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. 2009 Nov;83(22):11890-901.
doi: 10.1128/JVI.00864-09. Epub 2009 Sep 16.

Evolutionary dynamics of GII.4 noroviruses over a 34-year period

Karin Bok  1 , Eugenio J Abente, Mauricio Realpe-Quintero, Tanaji Mitra, Stanislav V Sosnovtsev, Albert Z Kapikian, Kim Y Green
Affiliations

Evolutionary dynamics of GII.4 noroviruses over a 34-year period

Karin Bok et al. J Virol. 2009 Nov.

Abstract

Noroviruses are a major cause of epidemic gastroenteritis in children and adults, and GII.4 has been the predominant genotype since its first documented occurrence in 1987. This study examined the evolutionary dynamics of GII.4 noroviruses over more than three decades to investigate possible mechanisms by which these viruses have emerged to become predominant. Stool samples (n = 5,424) from children hospitalized at the Children's Hospital in Washington, DC, between 1974 and 1991 were screened for the presence of noroviruses by a custom multiplex real-time reverse transcription-PCR. The complete genome sequences of five GII.4 noroviruses (three of which predate 1987 by more than a decade) in this archival collection were determined and compared to the sequences of contemporary strains. Evolutionary analysis determined that the GII.4 VP1 capsid gene evolved at a rate of 4.3 x 10(-3) nucleotide substitutions/site/year. Only six sites in the VP1 capsid protein were found to evolve under positive selection, most of them located in the shell domain. No unique mutations were observed in or around the two histoblood group antigen (HBGA) binding sites in the P region, indicating that this site has been conserved since the 1970s. The VP1 proteins from the 1974 to 1977 noroviruses contained a unique sequence of four consecutive amino acids in the P2 region, which formed an exposed protrusion on the modeled capsid structure. This protrusion and other observed sequence variations did not affect the HBGA binding profiles of recombinant virus-like particles derived from representative 1974 and 1977 noroviruses compared with more recent noroviruses. Our analysis of archival GII.4 norovirus strains suggests that this genotype has been circulating for more than three decades and provides new ancestral strain sequences for the analysis of GII.4 evolution.

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Figures

FIG. 1.
FIG. 1.
Distribution of genotypes in the norovirus-positive samples of the study of Children's Hospital in Washington, DC. A specific norovirus genotype was assigned to 48 of the 50 norovirus-positive samples on the basis of partial polymerase coding sequence. The GII.4 norovirus strains selected for complete genome sequencing in this study are indicated in boldface type. ND*, a specific norovirus genotype could not be determined on the basis of the sequence available.
FIG. 2.
FIG. 2.
Maximum likelihood analysis of complete genome sequences from noroviruses of different genogroups. Complete genomic sequences of norovirus genogroups I, II, III, and V obtained from the GenBank database (see Table S1 in the supplemental material) were compared with five GII.4 CHDC samples from this study (shown in boldface type). Bootstrap values are shown at the nodes of each selected phylogenetic group only. FHills-2002, Hu/NoV/GII.4/Farmington Hills/2002/US; Camberwell-1994, Hu/NoV/GII.4/Camberwell/1994/AUS; CHDC3967-1988, Hu/NoV/GII.4/CHDC3967/1988/US; CHDC4108, Hu/NoV/GII.4/CHDC4108/1987/US.
FIG. 3.
FIG. 3.
Maximum credibility clade of GII.4 norovirus VP1 sequences, as a result of Bayesian estimation using the BEAST package. GII.4 sequences from 1987 to 2007 are shown colored in the different clusters previously proposed for GII.4 evolution (32). The CHDC sequences from the 1970s are shown in a separate cluster. The percent nucleotide (nt) and percent amino acid (aa) identities are shown in parentheses. Camberwell-1994, Hu/NoV/GII.4/Camberwell/1994/AUS; Grimsby, Hu/NoV/GII.4/Grimsby/1995/UK; Farmington Hills, Hu/NoV/GII.4/Farmington Hills/2002/US; Hunter, Hu/NoV/GII.4/Hunter/2004/AUS; Sakai, Hu/NoV/GII.4/Sakai/2005/JP.
FIG. 4.
FIG. 4.
Amino acid variation of the VP1 capsid region of representative strains over time. The GII.4 strains from the study of the Children's Hospital in Washington, DC, were compared with representative strains from the clusters previously proposed for GII.4 evolution (32) as follows: CHDC2094, Hu/NoV/GII.4/CHDC2094/1974/US; CHDC5195, Hu/NoV/GII.4/CHDC5191/1974/US; CHDC4871, Hu/NoV/GII.4/CHDC4871/1977/US; CHDC4108, Hu/NoV/GII.4/CHDC4108/1987/US; CHDC3967, Hu/NoV/GII.4/CHDC3967/1988/US; Camberwell, Hu/NoV/GII.4/Camberwell/1994/AUS; Grimsby, Hu/NoV/GII.4/Grimsby/1995/UK; F. Hills, Hu/NoV/GII.4/Farmington Hills/2002/US; Hunter, Hu/NoV/GII.4/Hunter/2004/AUS; Sakai, Hu/NoV/GII.4/Sakai/2005/JP. VA387, Hu/NoV/GII.4/VA387/1998/US was included in the P2 region alignment for comparison (see Table S1 in the supplemental material for GenBank accession numbers). Amino acids labeled with an asterisk indicate changes that are unique to the sequences of the CHDC study. Positions predicted to evolve under positive selection are highlighted in green.
FIG. 5.
FIG. 5.
Amino acid variation in the VP2 minor capsid protein of representative strains over time. The GII.4 strains from the study of the Children's Hospital in Washington, DC, were compared with representative strains from the clusters previously proposed for GII.4 VP1 evolution (32) as follows: CHDC2094, Hu/NoV/GII.4/CHDC2094/1974/US; CHDC5195, Hu/NoV/GII.4/CHDC5191/1974/US; CHDC4871, Hu/NoV/GII.4/CHDC4871/1977/US; CHDC4108, Hu/NoV/GII.4/CHDC4108/1987/US; CHDC3967, Hu/NoV/GII.4/CHDC3967/1988/US; Camberwell, Hu/NoV/GII.4/Camberwell/1994/AUS; Dresden, Hu/NoV/GII.4/Dresden/1997/GER; F. Hills, Hu/NoV/GII.4/Farmington Hills/2002/US; GII.4/2004, Hu/NoV/GII.4/MD-2004/2004/US; Sakai, Hu/NoV/GII.4/Sakai/2005/JP. VA387, Hu/NoV/GII.4/VA387/1998/US was included for comparison (see Table S1 in the supplemental material for GenBank accession numbers). Positions predicted to evolve under positive selection are highlighted in green.
FIG. 6.
FIG. 6.
(A) Minimized structure of the P region of Hu/NoV/GII.4/CHDC2094/1974/US based on the protein structure of Hu/NoV/GII.4/VA387/1998/US. The corresponding sequence changes were added to the original structure, and the resulting P-region structure was minimized. The unique amino acid changes found in CHDC2094/1974 (marked with an asterisk in Fig. 4) were mapped into the new structure (purple). The two carbohydrate binding sites are shown in yellow (site 1) and orange (site 2). (B) Superimposed structures of CHDC2094/1974 and VA387/1998. The experimentally determined structure of VA387/1998 (pink) and the minimized structure of CHDC2094/1974 (orange) were superimposed and shown in ribbon format. The amino acid differences between the two structures and surrounding the carbohydrate binding site are highlighted in surface format. The trisaccharide molecule is colored in cyan blue.
FIG. 7.
FIG. 7.
Binding of 1974 and 1977 norovirus recombinant VLPs with synthetic HBGAs. The binding of rVLPs from archival noroviruses Hu/NoV/GII.4/CHDC5191/1974/US (GII.4/1974) and Hu/NoV/GII.4/CHDC4871/1977/US (GII. 4/1977) to synthetic HBGA oligosaccharides was determined by optical density at 405 nm (OD 405 nm) at room temperature. Hu/NoV/GI.1/Norwalk virus/1968/US (GI.1/NV) and Hu/NoV/GII.1/Hawaii/1971/USA (GII.1/HV) rVLPs were utilized as controls. (A) rVLP binding assays to Lewis antigens a, b, x, and y. (B) rVLP binding assay to H1, H3, and H3 oligosaccharides. (C) rVLP binding assay to blood group A and B synthetic antigens.
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