Comparative evolution of GII.3 and GII.4 norovirus over a 31-year period

Abstract

Noroviruses are the most common cause of epidemic gastroenteritis. Genotype II.3 is one of the most frequently detected noroviruses associated with sporadic infections. We studied the evolution of the major capsid gene from seven archival GII.3 noroviruses collected during a cross-sectional study at the Children's Hospital in Washington, DC, from 1975 through 1991, together with capsid sequence from 56 strains available in GenBank. Evolutionary analysis concluded that GII.3 viruses evolved at a rate of 4.16 × 10(-3) nucleotide substitutions/site/year (strict clock), which is similar to that described for the more prevalent GII.4 noroviruses. The analysis of the amino acid changes over the 31-year period found that GII.3 viruses evolve at a relatively steady state, maintaining 4% distance, and have a tendency to revert back to previously used residues while preserving the same carbohydrate binding profile. In contrast, GII.4 viruses demonstrate increasing rates of distance over time because of the continued integration of new amino acids and changing HBGA binding patterns. In GII.3 strains, seven sites acting under positive selection were predicted to be surface-exposed residues in the P2 domain, in contrast to GII.4 positively selected sites located primarily in the shell domain. Our study suggests that GII.3 noroviruses caused disease as early as 1975 and that they evolve via a specific pattern, responding to selective pressures induced by the host rather than presenting a nucleotide evolution rate lower than that of GII.4 noroviruses, as previously proposed. Understanding the evolutionary dynamics of prevalent noroviruses is relevant to the development of effective prevention and control strategies.

Fig. 1.

Distribution of genotypes in the NoV-positive samples of the CHDC study by year of collection.

Fig. 2.

Maximum-likelihood analysis of VP1 region sequences from GII.3 NoVs. GII.3 VP1 sequences from 1975 to 2006 (for GenBank accession numbers, see Fig. S2 in the supplemental material), including the seven new CHDC sequences, are shown, circled within proposed clusters as a result of the analysis. The percent nucleotide (nt) and percent amino acid (aa) variation within each cluster are shown in parentheses, and between-cluster variation is shown in the inset table.

Fig. 3.

Bayesian skyline plots of GII.4 and GII.3 NoV VP1 sequences. The plots illustrate the relative genetic diversity of circulating GII.4 and GII.3 NoVs through time. The black line represents the median posterior value and the gray lines the 95% highest probability density (HPD) intervals.

Fig. 4.

Amino acid evolution of GII.3 and GII.4 NoV sequences compared to sequences of the most ancestral CHDC strains. Amino acid distance from most ancestral GII.3 (Hu/NoV/GII.3/CHDC2005/1975/US) and GII.4 (Hu/NoV/GII.4/CHDC2094/1974/US) strains was calculated for all VP1 sequences available in GenBank (GenBank accession numbers for GII.3 are in Fig. S2 in the supplemental material; those for GII.4 are in reference 2). Samples are marked by cluster from the phylogenetic analysis.

Fig. 5.

Amino acid variation of the VP1 capsid region of GII.3 NoVs over time. The VP1 amino acid sequences of the GII.3 strains from the CHDC study were compared to sequences of representative strains from all three clusters predicted for GII.3 evolution. Amino acids predicted to be under positive selective pressure are highlighted in green and were determined using the single-likelihood ancestor counting (SLAC) method available in the HyPhy package.

Fig. 6.

Structural modeling of GII.3 P2 dimer. Homology modeling was performed to predict the P2 protein structure of the GII.3 P region using the amino acid sequence of Hu/NoV/GII.3/CHDC2005/1975/US. (A) Sites predicted to undergo positive selection (highlighted in green in Fig. 5) were highlighted in purple on this minimized structure. (B) The same residues were mapped on the protein structure of the Hu/NoV/GII.4/VA387/1998/US dimer and are highlighted in purple. The two HBGA binding sites are shown in yellow (site 1) and orange (site 2), with the trisaccharide B molecule shown in purple. (C) An overlay of the two minimized ribbon structures predicted for Hu/NoV/GII.3/CHDC2005/1975/US P domain (purple) and Hu/NoV/GII.4/VA387/1998/US P domain (teal) was performed. The inset table shows the number and locations of positively selected sites.

Fig. 7.

Detection of HBGA blocking antibodies in sera from GII.4 clusters. Sera from three different GII.4 clusters (CHDC, Camberwell, and F. Hills) were tested for the ability to block the interaction between recombinant F. Hills VLPs and H3 carbohydrates.