Lisa C. Lindesmith

Norwalk virus: How infectious is it?†

Noroviruses are major agents of viral gastroenteritis worldwide. The infectivity of Norwalk virus, the prototype norovirus, has been studied in susceptible human volunteers. A new variant of the hit theory model of microbial infection was developed to estimate the variation in Norwalk virus infectivity, as well as the degree of virus aggregation, consistent with independent (electron microscopic) observations. Explicit modeling of viral aggregation allows us to express virus infectivity per single infectious unit (particle). Comparison of a primary and a secondary inoculum showed that passage through a human host does not change Norwalk virus infectivity. We estimate the average probability of infection for a single Norwalk virus particle to be close to 0.5, exceeding that reported for any other virus studied to date. Infected subjects had a dose‐dependent probability of becoming ill, ranging from 0.1 (at a dose of 103 NV genomes) to 0.7 (at 108 virus genomes). A norovirus dose response model is important for understanding its transmission and essential for development of a quantitative risk model. Norwalk virus is a valuable model system to study virulence because genetic factors are known for both complete and partial protection; the latter can be quantitatively described as heterogeneity in dose response models. J. Med. Virol. 80:1468–1476, 2008.

Human susceptibility and resistance to Norwalk virus infection

Infectious diseases have influenced population genetics and the evolution of the structure of the human genome in part by selecting for host susceptibility alleles that modify pathogenesis. Norovirus infection is associated with ∼90% of epidemic non-bacterial acute gastroenteritis worldwide. Here, we show that resistance to Norwalk virus infection is multifactorial. Using a human challenge model, we showed that 29% of our study population was homozygous recessive for the α(1,2)fucosyltransferase gene (FUT2) in the ABH histo-blood group family and did not express the H type-1 oligosaccharide ligand required for Norwalk virus binding. The FUT2 susceptibility allele was fully penetrant against Norwalk virus infection as none of these individuals developed an infection after challenge, regardless of dose. Of the susceptible population that encoded a functional FUT2 gene, a portion was resistant to infection, suggesting that a memory immune response or some other unidentified factor also affords protection from Norwalk virus infection.

Mechanisms of GII.4 norovirus persistence in human populations.

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.

Binding of Norwalk Virus-Like Particles to ABH Histo-Blood Group Antigens Is Blocked by Antisera from Infected Human Volunteers or Experimentally Vaccinated Mice

ABSTRACT Attachment of Norwalk (NV), Snow Mountain (SMV), and Hawaii (HV) virus-like particles (VLPs) to specific ABH histo-blood group antigens was investigated by using human saliva and synthetic biotinylated carbohydrates. The three distinct Norwalk-like viruses (NLVs) have various capacities for binding ABH histo-blood group antigens, suggesting that different mechanisms for NLV attachment likely exist. Importantly, antisera from NV-infected human volunteers, as well as from mice inoculated with packaged Venezuelan equine encephalitis virus replicons expressing NV VLPs, blocked the ability of NV VLPs to bind synthetic H type 1, Leb, and H type 3, suggesting a potential mechanism for antibody-mediated neutralization of NV.

Norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations

Summary: Noroviruses are important human pathogens known to cause epidemic outbreaks of severe gastroenteritis in communities, military barracks, cruise ships, hospitals, and assisted living communities, resulting in over 267 000 000 annual infections worldwide. Diversity within the norovirus genus allows this virus to persist in human populations, although a single genocluster, the GII.4 noroviruses, currently accounts for ∼80% of all infections. Noroviruses bind to the polymorphic histoblood group antigens (HBGAs), which act as the putative cellular receptor, and strains from different genoclusters bind various HBGAs. Human challenge studies using viruses from different genoclusters have demonstrated that norovirus immunity is complicated and probably confounded by pre‐existing exposure histories and variable immune responses. Evidence for both short‐term and long‐term immunity has been demonstrated, but the molecular mechanisms mediating differential immune responses in the face of infection remain unclear. Studies with virus‐like particles from the GII.4 genocluster demonstrated that variation in and around the receptor‐binding domain results in differential HBGA binding and altered antigenicity. These observations suggest that the norovirus capsid evolves to evade the memory immune response while retaining its ability to bind any of several HBGAs. In this review, we discuss how evolution within the capsid drives receptor switching and allows escape from herd immunity.

Viral shape-shifting: norovirus evasion of the human immune system

Noroviruses are the most common cause of food-borne gastroenteritis worldwide, and explosive outbreaks frequently occur in community settings, where the virus can immobilize large numbers of infected individuals for 24–48 hours, making the development of effective vaccines and antiviral therapies a priority. However, several challenges have hampered therapeutic design, including: the limitations of cell culture and small-animal model systems; the complex effects of host pre-exposure histories; differential host susceptibility, which is correlated with blood group and secretor status; and the evolution of novel immune escape variants. In this Review, we discuss the molecular and structural mechanisms that facilitate the persistence of noroviruses in human populations.

Cellular and humoral immunity following snow mountain virus challenge

ABSTRACT Little is known about the immune response to noroviruses. To elucidate the immunobiology of norovirus infection in humans, 15 volunteers were challenged with Snow Mountain virus (SMV), a genogroup 2 norovirus. We assessed the cellular and humoral immune response and infection by analyzing stool, serum, saliva, and peripheral blood mononuclear cell (PBMC) responses pre- and postchallenge. In contrast to Norwalk virus (NV), SMV infection was not dependent upon blood group secretor status. Nine of 15 volunteers were infected and showed a ≥4-fold increase over the prechallenge anti-SMV serum immunoglobulin G (IgG) titer, mostly subclass IgG1. Although serum IgG elicited by SMV infection was cross-reactive with Hawaii virus (HV), another genogroup 2 norovirus, salivary IgA was less cross-reactive. Neither SMV-elicited serum IgG nor salivary IgA cross-reacted with NV, a genogroup 1 norovirus. Significant increases in serum gamma interferon (IFN-γ) and IL-2, but not IL-6 or IL-10, were noted on day 2 postchallenge. For the majority of volunteers, both infected and uninfected, PBMCs stimulated with norovirus virus-like particles secreted IFN-γ and other Th1 cytokines, suggesting previous norovirus exposure in most volunteers. Like the IgG antibodies, the SMV-activated T cells were cross-reactive with HV but not NV. IFN-γ production was dependent upon CD4+ cells, consistent with a predominant, but not exclusive, Th1 response. To our knowledge, this is the first report characterizing T-cell and cytokine responses following live norovirus challenge.

Immunogenetic Mechanisms Driving Norovirus GII.4 Antigenic Variation

Noroviruses are the principal cause of epidemic gastroenteritis worldwide with GII.4 strains accounting for 80% of infections. The major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered antigenic potentials. To test if antigenic drift may contribute to GII.4 persistence, human memory B cells were immortalized and the resulting human monoclonal antibodies (mAbs) characterized for reactivity to a panel of time-ordered GII.4 virus-like particles (VLPs). Reflecting the complex exposure history of the volunteer, human anti-GII.4 mAbs grouped into three VLP reactivity patterns; ancestral (1987–1997), contemporary (2004–2009), and broad (1987–2009). NVB 114 reacted exclusively to the earliest GII.4 VLPs by EIA and blockade. NVB 97 specifically bound and blocked only contemporary GII.4 VLPs, while NBV 111 and 43.9 exclusively reacted with and blocked variants of the GII.4.2006 Minerva strain. Three mAbs had broad GII.4 reactivity. Two, NVB 37.10 and 61.3, also detected other genogroup II VLPs by EIA but did not block any VLP interactions with carbohydrate ligands. NVB 71.4 cross-neutralized the panel of time-ordered GII.4 VLPs, as measured by VLP-carbohydrate blockade assays. Using mutant VLPs designed to alter predicted antigenic epitopes, two evolving, GII.4-specific, blockade epitopes were mapped. Amino acids 294–298 and 368–372 were required for binding NVB 114, 111 and 43.9 mAbs. Amino acids 393–395 were essential for binding NVB 97, supporting earlier correlations between antibody blockade escape and carbohydrate binding variation. These data inform VLP vaccine design, provide a strategy for expanding the cross-blockade potential of chimeric VLP vaccines, and identify an antibody with broadly neutralizing therapeutic potential for the treatment of human disease. Moreover, these data support the hypothesis that GII.4 norovirus evolution is heavily influenced by antigenic variation of neutralizing epitopes and consequently, antibody-driven receptor switching; thus, protective herd immunity is a driving force in norovirus molecular evolution.

Norovirus Infectivity in Humans and Persistence in Water

ABSTRACT To examine the long-term infectivity of human norovirus in water, 13 study subjects were challenged at different time points with groundwater spiked with the prototype human norovirus, Norwalk virus. Norwalk virus spiked in groundwater remained infectious after storage at room temperature in the dark for 61 days (the last time point tested). The Norwalk virus-seeded groundwater was stored for 1,266 days and analyzed, after RNase treatment, by reverse transcription-quantitative PCR (RT-qPCR) to detect Norwalk virus RNA contained within intact capsids. Norwalk virus RNA within intact capsids was detected in groundwater for 1,266 days, with no significant log10 reduction throughout 427 days and a significant 1.10-log10 reduction by day 1266. Purified Norwalk virus RNA (extracted from Norwalk virus virions) persisted for 14 days in groundwater, tap water, and reagent-grade water. This study demonstrates that Norwalk virus in groundwater can remain detectable for over 3 years and can remain infectious for at least 61 days. (ClinicalTrials.gov identifier NCT00313404.)

Norovirus GII.4 strain antigenic variation.

ABSTRACT Noroviruses are the principal cause of epidemic gastroenteritis worldwide. Multiple reports have concluded that the major capsid proteins of GII.4 strains, which cause 80% of norovirus infections worldwide, are evolving rapidly, resulting in new epidemic strains. Surrogate neutralization assays using sera from outbreaks and from immunized mice suggest that, as with influenza virus, antigenic variation maintains GII.4 persistence in the face of human population herd immunity. To test this hypothesis, mice were hyperimmunized with virus-like particles (VLPs) representing an early (GII.4-1987) and a contemporary (GII.4-2006) GII.4 strain. Anti-GII.4-1987 IgG monoclonal antibodies (MAbs) strongly reacted with GII.4 VLPs derived between only 1987 and 2002. Ligand binding blockade was more efficient with GII.4-1987 and GII.4-1997 VLPs than with GII.4-2002. Anti-GII.4-2006 IgG MAbs recognized either a broad panel of GII.4 VLPs (1987 to 2006) or a subset of contemporary (2004 to 2006) VLPs. Most 2006 antibodies did not recognize or only poorly recognized GII.4 VLPs of 2007 or 2008, documenting rapid antigenic evolution of GII.4 capsids. Generally, 2006 MAbs blocked homotypic VLP-ligand binding but were unable to block VLPs representing strains primarily circulating during or earlier than 2002. These analyses demonstrate that both subtle and significant evolutionary change has occurred within antibody epitopes between epidemic strains, providing direct evidence that the GII.4 noroviruses are undergoing antigenic variation, likely in response to herd immunity. As with influenza virus, HIV, and hepatitis C virus, norovirus antigenic variation will significantly influence the design of efficacious vaccines and immunotherapeutics against these important human pathogens.