral shedding, with evidence of increased transmission relative to HPhLA D1.1. Histopathological analysis revealed more extensive lung pathology in animals infected with HPhTX B3.13, consistent with increased viral loads and inflammatory responses. Importantly, both genotypes showed no significant differences in reactivity to ferret sera raised against candidate vaccine virus (CVV) strains, receptor binding properties, or neuraminidase (NA) activity and thermostability. Whole-genome sequencing revealed no adaptive mutations in HPhTX B3.13 following infection or transmission. In contrast, HPhLA D1.1 showed rapid acquisition of the mammalian-adaptive mutation E627K in infected ferrets and both E627K and Q194K in the only fatal contact animal. Both mutations were associated with enhanced polymerase activity and computational analyses suggested that they enhance interactions with the mammalian host factors ANP32A and B. Our findings indicate that B3.13 is already well adapted for mammalian infection and transmission whereas D1.1 retains evolutionary potential through the rapid acquisition of adaptive mutations, highlighting important genotype-specific differences relevant to zoonotic risk assessment and pandemic preparedness. SignificanceInfluenza H5N1 viruses continue to diversify genetically while expanding into mammalian hosts, increasing opportunities for viral adaptation and zoonotic transmission, including humans. However, whether the predominant clade 2.3.4.4b genotype differs in its capacity to infect, transmit, and evolve in mammals remains poorly understood. Using the ferret model of influenza infection and transmission, we demonstrated that the currently circulating B3.13 and D1.1 genotypes exhibit distinct pathogenic and transmission characteristics despite retaining similar receptor-binding characteristics, NA functions, and antigenic profiles. While B3.13 readily infects and transmits in ferrets and does not develop further adaptive mutations associated with increased replication and transmission, D1.1 rapidly acquires mammalian-adaptive mutations after a single infection and/or transmission event, highlighting its evolutionary potential. These findings show that genotype-specific biological properties can influence zoonotic risk independently of antigenic similarity and emphasize the importance of integrating phenotypic characterization with genomic surveillance to improve pandemic preparedness and guide public health risk assessment.

microbiology↗

bioRxiv2026

Chemoenzymatic Synthesis of 6-Sulfo Sialyl Lewisx Containing Glycans to Probe the Receptor Specificity of MERS Coronavirus

The initial attachment of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) to host cell sialosides is critical for infection, yet its precise receptor specificity remains poorly understood. Here, we describe a chemoenzymatic methodology to synthesize a comprehensive panel of 6-sulfo sialyl Lewisx (6-sulfo-SLex) containing glycans. Our approach entails the enzymatic assembly of an oligo-lactosamine chain modified at specific positions with N-trifluoroacetyl-glucosamine (GlcNTFA) moieties. Mild base treatment removes the TFA group to yield glucosamine, which effectively blocks enzymatic fucosylation. By leveraging this approach alongside the unique substrate selectivity of GlcNAc-6-O-sulfotransferases 2 (CHST-2), we achieved the selective preparation of fucosylated 6-sulfo-SLex glycans. Microarray screening of these printed glycans revealed that a 6-sulfo-SLex derivative presented on an extended LacNAc chain is the preferred host receptor for MERS-CoV. Conjugation of this lead compound to a polyglycerol-based dendrimer generated a multivalent inhibitor that potently blocks hemagglutination of human red blood cells by the MERS-CoV spike protein N-terminal domain (NTD). Furthermore, computational modeling demonstrated that the fucose moiety does not directly contact the viral spike protein. Instead, it pre-organizes the ligand into a favorable conformation, enabling a critical salt bridge between the glycans sulfate group and the guanidinium side chain of viral residue Arg307.

biochemistry↗

bioRxiv2026

Loss of hemagglutination ability by H3N2 influenza A virus, subclade K.

Seasonal human H3N2 influenza viruses, subclade K (J.2.4.1), have been the predominant influenza A viruses in the Northern hemisphere influenza season of 2025/2026. Since 2024, the vaccine virus A/Darwin/6/21 has emerged in different antigenic variants. Antigenic changes are frequently caused by amino acid substitutions near the hemagglutinin (HA) receptor-binding pocket, which can also affect receptor binding properties, such as hemagglutination. Hemagglutination is crucial for assessing antigenicity using the hemagglutination inhibition (HAI) assay, and a loss of binding to turkey erythrocytes could significantly hamper this process. In this study, we explored how substitutions in or around the HA receptor-binding site affect binding to glycans at the molecular level. We employed ELISA, glycan array, flow cytometry, hemagglutination assays, and tissue staining. Substitutions at positions 140, 192, and 223 establish clade J viruses that emerged in 2024. Computational analysis of HA in complex with an elongated glycan reveals that mutation F192 forms a CH-Pi interaction to stabilize the binding. Based on this background, substitutions in antigenic sites A and B within subclade K viruses exhibit a binding preference for elongated glycans, which are not displayed on turkey erythrocytes. Conversely, our previously established glyco-remodeled erythrocytes are efficiently bound by these subclade K H3N2 viruses and could support influenza surveillance and vaccine development.

microbiology↗

bioRxiv2026

Self-assembling nanoparticles to assess multivalent interactions between influenza A virus hemagglutinin and glycan surfaces

The multivalent display of surface glycoprotein hemagglutinin (HA) on Influenza A viruses (IAVs) enhances the overall binding avidity to sialylated glycans on host cell surfaces. While precomplexing HA trimers with antibodies increases multivalency and avidity, this method does not replicate the virions geometry and limits insights into the multivalent binding process. Here, we use perfectly controllable icosahedral protein nanoparticles to examine the multivalent HA receptor-binding properties. We compare three HA presentation systems with varying degrees of multivalency: single HA trimers, antibody-precomplexed HA trimers, and HA trimers on nanoparticles. Our results indicate that increasing HA valency enhances binding avidity across various glycan surfaces, including erythrocytes, cells, and lipid bilayers with varying glycan densities, while maintaining receptor specificity. By combining functional and non-functional HA trimers during nanoparticle formation, we create statistical mixtures of nanoparticles with varying valencies. At high receptor densities, nanoparticles with few functional trimers still bind strongly, whereas at low receptor densities, a patch of five HA trimers appears necessary for binding. As a key finding, we observe that such a statistical mixture of nanoparticles with functional and nonfunctional HAs binds to glycan surfaces in a stronger density-dependent manner than fully functional particles. We also observe differences in binding modes that correlate with the number of functional trimers, the glycan structure (linear vs branched), and the densities achievable with these glycans. Overall, our findings demonstrate that the presentation of multivalent HA plays an enormous role in the response to glycan receptor type and density, with implications for the future design of virus monitoring, viral inhibitors, and targeting vectors.

biochemistry↗

bioRxiv2026

Opposing cell type preferences for binding and replication shape influenza A virus infection in human airways

Influenza A viruses (IAVs) pose a persistent threat to human health through seasonal epidemics and zoonotic spillover from avian reservoirs. As respiratory pathogens, they primarily target the airway epithelium. However, it remains unclear how host cell-specific barriers jointly shape viral tropism and replication in primary human airway cultures. Here, we show that avian IAVs can infect ciliated and secretory cells but preferentially bind to ciliated cells, consistent with higher abundance of their receptor 2,3-linked sialic acids, specifically sialyl Lewis X glycans, present on the apical surface of ciliated cells. Replication levels were comparable between secretory and ciliated cells for the avian strains, resulting in an overall preference for ciliated cells. In contrast, human IAVs also preferentially bind to ciliated cells but independently of 2,6-linked sialic acid abundance. Human IAVs replicate more efficiently than avian IAVs due to their ability to utilize human ANP32 proteins, but they also exhibit cell type-specific differences due to ANP32, allowing for higher viral RNA levels in secretory cells. Thus, preferential binding to ciliated cells coupled with enhanced replication in secretory cells equalizes overall infection levels across cell types for human IAVs. Together, our findings highlight the spatiotemporal complexity and interplay of IAV infection dynamics in the airway epithelium and redefine current models of influenza A virus tropism.

microbiology↗

bioRxiv2026

Acquisition of specific human respiratory tract binding of 2.3.4.4b H5N1 hemagglutinins requires multiple mutations

It has been suggested that the hemagglutinin of the human-infecting cattle-derived 2.3.4.4b virus A/Texas/34 (H5TX) requires only one mutation, namely Q226L, to switch from binding avian-type to human-type receptor preference. In this study, we examined the binding of H5TX Q226L, along with other key mutations, to sections of human trachea. We conclude that, while H5TX Q226L can bind human-type receptors, more than a single mutation is required for this protein to bind to human respiratory tract tissue. We also report changes in receptor-binding specificity of another 2.3.4.4b HA mutant, H5FR Q226L, associated with the presence of a multibasic cleavage site. This study offers insight into the determinants of evolution towards human-type receptor binding in currently circulating H5Nx viruses. It also emphasizes the importance of testing individual strains using additional methods, including tissue-based approaches, alongside synthetic glycans. ImportanceCurrently, H5N1 influenza A viruses are responsible for numerous zoonotic spillover events, from infecting birds to other mammals, including dairy cattle. Although no human-to-human transmission has been observed, several people have been infected. This host range expansion is typically linked to changes in one of the viral surface proteins, hemagglutinin, which can switch its preference from avian-type to human-type receptors. To better understand the potential of the currently circulating H5N1 virus to transmit among humans, we evaluated the effects of the Q226L mutation, in combination with other amino acid substitutions, on binding to the human trachea. We also studied the effect of the multibasic cleavage site, a specific motif present in highly pathogenic influenza strains, on receptor binding properties. These findings provide insight into the role of receptor binding in influenza infections.

microbiology↗

bioRxiv2026

Structural insights into antibody responses against influenza A virus in its natural reservoir

While influenza A virus undergoes rapid antigenic drift in humans, at least some subtypes, such as H3, have relatively stable antigenicity in natural waterfowl reservoirs, despite the presence of immune pressure. However, the underlying mechanisms remain poorly understood. This study identified and characterized 187 antibodies to H3 hemagglutinin from experimentally infected mallard ducks, 18 of which were further analyzed by cryo-EM. Compared with human H3 antibodies, duck H3 antibodies exhibited higher glycan-binding propensity, more balanced immunodominance hierarchy, and targeted distinct epitopes. Other unique features of duck H3 antibodies included a convergent CDR H3-independent heavy chain-only binding mode and an N-glycosylated CDR H3 as decoy receptor. By annotating duck immunoglobulin germline genes, we also demonstrated the importance of gene conversion in duck H3 antibodies. Overall, our findings provide insights into how millennia of coevolution have shaped the interplay between influenza A virus antigenic drift and antibody responses in the natural reservoir.

immunology↗

bioRxiv2026

Emergence and antigenic characterisation of influenza A(H3N2) viruses with hemagglutinin substitutions N158K and K189R during the 2024/25 influenza season

BackgroundSeasonal human influenza viruses can escape from antibody-mediated neutralization when amino acid changes occur in the hemagglutinin protein. Routine surveillance identified circulation of an A(H3N2) virus variant in the Netherlands with amino acid substitutions at hemagglutinin positions 158 and 189. These amino acid positions were previously responsible for antigenic change of influenza A(H3N2) viruses and potentially lead to escape of this variant from vaccine-mediated immunity. AimTo characterize the emergence and antigenic properties of N158K and K189R double substitution virus variants. MethodsWe analyzed the geographical and temporal dynamics of the double-substitution variant using a phylogeographic approach and used hemagglutination inhibition assays and antigenic cartography methods to map its antigenic properties. ResultsA(H3N2) viruses carrying K189R were first detected in Guatemala in June 2024, before subsequently gaining the N158K substitution, which was intially detected in Colombia in November 2024, followed by detection in the Netherlands in December 2024. However, detections within Europe remained almost entirely confined to the Netherlands. The proportion of viruses carrying the N158K and K189R substitutions increased to 16% - 24% per collection week of sequenced Dutch viruses during the peak of the epidemic of the 2024-2025 respiratory season. Antigenic characterization of viruses with N158K and K189R substitutions indicated that these are antigenically distinct from the A(H3N2) components of 2025-2026 Northern Hemisphere vaccines, showing 8-192-fold reduction in hemagglutination inhibition titers with antisera against the vaccine strain compared to antisera against the homologous virus. ConclusionsInfluenza A(H3N2) viruses with N158K and K189R escaped recognition by antibodies raised against the 2024-2025 and 2025/2026 Northern Hemipshere vaccine strains in hemagglutination inhibition assays. These variants circulated widely in the Netherlands during the 2024-2025 influenza season, raising concerns about reduced vaccine-mediated protection if such variants would spread more broadly during 2025-2026 Northern Hemipshere season.

microbiology↗

bioRxiv2025

Asymmetrical glycoengineering of monoclonal antibodies: new insights in a-gal immunogenicity

Glycosylation heterogeneity in therapeutic monoclonal antibodies (mAbs) necessitates precise glycoengineering to optimize effector functions, such as those mediated by Fc{gamma}-receptors (Fc{gamma}Rs). Current enzymatic remodeling methods are limited in scope, producing only a narrow spectrum of glycoforms and struggling to achieve complete control over the synthesis of defined asymmetrical structures, which are often critical for enhanced mAb efficacy. We report a multi-step, fully enzymatic platform for the solution-phase synthesis of a comprehensive library of biantennary mAb glycoforms with complete control over the 1,3- and 1,6-mannose arm architecture. The strategy relies on two key catalytic steps: the use of the single domain of the {beta}-N-acetylglucosaminidase StrH (GH20-2) to achieve the regioselective hydrolysis of the GlcNAc residue on the 1,3-mannose arm of the intact antibody, thereby introducing asymmetry; and exploiting the intrinsic, broad acceptor flexibility of GnT-I to reinstall the 1,3-mannose arm GlcNAc even after the 1,6-mannose arm has been selectively extended and terminally capped with Neu5Ac or the -gal epitope. This robust, two-stage enzymatic remodeling methodology, demonstrated on infliximab and cetuximab, unlocks the complete chemical space of biantennary Fc-glycans for comprehensive, functional studies. We employed these well-defined glycovariants to determine the minimum structural requirements for anti--gal IgE binding and systematically evaluate binding affinities toward Fc{gamma}Rs, providing essential insights for the rational design of next-generation immunotherapeutics.

biochemistry↗

bioRxiv2025

Vaccine-induced antigenic drift of a human-origin H3N2 Influenza A virus in swine alters glycan binding and sialic acid avidity

Interspecies transmission of human influenza A viruses (FLUAV) to swine occurs frequently, yet the molecular factors driving adaptation remain poorly understood. Here we investigated how vaccine-induced immunity shapes the evolution of a human-origin H3N2 virus in pigs using an in vivo sustained transmission model. Pigs (seeders) were vaccinated with a commercial inactivated swine vaccine and then infected with an antigenically distinct FLUAV containing human-origin HA/NA. Contact pigs were introduced two days later. After 3 days, seeder pigs were removed, and new contacts introduced. This was repeated for a total of 4 contacts. Sequencing of nasal swab samples showed the emergence of mutations clustered near the HA receptor binding site, enabling immune escape and abolishing binding to N-glycolylneuraminic acid. Mutant viruses recognized 2,6-sialosides with 3 N-acetyllactosamine repeats, which are rare in swine lungs, while the parental virus bound structures with a minimum of 2 repeats. Adaptative HA mutations enhanced avidity for 2,6-linked sialic acid, likely compensating for the low abundance of extended glycans. Notably, residues outside the canonical HA binding pocket contribute to glycan binding, suggesting a trade-off between receptor breadth and avidity. These findings show that non-neutralizing immunity promotes viral adaptation by fine-tuning receptor engagement and immune evasion. SIGNIFICANCEUnderstanding how vaccination shapes influenza A virus (FLUAV) evolution across species barriers is critical for predicting and preventing zoonotic and reverse-zoonotic events. Our study demonstrates that vaccine-induced immune pressure can drive antigenic drift in a human-origin H3N2 virus, altering HA receptor binding properties that could inadvertently facilitate adaptation to swine. These changes shifted glycan specificity toward extended poly-LacNAc structures and enhanced 2,6-linked sialoglycans binding while abolishing Neu5Gc recognition. By revealing how non-neutralizing immunity fine-tunes HA-glycan interactions by engaging antigenically relevant residues in glycan binding, this work highlights vaccination as an underappreciated driver of host adaptation and viral evolution.

microbiology↗

bioRxiv2025

C-6 Modified 2-F-Fucose Derivatives as Inhibitors of Fucosyl Transferases

Fluorinated analogs of guanosine-diphosphate-{beta}-L-fucose (GDP-Fuc) have received considerable attention for the development of inhibitors of fucosyltransferases (FUTs). These compounds can be recognized by FUTs but do not or slowly transfer the fluorinated fucosyl residue because the electron-withdrawing fluorine(s) destabilize the oxocarbenium-like transition state. Fluorinated GDP-Fuc analogs can also act as feedback inhibitor of the de novo biosynthesis pathway of GDP-Fuc. To investigate the biological significance of distinct glycoconjugate classes, it is important to develop inhibitors that can selectively target specific FUT enzymes. Here, we report the design, synthesis, and biological evaluation of a series of GDP-2-F-Fuc analogs modified at C-6 of Fuc by various amides and ethers. We also prepared and examined corresponding prodrugs as potential FUT inhibitors of cellular glycosylation. Our findings reveal that two of the inhibitors potently inhibited FUT1, 3, 6, and 9, while displaying minimal activity against FUT8. However, the corresponding prodrugs did not inhibit cellular fucosylation, which is probably due to a lack of GDP-fucose pyrophosphorylase activity. The results demonstrate that modifications at the C-6 position of Fuc can confer selectivity, although further investigations of alternative functional groups are required to enhance cellular tolerance and efficacy.

biochemistry↗

bioRxiv2025

Chemoenzymatically Synthesized O-Acetylated GD3 Gangliosides to Examine Viral Receptor Specificities in a Cellular Context

Gangliosides are a class of sialic acid-containing glycosphingolipids involved in a wide range of biological processes. The terminal sialic acid of gangliosides can be O-acetylated at C7 and/or C9 hydroxyl, contributing to ganglioside structural complexity and function. It has been difficult to obtain panels of structurally well-defined O-acetylated gangliosides for binding and functional studies. We describe here a chemoenzymatic strategy that can provide, for the first time, 7-O-, 9-O-, and 7,9-di-O-acetylated GD3 gangliosides. It is based on the chemical assembly of a common tetrasaccharide precursor as -glycosyl fluoride that is coupled to sphingosine by a glycosynthase, followed by O-acetyl editing by coronaviral hemagglutinin-esterases. The resulting synthetic glycosphingolipids have been employed for cell surface remodeling of erythrocytes. Analysis by liquid chromatography and ion mobility mass spectrometry (LC-IM-MS) demonstrated successful integration of the glycosphingolipids into the plasma membrane with preservation of acetyl ester patterns. Using human coronavirus HKU1 spike-functionalized virus-like particles, we demonstrate that the resulting glycan-remodeled erythrocytes can be utilized in hemagglutination studies as a label free method to investigate viral protein binding to individual glycoforms in a cellular environment. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/670503v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@37de01org.highwire.dtl.DTLVardef@1d043faorg.highwire.dtl.DTLVardef@1ee57borg.highwire.dtl.DTLVardef@6bf474_HPS_FORMAT_FIGEXP M_FIG Table of Contents 7-O-, 9-O-, and 7,9-di-O-acetylated GD3 gangliosides can be prepared by chemical assembly of a tetrasaccharide as -glycosyl fluoride that is coupled to sphingosine using a glycosynthase, followed by O-acetyl editing by coronaviral hemagglutinin-esterases. Cell surface remodeling of erythrocytes using the GD3 derivatives provide a tool to examine viral receptor specificities. C_FIG

biochemistry↗

bioRxiv2025

Chemoenzymatic Synthesis of Asymmetric Bisecting Bi-, Tri-, and Tetra-Antennary N-Glycans

N-Acetylglucosaminyltransferase-III (GnT-III) is a glycosyltransferase that can install a {beta}1,4-linked N-acetylglucosamine (GlcNAc) residue at the central {beta}-mannoside of N-glycans. The resulting so-called bisecting GlcNAc is not further extended by glycosyl transferases and has been implicated a wide range of biological processes. The molecular mechanisms by which bisection modulates the biosynthesis of N-glycans and influences molecular recognition is not well understood, which is due to a lack of well-defined N-glycans with and without bisection. We describe a chemoenzymatic methodology that can readily provide a wide range of asymmetrical bisecting bi-, tri- and tetra-antennary N-glycans. It was found GnT-III can act on bi-, tri- and tetra-antennary N-glycans and can also accepts N-glycans having a {beta}1,2GlcNTFA or GlcN3 moiety at the 1,2Man- or 1,6Man-antenna making it possible to prepare panels of asymmetrical N-glycans with and without bisection and having different patterns of sialylation and fucosylation. Kinetic experiments showed GnT-III preferentially modifies bi-antennary glycans. The compounds were printed as a glycan microarray, which was screened for lectin binding. It was found that some lectins preferentially bind to bisecting glycans, whereas others do not tolerate or are not affected by this modification. We investigated receptor specificities of human H1N1 and H2N3 influenza viruses and animal H5N1 viruses that pose a pandemic threat including a virus that has become endemic in cattle. The H1N1 and H2N3 viruses did not tolerate bisection whereas it did not affect H5N1 viruses. A/bovine had the broadest receptor specificity providing a rationale for its wide host range.

biochemistry↗

bioRxiv2025

De novo sequencing of complex glycans by ion mobility-mass spectrometry using a self-expanding reference database

It is essential to determine exact structures of glycans in complex biological samples to understand their biology and exploit their diagnostics, therapeutics and nutraceuticals potential. An unresolved analytical challenge is the identification of isomeric glycan structures in complex biological samples. Ion mobility (IM) combined with MS enables separation of isomeric glycans and identification by comparing their intrinsic collision cross section (CCS) values with similar data of synthetic standards. To identify glycans without the need to synthesize all biologically occurring glycans, we describe here an IM-MS de novo sequencing method based on fragment identification and sequence assembly. CCS values of additional fragments from glycans in biological samples resulted in a self-expanding reference database, gradually facilitating the sequencing of glycans of increasing complexity and expanding the database from an initial 20 standards to 332 unique entries. The methodology was employed to determine exact structures of human milk oligosaccharides and N-glycans of biotherapeutics.

biochemistry↗

bioRxiv2025

Characterization of the glycoproteins of novel fish influenza B-like viruses

Novel influenza-like virus sequences previously identified in fish and amphibians were found to cluster as a sister clade of influenza B viruses, but have thus far remained uncharacterized. We demonstrate that salamander influenza-like virus (SILV) HA is functionally divergent from influenza B virus HA and does not bind to 2,3- and 2,6-linked sialic acids. However, the HAs of Siamese algae-eater influenza-like virus (SAEILV) and chum salmon influenza-like virus (CSILV) bind to 2,3 linked sialic acid. Furthermore, SAEILV HA binds to sialyated Lewis X, is activated by human airway enzymes and is fusogenic at a wide range of pH conditions. SAEILV NA has a highly conserved active site and a similar structure to other known NAs. We also determined the cryo-electron microscopy structure of the HA of a previously described virus from the same sister clade, the Wuhan spiny eel influenza virus (WSEIV). Importantly, no cross-reactive antibodies against these HAs or NAs were found in the human serum, suggesting that humans are immunologically naive to these viruses. One sentence summaryNovel influenza like-viruses, displayed different target receptor specificity and limited antigenic conservation of the HA and NA relative to influenza B Viruses.

microbiology↗

bioRxiv2025

The receptor binding properties of H5Nx influenza A viruses have evolved to promiscuously bind to avian-type mucin-like O-glycans

Highly pathogenic H5Nx influenza A viruses are causing unprecedented, season-independent outbreaks across avian and mammalian species, including dairy cattle, a novel reservoir. The sialoside-binding properties of influenza A hemagglutinin (HA) are strongly related to its ability to infect and transmit between hosts. Mucin-like O-glycans, omnipresent in respiratory tracts, have been understudied as viral receptors due to their complexity. To address this, we synthesized 25 O-linked glycans with diverse sialosides, including modifications by fucosides and sulfates. Our findings reveal that H5Nx 2.3.4.4b viruses uniquely bind core 3 sialyl-Lewisx and Sia-Gal-{beta}3GalNAc, glycans not recognized by classical H5 or other avian viruses. By determining its crystal structure, we resolved the structural features of both structures in an H5 hemagglutinin (HA) from a 2016 2.3.4.4b virus. While these viruses do not bind human-type receptors, their promiscuous receptor specificity enhances binding to human tracheal tissues, suggesting that O-glycan recognition contributes to their zoonotic potential.

microbiology↗

bioRxiv2025

Chemoenzymatic Synthesis of Glycosphingolipids having an HNK-1 Epitope for Erythrocyte Cell Surface Remodeling

Several neuropathies, such as Guillain-Barre syndrome and myelin-associated glycoprotein neuropathy (MAG), are caused by antibodies targeting glycosphingolipids. Several studies have indicated that MAG arises from pathogenic IgM autoantibodies targeting sulfoglucuronyl (HNK-1) containing glycosphingolipids. The exact mechanism by which IgM-neuropathy occurs has not been fully elucidated. Furthermore, no appropriate diagnostic tools are available for MAG using sulfoglucuronyl containing glycosphingolipids. To address these limitations, we describe here a synthetic strategy that makes it possible to prepare sulfoglucuronyl paraglobosides using a neochemo-enzymatic approach. It is based on the enzymatic assembly of N-acetyl-lactosamine (LacNAc) backbones as thioglycosides that were subjected to protecting group manipulations to give glycosyl acceptors for the chemical installation of a sulfated glucuronic acid moiety. A late-stage conversion of the thioglycosides into anomeric fluorides made it possible to enzymatically introduce sphingosine. The resulting compounds were acetylated to provide 3-sulfo-glucuronyl- and glucuronyl-containing glycosphingolipids, respectively. The glycosphingolipids were employed to remodel the surface of erythrocytes to examine complement mediated toxicity by an anti-HNK-1 antibody. It was found that erythrocytes remodeled with exogenous administered HNK-1 containing glycosphingolipid undergo complement dependent lysis when incubated with an IgM anti-CD57 IgM antibody, whereas a compound lacking a sulfate was not able to induce this effect. The approach could be extended to the gangliosides GM1a and GD1a, which have been implicated in Guillain-Barre syndrome. The results highlight that cell surface remodeling will be attractive for diagnosis, disease monitoring and immunological research of diseases associated with pathogenic antibodies targeting glycosphingolipids. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/642389v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1c5a4e9org.highwire.dtl.DTLVardef@cddba7org.highwire.dtl.DTLVardef@10fc7eborg.highwire.dtl.DTLVardef@10e5678_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗