Impact of chlorine dioxide and electron-beam irradiation for the reduction of murine norovirus in low-salted "jogaejeotgal", a traditional Korean salted and fermented clam.

Clam jeotgal, called "jogaejeotgal," is a Korean fermented seafood product with, generally, a high amount of added salt to inhibit the growth of pathogenic microorganisms. This study aimed to evaluate the efficacy of chlorine dioxide (ClO2) and sodium hypochlorite (NaOCl) against murine norovirus 1 (MNV-1), a surrogate for human norovirus, in salt-fermented clam, jogaejeotgal. The sequential effect of ClO2 and electron-beam (e-beam) irradiation on the inactivation of MNV-1 was also investigated. Treatments of up to 300 ppm ClO2 and 1000 ppm NaOCl were used to determine the disinfectant concentrations at which more than 1 log (90%) MNV-1 inactivation occurred. The sequential treatment of ClO2 (50-300 ppm) and e-beam (1-5.5 kGy) was performed after storage at 4 °C for 7 days. There was a 1.9-log reduction of the virus in seasoned clams irradiated at 5.5 kGy after ClO2 treatment at 300 ppm. No significant change (p > 0.05) in physicochemical quality was observed after the combined treatment, suggesting the potential for the use of a combined treatment using ClO2 (300 ppm) and e-beam (5.5 kGy) in the jeotgal manufacturing industry for the reduction of norovirus.

Electron Beam Susceptibility of Enteric Viruses and Surrogate Organisms on Fruit, Seed and Spice Matrices.

The objective of this study was to use high-energy electron beam (HEEB) treatments to find surrogate microorganisms for enteric viruses and to use the selected surrogates as proof of concept to investigate low-energy electron beam (LEEB) treatments for enteric virus inactivation at industrial scale on frozen blueberries. Six food matrices inoculated with HAV (hepatitis A virus), MNV S99 (murine norovirus), bacteriophages MS2 and Qß, and Geobacillus stearothermophilus spores were treated with HEEB at 10 MeV using 4, 8 and 16 kGy doses. G. stearothermophilus spores showed the highest inactivation on all matrices except on raisins, with a dose-dependent effect. HAV reached the maximum measurable log10 reduction (> 3.2 log10) when treated at 16 kGy on raisins. MNV showed the highest resistance of all tested microorganisms, independent of the dose, except on frozen blueberries. On frozen blueberries, freeze-dried raspberries, sesame seeds and black peppercorns, HAV showed a mean inactivation level in between those of MS2 and G. stearothermophilus. Based on this, we selected both surrogate organisms as first approximation to estimate HAV inactivation on frozen blueberries during LEEB treatment at 250 keV using 16 kGy. Reductions of 3.1 and 1.3 log10 were measured for G. stearothermophilus spores and MS2, respectively, suggesting that a minimum reduction of 1.4 log10 can be expected for HAV under the same conditions.

Assessment of human norovirus inhibition in cabbage kimchi by electron beam irradiation using RT-qPCR combined with immunomagnetic separation.

Cabbage Kimchi, a traditional Korean fermented food, has occasionally been related to acute gastroenteritis caused by human norovirus (HuNoV). The present study examined the inhibitory effects of electron beam (e-beam) irradiation (1, 3, 5, 7, and 10 kGy) on HuNoV GII.4 in suspension or cabbage Kimchi using reverse transcription quantitative polymerase chain reaction combined with immunomagnetic separation (IMS/RT-qPCR). In addition, physicochemical and sensorial analyses were conducted to assess any change in the quality of cabbage Kimchi following e-beam irradiation. Following e-beam irradiation at 1 to 10 kGy, HuNoV significantly decreased to 0.28 to 2.08 log10 copy number/mL in suspension (P < 0.05). HuNoV levels in cabbage Kimchi were also significantly reduced to 0.26 to 1.57 log10 copy number/mL following irradiation with 1 to 10 kGy (P < 0.05) compared to positive control (6.0 log10 copy number/mL). The D-values for 1 log10 reduction (90% inhibition) of HuNoV in suspension and cabbage Kimchi were 4.94 and 6.96 kGy of e-beam, respectively. The pH and acidity in the irradiated cabbage Kimchi were 4.41 to 4.58 and 0.61% to 0.71%, respectively, indicating that e-beam did not affect the optimal pH or acidity. Although a slight increase of greenness was observed in the leaf portion of cabbage Kimchi irradiated with 7 to 10 kGy of e-beam, this color change was minimal and went undetected by panelists in the sensorial evaluation. The five properties of sensorial quality assessed were no different in the irradiated Kimchi sample compared with the control sample (nonirradiated cabbage Kimchi). Therefore, this study suggests that ≥6.96 kGy of e-beam could be applied in the cabbage Kimchi industry to obtain >90% of HuNoV without affecting the quality. PRACTICAL APPLICATION: As the most representative food in Korea, Kimchi needs the sanitation technology that can inhibit viral infection. Our findings suggest that e-beam irradiation can be used to reduce HuNoV effectively in Kimchi without changes in sensorial quality.

Inactivation of hepatitis A virus and murine norovirus on surfaces of plastic, steel and raspberries using steam-ultrasound treatment.

The leading causes of foodborne viral disease outbreaks are human norovirus and hepatitis A virus (HAV). Their environmental persistence enables contamination of kitchen surfaces and crops often consumed raw, such as berries. Many decontamination procedures are inefficient and unsuitable for surfaces of industrial kitchen environments and soft fruits. In this study, we investigated the efficiency of a novel surface decontamination technology, combining steam and ultrasound (steam-ultrasound). Plastic, steel or raspberry surfaces were spiked with the norovirus surrogate, murine norovirus (MNV), and HAV, and steam-ultrasound treated at 85, 90 and 95 °C for 0-5 s. Post treatment viruses were titrated for survival by plaque assay and for genome stability by real-time quantitative PCR (RT-qPCR) of nucleic acid extracts. Survival of viruses were estimated in a log-linear model and the treatment time requirements for each decimal reduction (D value) in viral survival were calculated. The estimated D values of MNV or HAV were 0.4-0.2 or 1.1-0.8 s on plastic, 0.9-0.7 or 1.4-0.8 s on steel and 1.6-1.7 or 3.2-4.7 s on raspberries. No clear trend of genome reduction was observed with tested treatment parameters. Raspberries treated up to 4 s retained its natural texture and visual appeal similar to untreated controls whilst monitored for 7 days. In conclusion, steam-ultrasound treatment can within seconds reduce the titre of foodborne viruses on surfaces of plastic, steel and raspberries. This may particularly benefit industrial scale production of soft fruits for raw consumption and for swift non-hazardous decontamination of industrial kitchen surfaces.

The Effectiveness of Activated Sludge Procedure and UV-C254 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 effectiveness of secondary biological treatment using the activated sludge procedure and the UV-C254 tertiary treatment radiation using a UV disinfection prototype to upgrade the quality of the purified wastewater. A total of 140 sewage samples were collected from the two lines of sewage treatment procedures. Norovirus GI and Norovirus GII have been found and quantified using Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) in 66.4 and 86.4% of the collected wastewater samples. The Norovirus GI and GII mean concentration values got in the treated effluents showed a significant decrease of Norovirus viral content rates detected from upstream to downstream of activated sludge procedures and at the output of UV-C254 treatment. These findings characterise and denote for the first time the effectiveness of biological and UV-C254 treatment for Norovirus GI and Norovirus GII removal in Tunis City, northern Tunisia. Also, these data underlined the fact that purified sewage makes up a route of gastroenteritis virus transmission and a cause of viral gastroenteritis associated with water-borne and food-borne outbreaks.

Evaluation of gamma irradiation for human norovirus inactivation and its effect on strawberry cells.

Norovirus (NoV) is the leading cause of epidemic and sporadic gastroenteritis worldwide; a high number of those cases are attributed to the consumption of contaminated food. Crop producers have used several strategies to inactivate the virus present in these products and thus stop the NoV transmission chain. Physical methods such as gamma radiation show excellent results in the inactivation of bacteria, but its effect on NoV has been little studied. This study aimed to evaluate the effect of gamma radiation for NoV inactivation, and over the surface topographic characteristics of strawberry cells, as a prototype of soft fruit. A 10% suspension of GII norovirus-positive stool samples were treated with either 200 mg/L of sodium hypochlorite (NaClO) or gamma-irradiated at doses of 5, 10, 15 and 20 kilograys (kGy). Viral inactivation was determined by measuring the integrity of viral capsid using RNase A alone or in combination with proteinase K followed by RT-qPCR. The effect over cellular surface topology characteristics of the fruit was measured by atomic force microscopy (AFM) and confocal microscopy. High doses of radiation (20 kGy) were necessary to detect a significant (p < 0.05) decrease of up to 1.26 log10 viral copy number. This dose significantly (p < 0.05) raises the root means square roughness (Rq), which affects directly the quality and texture of the product. The gamma irradiation doses tested in this study were not enough to inactivate NoV. The allowed gamma irradiation doses for fresh produce does not alter the surface topology of the fruit, but they affect the content of fluorescent compounds, responsible for the antioxidant activity of the fruit.

Assessment of the Applicability of Capsid-Integrity Assays for Detecting Infectious Norovirus Inactivated by Heat or UV Irradiation.

Human noroviruses are the leading cause of viral gastroenteritis. In the absence of a practical culture technique for routine analysis of infectious noroviruses, several methods have been developed to discriminate between infectious and non-infectious viruses by removing non-viable viruses prior to analysis by RT-qPCR. In this study, two such methods (RNase and porcine gastric mucin) which were designed to remove viruses with compromised capsids (and therefore assumed to be non-viable), were assessed for their ability to quantify viable F-specific RNA bacteriophage (FRNAP) and human norovirus following inactivation by UV-C or heat. It was found that while both methods could remove a proportion of non-viable viruses, a large proportion of non-viable virus remained to be detected by RT-qPCR, leading to overestimations of the viable population. A model was then developed to determine the proportion of RT-qPCR detectable RNA from non-viable viruses that must be removed by such methods to reduce overestimation to acceptable levels. In most cases, nearly all non-viable virus must be removed to reduce the log overestimation of viability to within levels that might be considered acceptable (e.g. below 0.5 log10). This model could be applied when developing alternative pre-treatment methods to determine how well they should perform to be comparable to established infectivity assays.

Virucidal activity of gamma radiation on strawberries and raspberries.

The environmental stability of enteric viruses and resistance to conventional treatments and common disinfectants, leads to their persistence in waters and food, causing serious implications on public health. Among non-thermal treatment methods, ionizing radiation is recognized as a useful and effective mean of disinfection. The objective of this study was to estimate the inactivation of enteric virus by gamma radiation in raw berry fruits, in order to evaluate the potential of this technology to be applied as a disinfection treatment. Fresh strawberries and raspberries were inoculated either individually with murine norovirus type 1 (MuNoV; as a human norovirus surrogate) and human adenovirus type 5 (HAdV) or with a viral pool of both viruses, and irradiated in a Co-60 equipment at doses of 1 kGy up to 11 kGy. The infectivity of viral particles of MuNoV and HAdV was assessed by plaque assay using Raw 264.7 and A549 cells, respectively. A 2 log PFU/g reduction on MuNoV and HAdV titers was obtained after treatment with a dose of 4 kGy for both fruits. However, non-linear inactivation survival curves were obtained for MuNoV and HAdV in fresh fruits, leading to the detection of infective viral particles at a dose of 11 kGy. The irradiation process indicated virucidal potential, although the estimated gamma radiation dose to attain food safety (> 7 kGy) would compromise the preservation of food quality. Nevertheless, the irradiation technology could be an effective virus mitigation tool to treat polluted waters, which are a major vehicle of contamination for fresh produce.

Effect of UV-C light or hydrogen peroxide wipes on the inactivation of methicillin-resistant Staphylococcus aureus, Clostridium difficile spores and norovirus surrogate.

AIMS: The current study aimed to assess the potential of a new high dose ultraviolet (UV) disinfection device to inactivate methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile and a norovirus surrogate on handheld mobile devices, and to compare the efficacy of the UV-C device to hydrogen peroxide disinfection wipes. METHODS AND RESULTS: Suspensions of MRSA, C. difficile spores and a surrogate for norovirus (MS2) were inoculated onto glass or plastic coupons, with or without organic contamination and were exposed to continuous UV-C light for 15-60 s (165-646 mJ cm-2 ) in a self-contained UV-C chamber or treated with hydrogen peroxide wipes. Increasing the UV-C dose from 310 to 650 mJ cm-2 did not result in greater levels of inactivation. UV-C light inactivated all three micro-organisms, in the absence of organic contamination, by >2·9 log. Treatment of MRSA, C. difficile spores or MS2, in the presence of organic contamination, with UV-C light (310-646 mJ cm-2 ) resulted in 2·3-3·7 log reductions. Treatment of MRSA with UV-C light provided levels of inactivation comparable to treatment with hydrogen peroxide wipes used following the manufacturer's instructions. CONCLUSIONS: UV-C light and hydrogen peroxide wipes had strong antimicrobial activity against MRSA, C. difficile spores and a norovirus surrogate, in the presence or absence of organic contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: Chemical disinfection wipes are widely used in healthcare facilities, but they are not recommended for use on handheld mobile devices which may harbour pathogenic micro-organisms. The powerful bactericidal, sporicidal and virucidal activity of this high dose UV-C light device, shows that this technology is a promising alternative to chemical disinfectants, particularly for control of MRSA.

Modelling of ultraviolet light inactivation kinetics of methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, Clostridium difficile spores and murine norovirus on fomite surfaces.

AIMS: Quantitative data on the doses needed to inactivate micro-organisms on fomites are not available for ultraviolet applications. The goal of this study was to determine the doses of UV light needed to reduce bacteria and murine norovirus (MNV) on hard surface fomites through experimentation and to identify appropriate models for predicting targeted levels of reduction. METHODS AND RESULTS: Stainless steel and Formica laminate coupons were selected as they are common surfaces found in healthcare settings. Test organisms included methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Clostridium difficile and MNV. The fomites were inoculated with 105 -107 bacteria or virus and exposed to a range of UV doses. The order of resistance to UV irradiation was virus, bacterial spore and vegetative cell. The best fitting inactivation curves suggested nonlinear responses to increasing doses after a 3-4 log reduction in the test organisms. The average UV doses required for a 3 log reduction in the C. difficile, MRSA and VRE were 16 000, 6164 and 11 228 (mJ-s cm-2 ) for stainless steel, respectively, and 16 000, 11 727 and 12 441 (mJ-s cm-2 ) for Formica laminate, respectively. CONCLUSIONS: Higher UV light doses are required to inactivate bacteria and viruses on hard surfaces than in suspension. Greater doses are needed to inactivate bacterial spores and MNV compared to vegetative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantitative data and models on UV light doses needed to inactivate bacteria and MNV on hard surfaces are now available. The generalizable results of this study can be used to estimate required UV dosages to achieve targeted levels of inactivation based on estimated levels of contamination or to support quantitative microbial risk assessments.

Oxygen-dependent laser inactivation of murine norovirus using visible light lasers.

BACKGROUND: Previous work indicated that an ultrashort pulse (USP) 425 nm laser is capable of inactivating murine norovirus (MNV: Virol. J. 11:20), perhaps via an impulsive stimulated Raman scattering (ISRS) mechanism, and does not substantially damage human plasma proteins (PLOS One 9:11). Here, further investigation of virus inactivation by laser light is performed. METHODS: In this study, we evaluate whether inactivation of MNV is specific to the USP wavelength of 425 nm, or if it occurs at other visible wavelengths, using a tunable mode-locked Ti-Sapphire laser that has been frequency doubled to generate femtosecond pulses at wavelengths of 400, 408, 425, 450, 465, and 510 nm. Continuous Wave (CW) lasers are also applied. Singlet oxygen enhancers are used to evaluate the sensitivity of MNV to singlet oxygen and oxygen quenchers are used to evaluate effects on virus inactivation as compared to untreated controls. RESULTS: > 3 log10 inactivation of MNV pfu occurs after irradiation with an average power of 150 mW at wavelengths of 408, 425 or 450 nm femtosecond-pulsed light for 3 h. Thus results suggest that the mechanism by which a laser inactivates the virus is not wavelength-specific. Furthermore, we also show that irradiation using a continuous wave (CW) laser of similar power at 408 nm also yields substantial MNV inactivation indicating that inactivation does not require a USP. Use of photosensitizers, riboflavin, rose bengal and methylene blue that generate singlet oxygen substantially improves the efficiency of the inactivation. The results indicate a photochemical mechanism of the laser-induced inactivation where the action of relatively low power blue laser light generates singlet oxygen. CONCLUSION: Results suggest formation of short-lived reactive oxygen species such as singlet oxygen by visible laser light as the cause of virus inactivation rather than via an ISRS mechanism which induces resonant vibrations.

UV-C inactivation of foodborne bacterial and viral pathogens and surrogates on fresh and frozen berries.

Outbreaks of foodborne illness associated with berries often involve contamination with hepatitis A virus (HAV) and norovirus but also bacteria such as Escherichia coli O157:H7 and parasites such as Cyclospora caytanensis. We evaluated the applicability of UV-C to the inactivation of pathogens on strawberries, raspberries and blueberries. Our three-step approach consisted of assessing the chemical safety of UV-C-irradiated berries, evaluating the sensory quality after UV-C treatment and finally studying the inactivation of the target microorganisms. Treatments lasting up to 9 min (4000 mJ cm-2) did not produce detectable levels of furan (<5 µg/kg), a known photolysis product of fructose with genotoxic activity and thus were assessed to be toxicologically safe. No effect on taste or appearance was observed, unless treatment was excessively long. 20 s of treatment (an average fluence of ~ 212 mJ cm-2) reduced active HAV titer by >1 log10 unit in 95% of cases except on frozen raspberries, while 120 s were required to inactivate murine norovirus to this extent on fresh blueberries. The mean inactivation of HAV and MNV was greater on blueberries (2-3 log10) than on strawberries and raspberries (<2 log10). MNV was more sensitive on fresh than on frozen berries, unlike HAV. Inactivation of Salmonella, E. coli O157:H7 and Listeria monocytogenes was poor on all three berries, no treatment reducing viable counts by >1 log10 unit. In most matrices, prolonging the treatment did not improve the result to any significant degree. The effect was near its plateau after 20 s of treatment. These results provide insight into the effectiveness of UV-C irradiation for inactivating bacterial and viral pathogens and surrogates on fresh and frozen berries having different surface types, under different physical conditions and at different levels of contamination. Overall they show that UV-C as single processing step is unsuitable to inactivate significant numbers of foodborne pathogens on berries.

Human enteric viruses in a wastewater treatment plant: evaluation of activated sludge combined with UV disinfection process reveals different removal performances for viruses with different features.

This study assess the quality of wastewater through the detection and quantification of important viruses causing gastroenteritis at different stages of the wastewater treatment process in an activated-sludge wastewater treatment plant with ultraviolet disinfection. Ten sampling events were carried out in a campaign along a period of 18 months collecting wastewater samples from the influent, after the activated-sludge treatment, and after the final disinfection with UV radiation. Samples were concentrated through ultracentrifugation and analysed using retro-transcription, PCR and real time quantitative PCR protocols, for detection and quantification of Group A Rotavirus (RVA), Human Astrovirus (HAstV), Norovirus Genogroup II (NoV GII) and Human Adenovirus (HAdV). HAdV (100%), NoV GII (90%), RVA (70%) and HAstV (60%) were detected in influent samples with concentration from 1·4 (NoV GII) to 8·0 (RVA) log10  gc l-1 . Activated-sludge treatment reached well quality effluents with low organic material concentration, although nonstatistical significant differences were registered among influent and postactivated sludge treatment samples, regarding the presence and concentration for most viruses. All post-UV samples were negative for NoV GII and HAstV, although RVA and HAdV were detected in 38% and 63% of those samples respectively, with concentration ranging from 2·2 to 5·5 and 3·1 to 3·4 log10  gc l-1 . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that an activated-sludge wastewater treatment plant with UV disinfection reduces to levels below the detection limit those single-stranded RNA viruses as noroviruses and astroviruses and reach significant lower levels of rotaviruses and adenoviruses after the complete treatment process.

Evaluation of 405-nm monochromatic light for inactivation of Tulane virus on blueberry surfaces.

AIM: The study aim was to evaluate the potential of 405-nm light as a virus intervention for blueberries. METHODS AND RESULTS: Tulane virus (TV)-inoculated blueberries were treated with 4·2 mW cm-2 of 405-nm light for 5-30 min. To mitigate thermal heating due to the intense light, a dry ice-chilled, nitrogen-based cooling system was utilized. Blueberries were rotated to ensure exposure of all surfaces to 405-nm light. Five-, 15- and 30-min treatments resulted in little or no inactivation of TV on blueberries (average log reductions of -0·18; -0·02; and +0·06 respectively). Since 405-nm light's inactivation mechanism may involve singlet oxygen, two singlet oxygen enhancers, riboflavin and rose bengal, were used to coat the blueberries prior to 405-nm light treatment. When 0·1% riboflavin or rose bengal was added, resulting in an average PFU reduction of -0·51 and -1·01 logs respectively. However, it was noted that the addition of riboflavin and rose bengal in the absence of 405-nm light treatment produced some inactivation. Average untreated log reductions for riboflavin and rose bengal were -0·13 and -0·66 respectively. Also, 60-30-s 405-nm light pulses with 2-min ambient cooling periods without the dry ice nitrogen cooling system did not inactivate TV, suggesting that oxygen limitation by the nitrogen CO2 mixture was not the cause of limited inactivation. CONCLUSIONS: Overall results indicate that 405-nm light has some potential to inactivate viruses if singlet oxygen enhancers are present. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of visible monochromatic violet/blue light (405 nm) as a nonthermal intervention for viruses on foods, such as berries that are prone to norovirus contamination, had not been previously evaluated. Use of food-grade singlet oxygen enhancer compounds in combination with visible spectra light may offer a means to inactivate foodborne viruses.

Application of gamma radiation for the reduction of norovirus and the quality stability in optimally ripened cabbage kimchi.

Optimally ripened commercial cabbage kimchi is considered the main cause of enteric norovirus (NoV) outbreaks in Korea. This study investigated the effect of 1-10kGy gamma radiation on the inactivation of murine norovirus-1 (MNV-1; initial inoculum of 5-6log10PFU/ml), used as a human NoV surrogate, in kimchi. The effects of gamma radiation on the pH and acidity were also examined to address the index of quality and fermentation, respectively. Titers of MNV-1 significantly reduced (p<0.05) in kimchi subjected to increasing gamma radiation doses: MNV-1 titers in kimchi after 1, 3, 5, 7, and 10kGy were 4.82 (0.34-log10 reduction), 4.45 (0.71-log10 reduction), 4.18 (0.98-log10 reduction), 3.71 (1.45-log10 reduction), and 3.40 (1.76-log10 reduction) log10 PFU/ml, respectively. However, the values of pH (4.5-4.6) and acidity (0.6-0.7%) were not significantly different between non-irradiated and irradiated kimchi (p>0.05). The D-value (1-log reduction) for MNV-1 in kimchi, calculated using first-order kinetics, was 5.75kGy (R2=0.98, RMSE=0.10). Therefore, this study suggests that the use of ≥5.75kGy gamma radiation in the kimchi manufacturing industry could be very effective in reducing NoV contamination by >90% (1 log), without causing changes in quality and fermentation.

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.

Pulsed light inactivation of murine norovirus, Tulane virus, Escherichia coli O157:H7 and Salmonella in suspension and on berry surfaces.

Pulsed light (PL) inactivation of two human norovirus (HuNoV) surrogates, murine norovirus (MNV-1) and Tulane virus (TV), and two bacterial pathogens, Escherichia coli O157:H7 and Salmonella, were evaluated. The viruses and bacteria were suspended in phosphate buffered saline (PBS) to final populations of ∼6 log PFU/mL and ∼6 log CFU/mL, respectively. Both viral and bacterial suspensions were then irradiated by PL for different durations and the reductions of each microorganisms were determined. MNV-1 and TV were significantly (P < 0.05) more resistant to PL treatment than Salmonella and E. coli O157:H7 in PBS suspension. MNV-1, Salmonella and E. coli O157:H7 were also inoculated on strawberries and blueberries and the PL inactivation of each microorganism was determined. Lower inactivation of each microorganism was achieved on berry surfaces than in PBS suspension. This study shows that PL can induce rapid inactivation of MNV-1, TV, Salmonella and E. coli O157:H7 in clear suspension with viruses more resistant to PL treatment than bacteria. The efficacy of PL treatment is substantially influenced by food surface structure.

Inactivation efficiency and mechanism of UV-TiO2 photocatalysis against murine norovirus using a solidified agar matrix.

Human norovirus (HuNoV) is the primary cause of viral gastroenteritis worldwide. Fresh blueberries are among high risk foods associated with norovirus related outbreaks. Therefore, it is important to assess intervention strategies to reduce the risk of foodborne illness. The disinfection efficiency of decontamination methods is difficult to evaluate for fruits and vegetables due to an inconsistent degree of contamination and irregular surface characteristics. The inactivation efficiency and mechanism of murine norovirus 1 (MNV-1, a surrogate for HuNoV) was studied on an experimentally prepared solidified agar matrix (SAM) to simulate blueberries using different wavelengths (A, B, C) of UV light both with and without TiO2 photocatalysis (TP). MNV-1 was inoculated on exterior and interior of SAM and inactivation efficiencies of different treatments were investigated using a number of assays. Initial inoculum levels of MNV-1 on the SAM surface and interior were 5.2logPFU/mL. UVC with TiO2 (UVC-TP) achieved the highest level of viral reduction for both externally inoculated and internalized MNV-1. Externally inoculated MNV-1 was reduced to non-detectable levels after UVC-TP treatment for 5min while there was still a 0.9 log viral titer after UVC alone. For internalized MNV-1, 3.2 log and 2.7 log reductions were obtained with UVC-TP and UVC alone treatments for 10min, respectively. The Weibull model was applied to describe the inactivation behavior of MNV-1, and the model showed a good fit to the data. An excellent correlation between the steady-state concentration of OH radicals ([OH]ss) and viral inactivation was quantified using a para-chlorobenzoic acid (pCBA) probe compound, suggesting that OH radicals produced in the UV-TP reaction were the major species for MNV-1 inactivation. Transmission electron microscopy images showed that the structure of viral particles was completely disrupted with UVC-TP and UVC alone. SDS-PAGE analysis showed that the major capsid protein VP1 was degraded after UVC-TP and UVC alone. Real-time RT-qPCR analysis showed that UVC-TP and UVC alone caused a reduction in the level of viral genomic RNA. Propidium monoazide (PMA) pretreatment RT-qPCR analysis showed that UVC-TP caused damage to the viral capsid protein in addition to viral genomic RNA. UVC both with and without TiO2 was more effective for MNV-1 inactivation than UVB and UVA. Thus, UVC-TP disinfection aimed to reduce levels of food-borne viruses can inactivate viruses present on the surface and internalized in the interior of blueberries.

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.

Inactivation of human norovirus and Tulane virus in simple media and fresh whole strawberries by ionizing radiation.

Human norovirus (NoV) is a major cause of fresh produce-associated outbreaks and human NoV in irrigation water can potentially lead to viral internalization in fresh produce. Therefore, there is a need to develop novel intervention strategies to target internalized viral pathogens while maintaining fresh produce quality. In this study electron beam (E-beam) and gamma radiation were evaluated for efficacy against a human NoV GII.4 strain and Tulane virus (TV). Virus survival following ionizing radiation treatments was determined using direct quantitative reverse transcriptase PCR (RT-qPCR), the porcine gastric mucin magnetic bead (PGM-MB) binding assay followed by RT-qPCR, and plaque assay. In simple media, a high dose of E-beam treatment was required to completely abolish the receptor binding ability of human NoV (35.3kGy) and TV (19.5-24.1kGy), as assessed using the PGM-MB binding assay. Both human NoV and TV were more susceptible to gamma irradiation than E-beam, requiring 22.4kGy to achieve complete inactivation. In whole strawberries, no human NoV or TV RNA was detected following 28.7kGy of E-beam treatment using the PGM-MB binding assay. Overall, human NoV and TV are highly resistant to ionizing radiation and therefore the technology may not be suitable to eliminate viruses in fresh produce at the currently approved levels. In addition, the PGM-MB binding assay is an improved method to detect viral infectivity compared to direct RT-qPCR.