Effect of Plant-derived Proteases on Infectivity of Tulane Virus, Murine Norovirus, and Hepatitis A Virus

Journal of Food Protection ◽

10.4315/jfp-20-296 ◽

2020 ◽

Author(s):

Adrienne Shearer ◽

Kalmia Kniel

Keyword(s):

Time Course ◽

Hepatitis A ◽

Elevated Temperatures ◽

Hepatitis A Virus ◽

Plaque Assay ◽

Antimicrobial Properties ◽

Specific Treatment ◽

Murine Norovirus ◽

Current Application ◽

Tulane Virus

Plant-derived proteases, bromelain, papain, and ficin are broad-acting enzymes with generally recognized as safe (GRAS) status for foods and have current application in several food industries. These proteases have also been reported to have antimicrobial properties. This study investigated the efficacy of commercially-prepared bromelain, papain, and ficin, individually and combined (2500 ppm crude extract), for inactivation of hepatitis A virus (HAV) and HuNoV surrogates, Tulane virus (TV), and murine norovirus (MNV). Various treatment temperatures (45, 50, 55°C), times (10 or 60 min), and pH values (5.5, 7.0) in the presence of cysteine (2 mM) were evaluated. Inactivation was assessed by infectivity in plaque assay for TV and MNV and by TCID50 for HAV. No reduction in infectious TV or HAV was attributed to the plant-derived proteases at any of the conditions tested. Infectious MNV was reduced by one to 3 log10 PFU/ml; the most effective treatment was bromelain at pH 7 and 50°C for 10 minutes. A time course study with MNV in bromelain at 50°C indicated that a 2-log10 PFU/ml reduction could be achieved within 6 minutes, but extended treatment of 15 minutes was still insufficient to eliminate infectious MNV. The lack of or limited efficacy of bromelain, papain, and ficin on HAV, TV, and MNV even at elevated temperatures and exposure times suggests the plant-derived proteases are not commercially applicable for inactivation of virus on commodities or materials that could not also withstand mild heat treatment. The variable susceptibilities observed between TV and MNV illustrate limitations in utilization of surrogates for predicting pathogen behavior for a structure-specific treatment.

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Survival of Murine Norovirus, Tulane Virus, and Hepatitis A Virus on Alfalfa Seeds and Sprouts during Storage and Germination

Applied and Environmental Microbiology ◽

10.1128/aem.01704-13 ◽

2013 ◽

Vol 79 (22) ◽

pp. 7021-7027 ◽

Cited By ~ 22

Author(s):

Qing Wang ◽

Kirsten A. Hirneisen ◽

Sarah M. Markland ◽

Kalmia E. Kniel

Keyword(s):

Hepatitis A ◽

Hepatitis A Virus ◽

Storage Period ◽

Testing Time ◽

Murine Norovirus ◽

Food Borne ◽

Time Period ◽

Tulane Virus ◽

Viral Etiologies ◽

Alfalfa Seeds

ABSTRACTHuman norovirus (huNoV) and hepatitis A virus (HAV) have been involved in several produce-associated outbreaks and identified as major food-borne viral etiologies. In this study, the survival of huNoV surrogates (murine norovirus [MNV] and Tulane virus [TV]) and HAV was investigated on alfalfa seeds during storage and postgermination. Alfalfa seeds were inoculated with MNV, TV, or HAV with titers of 6.46 ± 0.06 log PFU/g, 3.87 ± 0.38 log PFU/g, or 7.01 ± 0.07 log 50% tissue culture infectious doses (TCID50)/g, respectively. Inoculated seeds were stored for up to 50 days at 22°C and sampled during that storage period on days 0, 2, 5, 10, and 15. Following storage, virus presence was monitored over a 1-week germination period. Viruses remained infectious after 50 days, with titers of 1.61 ± 0.19 log PFU/g, 0.85 ± 0.21 log PFU/g, and 3.43 ± 0.21 log TCID50/g for MNV, TV, and HAV, respectively. HAV demonstrated greater persistence than MNV and TV, without a statistically significant reduction over 20 days (<1 log TCID50/g); however, relatively high levels of genomic copies of all viruses persisted over the testing time period. Low titers of viruses were found on sprouts and were located in all tissues as well as in sprout-spent water sampled on days 1, 3, and 6 following seed planting. Results revealed the persistence of viruses in seeds for a prolonged period of time, and perhaps of greater importance these data suggest the ease of which virus may transfer from seeds to sprouts and spent water during germination. These findings highlight the importance of sanitation and prevention procedures before and during germination.

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Inactivation of Hepatitis A Virus by High-Pressure Processing: The Role of Temperature and Pressure Oscillation

Journal of Food Protection ◽

10.4315/0362-028x-69.10.2454 ◽

2006 ◽

Vol 69 (10) ◽

pp. 2454-2459 ◽

Cited By ~ 44

Author(s):

DAVID H. KINGSLEY ◽

DONGSHENG GUAN ◽

DALLAS G. HOOVER ◽

HAIQIANG CHEN

Keyword(s):

High Pressure ◽

Hepatitis A ◽

Elevated Temperatures ◽

Treatment Time ◽

Hepatitis A Virus ◽

Virus Titer ◽

Pressure Oscillation ◽

Weibull Model ◽

High Pressure Processing ◽

Pressure Application

Inactivation of hepatitis A virus (HAV) in Dulbecco's modified Eagle medium with 10% fetal bovine serum was studied at pressures of 300, 350, and 400 MPa and initial sample temperatures of −10, 0, 5, 10, 20, 30, 40, and 50°C. Sample temperature during pressure application strongly influenced the efficiency of HAV inactivation. Elevated temperature (>30°C) enhanced pressure inactivation of HAV, while lower temperatures resulted in less inactivation. For example, 1-min treatments of 400 MPa at −10, 20, and 50°C reduced titers of HAV by 1.0, 2.5, and 4.7 log PFU/ml, respectively. Pressure inactivation curves of HAV were obtained at 400 MPa and three temperatures (−10, 20, and 50°C). With increasing treatment time, all three temperatures showed a rapid initial drop in virus titer with a diminishing inactivation rate (or tailing effect). Analysis of inactivation data indicated that the Weibull model more adequately fitted the inactivation curves than the linear model. Oscillatory high-pressure processing for 2, 4, 6, and 8 cycles at 400 MPa and temperatures of 20 and 50°C did not considerably enhance pressure inactivation of HAV as compared with continuous high-pressure application. These results indicate that HAV exhibits, unlike other viruses examined to date, a reduced sensitivity to high pressure observed at cooler treatment temperatures. This work suggested that slightly elevated temperatures are advantageous for pressure inactivation of HAV within foods.

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Survival of Hepatitis A Virus in Spinach during Low Temperature Storage

Journal of Food Protection ◽

10.4315/0362-028x-72.11.2390 ◽

2009 ◽

Vol 72 (11) ◽

pp. 2390-2393 ◽

Cited By ~ 27

Author(s):

Y. CAROL SHIEH ◽

DIANA S. STEWART ◽

DAVID T. LAIRD

Keyword(s):

Hepatitis A ◽

Hepatitis A Virus ◽

Plaque Assay ◽

Storage Conditions ◽

Inactivation Rate ◽

Low Temperature Storage ◽

Foodborne Outbreaks ◽

Phosphate Buffered Saline ◽

Temperature Storage ◽

Spinach Leaves

Spinach leaves are frequently consumed raw and have been involved with past foodborne outbreaks. In this study, we examined the survival of hepatitis A virus (HAV) on fresh spinach leaves in moisture- and gas-permeable packages that were stored at 5.4 ± 1.2°C for up to 42 days. Different eluents including phosphate-buffered saline (PBS), pH 7.5 (with and without 2% serum), and 3% beef extract (pH 7.5 and 8) were compared for how efficiently they recovered viruses from spinach by using a simple elution procedure (<1 h). The recoveries were compared and determined by a plaque assay with FRhK-4 cells. Culture grade PBS containing 2% serum was found to be appropriate for HAV elution from spinach leaves, with an average recovery of 45% ± 10%. Over 4 weeks of storage at 5.4 ± 1.2°C, HAV in spinach decreased slightly more than 1 log, with 6.75% of the original titer remaining. HAV survived under refrigerated temperatures on spinach leaves with a D-value of 28.6 days (equivalent to an inactivation rate of 20.035 log of HAV per day, r2 = 0.88). In comparison, HAV in PBS containing 2% serum under the same storage conditions remained constant throughout 7 weeks. The inactivation rate of 20.035 log each day for HAV on spinach leaves was possibly due to the interaction of the virus and the leaf.

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Antiviral effects of grape seed extract against feline calicivirus, murine norovirus, and hepatitis A virus in model food systems and under gastric conditions

Food Microbiology ◽

10.1016/j.fm.2015.05.011 ◽

2015 ◽

Vol 52 ◽

pp. 1-10 ◽

Cited By ~ 28

Author(s):

Snehal S. Joshi ◽

Xiaowei Su ◽

Doris H. D'Souza

Keyword(s):

Food Systems ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Grape Seed Extract ◽

Grape Seed ◽

Seed Extract ◽

Feline Calicivirus ◽

Murine Norovirus ◽

Antiviral Effects ◽

Model Food

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Development of a plaque assay for a cytopathic, rapidly replicating isolate of hepatitis A virus

Journal of Medical Virology ◽

10.1002/jmv.1890220107 ◽

1987 ◽

Vol 22 (1) ◽

pp. 45-56 ◽

Cited By ~ 145

Author(s):

Theresa Cromeans ◽

Mark D. Sobsey ◽

Howard A. Fields

Keyword(s):

Hepatitis A ◽

Hepatitis A Virus ◽

Plaque Assay

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Comparing Human Norovirus Surrogates: Murine Norovirus and Tulane Virus

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-216 ◽

2013 ◽

Vol 76 (1) ◽

pp. 139-143 ◽

Cited By ~ 78

Author(s):

KIRSTEN A. HIRNEISEN ◽

KALMIA E. KNIEL

Keyword(s):

Tap Water ◽

Plaque Assay ◽

Heat Treatments ◽

Viral Infectivity ◽

Murine Norovirus ◽

Human Norovirus ◽

Human Noroviruses ◽

Ph Values ◽

Significant Difference ◽

Tulane Virus

Viral surrogates are widely used by researchers to predict human norovirus behavior. Murine norovirus (MNV) is currently accepted as the best surrogate and is assumed to mimic the survival and inactivation of human noroviruses. Recently, a new calicivirus, the Tulane virus (TV), was discovered, and its potential as a human norovirus surrogate is being explored. This study aimed to compare the behavior of the two potential surrogates under varying treatments of pH (2.0 to 10.0), chlorine (0.2 to 2,000 ppm), heat (50 to 75°C), and survival in tap water at room (20°C) and refrigeration (4°C) temperatures for up to 30 days. Viral infectivity was determined by the plaque assay for both MNV and TV. There was no significant difference between the inactivation of MNV and TV in all heat treatments, and for both MNV and TV survival in tap water at 20°C over 30 days. At 4°C, MNV remained infectious over 30 days at a titer of approximately 5 log PFU/ml, whereas TV titers decreased significantly by 5 days. MNV was more pH stable, as TV titers were reduced significantly at pH 2.0, 9.0, and 10.0, as compared with pH 7.0, whereas MNV titers were only significantly reduced at pH 10.0. After chlorine treatment, there was no significant difference in virus with the exception of at 2 ppm, where TV decreased significantly compared with MNV. Compared with TV, MNV is likely a better surrogate for human noroviruses, as MNV persisted over a wider range of pH values, at 2 ppm of chlorine, and without a loss of titer at 4°C.

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