Novel insights into the host cell glycan binding profile of human metapneumovirus.

Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galß1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates.IMPORTANCEAll cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease.

Antiviral strategies against human metapneumovirus: Targeting the fusion protein.

Human metapneumoviruses have emerged in the past decades as an important global pathogen that causes severe upper and lower respiratory tract infections. Children under the age of 2, the elderly and immunocompromised individuals are more susceptible to HMPV infection than the general population due to their suboptimal immune system. Despite the recent discovery of HMPV as a novel important respiratory virus, reports have rapidly described its epidemiology, biology, and pathogenesis. However, progress is still to be made in the development of vaccines and drugs against HMPV infection as none are currently available. Herein, we discuss the importance of HMPV and review the reported strategies for anti-HMPV drug candidates. We also present the fusion protein as a promising antiviral drug target due to its multiple roles in the HMPV lifecycle. This key viral protein has previously been targeted by a range of inhibitors, which will be discussed as they represent opportunities for future drug design.

Drug Repurposing for Therapeutic Discovery against Human Metapneumovirus Infection.

Human metapneumovirus (HMPV) is recognized as an important cause of pneumonia in infants, in the elderly, and in immunocompromised individuals worldwide. The absence of an antiviral treatment or vaccine strategy against HMPV infection creates a high burden on the global health care system. Drug repurposing has become increasingly attractive for the treatment of emerging and endemic diseases as it requires less research and development costs than traditional drug discovery. In this study, we developed an in vitro medium-throughput screening assay that allows for the identification of novel anti-HMPV drugs candidates. Out of ~2,400 compounds, we identified 11 candidates with a dose-dependent inhibitory activity against HMPV infection. Additionally, we further described the mode of action of five anti-HMPV candidates with low in vitro cytotoxicity. Two entry inhibitors, Evans Blue and aurintricarboxylic acid, and three post-entry inhibitors, mycophenolic acid, mycophenolate mofetil, and 2,3,4-trihydroxybenzaldehyde, were identified. Among them, the mycophenolic acid series displayed the highest levels of inhibition, due to the blockade of intracellular guanosine synthesis. Importantly, MPA has significant potential for drug repurposing as inhibitory levels are achieved below the approved human oral dose. Our drug-repurposing strategy proved to be useful for the rapid discovery of novel hit candidates to treat HMPV infection and provide promising novel templates for drug design.

Pneumococcal Phasevarions Control Multiple Virulence Traits, Including Vaccine Candidate Expression.

Streptococcus pneumoniae is the most common cause of bacterial illness worldwide. Current vaccines based on the polysaccharide capsule are only effective against a limited number of the >100 capsular serotypes. A universal vaccine based on conserved protein antigens requires a thorough understanding of gene expression in S. pneumoniae. All S. pneumoniae strains encode the SpnIII Restriction-Modification system. This system contains a phase-variable methyltransferase that switches specificity, and controls expression of multiple genes-a phasevarion. We examined the role of this phasevarion during pneumococcal pathobiology, and determined if phase variation resulted in differences in expression of currently investigated conserved protein antigens. Using locked strains that express a single methyltransferase specificity, we found differences in clinically relevant traits, including survival in blood, and adherence to and invasion of human cells. We also observed differences in expression of numerous proteinaceous vaccine candidates, which complicates selection of antigens for inclusion in a universal protein-based pneumococcal vaccine. This study will inform vaccine design against S. pneumoniae by ensuring only stably expressed candidates are included in a rationally designed vaccine. IMPORTANCE S. pneumoniae is the world's foremost bacterial pathogen. S. pneumoniae encodes a phasevarion (phase-variable regulon), that results in differential expression of multiple genes. Previous work demonstrated that the pneumococcal SpnIII phasevarion switches between six different expression states, generating six unique phenotypic variants in a pneumococcal population. Here, we show that this phasevarion generates multiple phenotypic differences relevant to pathobiology. Importantly, expression of conserved protein antigens varies with phasevarion switching. As capsule expression, a major pneumococcal virulence factor, is also controlled by the phasevarion, our work will inform the selection of the best candidates to include in a rationally designed, universal pneumococcal vaccine.

Characterization of the Phase-Variable Autotransporter Lav Reveals a Role in Host Cell Adherence and Biofilm Formation in Nontypeable Haemophilus influenzae.

Lav is an autotransporter protein found in pathogenic Haemophilus and Neisseria species. Lav in nontypeable Haemophilus influenzae (NTHi) is phase-variable: the gene reversibly switches ON-OFF via changes in length of a locus-located GCAA(n) simple DNA sequence repeat tract. The expression status of lav was examined in carriage and invasive collections of NTHi, where it was predominantly not expressed (OFF). Phenotypic study showed lav expression (ON) results in increased adherence to human lung cells and denser biofilm formation. A survey of Haemophilus species genome sequences showed lav is present in ∼60% of NTHi strains, but lav is not present in most typeable H. influenzae strains. Sequence analysis revealed a total of five distinct variants of the Lav passenger domain present in Haemophilus spp., with these five variants showing a distinct lineage distribution. Determining the role of Lav in NTHi will help understand the role of this protein during distinct pathologies.

A Portable Device for LAMP Based Detection of SARS-CoV-2.

This paper reports the design, development, and testing of a novel, yet simple and low-cost portable device for the rapid detection of SARS-CoV-2. The device performs loop mediated isothermal amplification (LAMP) and provides visually distinguishable images of the fluorescence emitted from the samples. The device utilises an aluminium block embedded with a cartridge heater for isothermal heating of the sample and a single-board computer and camera for fluorescence detection. The device demonstrates promising results within 20 min using clinically relevant starting concentrations of the synthetic template. Time-to-signal data for this device are considerably lower compared to standard quantitative Polymerase Chain Reaction(qPCR) machine (~10-20 min vs. >38 min) for 1 × 102 starting template copy number. The device in its fully optimized and characterized state can potentially be used as simple to operate, rapid, sensitive, and inexpensive platform for population screening as well as point-of-need severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) detection and patient management.

Multidisciplinary Approaches Identify Compounds that Bind to Human ACE2 or SARS-CoV-2 Spike Protein as Candidates to Block SARS-CoV-2-ACE2 Receptor Interactions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged virus that causes coronavirus infectious disease 2019 (COVID-19). SARS-CoV-2 spike protein, like SARS-CoV-1, uses the angiotensin converting enzyme 2 (ACE2) as a cellular receptor to initiate infection. Compounds that interfere with the SARS-CoV-2 spike protein receptor binding domain protein (RBD)-ACE2 receptor interaction may function as entry inhibitors. Here, we used a dual strategy of molecular docking and surface plasmon resonance (SPR) screening of compound libraries to identify those that bind to human ACE2 or the SARS-CoV-2 spike protein receptor binding domain (RBD). Molecular modeling screening interrogated 57,641 compounds and focused on the region of ACE2 that is engaged by RBD of the SARS-CoV-2 spike glycoprotein and vice versa. SPR screening used immobilized human ACE2 and SARS-CoV-2 Spike protein to evaluate the binding of these proteins to a library of 3,141 compounds. These combined screens identified compounds from these libraries that bind at KD (equilibrium dissociation constant) <3 µM affinity to their respective targets, 17 for ACE2 and 6 for SARS-CoV-2 RBD. Twelve ACE2 binders and six of the RBD binders compete with the RBD-ACE2 interaction in an SPR-based competition assay. These compounds included registered drugs and dyes used in biomedical applications. A Vero-E6 cell-based SARS-CoV-2 infection assay was used to evaluate infection blockade by candidate entry inhibitors. Three compounds demonstrated dose-dependent antiviral in vitro potency-Evans blue, sodium lifitegrast, and lumacaftor. This study has identified potential drugs for repurposing as SARS-CoV-2 entry inhibitors or as chemical scaffolds for drug development.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, has caused more than 60 million cases worldwide with almost 1.5 million deaths as of November 2020. Repurposing existing drugs is the most rapid path to clinical intervention for emerging diseases. Using an in silico screen of 57,641 compounds and a biophysical screen of 3,141 compounds, we identified 22 compounds that bound to either the angiotensin converting enzyme 2 (ACE2) and/or the SARS-CoV-2 spike protein receptor binding domain (SARS-CoV-2 spike protein RBD). Nine of these drugs were identified by both screening methods. Three of the identified compounds, Evans blue, sodium lifitegrast, and lumacaftor, were found to inhibit viral replication in a Vero-E6 cell-based SARS-CoV-2 infection assay and may have utility as repurposed therapeutics. All 22 identified compounds provide scaffolds for the development of new chemical entities for the treatment of COVID-19.

Targeting Human Parainfluenza Virus Type-1 Haemagglutinin-Neuraminidase with Mechanism-Based Inhibitors.

Human parainfluenza virus (hPIV) infections are a major cause of respiratory tract illnesses in children, with currently no available vaccine or drug treatment. The surface glycoprotein haemagglutinin-neuraminidase (HN) of hPIV has a central role in the viral life cycle, including neuraminic acid-recognising receptor binding activity (early stage) and receptor-destroying activity (late stage), which makes it an ideal target for antiviral drug disovery. In this study, we showed that targeting the catalytic mechanism of hPIV-1 HN by a 2α,3ß-difluoro derivative of the known hPIV-1 inhibitor, BCX 2798, produced more potent inhibition of the neuraminidase function which is reflected by a stronger inhibition of viral replication. The difluorosialic acid-based inhibitor efficiently blocked the neuraminidase activity of HN for a prolonged period of time relative to its unsaturated neuraminic acid (Neu2en) analogue, BCX 2798 and produced a more efficient inhibition of the HN neuraminidase activity as well as in vitro viral replication. This prolonged inhibition of the hPIV-1 HN protein suggests covalent binding of the inhibitor to a key catalytic amino acid, making this compound a new lead for a novel class of more potent hPIV-1 mechanism-based inhibitors.

New antiviral approaches for human parainfluenza: Inhibiting the haemagglutinin-neuraminidase.

Human parainfluenza viruses cause acute respiratory tract infections and disease predominantly in young children and immunocompromised individuals. Currently, there are no vaccines to prevent hPIV infections, nor licensed anti-hPIV drugs. There is therefore a need for specific antiviral therapies to decrease the morbidity and mortality associated with hPIV diseases. Haemagglutinin-neuraminidase (HN) is one of two hPIV surface proteins with critical roles in host receptor recognition, binding and cleavage; it has been explored as a key drug development target for the past few decades with variable success. Recent advancements in computational modelling and the availability of the X-ray crystal structure of hPIV3 HN have improved our understanding of the structural and mechanistic features of HN. This review explores structural features of the HN protein that are being exploited for structure-guided inhibitor design. We describe past and present hPIV HN inhibition strategies based on sialic acid scaffolds, together with other novel approaches that decrease hPIV infectivity. Although many HN inhibitors have been developed and evaluated as anti-hPIV agents, currently only a host-directed therapy (DAS181) has succeeded in phase II clinical drug trials. Hence, the review concludes with future considerations for targeting the specific function(s) of hPIV HN and suggestions for antiviral drug design.

Structural Insights into Human Parainfluenza Virus 3 Hemagglutinin-Neuraminidase Using Unsaturated 3- N-Substituted Sialic Acids as Probes.

A novel approach to human parainfluenza virus 3 (hPIV-3) inhibitor design has been evaluated by targeting an unexplored pocket within the active site region of the hemagglutinin-neuraminidase (HN) of the virus that is normally occluded upon ligand engagement. To explore this opportunity, we developed a highly efficient route to introduce nitrogen-based functionalities at the naturally unsubstituted C-3 position on the neuraminidase inhibitor template N-acyl-2,3-dehydro-2-deoxy-neuraminic acid ( N-acyl-Neu2en), via a regioselective 2,3-bromoazidation. Introduction of triazole substituents at C-3 on this template provided compounds with low micromolar inhibition of hPIV-3 HN neuraminidase activity, with the most potent having 48-fold improved potency over the corresponding C-3 unsubstituted analogue. However, the C-3-triazole N-acyl-Neu2en derivatives were significantly less active against the hemagglutinin function of the virus, with high micromolar IC50 values determined, and showed insignificant in vitro antiviral activity. Given the different pH optima of the HN protein's neuraminidase (acidic pH) and hemagglutinin (neutral pH) functions, the influence of pH on inhibitor binding was examined using X-ray crystallography and STD NMR spectroscopy, providing novel insights into the multifunctionality of hPIV-3 HN. While the 3-phenyltriazole- N-isobutyryl-Neu2en derivative could bind HN at pH 4.6, suitable for neuraminidase inhibition, at neutral pH binding of the inhibitor was substantially reduced. Importantly, this study clearly demonstrates for the first time that potent inhibition of HN neuraminidase activity is not necessarily directly correlated with a strong antiviral activity, and suggests that strong inhibition of the hemagglutinin function of hPIV HN is crucial for potent antiviral activity. This highlights the importance of designing hPIV inhibitors that primarily target the receptor-binding function of hPIV HN.

The impact of the butterfly effect on human parainfluenza virus haemagglutinin-neuraminidase inhibitor design.

Human parainfluenza viruses represent a leading cause of lower respiratory tract disease in children, with currently no available approved drug or vaccine. The viral surface glycoprotein haemagglutinin-neuraminidase (HN) represents an ideal antiviral target. Herein, we describe the first structure-based study on the rearrangement of key active site amino acid residues by an induced opening of the 216-loop, through the accommodation of appropriately functionalised neuraminic acid-based inhibitors. We discovered that the rearrangement is influenced by the degree of loop opening and is controlled by the neuraminic acid's C-4 substituent's size (large or small). In this study, we found that these rearrangements induce a butterfly effect of paramount importance in HN inhibitor design and define criteria for the ideal substituent size in two different categories of HN inhibitors and provide novel structural insight into the druggable viral HN protein.

Exploring inhibitor structural features required to engage the 216-loop of human parainfluenza virus type-3 hemagglutinin-neuraminidase.

Human parainfluenza virus type-3 is a leading cause of acute respiratory infection in infants and children. There is currently neither vaccine nor clinically effective treatment for parainfluenza virus infection. Hemagglutinin-neuraminidase glycoprotein is a key protein in viral infection, and its inhibition has been a target for inhibitor development. In this study, we explore the structural features required for Neu2en derivatives to efficiently lock-open the 216-loop of the human parainfluenza virus type-3 hemagglutinin-neuraminidase protein.

A dual drug regimen synergistically blocks human parainfluenza virus infection.

Human parainfluenza type-3 virus (hPIV-3) is one of the principal aetiological agents of acute respiratory illness in infants worldwide and also shows high disease severity in the elderly and immunocompromised, but neither therapies nor vaccines are available to treat or prevent infection, respectively. Using a multidisciplinary approach we report herein that the approved drug suramin acts as a non-competitive in vitro inhibitor of the hPIV-3 haemagglutinin-neuraminidase (HN). Furthermore, the drug inhibits viral replication in mammalian epithelial cells with an IC50 of 30 µM, when applied post-adsorption. Significantly, we show in cell-based drug-combination studies using virus infection blockade assays, that suramin acts synergistically with the anti-influenza virus drug zanamivir. Our data suggests that lower concentrations of both drugs can be used to yield high levels of inhibition. Finally, using NMR spectroscopy and in silico docking simulations we confirmed that suramin binds HN simultaneously with zanamivir. This binding event occurs most likely in the vicinity of the protein primary binding site, resulting in an enhancement of the inhibitory potential of the N-acetylneuraminic acid-based inhibitor. This study offers a potentially exciting avenue for the treatment of parainfluenza infection by a combinatorial repurposing approach of well-established approved drugs.

The catalytic mechanism of human parainfluenza virus type†3 haemagglutinin-neuraminidase revealed.

Human parainfluenza virus type 3 (hPIV-3) is one of the leading causes for lower respiratory tract disease in children, with neither an approved antiviral drug nor vaccine available to date. Understanding the catalytic mechanism of human parainfluenza virus haemagglutinin-neuraminidase (HN) protein is key to the design of specific inhibitors against this virus. Herein, we used (1) H NMR spectroscopy, X-ray crystallography, and virological assays to study the catalytic mechanism of the HN enzyme activity and have identified the conserved Tyr530 as a key amino acid involved in catalysis. A novel 2,3-difluorosialic acid derivative showed prolonged enzyme inhibition and was found to react and form a covalent bond with Tyr530. Furthermore, the novel derivative exhibited enhanced potency in virus blockade assays relative to its Neu2en analogue. These outcomes open the door for a new generation of potent inhibitors against hPIV-3 HN.

Structure-guided discovery of potent and dual-acting human parainfluenza virus haemagglutinin-neuraminidase inhibitors.

Human parainfluenza viruses (hPIVs) cause upper and lower respiratory tract disease in children that results in a significant number of hospitalizations and impacts health systems worldwide. To date, neither antiviral drugs nor vaccines are approved for clinical use against parainfluenza virus, which reinforces the urgent need for new therapeutic discovery strategies. Here we use a multidisciplinary approach to develop potent inhibitors that target a structural feature within the hPIV type 3 haemagglutinin-neuraminidase (hPIV-3 HN). These dual-acting designer inhibitors represent the most potent designer compounds and efficiently block both hPIV cell entry and virion progeny release. We also define the binding mode of these inhibitors in the presence of whole-inactivated hPIV and recombinantly expressed hPIV-3 HN by Saturation Transfer Difference NMR spectroscopy. Collectively, our study provides an antiviral preclinical candidate and a new direction towards the discovery of potential anti-parainfluenza drugs.

Exploring human parainfluenza virus type-1 hemagglutinin-neuraminidase as a target for inhibitor discovery.

Human parainfluenza virus type 1 is the major cause of croup in infants and young children. There is currently neither vaccine nor clinically effective treatment for parainfluenza virus infection. Hemagglutinin-neuraminidase glycoprotein is a key protein in viral infection, and its inhibition has been a target for 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid (Neu5Ac2en)-based inhibitor development. In this study, we explore the effect of C-5 modifications on the potency of Neu5Ac2en derivatives that target the human parainfluenza type-1 hemagglutinin-neuraminidase protein. Our study demonstrates that the replacement of the Neu5Ac2en C-5 acetamido moiety with more hydrophobic alkane-based moieties improves the inhibitory potency for both hemagglutinin-neuraminidase functions. These findings shed light on the importance of C-5 substitution on Neu5Ac2en in the design of novel sialic acid-based inhibitors that target human parainfluenza type-1 hemagglutinin-neuraminidase.

The approved pediatric drug suramin identified as a clinical candidate for the treatment of EV71 infection-suramin inhibits EV71 infection in vitro and in vivo.

Enterovirus 71 (EV71) causes severe central nervous system infections, leading to cardiopulmonary complications and death in young children. There is an urgent unmet medical need for new pharmaceutical agents to control EV71 infections. Using a multidisciplinary approach, we found that the approved pediatric antiparasitic drug suramin blocked EV71 infectivity by a novel mechanism of action that involves binding of the naphtalentrisulonic acid group of suramin to the viral capsid. Moreover, we demonstrate that when suramin is used in vivo at doses equivalent to or lower than the highest dose already used in humans, it significantly decreased mortality in mice challenged with a lethal dose of EV71 and peak viral load in adult rhesus monkeys. Thus, suramin inhibits EV71 infection by neutralizing virus particles prior to cell attachment. Consequently, these findings identify suramin as a clinical candidate for further development as a therapeutic or prophylactic treatment for severe EV71 infection.

Infection-associated FUT2 (Fucosyltransferase 2) genetic variation and impact on functionality assessed by in vivo studies.

The secretor (Se)/nonsecretor (se) histo-blood group variation depends on the action of the FUT2 enzyme and has major implications for human susceptibility to infections. To characterize the functionality of FUT2 variants, we assessed the correlation between saliva phenotypes and sequence variation at the FUT2 gene in sixty seven individuals from northern Portugal. While most non-secretor haplotypes were found to carry the 428G > A nonsense mutation in association with a 739G > A missense substitution, we have also identified a recombinant haplotype carrying the 739*A allele together with the efficient 428*G variant in individuals with the Se phenotype. This finding suggested, in contrast to previous results, that the 739*A allele encodes an efficient Se allele. To test this hypothesis we evaluated the in vivo enzyme activity of full coding expression constructs in transient transfection of CHO-K1 cells using FACS (fluorescence-activated cell sorting) analysis and expression of type 2 and type 3 chain H structures as read out. We detected FUT2 activity for the 739*A expression construct, demonstrating that the 739G > A substitution is indeed not inactivating. In accordance with the hypothesis that FUT2 is under long standing balancing selection, we estimated that the time depth of FUT2 global genetic variation is as old as 3 million years. Age estimates of specific variants suggest that the 428G > A mutation occurred at least 1.87 million years ago while the 739G > A substitution is about 816,000 years old. The 385A > T missense mutation underlying the non-secretor phenotype in East Asians appears to be more recent and is likely to have occurred about 256,000 years ago.

Widespread gene conversion of alpha-2-fucosyltransferase genes in mammals.

The alpha-2-fucosyltransferases (alpha2FTs) are enzymes involved in the biosynthesis of alpha2fucosylated glycan structures. In mammalian genomes, there are three alpha2FT genes located in tandem-FUT1, FUT2, and Sec1-each contained within a single exon. It has been suggested that these genes originated from two successive duplications, with FUT1 being generated first and FUT2 and Sec1 second. Despite gene conversion being considered the main mechanism of concerted evolution in gene families, previous studies of primates alpha2FTs failed to detect it, although the occurrence of gene conversion between FUT2 and Sec1 was recently reported in a human allele. The primary aim of our work was to initiate a broader study on the molecular evolution of mammalian alpha2FTs. Sequence comparison leads us to confirm that the three genes appeared by two rounds of duplication. In addition, we were able to detect multiple gene-conversion events at the base of primates and within several nonprimate species involving FUT2 and Sec1. Gene conversion involving FUT1 and either FUT2 or Sec1 was also detected in rabbit. The extent of gene conversion between the alpha2FTs genes appears to be species-specific, possibly related to functional differentiation of these genes. With the exception of rabbits, gene conversion was not observed in the region coding the C-terminal part of the catalytic domain. In this region, the number of amino acids that are identical between FUT1 and FUT2, but different in Sec1, is higher than in other parts of the protein. The biologic meaning of this observation may be related to functional constraints.

Association between expression of the H histo-blood group antigen, alpha1,2fucosyltransferases polymorphism of wild rabbits, and sensitivity to rabbit hemorrhagic disease virus.

RHDV (rabbit hemorrhagic disease virus) is a highly virulent calicivirus that has become a major cause of mortality in wild rabbit populations (Oryctolagus cuniculus). It binds to the histo-blood group antigen (HBGA) H type 2 which requires an alpha1,2fucosyltransferase for its synthesis. In rabbit, three alpha1,2fucosyltransferases genes are known, Fut1, Fut2, and Sec1. Nonfunctional alleles at any of these loci could potentially confer resistance to RHDV, similar to human FUT2 alleles that determine the nonsecretor phenotype and resistance to infection by various NoV strains. In this study, we looked for the presence of H type 2 on buccal epithelial cells of wild rabbits from two geographic areas under RHDV pressure and from one RHDV-free area. Some animals with diminished H type 2 expression were found in the three populations (nonsecretor-like phenotype). Their frequency markedly increased according to the RHDV impact, suggesting that outbreaks selected survivors with low expression of the virus ligand. Polymorphisms of the Fut1, Fut2, and Sec1 coding regions were determined among animals that either died or survived outbreaks. The Fut2 and Sec1 genes presented a high polymorphism and the frequency of one Sec1 allele was significantly elevated, over 6-fold, among survivors. Sec1 enzyme variants showed either moderate, low, or undetectable catalytic activity, whereas all variant Fut2 enzymes showed strong catalytic activity. This functional analysis of the enzymes encoded by each Fut2 and Sec1 allele suggests that the association between one Sec1 allele and survival might be explained by a deficit of alpha1,2fucosyltransferase expression rather than by impaired catalytic activity.