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Mechanisms of GII.4 norovirus persistence in human populations.

Lindesmith LC, Donaldson EF, Lobue AD, Cannon JL, Zheng DP, Vinje J, Baric RS - PLoS Med. (2008)

Bottom Line: Individuals with defects in the FUT2 gene are termed secretor-negative, do not express the appropriate HBGA necessary for docking, and are resistant to Norwalk infection.Our data suggest that the surface-exposed carbohydrate ligand binding domain in the norovirus capsid is under heavy immune selection and likely evolves by antigenic drift in the face of human herd immunity.Variation in the capsid carbohydrate-binding domain is tolerated because of the large repertoire of similar, yet distinct HBGA carbohydrate receptors available on mucosal surfaces that could interface with the remodeled architecture of the capsid ligand-binding pocket.

View Article: PubMed Central - PubMed

Affiliation: University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA.

ABSTRACT

Background: Noroviruses are the leading cause of viral acute gastroenteritis in humans, noted for causing epidemic outbreaks in communities, the military, cruise ships, hospitals, and assisted living communities. The evolutionary mechanisms governing the persistence and emergence of new norovirus strains in human populations are unknown. Primarily organized by sequence homology into two major human genogroups defined by multiple genoclusters, the majority of norovirus outbreaks are caused by viruses from the GII.4 genocluster, which was first recognized as the major epidemic strain in the mid-1990s. Previous studies by our laboratory and others indicate that some noroviruses readily infect individuals who carry a gene encoding a functional alpha-1,2-fucosyltransferase (FUT2) and are designated "secretor-positive" to indicate that they express ABH histo-blood group antigens (HBGAs), a highly heterogeneous group of related carbohydrates on mucosal surfaces. Individuals with defects in the FUT2 gene are termed secretor-negative, do not express the appropriate HBGA necessary for docking, and are resistant to Norwalk infection. These data argue that FUT2 and other genes encoding enzymes that regulate processing of the HBGA carbohydrates function as susceptibility alleles. However, secretor-negative individuals can be infected with other norovirus strains, and reinfection with the GII.4 strains is common in human populations. In this article, we analyze molecular mechanisms governing GII.4 epidemiology, susceptibility, and persistence in human populations.

Methods and findings: Phylogenetic analyses of the GII.4 capsid sequences suggested an epochal evolution over the last 20 y with periods of stasis followed by rapid evolution of novel epidemic strains. The epidemic strains show a linear relationship in time, whereby serial replacements emerge from the previous cluster. Five major evolutionary clusters were identified, and representative ORF2 capsid genes for each cluster were expressed as virus-like particles (VLPs). Using salivary and carbohydrate-binding assays, we showed that GII.4 VLP-carbohydrate ligand binding patterns have changed over time and include carbohydrates regulated by the human FUT2 and FUT3 pathways, suggesting that strain sensitivity to human susceptibility alleles will vary. Variation in surface-exposed residues and in residues that surround the fucose ligand interaction domain suggests that antigenic drift may promote GII.4 persistence in human populations. Evidence supporting antigenic drift was obtained by measuring the antigenic relatedness of GII.4 VLPs using murine and human sera and demonstrating strain-specific serologic and carbohydrate-binding blockade responses. These data suggest that the GII.4 noroviruses persist by altering their HBGA carbohydrate-binding targets over time, which not only allows for escape from highly penetrant host susceptibility alleles, but simultaneously allows for immune-driven selection in the receptor-binding region to facilitate escape from protective herd immunity.

Conclusions: Our data suggest that the surface-exposed carbohydrate ligand binding domain in the norovirus capsid is under heavy immune selection and likely evolves by antigenic drift in the face of human herd immunity. Variation in the capsid carbohydrate-binding domain is tolerated because of the large repertoire of similar, yet distinct HBGA carbohydrate receptors available on mucosal surfaces that could interface with the remodeled architecture of the capsid ligand-binding pocket. The continuing evolution of new replacement strains suggests that, as with influenza viruses, vaccines could be targeted that protect against norovirus infections, and that continued epidemiologic surveillance and reformulations of norovirus vaccines will be essential in the control of future outbreaks.

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GII.4 VLP–Carbohydrate Binding Patterns at Room TemperatureVLPs were assayed for ability to bind to synthetic biotinylated HBGA bound to avidin-coated plates. The mean optical density is indicated by the line in the box. The upper and lower boundaries of the box represent the maximum and minimum values.(A) VLP binding to core chains including an α-1,2-fucose.(B) VLP binding to either core chains or H antigens modified with the Lewis antigen.(C) VLP binding to A or B antigen trimer.(D) Comparison of binding of GII.4–1987, GII.4–1987 D393G, and GII.4–1997 VLPs to select HBGAs.
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pmed-0050031-g007: GII.4 VLP–Carbohydrate Binding Patterns at Room TemperatureVLPs were assayed for ability to bind to synthetic biotinylated HBGA bound to avidin-coated plates. The mean optical density is indicated by the line in the box. The upper and lower boundaries of the box represent the maximum and minimum values.(A) VLP binding to core chains including an α-1,2-fucose.(B) VLP binding to either core chains or H antigens modified with the Lewis antigen.(C) VLP binding to A or B antigen trimer.(D) Comparison of binding of GII.4–1987, GII.4–1987 D393G, and GII.4–1997 VLPs to select HBGAs.

Mentions: GII2.4–1987, GII.4–1997 and GII.4–2002/2002a demonstrated binding to secretor-positive saliva from individuals of blood types O, A, and B [60], although binding of GII.4–2002a was temperature dependent (Figure 6). These data are consistent with previously published salivary binding data for VLPs from Grimsby strains isolated in 1997 and 1998 [57,68]. GII.4–2002a also bound weakly to some secretor-negative saliva at 37 °C, suggesting that this strain may bind through the Lewis antigens. In contrast, GII.4–2004 and GII.4–2005 strains bound only weakly (2× background) to secretor-positive, blood type B saliva at room temperature. Since saliva is a complex biological fluid containing many carbohydrates in varying amounts depending on both the donor's genetics and sample integrity, this assay cannot positively identify specific carbohydrate binding partners or identify subtle differential binding patterns within the GII.4 VLP panel [69]. However, in agreement with salivary binding assays, synthetic HBGA (Figure S16) binding assays revealed three patterns of carbohydrate binding for the six VLPs (Figures 7 and S17). Although tested, none of the VLPs bound to any of the core chain precursor molecules (unpublished data). The first pattern exhibited by GII.4–1987, GII.4–1997, and GII.4–2002 utilized known FUT2-dependent HBGAs. GII.4–1987 VLPs bound strongly to H type 3 and less well to Ley; GII.4–1997 bound H type 3, but also bound efficiently to Ley; and A and B and GII.4–2002 bound to Ley and less efficiently to H type 3 and B. The second pattern exhibited by GII.4–2002a utilized the Lewis enzyme products Lea and Lex as well as the FUT2-dependent A antigen. GII.4–2002a is the first GII.4 strain reported to bind FUT2-independent products, indicating a possible pathway for infection of secretor-negative individuals. GII.4–2004 and GII.4–2005 did not bind strongly to any of the carbohydrates tested (Figure 7), depicting the third binding pattern. It was particularly interesting that GII.4–1987 D393G had an intermediate binding phenotype between GII.4–1987 and GII.4–1997. Replacement of GII.4–1987 Asp393 with GII.4–1997 Gly393 resulted in the addition of binding to B antigen to GII.4–1987 but not the ability to bind A antigen, indicating that position 393 is important in determining HBGA binding, but that other surrounding residues must also play a role. Concordant with the predicted remodeling of the receptor binding pocket, these data support the hypothesis that sequence variation in and around the second carbohydrate-stabilizing domain of ORF2 alters VLP structure and modulates HBGA binding patterns within a genotype, resulting in changes in VLP–carbohydrate ligand binding over time (Figure 7).


Mechanisms of GII.4 norovirus persistence in human populations.

Lindesmith LC, Donaldson EF, Lobue AD, Cannon JL, Zheng DP, Vinje J, Baric RS - PLoS Med. (2008)

GII.4 VLP–Carbohydrate Binding Patterns at Room TemperatureVLPs were assayed for ability to bind to synthetic biotinylated HBGA bound to avidin-coated plates. The mean optical density is indicated by the line in the box. The upper and lower boundaries of the box represent the maximum and minimum values.(A) VLP binding to core chains including an α-1,2-fucose.(B) VLP binding to either core chains or H antigens modified with the Lewis antigen.(C) VLP binding to A or B antigen trimer.(D) Comparison of binding of GII.4–1987, GII.4–1987 D393G, and GII.4–1997 VLPs to select HBGAs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2235898&req=5

pmed-0050031-g007: GII.4 VLP–Carbohydrate Binding Patterns at Room TemperatureVLPs were assayed for ability to bind to synthetic biotinylated HBGA bound to avidin-coated plates. The mean optical density is indicated by the line in the box. The upper and lower boundaries of the box represent the maximum and minimum values.(A) VLP binding to core chains including an α-1,2-fucose.(B) VLP binding to either core chains or H antigens modified with the Lewis antigen.(C) VLP binding to A or B antigen trimer.(D) Comparison of binding of GII.4–1987, GII.4–1987 D393G, and GII.4–1997 VLPs to select HBGAs.
Mentions: GII2.4–1987, GII.4–1997 and GII.4–2002/2002a demonstrated binding to secretor-positive saliva from individuals of blood types O, A, and B [60], although binding of GII.4–2002a was temperature dependent (Figure 6). These data are consistent with previously published salivary binding data for VLPs from Grimsby strains isolated in 1997 and 1998 [57,68]. GII.4–2002a also bound weakly to some secretor-negative saliva at 37 °C, suggesting that this strain may bind through the Lewis antigens. In contrast, GII.4–2004 and GII.4–2005 strains bound only weakly (2× background) to secretor-positive, blood type B saliva at room temperature. Since saliva is a complex biological fluid containing many carbohydrates in varying amounts depending on both the donor's genetics and sample integrity, this assay cannot positively identify specific carbohydrate binding partners or identify subtle differential binding patterns within the GII.4 VLP panel [69]. However, in agreement with salivary binding assays, synthetic HBGA (Figure S16) binding assays revealed three patterns of carbohydrate binding for the six VLPs (Figures 7 and S17). Although tested, none of the VLPs bound to any of the core chain precursor molecules (unpublished data). The first pattern exhibited by GII.4–1987, GII.4–1997, and GII.4–2002 utilized known FUT2-dependent HBGAs. GII.4–1987 VLPs bound strongly to H type 3 and less well to Ley; GII.4–1997 bound H type 3, but also bound efficiently to Ley; and A and B and GII.4–2002 bound to Ley and less efficiently to H type 3 and B. The second pattern exhibited by GII.4–2002a utilized the Lewis enzyme products Lea and Lex as well as the FUT2-dependent A antigen. GII.4–2002a is the first GII.4 strain reported to bind FUT2-independent products, indicating a possible pathway for infection of secretor-negative individuals. GII.4–2004 and GII.4–2005 did not bind strongly to any of the carbohydrates tested (Figure 7), depicting the third binding pattern. It was particularly interesting that GII.4–1987 D393G had an intermediate binding phenotype between GII.4–1987 and GII.4–1997. Replacement of GII.4–1987 Asp393 with GII.4–1997 Gly393 resulted in the addition of binding to B antigen to GII.4–1987 but not the ability to bind A antigen, indicating that position 393 is important in determining HBGA binding, but that other surrounding residues must also play a role. Concordant with the predicted remodeling of the receptor binding pocket, these data support the hypothesis that sequence variation in and around the second carbohydrate-stabilizing domain of ORF2 alters VLP structure and modulates HBGA binding patterns within a genotype, resulting in changes in VLP–carbohydrate ligand binding over time (Figure 7).

Bottom Line: Individuals with defects in the FUT2 gene are termed secretor-negative, do not express the appropriate HBGA necessary for docking, and are resistant to Norwalk infection.Our data suggest that the surface-exposed carbohydrate ligand binding domain in the norovirus capsid is under heavy immune selection and likely evolves by antigenic drift in the face of human herd immunity.Variation in the capsid carbohydrate-binding domain is tolerated because of the large repertoire of similar, yet distinct HBGA carbohydrate receptors available on mucosal surfaces that could interface with the remodeled architecture of the capsid ligand-binding pocket.

View Article: PubMed Central - PubMed

Affiliation: University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA.

ABSTRACT

Background: Noroviruses are the leading cause of viral acute gastroenteritis in humans, noted for causing epidemic outbreaks in communities, the military, cruise ships, hospitals, and assisted living communities. The evolutionary mechanisms governing the persistence and emergence of new norovirus strains in human populations are unknown. Primarily organized by sequence homology into two major human genogroups defined by multiple genoclusters, the majority of norovirus outbreaks are caused by viruses from the GII.4 genocluster, which was first recognized as the major epidemic strain in the mid-1990s. Previous studies by our laboratory and others indicate that some noroviruses readily infect individuals who carry a gene encoding a functional alpha-1,2-fucosyltransferase (FUT2) and are designated "secretor-positive" to indicate that they express ABH histo-blood group antigens (HBGAs), a highly heterogeneous group of related carbohydrates on mucosal surfaces. Individuals with defects in the FUT2 gene are termed secretor-negative, do not express the appropriate HBGA necessary for docking, and are resistant to Norwalk infection. These data argue that FUT2 and other genes encoding enzymes that regulate processing of the HBGA carbohydrates function as susceptibility alleles. However, secretor-negative individuals can be infected with other norovirus strains, and reinfection with the GII.4 strains is common in human populations. In this article, we analyze molecular mechanisms governing GII.4 epidemiology, susceptibility, and persistence in human populations.

Methods and findings: Phylogenetic analyses of the GII.4 capsid sequences suggested an epochal evolution over the last 20 y with periods of stasis followed by rapid evolution of novel epidemic strains. The epidemic strains show a linear relationship in time, whereby serial replacements emerge from the previous cluster. Five major evolutionary clusters were identified, and representative ORF2 capsid genes for each cluster were expressed as virus-like particles (VLPs). Using salivary and carbohydrate-binding assays, we showed that GII.4 VLP-carbohydrate ligand binding patterns have changed over time and include carbohydrates regulated by the human FUT2 and FUT3 pathways, suggesting that strain sensitivity to human susceptibility alleles will vary. Variation in surface-exposed residues and in residues that surround the fucose ligand interaction domain suggests that antigenic drift may promote GII.4 persistence in human populations. Evidence supporting antigenic drift was obtained by measuring the antigenic relatedness of GII.4 VLPs using murine and human sera and demonstrating strain-specific serologic and carbohydrate-binding blockade responses. These data suggest that the GII.4 noroviruses persist by altering their HBGA carbohydrate-binding targets over time, which not only allows for escape from highly penetrant host susceptibility alleles, but simultaneously allows for immune-driven selection in the receptor-binding region to facilitate escape from protective herd immunity.

Conclusions: Our data suggest that the surface-exposed carbohydrate ligand binding domain in the norovirus capsid is under heavy immune selection and likely evolves by antigenic drift in the face of human herd immunity. Variation in the capsid carbohydrate-binding domain is tolerated because of the large repertoire of similar, yet distinct HBGA carbohydrate receptors available on mucosal surfaces that could interface with the remodeled architecture of the capsid ligand-binding pocket. The continuing evolution of new replacement strains suggests that, as with influenza viruses, vaccines could be targeted that protect against norovirus infections, and that continued epidemiologic surveillance and reformulations of norovirus vaccines will be essential in the control of future outbreaks.

Show MeSH
Related in: MedlinePlus