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.

Evaluation of Steady-State Gaseous Chlorine Dioxide Treatment for the Inactivation of Tulane virus on Berry Fruits.

The effectiveness of steady-state levels of gaseous chlorine dioxide (ClO2) against Tulane virus (TV), a human norovirus surrogate, on berries was determined. The generated ClO2 was maintained at 1 mg/L inside a 269 L glove box to treat two 50 g batches of blueberries, raspberries, and blackberries, and two 100 g batches of strawberries that were immersion coated with TV. The standardized/normalized treatment concentrations of ClO2 ranging from 0.63 to 4.40 ppm-h/g berry were evaluated. When compared to untreated TV contaminated berries, log reductions of TV were in excess of 2.9 log PFU/g for all berry types and conditions tested, indicating that ClO2 was highly effective. In general, the efficacy of all ClO2 treatments on log reductions of TV on all berries was not significantly different (p < 0.05). The average log reduction with strawberries, raspberries, blueberries, and blackberries, treated with the lowest ClO2 concentration, 0.63 ppm-h/g, were 2.98, 3.40, 3.82, and 4.17 log PFU/g, respectively. Overall results suggest that constant levels of ClO2 could be quite effective against foodborne viruses.

Inhibition of Murine Norovirus and Feline Calicivirus by Edible Herbal Extracts.

Human noroviruses (HuNoVs) cause foodborne and waterborne viral gastroenteritis worldwide. Because HuNoV culture systems have not been developed thus far, no available medicines or vaccines preventing infection with HuNoVs exist. Some herbal extracts were considered as phytomedicines because of their bioactive components. In this study, the inhibitory effects of 29 edible herbal extracts against the norovirus surrogates murine norovirus (MNV) and feline calicivirus (FCV) were examined. FCV was significantly inhibited to 86.89 ± 2.01 and 48.71 ± 7.38% by 100 µg/mL of Camellia sinensis and Ficus carica, respectively. Similarly, ribavirin at a concentration of 100 µM significantly reduced the titer of FCV by 77.69 ± 10.40%. Pleuropterus multiflorus (20 µg/mL) showed antiviral activity of 53.33 ± 5.77, and 50.00 ± 16.67% inhibition was observed after treatment with 20 µg/mL of Alnus japonica. MNV was inhibited with ribavirin by 59.22 ± 16.28% at a concentration of 100 µM. Interestingly, MNV was significantly inhibited with 150 µg/mL Inonotus obliquus and 50 µg/mL Crataegus pinnatifida by 91.67 ± 5.05 and 57.66 ± 3.36%, respectively. Treatment with 20 µg/mL Coriandrum sativum slightly reduced MNV by 45.24 ± 4.12%. The seven herbal extracts of C. sinensis, F. carica, P. multiflorus, A. japonica, I. obliquus, C. pinnatifida, and C. sativum may have the potential to control noroviruses without cytotoxicity.

High hydrostatic pressure inactivation of murine norovirus and human noroviruses on green onions and in salsa.

In this study, high hydrostatic pressure (HHP) was evaluated as an intervention for human noroviruses (HuNoVs) in green onions and salsa. To determine the effect of water during HHP treatment on virus inactivation, a HuNoV surrogate, murine norovirus 1 (MNV-1), was inoculated onto green onions and then HHP-treated at 350MPa with or without water at 4 or 20°C. The presence of water enhanced HHP inactivation of MNV-1 on green onions at 4°C but not at 20°C. To test the temperature effect on HHP inactivation of MNV-1, inoculated green onions were HHP-treated at 300MPa at 1, 4 and 10°C. As the temperature decreased, MNV-1 became more sensitive to HHP treatment. HHP inactivation curves of MNV-1 on green onions and salsa were obtained at 300 or 350MPa for 0.5-3min at 1°C. All three inactivation curves showed a linear relationship between log reduction of MNV-1 and time. D values of HHP inactivation of MNV-1 on green onions were 1.10 and 0.61min at 300 and 350MPa, respectively. The D value of HHP inactivation of MNV-1 in salsa at 300MPa was 0.63min. HHP inactivation of HuNoV GI.1 and GII.4 on green onions and salsa was also conducted. To achieve >3 log reduction of HuNoV GI.1, HHP treatments for 2min at 1°C should be conducted at 600MPa and 500MPa for green onions and salsa, respectively. To achieve >3 log reduction of HuNoV GII.4, HHP treatments for 2min at 1°C should be conducted at 500MPa and 300MPa for green onions and salsa, respectively.

Antiviral effects of black raspberry (Rubus coreanus) seed extract and its polyphenolic compounds on norovirus surrogates.

Black raspberry seeds, a byproduct of wine and juice production, contain large quantities of polyphenolic compounds. The antiviral effects of black raspberry seed extract (RCS) and its fraction with molecular weight less than 1 kDa (RCS-F1) were examined against food-borne viral surrogates, murine norovirus-1 (MNV-1) and feline calicivirus-F9 (FCV-F9). The maximal antiviral effect was achieved when RCS or RCS-F1 was added simultaneously to cells with MNV-1 or FCV-F9, reaching complete inhibition at 0.1-1 mg/mL. Transmission electron microscopy (TEM) images showed enlarged viral capsids or disruption (from 35 nm to up to 100 nm) by RCS-F1. Our results thus suggest that RCS-F1 can interfere with the attachment of viral surface protein to host cells. Further, two polyphenolic compounds derived from RCS-F1, cyanidin-3-glucoside (C3G) and gallic acid, identified by liquid chromatography-tandem mass spectrometry, showed inhibitory effects against the viruses. C3G was suggested to bind to MNV-1 RNA polymerase and to enlarge viral capsids using differential scanning fluorimetry and TEM, respectively.

Aqueous Extracts of Hibiscus sabdariffa Calyces Decrease Hepatitis A Virus and Human Norovirus Surrogate Titers.

Hibiscus sabdariffa extract is known to have antioxidant, anti-diabetic, and antimicrobial properties. However, their effects against foodborne viruses are currently unknown. The objective of this study was to determine the antiviral effects of aqueous extracts of H. sabdariffa against human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) and hepatitis A virus (HAV) at 37 °C over 24 h. Individual viruses (~5 log PFU/ml) were incubated with 40 or 100 mg/ml of aqueous hibiscus extract (HE; pH 3.6), protocatechuic acid (PCA; 3 or 6 mg/ml, pH 3.6), ferulic acid (FA; 0.5 or 1 mg/ml; pH 4.0), malic acid (10 mM; pH 3.0), or phosphate buffered saline (pH 7.2 as control) at 37 °C over 24 h. Each treatment was replicated thrice and plaque assayed in duplicate. FCV-F9 titers were reduced to undetectable levels after 15 min with both 40 and 100 mg/ml HE. MNV-1 was reduced by 1.77 ± 0.10 and 1.88 ± 0.12 log PFU/ml after 6 h with 40 and 100 mg/ml HE, respectively, and to undetectable levels after 24 h by both concentrations. HAV was reduced to undetectable levels by both HE concentrations after 24 h. PCA at 3 mg/ml reduced FCV-F9 titers to undetectable levels after 6 h, MNV-1 by 0.53 ± 0.01 log PFU/ml after 6 h, and caused no significant change in HAV titers. FA reduced FCV-F9 to undetectable levels after 3 h and MNV-1 and HAV after 24 h. Transmission electron microscopy showed no conclusive results. The findings suggest that H. sabdariffa extracts have potential to prevent foodborne viral transmission.

Mechanisms of antiviral action of plant antimicrobials against murine norovirus.

Numerous plant compounds have antibacterial or antiviral properties; however, limited research has been conducted with nonenveloped viruses. The efficacies of allspice oil, lemongrass oil, and citral were evaluated against the nonenveloped murine norovirus (MNV), a human norovirus surrogate. The antiviral mechanisms of action were also examined using an RNase I protection assay, a host cell binding assay, and transmission electron microscopy. All three antimicrobials produced significant reductions (P ≤ 0.05) in viral infectivity within 6 h of exposure (0.90 log10 to 1.88 log10). After 24 h, the reductions were 2.74, 3.00, and 3.41 log10 for lemongrass oil, citral, and allspice oil, respectively. The antiviral effect of allspice oil was both time and concentration dependent; the effects of lemongrass oil and citral were time dependent. Based on the RNase I assay, allspice oil appeared to act directly upon the viral capsid and RNA. The capsids enlarged from ≤ 35 nm to up to 75 nm following treatment. MNV adsorption to host cells was not significantly affected. Alternatively, the capsid remained intact following exposure to lemongrass oil and citral, which appeared to coat the capsid, causing nonspecific and nonproductive binding to host cells that did not lead to successful infection. Such contrasting effects between allspice oil and both lemongrass oil and citral suggest that though different plant compounds may yield similar reductions in virus infectivity, the mechanisms of inactivation may be highly varied and specific to the antimicrobial. This study demonstrates the antiviral properties of allspice oil, lemongrass oil, and citral against MNV and thus indicates their potential as natural food and surface sanitizers to control noroviruses.

Comparative uptake of enteric viruses into spinach and green onions.

Root uptake of enteric pathogens and subsequent internalization has been a produce safety concern and is being investigated as a potential route of pre-harvest contamination. The objective of this study was to determine the ability of hepatitis A virus (HAV) and the human norovirus surrogate, murine norovirus (MNV), to internalize in spinach and green onions through root uptake in both soil and hydroponic systems. HAV or MNV was inoculated into soil matrices or into two hydroponic systems, floating and nutrient film technique systems. Viruses present within spinach and green onions were detected by RT-qPCR or infectivity assays after inactivating externally present viruses with Virkon(®). HAV and MNV were not detected in green onion plants grown up to 20 days and HAV was detected in only 1 of 64 spinach plants grown in contaminated soil substrate systems up to 20 days. Compared to soil systems, a drastic difference in virus internalization was observed in hydroponic systems; HAV or pressure-treated HAV and MNV were internalized up to 4 log RT-qPCR units and internalized MNV was shown to remain infectious. Understanding the interactions of human enteric viruses on produce can aid in the elucidation of the mechanisms of attachment and internalization, and aid in understanding risks associated with contamination events.

Antiviral effects of black raspberry (Rubus coreanus) juice on foodborne viral surrogates.

Abstract Human noroviruses (HuNoVs) are the most frequent cause of foodborne viral gastroenteritis, causing approximately 90% of non-bacterial epidemic outbreaks around the world. Rubus coreanus is a species of black raspberry, rich in polyphenols, and known to exert anti-inflammatory, antibacterial, and antiviral activities. In the present study, the antiviral effects of R. coreanus juice (black raspberry [BRB] juice) on foodborne viral surrogates, murine norovirus-1 (MNV-1) and feline calicivirus-F9 (FCV-F9), were compared with those of cranberry juice, grape juice, and orange juice by plaque assays. Among the four juices tested, BRB juice was the most effective in reducing plaques formation of these viruses. Time-of-addition experiments were designed to determine the mechanism of action of BRB juice on MNV-1 and FCV-F9. The maximal antiviral effect of BRB juice against MNV-1 was observed when it was added to RAW 264.7 cells (mouse leukemic monocyte macrophage cell line) simultaneously with the virus. Pre-treatment of either Crandell Reese Feline Kidney cells or FCV-F9 with BRB juice exhibited significant antiviral activity. The inhibition of viral infection by BRB juice on MNV-1 and FCV-F9 probably occurs at the internalization of virions into the cell or the attachment of the viral surface protein to the cellular receptor. The polyphenol components in BRB (i.e., gallic acid and quercetin), however, did not show any activity against these viruses. Our data provide great promise for the utilization of BRB in the prevention of foodborne viral outbreaks.

Inactivation of norovirus surrogates on surfaces and raspberries by steam-ultrasound treatment.

Human disease outbreaks caused by norovirus (NoV) following consumption of contaminated raspberries are an increasing problem. An efficient method to decontaminate the fragile raspberries and the equipment used for processing would be an important step in ensuring food safety. A potential surface treatment that combines pressurized steam and high-power ultrasound (steam-ultrasound) was assessed for its efficacy to inactivate human NoV surrogates: coliphage (MS2), feline calicivirus (FCV), and murine norovirus (MNV) inoculated on plastic surfaces and MS2 inoculated on fresh raspberries. The amounts of infectious virus and viral genomes were determined by plaque assay and reverse transcription-real time quantitative PCR (RT-qPCR), respectively. On plastic surfaces, an inactivation of >99.99% was obtained for both MS2 and FCV, corresponding to a 9.1-log and >4.8-log reduction after 1 or 3 s of treatment, respectively; while a 3.7-log (99.9%) reduction of MNV was reached after 3 s of treatment. However, on fresh raspberries only a 1-log reduction (∼89%) of MS2 could be achieved after 1 s of treatment, at which point damage to the texture of the fresh raspberries was evident. Increasing treatment time (0 to 3 s) resulted in negligible reductions of viral genome titers of MS2, FCV, and MNV on plastic surfaces as well as of MS2 inoculated on raspberries. Steam-ultrasound treatment in its current format does not appear to be an appropriate method to achieve sufficient decontamination of NoV-contaminated raspberries. However, steam-ultrasound may be used to decontaminate smooth surface areas and utensils in food production and processing environments.

Ozone inactivation of norovirus surrogates on fresh produce.

Preharvest contamination of produce by foodborne viruses can occur through a variety of agents, including animal feces/manures, soil, irrigation water, animals, and human handling. Problems of contamination are magnified by potential countrywide distribution. Postharvest processing of produce can involve spraying, washing, or immersion into water with disinfectants; however, disinfectants, including chlorine, have varying effects on viruses and harmful by-products pose a concern. The use of ozone as a disinfectant in produce washes has shown great promise for bacterial pathogens, but limited research exists on its efficacy on viruses. This study compares ozone inactivation of human norovirus surrogates (feline calicivirus [FCV] and murine norovirus [MNV]) on produce (green onions and lettuce) and in sterile water. Green onions and lettuce inoculated with FCV or MNV were treated with ozone (6.25 ppm) for 0.5- to 10-min time intervals. Infectivity was determined by 50% tissue culture infectious dose (TCID(50)) and plaque assay for FCV and MNV, respectively. After 5 min of ozone treatment, >6 log TCID(50)/ml of FCV was inactivated in water and ∼2-log TCID(50)/ml on lettuce and green onions. MNV inoculated onto green onions and lettuce showed a >2-log reduction after 1 min of ozone treatment. The food matrix played the largest role in protection against ozone inactivation. These results indicate that ozone is an alternative method to reduce viral contamination on the surface of fresh produce.

Inactivation of human and murine norovirus by high-pressure processing.

The effect of high hydrostatic pressure (HPP) was evaluated for inactivation of murine norovirus (MNV), a propagable norovirus (NoV), and human NoV genogroup II.4. Inactivation of MNV was assessed by viral culturing (50% tissue culture infectious dose [TCID(50)]) and real-time reverse-transcription-polymerase chain reaction (RT-qPCR), whereas NoV survival was determined only by RT-qPCR. A treatment of 450 MPa for 15 min at 45°C was sufficient to inactivate 6.5 log(10) of infectious MNV in culture medium as determined by TCID(50). Further, the inactivation of MNV was enhanced when pressure was applied at an initial temperature of 25°C. On the other hand, a baroprotective effect was observed when MNV suspensions were supplemented with 10 mM of CaCl(2). A 400 MPa treatment at 45°C inactivated >5 log(10) of infectious MNV, whereas the addition of CaCl(2) increased the pressure resistance of MNV, with <0.5 log(10) reduction observed. MNV decay as determined by TCID(50) was generally greater than that determined by RT-qPCR; for instance, MNV genomes were detected even after 15 min treatment at 450 MPa, with <0.5 log(10) reduction. Experiments with NoV suspensions showed that all tested HPP treatments reduced the numbers of NoV by <0.5 log(10) units as determined by RT-qPCR. Additionally, RNA of human NoV was more resistant to certain HPP treatments than the RNA of MNV.

Efficacy of sodium hypochlorite and peroxyacetic acid to reduce murine norovirus 1, B40-8, Listeria monocytogenes, and Escherichia coli O157:H7 on shredded iceberg lettuce and in residual wash water.

The efficiency of sodium hypochlorite (NaOCl) and peroxyacetic acid (PAA) to reduce murine norovirus 1 (MNV-1), a surrogate for human norovirus, and Bacteroides fragilis HSP40-infecting phage B40-8 on shredded iceberg lettuce was investigated. The levels of removal of viruses MNV-1 and B40-8 were compared with the reductions observed for bacterial pathogens Listeria monocytogenes and Escherichia coli O157:H7. Two inoculation levels, one with a high organic load and the other containing a 10-fold lower number of pathogens and organic matter, showed that the effectiveness of NaOCl was greatly influenced by the presence of organic material, which was not observed for PAA. Moreover, the present study showed that 200 mg/liter NaOCl or 250 mg/liter PAA is needed to obtain an additional reduction of 1 log (compared with tap water) of MNV-1 on shredded iceberg lettuce, whereas only 250 mg/liter PAA achieved this for bacterial pathogens. None of the treatments resulted in a supplementary 1-log PFU/g reduction of B40-8 compared with tap water. B40-8 could therefore be useful as an indicator of decontamination processes of shredded iceberg lettuce based on NaOCl or PAA. Neither MNV-1, B40-8, nor bacterial pathogens could be detected in residual wash water after shredded iceberg lettuce was treated with NaOCl and PAA, whereas considerable numbers of all these microorganisms were found in residual wash water consisting solely of tap water. This study illustrates the usefulness of PAA and NaOCl in preventing cross-contamination during the washing process rather than in causing a reduction of the number of pathogens present on lettuce.

Survival and transfer of murine norovirus 1, a surrogate for human noroviruses, during the production process of deep-frozen onions and spinach.

The reduction of murine norovirus 1 (MNV-1) on onions and spinach by washing was investigated as was the risk of contamination during the washing procedure. To decontaminate wash water, the industrial sanitizer peracetic acid (PAA) was added to the water, and the survival of MNV-1 was determined. In contrast to onions, spinach undergoes a heat treatment before freezing. Therefore, the resistance of MNV-1 to blanching of spinach was examined. MNV-1 genomic copies were detected with a real-time reverse transcription PCR assay in PAA-treated water and blanched spinach, and PFUs (representing infectious MNV-1 units) were determined with a plaque assay. A < or = 1-log reduction in MNV-1 PFUs was achieved by washing onion bulbs and spinach leaves. More than 3 log PFU of MNV-1 was transmitted to onion bulbs and spinach leaves when these vegetables were washed in water containing approximately 5 log PFU/ml. No decline of MNV-1 occurred in used industrial spinach wash water after 6 days at room temperature. A concentration of 20 ppm of PAA in demineralized water (pH 4.13) and in potable water (pH 7.70) resulted in reductions of 2.88 +/- 0.25 and 2.41 +/- 0.18 log PFU, respectively, after 5 min of exposure, but no decrease in number of genomic copies was observed. No reduction of MNV-1 PFUs was observed on frozen onions or spinach during storage for 6 months. Blanching spinach (80 degrees C for 1 min) resulted in at least 2.44-log reductions of infectious MNV-1, but many genomic copies were still present.