Efficacy of Chlorine Dioxide Gas Against Hepatitis A Virus on Blueberries, Blackberries, Raspberries, and Strawberries.

Seeking a means of sanitizing berries, the effectiveness of steady state levels of gaseous chlorine dioxide (ClO2) against hepatitis A virus (HAV) on laboratory-contaminated berries was determined. The generated ClO2 was maintained with 1 or 2 mg/l air inside a 269-l glove box to treat 50 g batches of blueberries, raspberries, and blackberries, and 100 g batches of strawberries that were immersion coated with HAV. Normalized data for ClO2 (ppm-h/g product) is reported as a function of ClO2 concentration, treatment time, and weight of treated product. Treatments of ClO2 ranging from 1.00 to 6.27 ppm-h/g berry were evaluated. When compared to untreated HAV-contaminated berries, log reductions of HAV were > 2.1 for all berry types and conditions tested indicating the gaseous ClO2 was effective. The average log reduction with strawberries, raspberries, blueberries and blackberries treated with 1.00 ppm-h/g, the lowest ClO2 treatment tested, were 2.44, 2.49, 3.23, and 3.45, respectively. The highest treatment of 6.27 ppm-h/g was applied at two different gas concentrations of 1 mg/l and 2 mg/l. Average log reductions for blueberries and strawberries treated with 6.27 ppm-h/g were 4.34 and 4.42, and 4.03 and 3.51, applied at 1 mg/l and 2 mg/l, respectively. For blackberries and raspberries 3.20 and 3.24, and 3.23 and 3.97 log reductions were observed for 6.27 ppm-h/g treatments applied at 1 mg/l and 2 mg/l, respectively. Results indicate that HAV contamination of berries can be substantially reduced by gaseous ClO2 and offer industry a waterless means of sanitizing berries against HAV.

Investigation of F-RNA Bacteriophage as a Tool in Re-Opening Australian Oyster Growing Areas Following Sewage Spills.

Oysters contaminated with human enteric viruses from sewage are implicated in foodborne outbreaks globally. Bacteriophages have been identified as potential indicators for these viruses, but have not been used in shellfish management outside of the USA. This study aimed to determine the background levels of F-RNA phage in five Australian oyster growing areas with a history of sewage spills and closures, over an 18-month period. In addition, oysters from five growing areas impacted by adverse sewage events were investigated for F-RNA phage, Escherichia coli, norovirus (NoV) and hepatitis A virus (HAV). F-RNA phage ≤ 60 pfu/100 gm shellfish flesh were found to represent a conservative background level in the surveyed areas. Following two of the five sewage spills, elevated phage levels were observed in most sample sites less than 4 days post spill. By 7 days, most sites from all events had phage < 30 pfu/100 gm. NoV was detected in day 1 and day 6 samples from one event when all phage were ≤ 30 pfu/100 gm. NoV was also detected in a day 3 sample from another event with < 30 phage pfu/100 gm, however, multiple replicate samples had elevated phage levels. The results of this study add evidence on the potential use of F-RNA phage as a tool in early re-opening of oyster harvest areas post sewage spills. However, it also highlights the need to better understand situations where phage testing may be ineffectual, and the importance of sampling at multiple sites and over multiple time points, to effectively capture evidence of contamination.

Survival and Inactivation by Advanced Oxidative Process of Foodborne Viruses in Model Low-Moisture Foods.

Enteric viruses, such as human norovirus (NoV) and hepatitis A virus (HAV), are the major causes of foodborne illnesses worldwide. These viruses have low infectious dose, and may remain infectious for weeks in the environment and food. Limited information is available regarding viral survival and transmission in low-moisture foods (LMF). LMFs are generally considered as ready-to-eat products, which undergo no or minimal pathogen reduction steps. However, numerous foodborne viral outbreaks associated with LMFs have been reported in recent years. The objective of this study was to examine the survival of foodborne viruses in LMFs during 4-week storage at ambient temperature and to evaluate the efficacy of advanced oxidative process (AOP) treatment in the inactivation of these viruses. For this purpose, select LMFs such as pistachios, chocolate, and cereal were inoculated with HAV and the norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), then viral survival on these food matrices was measured over a four-week incubation at ambient temperature, by both plaque assay and droplet-digital RT-PCR (ddRT-PCR) using the modified ISO-15216 method as well as the magnetic bead assay for viral recovery. We observed an approximately 0.5 log reduction in viral genome copies, and 1 log reduction in viral infectivity for all three tested viruses following storage of select inoculated LMFs for 4 weeks. Therefore, the present study shows that the examined foodborne viruses can persist for a long time in LMFs. Next, we examined the inactivation efficacy of AOP treatment, which combines UV-C, ozone, and hydrogen peroxide vapor, and observed that while approximately 100% (4 log) inactivation can be achieved for FCV, and MNV in chocolate, the inactivation efficiency diminishes to approximately 90% (1 log) in pistachios and 70% (< 1 log) in cereal. AOP treatment could therefore be a good candidate for risk reduction of foodborne viruses from certain LMFs depending on the food matrix and surface of treatment.

Mineral Waste Containing High Levels of Iron from an Environmental Disaster (Bento Rodrigues, Mariana, Brazil) is Associated with Higher Titers of Enteric Viruses.

Although the effects of heavy metals on the behavior, including infectivity, of bacteria have been studied, little information is available about their effects on enteric viruses. We report an investigation of effects on the biosynthesis of human adenoviruses (HAdV) and hepatitis A (HAV) of waters contaminated with mineral waste following an environmental disaster in Mariana City, Minas Gerais State, Brazil. The study area was affected on November 5, 2015, by 60 million m3 of mud (containing very high concentrations of iron salts) from a mining reservoir (Fundão), reaching the Gualaxo do Norte River (sites evaluated in this study), the "Rio Doce" River and finally the Atlantic Ocean. We found substantial counts of infectious HAdV and HAV (by qPCR) in all sampled sites from Gualaxo do Norte River, indicating poor basic sanitation in this area. The effects of iron on viral infection processes were evaluated using HAdV-2 and HAV-175, as DNA and RNA enteric virus models, respectively, propagated in the laboratory and exposed to this contaminated water. Experiments in field and laboratory scales found that the numbers of plaque forming units (PFU) of HAdV and HAV were significantly higher in contaminated water with high iron concentrations than in waters with low iron concentration (< 20 µg/L of iron). These findings indicate that iron can potentiate enteric virus infectivity, posing a potential risk to human and animal health, particularly during pollution disasters such as that described here in Mariana, Brazil.

[Markers of hepatitis A in the monkeys of the Adlers primate center.]

Hepatitis A is a widespread viral infection. The HAV strains of "human" and "monkey" origin are similar in their morphological and antigenic properties, but differ genotypically. OBJECTIVES: The aim of this research was a comparative study of serological and molecular-genetic markers of HAV infection in monkeys born at the Adler Primate Center and in those imported from different countries. MATERIAL AND METHODS: Fecal samples (n = 313) and serum (n = 266) from various species of monkey using ELISA and RT-PCR were studied. RESULTS AND DISCUSSION: The frequency of anti-HAV-IgG was high (78.9%) in imported animals (vervet monkeys from Tanzania and cynomolgus monkeys from Vietnam) and as well as in various species of monkeys (rhesus monkeys, cynomolgus monkeys, green monkeys and papio hamadryas) of the Center (88.6%). At the same time, in the imported monkeys, the markers of "fresh" HAV infection (IgM-27.2%, Ag-HAV-16.7%, RNA-22.0%) were detected significantly more often (p> 0.05) than in monkeys kept at the Colony (IgM-7.5%, HAV-Ag - 5.2%, RNA - 3.6%). In general, anti-IgG reactivity ranged from 1.064 to 2.073 OD450, anti-IgM ranged from 0.546 to 1.059 OD450. The number of HAV-Ag was 0.496 - 1.995 OD450. RNA HAV only in rhesus monkeys and cynomolgys monkeys born at the Colony, as well as in imported vervet monkeys was detected. CONCLUSIONS: The data obtained indicate a wide circulation of HAV among monkeys born in the Adler Primate Center and among the imported animals. Markers of "fresh" HAV infection varied depending on the species of monkeys and their origin.

Development of a Real-Time Cell Analysis (RTCA) Method as a Fast and Accurate Method for Detecting Infectious Particles of the Adapted Strain of Hepatitis A Virus.

Hepatitis A virus (HAV) is one of the most common agents causing acute liver disease worldwide. HAV has been increasingly reported as the cause of foodborne disease outbreaks. The standard method currently available for detection of the genome of HAV in vulnerable foodstuffs is by RT-qPCR (ISO 15216). Despite its usefulness in the investigation of foodborne viruses, the use of RT-qPCR in food virology has been shown to overestimate the quantity of infectious virus or to highly underestimate the effect of the treatment on virus inactivation. The gold standard methods currently used for evaluating the efficacy of inactivation treatments on the adapted strain of HAV (HM175/18f) are either the plaque assay or the end-point dilution assay (TCID50). However, both assays are labor-intensive and time-consuming. The aim of this study was to evaluate the use of the xCELLigence real-time cell analysis (RTCA) system for detecting the infectivity of the adapted strain of HAV. Kinetics of cell impedance showed that HAV induced a decrease in cell index (CI) correlated with the onset of HAV-induced cell death. In addition, the time to which the HAV-induced CI drop occurred was dependent on the viral concentration. An inverse linear relation could be established over a range of 5 log10 between the concentration of HAV and the time to reach 50% of CI decrease (TCI50), showing that the RTCA assay could be used as a titration method for HAV. In addition, the RTCA-based assay could be performed in less than 6 days instead of 12 to 14 days with the gold standard methods. Therefore, the RTCA-based titration method is a powerful and suitable tool for high-throughput screening of anti-viral treatments. Its usefulness in HAV inactivation studies will improve the assessment of viral risk in food virology, as controlling transmission of viruses through their removal from foodstuffs is also an important challenge in reducing the burden of viral foodborne illnesses.

Assessment of the Virological Quality of Marine and Running Surface Waters in NW Greece: A Case Study.

The virological quality of surface marine and running water samples collected from Igoumenitsa gulf and Kalamas river (NW Greece) was assessed from October 2012 to September 2013. Sampling sites were exposed to different land and/or anthropogenic effects. Seawater samples were collected monthly from five sampling stations (new harbor, old harbor, wastewater treatment plant outlet, protected Natura area, Drepano beach). Viral targets included human adenoviruses (hAdVs), as index human viruses, while noroviruses (NoVs) and hepatitis A virus (HAV) were also studied. Kalamas river samples were collected seasonally, from three sampling stations (Soulopoulo, Dam, Sagiada-estuaries), while viral targets included also porcine adenoviruses (pAdVs) and bovine polyoma viruses (bPyVs), as additional index viruses. All water samples were analyzed for standard bacterial indicators, as well. Physicochemical and meteorological data were also collected. Based on the standard bacterial indices, both sea and river water samples did not exceed the limits set according to Directive 2006/7/EU. However, positive samples for hAdVs were found occasionally in all sampling sites in Igoumenitsa gulf (23.3%, 14/60) showing fecal contamination of human origin. Moreover, HAV was detected once, in the sampling site of the old port (at 510 GC/L). Most of the Kalamas water samples were found positive for hAdVs (58.3%, 7/12), while human noroviruses GI (NoVGI) (8.3%, 1/12) and GII (NoVGII) (16.7%, 2/12) were also detected. HAV, pAdVs, and bovine polyomaviruses (bPyVs) were not detected in any of the analyzed samples. No statistically significant correlations were found between classic bacterial indicators and viral targets, nor between viruses and meteorological data. Overall, the present study contributed to the collection of useful data for the biomonitoring of the region, and the assessment of the overall impact of anthropogenic activities. It provided also valuable information for the evaluation of the risk of waterborne viral infections and the protection of public health. It was the first virological study in the area and one of the few in Greece.

Enzymatic and viability RT-qPCR assays for evaluation of enterovirus, hepatitis A virus and norovirus inactivation: Implications for public health risk assessment.

AIM: To assess the potential of a viability dye and an enzymatic reverse transcription quantitative PCR (RT-qPCR) pretreatment to discriminate between infectious and noninfectious enteric viruses. METHODS AND RESULTS: Enterovirus (EntV), norovirus (NoV) GII.4 and hepatitis A virus (HAV) were inactivated at 95°C for 10 min, and four methods were used to compare the efficiency of inactivation: (i) cell culture plaque assay for HAV and EntV, (ii) RT-qPCR alone, (iii) RT-qPCR assay preceded by RNase treatment, and (iv) pretreatment with a viability dye (reagent D (RD)) followed by RT-qPCR. In addition, heat-inactivated NoV was treated with RD coupled with surfactants to increase the efficiency of the viability dye. No treatment was able to completely discriminate infectious from noninfectious viruses. RD-RT-qPCR reduced more efficiently the detection of noninfectious viruses with little to no removal observed with RNase. RD-RT-qPCR method was the closest to cell culture assay. The combination of surfactants and RD did not show relevant improvements on the removal of inactivated viruses signal compared with viability RT-qPCR, with the exception of Triton X-100. CONCLUSION: The use of surfactant/RD-RT-qPCR, although not being able to completely remove the signal from noninfectious viral particles, yielded a better estimation of viral infectivity. SIGNIFICANCE AND IMPACT OF THE STUDY: Surfactant/RD-RT-qPCR may be an advantageous tool for a better detection of infectious viruses with potential significant impact in the risk assessment of the presence of enteric viruses.

Efficacy of Cinnamaldehyde Against Enteric Viruses and Its Activity After Incorporation Into Biodegradable Multilayer Systems of Interest in Food Packaging.

Cinnamaldehyde (CNMA), an organic compound that gives cinnamon its flavor and odor, was investigated for its virucidal activity on norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), and hepatitis A virus (HAV). Initially, different concentrations of CNMA (0.1, 0.5 and 1 %) were individually mixed with each virus at titers of ca. 6-7 log10 TCID50/ml and incubated 2 h at 4 and 37 °C. CNMA was effective in reducing the titers of norovirus surrogates in a dose-dependent manner after 2 h at 37 °C, while HAV titers were reduced by 1 log10 after treatment with 1 % of CNMA. When incubation time was extended, HAV titers were reduced by 3.4 and 2.7 log10 after overnight incubation at 37 °C with 1 and 0.5 % of CNMA, respectively. Moreover, this paper analyzed, for the first time, the antiviral activity of adding an active electrospun interlayer based on zein and CNMA to a polyhydroxybutyrate packaging material (PHB) in a multilayer form. Biodegradable multilayer systems prepared with 2.60 mg/cm(2) (~9.7 %) of CNMA completely inactivated FCV according to ISO 22196:2011, while MNV titers were reduced by 2.75 log10. When the developed multilayer films were evaluated after one month of preparation or at 25 °C, the antiviral activity was reduced as compared to freshly prepared multilayer films evaluated at 37 °C. The results show the excellent potential of this system for food contact applications as well as for active packaging technologies in order to maintain or extend food quality and safety.

Thermal inactivation kinetics of hepatitis A virus in homogenized clam meat (Mercenaria mercenaria).

AIMS: Epidemiological evidence suggests that hepatitis A virus (HAV) is the most common pathogen transmitted by bivalve molluscs such as clams, cockles, mussels and oysters. This study aimed to generate thermal inactivation kinetics for HAV as a first step to design adequate thermal processes to control clam-associated HAV outbreaks. METHODS AND RESULTS: Survivor curves and thermal death curves were generated for different treatment times (0-6 min) at different temperatures (50-72°C) and Weibull and first-order models were compared. D-values for HAV ranged from 47·37 ± 1·23 to 1·55 ± 0·12 min for the first-order model and 64·43 ± 3·47 to 1·25 ± 0·45 min for the Weibull model at temperatures from 50 to 72°C. z-Values for HAV in clams were 12·97 ± 0·59°C and 14·83 ± 0·0·28°C using the Weibull and first-order model respectively. The calculated activation energies for the first-order and Weibull model were 145 and 170 kJ mole(-1) respectively. CONCLUSION: The Weibull model described the thermal inactivation behaviour of HAV better than the first-order model. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides novel and precise information on thermal inactivation kinetics of HAV in homogenized clams. This will enable reliable thermal process calculations for HAV inactivation in clams and closely related seafood.

Thermal Inactivation of Foodborne Enteric Viruses and Their Viral Surrogates in Foods.

Foodborne viruses, in particular human norovirus and hepatitis A virus, are the most common causes of food-associated infections and foodborne illness outbreaks around the world. Since it is currently not possible to cultivate human noroviruses and the wild-type strain of hepatitis A virus in vitro, the use of a variety of viral surrogates is essential to determine appropriate thermal processing conditions to reduce the risk associated with their contamination of food. Therefore, the objectives of this review are to (i) present pertinent characteristics of enteric foodborne viruses and their viral surrogates, (ii) discuss the viral surrogates currently used in thermal inactivation studies and their significance and value, (iii) summarize available data on thermal inactivation kinetics of enteric viruses, (iv) discuss factors affecting the efficacy of thermal treatment, (v) discuss suggested mechanisms of thermal inactivation, and (vi) provide insights on foodborne enteric viruses and viral surrogates for future studies and industrial applications. The overall goal of this review is to contribute to the development of appropriate thermal processing protocols to ensure safe food for human consumption.

Aqueous Extracts of Hibiscus sabdariffa Calyces Decrease Hepatitis A Virus and Human Norovirus Surrogate Titers.

Hibiscus sabdariffa extract is known to have antioxidant, anti-diabetic, and antimicrobial properties. However, their effects against foodborne viruses are currently unknown. The objective of this study was to determine the antiviral effects of aqueous extracts of H. sabdariffa against human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) and hepatitis A virus (HAV) at 37 °C over 24 h. Individual viruses (~5 log PFU/ml) were incubated with 40 or 100 mg/ml of aqueous hibiscus extract (HE; pH 3.6), protocatechuic acid (PCA; 3 or 6 mg/ml, pH 3.6), ferulic acid (FA; 0.5 or 1 mg/ml; pH 4.0), malic acid (10 mM; pH 3.0), or phosphate buffered saline (pH 7.2 as control) at 37 °C over 24 h. Each treatment was replicated thrice and plaque assayed in duplicate. FCV-F9 titers were reduced to undetectable levels after 15 min with both 40 and 100 mg/ml HE. MNV-1 was reduced by 1.77 ± 0.10 and 1.88 ± 0.12 log PFU/ml after 6 h with 40 and 100 mg/ml HE, respectively, and to undetectable levels after 24 h by both concentrations. HAV was reduced to undetectable levels by both HE concentrations after 24 h. PCA at 3 mg/ml reduced FCV-F9 titers to undetectable levels after 6 h, MNV-1 by 0.53 ± 0.01 log PFU/ml after 6 h, and caused no significant change in HAV titers. FA reduced FCV-F9 to undetectable levels after 3 h and MNV-1 and HAV after 24 h. Transmission electron microscopy showed no conclusive results. The findings suggest that H. sabdariffa extracts have potential to prevent foodborne viral transmission.

Ultraviolet-C efficacy against a norovirus surrogate and hepatitis A virus on a stainless steel surface.

In this study, the effects of 10-300 mWs/cm(2) of ultraviolet radiation (UV-C) at 260 nm were investigated for the inactivation of two foodborne viruses: murine norovirus-1 (MNV-1; a human norovirus [NoV] surrogate) and hepatitis A virus (HAV). We used an experimentally contaminated stainless steel surface, a common food-contact surface, to examine the effects of low doses of UV-C radiation on MNV-1 and HAV titers. The modified Gompertz equation was used to generate non-linear survival curves and calculate dR-values as the UV-C dose of 90% reduction for MNV-1 (R(2)=0.95, RMSE=0.038) and HAV (R(2)=0.97, RMSE=0.016). Total MNV-1 and HAV titers significantly decreased (p<0.05) with higher doses of UV-C. MNV-1 and HAV were reduced to 0.0-4.4 and 0.0-2.6 log10PFU/ml, respectively, on the stainless steel surfaces by low-dose UV-C treatment. The dR-value, 33.3 mWs/cm(2) for MNV-1 was significantly (p<0.05) lower than 55.4 mWs/cm(2) of HAV. Therefore, the present study shows that HAV is more resistant to UV-C radiation than MNV-1. These data suggest that low doses of UV-C light on food contact surfaces could be effective to inactivate human NoV and HAV in restaurant, institutional, and industrial kitchens and facilities.

Mathematical model for viral depuration kinetics in shellfish: an useful tool to estimate the risk for the consumers.

Enteric virus depuration from shellfish is a complex biological process that may be influenced by biological properties of the mollusc and/or virus species. On the basis of previous experimental data, a mathematical model was developed to characterize the kinetics of viral elimination during the depuration process. The experimental data consisted on twenty depuration trials, each with 60 kg of Manila clams (Venerupis philippinarum) and mediterranean mussels (Mytilus galloprovincialis) previously subjected to bioaccumulation with HAV or MNV-1 (as a surrogate for human norovirus), that were performed in an experimental depuration system during 7 days. It was observed that although viral loads decay along depuration, a residual viral load remains at the end of the process suggesting a decomposition of viral load in a diluted load (susceptible of depuration) and a non-diluted load (unavailable to depurate). The model yielded a general equation, which can predict the viral load at any depuration time knowing the specific filtration rate, dependent on the bivalve species, and specific viral properties. The mathematical model can be combined with quantitative risk assessment calculations to determine the safety of the depurated shellfish, which can be very helpful not only for shellfish producers but also to public health authorities.

Synergistic effect of high hydrostatic pressure (HHP) and marination treatment on the inactivation of hepatitis a virus in mussels (Mytilus galloprovincialis).

Consumption of raw or insufficiently cooked mussels contaminated with hepatitis A virus (HAV) is a major cause of infection to humans. The origin of mussels commonly used for the preparation of marinated seafood salads is often unknown, since different producers worldwide undergo a precooking treatment at the original collection site with methods and parameters not always indicated. These treatments could be insufficient for the inactivation of HAV, which is characterized by a high temperature resistance. Both high hydrostatic pressure (HHP) and marinade treatments have been shown to affect HAV vitality. In this study, two treatments (HHP and marinating) were combined in order to assess a potential synergistic effect on the virus vitality. A kinetic test was conducted by subjecting the experimentally-contaminated mussels (HAV titre: 10(6)/ml TCID50) to marinating, and to different HHP treatment (4,000; 5,000; and 6,000 bar for 1, 5, and 9 min). Virus post-treatment vitality was assessed by its ability to grow on cell cultures and by quantitative real-time RT-PCR to evaluate virus resistance under such conditions. Marinating treatment alone (final pH 4.3, and NaCl 2 %) did not inactivate the virus. On the other hand, the use of HHP treatment alone on non-marinated HAV-contaminated mussels was effective only above 5,000 bar for 5 min. The results of the present study elucidate the synergistic effect of a combination between marination and HHP treatments on the inactivation of the virus.

A comparison of the thermal inactivation kinetics of human norovirus surrogates and hepatitis A virus in buffered cell culture medium.

Human noroviruses and hepatitis A virus (HAV) are considered as epidemiologically significant causes of foodborne disease. Therefore, studies are needed to bridge existing data gaps and determine appropriate parameters for thermal inactivation of human noroviruses and HAV. The objectives of this research were to compare the thermal inactivation kinetics of human norovirus surrogates (murine norovirus (MNV-1), and feline calicivirus (FCV-F9)) and HAV in buffered medium (2-ml vials), compare first-order and Weibull models to describe the data, calculate Arrhenius activation energy for each model, and evaluate model efficiency using selected statistical criteria. The D-values calculated from the first-order model (50-72 °C) ranged from 0.21-19.75 min for FCV-F9, 0.25-36.28 min for MNV-1, and 0.88-56.22 min for HAV. Using the Weibull model, the tD = 1 (time to destroy 1 log) for FCV-F9, MNV-1 and HAV at the same temperatures ranged from 0.10-13.27, 0.09-26.78, and 1.03-39.91 min, respectively. The z-values for FCV-F9, MNV-1, and HAV were 9.66 °C, 9.16 °C, and 14.50 °C, respectively, using the Weibull model. For the first order model, z-values were 9.36 °C, 9.32 °C, and 12.49 °C for FCV-F9, MNV-1, and HAV, respectively. For the Weibull model, estimated activation energies for FCV-F9, MNV-1, and HAV were 225, 278, and 182 kJ/mol, respectively, while the calculated activation energies for the first order model were 195, 202, and 171 kJ/mol, respectively. Knowledge of the thermal inactivation kinetics of norovirus surrogates and HAV will allow the development of processes that produce safer food products and improve consumer safety.

Determination of thermal inactivation kinetics of hepatitis A virus in blue mussel (Mytilus edulis) homogenate.

Hepatitis A virus (HAV) is a food-borne enteric virus responsible for outbreaks of hepatitis associated with shellfish consumption. The objectives of this study were to determine the thermal inactivation behavior of HAV in blue mussels, to compare the first-order and Weibull models to describe the data, to calculate Arrhenius activation energy for each model, and to evaluate model efficiency by using selected statistical criteria. The times required to reduce the population by 1 log cycle (D-values) calculated from the first-order model (50 to 72°C) ranged from 1.07 to 54.17 min for HAV. Using the Weibull model, the times required to destroy 1 log unit (tD = 1) of HAV at the same temperatures were 1.57 to 37.91 min. At 72°C, the treatment times required to achieve a 6-log reduction were 7.49 min for the first-order model and 8.47 min for the Weibull model. The z-values (changes in temperature required for a 90% change in the log D-values) calculated for HAV were 15.88 ± 3.97°C (R(2), 0.94) with the Weibull model and 12.97 ± 0.59°C (R(2), 0.93) with the first-order model. The calculated activation energies for the first-order model and the Weibull model were 165 and 153 kJ/mol, respectively. The results revealed that the Weibull model was more appropriate for representing the thermal inactivation behavior of HAV in blue mussels. Correct understanding of the thermal inactivation behavior of HAV could allow precise determination of the thermal process conditions to prevent food-borne viral outbreaks associated with the consumption of contaminated mussels.

Depuration kinetics of hepatitis A virus in clams.

The efficacy and dynamic of depuration for the removal of hepatitis A virus (HAV) contamination were evaluated under experimental conditions using Manila clams previously subjected to bioaccumulation with this virus. Five independent trials were assayed in a closed experimental system with a total volume of approximately 1750 l, using clam batches of 60 Kg. The reverse transcriptase-real time PCR (RT-qPCR) technique was utilized for viral quantification. Infectivity assays were conducted at the end of depuration. Although the final viral loads in shellfish after 7 days remained relatively high and still infectious, an average reduction in HAV levels of 1.44 log units (approx. 93.1%) was observed. This reduction showed a two-phase removal kinetic, with an initial rapid reduction of viruses during the first 72 h of depuration, with a 0.6 log units (69%) of average decrease in HAV RNA copies/g digestive tissue, and a subsequent stabilization with a slower depuration rate in the remaining days.

[The hepatitis A virus: structural and functional organization of the genome, its molecular diagnostic value and cultivation].

The analysis of recently published data on hepatitis A virus (HAV) genome clinical features, molecular diagnostic value and cell culture propagation are reviewed. The growing need in the study of the genetic diversity of HAV isolates and the search of its possible new antigenic variants are underlined. The results of the cultivation of different HAV strains are analyzed for possible application in vaccine and diagnostic kit production.

Survival of murine norovirus, Tulane virus, and hepatitis A virus on alfalfa seeds and sprouts during storage and germination.

Human 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.