Norovirus capture with histo-blood group antigens reveals novel virus-ligand interactions

Abstract

Noroviruses are genetically diverse, uncultivable, positive-sense RNA viruses and are the most common cause of epidemic acute gastroenteritis in humans in the United States. Recent studies of norovirus attachment in vitro by using recombinant virus-like particles (VLPs) suggest that various norovirus strains exhibit different patterns of attachment to ABH histo-blood group antigens, which are carbohydrate epitopes present in high concentrations on mucosal cell surfaces of the gut. However, attachment of live norovirus strains to histo-blood group antigens has not been investigated to date. Utilizing a newly designed magnetic bead-virus capture method, we characterized histo-blood group antigen attachment properties of various norovirus strains obtained from clinical stool specimens to compare the attachment properties of wild-type virus and VLPs and to further map norovirus attachment. Consistent with previous reports using VLPs, various strains of noroviruses exhibited different patterns of attachment to histo- blood group antigens. Norwalk virus bound specifically to H type 1, H type 3, and Le(b). Two genogroup II noroviruses, one representing the Toronto genotype and the other from a novel genotype, bound specifically to Le(b). A Desert Shield-like strain did not attach to H types 1, 2, or 3, H type 1 and 3 precursors, Le(a), or Le(b). Surprisingly, wild-type Snow Mountain virus (SMV) attached specifically to H type 3, which contradicted previous findings with SMV VLPs. On further investigation, we found that stool components promote this attachment, providing the first known observation that one or more components of human feces could promote and enhance norovirus attachment to histo-blood group antigens.

FIG. 1.

Magnetic bead method and carbohydrates used in this study. (A) A magnetic bead-based method was designed to characterize histo-blood group antigen attachment specificities of wild-type noroviruses obtained from clinical stool specimens. Streptavidin-precoated magnetic beads were coated with specific synthetic, biotinylated histo-blood group antigens and then blocked with 5% milk-PBS containing 0.25% Tween 20 to inhibit nonspecific binding. Stool extracts containing equal RT-PCR detectable levels of particular norovirus strains were then incubated with the magnetic beads. After extensive washing to remove unbound virus, the magnetic beads were 10-fold serially diluted and subjected to heat release to extract the RNA genome. The virus-bead suspensions were diluted for two reasons: (i) to generate a semiquantitative result of viral attachment and (ii) to dilute any possible inhibitors of the RT-PCR amplification. Finally, RT-PCR was performed to assay for the presence of viral RNA to detect the attachment of the virus to the specific histo-blood group antigens. (B) Type 1 and 3 chain histo-blood group antigens used in this study are shown in bold type. Enzymatic activities required to produce the various carbohydrates are shown in italics. H type 2 antigen, which is not present in high concentrations on the superficial surfaces of the gut (47), was also used in this study as a tissue-specific control.

FIG. 2.

Histo-blood group antigen attachment specificities of noroviruses obtained from clinical specimens. RT-PCR-positive products indicate attachment of norovirus strains to the specific synthetic histo-blood group antigens shown. RNA samples extracted from the same clinical stool specimens subjected to the attachment assay were used as positive controls (lane +), and sterile water was used for negative controls (lane −). “No Carbohydrate” represents mock-coated magnetic beads as a negative control for nonspecific viral binding.

FIG. 3.

Attachment of wild-type SMV to H type 3 in the presence and absence of VLPs. SMV from a clinical stool specimen was added to H type 3-coated magnetic beads and assayed for attachment by RT-PCR as described in the legend to Fig. 1. The effects of adding excess sucrose-purified SMV VLPs (10 μg) to the attachment reaction mixture (lanes +SMV VLPs) or preincubating and then washing the VLPs prior to adding the SMV clinical stool specimen (lanes + SMV VLPs, washed) were investigated.

FIG. 4.

Attachment of SMV VLPs to H type 3. The ability of SMV VLPs to attach to H type 3 in the presence or absence of the SMV clinical stool extract was investigated by using a microwell-based attachment assay. Wells were coated with H type 3 and blocked overnight, and the indicated protein concentrations of SMV VLPs (with or without 1% [vol/vol] stool extract in the sample buffer) were added. The wells were then washed, and VLP attachment was quantified by using human polyclonal anti-norovirus antiserum, goat anti-human IgG-alkaline phosphatase conjugate, and pNPP substrate. Net OD 405nm represents the OD₄₀₅ values corrected for blank OD₄₀₅ values (no VLPs added). Mean values from triplicate wells are shown, and error bars represent the standard deviation.

FIG. 5.

Effect of nonspecific stool extracts on SMV VLP attachment to H type 3. The effect of various archived stool extracts on SMV VLP attachment (10 μg/ml) to H type 3 was investigated by using the same microwell-based assay described in the legend to Fig. 4. Lanes Se+ Pre-challenge and Se− Pre-challenge contain virus-free stool extracts from secretor-positive and secretor-negative human volunteers, respectively, obtained prior to norovirus challenge.

FIG. 6.

SMV VLP attachment to other histo-blood group antigens in the presence of stool extract. Attachment of SMV VLPs in the presence of stool extract was investigated using the same microwell-based assay described in the legends to Fig. 4 and 5. A pool of no. 22, no. 27 (numbers are shown in Fig. 5), and SMV stool extracts was used at a final concentration of 1% in the sample buffer when added with the VLPs. Mean OD₄₀₅ values from duplicate wells are shown, and error bars represent the standard deviation.

FIG. 7.

Effect of stool components on NV and HV VLP attachment to histo-blood group antigens. (A) The effect of stool on NV VLP attachment to H type 1, H type 3, and Le^b was investigated using the same microwell-based assay described in the legends to Fig. 4 and 5. Note that different “1×” VLP concentrations of 0.2 μg/ml for H type 1 and 0.4 μg/ml for H type 3 and Le^b were used to account for previously determined differences in attachment avidity of the different carbohydrates with NV VLPs (27). (B) Effect of stool extract on HV VLP attachment. Data shown are based on using 10 μg of HV VLP per ml. The dashed line represents an arbitrary background cutoff equivalent to twice the average background OD₄₀₅. In all assays, a pool of no. 22, no. 27 (numbers from fig. 5), and SMV stool extracts was used at a final concentration of 1% in the sample buffer when added with the indicated VLPs.

FIG. 8.

Effect of denaturing stool extracts on SMV VLP attachment to H type 3. Stool extracts (no. 22, no. 27 [numbers from fig. 5], and SMV) were denatured by boiling for 10 min before being added to the sample buffer with SMV VLPs (10 μg of VLPs per ml plus 1% stool extract). Mean OD₄₀₅ values for SMV VLP attachment in the presence of each of the three different extracts are shown, and error bars represent the standard deviation.

FIG. 9.

Effect of pretreating VLPs or carbohydrate with stool extracts followed by washing on SMV VLP attachment to H type 3. (A) SMV VLPs were serially diluted and added to H type 3-coated microwells in either the absence or presence of 1% stool extracts or to coated wells that were pretreated for 2 h with sample buffer containing 1% stool extracts followed by washing before the addition of the VLPs. VLP attachment was quantified as described in the legends to Fig. 4 and 5. (B) Biotinylated H type 3 carbohydrates were serially diluted and added to SMV VLP-coated microwells in either the presence or absence of 1% stool extracts or to coated wells that were pretreated for 2 h with sample buffer containing 1% stool extracts followed by washing before the addition of adding biotinylated H type 3. Attachment of biotinylated H type 3 to SMV VLPs was quantified by adding streptavidin-alkaline phosphatase conjugate and pNPP substrate. Net OD 405nm indicates OD₄₀₅ values corrected for plate blank values (no H type 3 added). In both experiments, mean OD₄₀₅ values for attachment in the presence or absence of three different stool extracts (no. 22, no. 27 [numbers from Fig. 5], or SMV) are shown, and error bars represent the standard deviation.

FIG. 10.

Summary of norovirus attachment data in relation to genetic grouping. Genetic classifications of noroviruses used in this study (indicated by underlining) are based on previously described criteria and compared with known sequences of other noroviruses (genogroup.genotype indicated) (23, 24, 51). The nucleotide sequences that were compared are from a 177-nucleotide region in the ORF2 gene (region D) that has been shown to reliably classify Norovirus genotypes (52). Specific histo-blood group antigen attachment data from this study compiled with results from other studies are indicated. ¹Unpublished data obtained using VLP reagents. ²Previous results using HV VLPs alone showed no binding to any carbohydrates tested (27), whereas results from this study demonstrated slight binding of VLPs to Le^b, but only in the presence of stool extract. ³Observation made by Hutson et al. using baculovirus-expressed recombinant NV VLPs (33). ⁴Based on the observation that SMV VLPs attached specifically to saliva from secretor-positive, blood type B individuals (27).