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.
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.
Through phylogenetic analysis of norovirus isolates, Ralph Baric and colleagues show that new epidemic strains arise as the variety of available cellular receptors permits antigenic drift in the viral capsid.
Noroviruses are the leading cause of viral gastroenteritis (stomach flu), the symptoms of which include nausea, vomiting, and diarrhea. There is no treatment for infection with these highly contagious viruses. While most people recover within a few days, the very young and old may experience severe disease. Like influenza, large outbreaks (epidemics) of norovirus infection occur periodically (often in closed communities such as cruise ships), and most people have several norovirus infections during their lifetime. Currently, 100,000–200,000 people are being infected each week in England with a new GII.4 variant. There are several reasons for this pattern of infection and reinfection. First, the immune response induced by a norovirus infection is short-lived in some people, but not all. Second, there are many different noroviruses. Based on their genomes (genetic blueprints), noroviruses belong to five “genogroups,” which are further subdivided into “genotypes.” An immune response to one norovirus provides little protection against noroviruses of other genogroups or genotypes. Third, like influenza viruses, noroviruses frequently acquire small changes in their genome. This process is called antigenic drift (antigens are the molecules on the surface of infectious agents that stimulate the production of antibodies, proteins that help the immune system recognize and deal with foreign invaders). Norovirus epidemics occur when virus variants emerge to which the human population has no immunity.
Why Was This Study Done?
It is unknown exactly how noroviruses change over time or how they persist in human populations. In addition, little is known about susceptibility to norovirus infections except that secretor-positive individuals—people who express “histoblood group antigens” (HBGAs, a heterogeneous group of sugar molecules by which noroviruses attach themselves to human cells) on the cells that line their mouths and guts—are more susceptible than secretor-negative people, who express these antigens only on red blood cells. Information of this sort is needed to devise effective intervention strategies, therapies, and vaccines to reduce the illness and economic costs associated with norovirus outbreaks. In this study, the researchers investigate the molecular mechanisms governing the emergence and persistence of epidemic norovirus strains in human populations by analyzing how GII.4 norovirus strains (the genotype usually associated with epidemics) have changed over time.
What Did the Researchers Do and Find?
The researchers analyzed the relationships among the sequences of the gene encoding the capsid protein of GII.4 norovirus strains isolated over the past 20 years. The capsid protein forms a shell around noroviruses and is involved in their binding to HBGAs and their recognition by the human immune system. The researchers found that the virus evolved in fits and starts. That is, for several years, one cluster of strains was predominant but then new epidemic strains emerged rapidly from the cluster. In all, the researchers identified five major evolutionary clusters. They then created “virus-like particles” (VLPs) using representative capsid genes from each cluster and showed that these VLPs bound to different HBGAs. Finally they measured the antigenic relatedness of the different VLPs using human blood collected during a 1988 GII.4 outbreak. Antibodies in these samples recognized the VLPs representing early GII.4 strains better than VLPs representing recent GII.4 strains. The ability of the blood samples to block the interaction of VLPs with their matching HBGAs showed a similar pattern.
What Do These Findings Mean?
These findings suggest that the part of the norovirus capsid protein that binds to sugars on host cells is under heavy immune selection and evolves over time by antigenic drift. They show that, like influenza viruses, GII.4 viruses evolve through serial changes in the capsid sequence that occur sporadically after periods of stability, probably to evade the build up of immunity within the human population. Variation in this region of the viral genome is possible because human populations express a great variety of HBGA molecules so there is always likely to be a subpopulation of people that is susceptible to the altered virus. Overall, these findings suggest that it should be possible to develop vaccines to protect against norovirus infections but, just as with influenza virus, surveillance systems will have to monitor how the virus is changing and vaccines will need to be reformulated frequently to provide effective protection against norovirus outbreaks.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050031.
See a related PLoS Medicine Perspective article
The MedlinePlus encyclopedia has a page on viral gastroenteritis (in English and Spanish)
The US Centers for Disease Control and Prevention provides information on viral gastroenteritis (in English and Spanish) and on noroviruses
The UK National Health Service's health website (NHS Direct) provides information about noroviruses
The UK Health Protection Agency and the US Food & Drug Administration also provide information about noroviruses