The HBGA binding interfaces of NVs are known to be highly conserved among strains within each of the two major genogroups (GI and GII) (41
). This finding has led to the hypothesis that the human HBGAs play an important role in NV evolution. In this study, we provide further support for this hypothesis through systematic characterization of the genetic and antigenic variations and HBGA binding patterns of extended GII-4 viruses from GenBank and our collection. Sequence alignments demonstrated that the HBGA binding sites remain highly conserved among GII-4 viruses over time, which is consistent with recent reports by others (1
). The amino acid residues essential for HBGA recognition, according to the atomic resolution structures and mutagenesis studies (4
), remained virtually unchanged among 93 GII-4 sequences spanning 2 decades from 1987 to 2008. A recent study indicated that the conservation of the HBGA binding site has been traced back to the 1970s (1
). Our current study showed that the ability to bind the secretor HBGAs also did not change among 15 selected strains representing all six major genetic subclusters of GII-4 viruses isolated at different time points during the past decade, although certain levels of variation in binding affinity to some HBGAs by several GII-4 variants were noted.
The observed consensus phenotype of GII-4 binding to saliva of the A, B, and O secretors suggests a functional selection pressure on the capsid-ligand interface exerted by the human HBGAs. Structurally, the H epitope (α-1,2-fucose) plays the central role in recognition by the GII-4 viruses, as shown by VA387-HBGA interaction (7
). With a conformational multivalent interface, additional carbohydrates around the H epitopes, such as N
-acetyl-galactosamine, galactose, and α-1,3/4 fucose, may also participate in GII-4 binding. These epitopes form the A, B, Leb
, H type A, and H type B plus the H-1, H-2, and H-3 molecules which are made in the A, B, and/or O secretors that represent 80 to 85% of the general population (40
). According to these results we hypothesize that the persistence of such a broad spectrum of HBGA binding could be the major reason for GII-4 predominance over GI and other GII viruses that generally recognize a narrower spectrum of HBGAs (23
In our previous studies, we have described eight HBGA binding patterns among different GI and GII NVs studied (23
). Many of them recognize saliva of only the A and/or B secretors and do not recognize (or bind poorly to) the type O secretors, such as the GII-1, GII-2, GII-3, GII-5, GII-12, and GII-14 strains (23
; our unpublished data); thus, it would be logical to speculate why the GII-4 strains are so predominant among NV strains. In another study of NV gastroenteritis in China during 1999 to 2005 (74
), we observed a typical gradient prevalence among different GII viruses in the case of acute gastroenteritis in children, which fits well with the gradient spectra of HBGA binding observed in this study (Fig. ). This result is encouraging because it suggests that we may be able to predict NV prevalence for individual genotypes based on their HBGA binding spectra. Further studies in this direction are necessary.
FIG. 10. Phylogenetic tree of selected GII-4 and other GII viruses. The receptor binding patterns of strains are shown in the parentheses as HBGA epitopes. The capital letters A, B, and H indicate a strong binding, and the lowercase letters a, b, and h indicate (more ...)
The observed antigenic variations by different assays using antibodies from mice and GII-4-infected patients suggested that the host immunity continually drives antigenic changes within the GII-4 genotype. However, the high conservation of the HBGA binding interfaces, with variations only in surrounding regions, and the observed cross-blocking activities of HBGA binding among GII-4 from different years indicate that the HBGAs are involved and may act as an important counterbalancing factor in the selection process. This should be emphasized in the hypothesis of GII-4 epochal evolution, in which the carbohydrate variation may be only partially tolerated. Thus, antigenic variants that do not affect the HBGA binding interface and maintain the consensus phenotypes of HBGA binding are likely to be selected, contributing predominantly to the main stream of circulation in the population. On the other hand, antigenic variants with changes in the HBGA interface may have lost their survival advantage and, therefore, may not continue circulating in the population. The only nonbinder of GII-4 seen in this study was found to have an amino acid change in the HBGA binding interface and could be such an example.
It should be noted that our results are in conflict with recently published data of a study in which GII-4 variants with significant HBGA binding changes were reported (43
). As a result, an alternative hypothesis, that the variation of HBGA binding is tolerated and that selection via host immunity may result in antigenic variants acquiring new HBGA binding patterns, has been proposed (43
). Further studies are required to resolve this discrepancy. We would like to comment that the two studies were performed using different viral particles (VLPs versus P particles) as models. In retrospective analysis of our data, we do not anticipate that the use of P particles would pose a problem because our laboratory has developed over 40 P particles for variable GI and GII NVs and we have not observed significant antigenic and receptor binding changes in P particles compared with VLPs (59
; unpublished data). We also obtained the HBGA binding results of additional strains in the Hunter and Sakai clusters, which further confirmed the consensus binding phenotype of GII-4 viruses. Since this issue is fundamentally important, confirmatory studies and side-by-side comparison, as well as testing of additional GII-4 variants in more laboratories, will be necessary.
The antigenic variations described in our study are comparable to those reported in other studies, in which the surrogate neutralization assay to measure HBGA blocking activities was used (6
). In general, the blockade of GII-4 VLP/P particles binding to HBGAs was greater with serum samples from patients in outbreaks that occurred near the time of origin of the VLP/P-particle strain (6
), indicating a continual antigenic drift (possibly due to a selection by the host immunity). Whether these variants represent new escape mutants responsible for new epidemics or pandemics seen with flu virus remains to be confirmed. According to the epidemiology and transmission mode of NVs, such likelihood may be low. NVs are transmitted by the fecal/oral route, which commonly causes community outbreaks, and may not spread as widely as influenza viruses do. Consequently, NVs may not reach (or reach as quickly) the same level of herd immunity in the general population as that of flu virus. Thus, any sublineages of GII-4 may continue circulating somewhere in the world even if they may die off locally. Their chance to emerge in a new season would also be equal if the herd immunity is low.
The observed variation in A-antigen binding due to the I389V mutation may serve as a good example of the equal chance of circulation (random fluctuation). It is known that the European and North American populations are high A/low B, while the Pacific and Asian populations are low A/high B for their blood antigen types (15
). The high or low bindings to the A antigen by GII-4 variants suggest a fitness to either the A or non-A individuals by adaption (selection). Thus, a strain with high affinity for the A antigen is more likely to be derived from the European and North American population, while a low A binder is more likely to come from an Asian population. Since the binding to the A antigen is nonessential for GII-4 viruses, mutants with both phenotypes would have similar survival advantages. The discovery of a single mutation that could be the cause of such epidemic cluster-switching is highly significant.
The present study has several limitations. For example, significant variation in the binding affinities to HBGAs by 16 different GII-4 variants was observed even under strict control of the P particles used in the comparison. At this stage, we do not know whether the difference is due to microvariation of the receptor binding interfaces among the GII-4 variants or variation due to the in vitro
expression system used for making the P particles. Regrettably, due to the lack of a reliable in vivo
system for assessment, this issue cannot be resolved at this time. In addition, we also observed variation of results between the saliva- and the oligosaccharide-based assays. Such differences were also reported in our previous studies and in other laboratories (19
). We believe that both assays are important; while the oligosaccharide assays provide valuable information on the structure and target of the HBGAs, the saliva binding assays are more biologically relevant. Finally, the antigenic characterization described in our study remains preliminary and needs to be improved by using increasingly standardized assay conditions with more defined reagents and more human subjects.
In summary, both host immunity and HBGAs play a role in NV evolution. While the host immunity will continue to drive antigenic change, the HBGAs, as convergent factors, may enforce a functional selection through structural constraint. This may allow only partial tolerance of HBGA binding variation. The diverged lineages (genotypes) of NVs in the two genogroups seen today are the results of such selection by the polymorphic HBGA types. Each lineage may fit one or a combination of a few HBGAs as a stable genetic trait. The GII-4 viruses have the best fit to the secretor types that express the major determinants of the H antigens, which may be the reason for their predominance over other NVs. We hope that our results help to clarify some epidemiology and evolution issues of NVs. If the GII-4 viruses remain in a clone expanding stage and undergo only low levels of antigenic variation (drift), the vaccine strategy used to select vaccine strains for influenza viruses may not be suitable for NVs. It is also possible that a longer time may be needed for a new antigenic variant to emerge for NVs than for flu virus. However, these questions remain to be resolved in future studies.