Doris H. D'Souza

The effect of cranberry juice and cranberry proanthocyanidins on the infectivity of human enteric viral surrogates.

The effect of cranberry juice (CJ) and cranberry proanthocyanidins (PAC) on the infectivity of human enteric virus surrogates, murine norovirus (MNV-1), feline calicivirus (FCV-F9), MS2(ssRNA) bacteriophage, and phiX-174(ssDNA) bacteriophage was studied. Viruses at high (approximately 7 log(10) PFU/ml) or low (approximately 5 log(10) PFU/ml) titers were mixed with equal volumes of CJ, 0.30, 0.60, and 1.20 mg/ml final PAC concentration, or water and incubated for 1 h at room temperature. Viral infectivity after treatments was evaluated using standardized plaque assays. At low viral titers, FCV-F9 was undetectable after exposure to CJ or the three tested PAC solutions. MNV-1 was reduced by 2.06 log(10) PFU/ml with CJ, and 2.63, 2.75, and 2.95 log(10) PFU/ml with 0.15, 0.30, and 0.60 mg/ml PAC, respectively. MS2 titers were reduced by 1.14 log(10) PFU/ml with CJ, and 0.55, 0.80, and 0.96 log(10) PFU/ml with 0.15, 0.30, and 0.60 mg/ml PAC, respectively. phi-X174 titers were reduced by 1.79 log(10) PFU/ml with CJ, and 1.95, 3.67, and 4.98 log(10) PFU/ml with PAC at 0.15, 0.30, and 0.60 mg/ml, respectively. Experiments using high titers showed similar trends but with decreased effects. CJ and PAC show promise as natural antivirals that could potentially be exploited for foodborne viral illness treatment and prevention.

Grape seed extract for control of human enteric viruses.

ABSTRACT Grape seed extract (GSE) is reported to have many pharmacological benefits, including antioxidant, anti-inflammatory, anticarcinogenic, and antimicrobial properties. However, the effect of this inexpensive rich source of natural phenolic compounds on human enteric viruses has not been well documented. In the present study, the effect of commercial GSE, Gravinol-S, on the infectivity of human enteric virus surrogates (feline calicivirus, FCV-F9; murine norovirus, MNV-1; and bacteriophage MS2) and hepatitis A virus (HAV; strain HM175) was evaluated. GSE at concentrations of 0.5, 1, and 2 mg/ml was individually mixed with equal volumes of each virus at titers of ∼7 log10 PFU/ml or ∼5 log10 PFU/ml and incubated for 2 h at room temperature or 37°C. The infectivity of the recovered viruses after triplicate treatments was evaluated by standardized plaque assays. At high titers (∼7 log10 PFU/ml), FCV-F9 was significantly reduced by 3.64, 4.10, and 4.61 log10 PFU/ml; MNV-1 by 0.82, 1.35, and 1.73 log10 PFU/ml; MS2 by 1.13, 1.43, and 1.60 log10 PFU/ml; and HAV by 1.81, 2.66, and 3.20 log10 PFU/ml after treatment at 37°C with 0.25, 0.50, and 1 mg/ml GSE, respectively (P < 0.05) in a dose-dependent manner. GSE treatment of low titers (∼5 log10 PFU/ml) at 37°C also showed viral reductions. Room-temperature treatments with GSE caused significant reduction of the four viruses, with higher reduction for low-titer FCV-F9, MNV-1, and HAV compared to high titers. Our results indicate that GSE shows promise for application in the food industry as an inexpensive novel natural alternative to reduce viral contamination and enhance food safety.

Efficacy of Chemical Treatments Against Murine Norovirus, Feline Calicivirus, and MS2 Bacteriophage

Human noroviruses pose an emerging public health threat, and despite stringent control strategies, variant strains continue to spread and cause disease outbreaks. Routinely used chemical sanitizers, such as sodium hypochlorite though effective on food contact surfaces, require high concentrations to cause reduction in enteric viral titers. The aim of this study was to evaluate the efficacy of trisodium phosphate (TSP) against three human enteric virus surrogates, murine norovirus (MNV-1), feline calicivirus (FCV), and bacteriophage MS2 in comparison to routinely used sanitizers. Three concentrations of TSP (1%, 2%, and 5%) at different contact times (30 sec and 1 min) were evaluated against the surrogate viruses individually inoculated on formica coupons. Our results showed that 5% TSP was effective in obtaining a >or=6 log(10) PFU reduction for MNV-1, FCV, and MS2 after a contact time of only 30 sec or 1 min similar to 10% household bleach (0.6% sodium hypochlorite, 5000 ppm available chlorine) for high titers of FCV and MS2, and with approximately 5 log(10) reduction after either 30 sec or 1 min on low viral titers. However, 2% TSP for 1 min resulted in >or=6 log(10) PFU reduction for FCV and MS2, but only a 1.05 log(10) PFU reduction for MNV-1 at high titers, with similar results after 30 sec. Decreasing TSP levels to 1% reduced FCV by approximately 2.65 log(10) PFU, MS2 by 4.5 log(10) PFU at high titers, and no reduction for MNV-1 after 30-sec or 1 min contact. Glutaraldehyde (2%) reduced FCV and MNV-1 titers by approximately 6 log(10) PFU; however, MS2 was reduced by only 3.22 and 3.74 log(10) PFU after 30 sec and 1 min, respectively, while 70% ethanol was not effective in reducing the three viruses at either high or low titers at both contact times. Alternative control strategies using TSP with short contact times should benefit the food industry in reducing norovirus transmission.

In Vitro Effects of Pomegranate Juice and Pomegranate Polyphenols on Foodborne Viral Surrogates

Pomegranate juice (PJ) has gained popularity because of its associated antioxidant, antimicrobial, anticancer, and anti-inflammatory properties. However, its effects against epidemiologically significant foodborne viruses have not been investigated. In the absence of culturable human noroviruses, feline calicivirus (FCV-F9), murine norovirus (MNV-1), and MS2 (ssRNA) bacteriophage were used as foodborne viral surrogates. The aim of this research was to study the effects of PJ and pomegranate polyphenols (PP) on foodborne viral infectivity. Viruses at high (∼ 7 log(10) PFU/mL) or low (∼ 5 log(10) PFU/mL) titers were mixed with equal volumes of PJ, 8, 16, and 32 mg/mL of PP, or water (control) and incubated for 1 h at room temperature. Viral infectivity after treatments was evaluated using standardized plaque assays. PJ decreased the titer of FCV-F9, MNV-1, and MS2 by 2.56, 1.32, and 0.32 log(10) PFU/mL, respectively, for low titers and 1.20, 0.06, and 0.63 log(10) PFU/mL, respectively, for high titers. Interestingly, FCV-F9 was undetectable after exposure to the three tested PP solutions using both low and high titers. MNV-1 at low initial titers was reduced by 1.30, 2.11, and 3.61 log(10) PFU/mL and at high initial titers by 1.56, 1.48, and 1.54 log(10) PFU/mL with 4, 8, and 16 mg/mL of PP treatment, respectively. MS2 at low initial titers was reduced by 0.41, 0.45, and 0.93 log(10) PFU/mL and at high initial titers by 0.32, 0.41, and 0.72 log(10) PFU/mL after 4, 8, and 16 mg/mL of PP treatment, respectively. PJ and PP resulted in titer reductions of foodborne virus surrogates after 1 h exposure, showing promise for use in hurdle technologies and/or for therapeutic or preventive use. To suggest the use of PJ and PP as natural remedies for foodborne viral illness prevention, their mechanism of action against viral infectivity needs to be further investigated.

Antiviral effects of cranberry juice and cranberry proanthocyanidins on foodborne viral surrogates--a time dependence study in vitro.

Cranberry juice (CJ) and cranberry proanthocyanidins (PAC) are widely known for their antibacterial, antiviral, and pharmacological activities. The effect of CJ and cranberry PAC on the infectivity of foodborne viral surrogates, murine norovirus (MNV-1), feline calicivirus (FCV-F9), MS2 (ssRNA) bacteriophage, and ϕX-174 (ssDNA) bacteriophage after 0 min to 1h at room temperature was evaluated. Viruses at titers of ∼5log(10)PFU/ml were mixed with equal volumes of CJ at pH 2.6, CJ at pH 7.0, 0.30 mg/ml CJ PAC, 0.60mg/ml PAC, or water and incubated for 0, 10, 20, 30, 40, 50 min, and 1h at room temperature. Infectivity was determined using standard plaque assays. The viral reduction rates of the four tested viruses were found to vary considerably. Among the tested viruses, FCV-F9 titers were decreased the most by ∼5log(10)PFU/ml within 30 min. MS2 titers were decreased the least by only ∼1log(10)PFU/ml after 1h with CJ at pH 2.6 and 0.30 mg/ml PAC, and ∼0.5log(10)PFU/ml with CJ at pH 7.0 and 0.15 mg/ml PAC. MNV-1 and ϕ-X174 showed comparable titer reductions which was between that of FCV-F9 and MS2. In most cases, viral reduction within the first 10 min of treatment accounted for ≥50% of the total reduction. Transmission electron microscopy on FCV-F9 treated with CJ and PAC revealed structural changes. This study shows potential of using natural bioactive compounds for controlling foodborne viral diseases. Further studies are necessary to elucidate the mechanism of action of CJ components and to understand the differences in viral titer reduction profiles.

Time-dependent effects of pomegranate juice and pomegranate polyphenols on foodborne viral reduction.

Pomegranate juice (PJ) and pomegranate polyphenolic extracts (PP) have antiviral effects against HIV-1, influenza, herpes, and poxviruses, and we recently demonstrated their effect against human noroviral surrogates. In the present study, the time-dependent effects of two commercial brands of PJ and PP at two concentrations (2 and 4 mg/mL) on the infectivity of foodborne viral surrogates (feline calicivirus FCV-F9, murine norovirus MNV-1, and MS2 bacteriophage) at room temperature for up to 1 h were evaluated. Each virus at ∼5 log(10) plaque-forming units (PFU)/mL was mixed with equal volumes of PJ, or PP at 4 or 8 mg/mL, and incubated for 0, 10, 20, 30, 45, and 60 min at room temperature. Viral titers after each treatment were determined by standardized plaque assays and compared with untreated controls. Virus titer reduction by PJ and PP was found to be a rather rapid process, with ≥50% of titer reduction occurring within the first 20 min of treatment for all three tested viruses. Within the first 20 min, titer reductions of 3.12, 0.79, and 0.23 log(10) PFU/mL for FCV-F9, MNV-1, and MS2, respectively, were obtained using PJ. FCV-F9, MNV-1, and MS2 titers were reduced by 4.02, 0.68, and 0.18 log(10) PFU/mL with 2 mg/mL PP and 5.09, 1.14, and 0.19 log(10) PFU/mL with 4 mg/mL PP, respectively, after 20 min. The mechanism of viral reduction by PJ and PP needs to be elucidated and clinical trials should be undertaken before recommending for therapeutic or preventive purposes.

Loop-mediated isothermal amplification (LAMP) for the rapid and sensitive detection of Salmonella Typhimurium from pork.

UNLABELLED Loop-mediated isothermal amplification (LAMP) is a novel molecular detection method that is more rapid and simpler than PCR. Products can be detected by turbidity using one temperature without the need for expensive PCR equipment. Our objective was to sensitively detect Salmonella Typhimurium from pork products within 1 d using the LAMP assay. Pork chop and pork sausage samples (25 g) were inoculated with high (10(8) to 10(6) CFU) and low (10(5) to 10(0) CFU) inocula of S. Typhimurium. Serial dilutions in phosphate buffered saline were plated on XLT4 agar either immediately or after selective preenrichment in tetrathionate broth (225 mL) for 10-h at 37 degrees C. Nucleic acid was extracted using the TRIzol method from 1-mL samples. The LAMP assay using 6 specific invA gene primers and Bst DNA polymerase reaction mix was carried out at 62 degrees C for 90 min in a waterbath. Turbid products were detected visually and by agarose gel electrophoresis. Improved Salmonella detection at 10(2) CFU/25 g for both pork chop and sausage was obtained after 10-h enrichment and 10(6) CFU/25 g without enrichment for both products. This assay can detect Salmonella from pork within 1 d, significantly faster than traditional methods that take >5 d. This method shows tremendous potential for routine diagnostics and monitoring of Salmonella by the pork industry. PRACTICAL APPLICATION The novel loop-mediated isothermal amplification (LAMP) assay is a rapid, specific, and sensitive method that has potential application for routine diagnostics of Salmonella from pork products. The isothermal method does not require expensive equipment such as a PCR thermocycler but only a simple waterbath for amplification within 90 min. Detection is even simpler by visual eye or turbidimeters that are less expensive than fluorescent spectrophotometers or real-time PCR machines. All these advantages make it a practical approach for routine use by processing industries to rapidly detect Salmonella in their environment and to implement appropriate control strategies. To improve detection sensitivities, preenrichment followed by selective enrichment may be necessary. Even so, the entire assay can be completed at the most within two 8-h working shifts.

Determination of the thermal inactivation kinetics of the human norovirus surrogates, murine norovirus and feline calicivirus.

Studies are needed to bridge existing data gaps and determine appropriate parameters for thermal inactivation methods for human noroviruses. Cultivable surrogates, such as feline calicivirus (FCV-F9) and murine norovirus (MNV-1), have been used in the absence of human norovirus infectivity assays. This study aimed to characterize the thermal inactivation kinetics of MNV-1 and FCV-F9 at 50, 56, 60, 65, and 72°C for different treatment times (0 to 60 min). Thermal inactivation was performed using the capillary tube method with titers of 4.0 × 10(7) (MNV-1) and 5.8 × 10(8) (FCV-F9) PFU/ml in triplicate experiments, followed by standard plaque assays in duplicate for each experiment. Weibull and first-order models were compared to describe survival curve kinetics. Model fitness was investigated by comparing the regression coefficients (R(2)) and the chi-square (χ(2)) and root mean square error (RMSE) values. The D-values calculated from the first-order model (50 to 72°C) were 0.15 to 34.49 min for MNV-1 and 0.11 to 20.23 min for FCV-9. Using the Weibull model, the t(D) values needed to destroy 1 log PFU of MNV-1 and FCV-F9 at the same temperatures were 0.11 to 28.26 and 0.06 to 13.86 min, respectively. In terms of thermal resistance, MNV-1 was more sensitive than FCV-F9 up to 65°C. At 72°C, FCV-F9 was slightly more susceptible to heat inactivation. Results revealed that the Weibull model was more appropriate to represent the thermal inactivation behavior of both tested surrogates. The z-values were calculated using D-values for the first-order model and the t(D) values for the Weibull model. The z-values were 9.31 and 9.19°C for MNV-1 and 9.36 and 9.31°C for FCV-F9 for the first-order and Weibull models, respectively. This study provides more precise information than previous reports on the thermal inactivation kinetics of two norovirus surrogates for use in thermal process calculations.

Effect of chitosan on the infectivity of murine norovirus, feline calicivirus, and bacteriophage MS2.

Chitosan is known to inhibit microorganisms of concern to plants, animals, and humans. However, the effect of chitosan on human enteric viruses of public health concern has not been extensively investigated. The purpose of this study was to determine the effect of chitosan on three human enteric viral surrogates: murine norovirus 1 (MNV-1), feline calicivirus F-9 (FCV-F9), and (ssRNA) bacteriophage MS2 (MS2). Chitosan oligosaccharide lactate (molecular weight of 5,000) and water-soluble chitosan (molecular weight of 53,000) at concentrations of 1.4, 0.7, and 0.35% were incubated at 37 degrees C for 3 h with equal volumes of each virus at high (approximately 7 log PFU/ml) and low (approximately 5 log PFU/ml) titers. Chitosan effects on each treated virus were evaluated with standardized plaque assays in comparison to untreated virus controls. The water-soluble chitosan at 0.7% decreased the FCV-F9 titer by approximately 2.83 log PFU/ml, with decreasing effects at lower concentrations, and also decreased MS2 at high titers by approximately 1.18 to 1.41 log PFU/ml, regardless of the concentration used. Chitosan treatments at the concentrations studied had no effect on MNV-1 at high titers. Chitosan oligosaccharide showed similar trends against the viruses, but to a lesser extent compared with that of water-soluble chitosan. When lower virus titers (approximately 5 log PFU/ml) were used, plaque reduction was observed for FCV-F9 and MS2, but not MNV-1. The use of higher-molecular-weight chitosan and at higher concentrations with longer incubation may be necessary to inactivate MNV-1. These results in the plaque reduction of human enteric virus surrogates by chitosan treatment show promise for its potential application in the food environment.

Inactivation of human enteric virus surrogates by high-intensity ultrasound.

Foodborne viruses, especially human noroviruses, are recognized as leading causes of nonbacterial gastroenteritis worldwide. Development of effective inactivation methods is of great importance to control their spread. In this study, the effect of high-intensity ultrasound (HIUS) on the infectivity of three foodborne virus surrogates was investigated. The three surrogates, murine norovirus (MNV-1), feline calicivirus (FCV-F9), and MS2 bacteriophage, were diluted in phosphate-buffered saline (PBS) or orange juice to a titer of approximately 6 log(10) PFU/mL or approximately 4 log(10) PFU/mL. The ultrasound treatment was performed in duplicate by immersing the HIUS probe in virus-containing solution that was cooled in ice-water and sonicated at 20 kHz for 2, 5, 10, 15, 20, and 30 min with 30 sec on and 30 sec off. The infectivity of the recovered viruses after each ultrasound treatment was evaluated in duplicate using standardized plaque assays and compared to untreated controls. The results show that HIUS effectiveness depended on the virus type, the initial titer of the viruses, and the virus suspension solution. At titers of approximately 4 log(10) PFU/mL in PBS, feline calicivirus (FCV)-F9, MS2, and murine norovirus (MNV)-1 required 5-, 10-, and 30-min treatment, respectively, for complete inactivation. At initial titers of approximately 4 log(10) PFU/mL in orange juice, FCV-F9 required a 15-min treatment for complete inactivation and only a 1.55 log(10) PFU/mL reduction was achieved for MNV-1 in orange juice after 30-min treatment. Thus, inactivation by HIUS in orange juice was much lower than in PBS. Experiments using titers of approximately 6 log(10) PFU/mL showed decreased effects compared to those using titers of approximately 4 log(10) PFU/mL. These results indicate that HIUS alone is not sufficient to inactivate virus in food. Hurdle technologies that combine HIUS with antimicrobials, heat, or pressure should be explored for viral inactivation.