Hepatitis A Virus,Hepatitis E Virus,and Rotavirus in Foods of Animal Origin Traded at the Borders of Brazil,Argentina, and Uruguay

The aim of this study was to investigate hepatitis A virus (HAV), hepatitis E (HEV), and rotavirus (RV) in fresh and processed meat traded on the border of Brazil with Argentina and Uruguay. In total, 159 samples of raw and processed foods of animal origin were collected in Paso de los Libres, Argentina (n?=?53 raw meat, n?=?24 processed meat) and Rivera, Uruguay (n?=?55 raw meat, n?=?18 processed meat), or were seized by the Brazilian International Agricultural Surveillance System—VIGIAGRO (Brazil–Argentina border) (n?=?8 raw meat, n?=?1 bush meat). All samples were tested for the presence of HAV, HEV, and RV genomes. HAV genes were detected in 18.23% of samples and RV genes in 23.89%. No HEV-positive samples were detected. HAV was also detected in two of the VIGIAGRO samples. Processed meats from Argentina and Uruguay had a higher rate of HAV and RV than raw meat (P?>?0.05). The median HAV in the Argentinian and Uruguayan samples was 6.9?×?10⁴ and 3.5?×?10³ copies/g, respectively. The presence of RV viral genes in raw meats from Argentina was significant, and this was not observed in processed meats. The presence of HAV and RV genes in a significant portion of products from Argentina and Uruguay is a potential source of human infection. This also indicates precarious conditions of acquisition, processing, and manipulation, which could be improved by improved regulation of food across borders.     

Discrimination of Infectious Hepatitis A Viruses by Propidium Monoazide Real-Time RT-PCR

The discrimination of infectious and inactivated viruses remains a key obstacle when using quantitative RT-PCR (RT-qPCR) to quantify enteric viruses. In this study, propidium monoazide (PMA) and RNase pretreatments were evaluated for the detection and quantification of infectious hepatitis A virus (HAV). For thermally inactivated HAV, PMA treatment was more effective than RNase treatment for differentiating infectious and inactivated viruses, with HAV titers reduced by more than 2.4 log₁₀ units. Results showed that combining 50 μM of PMA and RT-qPCR selectively quantify infectious HAV in media suspensions. Therefore, PMA treatment previous to RT-qPCR detection is a promising alternative to assess HAV infectivity.     

Persistence of Hepatitis A Virus in Fresh Produce and Production Environments,and the Effect of Disinfection Procedures: A Review

Although information is limited, it is evident that prolonged persistence of infectious Hepatitis A virus (HAV) is a factor in the transmission of the virus via fresh produce. Consequently, data on persistence of the virus on produce, and in environments relevant to production, such as soils, water and surfaces, are required to fully understand the dynamics of transmission of HAV via foods. Furthermore, information on effective disinfection procedures is necessary to implement effective post-harvest control measures. This review summarises current information on HAV persistence in fresh produce and on relevant disinfection procedures. On vegetables, HAV can remain infectious for several days; on frozen berries, it can persist for several months. HAV can remain infectious on surfaces for months, depending on temperature and relative humidity, and can survive desiccation. It can survive for several hours on hands. Washing hands can remove the virus, but further data are required on the appropriate procedure. Chlorination is effective in water, but not when HAV is associated with foodstuffs. Bleach and other sodium hypochlorite disinfectants at high concentrations can reduce HAV on surfaces, but are not suitable for use on fresh produce. There is only limited information on the effects of heating regimes used in the food industry on HAV. HAV is resistant to mild pasteurisation. Some food components, e.g. fats and sugars, can increase the virus’ resistance to higher temperatures. HAV is completely eliminated by boiling. Quantitative prevalence data are needed to allow the setting of appropriate disinfection log reduction targets for fresh produce.     

Occurrence of Norovirus and Hepatitis A Virus in U.S. Oysters

Noroviruses (NoV) and hepatitis A virus (HAV) are the leading causes of non-bacterial gastroenteritis in shellfish consumers worldwide. This study determined the seasonal and geographical distribution of NoV (genogroups I and II) and HAV in live U.S. market oysters. Samples were analyzed to determine the occurrence and levels of NoV and HAV using RT-qPCR and conventional RT-PCR. NoV and HAV were detected in 3.9 and 4.4%, respectively. NoV genogroups I and II were detected, with genogroup II predominating. Sequencing identified genotypes II.4, II.3, and II.7. The GII.4 strain showed ≥98% similarity with 2006–2007 circulating strains, Minerva and Laurens. HAV sequences from the 5′ non-coding region (NCR) of the genome were from genotypes I, II, or III. The incidence of NoV in oysters harvested from Atlantic Coast states was higher than that in oysters from other regions and its occurrence was greatest during the cooler months (December to February). HAV was detected at a higher frequency in shellfish harvested from the Gulf Coast and also predominated during cooler months. The seasonal occurrence of viruses in this study corresponded to the reported incidence of shellfish-associated viral illnesses. This investigation provides an overview of the occurrence and distribution of NoV and HAV in U.S. market shellfish.     

Heat Inactivation of Hepatitis A Virus in Shellfish Using Steam

Shellfish are an important cause of foodborne viral illness. Consumer-friendly cooking recommendations for shellfish could improve food safety and decrease the risk for infection from contaminated products. Thermal inactivation parameters were established for hepatitis A virus (HAV) in mussels and validated with cooking experiments. Steaming for only 2–5 min was not sufficient to inactivate HAV in mussels in all layers of a steamer. Steaming mussels for 6 min was sufficient to inactivate HAV in all layers. These cooking guidelines produce shellfish with a reduced risk for foodborne virus transmission.     

Comparative Analysis of Viral Concentration Methods for Detecting the HAV Genome Using Real-Time RT-PCR Amplification

Hepatitis A is a major infectious disease epidemiologically associated with foodborne and waterborne outbreaks. Molecular detection using real-time RT-PCR to detect the hepatitis A virus (HAV) in contaminated vegetables can be hindered by low-virus recoveries during the concentration process and by natural PCR inhibitors in vegetables. This study evaluated three virus concentration methods from vegetables: polyethylene glycol (PEG) precipitation, ultrafiltration (UF), and immunomagnetic separation (IMS). UF was the most efficient concentration method, while PEG and IMS were very low for the recovery rate of HAV. These results demonstrate that UF is the most appropriate method for recovering HAV from contaminated vegetables and that this method combined with the real-time RT-PCR assay may be suitable for routine laboratory use.     

Comparative Uptake of Enteric Viruses into Spinach and Green Onions

Root uptake of enteric pathogens and subsequent internalization has been a produce safety concern and is being investigated as a potential route of pre-harvest contamination. The objective of this study was to determine the ability of hepatitis A virus (HAV) and the human norovirus surrogate, murine norovirus (MNV), to internalize in spinach and green onions through root uptake in both soil and hydroponic systems. HAV or MNV was inoculated into soil matrices or into two hydroponic systems, floating and nutrient film technique systems. Viruses present within spinach and green onions were detected by RT-qPCR or infectivity assays after inactivating externally present viruses with Virkon^®. HAV and MNV were not detected in green onion plants grown up to 20 days and HAV was detected in only 1 of 64 spinach plants grown in contaminated soil substrate systems up to 20 days. Compared to soil systems, a drastic difference in virus internalization was observed in hydroponic systems; HAV or pressure-treated HAV and MNV were internalized up to 4 log RT-qPCR units and internalized MNV was shown to remain infectious. Understanding the interactions of human enteric viruses on produce can aid in the elucidation of the mechanisms of attachment and internalization, and aid in understanding risks associated with contamination events.     

Optimization of Methods for Detecting Hepatitis A Virus in Food

Hepatitis A virus (HAV) is currently recognized as an important human food borne pathogen, and it is one of the most resistant enteric RNA viruses, is highly infectious, and may lead to widespread outbreaks. The aim of this study was to optimize the methods to detect HAV from artificially contaminated food. To this end, strawberry and lettuce were experimentally contaminated with HAV suspension containing 6 × 10⁶ copies/ml. After contamination, HAV persistence and washing procedure were evaluated at 0, 1, 3, 7, and 9 days of storage. Five elution buffers (PBS (pH 7.4)/0.1% Tween80; 50 mM glycine/3% (wt/vol) beef extract (pH 9.5); PBS (pH 7, 4); 25 mM glycine/0.1 Tween80; and 1 M sodium bicarbonate) were used to elute the virus, and qualitative and quantitative PCR were used for HAV detection. HAV was detected by qualitative and quantitative PCR using any of the five elution buffers, but PBS was the most effective. Even after washing, HAV was detected up to 9 days after contamination by quantitative PCR. Quantitative PCR was more sensitive than qualitative PCR since samples containing viral load lower than 1.4 × 10³ copies/ml could not be detected by qualitative PCR. Quantitative PCR can be used for rapid detection of food borne viruses and will help in the monitoring and control of food borne disease.     

Identification of Enteric Viruses in Foods from Mexico City

Foodborne viruses are a common and, probably, the most under-recognized cause of outbreaks of gastroenteritis. Among the main foods involved in the transmission of human enteric viruses are mollusks, and fruits and vegetables irrigated with wastewater and/or washed with non-potable water or contaminated by contact with surfaces or hands of the infected personnel during its preparation. In this study, 134 food samples were analyzed for the detection of Norovirus, Rotavirus, and Hepatitis A virus (HAV) by amplification of conserved regions of these viruses. From the 134 analyzed samples, 14 were positive for HAV, 6 for Norovirus, and 11 for Rotavirus. This is the first report in Mexico where emphasis is given to the presence of HAV and Norovirus on perishable foods and food from fisheries, as well as Rotavirus on frozen vegetables, confirming the role of vegetables and bivalve mollusks as transmitting vehicles of enteric viruses.     

渤海湾天津沿岸海水中甲肝病毒的检测和定量

甲肝病毒（hepatitis A virus, HAV）是能引起传染性甲型肝炎的单链RNA病毒.用常规RT-PCR（反转录PCR）和SYBR Green实时定量RT-PCR方法,根据甲肝病毒保守的VP1-VP2基因序列设计引物,对渤海湾天津沿岸海水中的甲肝病毒进行了检测和定量分析. 9个样品取自渤海湾天津塘沽以南沿岸海水,取样时间分别是2007年夏、秋、冬季和2008年春季. 海水样品先用小型超滤装置（Millipore Pellicon Mini TFF）或超滤离心管（Millipore Centricon Plus-70）浓缩,然后进行RT-PCR检测.结果表明,从9个海水样品中都能扩增出192 bp的HAV cDNA,这些cDNA的核苷酸序列与GenBank中的同源序列相似性为95%～100%. 用SYBR Green 实时定量RT-PCR检测了春季和冬季的6个海水样品,结果表明,海水中甲肝病毒的浓度范围为5.35×10⁶～4.51×10⁷ virus particles/L.     

Occurrence of Norovirus and Hepatitis A Virus in Wild Mussels Collected from the Baltic Sea

The aim of the study was to define the occurrence of human noroviruses of genogroup I and II (NoV GI and NoV GII) and hepatitis A virus (HAV) in the Baltic Sea mussels. The shellfish samples were taken at the sampling sites located on the Polish coast. In total, 120 shellfish were tested as pooled samples using RT-PCR and hybridisation with virus specific probes. NoV GI was detected in 22 (18.3 %), NoV GII in 28 (23.3 %), and HAV in 9 (7.5 %) of the shellfish. The nucleotide sequence analysis of the detected NoV GII strains showed a 97.3–99.3 % similarity to GII.4 virus strain. This is the first report describing the NoV and HAV occurrence in wild Baltic mussels and their possible role as bioindicators of seawater contamination with human enteric viruses.     

High Pressure Inactivation of HAV Within Mussels

The potential of high pressure processing to inactivate hepatitis A virus (HAV) within Mediterranean mussels (Mytilus galloprovincialis) and blue mussels (Mytilus edulis) was evaluated. HAV was bioaccumulated within mussels to approximately 6-log₁₀ PFU by exposure of mussels to HAV-contaminated seawater. After shucking, 5 min pressure treatments of 300, 325, 350, 375, and 400 MegaPascals (MPa) were performed at room temperature (18–22°C). For blue mussels, log₁₀ PFU reductions of HAV averaged 2.1 and 3.6 for treatments of 350 and 400 MPa, while for Mediterranean mussels reductions of 1.7 and 2.9 log₁₀ PFU MPa were observed for equivalent treatments. These results demonstrate that high pressure processing is capable of inactivating HAV within mussels.     

Thermal Inactivation of Human Norovirus Surrogates

We investigated the thermal inactivation profiles of murine norovirus (MNV), Hepatitis A virus (HAV), and feline calicivirus (FCV), which are surrogates for the study of human noroviruses. Thermal inactivation of MNV and FCV were evaluated at 37, 50, and 60°C and HAV at 37, 50, 60, and 70°C. All viral surrogates were relatively stable at 37°C. MNV and FCV decimal reduction times (D-values) at 50°C were statistically significantly different (P < 0.05) with MNV being more stable. Both surrogates had comparable, low D-values at 60°C. HAV had significantly higher (P < 0.05) D-values than both MNV and FCV at 50 and 60°C. Overall, the infectivity assay results indicate that HAV is resistant to thermal inactivation while MNV is moderately resistant and FCV is least resistant.     

Detection of Hepatitis A Virus in Strawberries Implicated in an Outbreak in the USA in 1997

Food and Environmental Virology - Hepatitis A virus (HAV) was detected in frozen strawberries which had been implicated in a large outbreak of hepatitis A in 1997. The sample was analysed after...     

The Fate of Murine Norovirus and Hepatitis A Virus During Preparation of Fresh Produce by Cutting and Grating

Human noroviruses and hepatitis A virus (HAV) are commonly associated with outbreaks occurring in restaurant establishments and catered events. Food handlers are major contributing factors to foodborne illnesses initiated in the kitchen setting. In this study, transfer of HAV and murine norovirus (MNV-1), a human norovirus surrogate, between produce (cucumbers, strawberries, tomatoes, cantaloupes, carrots, and honeydew melons) and common kitchen utensils (graters and knives) was investigated. The extent of virus transfer to produce during utensil application, in the presence and the absence of food residue, and the impact of knife surface properties (sharp, dull, serrated) was also investigated. Transfer of MNV-1 and HAV from produce items, initially contaminated with ~5.5 log PFU, to knives and graters during application ranged from 0.9 to 5.1 log PFU. MNV-1 transfer to knives was the greatest for cucumbers, strawberries, and tomatoes, and the least for honeydew melons, while transfer of HAV to knives was greater for tomatoes and honeydew melons than strawberries, cantaloupes, and cucumbers. After preparation of a contaminated produce item, knife cross-contamination easily occurred as viruses were detected on almost all of the seven produce items successively prepared. Produce residues on utensils often resulted in less virus transfer when compared to utensils without residue accumulation. Knife surface properties did not impact virus transfer. The ease of virus transfer between produce and utensils demonstrated by the current study highlights the importance of efforts aimed toward preventing cross-contamination in the kitchen environment.     

High Pressure Inactivation of HAV Within Oysters: Comparison of Shucked Oysters with Whole-In-Shell Meats

High pressure inactivation of hepatitis A virus (HAV) within oysters bioaccumulated under simulated natural conditions to levels >10⁵ PFU/oyster has been evaluated. Five minute treatments at 20°C were administered at 350, 375, and 400 MegaPascals (MPa). Shucked and whole-in-shell oysters were directly compared to determine if there were any differences in inactivation levels. For whole-in-shell oysters and shucked oysters, average values obtained were 2.56 and 2.96 log₁₀ inactivation of HAV, respectively, after a 400-MPa treatment. Results indicate that there is no significant inactivation difference (P = 0.05) between inactivation for whole-in-shell oysters as compared to shucked oysters observed for all pressure treatments. This study indicates that commercial high pressure processing applied to whole-in-shell oysters will be capable of inactivating HAV pathogens.     

Quantitative Real-Time PCR and Digital PCR to Evaluate Residual Quantity of HAV in Experimentally Depurated Mussels

Food and Environmental Virology - Kinetics of hepatitis A virus (HAV) accumulation and depuration from mussels (Mytilus galloprovincialis) was studied in an experimental depuration system....