Green tea extract assisted low-temperature pasteurization to inactivate enteric viruses in juices.

The current popularity of minimally processed foods is an opportunity for natural antimicrobial agents to be combined with mild heat treatments to act synergistically in reducing viral foodborne pathogens. Viral inactivation by heat-treatments (at 25, 40, 50 and 63 °C for 30 min) combined with aged green tea extract (aged-GTE) was initially evaluated in phosphate buffered saline (PBS) against murine norovirus (MNV-1) and hepatitis A virus (HAV) by cell culture, and against human norovirus by in situ capture RT-qPCR. The combination of aged-GTE and heat treatment at 50 °C for 30 min exerted strong antiviral activity, reducing by more than 5 log MNV-1 infectivity in PBS. Heating at 40 °C for …

Antiviral properties of silver nanoparticles against norovirus surrogates and their efficacy in coated polyhydroxyalkanoates systems

Silver nanoparticles (AgNP) have strong broad-spectrum antimicrobial activity and gained increased attention for the development of AgNP based products, including medical and food applications. Initially, the efficacy of AgNP and silver nitrate (AgNO3) was evaluated for inactivating norovirus surrogates, the feline calicivirus (FCV) and the murine norovirus (MNV). These norovirus surrogates were exposed to AgNO3 and AgNP solutions for 24 h at 25 °C and then analyzed by cell-culture assays. Both AgNP and silver ions significantly decreased FCV and MNV infectivity in a dose-dependent manner between concentrations of 2.1 and 21 mg/L. Furthermore, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (…

SARS-CoV-2 RNA and antibody detection in human milk from a prospective multicenter study in Spain

Background During the COVID-19 pandemic in 2020, breastfeeding in women positive for SARS-CoV-2 was compromised due to contradictory data regarding potential viral transmission. However, growing evidence confirms the relevant role of breast milk in providing passive immunity by generating and transmitting specific antibodies against the virus. Thus, our study aimed to develop and validate a specific protocol to detect SARS-CoV-2 in breast milk matrix as well as to determine the impact of maternal SARS-CoV-2 infection on presence, concentration, and persistence of specific SARS-CoV-2 antibodies. Study design/Methods A prospective multicenter longitudinal study in Spain was carried out from A…

High pressure treatment and green tea extract synergistically control enteric virus contamination in beverages

Consumers are driving food production toward the use of natural preservatives and minimal processing technologies. Green tea extract (GTE) at low concentration could be combined with high pressure processing (HPP) for reduced treatment times and quality impact on foods in a hurdle concept for synergistic effects on foodborne viral pathogens, specifically human norovirus and hepatitis A virus (HAV). Viral inactivation by HPP (at 300, 400, and 500 MPa for 5 min) combined with 3.3 mg/mL aged-GTE was initially evaluated in buffer (PBS) against murine norovirus (MNV), a culturable human norovirus surrogate, and HAV. Furthermore, human norovirus inactivation was evaluated by the novel human intes…

Bias of library preparation for virome characterization in untreated and treated wastewaters

The use of metagenomics for virome characterization and its implementation for wastewater analyses, including wastewater-based epidemiology, has increased in the last years. However, the lack of standardized methods can led to highly different results. The aim of this work was to analyze virome profiles in upstream and downstream wastewater samples collected from four wastewater treatment plants (WWTPs) using two different library preparation kits. Viral particles were enriched from wastewater concentrates using a filtration and nuclease digestion procedure prior to total nucleic acid (NA) extraction. Sequencing was performed using the ScriptSeq v2 RNA-Seq (LS) and the NEBNext Ultra II RNA …

On the use of carrageenan matrices for the development of antiviral edible coatings of interest in berries

Different film-forming dispersions (FFD) based on κ−, ι− and λ− carrageenans and green tea extract (GTE) have been developed as an innovative strategy to guarantee the food safety of blueberries and raspberries. First, the FFD were characterized (surface tension and viscosity) and the physicochemical properties (water vapour permeability-WVP-, water sorption, contact angle, mechanical properties) of the stand-alone films were evaluated. Then, the FFD were applied to refrigerated-stored raspberries and blueberries, and the antiviral activity against murine norovirus (MNV), a cultivable norovirus surrogate, and hepatitis A virus (HAV) of coated fruits was determined at refrigerated (10 °C) an…

Epidemiological Surveillance of Norovirus and Rotavirus in Sewage (2016–2017) in Valencia (Spain)

© 2020 by the authors.

Seasonal variations of antimicrobial activity and chemical composition of essential oils extracted from three Citrus limon L. Burm. cultivars

In order to investigate the seasonal variations of antimicrobial properties and chemical composition of essential oils (EOs), three different cultivars of Citrus limon L. Burm. spp. (Femminello Santa Teresa, Monachello and Femminello Continella) were collected at 6-week intervals, from December 2012 to April 2013, for a total of four harvests. The EOs were extracted from lemon peel by hydro-distillation. The antimicrobial activity, tested by paper disc diffusion method, was evaluated against common food-related pathogenic bacteria (Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Enterobacter spp.). EOs were more effective against Gram-positive than Gram-negative bacte…

Qualitative comparision of olive oil mediterranean

Effect of Salt Concentration and Extremely Halophilic Archaea on the Safety and Quality Characteristics of Traditional Salted Anchovies

Extremely halophilic archaea (EHA) might play an important role in salted fish production. So far, limited information has been available on the effect of EHA and salt concentration on the safety and quality characteristics of salted anchovies. Eight Halobacterium salinarum strains were isolated from different sea salt samples and subjected to phenotypic and genotypic characterization. The strains were then inoculated into fresh salt before addition to anchovies. A total of 18 experimental productions were performed. The inoculated trials showed the lowest counts of undesired microorganisms. In particular, salted anchovies produced with Hbt. salinarum H11 showed the lowest histamine concent…

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

The hepatitis E virus (HEV) is an emerging pathogen showing a considerable increase in the number of reported cases in Europe mainly related to the ingestion of contaminated food. As with other relevant viral foodborne pathogens, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) is the gold standard for HEV detection in clinical, food, and environmental samples, but these procedures cannot discriminate between inactivated and potentially infectious viruses. Thus, the aim of this study was to develop a viability PCR method to discriminate between native, heat-, and high-pressure processing (HPP)-treated HEV using the hepatitis A virus (HAV) as a cultivable surrogate. To thi…

Antiviral and antioxidant properties of active alginate edible films containing phenolic extracts

In this work, novel active edible films having antiviral activity were developed through the solvent casting method. Emulsified edible films were prepared by adding lipids into alginate films in order to improve their water vapour permeability. In addition, two natural extracts rich in phenolic compounds, specifically a green tea extract (GTE) and a grape seed extract (GSE) were used as active agents. Interestingly, incorporating GTE or GSE within the biopolymer matrix, the films exhibited significant antiviral activity against murine norovirus (MNV) and hepatitis A virus (HAV) since ∼2 log reduction were recorded for the 0.75 g extract/g alginate in the film, being GTE more efficient than …

HEV Occurrence in Waste and Drinking Water Treatment Plants

Hepatitis E virus (HEV), particularly zoonotic genotype 3, is present in environmental waters worldwide, especially in industrialized countries. Thus, monitoring the presence of HEV in wastewater treatment plants (WWTPs) is an emerging topic due to the importance of reusing water on a global level. Given the limited data, this study aimed to monitor the occurrence of HEV in influent and effluent water in waste- and drinking-water treatment plants (WWTPs and DWTPs). To this end, different procedures to concentrate HEV in influent and effluent water from WWTPs and DWTPs were initially evaluated. The evaluated procedures resulted in average HEV recoveries of 15.2, 19.9, and 16.9% in influent, …

Detection Of Genomic Variants Of SARS-CoV-2 Circulating In Wastewater By High-Throughput Sequencing

The use of SARS-CoV-2 metagenomics in wastewater can allow the detection of variants circulating at community level. After comparing with clinical databases, we identified three novel variants in the spike gene, and six new variants in the spike detected for the first time in Spain. We finally support the hypothesis that this approach allows the identification of unknown SARS-CoV-2 variants or detected at only low frequencies in clinical genomes.

SARS-CoV-2 RNA titers in wastewater anticipated COVID-19 occurrence in a low prevalence area

Postprint disponible en http://hdl.handle.net/10261/211707

Effetto antimicrobico della componente volatile dell’olio essenziale di mandarino.

Gli oli essenziali, estratti dalle piante, sono composti lipofili contenenti una ampia varietà di elementi volatili. Vengono normalmente ottenuti per distillazione o estrazione con solventi. Trovano impiego nella preparazione di cosmetici, per aggiungere sapore ai cibi e alle bevande e per la formulazione di profumi sia personali che per l’ambiente. Gli oli essenziali hanno recentemente trovato impiego nelle biotecnologie applicate al settore agroalimentare. Sono stati, infatti, utilizzati per ottimizzare le proprietà nutrizionali degli alimenti e per prolungarne la shelf life [1,2]. Gli oli essenziali ottenuti da mandarino sono tradizionalmente usati come antidolorifici, sedativi e curativ…

Evaluation of viability PCR performance for assessing norovirus infectivity in fresh-cut vegetables and irrigation water

Norovirus (NoV) detection in food and water is mainly carried out by quantitative RT-PCR (RT-qPCR). The inability to differentiate between infectious and inactivated viruses and the resulting overestimation of viral targets is considered a major disadvantage of RT-qPCR. Initially, conventional photoactivatable dyes (i.e. propidium monoazide, PMA and ethidium monoazide, EMA) and newly developed ones (i.e. PMAxx and PEMAX) were evaluated for the discrimination between infectious and thermally inactivated NoV genogroup I (GI) and II (GII) suspensions. Results showed that PMAxx was the best photoactivatable dye to assess NoV infectivity. This procedure was further optimized in artificially inoc…

Characterization of kefir-like beverages produced from vegetable juices

The aim of this work was to develop new non-dairy fermented beverages using vegetable juices as fermentable substrates. Carrot, fennel, melon, onion, tomato and strawberry juices underwent back-slopping fermentations, carried out by water kefir microorganisms. Results indicated that lactic acid bacteria and yeasts were capable of growing in the juices tested. Melon juice registered the highest numbers of microorganisms. Almost all juices underwent a lactic fermentation. After fermentation, there was observance of a decrease of the soluble solid content and an increase of the number of volatile organic compounds. In particular, esters were present in high amounts after the fermentation, espe…

Virus entéricos humanos en alimentos: detección y métodos de inactivación

[ES] Los principales patógenos víricos que podemos adquirir ingiriendo alimentos contaminados son los norovirus, el virus de la hepatitis A y el virus de la hepatitis E que se propagan principalmente a través de la vía fecal oral. En los últimos años, la incidencia de brotes de transmisión alimentaria causados por estos patógenos ha experimentado un aumento considerable, en parte debido al comercio globalizado y a los cambios en los hábitos de consumo. Las matrices alimentarias que mayor riesgo representan para el consumidor son los moluscos bivalvos, vegetales de IV gama, frutas tipo baya y platos listos para comer. Actualmente las técnicas moleculares son las más habituales para la detecc…

Fostering the antiviral activity of green tea extract for sanitizing purposes through controlled storage conditions

Food-contact surfaces is considered an important vehicle for the indirect transmission of foodborne viral diseases with enteric viruses, especially human norovirus (HuNoV) and hepatitis A virus (HAV). The aim of the present study was to evaluate the antiviral activity of green tea extract (GTE) at room temperature as a function of pH and storage time and to relate it with changes in composition as a consequence of degradation and epimerization reactions in the storage conditions. The obtained results revealed that freshly prepared GTE was very effective in inactivating murine norovirus (MNV) and HAV at neutral and alkaline pH but was ineffective at pH 5.5. Additionally, storage of the solut…

Sanitizing food contact surfaces by the use of essential oils

Chemical sanitizers continue to be widely used by the food industry to disinfect food contact surfaces. However, as some chemical disinfectants have been reported to produce unhealthy by-products, alternative and natural compounds need to be investigated. To this end, nine essential oils (EOs) were screened to develop a natural sanitizing solution (SAN) for disinfecting food contact surfaces. Once extracted, their antimicrobial activity and chemical composition were determined. An exploratory multivariate approach was used to investigate the relationships between the chemical and microbiological data sets. Among the tested EOs, Thymbra capitata EO, containing up to 93.31% oxygenated monoter…

Metropolitan wastewater analysis for COVID-19 epidemiological surveillance

The COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a rapidly emerging pandemic which has enforced extreme containment measures worldwide. In the absence of a vaccine or efficient treatment, cost-effective epidemiological surveillance strategies are urgently needed. Here, we have used RT-qPCR for SARS-CoV-2 detection in a series of longitudinal metropolitan wastewaters samples collected from February to April 2020, during the earliest stages of the epidemic in the Region of Valencia, Spain. We were able to consistently detect SARS-CoV-2 RNA in samples taken in late February, when communicated cases in that region were only incipient. We also find…

SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area

Preprint disponible en http://hdl.handle.net/10261/209125

Monitoring Human Viral Pathogens Reveals Potential Hazard for Treated Wastewater Discharge or Reuse

Wastewater discharge to the environment or its reuse after sanitization poses a concern for public health given the risk of transmission of human viral diseases. However, estimating the viral infectivity along the wastewater cycle presents technical challenges and still remains underexplored. Recently, human-associated crAssphage has been investigated to serve as viral pathogen indicator to monitor fecal impacted water bodies, even though its assessment as biomarker for infectious enteric viruses has not been explored yet. To this end, the occurrence of potentially infectious norovirus genogroup I (GI), norovirus GII, hepatitis A virus (HAV), rotavirus A (RV), and human astrovirus (HAstV) a…

Experimental and CFD evaluation of ozone efficacy against coronavirus and enteric virus contamination on public transport surfaces

The limited information about the routes of the transmission of SARS-CoV-2 within the ongoing pandemic scenario mobilized the administration, industry and academy to develop sanitation and disinfection systems for public and private spaces. Ozone has been proposed as an effective disinfection method against enveloped and non-enveloped viruses, including viruses with similar morphology to SARS-CoV-2. Due to this efficacy, numerous gaseous and aqueous phase ozone applications have emerged potentially to inhibit virus persistence in aerosols, surfaces, and water. In this work, a numerical model, a RANS CFD model for ozone dispersion inside tram and underground coach has been developed includin…

Effect of (−)-epigallocatechin gallate at different pH conditions on enteric viruses.

Epigallocatechin gallate (EGCG), a flavonoid from green tea, is said to have extensive antimicrobial activity in a wide range of food spoilage or pathogenic fungi, yeast and bacteria. In this work, the antiviral activity of EGCG was assessed against hepatitis A virus (HAV) and murine norovirus (MNV), a human norovirus surrogate, at different temperatures, contact times and pH conditions by cell-culture methods. EGCG was effective in reducing the titers of HAV and MNV in a dose-dependent manner at neutral pH and 25 and 37 °C, while no effect was reported at 4 °C. HAV and MNV infectivity was completely removed after overnight treatment with EGCG at 2.5 mg/mL at 37 °C. Furthermore, results als…

CARATTERIZZAZIONE DEL PROFILO AROMATICO E PROVE PRELIMINARI DI INIBIZIONE MICROBICA PER L’OLIO ESSENZIALE ESTRATTO DALLE BUCCE DI CITRUS RETICULATA cv. “TARDIVO DI CIACULLI”

Il mandarino denominato “Tardivo di Ciaculli” ha avuto origine e viene prodotto in provincia di Palermo (Sicilia) nei pressi della contrada Ciaculli. Ha un’epoca di maturazione tardiva che si concentra nei mesi da febbraio ad aprile. I frutti sono piccoli e con buccia di medio spessore [1]. In generale gli oli essenziali di mandarino sono particolarmente apprezzati per le loro proprietà chimico-fisiche costituendo un fondamentale ingrediente nell’industria cosmetica e alimentare come esaltatori di sapore in cibi e bevande. Inoltre trovano un impiego sempre maggiore nelle biotecnologie applicate al settore agroalimentare per ottimizzare le proprietà nutrizionali degli alimenti e prolungarne …

Transfer, composition and technological characterization of the lactic acid bacterial populations of the wooden vats used to produce traditional stretched cheeses

The biofilms of 12 wooden vats used for the production of the traditional stretched cheeses Caciocavallo Palermitano and PDO Vastedda della valle del Belìce were investigated. Salmonella spp. and Listeria monocytogenes were never detected. Total coliforms were at low numbers with Escherichia coli found only in three vats. Coagulase-positive staphylococci (CPS) were below the enumeration limit, whereas lactic acid bacteria (LAB) dominated the surfaces of all vats. In general, the dominance was showed by coccus LAB. Enterococci were estimated at high numbers, but usually between 1 and 2 Log cycles lower than other LAB. LAB populations were investigated at species and strain level and for thei…

Comparing analytical methods to detect SARS-CoV-2 in wastewater

Wastewater based epidemiology (WBE) has emerged as a reliable strategy to assess the coronavirus disease 2019 (COVID-19) pandemic. Recent publications suggest that SARS-CoV-2 detection in wastewater is technically feasible; however, many different protocols are available and most of the methods applied have not been properly validated. To this end, different procedures to concentrate and extract inactivated SARS-CoV-2 and surrogates were initially evaluated. Urban wastewater seeded with gamma-irradiated SARS-CoV-2, porcine epidemic diarrhea virus (PEDV), and mengovirus (MgV) was used to test the concentration efficiency of an aluminum-based adsorption-precipitation method and a polyethylene…

Attività antifungina e composizione chimica di oli essenziali estratti da agrumi coltivati in Sicilia.

Risulta comprovata nell’industria agroalimentare l’efficacia di oli essenziali di agrumi quale tecnologia per esaltare la sapidità di cibi e bevande. Ad oggi, il rinnovato interesse verso gli oli essenziali deriva dalle loro proprietà antibatteriche ed antifungine, che possono trovare ampia applicazione in sistemi hurdle-technology ai fini di prolungare la shelf-life di agroprodotti, specie in cibi minimally-processed e di IV gamma. Infatti, la necessità di ricercare nuove sostanze non di sintesi con proprietà antimicrobiche deriva dai cambiamenti legislativi in campo alimentare, dalla tendenza dei consumatori ad acquistare cibi processati naturalmente e dalla crescente resistenza dei patog…

Seasonal variations of antimicrobial activity and chemical composition of Citrus limon L. Burm. spp. essential oils

The interest towards the application of essential oils (EOs) is increasing due to the antimicrobial potential to be used as “natural” alternative to the chemical preservation of foods [1]. EOs are aromatic oily liquids produced as secondary metabolites in various plants exerting different biological properties, such as antibacterial, antiviral, antimycotic and insecticidal. EOs are complex mixtures of lipophilic substances and the chemical composition, thereby the biological activities, is influenced by the raw plant material (genotype, part of the plant, harvest time, geographical, ecological conditions and cultural techniques) and extraction method [2].

Platinum chloride-based viability RT-qPCR for SARS-CoV-2 detection in complex samples.

Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned. Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples. We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets. A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, stool, and urine. Ou…

Development of new non-dairy beverages from Mediterranean fruit juices fermented with water kefir microorganisms

The aim of this work was to explore the use of several Mediterranean fruit juices as fermentable substrates to develop new non-dairy fermented beverages. Microbiological, chemical and sensory features of kefir-like beverages obtained after the fermentation of juices extracted from fruits cultivated in Sicily (southern Italy) with water kefir microorganisms were investigated. Results indicated that both lactic acid bacteria and yeasts were able to develop in the fruit juices tested, but the highest levels were registered with prickly pear fruit juice. All fruit juices underwent a lactic fermentation, since a lactic acid content was detected in the resulting kefir-like beverages. Except kiwif…

Viability RT-PCR for SARS-CoV-2: a step forward to solve the infectivity quandary

Background Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned. Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples. Methods We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets. A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, s…

Lactococcal 949 group phages recognize a carbohydrate receptor on the host cell surface

ABSTRACT Lactococcal bacteriophages represent one of the leading causes of dairy fermentation failure and product inconsistencies. A new member of the lactococcal 949 phage group, named WRP3, was isolated from cheese whey from a Sicilian factory in 2011. The genome sequence of this phage was determined, and it constitutes the largest lactococcal phage genome currently known, at 130,008 bp. Detailed bioinformatic analysis of the genomic region encoding the presumed initiator complex and baseplate of WRP3 has aided in the functional assignment of several open reading frames (ORFs), particularly that for the receptor binding protein required for host recognition. Furthermore, we demonstrate th…

Norovirus: The Burden of the Unknown

Human noroviruses (HNoVs) are primarily transmitted by the fecal-oral route, either by person-to-person contact, or by ingestion of contaminated food or water as well as by aerosolization. Moreover, HNoVs significantly contribute to foodborne diseases being the causative agent of one-fifth of acute gastroenteritis worldwide. As a consequence of globalization, transnational outbreaks of foodborne infections are reported with increasing frequency. Therefore, in this review, state-of-the-art information regarding molecular procedures for human norovirus detection in food as well common food processing technologies have been summarized. Besides, the purpose of this chapter is to consolidate bas…

Antiviral activity of alginate-oleic acid based coatings incorporating green tea extract on strawberries and raspberries

Foodborne illnesses caused by the consumption of berries contaminated with human enteric viruses, namely human noroviruses (NoVs) and the hepatitis A virus (HAV), remain a significant food safety concern. The objective of this research was to investigate a food-grade edible coating composed of alginate/oleic and containing green tea extract (GTE) as an antiviral agent for the preservation of fresh strawberries and raspberries. Berries were stored at ambient (25 °C) temperature and refrigerated (10 °C) conditions. Initially, the effect of the pH of the film-forming dispersions (FFD) on their antioxidant and antiviral activity was analysed. Then, the physicochemical properties of edible algin…

Polymers and Biopolymers with Antiviral Activity: Potential Applications for Improving Food Safety

Gastroenteritis and hepatitis, caused by human noroviruses (HuNoVs) and hepatitis A virus (HAV), respectively, are the most common illnesses resulting from the consumption of food contaminated with human enteric viruses. Food-grade polymers can be tailor designed to improve food safety, either as novel food-packaging materials imparting active antimicrobial properties, applied in food contact surfaces to avoid cross-contamination, or as edible coatings to increase fresh produce's shelf life. The incorporation of antimicrobial agents into food-grade polymers can be used to control the food microbiota and even target specific foodborne pathogens to improve microbiological food safety and to e…

Hepatitis E virus in lettuce and water samples: A method-comparison study

The hepatitis E virus (HEV), which is an increasing cause of acute viral hepatitis in Europe, is a zoonotic virus that is mainly transmitted through contaminated water, consumption of raw or undercooked meat from pigs or wild boar, blood transfusion, and organ transplantation. Although the role of HEV transmission through contaminated produce has not been confirmed, the presence of HEV has been reported in irrigation waters and in vegetables. The present study used a World Health Organization (WHO) international standard and clinical samples to evaluate the performance characteristics of three RT-qPCR assays for detection and quantification of HEV. Two of the evaluated assays provided good …

Effect of green tea extract on enteric viruses and its application as natural sanitizer

In this work, the effect of green tea extract (GTE) was assessed against murine norovirus (MNV) and hepatitis A virus (HAV) at different temperatures, exposure times and pH conditions. Initially, GTE at 0.5 and 5 mg/ml were individually mixed with each virus at 5 log TCID50/ml and incubated 2 h at 37 °C at different pHs (from 5.5 to 8.5). GTE affected both viruses depending on pH with higher reductions observed in alkaline conditions. Secondly, different concentrations of GTE (0.5 and 5 mg/ml) were mixed with viral suspensions and incubated for 2 or 16 h at 4, 25 and 37 °C at pH 7.2. A concentration-, temperature- and exposure time-dependent response was showed by GTE in suspension tests, w…

Hepatitis A infections from food.

Food contaminated by hepatitis A virus (HAV) is responsible of the 2–7% of all HAV outbreaks worldwide. This review provides a description of the HAV characteristics, its infectivity and epidemiological features. In addition, this review compiles existing original papers reporting HAV prevalence, viral titres in foodstuffs and the risk associated with food contamination. The purpose of this revision is to conduct a structured and systematic review of the published molecular procedures for HAV detection in food, including the assessment of its infectivity.

Spatial and temporal distribution of SARS-CoV-2 diversity circulating in wastewater

Wastewater-based epidemiology (WBE) has proven to be an effective tool for epidemiological surveillance of SARS-CoV-2 during the current COVID-19 pandemic. Furthermore, combining WBE together with high-throughput sequencing techniques can be useful for the analysis of SARS-CoV-2 viral diversity present in a given sample. The present study focuses on the genomic analysis of SARS-CoV-2 in 76 sewage samples collected during the three epidemiological waves that occurred in Spain from 14 wastewater treatment plants distributed throughout the country. The results obtained demonstrate that the metagenomic analysis of SARS-CoV-2 in wastewater allows the detection of mutations that define the B.1.1.…

Human Intestinal Enteroids to Evaluate Human Norovirus GII.4 Inactivation by Aged-Green Tea

Human noroviruses are the leading cause of epidemic and sporadic acute gastroenteritis worldwide and the most common cause of foodborne illness in the United States. Several natural compounds, such as aged-green tea extract (aged-GTE), have been suggested as ingestible antiviral agents against human norovirus based on data using murine norovirus and feline calicivirus as surrogates. However, in vitro data showing their effectiveness against infectious human norovirus are lacking. We tested the activity of aged-GTE to inhibit human norovirus in a human intestinal enteroids (HIEs) model and Tulane virus in LLC-monkey kidney (LLC-MK2) cell culture. HIE monolayers pretreated with aged-GTE at di…

Essential oils as multi-target compounds for novel food safety strategies

This research work has been developed in the context of the potencial applications of essential oils as multi-target compounds for novel food safety strategies, such as active packaging technologies. Essential oils are aromatic oils derived from plants and usually extracted by hydrodistillation when intended to be used in food. There is an increasing interest in the antimicrobial properties of essential oils due to the possibility of using them to replace ‘synthetic’ preservatives in food or, in general, to reduce viable numbers of pathogens along the food chain.

Assessment of ISO Method 15216 to Quantify Hepatitis E Virus in Bottled Water

Hepatitis E virus (HEV) is one of the causative agents of water-borne human viral hepatitis and considered in Europe an emerging zoonotic pathogen. Analysis of bottled water through a standard method validated for HEV can contribute towards the risk management of this hazard. Putting some recent reports by the European Food Safety Authority in place, this study aimed to assess the performance of the concentration and extraction procedures described in ISO 15216-1:2017 for norovirus and hepatitis A virus on HEV detection. Following the ISO recommendation, the bottled water samples were spiked using serially diluted HEV fecal suspensions together with mengovirus as process control and concent…

Rapid Selective Detection of Potentially Infectious Porcine Epidemic Diarrhea Coronavirus Exposed to Heat Treatments Using Viability RT-qPCR

Coronaviruses (CoVs) cause severe respiratory, enteric, and systemic infections in a wide range of hosts, including humans and animals. Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of porcine epidemic diarrhea (PED), a highly contagious intestinal disease affecting pigs of all ages. In this study, we optimized a viability real-time reverse transcriptase polymerase chain reaction (RT-qPCR) for the selective detection of infectious and heat-inactivated PEDV. PEMAX™, EMA™, and PMAxx™ photoactivable dyes along with PtCl4 and CDDP platinum compounds were screened as viability markers using two RT-qPCR assays: firstly, on PEDV purified RNA…

Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples

Shellfish contamination by human noroviruses (HuNoVs) is a serious health and economic problem. Recently an ISO procedure based on RT-qPCR for the quantitative detection of HuNoVs in shellfish has been issued, but these procedures cannot discriminate between inactivated and potentially infectious viruses. The aim of the present study was to optimize a pretreatment using PMAxx to better discriminate between intact and heat-treated HuNoVs in shellfish and sewage. To this end, the optimal conditions (30 min incubation with 100 μM of PMAxx and 0.5% of Triton, and double photoactivation) were applied to mussels, oysters and cockles artificially inoculated with thermally-inactivated (99 °C for 5 …

Antiviral activity of aged green tea extract in model food systems and under gastric conditions.

Aged-green tea extract (GTE) is known to reduce the infectivity of hepatitis A virus (HAV) and murine norovirus (MNV), a human norovirus surrogate, in vitro and in washing solutions. Initially, the effect of aged-GTE was evaluated on virus like particles (VLPs) of human norovirus (HuNoV) genogroup I (GI) by a porcine gastric mucine (PGM)-enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM), and on HuNoV GI suspensions by an in situ capture-RT-qPCR method, suggesting that HuNoVs are very sensitive to aged-GTE treatment at 37 °C. Moreover, the potential application of aged-GTE was evaluated using model foods and simulated gastric conditions. Then, aged-GTE samp…

Jaminaea phylloscopi sp. nov. (Microstromatales), a basidiomycetous yeast isolated from migratory birds in the Mediterranean basin.

During a survey of yeasts vectored by migratory birds in the Mediterranean basin, isolations from the cloacae of members of the order Passeriformes collected in Ustica (Italy) were performed. Based on phylogenetic analysis of the D1/D2 domain of the 26S rRNA gene and the internal transcribed spacer ITS1-5.8S rRNA gene-ITS2 region, five yeast isolates clustered in a new lineage within the Microstromatales clade. The DNA sequences of these isolates differed from those of their closest relatives, Jaminaea angkorensis and Jaminaea lanaiensis, by 20 and 25 nt substitutions in the D1/D2 domain and 119 and 131 nt substitutions in the complete ITS region, respectively. In addition, the five isolate…

Recovering coronavirus from large volumes of water

The need for monitoring tools to better control the ongoing coronavirus disease (COVID-19) pandemic is extremely urgent and the contamination of water resources by excreted viral particles poses alarming questions to be answered. As a first step to overcome technical limitations in monitoring SARS-CoV-2 along the water cycle, we assessed the analytical performance of a dead end hollow fiber ultrafiltration coupled to different options for secondary concentrations to concentrate viral particles from large volume of spiked tap water, seawater and surface water together with two quantitative RT-qPCR detection kits. Spiking the porcine epidemic diarrhea virus (PEDV), an enveloped virus surrogat…

Discrimination of non-infectious SARS-CoV-2 particles from fomites by viability RT-qPCR.

The ongoing coronavirus 2019 (COVID-19) pandemic constitutes a concerning global threat to public health and economy. In the midst of this pandemic scenario, the role of environment-to-human COVID-19 spread is still a matter of debate because mixed results have been reported concerning SARS-CoV-2 stability on high-touch surfaces in real-life scenarios. Up to now, no alternative and accessible procedures for cell culture have been applied to evaluate SARS-CoV-2 infectivity on fomites. Several strategies based on viral capsid integrity have latterly been developed using viability markers to selectively remove false-positive qPCR signals resulting from free nucleic acids and damaged viruses. T…

Improving efficiency of viability-qPCR for selective detection of infectious HAV in food and water samples.

Aim: To improve the efficacy of intercalating dyes to distinguishing between infectious and inactivated hepatitis A virus (HAV) in food. Methods and Results: Different intercalating dyes were evaluated for the discrimination between infectious and thermally inactivated HAV suspensions combining with the RT‐qPCR proposed in the ISO 15216. Among them, PMAxx was the best dye in removing the RT‐qPCR signal from inactivated HAV. Applied to lettuce and spinach, PMAxx–Triton pretreatment resulted in complete removal of the RT‐qPCR signal from inactivated HAV. Likewise, this study demonstrates that this pretreatment is suitable for the discrimination of inactivated HAV in shellfish without further …

Antilisterial effect of citrus essential oils and their performance inedible film formulations

[EN] The antimicrobial activity of eight essential oils (EOs) extracted from the fruit peel of Citrus genotypes (orange, mandarin and lemon) was evaluated against 76 strains of Listeria monocytogenes, previously isolated from different food matrices. EOs showing the most (EO L2 and EO L8) and least (EO O3 and EO M7) effective inhibition activities were chemically characterized by gas chromatography coupled with mass spectrometry (GC/MS) to compare their composition. EO L2 and EO L8 were chosen to determine the MIC and to evaluate the cell viability of the most sensitive strains (L. monocytogenes LM35 and LM69) after 1, 2, 4 and 6 h of exposure. The effectiveness of chitosan (CH) and methylc…

Viability RT-qPCR to detect potentially infectious enteric viruses on heat-processed berries

Berries have frequently been cited as causing gastroenteritis and acute hepatitis outbreaks due to enteric virus contamination, including human norovirus and hepatitis A virus (HAV). Model experiments were performed to evaluate the potential use of viability RT-qPCR to assess the thermal inactivation of norovirus genotype I (GI), GII, and HAV on raspberries, blueberries and strawberries. Initially, two viability markers, platinum chloride and propidium monoazide (PMAxx™), were compared using thermally inactivated norovirus GI and GII suspensions. The results showed better performance of PMAxx™ pretreatment in discriminating native and inactivated viruses. Thus, the pretreatment was optimize…