Eric F. Donaldson

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.

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.

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.

A decade after SARS: strategies for controlling emerging coronaviruses

Two novel coronaviruses have emerged in humans in the twenty-first century: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), both of which cause acute respiratory distress syndrome (ARDS) and are associated with high mortality rates. There are no clinically approved vaccines or antiviral drugs available for either of these infections; thus, the development of effective therapeutic and preventive strategies that can be readily applied to new emergent strains is a research priority. In this Review, we describe the emergence and identification of novel human coronaviruses over the past 10 years, discuss their key biological features, including tropism and receptor use, and summarize approaches for developing broadly effective vaccines.

Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice

Defining prospective pathways by which zoonoses evolve and emerge as human pathogens is critical for anticipating and controlling both natural and deliberate pandemics. However, predicting tenable pathways of animal-to-human movement has been hindered by challenges in identifying reservoir species, cultivating zoonotic organisms in culture, and isolating full-length genomes for cloning and genetic studies. The ability to design and recover pathogens reconstituted from synthesized cDNAs has the potential to overcome these obstacles by allowing studies of replication and pathogenesis without identification of reservoir species or cultivation of primary isolates. Here, we report the design, synthesis, and recovery of the largest synthetic replicating life form, a 29.7-kb bat severe acute respiratory syndrome (SARS)-like coronavirus (Bat-SCoV), a likely progenitor to the SARS-CoV epidemic. To test a possible route of emergence from the noncultivable Bat-SCoV to human SARS-CoV, we designed a consensus Bat-SCoV genome and replaced the Bat-SCoV Spike receptor-binding domain (RBD) with the SARS-CoV RBD (Bat-SRBD). Bat-SRBD was infectious in cell culture and in mice and was efficiently neutralized by antibodies specific for both bat and human CoV Spike proteins. Rational design, synthesis, and recovery of hypothetical recombinant viruses can be used to investigate mechanisms of transspecies movement of zoonoses and has great potential to aid in rapid public health responses to known or predicted emerging microbial threats.

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.

Emergence of New Pandemic GII.4 Sydney Norovirus Strain Correlates with Escape from Herd Immunity

BACKGROUND GII.4 noroviruses are a significant source of acute gastroenteritis worldwide, causing the majority of human norovirus outbreaks. Evolution of the GII.4 major capsid protein occurs rapidly, resulting in the emergence of new strains that produce successive waves of pandemic disease. A new pandemic isolate, GII.4 2012 Sydney, largely replaced previously circulating strains in late 2012. We compare the antigenic properties of GII.4 2012 Sydney with previously circulating strains. METHODS To determine whether GII.4-2012 Sydney is antigenically different from recently circulating strains GII.4-2006 Minerva and GII.4-2009 New Orleans in previously identified blockade epitopes, we compared reactivity and blockade profiles of GII.4-2006, GII.4-2009, and GII.4-2012 virus-like particles in surrogate neutralization/blockade assays using monoclonal antibodies and human polyclonal sera. RESULTS Using monoclonal antibodies that map to known blockade epitopes in GII.4-2006 and GII.4-2009 and human outbreak polyclonal sera, we demonstrate either complete loss or significantly reduced reactivity and blockade of GII.4.2012 compared to GII.4-2006 and GII.4-2009. CONCLUSIONS GII.4-2012 Sydney is antigenically different from GII.4-2006 Minerva and GII.4-2009 New Orleans in at least 2 key blockade epitopes. Viral evolution in key potential neutralization epitopes likely allowed GII.4-2012 to escape from human herd immunity and emerge as the new predominant strain.

Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus

Significance The identification of a novel, emerging human coronavirus with ∼50% mortality, designated Middle East respiratory syndrome coronavirus (MERS-CoV), emphasizes the importance of the rapid development of reagents that can be used to (i) characterize the replication and pathogenesis of emerging pathogens and (ii) develop therapeutics for treatment. In this report, we describe the development of a cassette-based infectious cDNA clone of MERS-CoV and verify that it functions similarly to the wild-type isolate in terms of replication, protein and RNA expression, and spike attachment protein processing. We also show that the virus replicates preferentially in differentiated primary lung cells. Severe acute respiratory syndrome with high mortality rates (∼50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ∼0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3–5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3–5 showed 1–1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.

Genetic Mapping of a Highly Variable Norovirus GII.4 Blockade Epitope: Potential Role in Escape from Human Herd Immunity

ABSTRACT Noroviruses account for 96% of viral gastroenteritis cases worldwide, with GII.4 strains responsible >80% of norovirus outbreaks. Histo-blood group antigens (HBGAs) are norovirus binding ligands, and antigenic and preferential HBGA binding profiles vary over time as new GII.4 strains emerge. The capsid P2 subdomain facilitates HBGA binding, contains neutralizing antibody epitopes, and likely evolves in response to herd immunity. To identify amino acids regulating HBGA binding and antigenic differences over time, we created chimeric virus-like particles (VLPs) between the GII.4-1987 and GII.4-2006 strains by exchanging amino acids in putative epitopes and characterized their antigenic and HBGA binding profiles using anti-GII.4-1987 and -2006 mouse monoclonal antibodies (MAbs) and polyclonal sera, 1988 outbreak human sera, and synthetic HBGAs. The exchange of amino acids 393 to 395 between GII.4-1987 and GII.4-2006 resulted in altered synthetic HBGA binding compared to parental strains. Introduction of GII.4-1987 residues 294, 297 to 298, 368, and 372 (epitope A) into GII.4-2006 resulted in reactivity with three anti-GII.4-1987 MAbs and reduced reactivity with four anti-GII.4-2006 MAbs. The three anti-GII.4-1987 MAbs also blocked chimeric VLP-HBGA interaction, while an anti-GII.4-2006 blocking antibody did not, indicating that epitope A amino acids comprise a potential neutralizing epitope for GII.4-1987 and GII.4-2006. We also tested GII.4-1987-immunized mouse polyclonal sera and 1988 outbreak human sera for the ability to block chimeric VLP-HBGA interaction and found that epitope A amino acids contribute significantly to the GII.4-1987 blockade response. Our data provide insights that help explain the emergence of new GII.4 epidemic strains over time, may aid development of norovirus therapeutics, and may help predict the emergence of future epidemic strains.