Less severe clinical symptoms of Norwalk virus 8fIIb inoculum compared to its precursor 8fIIa from human challenge studies.

Noroviruses (NoV) are a leading cause of epidemic gastroenteritis. Human challenge studies have been used to examine the infectivity, pathogenicity, and host immune response to NoV as well as vaccine efficacy. The goal of this study was to conduct a meta-analysis of data from five previously completed human challenge trials and compare the response to the secondary NV inoculum (8fIIb) to its precursor (8fIIa). We investigated a total of 158 subjects: 76 subjects were experimentally challenged with NV inoculum 8fIIa, and 82 subjects were challenged with 8fIIb. We compared demographic characteristics, infection, illness, mean severity score, blood types, and duration of viral shedding between the two groups of subjects. There were no statistically significant differences in overall infection and illness rates between subjects inoculated with 8fIIa and 8fIIb. However, individuals challenged with 8fIIa had significantly higher severity scores (5.05 vs. 3.22, p = .008) compared with those challenged with 8fIIb. We also observed that infection with 8fIIb was associated with significantly longer duration of viral shedding compared with 8fIIa (11.0 days vs. 5.0 days, p = .0005). These results have serious implications for the development of new NoV inocula for human challenge studies to test candidate vaccine efficacy-where illness severity and duration of viral shedding are important outcomes.

Selection and Characterization of Rupintrivir-Resistant Norwalk Virus Replicon Cells In Vitro.

Human norovirus (HuNoV) is a major cause of nonbacterial gastroenteritis worldwide, yet despite its impact on society, vaccines and antivirals are currently lacking. A HuNoV replicon system has been widely applied to the evaluation of antiviral compounds and has thus accelerated the process of drug discovery against HuNoV infection. Rupintrivir, an irreversible inhibitor of the human rhinovirus 3C protease, has been reported to inhibit the replication of the Norwalk virus replicon via the inhibition of the norovirus protease. Here we report, for the first time, the generation of rupintrivir-resistant human Norwalk virus replicon cells in vitro Sequence analysis revealed that these replicon cells contained amino acid substitutions of alanine 105 to valine (A105V) and isoleucine 109 to valine (I109V) in the viral protease NS6. The application of a cell-based fluorescence resonance energy transfer (FRET) assay for protease activity demonstrated that these substitutions were involved in the enhanced resistance to rupintrivir. Furthermore, we validated the effect of these mutations using reverse genetics in murine norovirus (MNV), demonstrating that a recombinant MNV strain with a single I109V substitution in the protease also showed reduced susceptibility to rupintrivir. In summary, using a combination of different approaches, we have demonstrated that, under the correct conditions, mutations in the norovirus protease that lead to the generation of resistant mutants can rapidly occur.

Hospital-acquired rotavirus and norovirus acute gastroenteritis in a pediatric unit, in 2014-2015.

The occurrence of hospital-acquired acute gastroenteritis (AGE) is a major concern for public health. RotavirusA (RVA) and norovirus (NoV) are common causes of viral AGE in the pediatric population, and their role in nosocomial infections has been proven, remaining poorly investigated. To investigate RVA and NoV in hospital-acquired AGE, 55 stool samples from children with nosocomial AGE were collected between May 2014 and May 2015. To evaluate virus spreading routes, 51 environmental swabs were collected from staff and patients' rooms. Stools were tested for both RVA and NoV RNA by reverse-transcription-PCR. Nucleotide sequencing and phylogenetic analysis were performed to characterize the viruses. Forty-seven of 55 cases analyzed resulted positive for RVA. The predominant genotype was G4P[8] (18/55) followed by G1P[8] (14/55). Mixed RVA infections were also detected (7/55). Twenty-two samples were positive for NoV, and GII.4 was revealed to be the predominant genotype. Seventeen samples were positive for both RVA and NoV. This study aimed to evaluate the burden of norovirus and rotavirus nosocomial AGE, contributing to identify the environment source of infections and to activate effective strategies for intervention. The reduction in nosocomial AGE cases is an important aspect, considered the worsened disease course in transplant, cancer, and intensive care unit inpatients.

Mapping and modeling of a strain-specific epitope in the Norwalk virus capsid inner shell.

Noroviruses are diverse positive-strand RNA viruses associated with acute gastroenteritis. Cross-reactive epitopes have been mapped primarily to conserved sequences in the capsid VP1 Shell (S) domain, and strain-specific epitopes to the highly variable Protruding (P) domain. In this work, we investigated a strain-specific linear epitope defined by MAb NV10 that was raised against prototype (Genogroup I.1) strain Norwalk virus (NV). Using peptide scanning and mutagenesis, the epitope was mapped to amino acids 21-32 (LVPEVNASDPLA) of the NV S domain, and its specificity was verified by epitope transfer and reactivity with a recombinant MAb NV10 single-chain variable fragment (scFv). Comparative structural modeling of the NV10 strain-specific and the broadly cross-reactive TV20 epitopes identified two internal non-overlapping sites in the NV shell, corresponding to variable and conserved amino acid sequences among strains, respectively. The S domain, like the P domain, contains strain-specific epitopes that contribute to the antigenic diversity among the noroviruses.

Immunogenicity of Newcastle disease virus vectors expressing Norwalk virus capsid protein in the presence or absence of VP2 protein.

Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirus-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans.

Experimental human infection with Norwalk virus elicits a surrogate neutralizing antibody response with cross-genogroup activity.

The human noroviruses (NoVs) are genetically diverse, rapidly evolving RNA viruses and are the major cause of epidemic gastroenteritis of humans. Serum antibodies that block the interaction of NoVs and NoV viruslike particles (VLPs) with host attachment factors are considered surrogate neutralizing antibodies in the absence of cell culture and small-animal replication models for the human NoVs. A serological assay for NoV-blocking antibodies was used to assess the breadth of the heterotypic antibody response in the context of an experimental challenge study with a human NoV. Heterotypic histo-blood group antigen (HBGA)-blocking activity against GI.4, GI.7, and GII.4 NoVs increased significantly in the serum of individuals (n = 18) infected with Norwalk virus (GI.1). Although the fold increases and peak titers of heterotypic antibody were more modest than titers of antibody reactive with the challenge antigen, Norwalk virus infection elicited a serological rise even against the novel Sydney variant of GII.4 NoVs. These observations indicate that the development of a broadly cross-protective NoV vaccine containing a limited number of genotypes may be possible.

The p4-p2' amino acids surrounding human norovirus polyprotein cleavage sites define the core sequence regulating self-processing order.

UNLABELLED: Noroviruses (NoV) are members of the family Caliciviridae. The human NoV open reading frame 1 (ORF1) encodes a 200-kDa polyprotein which is cleaved by the viral 20-kDa 3C-like protease (Pro, NS6) into 6 nonstructural proteins that are necessary for viral replication. The NoV ORF1 polyprotein is processed in a specific order, with "early" sites (NS1/2-3 and NS3-4) being cleaved rapidly and three "late" sites (NS4-5, NS5-6, and NS6-7) processed subsequently and less efficiently. Previously, we demonstrated that the NoV polyprotein processing order is directly correlated with the efficiency of the enzyme, which is regulated by the primary amino acid sequences surrounding ORF1 cleavage sites. Using fluorescence resonance energy transfer (FRET) peptides representing the NS2-3 and NS6-7 ORF1 cleavage sites, we now demonstrate that the amino acids spanning positions P4 to P2' (P4-P2') surrounding each site comprise the core sequence controlling NoV protease enzyme efficiency. Furthermore, the NoV polyprotein self-processing order can be altered by interchanging this core sequence between NS2-3 and any of the three late sites in in vitro transcription-translation assays. We also demonstrate that the nature of the side chain at the P3 position for the NS1/2-3 (Nterm/NTPase) site confers significant influence on enzyme catalysis (kcat and kcat/Km), a feature overlooked in previous structural studies. Molecular modeling provides possible explanations for the P3 interactions with NoV protease. IMPORTANCE: Noroviruses (NoV) are the prevailing cause of nonbacterial acute gastroenteritis worldwide and pose a significant financial burden on health care systems. Proteolytic processing of the viral nonstructural polyprotein is required for norovirus replication. Previously, the core sequence of amino acids surrounding the scissile bonds responsible for governing the relative processing order had not been determined. Using both FRET-based peptides and full-length NoV polyprotein, we have successfully demonstrated that the core sequences spanning positions P4-P2' surrounding the NS2-3, NS4-5, NS5-6, and NS6-7 cleavage sites contain all of the structural information necessary to control processing order. We also provide insight into a previously overlooked role for the NS2-3 P3 residue in enzyme efficiency. This article builds upon our previous studies on NoV protease enzymatic activities and polyprotein processing order. Our work provides significant additional insight into understanding viral polyprotein processing and has important implications for improving the design of inhibitors targeting the NoV protease.

Development of a Gaussia luciferase-based human norovirus protease reporter system: cell type-specific profile of Norwalk virus protease precursors and evaluation of inhibitors.

UNLABELLED: Norwalk virus (NV) is the prototype strain of human noroviruses (HuNoVs), a group of positive-strand RNA viruses in the Caliciviridae family and the leading cause of epidemic gastroenteritis worldwide. Investigation of HuNoV replication and development of antiviral therapeutics in cell culture remain challenging tasks. Here, we present NoroGLuc, a HuNoV protease reporter system based on a fusion of NV p41 protein with a naturally secreted Gaussia luciferase (GLuc), linked by the p41/p22 cleavage site for NV protease (Pro). trans cleavage of NoroGLuc by NV Pro or Pro precursors results in release and secretion of an active GLuc. Using this system, we observed a cell type-specific activity profile of NV Pro and Pro precursors, suggesting that the activity of NV Pro is modulated by other viral proteins in the precursor forms and strongly influenced by cellular factors. NoroGLuc was also cleaved by Pro and Pro precursors generated from replication of NV stool RNA in transfected cells, resulting in a measurable increase of secreted GLuc. Truncation analysis revealed that the N-terminal membrane association domain of NV p41 is critical for NoroGLuc activity. Although designed for NV, a genogroup GI.1 norovirus, NoroGLuc also efficiently detects Pro activities from GII.3 and GII.4 noroviruses. At noncytotoxic concentrations, protease inhibitors ZnCl2 and Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK) exhibited dose-dependent inhibitory effects on a GII.4 Pro by NoroGLuc assay. These results establish NoroGLuc as a pan-genogroup HuNoV protease reporter system that can be used for the study of HuNoV proteases and precursors, monitoring of viral RNA replication, and evaluation of antiviral agents. IMPORTANCE: Human noroviruses are the leading cause of epidemic gastroenteritis worldwide. Currently, there are no vaccines or antiviral drugs available to counter these highly contagious viruses. These viruses are currently noncultivatable in cell culture. Here, we report the development of a novel cell-based reporter system called NoroGLuc that can be used for studying norovirus replication and also for screening/evaluation of antiviral agents. This system is based on the fusion between viral protein p41 and a naturally secreted Gaussia luciferase (GLuc) with a cleavage site that can be recognized by the viral protease. Cleavage of this fusion protein by the viral protease results in the release and secretion of an active GLuc. Using NoroGLuc, we demonstrated a cell type-specific activity profile of the viral protease and its precursors and dose-dependent inhibitory effects of two protease inhibitors. This novel reporter system should be useful in probing norovirus replication and evaluating antiviral agents.

The enterovirus protease inhibitor rupintrivir exerts cross-genotypic anti-norovirus activity and clears cells from the norovirus replicon.

Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II [GII]) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2'-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.

Expression and assembly of Norwalk virus-like particles in plants using a viral RNA silencing suppressor gene.

Binary vector-based transient expression of heterologous proteins in plants is a very attractive strategy due to the short time required for proceeding from planning to expression. However, this expression system is limited by comparatively lower yields due to strong post-transcriptional gene silencing (PTGS) in the host plants. The aim of this study was to optimize a procedure for expression of norovirus virus-like particles (VLPs) in plants using a binary vector with co-expression of a PTGS suppressor to increase the yield of the target protein. The effects of four plant viral PTGS suppressors on protein expression were evaluated using green fluorescent protein (GFP) as a reporter. Constructs for both GFP and PTGS suppressor genes were co-infiltrated in Nicotiana benthamiana plants, and the accumulation of GFP was evaluated. The most effective PTGS suppressor was the 126K protein of Pepper mild mottle virus. Therefore, this suppressor was selected as the norovirus capsid gene co-expression partner for subsequent studies. The construct containing the major (vp1) and minor capsid (vp2) genes with a 3'UTR produced a greater amount of protein than the construct with the major capsid gene alone. Thus, the vp1-vp2-3'UTR and 126K PTGS suppressor constructs were co-infiltrated at middle scale and VLPs were purified by sucrose gradient centrifugation. Proteins of the expected size, specific to the norovirus capsid antibody, were observed by Western blot. VLPs were observed by transmission electron microscopy. It was concluded that protein expression in a binary vector co-expressed with the 126K PTGS suppressor protein enabled superior expression and assembly of norovirus VLPs.

Norwalk Virus Minor Capsid Protein VP2 Associates within the VP1 Shell Domain.

The major capsid protein of norovirus VP1 assembles to form an icosahedral viral particle. Despite evidence that the Norwalk virus (NV) minor structural protein VP2 is present in infectious virions, the available crystallographic and electron cryomicroscopy structures of NV have not revealed the location of VP2. In this study, we determined that VP1 associates with VP2 at the interior surface of the capsid, specifically with the shell (S) domain of VP1. We mapped the interaction site to amino acid 52 of VP1, an isoleucine located within a sequence motif IDPWI in the S domain that is highly conserved across norovirus genogroups. Mutation of this isoleucine abrogated VP2 incorporation into virus-like particles without affecting the ability for VP1 to dimerize and form particles. The highly basic nature of VP2 and its location interior to the viral particle are consistent with its potential role in assisting capsid assembly and genome encapsidation.

Secretory pathway antagonism by calicivirus homologues of Norwalk virus nonstructural protein p22 is restricted to noroviruses.

BACKGROUND: Our previous report that the Norwalk virus nonstructural protein p22 is an antagonist of the cellular secretory pathway suggests a new aspect of norovirus/host interaction. To explore conservation of function of this highly divergent calicivirus protein, we examined the effects of p22 homologues from four human and two murine noroviruses, and feline calicivirus on the secretory pathway. FINDINGS: All human noroviruses examined induced Golgi disruption and inhibited protein secretion, with the genogroup II.4 Houston virus being the most potent antagonist. Genogroup II.6 viruses have a conserved mutation in the mimic of an Endoplasmic Reticulum export signal (MERES) motif that is highly conserved in human norovirus homologues of p22 and is critical for secretory pathway antagonism, and these viruses had reduced levels of Golgi disruption and inhibition of protein secretion. p22 homologues from both persistent and nonpersistent strains of murine norovirus induced Golgi disruption, but only mildly inhibited cellular protein secretion. Feline calicivirus p30 did not induce Golgi disruption or inhibit cellular protein secretion. CONCLUSIONS: These differences confirm a norovirus-specific effect on host cell secretory pathway antagonism by homologues of p22, which may affect viral replication and/or cellular pathogenesis.

Characterization and inhibition of norovirus proteases of genogroups I and II using a fluorescence resonance energy transfer assay.

Noroviruses are the major cause of food- or water-borne gastroenteritis outbreaks in humans. The norovirus protease that cleaves a large viral polyprotein to nonstructural proteins is essential for virus replication and an attractive target for antiviral drug development. Noroviruses show high genetic diversity with at least five genogroups, GI-GV, of which GI and GII are responsible for the majority of norovirus infections in humans. We cloned and expressed proteases of Norwalk virus (GI) and MD145 virus (GII) and characterized the enzymatic activities with fluorescence resonance energy transfer substrates. We demonstrated that the GI and GII proteases cleaved the substrates derived from the naturally occurring cleavage site in the open reading frame (ORF) 1 of G1 norovirus with similar efficiency, and that enzymatic activity of both proteases was inhibited by commercial protease inhibitors including chymostatin. The interaction of chymostatin to Norwalk virus protease was validated by nuclear magnetic resonance (NMR) spectroscopy.

Nucleolin interacts with the feline calicivirus 3' untranslated region and the protease-polymerase NS6 and NS7 proteins, playing a role in virus replication.

Cellular proteins play many important roles during the life cycle of all viruses. Specifically, host cell nucleic acid-binding proteins interact with viral components of positive-stranded RNA viruses and regulate viral translation, as well as RNA replication. Here, we report that nucleolin, a ubiquitous multifunctional nucleolar shuttling phosphoprotein, interacts with the Norwalk virus and feline calicivirus (FCV) genomic 3' untranslated regions (UTRs). Nucleolin can also form a complex in vitro with recombinant Norwalk virus NS6 and -7 (NS6/7) and can be copurified with the analogous protein from feline calicivirus (p76 or NS6/7) from infected feline kidney cells. Nucleolin RNA levels or protein were not modified during FCV infection; however, as a consequence of the infection, nucleolin was seen to relocalize from the nucleoli to the nucleoplasm, as well as to the perinuclear area where it colocalizes with the feline calicivirus NS6/7 protein. In addition, antibodies to nucleolin were able to precipitate viral RNA from feline calicivirus-infected cells, indicating a direct or indirect association of nucleolin with the viral RNA during virus replication. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a reduction of the cytopathic effect and virus yield in CrFK cells. Taken together, these results demonstrate that nucleolin is a nucleolar component that interacts with viral RNA and NS6/7 and is required for feline calicivirus replication.

Chimpanzees as an animal model for human norovirus infection and vaccine development.

Noroviruses are global agents of acute gastroenteritis, but the development of control strategies has been hampered by the absence of a robust animal model. Studies in chimpanzees have played a key role in the characterization of several fastidious hepatitis viruses, and we investigated the feasibility of such studies for the noroviruses. Seronegative chimpanzees inoculated i.v. with the human norovirus strain Norwalk virus (NV) did not show clinical signs of gastroenteritis, but the onset and duration of virus shedding in stool and serum antibody responses were similar to that observed in humans. NV RNA was detected in intestinal and liver biopsies concurrent with the detection of viral shedding in stool, and NV antigen expression was observed in cells of the small intestinal lamina propria. Two infected chimpanzees rechallenged 4, 10, or 24 mo later with NV were resistant to reinfection, and the presence of NV-specific serum antibodies correlated with protection. We evaluated the immunogenicity and efficacy of virus-like particles (VLPs) derived from NV (genogroup I, GI) and MD145 (genogroup II, GII) noroviruses as vaccines. Chimpanzees vaccinated intramuscularly with GI VLPs were protected from NV infection when challenged 2 and 18 mo after vaccination, whereas chimpanzees that received GII VLPs vaccine or a placebo were not. This study establishes the chimpanzee as a viable animal model for the study of norovirus replication and immunity, and shows that NV VLP vaccines could induce protective homologous immunity even after extended periods of time.

Quantification of Norwalk virus inocula: Comparison of endpoint titration and real-time reverse transcription-PCR methods.

Human noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis. In order to fully characterize features such as persistence and infectious dose, precise quantification of virus concentration is necessary. The purpose of this study was to compare two methods [endpoint titration RT-PCR and quantitative RT-PCR (RT-qPCR)] with respect to quantification of Norwalk virus (NV) in inocula made from purified stock suspensions of human fecal specimens. A full-length NV RNA transcript was developed to facilitate quantification using RT-qPCR and provided log linear detection in the range of 49-4.9 x 10(4) genome equivalent copies (GEC) per reaction. Endpoint titration RT-PCR was used to estimate PCR detection units, and RT-qPCR was used to estimate genome copies in two NV inocula (8fIIa and 8fIIb) used in previous human challenge studies. Overall, RT-qPCR was 1.1-1.6 log(10) more sensitive (lower detection limit) than endpoint titration RT-PCR when the same RNA release method, PCR primers and thermocycle program were used. These findings have important implications for many experimental interpretations, not the least of which is estimating the median infectious dose in human challenge studies.

Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples.

Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72 degrees C, 37 degrees C, and 19 degrees C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72 degrees C and 37 degrees C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19 degrees C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37 degrees C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.

Norwalk virus-like particles as vaccines.

Noroviruses (NoV) cause the great majority of epidemic nonbacterial gastroenteritis in humans. Expression of the capsid protein in recombinant systems, including insect and plant cells, yields assembly of virus-like particles (VLPs) that mimic the antigenic structure of authentic virions, and are relatively acid- and heat-stable. Norwalk virus (NV), the prototype NoV, has been studied extensively, and Norwalk virus-like particles (NVLPs) produced in insect cells and plants are immunogenic in mice and humans when delivered orally, stimulating the production of systemic and mucosal anti-NV antibodies. NVLPs are also highly immunogenic when delivered intranasally, provoking antibodies at levels similar to orally delivered VLP at much lower doses. Oral and nasal delivery of NVLPs efficiently produces antibodies at distal mucosal sites, which suggests that NVLPs could be used to deliver heterologous peptide antigens by production of genetic fusion chimeric capsid proteins. Examination of norovirus VLP surface structures and receptor binding motifs facilitates identification of potential sites for insertion of foreign peptides that will minimally affect the efficiency of VLP assembly and receptor binding. Thus, there is strong potential to use norovirus VLPs as vaccine-delivery vehicles.

Effectiveness of liquid soap and hand sanitizer against Norwalk virus on contaminated hands.

Disinfection is an essential measure for interrupting human norovirus (HuNoV) transmission, but it is difficult to evaluate the efficacy of disinfectants due to the absence of a practicable cell culture system for these viruses. The purpose of this study was to screen sodium hypochlorite and ethanol for efficacy against Norwalk virus (NV) and expand the studies to evaluate the efficacy of antibacterial liquid soap and alcohol-based hand sanitizer for the inactivation of NV on human finger pads. Samples were tested by real-time reverse transcription-quantitative PCR (RT-qPCR) both with and without a prior RNase treatment. In suspension assay, sodium hypochlorite concentrations of >or=160 ppm effectively eliminated RT-qPCR detection signal, while ethanol, regardless of concentration, was relatively ineffective, giving at most a 0.5 log(10) reduction in genomic copies of NV cDNA. Using the American Society for Testing and Materials (ASTM) standard finger pad method and a modification thereof (with rubbing), we observed the greatest reduction in genomic copies of NV cDNA with the antibacterial liquid soap treatment (0.67 to 1.20 log(10) reduction) and water rinse only (0.58 to 1.58 log(10) reduction). The alcohol-based hand sanitizer was relatively ineffective, reducing the genomic copies of NV cDNA by only 0.14 to 0.34 log(10) compared to baseline. Although the concentrations of genomic copies of NV cDNA were consistently lower on finger pad eluates pretreated with RNase compared to those without prior RNase treatment, these differences were not statistically significant. Despite the promise of alcohol-based sanitizers for the control of pathogen transmission, they may be relatively ineffective against the HuNoV, reinforcing the need to develop and evaluate new products against this important group of viruses.

Heterotypic humoral and cellular immune responses following Norwalk virus infection.

Norovirus immunity is poorly understood as the limited data available on protection after infection are often contradictory. In contrast to the more prominent GII noroviruses, GI norovirus infections are less frequent in outbreaks. The GI noroviruses display very complex patterns of heterotypic immune responses following infection, and many individuals are highly susceptible to reinfection. To study the immune responses and mechanisms of GI.1 persistence, we built structural models and recombinant virus-like particles (VLPs) of five GI strains: GI.1-1968, GI.1-2001, GI.2-1999, GI.3-1999, and GI.4-2000. Structural models of four GI genotype capsid P domain dimers suggested that intragenotype structural variation is limited, that the GI binding pocket is mostly preserved between genotypes, and that a conserved, surface-exposed epitope may allow for highly cross-reactive immune responses. GI VLPs bound to histo-blood group antigens (HBGAs) including fucose, Lewis, and A antigens. Volunteers infected with GI.1-1968 (n = 10) had significant increases between prechallenge and convalescent reactive IgG for all five GI VLPs measured by enzyme immunoassay. Potential cross-neutralization of GI VLPs was demonstrated by convalescent-phase serum cross-blockade of GI VLP-HBGA interaction. Although group responses were significant for all GI VLPs, each individual volunteer demonstrated a unique VLP blockade pattern. Further, peripheral blood mononuclear cells (PBMCs) were stimulated with each of the VLPs, and secretion of gamma interferon (IFN-gamma) was measured. As seen with blockade responses, IFN-gamma secretion responses differed by individual. Sixty percent responded to at least one GI VLP, with only two volunteers responding to GI.1 VLP. Importantly, four of five individuals with sufficient PBMCs for cross-reactivity studies responded more robustly to other GI VLPs. These data suggest that preexposure history and deceptive imprinting may complicate PBMC and B-cell immune responses in some GI.1-1968-challenged individuals and highlight a potential complication in the design of efficacious norovirus vaccines.