Curcumin-Mediated Photodynamic Inactivation of Norovirus Surrogates

Photodynamic inactivation (PDI) is extensively used to inactivate different type of pathogens through the use of photosensitizers (PS). Curcumin has been identified as an excellent natural photosensitizer with some potential applications in the food industry. The aim of this study was to assess the antiviral activity of photoactivated curcumin on norovirus surrogates, feline calicivirus (FCV), and murine norovirus (MNV). Initially, different concentrations of curcumin (13.5–1358 µM) were individually mixed with each virus at titers of ca. 6–7 log TCID₅₀/ml and photoactivated by LED blue light with light dose of 3?J/cm². Results showed that photoactivated curcumin at 50 µg/mL reduced FCV titers by almost 5 log after incubation at 37 °C for 30 min. Lower antiviral activity (0.73 log TCID₅₀/mL reduction) was reported for MNV. At room temperature, curcumin at 5 µg/mL reduced FCV titers by 1.75 log TCID₅₀/mL. These results represent a step forward in improving food safety using photoactivated curcumin as an alternative natural additive to reduce viral contamination.     

Naturally Occurring Flavonoids Against Human Norovirus Surrogates

Naturally occurring plant-derived flavonoids are reported to have antibacterial, antiviral, and pharmacological activities. The objectives of this study were to determine the antiviral effects of four flavonoids (myricetin, l-epicatechin, tangeretin, and naringenin) on the infectivity of food borne norovirus surrogates after 2 h at 37 °C. The lab-culturable surrogates, feline calicivirus (FCV-F9) at titers of ~7 log₁₀ PFU/ml (high titer) or ~5 log₁₀ PFU/ml (low titer) and murine norovirus (MNV-1) at ~5 log₁₀ PFU/ml, were mixed with equal volumes of myricetin, l-epicatechin, tangeretin, or naringenin at concentrations of 0.5 or 1 mM, and incubated for 2 h at 37 °C. Treatments of viruses were neutralized in cell culture medium containing 10 % heat-inactivated fetal bovine serum, serially diluted, and plaque assayed. Each treatment was replicated thrice and assayed in duplicate. FCV-F9 (low titer) was not found to be reduced by tangeretin or naringenin, but was reduced to undetectable levels by myricetin at both concentrations. Low titer FCV-F9 was also decreased by 1.40 log₁₀ PFU/ml with l-epicatechin at 0.5 mM. FCV-F9 at high titers was decreased by 3.17 and 0.72 log₁₀ PFU/ml with myricetin and l-epicatechin at 0.5 mM, and 1.73 log₁₀ PFU/ml with myricetin at 0.25 mM, respectively. However, MNV-1 showed no significant inactivation by the four tested treatments. The antiviral effects of the tested flavonoids are dependent on the virus type, titer, and dose. Further research will focus on understanding the antiviral mechanism of myricetin and l-epicatechin.     

Comparative Virucidal Efficacy of Seven Disinfectants Against Murine Norovirus and Feline Calicivirus,Surrogates of Human Norovirus

Human noroviruses (HuNoV) are the leading cause of acute non-bacterial gastroenteritis in humans and can be transmitted either by person-to-person contact or by consumption of contaminated food. A knowledge of an efficient disinfection for both hands and food-contact surfaces is helpful for the food sector and provides precious information for public health. The aim of this study was to evaluate the effect of seven disinfectants belonging to different groups of biocides (alcohol, halogen, oxidizing agents, quaternary ammonium compounds, aldehyde and biguanide) on infectious viral titre and on genomic copy number. Due to the absence of a cell culture system for HuNoV, two HuNoV surrogates, such as murine norovirus and feline calicivirus, were used and the tests were performed in suspension, on gloves and on stainless steel discs. When, as criteria of efficacy, a log reduction >3 of the infectious viral titre on both surrogates and in the three tests is used, the most efficacious disinfectants in this study appear to be biocidal products B, C and D, representing the halogens, the oxidizing agents group and a mix of QAC, alcohol and aldehyde, respectively. In addition, these three disinfectants also elicited a significant effect on genomic copy number for both surrogate viruses and in all three tests. The results of this study demonstrate that a halogen compound, oxidizing agents and a mix of QAC, alcohol and aldehyde are advisable for HuNoV disinfection of either potentially contaminated surfaces or materials in contact with foodstuffs.     

Virucidal Efficacy of a Hydrogen Peroxide Nebulization Against Murine Norovirus and Feline Calicivirus,Two Surrogates of Human Norovirus

Human noroviruses (HuNoV) are amongst the leading causes of acute non-bacterial gastroenteritis in humans and can be transmitted via person-to-person contact, via contact with contaminated surfaces or by consumption of contaminated food. Contaminated surfaces in healthcare settings contribute to the transmission of viruses. No-touch automated room disinfection systems might prevent such a spread of contamination and thus their virucidal effect needs to be evaluated. The aim of this study was to assess the efficacy of a nebulization system spraying hydrogen peroxide on two main surrogates of HuNoV, namely murine norovirus (MNV) and feline calicivirus (FCV). The viruses were dried on cover glasses and on stainless steel discs and exposed to nebulization. The number of infectious viral particles and genomic copies before and after the nebulization was compared. The efficacy in reducing infectivity of both surrogates was demonstrated. For the infectious viral titre of MNV and FCV, a log₁₀ reduction factor ≥4.84 and 4.85 was observed after nebulization, respectively, for tests on cover glasses and ≥3.90 and 5.30, respectively, for tests on stainless steel discs. Only low reductions in genomic copy numbers were observed for both surrogates. The nebulization of hydrogen peroxide showed a clear virucidal effect on both HuNoV surrogates, MNV and FCV, on two different carriers and the use of nebulization should be promoted in complementarity with conventional disinfection methods in healthcare settings and food processing facilities to reduce viral load and spread of contamination.     

The Influence of Temperature on Norovirus Inactivation by Monochloramine in Potable Waters: Testing with Murine Norovirus as a Surrogate for Human Norovirus

Human noroviral infections are generally more common during winters in temperate regions. This study used a murine norovirus (MNV) as a human norovirus surrogate to test the effect of water temperature (4 and 25°C) on virus survival and its susceptibility to the levels of monochloramine (~1.89 ppm) to terminally disinfect municipally treated potable waters. The titre of MNV remained essentially unchanged for at least 24 h in raw river water at both temperatures. The virus became undetectable in <2 h in monochloramine-containing samples held at 25°C, but its titre remained virtually unaltered at 4°C (P < 0.05) under the same conditions. These findings strongly suggest that water temperature can influence the norovirucidal activity of monochloramine and its possible impact on the seasonality of outbreaks of noroviral infections.     

Critical Review of Norovirus Surrogates in Food Safety Research: Rationale for Considering Volunteer Studies

The inability to propagate human norovirus (NoV) or to clearly differentiate infectious from noninfectious virus particles has led to the use of surrogate viruses, like feline calicivirus (FCV) and murine norovirus-1 (MNV), which are propagatable in cell culture. The use of surrogates is predicated on the assumption that they generally mimic the viruses they represent; however, studies are proving this concept invalid. In direct comparisons between FCV and MNV, their susceptibility to temperatures, environmental and food processing conditions, and disinfectants are dramatically different. Differences have also been noted between the inactivation of NoV and its surrogates, thus questioning the validity of surrogates. Considerable research funding is provided globally each year to conduct surrogate studies on NoVs; however, there is little demonstrated benefit derived from these studies in regard to the development of virus inactivation techniques or food processing strategies. Human challenge studies are needed to determine which processing techniques are effective in reducing NoVs in foods. A major obstacle to clinical trials on NoVs is the perception that such trials are too costly and risky, but in reality, there is far more cost and risk in allowing millions of unsuspecting consumers to contract NoV illness each year, when practical interventions are only a few volunteer studies away. A number of clinical trials have been conducted, providing important insights into NoV inactivation. A shift in research priorities from surrogate research to volunteer studies is essential if we are to identify realistic, practical, and scientifically valid processing approaches to improve food safety.     

Optimizing Human Intestinal Enteroids for Environmental Monitoring of Human Norovirus

Human noroviruses (HuNoV) are the leading cause of gastrointestinal illness and environmental monitoring is crucial to prevent HuNoV outbreaks. The recent development of a HuNoV cell culture assay in human intestinal enteroids (HIEs) has enabled detection of infectious HuNoV. However, this complex approach requires adaptation of HIEs to facilitate HuNoV replication from environmental matrixes. Integrating data from 200 experiments, we examined six variables: HIE age, HIE basement membrane compounds (BMC), HuNoV inoculum processing, HuNoV inoculum volume, treatment of data below limit of detection (LOD), and cutoff criteria for determining positive HuNoV growth. We infected HIEs with HuNoV GII.4 Sydney positive stool and determined 1.4 × 10³ genome equivalents per HIE well were required for HuNoV replication. HIE age had minimal effect on assay outcomes. LOD replacement and cutoff affected data interpretation, with lower values resulting in higher estimated HuNoV detection. Higher inoculum volumes lead to minimal decreases in HuNoV growth, with an optimal volume of 250uL facilitating capture of low concentrations of HuNoVs present in environmental isolates. Processing of HuNoV inoculum is valuable for disinfection studies and concentrating samples but is not necessary for all HIE applications. This work enhances the HuNoV HIE cell culture approach for environmental monitoring. Future HIE research should report cell age as days of growth and should clearly describe BMC choice, LOD handling, and positive cutoff.

     

Inactivation of Murine Norovirus Suspended in Organic Matter Simulating Actual Conditions of Viral Contamination

Food and Environmental Virology - Foodborne viral illnesses are frequent worldwide and costly for the society. Human norovirus is one of the most common causal agents. Although some norovirus...     

Reduction of Norovirus Surrogates Alone and in Association with Bacteria on Leaf Lettuce and Tomatoes During Application of Aqueous Ozone

Food and Environmental Virology - Retail foodservice establishments (FSE) frequently utilize washes with sanitizing agents during fresh produce preparation. This study evaluated the efficacy of...     

Norovirus and Other Human Enteric Viruses in Moroccan Shellfish

The aim of this study was to evaluate the presence of human enteric viruses in shellfish collected along the Mediterranean Sea and Atlantic Coast of Morocco. A total of 77 samples were collected from areas potentially contaminated by human sewage. Noroviruses were detected in 30 % of samples, with an equal representation of GI and GII strains, but were much more frequently found in cockles or clams than in oysters. The method used, including extraction efficiency controls, allowed the quantification of virus concentration. As in previous reports, results showed levels of contamination between 100 and 1,000 copies/g of digestive tissues. Sapoviruses were detected in 13 % of samples mainly in oyster and clam samples. Hepatitis A virus was detected in two samples, with concentrations around 100 RNA copies/g of digestive tissues. Only two samples were contaminated with enterovirus and none with norovirus GIV or Aichi virus. This study highlights the interest of studying shellfish samples from different countries and different production areas. A better knowledge of shellfish contamination helps us to understand virus levels in shellfish and to improve shellfish safety, thus protecting consumers.     

Virus Genome Quantification Does not Predict Norovirus Infectivity After Application of Food Inactivation Processing Technologies

When determining the effect of food processing on the infectivity of any contaminating virus, it is necessary to distinguish unambiguously between infectious and non-infectious viruses present. However, this can be difficult in the particular case of noroviruses (NoVs) because no reliable cell culture model is available. The aim of this study was to assess the use of molecular methods—RT real-time PCR (RT-qPCR) and enzymatic treatment (ET) coupled to RT-qPCR—to quantify the infectivity of NoV after application of various inactivating food-processing technologies. RT-qPCR and ET-RT-qPCR gave significantly different (P < 0.01) results concerning the reduction in viral genome counts by all inactivation procedures and conditions used, except for HHP treatment at 600 MPa for 5 min. These findings indicate that the ET prior to RT-qPCR has an effect on the estimation of the reduction of virus genome counts, and may eliminate genomes of affected virus particles. However, no correlation was found between the results obtained by ET-RT-qPCR and those obtained by cell culture. Therefore, the effect is presumably only partial, and not adequate to allow accurate estimation of virus inactivation. Consequently, our results indicate that the quantification of virus genomes by PCR, regardless of prior ET, is not adequate for establishing virus inactivation and/or infectivity. In addition, our results also illustrate that the general effect of virus inactivation is not directly correlated to effects on the integrity of virus genome and protein capsid. Presumably, inactivation by food processing is the consequence of effects on proteins involved in adhesion and invasion stages.     

Evaluating Efficacy of Field-Generated Electrochemical Oxidants on Disinfection of Fomites Using Bacteriophage MS2 and Mouse Norovirus MNV-1 as Pathogenic Virus Surrogates

Surface disinfection, as part of environmental hygiene practices, is an efficient barrier to gastroenteritis transmission. However, surface disinfectants may be difficult to obtain in remote, resource-limited, or disaster relief settings. Electrochemical oxidants (ECO) are chlorine-based disinfectants that can be generated using battery power to electrolyze brine (NaCl) solutions. Electrolysis generates a mixed-oxidant solution that contains both chlorine (HOCl, OCl^?) and reactive oxygen species (e.g., ·OH, O₃, H₂O₂, and ·O_2?) capable of inactivating pathogens. One onsite generator of ECO is the Smart Electrochlorinator 200 (SE-200, Cascade Designs, Inc.). In a laboratory study, we assessed ECO surface disinfection efficacy for two gastrointestinal virus surrogates: bacteriophage MS2 and murine norovirus MNV-1. We quantified both infectivity and nucleic acid inactivation using culture-dependent and independent assays. At free available chlorine concentrations of 2,500 ppm and a contact time of 30 s, ECO inactivation of infective MS2 bacteriophage exceeded 7 log₁₀ compared to MNV-1 disinfection of approximately 2 log₁₀. Genomic RNA inactivation was less than infective virus inactivation: MS2 RNA inactivation was approximately 5 log₁₀ compared to MNV-1 RNA inactivation of approximately 1.5 log₁₀. The results are similar to inactivation efficacy of household bleach when used at similar free available chlorine concentrations. Our work demonstrates the potential of ECO solutions, generated onsite, to be used for surface disinfection.     

Microgreen Variety Impacts Leaf Surface Persistence of a Human Norovirus Surrogate

Food and Environmental Virology - Human norovirus (HuNoV) is a pathogenic agent that is frequently associated with foodborne disease outbreaks linked to fresh produce. Within microgreen production...     

Molecular Study of the Persistence of Infectious Human Norovirus on Food-Contact Surfaces

The aim of this study was to investigate the effect of relative humidity (RH) and temperature on norovirus (NoV) persistence as infectious particles on food-contact surfaces such as stainless steel and polyvinyl chloride (PVC). For this purpose, a new method combining enzymatic digestion with molecular beacon-based NASBA targeting the ORF1-ORF2 domain was developed to discriminate between infectious and noninfectious NoV. Stainless steel and PVC disks were contaminated with known amounts of human NoV and kept for 56 days at 7 and 20°C at high (86% ± 4%) and low (30% ± 10%) RH. NoV retained its putative infectivity for 56 days on PVC and for 49 days on stainless steel at 7°C and for 7 and 28 days, respectively, at low and high RH at 20°C on both tested surfaces. These results confirm that NoV persists in an infective state on inert surfaces for long periods of time and consequently may cause illness. The new molecular approach to detecting infectious NoV on inert surfaces may provide valuable information for evaluating environmental surface decontamination strategies.     

Virucidal Efficacy of Olanexidine Gluconate as a Hand Antiseptic Against Human Norovirus

Food and Environmental Virology - Human noroviruses are the major cause of non-bacterial acute gastroenteritis worldwide. Since no therapeutic agent has been proven to prevent human norovirus...     

Antiviral Effects of Lactococcus lactis on Feline Calicivirus,A Human Norovirus Surrogate

Foodborne viruses, particularly human norovirus (NV) and hepatitis virus type A, are a cause of concern for public health making it necessary to explore novel and effective techniques for prevention of foodborne viral contamination, especially in minimally processed and ready-to-eat foods. This study aimed to determine the antiviral activity of a probiotic lactic acid bacterium (LAB) against feline calicivirus (FCV), a surrogate of human NV. Bacterial growth medium filtrate (BGMF) of Lactococcus lactis subsp. lactis LM0230 and its bacterial cell suspension (BCS) were evaluated separately for their antiviral activity against FCV grown in Crandell–Reese feline kidney (CRFK) cells. No significant antiviral effect was seen when CRFK cells were pre-treated with either BGMF (raw or pH 7-adjusted BGMF) or BCS. However, pre-treatment of FCV with BGMF and BCS resulted in a reduction in virus titers of 1.3 log₁₀ tissue culture infectious dose (TCID)₅₀ and 1.8 log₁₀ TCID₅₀, respectively. The highest reductions in FCV infectivity were obtained when CRFK cells were co-treated with FCV and pH 7-adjusted BGMF or with FCV and BCS (7.5 log₁₀ TCID₅₀ and 6.0 log₁₀ TCID₅₀, respectively). These preliminary results are encouraging and indicate the need for continued studies on the role of probiotics and LAB on inactivation of viruses in various types of foods.     

The Effectiveness of Activated Sludge Procedure and UV-C 254 in Norovirus Inactivation in a Tunisian Industrial Wastewater Treatment Plant

The molecular detection of Norovirus GI and Norovirus GII in the Tunisian industrial wastewater treatment plant of Charguia I was conducted to test the eff