Isolation and Genomic Sequence of Hepatitis A Virus from Mixed Frozen Berries in Italy

Hepatitis A virus (HAV) was detected in two samples of mixed frozen berries linked to Italian hepatitis A outbreak in April and September 2013. Both viruses were fully sequenced by next-generation sequencing and the genomes clustered with HAV complete genomes of sub-genotype IA with nucleotide identities of 95–97 %.     

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...     

Successful Use of Advertisement Pictures to Assist Recall in a Food-Borne Hepatitis A Outbreak in The Netherlands, 2017

This study describes an outbreak investigation of 14 hepatitis A cases in the Netherlands. The hepatitis A virus (HAV) genotype IB sequences in cases were highly similar (459/460 nt). The origin of strains could be narrowed to Bulgaria based on information from EPIS-FWD. As an association with consumption of soft fruit was suspected, a case–control study was initiated using a questionnaire and a list of pictures of soft fruit available at the supermarket chain involved. Twelve out of 13 cases consumed a specific frozen raspberry/blueberry product shown on the list (OR 46.0, 95% CI 5.0–27). In multivariable regression analysis this product was the only risk factor (aOR 26.6, 95% CI 2.0–263). Laboratory analyses could not demonstrate HAV-RNA in batches that had been on the market in the incubation period of patients. Trace back of frozen fruit showed that raspberries had been traded by a producer in Bulgaria. After withdrawal of the product from the supermarket no new cases were reported. Use of advertisement pictures of consumed food was helpful in this investigation. Suspicion of the source was strengthened by data from molecular typing and food trace back activities, underlining the importance of good (inter)national cooperation between public health and food safety organisations.     

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.     

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.     

Detection of Potential Infectious Enteric Viruses in Fresh Produce by (RT)-qPCR Preceded by Nuclease Treatment

Foodborne illnesses associated with contaminated fresh produce are a common public health problem and there is an upward trend of outbreaks caused by enteric viruses, especially human noroviruses (HNoVs) and hepatitis A virus (HAV). This study aimed to assess the use of DNase and RNase coupled to qPCR and RT-qPCR, respectively, to detect intact particles of human adenoviruses (HAdVs), HNoV GI and GII and HAV in fresh produce. Different concentrations of DNase and RNase were tested to optimize the degradation of free DNA and RNA from inactivated HAdV and murine norovirus (MNV), respectively. Results indicated that 10 µg/ml of RNase was able to degrade more than 4 log₁₀ (99.99%) of free RNA, and 1 U of DNase degraded the range of 0.84–2.5 log₁₀ of free DNA depending on the fresh produce analysed. The treatment with nucleases coupled to (RT)-qPCR was applied to detect potential infectious virus in organic lettuce, green onions and strawberries collected in different seasons. As a result, no intact particles of HNoV GI and GII were detected in the 36 samples analysed, HAdV was found in one sample and HAV was present in 33.3% of the samples, without any reasonable distribution pattern among seasons. In conclusion, RT-qPCR preceded by RNase treatment of eluted samples from fresh produce is a good alternative to detect undamaged RNA viruses and therefore, potential infectious viruses. Moreover, this study provides data about the prevalence of enteric viruses in organic fresh produce from Brazil.     

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.     

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.     

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.     

Application of Pesticide Sprays to Fresh Produce: A Risk Assessment for Hepatitis A and <Emphasis Type="Italic">Salmonella</Emphasis>

The purpose of this study was to quantify the transfer of viral and bacterial pathogens in water used to dilute pesticides sprayed onto the surfaces of cantaloupe, iceberg lettuce, and bell peppers. The average percent transfer of bacteria was estimated to range from 0.00021 to 9.4%, while average viral transfer ranged from 0.055 to 4.2%, depending on the type of produce. Based on these values the concentrations of hepatitis A virus (HAV) and Salmonella in water necessary to achieve a 1:10,000 annual risk of infection were calculated. Under worst case scenario assumptions, in which a pesticide is applied on the same day that the produce is harvested and when maximum transfer values are used, concentrations of 1.5 × 10⁻³ CFU Salmonella or 2.7 × 10⁻⁷ MPN HAV per 100 ml of the water used for application would result in 1:10,000 annual infection risk to anyone who consumes the fresh produce. If harvesting does not occur until at least 14 days after the application, to produce the same risk of infection, the numbers of Salmonella in 100 ml of water used to dilute the pesticides will be greater by up to five orders of magnitude, while the HAV numbers will have increased by up to two orders of magnitude. Based on the reported concentrations of enteric viruses in surface and ground waters in the United States, a 1:10,000 annual risk of infection could easily be exceeded with some groundwater sources used in the United States. To reduce the risks associated with the consumption of fresh produce, water used to prepare pesticides in spray applications should be evaluated for its microbiological quality.     

Qualitative and Quantitative Assessment of Hepatitis A Virus in Wastewaters in Tunisia

Hepatitis A causes substantial morbidity in both industrialized and non-industrialized countries and represents an important health problem in several southern Mediterranean countries. The objectives of the study were as follows: (a) to assess the occurrence of hepatitis A virus (HAV) in Tunisia through the monitoring of urban wastewaters collected at wastewater treatment plants (WTPs); (b) to characterize environmental strains; and (c) to estimate the viral load in raw and treated sewages, in order to evaluate the potential impact on superficial waters receiving discharges. A total of 150 raw and treated wastewaters were collected from three WTPs and analyzed by both qualitative (RT-PCR/nested) and quantitative (qRT-PCR) methods. Of these, 100 (66 %) were found to be positive for HAV by the qualitative assay: 68.3 % in influents and 64.7 % in effluents. The vast majority of HAV sequences belonged to sub-genotype IA, with 11 different strains detected found to be identical to clinical strains isolated from Tunisian patients with acute hepatitis. Five unique variants were also detected, not previously reported in clinical cases. Only two IB strains were found, confirming the rarity of this sub-genotype in this country. The results of the present study indicate a wide circulation of the pathogen in the population, most probably in the form of asymptomatic infections, a finding consistent with the classification of the country as having intermediate/high endemicity. Quantitative data showed high viral loads in influents (3.5E+05 genome copies/liter, mean value) as well as effluents (2.5E+05 genome copies/liter, mean value), suggesting that contaminated water could be a critical element in transmission.     

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.     

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.     

Hepatitis A Outbreaks and Environmental Circulation of Genotype IA Strains in the São Paulo City, 2017–2018

Food and Environmental Virology - Hepatitis A virus (HAV) is the major cause of enterically transmitted infectious hepatitis. Between 2016 and 2017, the number of confirmed cases of hepatitis A...     

Environmental Surveillance of SARS-CoV-2 RNA in Wastewater and Groundwater in Quintana Roo,Mexico

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater has been reported as a result of fecal shedding of infected individuals. In this study, the occurrence of SARS-CoV-2 RNA was explored in primary-treated wastewater from two municipal wastewater treatment plants in Quintana Roo, Mexico, along with groundwater from sinkholes, a household well, and submarine groundwater discharges. Physicochemical variables were obtained in situ, and coliphage densities were determined. Three virus concentration methods based on adsorption-elution and sequential filtration were used followed by RNA isolation. Quantification of SARS-CoV-2 was done by RT-qPCR using the CDC 2020 assay, 2019-nCoV_N1 and 2019-nCoV_N2. The Pepper mild mottle virus, one of the most abundant RNA viruses in wastewater was quantified by RT-qPCR and compared to SARS-CoV-2 concentrations. The use of three combined virus concentration methods together with two qPCR assays allowed the detection of SARS-CoV-2 RNA in 58% of the wastewater samples analyzed, whereas none of the groundwater samples were positive for SARS-CoV-2 RNA. Concentrations of SARS-CoV-2 in wastewater were from 1.8 × 10³ to 7.5 × 10³ genome copies per liter (GC l⁻¹), using the N1 RT-qPCR assay, and from 2.4 × 10² to 5.9 × 10³ GC l⁻¹ using the N2 RT-qPCR assay. Based on PMMoV prevalence detected in all wastewater and groundwater samples tested, the three viral concentration methods used could be successfully applied for SARS-CoV-2 RNA detection in further studies. This study represents the first detection of SARS-CoV-2 RNA in wastewater in southeast Mexico and provides a baseline for developing a wastewater-based epidemiology approach in the area.

     

Hepatitis A Outbreak in Europe: Imported Frozen Berry Mix Suspected to be the Source of At least One Infection in Austria in 2013

We tested 19 sera from Austrian patients with acute hepatitis A. A serum from a 48-year-old female patient yielded HAV-nucleic acid that showed 99.7 % homology to the HAV-sequence obtained from samples taken during the current outbreak in several European countries, which is associated with consumption of frozen berries. So far, Austria was considered not to be affected by this hepatitis A outbreak.     

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.

     

Detection of Hepatitis A Virus and Norovirus in Different Food Categories: A 6-Year Survey in Italy

Food and Environmental Virology - To observe the prevalence of contamination by hepatitis A virus (HAV) and norovirus (NoV) in different food types, 9242 samples were analyzed over a 6-year period...     

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.