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

VLPs of noroviruses.

VLPs of 14 strains representing 13 genetic clusters of norovirus produced from baculovirus were used in this study, as follows: in genogroup I, Boxer (AF538679), C59 (AF435807) (2), Norwalk virus (M87661) (12, 13), Desert Shield virus (DSV; U044469), and VA115 (AY038598), and in genogroup II, BUDS (AY660568), Grimsby (GrV; AJ004864) (4), Hawaii (HV; U07611) (16), Mexico (MxV; U22498) (11), MOH (AF397156) (1), Parris Island (PiV; AY652979) (22), VA387 (AY038600) (14), VA207 (AY038599) (14), and Operation Iraqi Freedom 031998 (OIF, AY675554) (22). Among them, Norwalk, VA387, MOH, and VA207 represent the four previously published receptor binding patterns (8). The procedures of production of norovirus VLPs in insect cell culture have been published previously (11, 12, 14). Briefly, a cDNA from the 3′ end of the genome containing the viral capsid gene (open reading frame 2 [ORF2]) was cloned from the viral RNA extracted from stool specimens. The recombinant baculoviruses carrying the viral capsid genes were constructed from the cloned cDNAs using the Bac to Bac expression system according to the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, CA). Norovirus VLPs were produced in Sf9 or H5 insect cell cultures. VLPs were partially purified by sucrose gradient centrifugation and stored at −70°C. Protein concentrations were determined by measuring the optical density at 280 nm (OD₂₈₀) and by comparison with a bovine serum albumin standard in a sodium dodecyl sulfate-polyacrylamide gel (21).

Binding of norovirus VLPs to saliva samples.

Saliva samples from healthy adult volunteers were collected under an approval of human subject research protocol by the Institutional Review Board at the Cincinnati Children's Hospital Medical Center. Additional saliva samples were collected from U.S. military personnel as part of an outbreak surveillance of acute gastroenteritis under a Department of Defense project (Scott Thornton). The saliva-binding assays were performed as described previously (8). Briefly, microtiter plates (Dynex Immulon; Dynatech, Franklin, MA) were coated with saliva at a dilution of 1:1,000 to 1:5,000 in phosphate-buffered saline (pH 7.4). To avoid potential norovirus-specific antibodies in the saliva that may interfere in the receptor binding assays, saliva samples were boiled before being used in the assays. After removing the unbound saliva and blocking with 5% dried milk (Blotto), baculovirus-expressed norovirus VLPs at a concentration of 0.4 to 1.0 μg/ml in phosphate-buffered saline were added. The bound VLPs were detected using a pooled guinea pig anti-norovirus antiserum (dilution of 1:3,000), followed by the addition of horseradish peroxidase (HRP)-conjugated goat anti-guinea pig immunoglobulin G (IgG; ICN, Aurora, OH).

The pooled guinea pig anti-norovirus antiserum was a mixture of the hyperimmune sera of three groups of guinea pigs cross-immunized with three sets of recombinant norovirus capsid antigens: set 1, Norwalk virus, C59, and VA115; set 2, VA387, MxV, GrV, and HV; and set 3, VA207 and MOH. The pooled guinea pig anti-norovirus antiserum recognized recombined capsid proteins of all 14 norovirus strains studied.

Binding of VLP to synthetic oligosaccharides containing human HBGA epitopes.

Microtiter plates were coated with synthetic oligosaccharide-bovine serum albumin (BSA) conjugates containing human HBGA epitopes at a concentration of 20 μg/ml at 4°C overnight. Oligosaccharide-PAA-biotin conjugates (2 μg/ml) were coated to a microtiter plate by using streptavidin. After blocking with 5% Blotto, norovirus VLPs were added at 0.4 to 1.0 μg/ml. The captured VLPs were detected by the same procedures described above. Oligosaccharides conjugated with two types of carriers were used in this study, including the polyacrylamide (PAA)-biotin conjugates H type 3, type A, and type B disaccharides; Le^a, Le^x, H type 1, H type 2, type A, and type B trisaccharides; and Le^b, Le^y, sLe^a, and sLe^x tetrasaccharides (GlycoTech Corporation, Rockville, MD); and BSA conjugates A-trisaccharide-BSA {[GalNAc α1-3 (Fuc α1-2) Gal β-O-spacer]_n-BSA} and B-trisaccharide-BSA {[Gal α1-3 (Fuc α1-2) Gal β-O-spacer]_n-BSA}, both BSA conjugates containing a 5-atom spacer (Glycorex AB; Lund Sweden). Additional A-trisaccharide-BSA and B-trisaccharide-BSA, with 20- and 3-atom spacers, respectively, were also used (V-Labs, Inc., Covington, LA).

Validation of synthetic oligosaccharide conjugates by MAbs.

To determine the specificity of individual oligosaccharide products, we performed binding assays of the products with MAbs specific for HBGAs. Oligosaccharide-BSA conjugates (20 μg/ml) were coated directly, while oligosaccharide-PAA-biotin conjugates (2 μg/ml) were coated through streptavidin (10 μg/ml) as anchor. After removal of the uncoated conjugate and blocking with 5% Blotto, MAbs were added at a dilution of 1:100 to 1:200. The captured MAbs were detected using HRP-conjugated goat anti-mouse IgG or IgM (ICN, Aurora, OH). MAbs used in this study included MAbs anti-B (BG-3), anti-H type 1 (BG-4), anti-Le^a (BG-5), anti-Le^b (BG-6), anti-Le^x (BG-7), and anti-Le^y (BG-8) (Signet Laboratories, Inc., Dedham, MA) and MAbs anti-H type 2 (BCR9031), anti-A (BCR9010), and anti-B (BCRM 11007) (Accurate Chemical & Scientific Corporation, Westburg, NY).

Blocking of norovirus binding to saliva by MAbs.

The same conditions of saliva binding assays described above were used. For blocking, saliva-coated plates were preincubated with MAbs at dilutions of 1:5 to 1:10 for 1 h at 37°C before adding norovirus VLPs to the plate. The levels (%) of blocking were calculated by comparing the OD₄₅₀ values between wells with or without incubation with a MAb.

Cross-blocking assays with synthetic oligosaccharide conjugates.

The same conditions of oligosaccharide binding assays described above were used. For blocking, norovirus VLPs were preincubated with 20 μg/ml of BSA-oligosaccharide conjugates or 2 μg/ml of PAA-biotin oligosaccharide conjugates for 1 h at 37°C before transferring to the oligosaccharide-coated plate. The levels (%) of blocking were calculated from the OD₄₅₀ values between wells with or without preincubation with an oligosaccharide.

Cross-blocking assays with saliva samples.

The same conditions of saliva binding assays with the principle of the oligosaccharide-blocking assays described above were used, except saliva samples were used for both coating and blocking. Saliva samples containing specific HBGAs were selected from our saliva bank, and the blood types of the saliva samples were determined by the MAb typing assays.

Sequencing and phylogenetic analysis.

Sequencing was performed either directly with RT-PCR products or after cloning into the pGEM-T vector (Promega, Madison, WI). Sequencing reactions were read on an ABI PRIZM 3700 DNA analyzer. Besides those previously mentioned, the following capsid sequences published in the GenBank were used in the phylogenic analysis: Alphatron (AF195847), Amsterdam (AF195848), Arg320 (AF190817), Chiba (AB022679), Hesse (AF093797), Hillingdon (AJ277607), Jena (AJ011099), Leeds (AJ277608), Lordsdale (X86557), Melksham (X81879), Musgrove (AJ277614), Snow Mountain (AY134748), Southampton (L07418), Winchester (AJ277609), and Fayetteville (AY113106). Multiple alignments of deduced amino acid sequences of the capsid proteins were created by using Omiga v2.0 software (Oxford Molecular Ltd., Oxford, United Kingdom). Alignments were edited in GeneDoc v2.5 (19). Dendrograms were constructed by using the UPGMA clustering method of Molecular Evolutionary Genetics Analysis (MEGA version 2.1) with Poisson correction distance calculations and 125 bootstrap analyses (15).

A broad spectrum of norovirus binding specificity to human HBGAs.

In our previous studies, we described four major HBGA binding patterns represented by strains VA387, MOH, Norwalk, and VA207 based on a study of eight strains (VA387, GrV, Norwalk, C59, MxV, MOH, VA207, and VA115) representing seven genetic clusters of noroviruses (8). In this study, we characterized six additional strains representing six genetic clusters of noroviruses (PiV, BUDS, Boxer, OIF, DSV, and HV) (Fig. 1). When the binding results of all 14 strains with the 81 saliva samples were compared, three new binding patterns were revealed in addition to the four patterns previously described (Fig. 1).

Strain PiV revealed a binding pattern identical to that of MOH and MxV, which reacted with the type A and B secretors but not with the type O secretors and nonsecretors. Strain BUDS revealed a similar binding pattern but recognized only saliva of type A, not type B, secretors (Fig. 1). Boxer showed a binding pattern similar to that of VA207 (8), both reacting with nonsecretors, type O, and type A secretors, but not type B secretors; however, Boxer had an equal or even higher binding activity to type O secretors than to nonsecretors. OIF was another strain that bound the saliva of nonsecretors but with weaker binding activities to type O secretors than to nonsecretors (Fig. 1). In later studies using oligosaccharide- and MAb-based assays, significant differences in binding specificities to the Lewis and H epitopes were observed among these nonsecretor binding strains (VA207, Boxer, and OIF) (see Results).

Two strains (HV and DSV) did not react with any human HBGAs of the 81 saliva samples tested (Fig. 1), even at a high concentration of both saliva (1:200 dilution) and VLPs (5 μg/ml), the same as that of VA115 described in our previous study (8). DSV is genetically close to VA115 that belongs to genogroup I of the noroviruses, while HV belongs to genogroup II. Using oligosaccharide binding assays, however, HV revealed significant binding with types A, B, and Le^b oligosaccharides (see Results), but none of these oligosaccharides reacted with DSV and VA115 (data not shown).

Binding specificity to synthetic oligosaccharides.

Before each oligosaccharide was used, we performed a validation experiment to test its specificity using HBGA-specific MAbs and 13 oligosaccharide conjugates that contain the major ABH and Lewis epitopes selected in this study (Table 1). Another two oligosaccharide conjugates, H-1 and H-3 (trisaccharides), which either did not react with the corresponding MAb or did not have a corresponding MAb available but reacted with norovirus VLPs, were included in this study. Most of the reactive oligosaccharide conjugates were highly specific to corresponding MAbs, although cross-reactions were observed in a few cases (Table 1).

Following the validation experiments, we performed the oligosaccharide binding assays with norovirus VLPs representing variable binding patterns using the same conditions of the saliva-binding assays. As summarized in Table 2, most of the binding patterns obtained by the saliva-binding assays were confirmed by the oligosaccharide binding assays. For examples, Norwalk virus binds to the saliva samples of types A and O secretors, but not type B secretors and nonsecretors (8). Oligosaccharide binding assays revealed that it reacted with the synthetic A, but not B, oligosaccharides, and it also reacted with Le^b, Le^y, H-1, and H-3, which are expected HBGA products in the type O secretors. Corresponding results also were obtained for VA387 that reacted with most of the HBGA molecules, including types A, B, Le^b, Le^y, H-1, and H-3, which are expected products in the A, B, and O secretors (8). The fact that MOH reacted to types A and B but not to other oligosaccharides also confirmed the binding specificity of this strain that recognizes HBGAs in saliva of types A and B but not type O secretors (8).

The overall binding activities of the Lewis binding strains also agreed with our prediction. For examples, OIF reacted with Le^a only, VA207 reacted with Le^x and Le^y, and Boxer reacted with Le^b and Le^y, but none of them reacted with the A and B antigens. We also noticed that not all Lewis binding strains reacted with all Lewis epitope-containing antigens (Le^a, Le^b, Le^x, or Le^y). In our later study using MAbs to block binding, we also observed a difference in binding to these epitopes among the three strains (see Results). Thus, the difference in reactions with the Lewis-containing oligosaccharides might be a reflection of strain-specific variations among these strains.

More interestingly, the oligosaccharide-binding assays also detected binding activities that were not observed in the saliva-binding assays (Table 2). For example, strain HV, which did not bind to any HBGAs in the saliva-binding assays, reacted with synthetic A, B, and Le^b antigens (Fig. 1 and Table 2). Similarly, BUDS reacted with both synthetic A and B antigens, while it bound to only type A saliva (Fig. 1 and Table 2). Furthermore, none of the PiV, MxV, and HV VLPs bound to type O saliva, but all three strains bound to synthetic Le^b antigen (Fig. 1 and Table 2). It was noted that all these binding activities were dose related but with a low OD value (below 1.0). In fact, the binding of HV to synthetic Le^b also has been reported by others (6). Thus, we believe that these reactions are strain specific, not an artifact. Possible reasons for the difference between the results obtained by the saliva- and oligosaccharide-based assays are considered in the Discussion.

The Lewis binding strains recognize the Lewis epitopes, but each may have a unique binding specificity.

Since the MAb-based assays have already confirmed the involvement of the Lewis epitopes in the binding of VA207 (8), we performed additional blocking experiments on the three nonsecretor binding strains with MAbs against the four major Lewis epitope-containing antigens (Le^a, Le^x, Le^b, and Le^y). As expected, MAbs against Le^a and Le^x strongly blocked VA207 binding to saliva containing Le^a and Le^x (nonsecretor donors) (Table 3). However, they did not block Boxer and OIF binding to the same nonsecretor saliva samples. MAbs against Le^b and Le^y blocked VA207 and Boxer binding to saliva containing Le^b and Le^y (secretor donors) but did not block their binding to saliva containing Le^a and Le^x (nonsecretor donors) (Table 3). None of the four MAbs blocked OIF binding to the saliva of nonsecretors. Thus, we concluded that, although the MAb-based assays did not confirm all results by the saliva and oligosaccharide assays, the fact that all three strains bound to the saliva of the nonsecretors indicates that these strains are related by binding to the Lewis epitope (1,3/4 fucosyl residue). However, these strains are distinct, possibly by their variable affinity to the Lewis and H epitopes (see Discussion).

The A/B and H epitopes are independent but can act together in binding to A/B binding strains.

Since most of the strains binding the A/B epitopes reacted with more than one epitope, we particularly studied the role of individual epitopes in association with other epitopes for binding VLPs. Specifically, the VA387-like strains (VA387, GrV, Norwalk, and C59) can react with almost all secretor-related antigens containing the A/B and H epitopes (Table 2). The A/B and H epitopes are linked to the same galactose molecule with α1-3 and α1-2 linkages, respectively, that are physically next to each other, suggesting that they are likely to act together. To test this hypothesis, we performed blocking experiments using oligosaccharides or saliva as the blocking agents.

Using oligosaccharides to block norovirus binding to oligosaccharides, we observed significant homologous blocking activities among oligosaccharide conjugates containing the A, B, and H epitopes (Tables 4 and 5). In addition, significant heterologous blocking activities among different oligosaccharide conjugates have also been observed in strains of the A/B binding group (Tables 4 and 5). For examples, the A and B antigens cross-blocked each other for their binding to VA387 (Table 4), MOH, and other MOH-like strains (BUDS, MxV, and PiV) (data not shown). The A and/or B trisaccharides also blocked the binding of VA387 and Norwalk virus to the H-containing antigens (H-1, H-3, Le^b, and Le^y), possibly at the common H recognition site of the capsids (Table 5). Similarly, the H-containing antigens also blocked the binding of VA387 and Norwalk virus to the A and B antigens (trisaccharides) with variable efficiencies. However, the Lewis epitopes (Le^a and Le^x) did not block the binding of VA387, Norwalk virus, MOH, or the MOH-like strains to the A/B and H epitopes, nor did the A and B trisaccharides block the binding of VA207, Boxer, or OIF to the nonsecretor saliva samples (data not shown).

To further confirm these results, we also performed saliva-saliva blocking experiments using a panel of saliva samples representing different HBGA types for the coating and the blocking steps, respectively. Figure 2 shows the typical results of the blocking assays for VA387. In summary of the results, the following conclusions have been made: (i) significant blocking of binding was observed among homologous blood types, (ii) significant heterologous blocking activities were also observed among secretor saliva (types A, B, and O), (iii) saliva of nonsecretors did not block the binding to the A, B, and H antigens, and (iv) saliva samples with more complex antigens (e.g., type A and B secretors) were stronger blockers than saliva samples with less complex antigens (e.g., type O secretors).

In conclusion, both A/B and H are independent epitopes but they can act together depending on the structure of the binding interface of norovirus capsids. Strains MOH and BUDS apparently have one binding site to the A/B epitopes, while the remaining strains of the A/B binding group may contain two sites within the binding interface. The A and B epitopes may share the same binding site for the majority of the A/B binding group, except for Norwalk and C59, which recognize only the A but not B antigens. The H epitope is associated with the A/B epitopes in the binding, but it can act alone in the type O secretors. The fact that the A/B and Lewis epitopes do not cross-block each others indicates that strains in these two groups have distinct binding interfaces on the viral capsids.

Strains with similar binding patterns tend to be genetically clustered.

The fact that the two groups mutually exclude the A/B or Lewis epitopes raised the possibility that the human HBGAs may play a role in norovirus evolution. However, we did not observe a clear segregation of the two binding groups with the genogroups of noroviruses when the entire capsid sequences were analyzed (Fig. 3). In our recent study, we demonstrated that the P domain is responsible and contains the essential elements for the binding of norovirus VLPs to HBGAs (20). Therefore, we performed a phylogenetic analysis focusing on the P domain, but still no genetic correlation was observed between the two binding groups (data not shown).

However, when the 14 strains were listed on the phylogenetic tree in relation to their binding patterns, clear relationships of genetic identities with binding patterns were observed among some strains (Fig. 3). For example, GrV and VA387 are the only two strains that bound types A, B, and O secretors and both belong to the same cluster (GII4) of genogroup II that shares a 98% overall amino acid sequence identity. Norwalk virus and C59 are two strains that bound to the A and O, but not B, secretors, and they are genetically close to each other. In addition, both BUDS and MOH bound only the A/B epitopes and they are more related to each other than to any other strains studied. Furthermore, HV, MxV, and PiV revealed similar binding patterns to the A/B and H antigens and they are related with the shortest phylogenetic distances to each other. Finally, strain VA115 and DSV are the only two strains that did not react with any HBGAs and they are genetically close to each other. Thus, we conclude that the strain specificities are genetically related, indicating that human HBGAs are involved in norovirus evolution. The scattering of strains of the two binding groups in both genogroups indicates that other factor(s) may also be involved in norovirus evolution.