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

1987 ◽  
Vol 22 (1) ◽  
pp. 45-56 ◽  
Author(s):  
Theresa Cromeans ◽  
Mark D. Sobsey ◽  
Howard A. Fields
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Plaque Assay

Survival of Hepatitis A Virus in Spinach during Low Temperature Storage

2009 ◽  
Vol 72 (11) ◽  
pp. 2390-2393 ◽  
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.

Concentration of Hepatitis a Virus in Environmental Samples

1991 ◽  
Vol 24 (2) ◽  
pp. 229-234 ◽  
Author(s):  
A. Bosch ◽  
R. Gajardo ◽  
F. X. Abad ◽  
J. M. Diez ◽  
J. Jofre
Keyword(s):  
Environmental Samples ◽  
Hepatitis A ◽  
Glass Powder ◽  
Hepatitis A Virus ◽  
Tap Water ◽  
Plaque Assay ◽  
Wild Type ◽  
Filter Aid ◽  
Treatment Concentration ◽  
Positively Charged

The cytopathogenic pHM-175 strain of hepatitis A virus was used to develop different procedures for the concentration of HAV in tap water, fresh water, seawater and raw sewage, HAV was quantified by a plaque assay in the FRhK-4 cell line. Water samples were concentrated by a modification of the adsorption to and elution from glass powder (GPAE) method, by adsorption to and elution from filter aid, and by ammonium sulfate flocculation (ASF). The GPAE method consistently yielded greater HAV recoveries than filtration through filter aid, or ASF. HAV was concentrated by GPAE from 20-litre samples with satisfactory efficiencies in all kinds of water: 100% for tap water, 80% for freshwater, 75% for seawater and 61% for sewage. Concentration efficiencies for filter aid and ASF were always lower than 25% and 40%, respectively, in any kind of water. The charge of glass powder was modified by polyethylenimine treatment. Concentration efficiencies of HAV in 20 1 samples through adsorption to and elution from positively charged glass powder (PGPAE) were 100% for tap water, 94% for seawater, and 61% for freshwater and sewage. The presence of wild-type HAV in sewage samples could be monitored by molecular hybridization with cDNA probes after GPAE concentration.

scholarly journals Hepatitis A Virus Survival on Drug Paraphernalia

2020 ◽  
Vol 41 (S1) ◽  
pp. s248-s248
Author(s):  
Magdalena Medrzycki ◽  
Michael A. Purdy
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Plaque Assay ◽  
The United States ◽  
Dead Volume ◽  
Persons Who Inject Drugs ◽  
Dry Conditions ◽  
Drug Paraphernalia ◽  
Liquid Suspensions

Background: The ongoing hepatitis A outbreak in the United States has concerned public health authorities since March 2017. The outbreak has already spread throughout 30 states and includes primarily homeless individuals and persons who use drugs, including persons who inject drugs (PWIDs). Contaminated drug injection paraphernalia and sharing of these items are suspected to be one of multiple causes of hepatitis A virus (HAV) transmission in those populations. Methods: We used a standard plaque assay to investigate HAV infectivity. Liquid suspensions of HAV were tested to examine the effects of time and temperature on viral infectivity. We also examined HAV survival on commonly used drug paraphernalia, such as needles, syringes, cookers, tourniquets, and cotton balls/filters frequently shared among PWIDs. We investigated the effect of low pH on HAV survival using citric acid, which is frequently used by PWIDs during dose preparation. We also compared the plaque assay results with those concurrently obtained by RT-PCR to establish whether viral HAV RNA levels could be used as surrogates for plaque assay results. Results: We found that HAV suspended in PBS at room temperature was able to infect FRhk4 cells for >17 weeks. HAV remained viable in syringes and needles (ie, semidry conditions) for up to 10 weeks depending on the size of the needles and the syringe dead volume. HAV survival in dry conditions on cooker, tourniquet, and cotton balls/filter surfaces did not exceed 4 weeks. HAV retained its infectivity for >10 weeks at pH as low as 2. PCR results suggest that RNA is amplified from both infectious and noninfectious HAV. Conclusions: Our findings show that HAV can survive and remain infective in the PWID setting for 4–10 weeks depending on the type of paraphernalia examined. These findings suggest that sharing drug paraphernalia by the homeless and PWIDs can potentially facilitate the transmission of HAV within these populations. Moreover, our results confirm that the plaque assay is currently the only reliable method to determine the infectivity of HAV in vitro.Funding: NoneDisclosures: None

Heat Inactivation of Hepatitis A Virus in Dairy Foods†

2000 ◽  
Vol 63 (4) ◽  
pp. 522-528 ◽  
Author(s):  
S. BIDAWID ◽  
J. M. FARBER ◽  
S. A. SATTAR ◽  
S. HAYWARD
Keyword(s):  
Dairy Products ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Plaque Assay ◽  
Virus Titer ◽  
Skim Milk ◽  
Fat Content ◽  
Heat Inactivation ◽  
Log Reduction ◽  
Custom Made

Experiments were performed to determine the thermal resistance of hepatitis A virus (HAV) in three types of dairy products containing increased amounts of fat content (skim milk, homogenized milk; 3.5% MFG, and table cream; 18% MFG). HAV-inoculated dairy products were introduced into custom-made U-shaped microcapillary tubes that in turn were simultaneously immersed in a waterbath, using custom-made floating boats and a carrying platform. Following exposure to the desired time and temperature combinations, the contents of each of the tubes was retrieved and was tested by plaque assay to determine the reduction in virus titer. Our data indicated that <0.5 min at 85°C was sufficient to cause a 5-log reduction in HAV titer in all three dairy products, whereas at 80°C, ≤0.68 min (for skim and homogenized milk), and 1.24 min (for cream) were needed to cause a similar log reduction. Using a nonlinear two-phase negative exponential model (two-compartment model) to analyze the data, it was found that at temperatures of 65, 67, 69, 71, and 75°C, significantly (P < 0.05) higher exposure times were needed to achieve a 1-log reduction in virus titer in cream, as compared to skim and homogenized milk. For example, at 71°C, a significantly (P < 0.05) higher exposure time of 0.52 min (for cream) was needed as compared to ≤0.18 min (for skim and homogenized milk) to achieve a 1-log reduction in virus titer. A similar trend of inactivation was observed at 73 and 75°C where significantly (P < 0.05) higher exposure times of 0.29 to 0.36 min for cream were needed to cause a 1-log reduction in HAV in cream, as compared to ≤0.17 min for skim and homogenized milk. This study has provided information on the heat resistance of HAV in skim milk, homogenized milk, and table cream and demonstrated that an increase in fat content appears to play a protective role and contributes to the heat stability of HAV.

Pressure Inactivation of Hepatitis A Virus in Strawberry Puree and Sliced Green Onions†

2005 ◽  
Vol 68 (8) ◽  
pp. 1748-1751 ◽  
Author(s):  
DAVID H. KINGSLEY ◽  
DONGSHENG GUAN ◽  
DALLAS G. HOOVER
Keyword(s):  
High Pressure ◽  
Ambient Temperature ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Intervention Strategy ◽  
Plaque Assay ◽  
High Pressure Processing

Hepatitis A can be acquired by ingesting contaminated produce. To investigate the potential of high-pressure processing as an intervention strategy for virus in produce, strawberry puree and sliced green onions were inoculated with >106 PFU of hepatitis A virus and treated with pressures ranging from 225 to 375 megapascals (MPa) in 25-MPa increments at ambient temperature. Subsequent virus extraction and plaque assay determined that hepatitis A virus was inactivated in strawberry puree and sliced green onions after 5-min exposures to pressures of 375 MPa with log PFU reductions of 4.32 and 4.75, respectively. Hepatitis A virus was equally sensitive in puree and onions at pressures ≥350 MPa. For treatments of <325 MPa, the virus was more sensitive to pressure in strawberry puree than in sliced onions with log reductions of 1.2, 2.06, and 3.13 observed for strawberries and 0.28, 0.72, and 1.42 observed for onions after 5-min treatments at 250, 275, and 300 MPa, respectively. Although high-pressure processing may cause some organoleptic alterations to strawberries and onions, results show high-pressure processing will inactivate hepatitis A virus in these foods.

scholarly journals Temperature-Dependent Survival of Hepatitis A Virus during Storage of Contaminated Onions

2012 ◽  
Vol 78 (14) ◽  
pp. 4976-4983 ◽  
Author(s):  
Y. Sun ◽  
D. T. Laird ◽  
Y. C. Shieh
Keyword(s):  
Hepatitis A ◽  
Storage Temperature ◽  
Hepatitis A Virus ◽  
Fresh Produce ◽  
Plaque Assay ◽  
Preventive Strategy ◽  
Decimal Reduction Time ◽  
Produce Safety ◽  
Food Borne ◽  
Large Numbers

ABSTRACTPre- or postharvest contamination of green onions by hepatitis A virus (HAV) has been linked to large numbers of food-borne illnesses. Understanding HAV survival in onions would assist in projecting the risk of the disease associated with their consumption. This study defined HAV inactivation rates in contaminated green onions contained in air-permeable, moisture-retaining high-density polyethylene packages that were stored at 3, 10, 14, 20, 21, 22, and 23°C. A protocol was established to recover HAV from whole green onions, with 31% as the average recovery by infectivity assay. Viruses in eluates were primarily analyzed by a 6-well plaque assay on FRhK-4 cells. Eight storage trials, including two trials at 3°C, were conducted, with 3 to 7 onion samples per sampling and 4 to 7 samplings per trial. Linear regression correlation (r2= 0.80 to 0.98) was observed between HAV survival and storage time for each of the 8 trials, held at specific temperatures. Increases in the storage temperature resulted in greater HAV inactivation rates, e.g., a reduction of 0.033 log PFU/day at 3.4 ± 0.3°C versus 0.185 log PFU/day at 23.4 ± 0.7°C. Thus, decimal reduction time (D) values of 30, 14, 11, and 5 days, respectively, were obtained for HAV in onions stored at 3, 10, 14, and 23°C. Further regression analysis determined that 1 degree Celsius increase would increase inactivation of HAV by 0.007 log PFU/day in onions (r2= 0.97). The data suggest that natural degradation of HAV in contaminated fresh produce is minimal and that a preventive strategy is critical to produce safety. The results are useful in predicting the risks associated with HAV contamination in fresh produce.

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

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.

scholarly journals Effect of some wild egyptian medicinal plants on HAV

2021 ◽  
Vol 12 (9) ◽  
pp. 142-149
Author(s):  
Wafaa Kamal Taia ◽  
Ahmed Adel Helme Zayed
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Plaque Assay ◽  
Vero Cells ◽  
Elisa Test ◽  
Wild Plants ◽  
Old Patients ◽  
Negative Results ◽  
Pcr Techniques ◽  
Positive Results

Background: Folk Egyptian medicine considered from the most important source of the use of wild plants in medication. Wild plants are considered a perfect source of natural compounds that have been used as antimicrobial andante virus activities. Aims and Objective: The aim of the current study was to investigate the effect of the commonly used wild plants in the treatment of jaundice and they effect on HAV. This article provides much-needed insight into the effect of wild plants on the hepatitis A virus to shed more light on the important subject, which is unfortunately poorly investigated. Materials and Methods: In this investigation, the aqueous plant extracts of twenty-five wild Egyptian species, 16 perennials, and 9 annuals; three concentrations, 1%, 3% & 5%; were tested for HAV replication by using PA and PCR techniques. Questionnaires and interviews with Bedouins have been carried to know the most used species in the treatment of jaundice, and the usage values were calculated. Those species were collected from their habitats, rare ones purchased and reviewed their identifications and used in this work. Fecal and blood samples were taken from 9-10Y old patients, 35 girls and 25 boys. Results: The PA test revealed that 16 species out of twenty-five gave positive results, while the rest of the species gave negative results on Vero cells. Twelve of the sixteen species were perennial species, and the rest are annuals. The Plaque assay results showed that the most effective aqueous extract species, with the three concentrations, on HAV activity were those of Salvia verbennaca, Mentha microphylla, Ocimum basilicum, Cassia senna, Solenostemma argel, and Thymus capitatus, respectively. PCR test has been carried for the first three species and gave very faint nucleic acid tapes, which means that the plant extract of these species, the three concentrations, minimize the virus activity by a way or another. Samples from the patients tested for the antibodies for IgM and IgG using the ELISA test. Within both the females (58%) and males (42%), 6% have positive antibodies for IgM, while 36% in females and 34% in males’ have positive antibodies for IgG and 8% females and 10% males have both antibodies (IgM & IgG). Conclusion: Wild plants, especially those with essential oils can tolerate the Hepatitis A virus (HAV) activity and reduce its symptoms.

Application of solid-phase immune electron microscopy to study physical and stability characteristics of enterically transmitted non-a, non-b hepatitis virus

1988 ◽  
Vol 46 ◽  
pp. 80-81
Author(s):  
Charles D. Humphrey ◽  
E. H. Cook ◽  
Karen A. McCaustland ◽  
Daniel W. Bradley
Keyword(s):  
Electron Microscopy ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Solid Phase ◽  
Etiologic Agent ◽  
World Health ◽  
Health Concern ◽  
Morphological Identification ◽  
Immune Electron Microscopy

Enterically transmitted non-A, non-B hepatitis (ET-NANBH) is a type of hepatitis which is increasingly becoming a significant world health concern. As with hepatitis A virus (HAV), spread is by the fecal-oral mode of transmission. Until recently, the etiologic agent had not been isolated and identified. We have succeeded in the isolation and preliminary characterization of this virus and demonstrating that this agent can cause hepatic disease and seroconversion in experimental primates. Our characterization of this virus was facilitated by immune (IEM) and solid phase immune electron microscopic (SPIEM) methodologies.Many immune electron microscopy methodologies have been used for morphological identification and characterization of viruses. We have previously reported a highly effective solid phase immune electron microscopy procedure which facilitated identification of hepatitis A virus (HAV) in crude cell culture extracts. More recently we have reported utilization of the method for identification of an etiologic agent responsible for (ET-NANBH).

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