Radiosensitivity increase in FCV-F9 virus using combined treatments with natural antimicrobials and γ-irradiation.

AIMS: The objective was to evaluate the possible synergistic effect of cranberry juice (CJ) and commercial citrus extract (BS) against FCV-F9 viral titre in vitro in combination with γ-irradiation and to determinate the D10 values and radiosensitivity increase. METHODS AND RESULTS: Virus samples were treated with a formulation containing a mixture of BS or CJ. Results showed a D10 of 0·05, 0·42% and 1·34 kGy for the virus treated with the BS, the CJ and the irradiation alone respectively. Concentrations needed to reduce 6 log TCID50  ml-1 of viral titre were BS-0·3%, CJ-2·52% and 8·04 kGy. Irradiation combined with BS-0·01% and CJ-0·1% against FCV-F9 virus showed D10 values of 0·74 and 0·72 kGy, respectively, resulting in a viral radiosensitization of 1·28 and 1·50 for respective treatments. CONCLUSION: The higher viral radiosensitization observed after combining γ-irradiation with BS-0·01% and CJ-0·1% indicates that CJ and BS could be used as antiviral agents alone or in combination with γ-irradiation to prevent NoV outbreaks. SIGNIFICANCE AND IMPACT OF THE STUDY: Cranberry juice and BS could be used in hurdle approaches in combined treatment with γ-irradiation to assure food safety without a detrimental effect on nutritional value and maintain low processing cost.

Ultraviolet C light efficiently inactivates nonenveloped hepatitis A virus and feline calicivirus in platelet concentrates.

BACKGROUND: Nonenveloped transfusion-transmissible viruses such as hepatitis A virus (HAV) and hepatitis E virus (HEV) are resistant to many of the common virus inactivation procedures for blood products. This study investigated the pathogen inactivation (PI) efficacy of the THERAFLEX UV-Platelets system against two nonenveloped viruses: HAV and feline calicivirus (FCV), in platelet concentrates (PCs). STUDY DESIGN AND METHODS: PCs in additive solution were spiked with high titers of cell culture-derived HAV and FCV, and treated with ultraviolet C at various doses. Pre- and posttreatment samples were taken and the level of viral infectivity determined at each dose. For some samples, large-volume plating was performed to improve the detection limit of the virus assay. RESULTS: THERAFLEX UV-Platelets reduced HAV titers in PCs to the limit of detection, resulting in a virus reduction factor of greater than 4.2 log steps, and reduced FCV infectivity in PCs by 3.0 ± 0.2 log steps. CONCLUSIONS: THERAFLEX UV-Platelets effectively inactivates HAV and FCV in platelet units.

Inactivation of feline calicivirus using ultraviolet light-emitting diodes.

Ultraviolet light-emitting diodes (UV-LEDs) with peak emission wavelengths of 265, 280 and 300 nm were applied for the inactivation of feline calicivirus (FCV) in water, and the results were compared to those derived with a common viral surrogate coliphage MS2. The fluence response profiles indicated that the log10-based inactivation rate constant of FCV was 0.113, 0.101 and 0.007 cm2 mJ-1 for the 265, 280 and 300 nm UV-LEDs, respectively, while that of MS2 was 0.034, 0.033 and 0.003 cm2 mJ-1 for the 265, 280 and 300 nm UV-LEDs, respectively. Namely, FCV was about two to three times more sensitive than MS2 to germicidal UV emissions adopted in this study, and the 265 nm and 280 nm UV-LEDs were particularly effective to inactivate FCV. Results of this study are to be a part of database on fluence response profiles of various microorganisms, which would foster the development of disinfection apparatuses equipped with UV-LEDs.

Integrated molecular analysis of the inactivation of a non-enveloped virus, feline calicivirus, by UV-C radiation.

UV-C treatment has been shown to be a powerful way to inactivate non-enveloped viruses in water samples. However, little is known about how the viruses were inactivated by UV-C radiation. In this study, we investigated the inactivation mechanism of a single-stranded RNA (ssRNA) non-enveloped virus, feline calicivirus (FCV), as a surrogate for the human norovirus, using UV-C radiation with different wavelengths. Integrated molecular analyses using RT-qPCR, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and mass spectrometry were employed to evaluate the extent of ssRNA genome and protein degradation. UV-C radiation of FCV efficiently impaired the infectivity of FCV in mammalian cells. We also identified degradation of the RNA genome, whose copy numbers decreased from 48% to 56% following UV255 or UV281 radiation. Significant degradation of capsid protein was not observed, whereas oxidation of amino acid residues in the major capsid protein VP-1 was determined. Our results suggest that damage to the RNA genome is primarily responsible for the observed decrease in FCV infectivity of CRFK cells. This study provides not only relevant baseline data but also an overview and possible mechanism for the disinfection of non-enveloped ssRNA viruses using UV-C radiation.

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.

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.

Negative effect of heat shock on feline calicivirus release from infected cells is associated with the control of apoptosis.

FCV infection causes rapid cytopathic effects, and its replication results in the induction of apoptosis changes in cultured cells. It is well established that the survival of apoptotic cells can be enhanced by the expression of heat-shock proteins (Hsp) to prevent damage or facilitate recovery. Hsps can act as molecular chaperones, but they can also have anti-apoptotic roles by binding to apoptotic proteins and inhibiting the activation of caspases, the primary mediators of apoptosis. Because apoptosis occurs during FCV infection and heat shock (HS) treatment has a cytoprotective role due to the expression of Hsps, we studied the effect of the HS response to hyperthermia during FCV infection in cultured cells. We found that FCV infection does not inhibit the expression of Hsp70 induced by HS and that non-structural and structural protein synthesis was not modified during HS treatment. However, HS caused a delay in the appearance of a cytopathic effect in infected cells, as well as a reduction in the extracellular but not in the cell-associated viral yield. This antiviral effect of HS correlates with the inhibition of caspase-3 activation. Thus, the HS-induced reduction in virus production appeared to be associated with the control of apoptosis, supporting previous data that indicate that apoptosis is necessary for FCV release.

Inactivation of feline calicivirus as a surrogate for norovirus on lettuce by electron beam irradiation.

Caliciviridae, including norovirus, are considered important sources of human gastroenteritis. As leafy green vegetables are commonly consumed without additional processing, it is important to evaluate interventions to reduce the presence of human pathogens in these products. Feline calicivirus was used as a model for small round structured viruses on lettuce. The lettuce was inoculated by immersion to simulate contamination from irrigation or wash water. The inoculated lettuce was then exposed to electron beam irradiation at various dose levels to determine survival. The D10-value of the calicivirus on lettuce was determined to be 2.95 kGy. Irradiation to reduce bacterial pathogens on cut lettuce could also reduce the risk associated with small round structured viruses on lettuce.

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation.

AIMS: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. METHODS AND RESULTS: The inactivation rate for cell-free virus or intracellular echovirus 12 was determined by exposure to 254-nm UV light at fluence up to 100 mJ cm(-2) . The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4-log(10) inactivation were 25, 29, 30 and 70 (mJ cm(-2) ) for cell-free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm(-2) was needed to inactivate intracellular echovirus 12 by 4 log(10) . CONCLUSIONS: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8-fold more resistant to UV irradiation than cell-free one. SIGNIFICANCE AND IMPACT OF THE STUDY: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.

UV light inactivation of hepatitis A virus, Aichi virus, and feline calicivirus on strawberries, green onions, and lettuce.

A majority of illnesses caused by foodborne viruses are associated with fresh produce. Fruits and vegetables may be considered high-risk foods, as they are often consumed raw without a specific inactivation step. Therefore, there is a need to evaluate nonthermal treatments for the inactivation of foodborne pathogens. This study investigates the UV inactivation of three viruses: feline calicivirus (a surrogate for norovirus), and two picornaviruses, hepatitis A virus and Aichi virus. Three produce types were selected for their different surface topographies and association with outbreaks. Green onions, lettuce, and strawberries were individually spot inoculated with 10(7) to 10(9) 50% tissue culture infective doses (TCID50) of each virus per ml and exposed to UV light at various doses (< or = 240 mW s/cm2), and viruses were eluted using an optimized recovery strategy. Virus infection was quantified by TCID50 in mammalian cell culture and compared with untreated recovered virus. UV light applied to contaminated lettuce resulted in inactivation of 4.5 to 4.6 log TCID50/ml; for contaminated green onions, inactivation ranged from 2.5 to 5.6 log TCID50/ml; and for contaminated strawberries, inactivation ranged from 1.9 to 2.6 log TCID50/ml for the three viruses tested. UV light inactivation on the surface of lettuce is more effective than inactivation on the other two produce items. Consistently, the lowest results were observed in the inactivation of viruses on strawberries. No significant differences (P > 0.05) for virus inactivation were observed among the three doses applied (40, 120, and 240 mW s/cm2) on the produce, with the exception of hepatitis A virus and Aichi virus inactivation on green onions, where inactivation continued at 120 mW s/cm2 (P < 0.05).

Photocatalytic inactivation of diarrheal viruses by visible-light-catalytic titanium dioxide.

Titanium dioxide (TiO2) that had been irradiated with visible light (VL) was demonstrated to inactivate rotavirus, astrovirus, and feline calicivirus (FCV). The virus titers were dramatically reduced after exposure for 24 hrs to the VL-catalytic TiO2. The addition of bovine serum albumin could protect the virus against inactivation by VL-catalytic TiO2 in a dose-dependent manner. This finding implied that the VL-catalytic TiO2 products might somehow interact initially with the viral proteins in the process of virus inactivation. Moreover, we showed partial degradation of the rotaviral dsRNA genome. This was more prominent when the virus was exposed to the VL-catalytic TiO2 treatment for at least 2 days. An attempt was made to elucidate the mechanism underlying the inactivation of the viruses. It was found that upon activation of TiO2 with VL by using a white fluorescent lamp, the reactive oxygen species such as superoxide anions (O2-) and hydroxyl radicals (*OH) were generated in a significant amount after stimulation for 8, 16, and 24 hrs. We therefore assume that virus inactivation by VL-catalytic TiO2 might occur through the generation of O2- and *OH followed by damage to the viral protein and genome. This is the first report, to the best of our knowledge, demonstrating the inactivation of rotavirus, astrovirus and FCV by the presence of TiO2 film under VL as well as describing its mechanism.

Calicivirus inactivation by nonionizing (253.7-nanometer-wavelength [UV]) and ionizing (gamma) radiation.

Noroviruses (previously Norwalk-like viruses) are the most common viral agents associated with food- and waterborne outbreaks of gastroenteritis. In the absence of culture methods for noroviruses, animal caliciviruses were used as model viruses to study inactivation by nonionizing (253.7-nm-wavelength [UV]) and ionizing (gamma) radiation. Here, we studied the respiratory feline calicivirus (FeCV) and the presumed enteric canine calicivirus (CaCV) and compared them with the well-studied bacteriophage MS2. When UV irradiation was used, a 3-log(10) reduction was observed at a fluence of 120 J/m(2) in the FeCV suspension and at a fluence of 200 J/m(2) for CaCV; for the more resistant phage MS2 there was a 3-log(10) reduction at a fluence of 650 J/m(2). Few or no differences were observed between levels of UV inactivation in high- and low-protein-content virus stocks. In contrast, ionizing radiation could readily inactivate MS2 in water, and there was a 3-log(10) reduction at a dose of 100 Gy, although this did not occur when the phage was diluted in high-protein-content stocks of CaCV or FeCV. The low-protein-content stocks showed 3-log(10) reductions at a dose of 500 Gy for FeCV and at a dose of 300 for CaCV. The inactivation rates for both caliciviruses with ionizing and nonionizing radiation were comparable but different from the inactivation rates for MS2. Although most FeCV and CaCV characteristics, such as overall particle and genome size and structure, are similar, the capsid sequences differ significantly, making it difficult to predict human norovirus inactivation. Adequate management of UV and gamma radiation processes for virus inactivation should limit public health risks.

Inactivation of caliciviruses.

The viruses most commonly associated with food- and waterborne outbreaks of gastroenteritis are the noroviruses. The lack of a culture method for noroviruses warrants the use of cultivable model viruses to gain more insight on their transmission routes and inactivation methods. We studied the inactivation of the reported enteric canine calicivirus no. 48 (CaCV) and the respiratory feline calicivirus F9 (FeCV) and correlated inactivation to reduction in PCR units of FeCV, CaCV, and a norovirus. Inactivation of suspended viruses was temperature and time dependent in the range from 0 to 100 degrees C. UV-B radiation from 0 to 150 mJ/cm(2) caused dose-dependent inactivation, with a 3 D (D = 1 log(10)) reduction in infectivity at 34 mJ/cm(2) for both viruses. Inactivation by 70% ethanol was inefficient, with only 3 D reduction after 30 min. Sodium hypochlorite solutions were only effective at >300 ppm. FeCV showed a higher stability at pH <3 and pH >7 than CaCV. For all treatments, detection of viral RNA underestimated the reduction in viral infectivity. Norovirus was never more sensitive than the animal caliciviruses and profoundly more resistant to low and high pH. Overall, both animal viruses showed similar inactivation profiles when exposed to heat or UV-B radiation or when incubated in ethanol or hypochlorite. The low stability of CaCV at low pH suggests that this is not a typical enteric (calici-) virus. The incomplete inactivation by ethanol and the high hypochlorite concentration needed for sufficient virus inactivation point to a concern for decontamination of fomites and surfaces contaminated with noroviruses and virus-safe water.

Inactivation of feline calicivirus and adenovirus type 40 by UV radiation.

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm(2), respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm(2) in treated groundwater. A dose of 103 mJ/cm(2) was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.

Ultraviolet inactivation of feline calicivirus, human enteric viruses and coliphages.

Norwalk and Norwalk-like viruses (NLV) are major causes of food- and water-related disease in the United States. There is no host cell line in which the NLV can be tested for infectivity. Feline calicivirus (FCV) and NLV both belong to the family Caliciviridae. FCV can be assayed for infectivity in the Crandell Reese feline kidney cell line, so FCV serves as a surrogate for NLV. This study is the first report of UV inactivation of FCV and also of using the plaque technique, in contrast to the 50% tissue culture infectious dose end point technique, to determine the FCV infectivity titer. The infectivity titers (log10 plaque-forming units/mL) of UV-inactivated FCV, hepatitis A virus (HAV), poliovirus type 1 (PV1) and two small, round coliphages were plotted as a function of UV dose and analyzed by regression analysis and analysis of variance. These fitted straight-line curves represent exponential inactivation, so UV inactivation can be said to show "one-hit kinetics." The decimal inactivation doses of UV for FCV, HAV, PV1, MS2 and phiX174 were 47.85, 36.50, 24.10, 23.04 and 15.48 mW s/cm2, respectively. FCV appears to be the most UV resistant among the tested viruses.