Evaluation of a new automated viral RNA extraction platform for hepatitis A virus and human norovirus in testing of berries, lettuce, and oysters.

Fruits, vegetables, and shellfish are often associated with outbreaks of illness caused particularly by human norovirus (HuNoV) and hepatitis A virus (HAV), the leading causative agents of foodborne illness worldwide. The aim of this study was to evaluate a new automated nucleic acid extraction platform (EGENE-UP EASYPREP) for enteric viruses in several at-risk food matrices and to test its limit of detection in comparison to a semi-automated method (EGENE-UP) using Boom methodology for nucleic acid extraction as suggested in the reference method ISO 15216-2:2019. Fresh and frozen raspberries, frozen blackberries, romaine lettuce and oyster digestive glands were artificially contaminated with HAV, HuNoV GII.4 or HuNoV GI.7 at 102, 103 or 104 genome copies/sample. Virus was then recovered from the food matrix using the ISO method. Viral RNA extracted from frozen berry samples by the automated system was purified on a column for additional removal of RT-qPCR inhibitors. For fresh raspberry, oysters, and romaine lettuce, the two extraction platforms were deemed equivalent. For frozen raspberry, the automated platform appeared to be more efficient for viral recovery, particularly for HAV and HuNoV GI at lower concentrations. With frozen blackberries, the two platforms may be considered equivalent for all targeted viruses. However, the automated method led to less sample-associated inhibition of the PCR, 56.5 % of samples versus 95.0 % for the semi-automated. We thus found that the automated extraction can be performed easily by users while obtaining equivalent or even superior results to the ISO 15216-2:2019 method, and therefore appears to be suitable for routine sanitary monitoring in food processing and for tracing outbreaks of illness.

Attitudes toward a future norovirus vaccine among members of an integrated healthcare delivery system in Portland, Oregon, 2016-2017.

With recent advances in U.S. clinical trials for norovirus vaccines, it is an opportune time to examine what is known about the public receptivity to this novel vaccine. From October 2016-September 2017, we surveyed Kaiser Permanente Northwest members in Portland, Oregon, to ask their level of agreement on a 5-point scale with statements about the need for and willingness to get a potential norovirus vaccine for themselves or their child and analyzed their responses according to age, occupational status, prior vaccine uptake, and history of prior norovirus diagnoses. The survey response rate was 13.5% (n = 3,894); 807 (21%) responded as legal guardians, on behalf of a child <18 y of age and 3,087 (79%) were adults aged 18+ y. The majority of respondents were in agreement about getting the norovirus vaccine, if available (60% of legal guardians, 52% of adults aged 18-64 y, and 55% of adults aged 65+ y). Prior vaccination for influenza and rotavirus (among children) was the only correlate significantly associated with more positive attitudes toward receiving norovirus vaccine. Pre-pandemic attitudes in our all-ages study population reveal generally positive attitudes toward willingness to get a norovirus vaccine, particularly among those who previously received influenza or rotavirus vaccines.

Screening of natural extracts with anti-norovirus effects and analysis of this mechanism in grape seed extract.

Norovirus (NoV）is a major causative virus of viral gastroenteritis and requires a general disinfection method because it is resistant to common disinfectants such as ethanol and chlorhexidine. This study aimed to find natural extracts as candidates for versatile disinfectant ingredients. The antiviral effect of natural extracts against NoV can be evaluated using the feline calicivirus (FCV）-inactivation test and NoV virus-like particle (NoV-VLP）-binding inhibition test. In this study, screening of natural extracts with anti- NoV effects was performed using these two methods. Of the 63 natural extracts examined, 14 were found to have high FCV-inactivation and NoV-VLP-binding inhibitory effects. In addition, we evaluated the NoV-VLPbinding inhibitory effect of grape seed extract（GSE）containing proanthocyanidins under multiple concentration conditions and treatment times and determined that the binding inhibitory effect of GSE was concentration- and time-dependent. Electron microscopy showed that GSE-treated NoV-VLPs aggregated, distorted, and swelled, suggesting that GSE directly interacts with NoV particles. The results suggest that some natural extracts containing GSE can be used as components of disinfectants against NoV.

Simulation of contamination and elimination of Escherichia coli, Listeria monocytogenes, and Murine norovirus 1 (MNV-1) from the washing process when handling of potatoes.

Root vegetables, which are in close contact with soil, are particularly vulnerable to soil contamination or decay as they can be contaminated from multiple sources, including primary production and processing. This study investigated effective washing conditions to reduce the microbial contamination of potatoes by using soaking and shaking in the washing process. The reduction of Escherichia coli, Listeria monocytogenes, and Murine norovirus 1 (MNV-1) in four washing processes (soaking only, shaking only, combined soaking-shaking I, and combined soaking-shaking I-shaking II) were compared. The numbers of E. coli and L. monocytogenes decreased by 0.55 and 0.49 log CFU/g after shaking only, 1.96 and 1.80 log CFU/g after soaking, 2.07 and 1.67 log CFU/g after soaking-shaking I, and 2.42 and 1.90 log CFU/g after soaking-shaking I-shaking II, respectively. The combined process reduced the microbial contamination more efficiently than shaking only. The reduction of E. coli in the washing process was higher than that of L. monocytogenes by approximately 0.5 logs. MNV-1 showed a reduction in the soaking and shaking steps by 1.34 and 1.98 log GC/100 g, with no significant reduction observed after the combination process. A combined process of soaking-shaking I-shaking II was effective to eliminate E. coli, L. monocytogenes, and MNV-1 from potatoes during the handling and washing process.

A "peptide-target-aptamer" electrochemical biosensor for norovirus detection using a black phosphorous nanosheet@Ti3C2-Mxene nanohybrid and magnetic covalent organic framework.

Norovirus (NoV) is a major foodborne pathogen responsible for acute gastroenteritis epidemics, and establishing a robust detection method for the timely identification and monitoring of NoV contamination is of great significance. In this study, a peptide-target-aptamer sandwich electrochemical biosensor for NoV was fabricated using Au@BP@Ti3C2-MXene and magnetic Au@ZnFe2O4@COF nanocomposites. The response currents of the electrochemical biosensor were proportional to the NoV concentrations ranging from 0.01-105 copies/mL with a detection limit (LOD) of 0.003 copies/mL (S/N = 3). To our best knowledge, this LOD was the lowest among published assays to date, due to the specific recognition of the affinity peptide and aptamer for NoV and the outstanding catalytic activity of nanomaterials. Furthermore, the biosensor showed excellent selectivity, anti-interference performance, and satisfactory stability. The NoV concentrations in simulative food matrixes were successfully detected using the constructed biosensor. Meanwhile, NoV in stool samples was also successfully quantified without complex pretreatment. The designed biosensor had the potential to detect NoV (even at a low level) in foods, clinical samples, and environmental samples, providing a new method for NoV detection in food safety and diagnosing foodborne pathogens.

An oral NoV-rAd5 vaccine with built-in dsRNA adjuvant elicits systemic immune responses in mice.

Norovirus (NoV) is an enteric pathogen notorious for causing epidemics of acute gastroenteritis. An effective vaccine against NoV is therefore urgently needed. A short double-stranded RNA (dsRNA) has been described that acts as a retinoic-acid-inducible gene-I agonist to induce the production of type I interferon; it also exhibits adjuvant activity. Using built-in dsRNA of different lengths (DS1 and DS2), we developed a recombinant adenovirus 5 (rAd5) expressing NoV VP1, and evaluated its immunogenicity following oral administration in a mouse model. An in vitro study demonstrated that the dsRNA adjuvants significantly enhanced VP1 protein expression in infected cells. The oral administration of both rAd5-VP1-DS vaccines elicited high serum levels of VP1-specific IgG and blocking antibodies, as well as strong and long-lasting mucosal immunity. There was no apparent difference in immunostimulatory effects in immunised mice between the two dsRNA adjuvants. This study indicates that an oral NoV-rAd5 vaccine with a built-in dsRNA adjuvant may be developed to prevent NoV infection in humans.

Risk of Gastroenteritis from Swimming at a Wastewater-Impacted Tropical Beach Varies across Localized Scales.

Population growth and changing climate are expected to increase human exposure to pathogens in tropical coastal waters. We examined microbiological water quality in three rivers within 2.3 km of each other that impact a Costa Rican beach and in the ocean outside their plumes during the rainy and dry seasons. We performed quantitative microbial risk assessment (QMRA) to predict the risk of gastroenteritis associated with swimming and the amount of pathogen reduction needed to achieve safe conditions. Recreational water quality criteria based on enterococci were exceeded in >90% of river samples but in only 13% of ocean samples. Multivariate analysis grouped microbial observations by subwatershed and season in river samples but only by subwatershed in the ocean. The modeled median risk from all pathogens in river samples was between 0.345 and 0.577, 10-fold above the U.S. Environmental Protection Agency (U.S. EPA) benchmark of 0.036 (36 illnesses/1,000 swimmers). Norovirus genogroup I (NoVGI) contributed most to risk, but adenoviruses raised risk above the threshold in the two most urban subwatersheds. The risk was greater in the dry compared to the rainy season, due largely to the greater frequency of NoVGI detection (100% versus 41%). Viral log10 reduction needed to ensure safe swimming conditions varied by subwatershed and season and was greatest in the dry season (3.8 to 4.1 dry; 2.7 to 3.2 rainy). QMRA that accounts for seasonal and local variability of water quality contributes to understanding the complex influences of hydrology, land use, and environment on human health risk in tropical coastal areas and can contribute to improved beach management. IMPORTANCE This holistic investigation of sanitary water quality at a Costa Rican beach assessed microbial source tracking (MST) marker genes, pathogens, and indicators of sewage. Such studies are still rare in tropical climates. Quantitative microbial risk assessment (QMRA) found that rivers impacting the beach consistently exceeded the U.S. EPA risk threshold for gastroenteritis of 36/1,000 swimmers. The study improves upon many QMRA studies by measuring specific pathogens, rather than relying on surrogates (indicator organisms or MST markers) or estimating pathogen concentrations from the literature. By analyzing microbial levels and estimating the risk of gastrointestinal illness in each river, we were able to discern differences in pathogen levels and human health risks even though all rivers were highly polluted by wastewater and were located less than 2.5 km from one another. This variability on a localized scale has not, to our knowledge, previously been demonstrated.

Phenol Derivatives Obtained from Grape Seed Extract Show Virucidal Activity against Murine Norovirus.

Human noroviruses are the most common pathogens known to cause acute gastroenteritis, a condition that can lead to severe illness among immunocompromised individuals such as organ transplant recipients and the elderly. To date, no safe and effective vaccines or therapeutic agents have been approved for treating norovirus infections. Therefore, we aimed to demonstrate the virucidal activity of grape seed extract (GSE), which contains >83% proanthocyanidins, against murine norovirus (MNV), a surrogate for human norovirus. GSE showed virucidal activity against MNV in a dose- and time-dependent manner. Atomic force microscopic analysis showed viral particle aggregates after treatment of MNV with GSE. MNV treated with 50 µg/mL of GSE for 10 min resulted in the absence of pathogenicity in an animal model of infection, indicating that GSE has irreversible virucidal activity against MNV particles. Thus, GSE may aid in the development of treatments for norovirus infections.

Antiviral Activities of Asarones and Rhizomes of Acorus gramineus on Murine Norovirus.

Noroviruses (NVs) are a major cause of foodborne diseases worldwide. The rhizomes of Acorus gramineus (AGR) have been used as a traditional medicinal plant and a food additive. In this study, AGR and its bioactive components-α-asarone and ß-asarone-showed significant antiviral activities against murine NV (MNV) with pre-treatment, with more than two log reductions in viral plaques. They also demonstrated strong inhibition on binding to A- and O-type saliva by the recombinant P domain derived from human NV (HuNV) GII.4. Both α- and ß-asarones also inhibited the binding of the P domain to the receptor at 0.125-1 mM in a concentration-dependent manner and induced a marked reduction in Tm, suggesting that they may reduce structural stability and block receptor binding by the P domain. In simulated digestive conditions, the AGR extract, α-asarone, or ß-asarone further showed a significant reduction of MNV plaques by 1.5-2.8 logs. The asarones show a potential for development as a scaffold for anti-NV agents.

Natural extracts, honey, and propolis as human norovirus inhibitors.

Norovirus is the most important cause of acute gastroenteritis, yet there are still no antivirals, vaccines, or treatments available. Several studies have shown that norovirus-specific monoclonal antibodies, Nanobodies, and natural extracts might function as inhibitors. Therefore, the objective of this study was to determine the antiviral potential of additional natural extracts, honeys, and propolis samples. Norovirus GII.4 and GII.10 virus-like particles (VLPs) were treated with different natural samples and analyzed for their ability to block VLP binding to histo-blood group antigens (HBGAs), which are important norovirus co-factors. Of the 21 natural samples screened, date syrup and one propolis sample showed promising blocking potential. Dynamic light scattering indicated that VLPs treated with the date syrup and propolis caused particle aggregation, which was confirmed using electron microscopy. Several honey samples also showed weaker HBGA blocking potential. Taken together, our results found that natural samples might function as norovirus inhibitors.

Inactivation Mechanism and Efficacy of Grape Seed Extract for Human Norovirus Surrogate.

Proper disinfection of harvested food and water is critical to minimize infectious disease. Grape seed extract (GSE), a commonly used health supplement, is a mixture of plant-derived polyphenols. Polyphenols possess antimicrobial and antifungal properties, but antiviral effects are not well-known. Here we show that GSE outperformed chemical disinfectants (e.g., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. GSE induced virus aggregation, a process that correlated with a decrease in virus titers. This aggregation and disinfection were not reversible. Molecular docking simulations indicate that polyphenols potentially formed hydrogen bonds and strong hydrophobic interactions with specific residues in viral capsid proteins. Together, these data suggest that polyphenols physically associate with viral capsid proteins to aggregate viruses as a means to inhibit virus entry into the host cell. Plant-based polyphenols like GSE are an attractive alternative to chemical disinfectants to remove infectious viruses from water or food. IMPORTANCE Human noroviruses are major food- and waterborne pathogens, causing approximately 20% of all cases of acute gastroenteritis cases in developing and developed countries. Proper sanitation or disinfection are critical strategies to minimize human norovirus-caused disease until a reliable vaccine is created. Grape seed extract (GSE) is a mixture of plant-derived polyphenols used as a health supplement. Polyphenols are known for antimicrobial, antifungal, and antibiofilm activities, but antiviral effects are not well-known. In studies presented here, plant-derived polyphenols outperformed chemical disinfectants (i.e., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. Based on data from molecular assays and molecular docking simulations, the current model is that the polyphenols in GSE bind to the Tulane virus capsid, an event that triggers virion aggregation. It is thought that this aggregation prevents Tulane virus from entering host cells.

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.

Norovirus and Other Viral Causes of Medically Attended Acute Gastroenteritis Across the Age Spectrum: Results from the Medically Attended Acute Gastroenteritis Study in the United States.

BACKGROUND: Acute gastroenteritis (AGE) causes a substantial burden in the United States, but its etiology frequently remains undetermined. Active surveillance within an integrated healthcare delivery system was used to estimate the prevalence and incidence of medically attended norovirus, rotavirus, sapovirus, and astrovirus. METHODS: Active surveillance was conducted among all enrolled members of Kaiser Permanente Northwest during July 2014-June 2016. An age-stratified, representative sample of AGE-associated medical encounters were recruited to provide a stool specimen to be tested for norovirus, rotavirus, sapovirus, and astrovirus. Medically attended AGE (MAAGE) encounters for a patient occurring within 30 days were grouped into 1 episode, and all-cause MAAGE incidence was calculated. Pathogen- and healthcare setting-specific incidence estimates were calculated using age-stratified bootstrapping. RESULTS: The overall incidence of MAAGE was 40.6 episodes per 1000 person-years (PY), with most episodes requiring no more than outpatient care. Norovirus was the most frequently detected pathogen, with an incidence of 5.5 medically attended episodes per 1000 PY. Incidence of norovirus MAAGE was highest among children aged < 5 years (20.4 episodes per 1000 PY), followed by adults aged ≥ 65 years (4.5 episodes per 1000 PY). Other study pathogens showed similar patterns by age, but lower overall incidence (sapovirus: 2.4 per 1000 PY; astrovirus: 1.3 per 1000 PY; rotavirus: 0.5 per 1000 PY). CONCLUSIONS: Viral enteropathogens, particularly norovirus, are important contributors to MAAGE, especially among children < 5 years of age. The present findings underline the importance of judicious antibiotics use for pediatric AGE and suggest that an effective norovirus vaccine could substantially reduce MAAGE.

Antiviral Potential of Plants against Noroviruses.

Human noroviruses, which belong to the enterovirus family, are one of the most common etiological agents of food-borne diseases. In recent years, intensive research has been carried out regarding the antiviral activity of plant metabolites that could be used for the preservation of fresh food, because they are safer for consumption when compared to synthetic chemicals. Plant preparations with proven antimicrobial activity differ in their chemical compositions, which significantly affects their biological activity. Our review aimed to present the results of research related to the characteristics, applicability, and mechanisms of the action of various plant-based preparations and metabolites against norovirus. New strategies to combat intestinal viruses are necessary, not only to ensure food safety and reduce infections in humans but also to lower the direct health costs associated with them.

Bioactive extracts from persimmon waste: influence of extraction conditions and ripeness.

In this work, a bioactive persimmon extract was produced from discarded fruits. A central composite design was used to evaluate the effect of different extraction parameters and ripeness stages of persimmon fruits on the total phenolic content and antioxidant activity of the resulting extracts. Significantly greater phenolic contents were obtained from immature persimmon (IP) fruits. The optimum IP extract with the conditions set by the experimental design was industrially up-scaled and its composition and functional properties were evaluated and compared with those obtained under lab-scale conditions. Both extracts contained significant protein (>20%) and phenolic contents (∼11-27 mg GA/g dry extract) and displayed significant antiviral activity against murine norovirus and hepatitis A virus. Moreover, the extract showed no toxicity and significantly reduced the fat content and the cellular ageing of Caenorhabditis elegans (C. elegans) without affecting the worm development. These effects were mediated by down-regulation of fat-7, suggesting an anti-lipogenic activity of this extract.

Protective effect of pogostone on murine norovirus infected-RAW264.7 macrophages through inhibition of NF-κB/NLRP3-dependent pyroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Pogostemon cablin, the dry overground parts of Pogostemon cablin (Blanco) Benth, has been widely used in the treatment of gastrointestinal dysfunction, such as nausea, diarrhea, headaches and fever. Pogostone (PO) is a major component of Pogostemon cablin which has a variety of pharmacological properties, including antiinflammatory, and immunosuppressive activities, and antioxidant. However, the effect of PO on norovirus gastroenteritis and the underlying molecular mechanism remain unclear. AIM OF THE STUDY: The purpose of our study is to investigate the effects of PO against MNV infection using RAW264.7 cells and to elucidate its active mechanisms. MATERIALS AND METHODS: The cell viability was assessed using Cell Counting Kit-8 (CCK-8) assay and Fluorescein diacetate (FDA) staining. The activation of nuclear factor kappa B (NF-κB) signaling and NOD-like receptor 3 (NLRP3) inflammasome was evaluated by assessing the level of phospho-NF-κB p65, interleukin (IL)-6, TNF-α, NLRP3, cleaved caspase-1, IL-18, IL-1ß using Western blot and quantitative real-time PCR (qPCR), respectively. The number of infected cells were determined by immunofluorescence microscopic assay. RESULTS: PO did not possess a cytotoxic effect toward RAW264.7 cells. The cytotoxic damage caused by MNV infection in RAW264.7 cells decreased significantly in the presence of PO. Cell viability assays showed that pyroptosis is the major mechanism of death in MNV-infected RAW264.7 cells. PO could decreased the expression levels of p-p65, IL-6, TNF-α, NLRP3, cleaved caspase-1, IL-1ß, and IL-18. CONCLUSIONS: These results demonstrate that PO decreases MNV-induced RAW264.7 macrophages death and MNV replication through repressing NF-κB/NLRP3-dependent pyroptosis. Therefore PO may be considered as a potential therapeutic agent for preventing and treating norovirus gastroenteritis.

Antiviral Effects of Pomegranate Peel Extracts on Human Norovirus in Food Models and Simulated Gastrointestinal Fluids.

Human noroviruses (HuNoV) are the dominant cause of viral gastroenteritis in all age groups worldwide. In this study, we investigated the effects of pomegranate peel extract (PPE) on the reduction of HuNoV in different food models, on surfaces of fresh produce (green onion and cherry tomato), in low-fat milk, and simulated gastrointestinal fluids. The antiviral efficacy of PPE against HuNoV was evaluated by quantifying the number of residual virus genomes using a quantitative reverse transcription PCR (qRT-PCR) assay. Pomegranate peel, considered as a waste product of industrial processing, is known for beneficial health effects and broad antimicrobial activity due to the high content of phenolic compounds and tannins. PPE showed significant antiviral properties against HuNoV both in phosphate-buffered saline (PBS) and simulated gastric fluid. The reduction of HuNoV by pomegranate juice was lower than with PPE, which could be attributed to the lower content of antimicrobial compounds. A pretreatment of cherry tomato and green onion surfaces with PPE significantly reduced the amount of HuNoV particles that adhered to those surfaces during subsequent virus suspension treatment. A detrimental effect of PPE on HuNoV structure was confirmed by transmission electron microscopy. Our results indicate that PPE is a natural antiviral agent effective against food-borne noroviruses.

Magnolia officinalis and Its Honokiol and Magnolol Constituents Inhibit Human Norovirus Surrogates.

Norovirus is a major cause of foodborne disease and nonbacterial gastroenteritis globally. This study evaluated the antiviral effects of Magnolia officinalis extract and its honokiol and magnolol constituents against human norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV) in vitro, and in model food systems. Pretreatment or cotreatment of M. officinalis extract at 1 mg/mL reduced MNV and FCV titers by 0.6-1.8 log. Honokiol and magnolol, which are the major polyphenols in the extract, showed significant antiviral effects against MNV and FCV. The virus-infected cells that were treated with M. officinalis extract exhibited significantly increased glutathione levels (p < 0.05). The extract, honokiol, and magnolol revealed ferric ion-reducing and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities in a dose-dependent manner. Furthermore, MNV and FCV titers were reduced by >1.6 log or to undetectable levels in apple, orange, and plum juices and by 0.9 and 1.6 log in milk, respectively, when they were treated with the extract at 5 mg/mL. Therefore, the present study suggests that M. officinalis extract can be used as an antiviral food material to control norovirus foodborne diseases.

Effect of Plant-Derived Proteases on Infectivity of Tulane Virus, Murine Norovirus, and Hepatitis A Virus.

ABSTRACT: Plant-derived proteases, bromelain, papain, and ficin, are broad-acting enzymes with generally recognized as safe status for foods and have current application in several food industries. These proteases have also been reported to have antimicrobial properties. This study investigated the efficacy of commercially prepared bromelain, papain, and ficin, individually and combined (2,500 ppm of crude extract), for inactivation of hepatitis A virus (HAV) and human norovirus surrogates, Tulane virus (TV), and murine norovirus (MNV). Various treatment temperatures (45, 50, or 55°C), times (10 or 60 min), and pH values (5.5 or 7.0) in the presence of cysteine (2 mM) were evaluated. Inactivation was assessed by infectivity in plaque assay for TV and MNV and by median tissue culture infective dose for HAV. No reduction in infectious TV or HAV was attributed to the plant-derived proteases at any of the conditions tested. Infectious MNV was reduced by 1 to 3 log PFU/mL; the most effective treatment was bromelain at pH 7 and 50°C for 10 min. A time course study with MNV in bromelain at 50°C indicated that a 2-log PFU/mL reduction could be achieved within 6 min, but extended treatment of 15 min was still insufficient to eliminate infectious MNV. The lack of or limited efficacy of bromelain, papain, and ficin on HAV, TV, and MNV, even at elevated temperatures and exposure times, suggests the plant-derived proteases are not commercially applicable for inactivation of virus on commodities or materials that could not also withstand mild heat treatment. The variable susceptibilities observed between TV and MNV illustrate limitations in utilization of surrogates for predicting pathogen behavior for a structure-specific treatment.

Efficacy of alcohol-based hand sanitizers against human norovirus using RNase-RT-qPCR with validation by human intestinal enteroid replication.

Successful human norovirus (HuNoV) cultivation in stem cell-derived human intestinal enteroids (HIE) was recently reported. The purpose of this study was to evaluate the anti-HuNoV efficacy of two alcohol-based commercial hand sanitizers and 60% ethanol by suspension assay using RNase-RT-qPCR, with subsequent validation of efficacy by HuNoV cultivation using the HIE model. In suspension, when evaluated by RNase-RT-qPCR, 60% ethanol resulted in less than one log10 reduction in HuNoV genome equivalent copies (GEC) regardless of contact time (30 or 60s) or soil load. The two commercial products outperformed 60% ethanol regardless of contact time or soil load, providing 2·2-3·2 log10 HuNoV GEC reductions by suspension assay. Product B could not be validated in the HIE model due to cytotoxicity. Following a 60s exposure, viral replication in the HIE model increased 1·9 ± 0·2 log10 HuNoV GEC for the neutralization (positive) control and increased 0·9 ± 0·2 log10 HuNoV GEC in challenged HIE after treatment with 60% ethanol. No HuNoV replication in HIE was observed after a 60 s exposure to Product A.