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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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Related in: MedlinePlus

Phylogenic Reconstruction of the GII.4 Shell Domain and P1 and P2 SubdomainsIndependent multiple alignments were generated using 176 amino acid sequences divided into the S, P1, and P2 regions of the capsid, and trees were generated for each alignment using three different methods, with clusters marked as follows: yellow, sequences from Camberwell cluster; red, sequences from Grimsby cluster; blue, sequences from the Farmington Hills cluster; green, sequences from the Hunter cluster; orange, sequences from the Sakai cluster; and purple, sequences from the Den Haag cluster. Unmarked branches represent sequences that did not group with any specific cluster. The trees are drawn to similar scales and are rooted with the earliest Camberwell cluster.(A) BI of the S domain predicts only two distinct clusters with Camberwell as the first, while everything else groups into a single large cluster (for full tree see Figure S5).(B) The MEGA 4.0 MP tree of the S predicted similar results as the Bayesian tree, although there are two nondistinct clusters arising from the LCA derived from the Camberwell cluster (for full tree see Figure S6). A MP tree generated by PAUP 4.0b10 predicted similar results (Figure S7).(C) BI of the P1 domain predicted that the Camberwell cluster gave rise to the Grimsby cluster from which the Farmington Hills and all later clusters emerged, although the extant clusters were not fully resolved (for full tree see Figure S8).(D) The MEGA 4.0 MP tree of P1 predicted similar results, although it showed that Grimsby gave rise to the Farmington Hills cluster, from which the later clusters emerged. However, the Den Haag cluster falls within the Farmington Hills cluster (for full tree see Figure S9). An MP tree generated by PAUP 4.0b10 predicted similar results (Figure S10).(E) BI of the P2 subdomain predicts that Camberwell gave rise to Grimsby, which in turn gave rise to LCAs from which Farmington Hills and the three extant clusters evolved independently. All six clusters are distinct (for full tree see Figure S11).(F) The MEGA 4.0 MP bootstrapped tree agreed with the Bayesian tree (for full tree see Figure S12).(G) The MP bootstrapped tree generated using PAUP 4.0b10 predicted a nearly identical tree as the Bayesian tree (for full tree see Figure S13). The fact that all three methods generated similar trees with distinct clusters suggests that the P2 subdomain is the most appropriate region with which to determine phylogeny for the GII.4 noroviruses.
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pmed-0050031-g003: Phylogenic Reconstruction of the GII.4 Shell Domain and P1 and P2 SubdomainsIndependent multiple alignments were generated using 176 amino acid sequences divided into the S, P1, and P2 regions of the capsid, and trees were generated for each alignment using three different methods, with clusters marked as follows: yellow, sequences from Camberwell cluster; red, sequences from Grimsby cluster; blue, sequences from the Farmington Hills cluster; green, sequences from the Hunter cluster; orange, sequences from the Sakai cluster; and purple, sequences from the Den Haag cluster. Unmarked branches represent sequences that did not group with any specific cluster. The trees are drawn to similar scales and are rooted with the earliest Camberwell cluster.(A) BI of the S domain predicts only two distinct clusters with Camberwell as the first, while everything else groups into a single large cluster (for full tree see Figure S5).(B) The MEGA 4.0 MP tree of the S predicted similar results as the Bayesian tree, although there are two nondistinct clusters arising from the LCA derived from the Camberwell cluster (for full tree see Figure S6). A MP tree generated by PAUP 4.0b10 predicted similar results (Figure S7).(C) BI of the P1 domain predicted that the Camberwell cluster gave rise to the Grimsby cluster from which the Farmington Hills and all later clusters emerged, although the extant clusters were not fully resolved (for full tree see Figure S8).(D) The MEGA 4.0 MP tree of P1 predicted similar results, although it showed that Grimsby gave rise to the Farmington Hills cluster, from which the later clusters emerged. However, the Den Haag cluster falls within the Farmington Hills cluster (for full tree see Figure S9). An MP tree generated by PAUP 4.0b10 predicted similar results (Figure S10).(E) BI of the P2 subdomain predicts that Camberwell gave rise to Grimsby, which in turn gave rise to LCAs from which Farmington Hills and the three extant clusters evolved independently. All six clusters are distinct (for full tree see Figure S11).(F) The MEGA 4.0 MP bootstrapped tree agreed with the Bayesian tree (for full tree see Figure S12).(G) The MP bootstrapped tree generated using PAUP 4.0b10 predicted a nearly identical tree as the Bayesian tree (for full tree see Figure S13). The fact that all three methods generated similar trees with distinct clusters suggests that the P2 subdomain is the most appropriate region with which to determine phylogeny for the GII.4 noroviruses.

Mentions: Virus proteins contain multiple functional domains, and protein domains are recognized as the units of molecular evolution [61]. Not surprisingly, different domains within a protein may evolve at different rates based on structural and functional constraints of the specific domain, potentially masking informative evolutionary patterns [61]. Analysis of the sequence variation of the three different domains of the GII.4 capsid showed that of the 59 informative sites noted (Table S3), 11 occurred in the S domain (5% of sites; 11/225 S alterations), 18 occurred in the P1 subdomain (11% of sites; 18/166 P1 alterations), and 30 occurred in the P2 subdomain (24% of sites; 30/126 P2 alterations), the surface exposed region of the capsid protein. This observation suggested that the different domains were evolving under different evolutionary constraints. Therefore, we employed a more sophisticated approach whereby the capsid amino acid sequences were divided into the three structurally defined domains and subdomains of S, P1, and P2, and each region was analyzed separately. Analysis of the phylogeny of S, which is the most conserved domain, showed only two predominant clusters represented by the Camberwell-like sequences as one cluster, while the second cluster was composed of all other sequences (Figures 3A, 3B, S5–S7). The P1 domain phylogeny indicated a linear progression from Camberwell to Grimsby to later strains, but the tree did not resolve the evolution of the later clusters of Farmington Hills, Hunter, Sakai, and Den Haag, and more information is necessary to resolve contemporary patterns (Figures 3C, 3D, S8–S10). Intriguingly, the surface-exposed P2 domain phylogeny suggests that the P2 region has evolved in a linear fashion, punctuated by periods of stasis, over the last 20 y in a pattern similar to influenza viruses [64]. Both BI and MP confirmed that the evolution of each cluster was correlated with time, with the Camberwell cluster being near the root of the tree, and the Grimsby cluster having origins in the Camberwell cluster (Figures 3E–3G, S11–S13). Further, the Farmington Hills and all later clusters appeared to have arisen from the Grimsby cluster. Evolution beyond the Farmington Hills cluster is less clear, as it appears that a LCA, which arose from the Grimsby cluster, gave rise to all four later clusters. However, the Bayesian posterior probability at this node was only 57/100 (Figure S11), and an MP (PAUP) bootstrap value was 65/100 (Figure S13), suggesting that there was not enough information to fully define this branching order. Therefore, we computed the predicted ancestral sequence for this node and compared this sequence to the Farmington Hills, Hunter, and Sakai cluster sequences. The LCA sequence was definitively more Farmington Hills-like, which implies that the Farmington Hills cluster is ancestral to the extant clusters (Table S3). This implication further suggests that the GII.4 viruses evolved in a linear manner, with each subsequent cluster giving rise to the next (Figure 1; Table S3) from Camberwell to Farmington Hills.


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)

Phylogenic Reconstruction of the GII.4 Shell Domain and P1 and P2 SubdomainsIndependent multiple alignments were generated using 176 amino acid sequences divided into the S, P1, and P2 regions of the capsid, and trees were generated for each alignment using three different methods, with clusters marked as follows: yellow, sequences from Camberwell cluster; red, sequences from Grimsby cluster; blue, sequences from the Farmington Hills cluster; green, sequences from the Hunter cluster; orange, sequences from the Sakai cluster; and purple, sequences from the Den Haag cluster. Unmarked branches represent sequences that did not group with any specific cluster. The trees are drawn to similar scales and are rooted with the earliest Camberwell cluster.(A) BI of the S domain predicts only two distinct clusters with Camberwell as the first, while everything else groups into a single large cluster (for full tree see Figure S5).(B) The MEGA 4.0 MP tree of the S predicted similar results as the Bayesian tree, although there are two nondistinct clusters arising from the LCA derived from the Camberwell cluster (for full tree see Figure S6). A MP tree generated by PAUP 4.0b10 predicted similar results (Figure S7).(C) BI of the P1 domain predicted that the Camberwell cluster gave rise to the Grimsby cluster from which the Farmington Hills and all later clusters emerged, although the extant clusters were not fully resolved (for full tree see Figure S8).(D) The MEGA 4.0 MP tree of P1 predicted similar results, although it showed that Grimsby gave rise to the Farmington Hills cluster, from which the later clusters emerged. However, the Den Haag cluster falls within the Farmington Hills cluster (for full tree see Figure S9). An MP tree generated by PAUP 4.0b10 predicted similar results (Figure S10).(E) BI of the P2 subdomain predicts that Camberwell gave rise to Grimsby, which in turn gave rise to LCAs from which Farmington Hills and the three extant clusters evolved independently. All six clusters are distinct (for full tree see Figure S11).(F) The MEGA 4.0 MP bootstrapped tree agreed with the Bayesian tree (for full tree see Figure S12).(G) The MP bootstrapped tree generated using PAUP 4.0b10 predicted a nearly identical tree as the Bayesian tree (for full tree see Figure S13). The fact that all three methods generated similar trees with distinct clusters suggests that the P2 subdomain is the most appropriate region with which to determine phylogeny for the GII.4 noroviruses.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2235898&req=5

pmed-0050031-g003: Phylogenic Reconstruction of the GII.4 Shell Domain and P1 and P2 SubdomainsIndependent multiple alignments were generated using 176 amino acid sequences divided into the S, P1, and P2 regions of the capsid, and trees were generated for each alignment using three different methods, with clusters marked as follows: yellow, sequences from Camberwell cluster; red, sequences from Grimsby cluster; blue, sequences from the Farmington Hills cluster; green, sequences from the Hunter cluster; orange, sequences from the Sakai cluster; and purple, sequences from the Den Haag cluster. Unmarked branches represent sequences that did not group with any specific cluster. The trees are drawn to similar scales and are rooted with the earliest Camberwell cluster.(A) BI of the S domain predicts only two distinct clusters with Camberwell as the first, while everything else groups into a single large cluster (for full tree see Figure S5).(B) The MEGA 4.0 MP tree of the S predicted similar results as the Bayesian tree, although there are two nondistinct clusters arising from the LCA derived from the Camberwell cluster (for full tree see Figure S6). A MP tree generated by PAUP 4.0b10 predicted similar results (Figure S7).(C) BI of the P1 domain predicted that the Camberwell cluster gave rise to the Grimsby cluster from which the Farmington Hills and all later clusters emerged, although the extant clusters were not fully resolved (for full tree see Figure S8).(D) The MEGA 4.0 MP tree of P1 predicted similar results, although it showed that Grimsby gave rise to the Farmington Hills cluster, from which the later clusters emerged. However, the Den Haag cluster falls within the Farmington Hills cluster (for full tree see Figure S9). An MP tree generated by PAUP 4.0b10 predicted similar results (Figure S10).(E) BI of the P2 subdomain predicts that Camberwell gave rise to Grimsby, which in turn gave rise to LCAs from which Farmington Hills and the three extant clusters evolved independently. All six clusters are distinct (for full tree see Figure S11).(F) The MEGA 4.0 MP bootstrapped tree agreed with the Bayesian tree (for full tree see Figure S12).(G) The MP bootstrapped tree generated using PAUP 4.0b10 predicted a nearly identical tree as the Bayesian tree (for full tree see Figure S13). The fact that all three methods generated similar trees with distinct clusters suggests that the P2 subdomain is the most appropriate region with which to determine phylogeny for the GII.4 noroviruses.
Mentions: Virus proteins contain multiple functional domains, and protein domains are recognized as the units of molecular evolution [61]. Not surprisingly, different domains within a protein may evolve at different rates based on structural and functional constraints of the specific domain, potentially masking informative evolutionary patterns [61]. Analysis of the sequence variation of the three different domains of the GII.4 capsid showed that of the 59 informative sites noted (Table S3), 11 occurred in the S domain (5% of sites; 11/225 S alterations), 18 occurred in the P1 subdomain (11% of sites; 18/166 P1 alterations), and 30 occurred in the P2 subdomain (24% of sites; 30/126 P2 alterations), the surface exposed region of the capsid protein. This observation suggested that the different domains were evolving under different evolutionary constraints. Therefore, we employed a more sophisticated approach whereby the capsid amino acid sequences were divided into the three structurally defined domains and subdomains of S, P1, and P2, and each region was analyzed separately. Analysis of the phylogeny of S, which is the most conserved domain, showed only two predominant clusters represented by the Camberwell-like sequences as one cluster, while the second cluster was composed of all other sequences (Figures 3A, 3B, S5–S7). The P1 domain phylogeny indicated a linear progression from Camberwell to Grimsby to later strains, but the tree did not resolve the evolution of the later clusters of Farmington Hills, Hunter, Sakai, and Den Haag, and more information is necessary to resolve contemporary patterns (Figures 3C, 3D, S8–S10). Intriguingly, the surface-exposed P2 domain phylogeny suggests that the P2 region has evolved in a linear fashion, punctuated by periods of stasis, over the last 20 y in a pattern similar to influenza viruses [64]. Both BI and MP confirmed that the evolution of each cluster was correlated with time, with the Camberwell cluster being near the root of the tree, and the Grimsby cluster having origins in the Camberwell cluster (Figures 3E–3G, S11–S13). Further, the Farmington Hills and all later clusters appeared to have arisen from the Grimsby cluster. Evolution beyond the Farmington Hills cluster is less clear, as it appears that a LCA, which arose from the Grimsby cluster, gave rise to all four later clusters. However, the Bayesian posterior probability at this node was only 57/100 (Figure S11), and an MP (PAUP) bootstrap value was 65/100 (Figure S13), suggesting that there was not enough information to fully define this branching order. Therefore, we computed the predicted ancestral sequence for this node and compared this sequence to the Farmington Hills, Hunter, and Sakai cluster sequences. The LCA sequence was definitively more Farmington Hills-like, which implies that the Farmington Hills cluster is ancestral to the extant clusters (Table S3). This implication further suggests that the GII.4 viruses evolved in a linear manner, with each subsequent cluster giving rise to the next (Figure 1; Table S3) from Camberwell to Farmington Hills.

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