Spike protein VP8* of human rotavirus recognizes histo-blood group antigens in a type-specific manner.

Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Le(b)) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Le(b) and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells.

Effect of grape seed extract on human norovirus GII.4 and murine norovirus 1 in viral suspensions, on stainless steel discs, and in lettuce wash water.

The anti-norovirus (anti-NoV) effect of grape seed extract (GSE) was examined by plaque assay for murine norovirus 1 (MNV-1), cell-binding reverse transcription-PCR for human NoV GII.4, and saliva-binding enzyme-linked immunosorbent assay for human NoV GII.4 P particles, with or without the presence of interfering substances (dried milk and lettuce extract). GSE at 0.2 and 2 mg/ml was shown to reduce the infectivity of MNV-1 (>3-log PFU/ml) and the specific binding ability of NoV GII.4 to Caco-2 cells (>1-log genomic copies/ml), as well as of its P particles to salivary human histo-blood group antigen receptors (optical density at 450 nm of >0.8). These effects were decreased as increasing concentrations of dried milk (0.02 and 0.2%) or lettuce extract were added. Under an electron microscope, human NoV GII.4 virus-like particles showed inflation and deformation after treatment with GSE. Under conditions that simulated applications in the food industry, the anti-NoV effect of GSE using MNV-1 as a target organism was shown to be limited in surface disinfection (<1-log PFU/ml, analyzed in accordance with EN 13697:2001). However, a 1.5- to 2-log PFU/ml reduction in MNV-1 infectivity was noted when 2 mg of GSE/ml was used to sanitize water in the washing bath of fresh-cut lettuce, and this occurred regardless of the chemical oxygen demand (0 to 1,500 mg/ml) of the processing water.

Genetic and phenotypic characterization of GII-4 noroviruses that circulated during 1987 to 2008.

The predominance and continual emergence of new variants in GII-4 noroviruses (NVs) in recent years have raised questions about the role of host immunity and histo-blood group antigens (HBGAs) in NV evolution. To address these questions, we performed a genetic and phenotypic characterization of GII-4 variants circulating in the past decade (1998 to 2008). Ninety-three GII-4 sequences were analyzed, and of them, 16 strains representing 6 genetic clusters were selected for further characterization. The HBGA binding properties were determined by both saliva- and oligosaccharide-binding assays using P particles as a model of NV capsid. The antigenic properties were also examined by enzyme immunoassay (EIA), Western blot analysis, and receptor blocking assay, using P-particle-specific antibodies from immunized mice and GII-4 virus-infected patients. Our results showed that 15 of the 16 GII-4 viruses bound to saliva of all A, B, and O secretors. Oligosaccharide binding assays yielded largely consistent results, although the binding affinities to some oligosaccharides varied among some strains. The only nonbinder had a mutation in the binding site. While antigenic variations were detected among the 16 strains, significant cross-blocking on the HBGA binding was also noted. Sequence alignment revealed high conservation of HBGA binding interfaces with some variations in adjacent regions. Taken together, our data suggested that the ability of GII-4 to recognize different secretor HBGAs persisted over the past decade, which may explain the predominance of GII-4 over other genotypes. Our data also indicated that both the host immunity and HBGAs play a role in NV evolution. While host immunity may continue driving NV for antigenic change, the functional selection by the HBGAs tends to lock the architecture of the capsid/HBGA interfaces and allows only limited variations outside the HBGA binding sites. A potential outcome of such counterselection between theses two factors in NV evolution is discussed.

Saliva as a source of reagent to study human susceptibility to avian influenza H7N9 virus infection.

Avian influenza H7N9 viruses are an important public health concern due to their high mortality rate and potentials for future pandemics. We investigated human susceptibility to H7N9 viruses using recombinant H7N9 hemagglutinin (HA) proteins as a probe and found a strong association between H7N9 infections and HA binding among saliva samples from 32 patients and 60 uninfected controls in Jiangsu province, China, during the 2016 epidemic season. We also found that sialyl Lex (SLex) antigen that was recognized by H7N9 HA was associated with H7N9 virus infection. Further analysis suggested that additional saccharide residues adjacent to the SLex moiety may affect the H7N9-binding specificity. Our data suggested that saliva may be a useful reagent to study human susceptibility to avian influenza H7N9 virus, which may impact the disease control and prevention of avian influenza viruses as important human pathogens.

Two gastroenteritis outbreaks caused by GII Noroviruses: host susceptibility and HBGA phenotypes.

Noroviruses (NoVs) cause epidemic acute gastroenteritis, in which histo-blood group antigens (HBGAs) may play an important role in the host susceptibility. To further explore this issue, two outbreaks of acute gastroenteritis caused by a GII.4 and a GII.3 NoV, respectively, in China in 2009 were studied. Stool and saliva samples from symptomatic patients and water samples from the outbreak facilities were collected. RT-PCR showed that 23 out of 33 (GII.4 outbreak) and 12 out of 13 (GII.3outbreak) stool samples were NoV positive. For the GII.4 outbreak the NoV sequences of stool and water samples were from an identical GII.4 strain, while the same GII.3 NoV sequences were found in five stool samples from the GII.3 outbreak. The HBGA phenotypes (A, B, Le(a), Le(b), Le(x), and Le(y)) of all saliva samples were determined, which revealed both secretors and nonsecretors in the symptomatic groups of the two outbreaks. In the GII.3 outbreak, type O individuals appeared less susceptible, while the type A may be more at risk of infection. However, No preference of HBGAs was observed in the GII.4 outbreak. The observation that nonsecretors were infected in both outbreaks differed from the previous results that nonsecretors are resistant to these two GII NoVs.

Norovirus and histo-blood group antigens: demonstration of a wide spectrum of strain specificities and classification of two major binding groups among multiple binding patterns.

Noroviruses, an important cause of acute gastroenteritis, have been found to recognize human histo-blood group antigens (HBGAs) as receptors. Four strain-specific binding patterns to HBGAs have been described in our previous report. In this study, we have extended the binding patterns to seven based on 14 noroviruses examined. The oligosaccharide-based assays revealed additional epitopes that were not detected by the saliva-based assays. The seven patterns have been classified into two groups according to their interactions with three major epitopes (A/B, H, and Lewis) of human HBGAs: the A/B-binding group and the Lewis-binding group. Strains in the A/B binding group recognize the A and/or B and H antigens, but not the Lewis antigens, while strains in the Lewis-binding group react only to the Lewis and/or H antigens. This classification also resulted in a model of the norovirus/HBGA interaction. Phylogenetic analyses showed that strains with identical or closely related binding patterns tend to be clustered, but strains in both binding group can be found in both genogroups I and II. Our results suggest that noroviruses have a wide spectrum of host range and that human HBGAs play an important role in norovirus evolution. The high polymorphism of the human HBGA system, the involvement of multiple epitopes, and the typical protein/carbohydrate interaction between norovirus VLPs and HBGAs provide an explanation for the virus-ligand binding diversities.

Human milk contains elements that block binding of noroviruses to human histo-blood group antigens in saliva.

Noroviruses (NVs) recognize human histo-blood group antigens (HBGAs) as receptors. We characterized the interaction of human milk samples with recombinant virus-like particles representing VA387, Norwalk, VA207, and MOH. Milk samples from 60 healthy women were tested for human HBGAs and for their ability to block the binding of NVs. Fifty-four women were secretors (Se+), and 6 were nonsecretors (Se-). No women had detectable A or B antigens in their milk samples. All 54 Se+ milk samples, but 0 of 6 Se- milk samples, blocked VA387 and Norwalk virus (Se+ binders) from binding to saliva samples. All 6 Lewis-positive Se- milk samples blocked binding to VA207, and variable blocking activities were exhibited by the Se+ milk samples. No milk samples blocked the binding of MOH to A and B antigens. Secretor and Lewis, but not A or B antigens, were present in human milk and were responsible for blocking NV binding to receptors and therefore are likely to be decoy receptors that protect breast-fed infants from NV infection.

Noroviruses bind to human ABO, Lewis, and secretor histo-blood group antigens: identification of 4 distinct strain-specific patterns.

We characterized the binding of 8 Noroviruses (NORs) to histo-blood group antigens (HBGAs) in human saliva using recombinant NOR (rNOR) capsid proteins. Among the 8 rNORs tested, 6 formed viruslike particles (VLPs) when the capsid proteins were expressed in insect cells, all of which revealed variable binding activities with saliva; the remaining 2 rNORs did not form VLPs, and the proteins did not bind, or bound weakly, to saliva. Four distinct binding patterns were associated with different histo-blood types, defined by Lewis, secretor, and ABO types. Three patterns (VA387, NV, and MOH) recognized secretors, and 1 pattern (VA207) recognized Lewis-positive nonsecretors. The 3 secretor-recognizing patterns were defined as A/B (MOH), A/O (NV), and A/B/O (VA387) binders. Oligosaccharides containing the Lewis and ABH antigenic epitopes were involved in binding. Our findings suggest that different strains of NORs may recognize different human HBGAs on intestinal epithelial cells as receptors for infection.