Murine Norovirus Research Articles

Effects of Epigallocatechin-3-Gallate-Palmitate (EC16) on In Vitro Norovirus Infection.

Norovirus is the world-leading cause of acute gastroenteritis associated with severe symptoms and deaths. However, vaccines against norovirus are currently not available, and medications that specifically target human norovirus infection are still under development. The current study evaluated the virucidal and antiviral activities of epigallocatechin-3-gallate-palmitate (EC16), a compound derived from green tea polyphenols, against murine norovirus (MNV S99, a surrogate for human norovirus). Initially, formulation suitability tests were conducted to compare EGCG (epigallocatechin-3-gallate), EC16 and tea polyphenol-palmitate in alcohol solution and hand hygiene formulations. The virucidal activity of EC16 was then tested in hand sanitizer gel and hand sanitizer foam formulations using a TCID50 time-kill suspension assay. In vitro treatment and prevention tests were performed using a 1-hour incubation of EC16 or EGCG with RAW264.7 cells, either post-infection or pre-infection with MNV. Statistical analysis employed two-tailed student t test (alpha=0.05). Unlike EC16, both EGCG and tea polyphenol-palmitate showed auto-oxidation (color change) and precipitation in alcohol solution and hand hygiene formulations, and thus less suitable for potential hand hygiene product or new drug development. The time-kill suspension test results demonstrated that EC16 in both sanitizer gel and foam formulations reduced MNV by >99.99% (>log10 4) after 60 sec direct contact. One-hour incubation of EC16 with RAW264.7 cells either before or after MNV infection (i.e., without direct contact with MNV), resulted in >99% (>log10 2) reduction of MNV infectivity. EC16 is a candidate for use as a virucidal and antiviral compound to prevent and treat norovirus infection, with potential to be developed as a new drug against norovirus, pending in vivo and clinical tests.

High-Efficiency Biocidal Solution Based on Radiochemically Synthesized Cu-Au Alloy Nanoparticles.

The use of nanotechnologies in the applied biomedical sciences can offer a new way to treat infections and disinfect surfaces, materials, and products contaminated with various types of viruses, bacteria, and fungi. The Cu-Au nanoparticles (NPs) were obtained by an eco-friendly method that allowed the obtaining in a one-step process of size controlled, well dispersed, fully reduced, highly stable NPs at very mild conditions, using high energy ionizing radiations. The gamma irradiation was performed in an aqueous system of Cu2+/Au3+/Sodium Dodecyl Sulfate (SDS)/Ethylene Glycol. After irradiation, the change of color to ruby-red was the first indicator for the formation of NPs. Moreover, the UV-Vis spectra showed a maximum absorption peak between 524 and 540 nm, depending on the copper amount. The Cu-Au NPs presented nearly spherical shapes, sizes between 20 and 90 nm, and a zeta potential of about −44 mV indicating a good electrostatic stability. The biocidal properties performed according to various standards applied in the medical area, in dirty conditions, showed a 5 lg reduction for Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus hirae, a 5 lg reduction for both enveloped and non-enveloped viruses such as Adenovirus type 5, Murine Norovirus, and human Coronavirus 229E, and a 4 lg reduction for Candida albicans, respectively. Thus, the radiochemically synthesized Cu-Au alloy NPs proved to have high biocide efficiency against the tested bacteria, fungi, and viruses (both encapsulated and non-encapsulated). Therefore, these nanoparticle solutions are suitable to be used as disinfectants in the decontamination of hospital surfaces or public areas characterized by high levels of microbiological contamination.

Facile and foldable point-of-care biochip for nucleic acid based-colorimetric detection of murine norovirus in fecal samples using G-quadruplex and graphene oxide coated microbeads

Norovirus is one of the most common causes of gastroenteritis, a disease characterized by diarrhea, vomiting, and stomach pain. A rapid on-site identification of the virus from fecal samples of patients is a prerequisite for accurate medical management. Here, we demonstrate a rapid nucleic acid-based detection platform as an on-site biosensing tool that can concentrate viruses from fecal samples. Moreover, it can perform RNA extraction and identification, and signal amplification using G-quadruplex and hemin containing DNA probes (G-DNA probes) and graphene oxide (GO)-coated microbeads. Briefly, murine noroviruses are lysed without chemicals on the surface of the GO microbeads. Subsequently, the target RNA is hybridized with G-DNA probes, and the resultant RNA/G-DNA probe complex is separated from unbound G-DNA probes using GO beads and is mixed with the detection buffer (ABTS/H2O2). Presence of murine noroviruses causes a colorimetric change of the buffer from colorless to green. Thus, we integrated all processes required to detect murine noroviruses in stool samples in a simple foldable microfluidic chip. Moreover, it can detect 101 pfu of the virus in 30 min in a fecal sample.

Determination of murine norovirus aerosol concentration during toilet flushing

Murine norovirus (MNV) was used as a surrogate for human viral pathogens (e.g., norovirus) to determine if toilet flushing resulted in the aerosolization of virus. A flushometer type toilet was seeded with a viral solution of 105 and 106 PFU mL-1 of MNV and then flushed. Upon flushing, two bioaerosol samplers were activated to collect aerosolized MNV. Prior to the experiment, two optical particle counters monitored particle size and number distribution of aerosol produced from flushing a toilet across height, position, and side. The location with the highest mean particle concentration, was behind the toilet and 0.15 m above the toilet bowl rim, which is where bioaerosol sampling occurred. Bioaerosol and toilet water samples were collected, extracted and then quantified using RT-ddPCR. The concentration of MNV collected after seeding the toilet water ranged from 2.18 × 105 to 9.65 × 106 total copies of MNV. Positive samples of airborne MNV were detected with collected concentrations ranging from 383 to 684 RNA copies/m3 of air. This study provides evidence that viral pathogens may be aerosolized when a toilet is flushed. Furthermore, the MNV used in this study is a model organism for human norovirus and may be generalizable to other viral pathogens (e.g., coronavirus). This study suggests that virus is aerosolized from toilet flushing and may contribute to human exposure to viral pathogens.

Impact of ultraviolet-C and peroxyacetic acid against murine norovirus on stainless steel and lettuce

The aim of this study was to evaluate the efficacy of the representative method of physical (ultraviolet-C [UV-C] irradiation) and chemical disinfectant treatment (peroxyacetic acid, PAA) against murine norovirus (MNV-1) in suspension and on a food-contact surface (stainless steel) and food (lettuce). Stainless steel and lettuce displayed less than 1 log10 reduction at the highest UV-C doses. More than 1 log10 reduction was possible on stainless steel and lettuce treated with the highest concentration of PAA. For sequential treatment with UV-C doses of 0–600 mJ/cm2 and a fixed PAA concentration (75 ppm for 1, 3, and 5 min) on lettuce, the MNV-1 titer was significantly decreased with increased doses of UV-C (p < 0.05). However, sequential treatment did not show any synergistic effect on MNV-1 reduction in this study. Therefore, single UV or PAA treatment had effective virucidal activities on a fresh vegetable sample.

Antibody Production Remains Intact Despite Loss of Bone Marrow B cells in Murine Norovirus Infected Stat1-/- Mice.

Murine norovirus (MNV), which can be used as a model system to study human noroviruses, can infect macrophages/ monocytes, neutrophils, dendritic, intestinal epithelial, T and B cells, and is highly prevalent in laboratory mice. We previously showed that MNV infection significantly reduces bone marrow B cell populations in a Stat1-dependent manner. We show here that while MNV-infected Stat1-/- mice have significant losses of bone marrow B cells, splenic B cells capable of mounting an antibody response to novel antigens retain the ability to expand. We also investigated whether increased granulopoiesis after MNV infection was causing B cell loss. We found that administration of anti-G-CSF antibody inhibits the pronounced bone marrow granulopoiesis induced by MNV infection of Stat1-/- mice, but this inhibition did not rescue bone marrow B cell losses. Therefore, MNV-infected Stat1-/- mice can still mount a robust humoral immune response despite decreased bone marrow B cells. This suggests that further investigation will be needed to identify other indirect factors or mechanisms that are responsible for the bone marrow B cell losses seen after MNV infection. In addition, this work contributes to our understanding of the potential physiologic effects of Stat1-related disruptions in research mouse colonies that may be endemically infected with MNV.

Selected Mechanistic Aspects of Viral Inactivation by Peracetic Acid.

Peracetic acid (PAA) is an alternative to traditional wastewater disinfection as it has a high oxidation potential without producing chlorinated disinfection byproducts. Reports have shown the effectiveness of PAA to reduce waterborne viruses, but the mechanism of inactivation is understudied. This study evaluated PAA consumption by amino acids and nucleotides that are the building blocks of both viral capsids and genomes. Cysteine (>1.7 min-1) and methionine (>1.2 min-1) rapidly consumed PAA, while cystine (1.9 × 10-2 min-1) and tryptophan (1.4 × 10-4 min-1) reactions occurred at a slower rate. All other amino acids and nucleotides did not react significantly (p < 0.05) with PAA during experiments. Also, PAA treatment did not result in significant (p < 0.05) reductions of purified RNA from MS2 bacteriophage and murine norovirus. Data in this study suggest that PAA effectively inactivates viruses by targeting susceptible amino acids on capsid proteins and does not readily damage viral genomes. Knowledge of virus capsid structures and protein compositions can be used to qualitatively predict the relative resistance or susceptibility of virus types to PAA. Capsid structures containing a higher total number of target amino acids may be more susceptible to PAA reactions that damage structural integrity resulting in inactivation.

Akt Plays Differential Roles during the Life Cycles of Acute and Persistent Murine Norovirus Strains in Macrophages.

Akt (protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes, such as glucose metabolism and cell proliferation, for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or altered cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. IMPORTANCE Human noroviruses (HNoV) are a leading cause of viral gastroenteritis, resulting in high annual economic burden and morbidity, yet there are no small-animal models supporting productive HNoV infection or robust culture systems producing cell culture-derived virus stocks. As a result, research on drug discovery and vaccine development against norovirus infection has been challenging, and no targeted antivirals or vaccines against HNoV are approved. On the other hand, murine norovirus (MNV) replicates to high titers in cell culture and is a convenient and widespread model in norovirus research. Our data demonstrate the importance of Akt signaling during the late stage of the MNV life cycle. Notably, the effect of Akt signaling on genome replication, virus assembly, and cellular egress is virus strain specific, highlighting the diversity of biological phenotypes despite small genetic variability among norovirus strains. This study is the first to demonstrate a role for Akt in viral assembly.

Structural Studies on the Shapeshifting Murine Norovirus.

Noroviruses are responsible for almost a fifth of all cases of gastroenteritis worldwide. The calicivirus capsid is composed of 180 copies of VP1 with a molecular weight of ~58 kDa. This coat protein is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. Reviewed here are studies on murine norovirus (MNV) showing that the capsid responds to several physiologically relevant cues; bile, pH, Mg2+, and Ca2+. In the initial site of infection, the intestinal tract, high bile and metal concentrations and low pH cause two significant conformational changes: (1) the P domain contracts onto the shell domain and (2) several conformational changes within the P domain lead to enhanced receptor binding while blocking antibody neutralization. In contrast, the pH is neutral, and the concentrations of bile and metals are low in the serum. Under these conditions, the loops at the tip of the P domain are in the open conformation with the P domain floating on a linker or tether above the shell. This conformational state favors antibody binding but reduces interactions with the receptor. In this way, MNV uses metabolites and environmental cues in the intestine to optimize cellular attachment and escape antibody binding but presents a wholly different structure to the immune system in the serum. To our knowledge, this is the first example of a virus shapeshifting in this manner to escape the immune response.

Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11–6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10–100 PFU for the MNV-1 and 1–10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Comparison of five polyethylene glycol precipitation procedures for the RT-qPCR based recovery of murine hepatitis virus, bacteriophage phi6, and pepper mild mottle virus as a surrogate for SARS-CoV-2 from wastewater

Polyethylene glycol (PEG) precipitation is one of the conventional methods for virus concentration. This technique has been used to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. The procedures and seeded surrogate viruses were different among implementers; thus, the reported whole process recovery efficiencies considerably varied among studies. The present study compared five PEG precipitation procedures, with different operational parameters, for the RT-qPCR-based whole process recovery efficiency of murine hepatitis virus (MHV), bacteriophage phi6, and pepper mild mottle virus (PMMoV), and molecular process recovery efficiency of murine norovirus using 34 raw wastewater samples collected in Japan. The five procedures yielded significantly different whole process recovery efficiency of MHV (0.070%–2.6%) and phi6 (0.071%–0.51%). The observed concentration of indigenous PMMoV ranged from 8.9 to 9.7 log (8.2 × 108 to 5.6 × 109) copies/L. Interestingly, PEG precipitation with 2-h incubation outperformed that with overnight incubation partially due to the difference in molecular process recovery efficiency. The recovery load of MHV exhibited a positive correlation (r = 0.70) with that of PMMoV, suggesting that PMMoV is the potential indicator of the recovery efficiency of SARS-CoV-2. In addition, we reviewed 13 published studies and found considerable variability between different studies in the whole process recovery efficiency of enveloped viruses by PEG precipitation. This was due to the differences in operational parameters and surrogate viruses as well as the differences in wastewater quality and bias in the measurement of the seeded load of surrogate viruses, resulting from the use of different analytes and RNA extraction methods. Overall, the operational parameters (e.g., incubation time and pretreatment) should be optimized for PEG precipitation. Co-quantification of PMMoV may allow for the normalization of SARS-CoV-2 RNA concentration by correcting for the differences in whole process recovery efficiency and fecal load among samples.

Antiviral effect of a bacteriophage on murine norovirus replication via modulation of the innate immune response

Bacteriophages (phages) are viruses of bacteria. Despite the growing progress in research on phage interactions with eukaryotic cells, our understanding of the roles of phages and their potential implications remains incomplete. The objective of this study was to investigate the effects of the Staphylococcus aureus phage vB_SauM_JS25 on murine norovirus (MNV) replication. Experiments were performed using the RAW 264.7 cell line. After phage treatment, MNV multiplication was significantly inhibited, as indicated by real-time quantitative polymerase chain reaction (RT-qPCR) analysis, western blotting, the 50% tissue culture infectious dose and immunofluorescence. Furthermore, we revealed transcriptional changes in phage/MNV co-incubated RAW 264.7 cells through RNA sequencing (RNA-seq) and bioinformatic analysis. Our subsequent analyses revealed that the innate immune response might play an important role in restriction of MNV replication, such as the cellular response to IFN-γ and response to IFN-γ. Additionally, gene expression of IL-10, Arg-1, Ccl22, GBP2, GBP3, GBP5, and GBP7 was increased significantly, which indicated a strong correlation between RT-qPCR and RNA-seq results. Furthermore, phage treatment activated guanylate binding proteins (GBPs), as revealed by RT-qPCR analysis, western blotting, and confocal microscopy. Taken together, these data suggest that the phage affects the innate response, such as the IFN-inducible GTPases and GBPs, and therefore exerts an antiviral effect in vitro. Collectively, our findings provide insights into the interactions of immune cells and phages, which establish phage-based antiviral effects.

A Novel Class of Norovirus Inhibitors Targeting the Viral Protease with Potent Antiviral Activity In Vitro and In Vivo.

Human noroviruses (HuNoVs) are the most common cause of viral gastroenteritis resulting annually in ~219,000 deaths and a societal cost of ~USD 60 billion, and no antivirals or vaccines are available. Here, we assess the anti-norovirus activity of new peptidomimetic aldehydes related to the protease inhibitor rupintrivir. The early hit compound 4 inhibited the replication of murine norovirus (MNV) and the HuNoV GI.1 replicon in vitro (EC50 ~1 µM) and swiftly cleared the HuNoV GI.1 replicon from the cells. Compound 4 still inhibits the proteolytic activity. We selected a resistant GI.1 replicon, with a mutation (I109V) in a highly conserved region of the viral protease, conferring a low yield of resistance against compound 4 and rupintrivir. After testing new derivatives, compound 10d was the most potent (EC50 nanomolar range). Molecular docking indicated that the aldehyde group of compounds 4 and 10d bind with Cys139 in the HuNoV 3CL protease by a covalent linkage. Finally, compound 10d inhibited the replication of HuNoV GII.4 in infected zebrafish larvae, and PK studies in mice showed an adequate profile.

Murine norovirus virulence factor 1 (VF1) protein contributes to viral fitness during persistent infection.

Murine norovirus (MNV) is widely used as a model for studying norovirus biology. While MNV isolates vary in their pathogenesis, infection of immunocompetent mice mostly results in persistent infection. The ability of a virus to establish a persistent infection is dependent on its ability to subvert or avoid the host immune response. Previously, we described the identification and characterization of virulence factor 1 (VF1) in MNV, and demonstrated its role as an innate immune antagonist. Here, we explore the role of VF1 during persistent MNV infection in an immunocompetent host. Using reverse genetics, we generated MNV-3 viruses carrying a single or a triple termination codon inserted in the VF1 ORF. VF1-deleted MNV-3 replicated to comparable levels to the wildtype virus in tissue culture. Comparative studies between MNV-3 and an acute MNV-1 strain show that MNV-3 VF1 exerts the same functions as MNV-1 VF1, but with reduced potency. C57BL/6 mice infected with VF1-deleted MNV-3 showed significantly reduced replication kinetics during the acute phase of the infection, but viral loads rapidly reached the levels seen in mice infected with wildtype virus after phenotypic restoration of VF1 expression. Infection with an MNV-3 mutant that had three termination codons inserted into VF1, in which reversion was suppressed, resulted in consistently lower replication throughout a 3 month persistent infection in mice, suggesting a role for VF1 in viral fitness in vivo. Our results indicate that VF1 expressed by a persistent strain of MNV also functions to antagonize the innate response to infection. We found that VF1 is not essential for viral persistence, but instead contributes to viral fitness in mice. These data fit with the hypothesis that noroviruses utilize multiple mechanisms to avoid and/or control the host response to infection and that VF1 is just one component of this.

Eliminating murine norovirus, Helicobacter hepaticus, and intestinal protozoa by embryo transfer for an entire mouse barrier facility.

Pathogens can affect physiological and immunological reactions in immunocompromised animals and genetically engineered mice. Specifically, murine norovirus (MNV), Helicobacter, and intestinal protozoa are prevalent in rodent laboratory facilities worldwide. In this study, microbiological test results of the soiled bedding of sentinel mice showed the prevalence of MNV (50.9%, 28/55), Helicobacter hepaticus (29.1%, 16/55), Trichomonas spp. (14.5%, 8/55), and Entamoeba spp. (32.7%, 18/55). No single infections were detected as all cases were confirmed to have complex infections with two or four pathogens. In previous studies, the success rate of the cross-fostering method was not perfect; therefore, in this study, the entire mouse strain of the SPF rodent facility was rederived using embryo transfer. For up to three years, we confirmed that the results were negative with regular health surveillance tests. Embryo transfer was, thus, determined to be an effective method for the rederivation of specific pathogen free (SPF) barrier mouse facilities. This is the report for the effectiveness of embryo transfer as an example of successful microbiological clean-up of a mouse colony with multiple infections in an entire SPF mouse facility and embryo transfer may be useful for rederiving.

Mori Cortex Radicis extract inhibits human norovirus surrogate in simulated digestive conditions.

Norovirus is a major cause of acute gastroenteritis globally, resulting in enormous health and societal costs. In this study, the antiviral activities of Mori Cortex Radicis (MCR) extract and its bioactive flavonoids, morusin and kuwanon G, were tested against murine norovirus (MNV), a human norovirus surrogate, using plaque assay. The antiviral activity was confirmed in simulated digestive conditions, including simulated saliva fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF). Pre-treatment of MNV with MCR extract at 1000 µg/mL showed antiviral activity with a 1.1-log reduction. Morusin and kuwanon G also demonstrated a 1.0-log and 0.6-log reductions of MNV titers, respectively, at 100 µM. MCR extract at a concentration of 2 mg/mL in SSF, SGF, and SIF markedly reduced MNV titers by 1.8, 1.9, and 1.5 logs, respectively. Therefore, these data suggest that MCR extract can be used to control norovirus infectivity.

Structure-Activity Relationship Studies on Novel Antiviral Agents for Norovirus Infections.

Human norovirus is the leading cause of acute gastroenteritis worldwide, affecting every year 685 million people. Norovirus outbreaks are associated with very significant economic losses, with an estimated societal cost of 60 billion USD per year. Despite this, no therapeutic options or vaccines are currently available to treat or prevent this infection. An antiviral therapy that can be used as treatment and as a prophylactic measure in the case of outbreaks is urgently needed. We previously described the computer-aided design and synthesis of novel small-molecule agents able to inhibit the replication of human norovirus in cell-based systems. These compounds are non-nucleoside inhibitors of the viral polymerase and are characterized by a terminal para-substituted phenyl group connected to a central phenyl ring by an amide-thioamide linker, and a terminal thiophene ring. Here we describe new modifications of these scaffolds focused on exploring the role of the substituent at the para position of the terminal phenyl ring and on removing the thioamide portion of the amide-thioamide linker, to further explore structure-activity relationships (SARs) and improve antiviral properties. According to three to four-step synthetic routes, we prepared thirty novel compounds, which were then evaluated against the replication of both murine (MNV) and human (HuNoV) norovirus in cells. Derivatives in which the terminal phenyl group has been replaced by an unsubstituted benzoxazole or indole, and the thioamide component of the amide-thioamide linker has been removed, showed promising results in inhibiting HuNoV replication at low micromolar concentrations. Particularly, compound 28 was found to have an EC50 against HuNoV of 0.9 µM. Although the most active novel derivatives were also associated with an increased cytotoxicity in the human cell line, these compounds represent a very promising starting point for the development of new analogues with reduced cytotoxicity and improved selectivity indexes. In addition, the experimental biological data have been used to create an initial 3D quantitative structure-activity relationship model, which could be used to guide the future design of novel potential anti-norovirus agents.

Experimental and CFD evaluation of ozone efficacy against coronavirus and enteric virus contamination on public transport surfaces

The limited information about the routes of the transmission of SARS-CoV-2 within the ongoing pandemic scenario mobilized the administration, industry and academy to develop sanitation and disinfection systems for public and private spaces. Ozone has been proposed as an effective disinfection method against enveloped and non-enveloped viruses, including viruses with similar morphology to SARS-CoV-2. Due to this efficacy, numerous gaseous and aqueous phase ozone applications have emerged potentially to inhibit virus persistence in aerosols, surfaces, and water. In this work, a numerical model, a RANS CFD model for ozone dispersion inside tram and underground coach has been developed including the chemical self-decomposition and surface reactions of the ozone. The CFD model has been developed for a real tram coach of 28.6 × 2.4 × 2.2 m (L × W × H) using 1.76 million nodes and the Menter’s shear stress transport turbulence model. The model predicts the O3 concentration needed to meet disinfection criteria and the fluid dynamics inside the public transport coach. The effectiveness of the system has been validated with laboratory and field tests in real full-scale coach using porcine epidemic diarrhea virus (PEDV) and murine norovirus (MNV-1) as SARS-CoV-2 and human norovirus surrogates, respectively. Lab-scale experiments on plastic surfaces demonstrated O3 disinfection (100 ppm, 95% RH, 25 min) inactivate > 99.8% MNV-1 and PEDV. Additionally, field tests in real full-scale coach demostrate the efficacy of the system as > 98.6% of infectious MNV-1 and > 96.3% PEDV were inactivated.

Development of a Specific Anti-capsid Antibody- and Magnetic Bead-Based Immunoassay to Detect Human Norovirus Particles in Stool Samples and Spiked Mussels via Flow Cytometry.

Human noroviruses impose a considerable health burden globally. Here, a flow cytometry approach designed for their detection in biological waste and food samples was developed using antibody-coated magnetic beads. Antipeptide antibodies against murine norovirus and various human norovirus genotypes were generated for capture and coated onto magnetic beads. A flow cytometry assay was then implemented to detect bead-bound human norovirus GI.3 in patient stool samples and in norovirus-spiked mussel digestive tissues. The detection limit for stool samples was 105gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was ten-fold higher than in stool samples. Further assays showed a decrease in fluorescence intensity for heat- or UV-inactivated virus particles. Overall, we demonstrate the application of a flow cytometry approach for direct detection of small non-enveloped virus particles such as noroviruses. An adaptation of the technology to routine diagnostics has the potential to contribute a rapid and sensitive tool to norovirus outbreak investigations. Further improvements to the method, notably decreasing the detection limit of the approach, may allow the analysis of naturally contaminated food and environmental samples.

Development of an RNA Extraction Protocol for Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR.

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.