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

Abstract

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.

FIG. 1.

Binding of recombinant VLPs of 14 noroviruses to saliva samples from individuals with different HBGA types. Saliva samples were tested at dilutions of 1:1,000. The HBGA types of the individuals are shown at the bottoms of the panels (Non-sec, Non-secretors; O, type O secretors; A, type A secretors; B, type B secretors). Among the 14 strains compared, binding patterns of six strains (PiV, BUDS, HV, Boxer, OIF, and DSV) were characterized in this study. Four strains representing the four norovirus/HBGA binding patterns described previously are Norwalk, VA387, MOH, and VA207, respectively. Strain PiV was tested on a different set of saliva samples (52 in total) because of the consumption of the original set of the 81 saliva samples.

FIG. 2.

Blocking of VA387 binding to saliva by saliva. The standard saliva binding assays were performed except for one additional step of preincubation of the VA387 VLPs with blocking saliva. A set of five saliva samples with different HBGA types was used for the coating. The HBGA types of five saliva samples are shown, including type O, H positive; type O, Le^b and H positive; type O, Le^x and H positive; type A, H positive; and type A, Le^b and H positive. For blocking, another set of seven saliva samples was used. The HBGA types of seven saliva samples are shown on the x axis (from left to right): type O, Le^a positive (a+, O); type O, Le^x positive (x+, O); type O, H antigen positive (H+, O); type O, Le^b and H positive (b+H+, O); type A, H positive (H+, A); type A, Le^b and H positive (b+H+, A); and type B, H positive (H+, B). The levels of blocking (%) of the binding were calculated by the OD values between wells with or without preincubation with a saliva sample. The original OD values of VA387 to the five saliva samples without blocking were >3.0.

FIG. 3.

Phylogenetic tree and prediction of HBGA targets of noroviruses determined by binding and blocking experiments. The phylogenetic tree was constructed based on the amino acid sequences of the entire capsid genes using the UPGMA clustering method (MEGA v2.1) with Poisson correction distance calculations. Scale bar represents the phylogenetic distances expressed as units of expected amino acid substitutions per site. Bootstrap values are indicated as percentages of 125 replicates. Strains characterized in this study and strains representing the four previously described binding patterns are in bold. Strain SMV is characterized by Harrington et al. based on oligosaccharide binding assays (5). The potential HBGA targets for individual strains are shown on the right side of the panel. The binding results of each strain were assigned based on reactions in assays with saliva and/or oligosaccharide conjugates. “+” indicates a positive binding observed in any of the assays, “++, +++” indicate higher binding signals, and “−” indicates no binding.

FIG. 4.

A model of the norovirus/HBGA interaction. This model is developed based on the classification of two binding groups of norovirus to human HBGAs, the A/B binding group, and the Lewis binding group. The five-circle structure shown on the top right of the figure represents a pentasaccharide as the final product (ALe^b or BLe^b) of the human HBGAs. Thirteen norovirus strains are indicated according to their relative interaction levels with the A/B, H, and Lewis epitopes shown in Fig. 3. Strain SMV was reported to recognize the type B antigen (5). The potential binding sites for each of the three HBGA epitopes on the capsid are indicated. The specificities of individual binding domains may be different for the A and B epitopes, in the cases of Norwalk virus, C59, BUDS, and SMV, but the same shape of the binding sites was used for convenience.