Multiple antigenic sites are involved in blocking the interaction of GII.4 norovirus capsid with ABH histo-blood group antigens

Noroviruses are major etiological agents of acute viral gastroenteritis. In 2002, a GII.4 variant (Farmington Hills cluster) spread so rapidly in the human population that it predominated worldwide and displaced previous GII.4 strains. We developed and characterized a panel of six monoclonal antibodies (MAbs) directed against the capsid protein of a Farmington Hills-like GII.4 norovirus strain that was associated with a large hospital outbreak in Maryland in 2004. The six MAbs reacted with high titers against homologous virus-like particles (VLPs) by enzyme-linked immunoassay but did not react with denatured capsid protein in immunoblots. The expression and self-assembly of newly developed genogroup I/II chimeric VLPs showed that five MAbs bound to the GII.4 protruding (P) domain of the capsid protein, while one recognized the GII.4 shell (S) domain. Cross-competition assays and mutational analyses showed evidence for at least three distinct antigenic sites in the P domain and one in the S domain. MAbs that mapped to the P domain but not the S domain were able to block the interaction of VLPs with ABH histo-blood group antigens (HBGA), suggesting that multiple antigenic sites of the P domain are involved in HBGA blocking. Further analysis showed that two MAbs mapped to regions of the capsid that had been associated with the emergence of new GII.4 variants. Taken together, our data map antibody and HBGA carbohydrate binding to proximal regions of the norovirus capsid, showing that evolutionary pressures on the norovirus capsid protein may affect both antigenic and carbohydrate recognition phenotypes.     

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.     

Genetic mapping of a highly variable norovirus GII.4 blockade epitope: potential role in escape from human herd immunity

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.     

Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica)

Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin–magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.     

Binding patterns of human norovirus-like particles to buccal and intestinal tissues of gnotobiotic pigs in relation to A/H histo-blood group antigen expression

Histo-blood group antigen (HBGA) phenotypes have been associated with susceptibility to human noroviruses (HuNoVs). Our aims were: (i) to determine the patterns of A/H HBGA expression in buccal and intestinal tissues of gnotobiotic (Gn) pigs; (ii) to determine if virus-like particles (VLPs) of HuNoV genogroup I (GI) and GII bind to A- or H-type tissues; (iii) to compare A/H expression and VLP binding patterns and confirm their binding specificities by blocking assays; (iv) to develop a hemagglutination inhibition test using buccal cells from live pigs to determine the Gn pig's A/H phenotype and to match viral strains with previously determined HuNoV VLP binding specificities; and (v) to determine the A/H phenotypes and compare these data to the infection outcomes of a previous study of 65 Gn pigs inoculated with HuNoV GII/4 strain HS66 and expressing A and/or H or neither antigen on their buccal and intestinal tissues (S. Cheetham, M. Souza, T. Meulia, S. Grimes, M. G. Han, and L. J. Saif, J. Virol. 80:10372-10381, 2006). We found that the HuNoV GI/GII VLPs of different clusters bound to tissues from four pigs tested (two A+ and two H+). The GI/1 and GII/4 VLPs bound extensively to duodenal and buccal tissues from either A+ or H+ pigs, but surprisingly, GII/1 and GII/3 VLPs bound minimally to the duodenum of an A+ pig. The VLP binding was partially inhibited by A-, H1-, or H2-specific monoclonal antibodies, but was completely blocked by porcine mucin. Comparing the A/H phenotypes of 65 HS66-inoculated Gn pigs from our previous study, we found that significantly more A+ and H(+) pigs (51%) than non-A+ and non-H+ pigs (12.5%) shed virus. From the 22 convalescent pigs, significantly more A+ or H+ pigs (66%) than non-A+ or H+ pigs (25%) seroconverted.     

Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability

Noroviruses are the dominant cause of outbreaks of gastroenteritis worldwide, and interactions with human histo-blood group antigens (HBGAs) are thought to play a critical role in their entry mechanism. Structures of noroviruses from genogroups GI and GII in complex with HBGAs, however, reveal different modes of interaction. To gain insight into norovirus recognition of HBGAs, we determined crystal structures of norovirus protruding domains from two rarely detected GII genotypes, GII.10 and GII.12, alone and in complex with a panel of HBGAs, and analyzed structure-function implications related to conservation of the HBGA binding pocket. The GII.10- and GII.12-apo structures as well as the previously solved GII.4-apo structure resembled each other more closely than the GI.1-derived structure, and all three GII structures showed similar modes of HBGA recognition. The primary GII norovirus-HBGA interaction involved six hydrogen bonds between a terminal αfucose1-2 of the HBGAs and a dimeric capsid interface, which was composed of elements from two protruding subdomains. Norovirus interactions with other saccharide units of the HBGAs were variable and involved fewer hydrogen bonds. Sequence analysis revealed a site of GII norovirus sequence conservation to reside under the critical αfucose1-2 and to be one of the few patches of conserved residues on the outer virion-capsid surface. The site was smaller than that involved in full HBGA recognition, a consequence of variable recognition of peripheral saccharides. Despite this evasion tactic, the HBGA site of viral vulnerability may provide a viable target for small molecule- and antibody-mediated neutralization of GII norovirus.     

Llama Nanoantibodies with Therapeutic Potential against Human Norovirus Diarrhea

Noroviruses are a major cause of acute gastroenteritis, but no vaccines or therapeutic drugs are available. Llama-derived single chain antibody fragments (also called VHH) are small, recombinant monoclonal antibodies of 15 kDa with several advantages over conventional antibodies. The aim of this study was to generate recombinant monoclonal VHH specific for the two major norovirus (NoV) genogroups (GI and GII) in order to investigate their potential as immunotherapy for the treatment of NoV diarrhea. To accomplish this objective, two llamas were immunized with either GI.1 (Norwalk-1968) or GII.4 (MD2004) VLPs. After immunization, peripheral blood lymphocytes were collected and used to generate two VHH libraries. Using phage display technology, 10 VHH clones specific for GI.1, and 8 specific for GII.4 were selected for further characterization. All VHH recognized conformational epitopes in the P domain of the immunizing VP1 capsid protein, with the exception of one GII.4 VHH that recognized a linear P domain epitope. The GI.1 VHHs were highly specific for the immunizing GI.1 genotype, with only one VHH cross-reacting with GI.3 genotype. The GII.4 VHHs reacted with the immunizing GII.4 strain and showed a varying reactivity profile among different GII genotypes. One VHH specific for GI.1 and three specific for GII.4 could block the binding of homologous VLPs to synthetic HBGA carbohydrates, saliva, and pig gastric mucin, and in addition, could inhibit the hemagglutination of red blood cells by homologous VLPs. The ability of Nov-specific VHHs to perform well in these surrogate neutralization assays supports their further development as immunotherapy for NoV treatment and immunoprophylaxis.     

Selection,Characterization and Application of Nucleic Acid Aptamers for the Capture and Detection of Human Norovirus Strains

Human noroviruses (HuNoV) are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ss)DNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV). Four aptamer candidates (designated 19, 21, 25 and 26) were identified and screened for binding affinity to 14 different virus-like particles (VLPs) corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA). Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV) and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC) method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5–36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.     

Antiviral Activity of Gold/Copper Sulfide Core/Shell Nanoparticles against Human Norovirus Virus-Like Particles

Human norovirus is a leading cause of acute gastroenteritis worldwide in a plethora of residential and commercial settings, including restaurants, schools, and hospitals. Methods for easily detecting the virus and for treating and preventing infection are critical to stopping norovirus outbreaks, and inactivation via nanoparticles (NPs) is a more universal and attractive alternative to other physical and chemical approaches. Using norovirus GI.1 (Norwalk) virus-like particles (VLPs) as a model viral system, this study characterized the antiviral activity of Au/CuS core/shell nanoparticles (NPs) against GI.1 VLPs for the rapid inactivation of HuNoV. Inactivation of VLPs (GI.1) by Au/CuS NPs evaluated using an absorbance-based ELISA indicated that treatment with 0.083 μM NPs for 10 min inactivated ~50% VLPs in a 0.37 μg/ml VLP solution and 0.83 μM NPs for 10 min completely inactivated the VLPs. Increasing nanoparticle concentration and/or VLP-NP contact time significantly increased the virucidal efficacy of Au/CuS NPs. Changes to the VLP particle morphology, size, and capsid protein were characterized using dynamic light scattering, transmission electron microscopy, and Western blot analysis. The strategy reported here provides the first reported proof-of-concept Au/CuS NPs-based virucide for rapidly inactivating human norovirus.     

Vesicular stomatitis virus as a vector to deliver virus-like particles of human norovirus: a new vaccine candidate against an important noncultivable virus

Human norovirus (HuNoV) is a major causative agent of food-borne gastroenteritis worldwide. Currently, there are no vaccines or effective therapeutic interventions for this virus. Development of an attenuated vaccine for HuNoV has been hampered by the inability to grow the virus in cell culture. Thus, a vector-based vaccine may be ideal. In this study, we constructed a recombinant vesicular stomatitis virus (rVSV-VP1) expressing VP1, the major capsid protein of HuNoV. Expression of the capsid protein by VSV resulted in the formation of HuNoV virus-like particles (VLPs) that are morphologically and antigenically similar to native virions. Recombinant rVSV-VP1 was attenuated in cultured mammalian cells as well as in mice. Mice inoculated with a single dose of rVSV-VP1 through intranasal and oral routes stimulated a significantly stronger humoral and cellular immune response than baculovirus-expressed VLP vaccination. Moreover, we demonstrated that mice inoculated with rVSV-VP1 triggered a comparable level of fecal and vaginal IgA antibody. Taken together, the VSV recombinant system not only provides a new approach to generate HuNoV VLPs in vitro but also a new avenue for the development of vectored vaccines against norovirus and other noncultivable viruses.     

Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces

Inanimate surfaces are regarded as key vehicles for the spread of human norovirus during outbreaks. ISO method 15216 involves the use of cotton swabs for environmental sampling from food surfaces and fomites for the detection of norovirus genogroup I (GI) and GII. We evaluated the effects of the virus drying time (1, 8, 24, or 48 h), swab material (cotton, polyester, rayon, macrofoam, or an antistatic wipe), surface (stainless steel or a toilet seat), and area of the swabbed surface (25.8 cm² to 645.0 cm²) on the recovery of human norovirus. Macrofoam swabs produced the highest rate of recovery of norovirus from surfaces as large as 645 cm². The rates of recovery ranged from 2.2 to 36.0% for virus seeded on stainless-steel coupons (645.0 cm²) to 1.2 to 33.6% for toilet seat surfaces (700 cm²), with detection limits of 3.5 log₁₀ and 4.0 log₁₀ RNA copies. We used macrofoam swabs to collect environmental samples from several case cabins and common areas of a cruise ship where passengers had reported viral gastroenteritis symptoms. Seventeen (18.5%) of 92 samples tested positive for norovirus GII, and 4 samples could be sequenced and had identical GII.1 sequences. The viral loads of the swab samples from the cabins of the sick passengers ranged from 80 to 31,217 RNA copies, compared with 16 to 113 RNA copies for swab samples from public spaces. In conclusion, our swab protocol for norovirus may be a useful tool for outbreak investigations when no clinical samples are available to confirm the etiology.     

Chlorine inactivation of human norovirus,murine norovirus and poliovirus in drinking water

Aims: To evaluate the reduction of human norovirus (HuNoV) by chlorine disinfection under typical drinking water treatment conditions. Methods and Results: HuNoV, murine norovirus (MNV) and poliovirus type 1 (PV1) were inoculated into treated water before chlorination, collected from a drinking water treatment plant, and bench‐scale free chlorine disinfection experiments were performed for two initial free chlorine concentrations, 0·1 and 0·5 mg l^?1. Inactivation of MNV reached more than 4 log₁₀ after 120 and 0·5 min contact time to chlorine at the initial free chlorine concentrations of 0·1 and 0·5 mg l^?1, respectively. Conclusions: MNV was inactivated faster than PV1, and there was no significant difference in the viral RNA reduction rate between HuNoV and MNV. The results suggest that appropriate water treatment process with chlorination can manage the risk of HuNoV infection via drinking water supply systems. Significance and Impact of the Study: The data obtained in this study would be useful for assessing or managing the risk of HuNoV infections from drinking water exposure.     

Norovirus GII.4 strain antigenic variation

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.     

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.     

High-pressure inactivation of human norovirus virus-like particles provides evidence that the capsid of human norovirus is highly pressure resistant

Human norovirus (NoV) is the leading cause of nonbacterial acute gastroenteritis epidemics worldwide. High-pressure processing (HPP) has been considered a promising nonthermal processing technology to inactivate food- and waterborne viral pathogens. Due to the lack of an effective cell culture method for human NoV, the effectiveness of HPP in inactivating human NoV remains poorly understood. In this study, we evaluated the effectiveness of HPP in disrupting the capsid of human NoV based on the structural and functional integrity of virus-like particles (VLPs) and histo-blood group antigen (HBGA) receptor binding assays. We found that pressurization at 500 to 600 MPa for 2 min, a pressure level that completely inactivates murine norovirus and feline calicivirus, was not sufficient to disrupt the structure and function of human NoV VLPs, even with a holding time of 60 min. Degradation of VLPs increased commensurate with increasing pressure levels more than increasing time. The times required for complete disruption of human NoV VLPs at 700, 800, and 900 MPa were 45, 15, and 2 min, respectively. Human NoV VLPs were more resistant to HPP in their ability to bind type A than type B and O HBGAs. Additionally, the 23-nm VLPs appeared to be much more stable than the 38-nm VLPs. Taken together, our results demonstrated that the human NoV capsid is highly resistant to HPP. While human NoV VLPs may not be fully representative of viable human NoV, destruction of the VLP capsid is highly suggestive of a typical response for viable human NoV.     

Inactivation of Murine Norovirus on a Range of Copper Alloy Surfaces Is Accompanied by Loss of Capsid Integrity

Norovirus is one of the most common causes of acute viral gastroenteritis. The virus is spread via the fecal-oral route, most commonly from infected food and water, but several outbreaks have originated from contamination of surfaces with infectious virus. In this study, a close surrogate of human norovirus causing gastrointestinal disease in mice, murine norovirus type 1 (MNV-1), retained infectivity for more than 2 weeks following contact with a range of surface materials, including Teflon (polytetrafluoroethylene [PTFE]), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. Persistence was slightly prolonged on ceramic surfaces. A previous study in our laboratory observed that dry copper and copper alloy surfaces rapidly inactivated MNV-1 and destroyed the viral genome. In this new study, we have observed that a relatively small change in the percentage of copper, between 70 and 80% in copper nickels and 60 and 70% in brasses, had a significant influence on the ability of the alloy to inactivate norovirus. Nickel alone did not affect virus, but zinc did have some antiviral effect, which was synergistic with copper and resulted in an increased efficacy of brasses with lower percentages of copper. Electron microscopy of purified MNV-1 that had been exposed to copper and stainless steel surfaces suggested that a massive breakdown of the viral capsid had occurred on copper. In addition, MNV-1 that had been exposed to copper and treated with RNase demonstrated a reduction in viral gene copy number. This suggests that capsid integrity is compromised upon contact with copper, allowing copper ion access to the viral genome.