Species-Specific N-Glycomes and Methylation Patterns of Oysters Crassostrea gigas and Ostrea edulis and Their Possible Consequences for the Norovirus-HBGA Interaction.

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles.

Macrobrachium rosenbergii nodavirus virus-like particles attach to fucosylated glycans in the gills of the giant freshwater prawn.

The Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white-tail disease (WTD) in many species of shrimp and prawn, has been shown to infect hemocytes and tissues such as the gills and muscles. However, little is known about the host surface molecules to which MrNV attach to initiate infection. Therefore, the present study investigated the role of glycans as binding molecules for virus attachment in susceptible tissues such as the gills. We established that MrNV in their virus-like particle (MrNV-VLP) form exhibited strong binding to gill tissues and lysates, which was highly reduced by the glycan-reducing periodate and PNGase F. The broad, fucose-binding Aleuria Aurantia lectin (AAL) highly reduced MrNV-VLPs binding to gill tissue sections and lysates, and efficiently disrupted the specific interactions between the VLPs and gill glycoproteins. Furthermore, mass spectroscopy revealed the existence of unique fucosylated LacdiNAc-extended N-linked and O-linked glycans in the gill tissues, whereas beta-elimination experiments showed that MrNV-VLPs demonstrated a binding preference for N-glycans. Therefore, the results from this study highly suggested that MrNV-VLPs preferentially attach to fucosylated N-glycans in the susceptible gill tissues, and these findings could lead to the development of strategies that target virus-host surface glycan interactions to reduce MrNV infections.

Host-Range Shift Between Emerging P[8]-4 Rotavirus and Common P[8] and P[4] Strains.

In Tunisia, we observed that rotavirus P[8]-3 and P[4] strains in young children with gastroenteritis associate with secretor histo-blood group phenotype. In contrast, the emerging P[8]-4 strain, representing 10% of cases, was exclusively found in nonsecretor patients. Unlike VP8* from P[8]-3 and P[4] strains, the P[8]-4 VP8* protein attached to glycans from saliva samples regardless of the donor's secretor status. Interestingly, a high frequency of FUT2 enzyme deficiency (nonsecretor phenotype) was observed in the population. This may allow cocirculation of P[8]-3 and P[8]-4 strains in secretor and nonsecretor children, respectively.

Fondness for sugars of enteric viruses confronts them with human glycans genetic diversity.

Together, norovirus and rotavirus are responsible for the majority of gastroenteritis cases worldwide, leading to a large number of deaths of children in low-income countries. Both attach to glycans of the histo-blood group antigen type (HBGAs) widely expressed in the digestive tract of vertebrates, albeit with interspecies differences. In humans, their synthesis is performed by glycosyltransferases encoded by the highly polymorphic ABO, FUT2 and FUT3 genes that are under long-term balanced selection. The combination of functional and null or weak alleles at these loci provides a diversity of glycan structures that define the ABO, Secretor and Lewis phenotypes. At the initial stage of infection norovirus and rotavirus attach to these glycans, although distinct strains of each virus present different specificities for individual glycans, hence exhibiting preferences for different human phenotypes. Absence or low expression of the recognized glycan motifs due to genetic polymorphism is associated with resistance to the disease, showing that the HBGA polymorphisms provide a population-based innate protection. Epidemiologically dominant strains of either norovirus or rotavirus display specificity for glycan motifs present in large fractions of the population, which may differ between geographical areas in accordance with the frequency of the ABO, FUT2, FUT3 gene polymorphisms. Evidence for virus adaptation to these geographical differences is amounting, indicative of a host-pathogen co-evolution and suggesting that enteric pathogens such as norovirus and rotavirus are likely the driving forces behind the balanced HBGA polymorphisms.

The Coxsackievirus and Adenovirus Receptor, a Required Host Factor for Recovirus Infection, Is a Putative Enteric Calicivirus Receptor.

Human norovirus (HuNoV) is a leading cause of acute gastroenteritis in both developed and developing countries. Studies of HuNoV host cell interactions are limited by the lack of a simple, robust cell culture system. Due to their diverse HuNoV-like biological features, including histo-blood group antigen (HBGA) binding, rhesus enteric caliciviruses (ReCVs) are viable surrogate models for HuNoVs. In addition, several ReCV strains can be propagated to high titers in standard nonhuman primate cell lines while causing lytic infection and cell death. To identify the ReCV entry receptor, we performed CRISPR/Cas9 library screening in Vero cells, which identified the coxsackievirus and adenovirus receptor (CAR) as a candidate ReCV entry receptor. We showed that short interfering RNA, anti-human CAR (hCAR) monoclonal antibody RmcB treatment, and recombinant hCAR ectodomain blocked ReCV replication in LLC-MK2 cells. CRISPR/Cas9-targeted knockout of CAR in LLC-MK2 and Vero cells made these cell lines resistant to ReCV infection, and susceptibility to infection could be restored by transient expression of CAR. CHO cells do not express CAR or HBGAs and are resistant to ReCV infection. Recombinant CHO cells stably expressing hCAR or the type B HBGA alone did not support ReCV infection. However, CHO cells expressing both hCAR and the type B HBGA were susceptible to ReCV infection. In summary, we have demonstrated that CAR is required for ReCV infection and most likely is a functional ReCV receptor, but HBGAs are also necessary for infection.IMPORTANCE Because of the lack of a simple and robust human norovirus (HuNoV) cell culture system surrogate, caliciviruses still represent valuable research tools for norovirus research. Due to their remarkable biological similarities to HuNoVs, including the utilization of HBGAs as putative attachment receptors, we used rhesus enteric caliciviruses (ReCVs) to study enteric calicivirus host cell interactions. Using CRISPR/Cas9 library screening and functional assays, we identified and validated the coxsackievirus and adenovirus receptor (CAR) as a functional proteinaceous receptor for ReCVs. Our work demonstrated that CAR and HBGAs both are necessary to convert a nonsusceptible cell line to being susceptible to ReCV infection. Follow-up studies to evaluate the involvement of CAR in HuNoV infections are ongoing.

Host-Specific Glycans Are Correlated with Susceptibility to Infection by Lagoviruses, but Not with Their Virulence.

Rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV) are two lagoviruses from the family Caliciviridae that cause fatal diseases in two leporid genera, Oryctolagus and Lepus, respectively. In the last few years, several examples of host jumps of lagoviruses among leporids were recorded. In addition, a new pathogenic genotype of RHDV emerged, and many nonpathogenic strains of lagoviruses have been described. The molecular mechanisms behind host shifts and the emergence of virulence are unknown. Since RHDV uses glycans of the histo-blood group antigen type as attachment factors to initiate infection, we studied if glycan specificities of the new pathogenic RHDV genotype, nonpathogenic lagoviruses, and EBHSV potentially play a role in determining the host range and virulence of lagoviruses. We observed binding to A, B, or H antigens of the histo-blood group family for all strains known to primarily infect European rabbits (Oryctolagus cuniculus), which have recently been classified as GI strains. However, we could not explain the emergence of virulence, since similar glycan specificities were found in several pathogenic and nonpathogenic strains. In contrast, EBHSV, recently classified as GII.1, bound to terminal ß-linked N-acetylglucosamine residues of O-glycans. Expression of these attachment factors in the upper respiratory and digestive tracts in three lagomorph species (Oryctolagus cuniculus, Lepuseuropaeus, and Sylvilagus floridanus) showed species-specific patterns regarding susceptibility to infection by these viruses, indicating that species-specific glycan expression is likely a major contributor to lagovirus host specificity and range.IMPORTANCE Lagoviruses constitute a genus of the family Caliciviridae comprising highly pathogenic viruses, RHDV and EBHSV, that infect rabbits and hares, respectively. Recently, nonpathogenic strains were discovered and new pathogenic strains have emerged. In addition, host jumps between lagomorphs have been observed. The mechanisms responsible for the emergence of pathogenicity and host species range are unknown. Previous studies showed that RHDV strains attach to glycans expressed in the upper respiratory and digestive tracts of rabbits, the likely portals of virus entry. Here, we studied the glycan-binding properties of novel pathogenic and nonpathogenic strains looking for a link between glycan binding and virulence or between glycan specificity and host range. We found that glycan binding did not correlate with virulence. However, expression of glycan motifs in the upper respiratory and digestive tracts of lagomorphs revealed species-specific patterns associated with the host ranges of the virus strains, suggesting that glycan diversity contributes to lagovirus host ranges.

Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens.

Some viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs), and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract and are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to a large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked fucose and α1,3/4-linked fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines, and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2-linked fucose and α1,3/4-linked fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored.IMPORTANCE Enteric caliciviruses such as noroviruses, sapoviruses, and recoviruses are the most important etiological agents of severe acute gastroenteritis in humans and many other mammalian host species. They initiate infection by attachment to cell surface carbohydrate moieties, HBGAs, and/or terminal SAs. However, the attachment factor(s) for BNeV, a recently classified enteric calicivirus genus/type species, remains unexplored. Here, we demonstrate that BNeV VLPs have a wide spectrum of binding to synthetic HBGAs, bovine and human saliva samples, and bovine duodenal sections. We further discovered that α1,2-linked fucose and α1,3/4-linked fucose epitopes are essential for binding of BNeV VLPs. However, BNeV VLPs do not bind to terminal SAs on cell carbohydrates. Continued investigation regarding the proteinaceous receptor(s) will be necessary for better understanding of the tropism, pathogenesis, and host range of this important viral genus.

Alterations of the iNKT cell compartment in brain-injured patients.

BACKGROUND: Brain injury (BI) induces a state of immunodepression leading to pneumonia. We investigated the invariant natural killer T (iNKT) cell compartment. METHODS: This is an observational study in two surgical intensive care units (ICUs) of a single institution and a research laboratory. Clinical data and samples from a prospective cohort were extracted. Severe brain-injured patients (n = 33) and sex- and age-matched healthy donors (n = 40) were studied. RESULTS: We observed the presence of IL-10 in serum, a loss of IFN-γ and IL-13 production by peripheral blood mononuclear cells (PBMCs) following IL-2 stimulation, and downregulation of HLA-DR expression on both monocytes and B cells early after BI. Inversely, CD1d, the HLA class I-like molecule involved in antigen presentation to iNKT cells, was over-expressed on patients' monocytes and B cells. The antigen-presenting activity to iNKT cells of PBMCs was increased in the patients who developed pneumonia, but not in those who remained free of infection. Frequencies of iNKT cells among PBMCs were dramatically decreased in patients regardless of their infection status. Following amplification, an increased frequency of CD4+ iNKT cells producing IL-4 was noticed in the group of patients free of infection compared with those who became infected and with healthy donors. Finally, serum from BI patients inhibited the iNKT cells' specific response as well as the non-specific IL-2 stimulation of PBMCs, and the expression of the beta-2 adrenergic receptor was elevated at the surface of patients T lymphocytes. CONCLUSIONS: We observed severe alterations of the iNKT cell compartment, including the presence of inhibitory serum factors. We demonstrate for the first time that the decreased capacity to present antigens is not a generalized phenomenon because whereas the expression of HLA-DR molecules is decreased, the capacity for presenting glycolipids through CD1d expression is higher in patients.

Cell attachment protein VP8* of a human rotavirus specifically interacts with A-type histo-blood group antigen.

As with many other viruses, the initial cell attachment of rotaviruses, which are the major causative agent of infantile gastroenteritis, is mediated by interactions with specific cellular glycans. The distally located VP8* domain of the rotavirus spike protein VP4 (ref. 5) mediates such interactions. The existing paradigm is that 'sialidase-sensitive' animal rotavirus strains bind to glycans with terminal sialic acid (Sia), whereas 'sialidase-insensitive' human rotavirus strains bind to glycans with internal Sia such as GM1 (ref. 3). Although the involvement of Sia in the animal strains is firmly supported by crystallographic studies, it is not yet known how VP8* of human rotaviruses interacts with Sia and whether their cell attachment necessarily involves sialoglycans. Here we show that VP8* of a human rotavirus strain specifically recognizes A-type histo-blood group antigen (HBGA) using a glycan array screen comprised of 511 glycans, and that virus infectivity in HT-29 cells is abrogated by anti-A-type antibodies as well as significantly enhanced in Chinese hamster ovary cells genetically modified to express the A-type HBGA, providing a novel paradigm for initial cell attachment of human rotavirus. HBGAs are genetically determined glycoconjugates present in mucosal secretions, epithelia and on red blood cells, and are recognized as susceptibility and cell attachment factors for gastric pathogens like Helicobacter pylori and noroviruses. Our crystallographic studies show that the A-type HBGA binds to the human rotavirus VP8* at the same location as the Sia in the VP8* of animal rotavirus, and suggest how subtle changes within the same structural framework allow for such receptor switching. These results raise the possibility that host susceptibility to specific human rotavirus strains and pathogenesis are influenced by genetically controlled expression of different HBGAs among the world's population.

Proposal for a unified classification system and nomenclature of lagoviruses.

Lagoviruses belong to the Caliciviridae family. They were first recognized as highly pathogenic viruses of the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus) that emerged in the 1970-1980s, namely, rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), according to the host species from which they had been first detected. However, the diversity of lagoviruses has recently expanded to include new related viruses with varying pathogenicity, geographic distribution and host ranges. Together with the frequent recombination observed amongst circulating viruses, there is a clear need to establish precise guidelines for classifying and naming lagovirus strains. Therefore, here we propose a new nomenclature based on phylogenetic relationships. In this new nomenclature, a single species of lagovirus would be recognized and called Lagovirus europaeus. The species would be divided into two genogroups that correspond to RHDV- and EBHSV-related viruses, respectively. Genogroups could be subdivided into genotypes, which could themselves be subdivided into phylogenetically well-supported variants. Based on available sequences, pairwise distance cutoffs have been defined, but with the accumulation of new sequences these cutoffs may need to be revised. We propose that an international working group could coordinate the nomenclature of lagoviruses and any proposals for revision.

Neofunctionalization of the Sec1 α1,2fucosyltransferase paralogue in leporids contributes to glycan polymorphism and resistance to rabbit hemorrhagic disease virus.

RHDV (rabbit hemorrhagic disease virus), a virulent calicivirus, causes high mortalities in European rabbit populations (Oryctolagus cuniculus). It uses α1,2fucosylated glycans, histo-blood group antigens (HBGAs), as attachment factors, with their absence or low expression generating resistance to the disease. Synthesis of these glycans requires an α1,2fucosyltransferase. In mammals, there are three closely located α1,2fucosyltransferase genes rSec1, rFut2 and rFut1 that arose through two rounds of duplications. In most mammalian species, Sec1 has clearly become a pseudogene. Yet, in leporids, it does not suffer gross alterations, although we previously observed that rabbit Sec1 variants present either low or no activity. Still, a low activity rSec1 allele correlated with survival to an RHDV outbreak. We now confirm the association between the α1,2fucosyltransferase loci and survival. In addition, we show that rabbits express homogenous rFut1 and rFut2 levels in the small intestine. Comparison of rFut1 and rFut2 activity showed that type 2 A, B and H antigens recognized by RHDV strains were mainly synthesized by rFut1, and all rFut1 variants detected in wild animals were equally active. Interestingly, rSec1 RNA levels were highly variable between individuals and high expression was associated with low binding of RHDV strains to the mucosa. Co-transfection of rFut1 and rSec1 caused a decrease in rFut1-generated RHDV binding sites, indicating that in rabbits, the catalytically inactive rSec1 protein acts as a dominant-negative of rFut1. Consistent with neofunctionalization of Sec1 in leporids, gene conversion analysis showed extensive homogenization between Sec1 and Fut2 in leporids, at variance with its limited degree in other mammals. Gene conversion additionally involving Fut1 was also observed at the C-terminus. Thus, in leporids, unlike in most other mammals where it became extinct, Sec1 evolved a new function with a dominant-negative effect on rFut1, contributing to fucosylated glycan diversity, and allowing herd protection from pathogens such as RHDV.

Evidence for human norovirus infection of dogs in the United kingdom.

Human noroviruses (HuNoVs) are a major cause of viral gastroenteritis, with an estimated 3 million cases per year in the United Kingdom. HuNoVs have recently been isolated from pet dogs in Europe (M. Summa, C.-H. von Bonsdorff, and L. Maunula, J Clin Virol 53:244-247, 2012, http://dx.doi.org/10.1016/j.jcv.2011.12.014), raising concerns about potential zoonotic infections. With 31% of United Kingdom households owning a dog, this could prove to be an important transmission route. To examine this risk, canine tissues were studied for their ability to bind to HuNoV in vitro. In addition, canine stool samples were analyzed for the presence of viral nucleic acid, and canine serum samples were tested for the presence of anti-HuNoV antibodies. The results showed that seven different genotypes of HuNoV virus-like particles (VLPs) can bind to canine gastrointestinal tissue, suggesting that infection is at least theoretically possible. Although HuNoV RNA was not identified in stool samples from 248 dogs, serological evidence of previous exposure to HuNoV was obtained in 43/325 canine serum samples. Remarkably, canine seroprevalence for different HuNoV genotypes mirrored the seroprevalence in the human population. Though entry and replication within cells have not been demonstrated, the canine serological data indicate that dogs produce an immune response to HuNoV, implying productive infection. In conclusion, this study reveals zoonotic implications for HuNoV, and to elucidate the significance of this finding, further epidemiological and molecular investigations will be essential.

Genogroup IV and VI canine noroviruses interact with histo-blood group antigens.

UNLABELLED: Human noroviruses (HuNV) are a significant cause of viral gastroenteritis in humans worldwide. HuNV attaches to cell surface carbohydrate structures known as histo-blood group antigens (HBGAs) prior to internalization, and HBGA polymorphism among human populations is closely linked to susceptibility to HuNV. Noroviruses are divided into 6 genogroups, with human strains grouped into genogroups I (GI), II, and IV. Canine norovirus (CNV) is a recently discovered pathogen in dogs, with strains classified into genogroups IV and VI. Whereas it is known that GI to GIII noroviruses bind to HBGAs and GV noroviruses recognize terminal sialic acid residues, the attachment factors for GIV and GVI noroviruses have not been reported. This study sought to determine the carbohydrate binding specificity of CNV and to compare it to the binding specificities of noroviruses from other genogroups. A panel of synthetic oligosaccharides were used to assess the binding specificity of CNV virus-like particles (VLPs) and identified α1,2-fucose as a key attachment factor. CNV VLP binding to canine saliva and tissue samples using enzyme-linked immunosorbent assays (ELISAs) and immunohistochemistry confirmed that α1,2-fucose-containing H and A antigens of the HBGA family were recognized by CNV. Phenotyping studies demonstrated expression of these antigens in a population of dogs. The virus-ligand interaction was further characterized using blockade studies, cell lines expressing HBGAs, and enzymatic removal of candidate carbohydrates from tissue sections. Recognition of HBGAs by CNV provides new insights into the evolution of noroviruses and raises concerns regarding the potential for zoonotic transmission of CNV to humans. IMPORTANCE: Infections with human norovirus cause acute gastroenteritis in millions of people each year worldwide. Noroviruses can also affect nonhuman species and are divided into 6 different groups based on their capsid sequences. Human noroviruses in genogroups I and II interact with histo-blood group antigen carbohydrates, bovine noroviruses (genogroup III) interact with alpha-galactosidase (α-Gal) carbohydrates, and murine norovirus (genogroup V) recognizes sialic acids. The canine-specific strains of norovirus are grouped into genogroups IV and VI, and this study is the first to characterize which carbohydrate structures they can recognize. Using canine norovirus virus-like particles, this work shows that representative genogroup IV and VI viruses can interact with histo-blood group antigens. The binding specificity of canine noroviruses is therefore very similar to that of the human norovirus strains classified into genogroups I and II. This raises interesting questions about the evolution of noroviruses and suggests it may be possible for canine norovirus to infect humans.

Tulane Virus as a Potential Surrogate To Mimic Norovirus Behavior in Oysters.

Oyster contamination by noroviruses is an important health and economic problem. The present study aimed to compare the behaviors of Norwalk virus (the prototype genogroup I norovirus) and two culturable viruses: Tulane virus and mengovirus. After bioaccumulation, tissue distributions were quite similar for Norwalk virus and Tulane virus, with the majority of viral particles detected in digestive tissues, while mengovirus was detected in large amounts in the gills and mantle as well as in digestive tissues. The levels of persistence of all three viruses over 8 days were comparable, but clear differences were observed over longer periods, with Norwalk and Tulane viruses displaying rather similar half-lives, unlike mengovirus, which was cleared more rapidly. These results indicate that Tulane virus may be a good surrogate for studying norovirus behavior in oysters, and they confirm the prolonged persistence of Norwalk virus in oyster tissues.

Increase in genogroup II.4 norovirus host spectrum by CagA-positive Helicobacter pylori infection.

BACKGROUND: Noroviruses (NoVs) represent a considerable public health burden. Despite their enormous genetic diversity, most outbreaks are due to the single GII.4 genotype, but the reasons for this are poorly understood. NoVs use histo-blood group antigens (HBGAs) as attachment factors. Since HBGAs are present in saliva, binding of strains to saliva is commonly used as a surrogate for recognition of the gut surface by specific strains, although the relationship between saliva and gut tissue expression of HBGAs is not well defined. METHODS: The presence of fucosylated HBGAs in saliva and stomach biopsy specimens, as well as that of genogroup I.1 and genogroup II.4 virus-like particles, were compared in a series of 109 donors from Portugal. RESULTS: An overall good concordance between HBGA expression in saliva and stomach surface mucosa was observed. However, unexpected mucosal expression of α(1,2)fucosylated epitopes in nonsecretor individuals was frequently detected, allowing for GII.4 attachment. Although all individuals were infected with Helicobacter pylori, abnormal expression of α(1,2)fucosylated motifs and binding of GII.4 virus-like particles in nonsecretors' mucosa were associated with positivity for the H. pylori CagA virulence factor. CONCLUSIONS: Infection by CagA-positive H. pylori induces expression of GII.4 attachment factors in nonsecretors' mucosa, expanding the host range of these strains and thereby possibly contributing to their epidemiological dominance.

A FUT2 gene common polymorphism determines resistance to rotavirus A of the P[8] genotype.

Attachment to carbohydrates of the histo-blood group type of several human Rotavirus strains (RVA) has recently been described. Synthesis of these ligands requires a functional FUT2 enzyme, suggesting that FUT2 null homozygote (ie, nonsecretor) individuals may not be recognized by most human RVA strains. Whereas such individuals represent 20% of the control population, this retrospective study determined that none of 51 patients infected by P[8] rotavirus strains were nonsecretors. The lack of α1,2fucosylated carbohydrate motifs in the gut surface mucosa is thus associated with resistance to symptomatic infection and virus attachment to such motifs is essential to the infection process.

The VP8* domain of neonatal rotavirus strain G10P[11] binds to type II precursor glycans.

Naturally occurring bovine-human reassortant rotaviruses with a P[11] VP4 genotype exhibit a tropism for neonates. Interaction of the VP8* domain of the spike protein VP4 with sialic acid was thought to be the key mediator for rotavirus infectivity. However, recent studies have indicated a role for nonsialylated glycoconjugates, including histo-blood group antigens (HBGAs), in the infectivity of human rotaviruses. We sought to determine if the bovine rotavirus-derived VP8* of a reassortant neonatal G10P[11] virus interacts with hitherto uncharacterized glycans. In an array screen of >600 glycans, VP8* P[11] showed specific binding to glycans with the Galß1-4GlcNAc motif, which forms the core structure of type II glycans and is the precursor of H type II HBGA. The specificity of glycan binding was confirmed through hemagglutination assays; GST-VP8* P[11] hemagglutinates type O, A, and B red blood cells as well as pooled umbilical cord blood erythrocytes. Further, G10P[11] infectivity was significantly enhanced by the expression of H type II HBGA in CHO cells. The bovine-origin VP4 was confirmed to be essential for this increased infectivity, using laboratory-derived reassortant viruses generated from sialic acid binding rotavirus SA11-4F and a bovine G10P[11] rotavirus, B223. The binding to a core glycan unit has not been reported for any rotavirus VP4. Core glycan synthesis is constitutive in most cell types, and modification of these glycans is thought to be developmentally regulated. These studies provide the first molecular basis for understanding neonatal rotavirus infections, indicating that glycan modification during neonatal development may mediate the age-restricted infectivity of neonatal viruses.

Bioaccumulation efficiency, tissue distribution, and environmental occurrence of hepatitis E virus in bivalve shellfish from France.

Hepatitis E virus (HEV), an enteric pathogen of both humans and animals, is excreted by infected individuals and is therefore present in wastewaters and coastal waters. As bivalve molluscan shellfish are known to concentrate viral particles during the process of filter feeding, they may accumulate this virus. The bioaccumulation efficiencies of oysters (Crassostrea gigas), flat oysters (Ostrea edulis), mussels (Mytilus edulis), and clams (Ruditapes philippinarum) were compared at different time points during the year. Tissue distribution analysis showed that most of the viruses were concentrated in the digestive tissues of the four species. Mussels and clams were found to be more sensitive to sporadic contamination events, as demonstrated by rapid bioaccumulation in less than 1 h compared to species of oysters. For oysters, concentrations increased during the 24-h bioaccumulation period. Additionally, to evaluate environmental occurrence of HEV in shellfish, an environmental investigation was undertaken at sites potentially impacted by pigs, wild boars, and human waste. Of the 286 samples collected, none were contaminated with hepatitis E virus, despite evidence that this virus is circulating in some French areas. It is possible that the number of hepatitis E viral particles discharged into the environment is too low to detect or that the virus may have a very short period of persistence in pig manure and human waste.

Detection of RHDV strains in the Iberian hare (Lepus granatensis): earliest evidence of rabbit lagovirus cross-species infection.

Rabbit hemorrhagic disease virus (RHDV) is a highly lethal Lagovirus, family Caliciviridae, that threatens European rabbits (Oryctolagus cuniculus). Although a related virus severely affects hares, cross-species infection was only recently described for new variant RHDV in Cape hares (Lepus capensis mediterraneus). We sequenced two strains from dead Iberian hares (Lepus granatensis) collected in the 1990s in Portugal. Clinical signs were compatible with a Lagovirus infection. Phylogenetic analysis of the complete capsid gene positioned them in the RHDV genogroup that circulated on the Iberian Peninsula at that time. This is the earliest evidence of RHDV affecting a species other than European rabbits.