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.     

Thermal Inactivation of Human Norovirus Surrogates

We investigated the thermal inactivation profiles of murine norovirus (MNV), Hepatitis A virus (HAV), and feline calicivirus (FCV), which are surrogates for the study of human noroviruses. Thermal inactivation of MNV and FCV were evaluated at 37, 50, and 60°C and HAV at 37, 50, 60, and 70°C. All viral surrogates were relatively stable at 37°C. MNV and FCV decimal reduction times (D-values) at 50°C were statistically significantly different (P < 0.05) with MNV being more stable. Both surrogates had comparable, low D-values at 60°C. HAV had significantly higher (P < 0.05) D-values than both MNV and FCV at 50 and 60°C. Overall, the infectivity assay results indicate that HAV is resistant to thermal inactivation while MNV is moderately resistant and FCV is least resistant.     

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.     

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.     

Survival and Inactivation by Advanced Oxidative Process of Foodborne Viruses in Model Low-Moisture Foods

Enteric viruses, such as human norovirus (NoV) and hepatitis A virus (HAV), are the major causes of foodborne illnesses worldwide. These viruses have low infectious dose, and may remain infectious for weeks in the environment and food. Limited information is available regarding viral survival and transmission in low-moisture foods (LMF). LMFs are generally considered as ready-to-eat products, which undergo no or minimal pathogen reduction steps. However, numerous foodborne viral outbreaks associated with LMFs have been reported in recent years. The objective of this study was to examine the survival of foodborne viruses in LMFs during 4-week storage at ambient temperature and to evaluate the efficacy of advanced oxidative process (AOP) treatment in the inactivation of these viruses. For this purpose, select LMFs such as pistachios, chocolate, and cereal were inoculated with HAV and the norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), then viral survival on these food matrices was measured over a four-week incubation at ambient temperature, by both plaque assay and droplet-digital RT-PCR (ddRT-PCR) using the modified ISO-15216 method as well as the magnetic bead assay for viral recovery. We observed an approximately 0.5 log reduction in viral genome copies, and 1 log reduction in viral infectivity for all three tested viruses following storage of select inoculated LMFs for 4 weeks. Therefore, the present study shows that the examined foodborne viruses can persist for a long time in LMFs. Next, we examined the inactivation efficacy of AOP treatment, which combines UV-C, ozone, and hydrogen peroxide vapor, and observed that while approximately 100% (4 log) inactivation can be achieved for FCV, and MNV in chocolate, the inactivation efficiency diminishes to approximately 90% (1 log) in pistachios and 70% (< 1 log) in cereal. AOP treatment could therefore be a good candidate for risk reduction of foodborne viruses from certain LMFs depending on the food matrix and surface of treatment.

     

Estimation of Human Norovirus Infectivity from Environmental Water Samples by In Situ Capture RT-qPCR Method

Human noroviruses (HuNoVs) are highly infectious viruses for which water is an important medium of transmission. In this study, we explored a new in situ capture RT-qPCR (ISC-RT-qPCR) methodology to estimate the infectivity of HuNoV in environmental water samples. This assay was based on capturing encapsidated HuNoV by viral receptors, followed by in situ amplification of the captured viral genomes by RT-qPCR. We demonstrated that the ISC-RT-qPCR did not capture and enable signal amplification of heat-denatured Tulane Virus (TV) and HuNoVs. We further demonstrated that the sensitivity of ISC-RT-qPCR was equal or better than that of conventional RT-qPCR procedures for the detection of HuNoV GI and GII. We then utilized the ISC-RT-qPCR to detect HuNoV in environmental water samples for comparison against that from a conventional RT-qPCR procedure. TV was used as a process control virus. While complete inhibition of TV genomic signal was observed in 27% of samples tested by RT-qPCR, no inhibition of TV genomic signal was observed by ISC-RT-qPCR. From 72 samples tested positive for HuNoV GI signal by RT-qPCR, only 20 (27.8%) of these samples tested positive by ISC-RT-qPCR, suggesting that 72.2% of RT-qPCR-positive samples were unlikely to be infectious. From 16 samples tested positive for HuNoV GII signal by RT-qPCR, only one of these samples tested positive by ISC-RT-qPCR. Five samples that had initially tested negative for HuNoV GII signal by RT-qPCR, was tested as positive by ISC-RT-qPCR. Overall, ISC-RT-qPCR method provided an alternative assay to estimate infectivity of HuNoV in environmental samples.     

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.     

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.     

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.

     

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.     

New Proof-of-Concept in Viral Inactivation: Virucidal Efficacy of 405 nm Light Against Feline Calicivirus as a Model for Norovirus Decontamination

The requirement for novel decontamination technologies for use in hospitals is ever present. One such system uses 405 nm visible light to inactivate microorganisms via ROS-generated oxidative damage. Although effective for bacterial and fungal inactivation, little is known about the virucidal effects of 405 nm light. Norovirus (NoV) gastroenteritis outbreaks often occur in the clinical setting, and this study was designed to investigate potential inactivation effects of 405 nm light on the NoV surrogate, feline calicivirus (FCV). FCV was exposed to 405 nm light whilst suspended in minimal and organically-rich media to establish the virucidal efficacy and the effect biologically-relevant material may play in viral susceptibility. Antiviral activity was successfully demonstrated with a 4 Log₁₀ (99.99%) reduction in infectivity when suspended in minimal media evident after a dose of 2.8 kJ cm^?2. FCV exposed in artificial faeces, artificial saliva, blood plasma and other organically rich media exhibited an equivalent level of inactivation using between 50–85% less dose of the light, indicating enhanced inactivation when the virus is present in organically-rich biologically-relevant media. Further research in this area could aid in the development of 405 nm light technology for effective NoV decontamination within the hospital environment.     

Inactivation of Viruses and Bacteriophages as Models for Swine Hepatitis E Virus in Food Matrices

Hepatitis E virus has been recognised as a food-borne virus hazard in pork products, due to its zoonotic properties. This risk can be reduced by adequate treatment of the food to inactivate food-borne viruses. We used a spectrum of viruses and bacteriophages to evaluate the effect of three food treatments: high pressure processing (HPP), lactic acid (LA) and intense light pulse (ILP) treatments. On swine liver at 400 MPa for 10 min, HPP gave log₁₀ reductions of ≥4.2, ≥5.0 and 3.4 for feline calicivirus (FCV) 2280, FCV wildtype (wt) and murine norovirus 1 (MNV 1), respectively. Escherichia coli coliphage ?X174 displayed a lower reduction of 1.1, while Escherichia coli coliphage MS2 was unaffected. For ham at 600 MPa, the corresponding reductions were 4.1, 4.4, 2.9, 1.7 and 1.3 log₁₀. LA treatment at 2.2 M gave log₁₀ reductions in the viral spectrum of 0.29–2.1 for swine liver and 0.87–3.1 for ham, with ?X174 and MNV 1, respectively, as the most stable microorganisms. The ILP treatment gave log₁₀ reductions of 1.6–2.8 for swine liver, 0.97–2.2 for ham and 1.3–2.3 for sausage, at 15–60 J cm^?2, with MS2 as the most stable microorganism. The HPP treatment gave significantly (p < 0.05) greater virus reduction on swine liver than ham for the viruses at equivalent pressure/time combinations. For ILP treatment, reductions on swine liver were significantly (p < 0.05) greater than on ham for all microorganisms. The results presented here could be used in assessments of different strategies to protect consumers against virus contamination and in advice to food producers. Conservative model indicators for the pathogenic viruses could be suggested.     

Efficacy of Cinnamaldehyde Against Enteric Viruses and Its Activity After Incorporation Into Biodegradable Multilayer Systems of Interest in Food Packaging

Cinnamaldehyde (CNMA), an organic compound that gives cinnamon its flavor and odor, was investigated for its virucidal activity on norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), and hepatitis A virus (HAV). Initially, different concentrations of CNMA (0.1, 0.5 and 1 %) were individually mixed with each virus at titers of ca. 6–7 log₁₀ TCID₅₀/ml and incubated 2 h at 4 and 37 °C. CNMA was effective in reducing the titers of norovirus surrogates in a dose-dependent manner after 2 h at 37 °C, while HAV titers were reduced by 1 log₁₀ after treatment with 1 % of CNMA. When incubation time was extended, HAV titers were reduced by 3.4 and 2.7 log₁₀ after overnight incubation at 37 °C with 1 and 0.5 % of CNMA, respectively. Moreover, this paper analyzed, for the first time, the antiviral activity of adding an active electrospun interlayer based on zein and CNMA to a polyhydroxybutyrate packaging material (PHB) in a multilayer form. Biodegradable multilayer systems prepared with 2.60 mg/cm² (~9.7 %) of CNMA completely inactivated FCV according to ISO 22196:2011, while MNV titers were reduced by 2.75 log₁₀. When the developed multilayer films were evaluated after one month of preparation or at 25 °C, the antiviral activity was reduced as compared to freshly prepared multilayer films evaluated at 37 °C. The results show the excellent potential of this system for food contact applications as well as for active packaging technologies in order to maintain or extend food quality and safety.     

Survival of Norovirus Surrogate on Various Food-Contact Surfaces

Norovirus (NoV) is an environmental threat to humans, which spreads easily from one infected person to another, causing foodborne and waterborne diseases. Therefore, precautions against NoV infection are important in the preparation of food. The aim of this study was to investigate the survival of murine norovirus (MNV), as a NoV surrogate, on six different food-contact surfaces: ceramic, wood, rubber, glass, stainless steel, and plastic. We inoculated 10⁵ PFU of MNV onto the six different surface coupons that were then kept at room temperature for 28 days. On the food-contact surfaces, the greatest reduction in MNV was 2.28 log₁₀ PFU/coupon, observed on stainless steel, while the lowest MNV reduction was 1.29 log₁₀ PFU/coupon, observed on wood. The rank order of MNV reduction, from highest to lowest, was stainless steel, plastic, rubber, glass, ceramic, and wood. The values of d _R (time required to reduce the virus by 90 %) on survival plots of MNV determined by a modified Weibull model were 277.60 h (R ² = 0.99) on ceramic, 492.59 h (R ² = 0.98) on wood, 173.56 h on rubber (R ² = 0.98), 97.18 h (R ² = 0.94) on glass, 91.76 h (R ² = 0.97) on stainless steel, and 137.74 h (R ² = 0.97) on plastic. The infectivity of MNV on all food-contact surfaces remained after 28 days. These results show that MNV persists in an infective state on various food-contact surfaces for long periods. This study may provide valuable information for the control of NoV on various food-contact surfaces, in order to prevent foodborne disease.     

Impact of Sodium Chloride, Sucrose and Milk on Heat Stability of the Murine Norovirus and the MS2 Phage

Until now, little is known about the influence of food additives on heat inactivation of noroviruses. Only a few studies have shown a protective or inhibiting effect on virus infectivity caused by the food matrix. Therefore, the aim of this study was to examine the influence of sodium chloride, sucrose and milk on heat stability of the surrogates murine norovirus (MNV) and MS2 phage at 60 °C for 1–5 min in PBS for MNV and for 5–120 min in suspension medium buffer for MS2 phage. Different concentrations of sodium chloride (5, 10 %) and sucrose (5, 50 %) were added to the respective buffers. In addition, commercially available milk with different fat concentrations (0.3, 1.5, 3.5 %) was investigated in this study. In general, a linear titre reduction for MNV and MS2 phage could be observed, except for the heat treatment of MNV in PBS with 50 % sucrose. A protective effect of PBS with 50 % sucrose and of the matrix milk on MNV could be concluded. All other tested conditions did not show any influence on virus inactivation. However, MS2 phage did show a higher heat resistance throughout the experiments compared to MNV. In future investigations, it should be tested, whether the achieved data may be considered in risk assessments of heat-treated food products with high concentrations of sugar. Furthermore, it should be clarified, whether these results can also be referred to complex food matrices.     

Minimizing Bias in Virally Seeded Water Treatment Studies: Evaluation of Optimal Bacteriophage and Mammalian Virus Preparation Methodologies

One key assumption impacting data quality in viral inactivation studies is that reduction estimates are not altered by the virus seeding process. However, seeding viruses often involves the inadvertent addition of co-constituents such as cell culture components or additives used during preparation steps which can impact viral reduction estimates by inducing non-representative oxidant demand in disinfection studies and fouling in membrane assessments. The objective of this study was therefore to characterize a mammalian norovirus surrogate, murine norovirus (MNV), and bacteriophage MS2 at sequential stages of viral purification and to quantify their potential contribution to artificial oxidant demand and non-representative membrane fouling. Our results demonstrate that seeding solvent extracted and 0.1 micron filtered MNV to ~10⁵ PFU/mL in an experimental water matrix will result in additional total organic carbon (TOC) and 30 min chlorine demand of 39.2 mg/L and 53.5 mg/L as Cl₂, respectively. Performing sucrose cushion purification on the MNV stock prior to seeding reduces the impacts of TOC and chlorine demand to 1.6 and 0.15 mg/L as Cl₂, respectively. The findings for MNV are likely relevant for other mammalian viruses propagated in serum-based media. Thus, advanced purification of mammalian virus stocks by sucrose cushion purification (or equivalent density-based separation approach) is warranted prior to seeding in water treatment assessments. Studies employing bacteriophage MS2 as a surrogate virus may not need virus purification, since seeding MS2 at a concentration of ~10⁶ PFU/mL will introduce only ~1 mg/L of TOC and ~1 mg/L as Cl₂ of chlorine demand to experimental water matrices.     

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.