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.Noroviruses (NVs) have been recognized as the most important cause of nonbacterial acute gastroenteritis in both developed and developing countries, affecting people of all ages (13, 35, 39, 44, 48, 56). They are single-stranded positive-sense RNA viruses belonging to the family Caliciviridae. NVs are highly contagious, spreading by a fecal/oral pathway through person-to-person contact and by contaminated food and/or water and usually causing large outbreaks within closed communities in a variety of settings, such as hospitals, nursing homes, schools, childcare centers, restaurants, cruise ships, and the military (11, 63). Human NVs have been difficult to study due to diverse members and the lack of an efficient cell culture and animal model for human NVs. The cloning of the NV genomes (33, 36, 73) and subsequent expression of the viral capsid proteins in baculovirus and other expression systems (3, 31, 32) have greatly advanced the research of NVs, including host-virus interaction, immunology, diagnosis, molecular virology, and epidemiology (16, 17, 19, 20, 25, 28-30, 46, 51, 59, 73).Several lines of evidence indicate that NVs recognize human histo-blood group antigens (HBGAs) as a ligand or receptor in a strain-specific manner (63, 64). HBGAs are complex carbohydrates presenting on red blood cells and on the epithelia of digestive, respiratory, and genitourinary tracts. They also exist in biologic fluid, such as milk and saliva. NVs are highly diverse in recognizing the human HBGAs, and a number of HBGA-binding patterns involving the ABO, secretor, and Lewis families of human HBGAs have been described (19, 20, 23, 24, 26, 28, 43, 45, 55). The association of HBGA binding with clinical infection and illness has been demonstrated by volunteer challenge studies and outbreak investigations (25, 27, 42, 62, 66), although exceptions also have been reported (41, 50, 53). Further study has mapped the HBGA binding site in the protruding (P) domain of the viral capsid protein (60). Using the P domain as a model, the atomic structures of the HBGA binding interfaces have been resolved by X-ray crystallography (5, 7, 9). The interfaces are comprised of several amino acids located on the top of the P dimer, within the outermost surface of the viral capsid. Extensive hydrogen bond networks between the P dimer and the HBGAs were elucidated and further confirmed by mutagenesis analyses (61, 68, 69). Despite significant differences in genetics and HBGA binding patterns, the sequences of the HBGA-binding interfaces are highly conserved within, but not between, the two major human-related genogroups (GI and GII) of NVs, suggesting that HBGAs are important factors in NV evolution (9, 69).The NV capsid is composed of a single major structural protein, the capsid protein (VP1), which can be divided into two major domains: the shell (S) and the protruding (P) domains (52). Expression of the full-length VP1 by a eukaryotic system forms empty virus-like particles (VLPs) that have been used as a surrogate for NVs for many years, e.g., in diagnostic tests. Recent studies showed that expression of the P domain alone results in the formation of a subviral particle, the P particle (54, 60). Owing to its easy production in an Escherichia coli system and the same HBGA-binding properties and antigenicity as its parental VLP, the P particle has been used as a research tool of NV-HBGA interaction in a number of studies (54, 59, 60, 67, 68, 69). This report took advantage of the convenient P particle model to study the phenotypic HBGA-binding properties and antigenicity of GII-4 NVs that have circulated in the past decade.NVs are grouped into five genogroups (GI to GV), of which GI and GII are involved in the majority of acute viral gastroenteritis cases in humans. Strains within each genogroup can be further divided into genotypes, and up to 30 genotypes of GI and GII NVs have been described (75). NVs can be detected throughout the year, with peaks during the fall and winter seasons. Strains representing multiple genotypes can be found cocirculating in the same geographical area during a season. However, a single genotype of NVs, GII-4 (genogroup II genotype 4), has been the predominant cause of major acute gastroenteritis epidemics in many countries since the mid-1990s, and the number of GII-4 epidemics has increased in recent years (49). Overall, the GII-4 genotype is estimated to be responsible for 60 to 80% of all NV-associated outbreaks worldwide (43).Molecular surveillance has found that the GII-4 viruses are continuously changing, with new variants emerging every 2 or 3 years (1, 2, 57, 71, 72). One hypothesis suggests that the GII-4 viruses might be under selection pressure of the herd immunity, similar to the epochal evolution model used to describe the evolution of influenza (flu) viruses (56). New antigenic variants of GII-4 derived by genetic shift (replacement) accompanied by changes of HBGA binding specificities have been reported (43). However, the HBGA-binding interfaces of NVs have been found to be highly conserved among NVs within each of the two major genogroups, supporting HBGAs as an important factor in NV evolution (69). In fact, it has been shown that the major HBGA-binding pattern of GII-4 viruses to the H3, Le^b, and Le^y antigens has remained unchanged from 1974 to 1997 (4, 23, 24).The objective of this study was to elucidate the roles of HBGAs and host immunity in NV evolution using GII-4 viruses as a model. Since most of the studies on the epochal evolution of GII-4 were based on genetic analysis and focused on GII-4 variants identified in the past decade, we performed a study on the GII-4 variants in the same period by both genetic and phenotypic characterizations. Phylogenetic analysis revealed 6 genetic clusters of GII-4 viruses similar to those reported before. Characterization of HBGA-binding patterns of the GII-4 viruses revealed a consensus phenotype of binding to all A, B, and O secretor HBGAs, with some variations in affinity to these antigens. We also discussed the role of both host immunity and HBGAs in NV evolution. While the host immunity may drive NVs for change, as a functional selection factor, the HBGAs may restrict variation. This counterselection mechanism may help in understanding the epochal evolution hypothesis. The principles found through the study of GII-4 NVs can also be applied to other genotypes, which may eventually lead to a refined functional classification of all NVs.