Virucidal activity of three ethanol-based hand rubs against murine norovirus in a hand hygiene clinical simulation study.

Aim: We evaluated the efficacy of three ethanol-based hand rubs against murine norovirus in a proposed clinical simulation test (prEN 17430). Materials & methods: Virucidal activity was determined in 18 volunteers using three hand rubs: ethanol 72.4 and 89.5% v/v solutions, and 86% v/v gel. Subjects underwent testing with each product (3/6 ml for 15/30 s) and a reference solution (6 ml 70% v/v ethanol for 60 s). Results: Against murine norovirus, the reduction factors (RF; RF mean ± standard deviation log10 reduction of postsampling) for ethanol gel 86% v/v (RF 1.96 ± 0.64), ethanol 89.5% v/v (RF 2.49 ± 0.59) and ethanol 72.4% v/v (RF 2.61 ± 0.50), were all significantly superior to that of the reference solution. Conclusion: All three hand rubs passed the criteria set out in prEN 17430 and exhibited excellent virucidal efficacy.

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.

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.

Chemical derivatives of a small molecule deubiquitinase inhibitor have antiviral activity against several RNA viruses.

Most antiviral treatment options target the invading pathogen and unavoidably encounter loss of efficacy as the pathogen mutates to overcome replication restrictions. A good strategy for circumventing drug resistance, or for pathogens without treatment options, is to target host cell proteins that are utilized by viruses during infection. The small molecule WP1130 is a selective deubiquitinase inhibitor shown previously to successfully reduce replication of noroviruses and some other RNA viruses. In this study, we screened a library of 31 small molecule derivatives of WP1130 to identify compounds that retained the broad-spectrum antiviral activity of the parent compound in vitro but exhibited improved drug-like properties, particularly increased aqueous solubility. Seventeen compounds significantly reduced murine norovirus infection in murine macrophage RAW 264.7 cells, with four causing decreases in viral titers that were similar or slightly better than WP1130 (1.9 to 2.6 log scale). Antiviral activity was observed following pre-treatment and up to 1 hour postinfection in RAW 264.7 cells as well as in primary bone marrow-derived macrophages. Treatment of the human norovirus replicon system cell line with the same four compounds also decreased levels of Norwalk virus RNA. No significant cytotoxicity was observed at the working concentration of 5 µM for all compounds tested. In addition, the WP1130 derivatives maintained their broad-spectrum antiviral activity against other RNA viruses, Sindbis virus, LaCrosse virus, encephalomyocarditis virus, and Tulane virus. Thus, altering structural characteristics of WP1130 can maintain effective broad-spectrum antiviral activity while increasing aqueous solubility.

The viral polymerase inhibitor 2'-C-methylcytidine inhibits Norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model.

Human noroviruses are a major cause of food-borne illness, accountable for 50% of all-etiologies outbreaks of acute gastroenteritis (in both developing and developed countries). There is no vaccine or antiviral drug for the prophylaxis or treatment of norovirus-induced gastroenteritis. We recently reported the inhibitory effect of 2'-C-methylcytidine (2CMC), a hepatitis C virus polymerase inhibitor, on the in vitro replication of murine norovirus (MNV). Here we evaluated the inhibitory effect of 2CMC on in vitro human norovirus replication through a Norwalk virus replicon model and in a mouse model by using AG129 mice orally infected with MNV. Survival, weight, and fecal consistency were monitored, and viral loads in stool samples and organs were quantified. Intestines were examined histologically. 2CMC reduced Norwalk virus replicon replication in a dose-dependent manner and was able to clear cells of the replicon. Treatment of MNV-infected AG129 mice with 2CMC (i) prevented norovirus-induced diarrhea; (ii) markedly delayed the appearance of viral RNA and reduced viral RNA titers in the intestine, mesenteric lymph nodes, spleen, lungs, and stool; (iii) completely prevented virus-induced mortality; and (iv) resulted in protective immunity against a rechallenge. We demonstrate for the first time that a small-molecule inhibitor of norovirus replication protects from virus-induced disease and mortality in a relevant animal model. These findings pave the way for the development of potent and safe antivirals as prophylaxis and therapy of norovirus infection.

Structural basis of substrate specificity and protease inhibition in Norwalk virus.

Norwalk virus (NV), the prototype human calicivirus, is the leading cause of nonbacterial acute gastroenteritis. The NV protease cleaves the polyprotein encoded by open reading frame 1 of the viral genome at five nonhomologous sites, releasing six nonstructural proteins that are essential for viral replication. The structural details of how NV protease recognizes multiple substrates are unclear. In our X-ray structure of an NV protease construct, we observed that the C-terminal tail, representing the native substrate positions P5 to P1, is inserted into the active site cleft of the neighboring protease molecule, providing atomic details of how NV protease recognizes a substrate. The crystallographic structure of NV protease with the C-terminal tail redesigned to mimic P4 to P1 of another substrate site provided further structural details on how the active site accommodates sequence variations in the substrates. Based on these structural analyses, substrate-based aldehyde inhibitors were synthesized and screened for inhibition potency. Crystallographic structures of the protease in complex with each of the three most potent inhibitors were determined. These structures showed concerted conformational changes in the S4 and S2 pockets of the protease to accommodate variations in the P4 and P2 residues of the substrate/inhibitor, which could be a mechanism for how the NV protease recognizes multiple sites in the polyprotein with differential affinities during virus replication. These structures further indicate that the mechanism of inhibition by these inhibitors involves covalent bond formation with the side chain of the conserved cysteine in the active site by nucleophilic addition, and such substrate-based aldehydes could be effective protease inhibitors.

Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response.

Ubiquitin (Ub) is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub) cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs). However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1), a critical mediator of the unfolded protein response (UPR). WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1) through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies.

Backbone and side-chain ¹H, ¹5N, and ¹³C resonance assignments of Norwalk virus protease.

Norovirus protease cleaves the virus-encoded polyprotein into six mature nonstructural proteins, presenting itself as an essential enzyme for the viral replication as well as an attractive target for the antiviral drug development. A deeper understanding of the structural mechanism of the protease-substrates/inhibitors interactions by means of solution NMR methods would facilitate a rational design of the virus protease inhibitor. We here report the backbone and side-chain resonance assignment of the protease from Norwalk virus, which is the prototype strain of norovirus. The assignment data has been deposited in the BMRB database under the accession number 17523.

Potent inhibition of Norwalk virus by cyclic sulfamide derivatives.

A new class of compounds that exhibit anti-norovirus activity in a cell-based system and embody in their structure a cyclosulfamide scaffold has been identified. The structure of the initial hit (compound 2a, ED(50) 4 µM, TD(50) 50 µM) has been prospected by exploiting multiple points of diversity and generating appropriate structure-activity relationships.

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.

Inactivation of norovirus by chlorine disinfection of water.

In an effort to validate previous research suggesting remarkable resistance of norovirus to free chlorine disinfection, we characterized the disinfection response of purified and dispersed Norwalk virus (NV) by bench-scale free chlorine disinfection using RT-PCR for virus assays. The inactivation of NV by two doses of free chlorine (1 and 5mg/L) at pH 6 and 5 degrees C based on two RT-PCR assays was similar to that of coliphage MS2, but much faster than that of poliovirus 1. Despite the underestimation of virus inactivation by RT-PCR assays, the predicted CT values for NV based on RT-PCR assays are lower than the ones for most other important waterborne viruses and the CT guidelines for chlorine disinfection of viruses under the Surface Water Treatment Rule by the United States Environmental Protection Agency. Overall, the results of this study indicate that NV is not highly resistant to free chlorine disinfection as suggested by previous research and it is likely that NV contamination of drinking water can be controlled by adequate free chlorine disinfection practices with provision of proper pre-treatment processes before chlorination.

Interferons and ribavirin effectively inhibit Norwalk virus replication in replicon-bearing cells.

The development of effective therapies for noroviral gastroenteritis has been hampered by the absence of a cell culture system. Recently, we reported the generation of Norwalk virus (NV) replicon-bearing cells in BHK21 and Huh-7 cells and demonstrated that alpha interferon (IFN-alpha) effectively inhibited the replication of NV in these cells. In continuing studies for screening potential antinoroviral agents, we tested IFN-gamma and ribavirin for their effects on NV replication in the cells. Like IFN-alpha, IFN-gamma inhibited the replication of NV in the replicon-bearing cells, showing the reduction of the NV genome and proteins in a dose-dependent manner. The effective dose for reducing 50% (ED(50)) of the NV genome and protein was calculated to be approximately 40 units/ml. When ribavirin was applied to the cells, it effectively reduced the NV genome and protein with the ED(50) calculated as approximately 40 microM. The combination of IFN-alpha and ribavirin showed additive effects on the inhibition of NV replication. With the addition of guanosine to the ribavirin treatment, moderately reversed antiviral effects were observed, suggesting that the ribavirin effect may be associated with the depletion of GTP in the cells. Sequencing analysis of the conserved polymerase regions of NV in the ribavirin-treated (100 microM) and nontreated groups showed that the mutation rates were similar and indicated that ribavirin did not induce catastrophic mutations. The NV replicon-bearing cells provide an excellent tool for screening potential antinoroviral agents, and our results indicated that IFNs and ribavirin may be good therapeutic options for noroviral gastroenteritis.

Ultrastructure changes induced by dry film formation of a trisodium phosphate blend, antimicrobial solution.

Trisodium phosphate (TSP) has been reported to have antimicrobial activity and is approved by the U.S. Department of Agriculture for use in food processing applications. A novel (U.S. Patent 6,184,198), antimicrobial solution containing a blend of TSP, sodium bicarbonate, and sodium carbonate (TSP blend) has demonstrated effective inhibition of microbial contamination in a broad spectrum of applications. This high-resolution cold field emission scanning electron microcopy (LVSEM) investigation details structural changes and dry film formation in various classes of microbes as a mechanism for antimicrobial activity of this solution. The results showed that this TSP blend solution completely inhibited the growth of pathogenic E. coli O157:H7, and Salmonella bacteria, the mold Cryptococcus, as well as a Norwalk virus surrogate-feline calicivirus. Results by LVSEM confirmed that the antimicrobial effect was induced when encapsulating the target microbes in a high lubricity film that is formed around these organisms as the solution dries. The thickness of the film was estimated to be approximately 60 nm.

Inhibition of attachment of virions of Norwalk virus to mammalian cells by soluble histone molecules.

Viral infection is usually initiated by the binding of virus particles to specific receptor molecule(s) on the host cell surface. Blocking of this step prevents the following step, penetration into the cell. In the present study, we investigated the virus-cell interactions of virions of Norwalk virus (NV), a major etiological agent for viral diarrhea. We found that histone was an extremely strong NV-binding protein. Histone H1, a heterologous histone molecule, appeared to be able to interact not only with NV particles, but also with the cell surface. Histone H1 appeared capable of effectively preventing the attachment of NV to intestinal cells, but not of other viruses. No cytotoxic effects of histone were observed under the assay conditions. These results indicate that nonsecretory histone molecules may inhibit the attachment of viruses to cells like lactoferrins. Our results suggest that by searching virus-binding molecules, we might find antiviral agents, even for new viruses.

Reduction of Norwalk virus, poliovirus 1, and bacteriophage MS2 by ozone disinfection of water.

Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5 degrees C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log(10) within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods. Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone.

Inactivation of feline calicivirus, a Norwalk virus surrogate.

Norwalk and Norwalk virus-like particles (NVLPs) [also known as small round structured viruses (SRSVs)] are members of the family Caliciviridae and are important causes of gastroenteritis in humans. Little is known about their survival in the environment or the disinfection procedures necessary to remove them from contaminated settings. As NVLPs cannot be grown in tissue culture, survival studies require the use of a closely related cultivable virus. This study assesses the survival of the surrogate feline calicivirus (FCV) after exposure to commercially available disinfectants and a range of environmental conditions. Disinfectants tested included glutaraldehyde, iodine, hypochlorite, a quaternary ammonium-based product, an anionic detergent and ethanol. Complete inactivation of FCV required exposure to 1000 ppm freshly reconstituted granular hypochlorite, or 5000 ppm pre-reconstituted hypochlorite solution. Glutaraldehyde and the iodine-based product effectively inactivated FCV whereas the quaternary ammonium product, detergent and ethanol failed to completely inactivate the virus. The stability of FCV in suspension and in a dried state was assessed after exposure to 4 degrees C, room temperature (20 degrees C) and 37 degrees C. With increasing temperature, the stability of FCV was found to diminish both in suspension and in the dried state. FCV in the dried state did not survive for one day at 37 degrees C. This study provides a basis for establishing guidelines for disinfection protocols to decrease the spread of NVLPs in a community setting.

Inactivation of Norwalk virus in drinking water by chlorine.

Norwalk virus in water was found to be more resistant to chlorine inactivation than poliovirus type 1 (LSc2Ab), human rotavirus (Wa), simian rotavirus (SA11), or f2 bacteriophage. A 3.75 mg/liter dose of chlorine was found to be effective against other viruses but failed to inactivate Norwalk virus. The Norwalk virus inoculum remained infectious for five of eight volunteers, despite the initial presence of free residual chlorine. Infectivity in volunteers was demonstrated by seroconversion to Norwalk virus. Fourteen of 16 subjects receiving untreated inoculum seroconverted to Norwalk virus. Illness was produced in four of the eight volunteers and in 11 of 16 control subjects. A similar Norwalk virus inoculum treated with a 10 mg/liter dose of chlorine produced illness in only one and failed to induce seroconversion in any of eight volunteers. Free chlorine (5 to 6 mg/liter) was measured in the reaction vessel after a 30-minute contact period. Norwalk virus appears to be very resistant to chlorine which may explain its importance in outbreaks of waterborne disease.