An agent-based model to simulate SARS-CoV-2 contamination of surfaces and meat cuts in processing plants.

At the beginning of the COVID-19 pandemic, several contamination clusters were reported in food-processing plants in France and several countries worldwide. Therefore, a need arose to better understand viral transmission in such occupational environments from multiple perspectives: the protection of workers in hotspots of viral circulation; the prevention of supply disruption due to the closure of plants; and the prevention of cluster expansion due to exports of food products contaminated by the virus to other locations. This paper outlines a simulation-based approach (using agent-based models) to study the effects of measures taken to prevent the contamination of workers, surfaces, and food products. The model includes user-defined parameters to integrate characteristics relating to SARS-CoV-2 (variant of concern to be considered, symptom onset…), food-processing plants (dimensions, ventilation…), and other sociodemographic transmission factors based on laboratory experiments as well as industrial and epidemiological investigations. Simulations were performed for a typical meat-processing plant in different scenarios for illustration purposes. The results suggested that increasing the mask-wearing ratio led to great reductions in the probability of observing clusters of more than 25 infections. In the case of clusters, masks being worn by all workers limited the presence of contamination (defined as levels of at least 5 log10 viral RNA copies) on meat cuts at less than 0.05 % and maintained the production capacity of the plant at optimal levels. Increasing the average distance between two workers from less than 1 m to more than 2 m decreased the cluster-occurrence probability by up to 15 % as well as contamination of food products during cluster situations. The developed approach can open up several perspectives in terms of potential communication-support tools for the agri-food sector and further reuses or adaptations for other hazards and occupational environments.

Clustered Cases of Waterborne Hepatitis E Virus Infection, France.

The identification of seven cases of hepatitis E virus infection in a French rural hamlet in April 2015 led to investigations confirming the clustering and identifying the source of the infection. Laboratories and general practitioners in the area actively searched for other cases based on RT-PCR and serological tests. The environment, including water sources, was also checked for HEV RNA. Phylogenetic analyses were performed to compare HEV sequences. No other cases were found. Six of the seven patients lived in the same hamlet, and the seventh used to visit his family who lived there. All HEV strains were very similar and belonged to the HEV3f subgenotype, confirming the clustering of these cases. All the patients drank water from the public network. A break in the water supply to the hamlet was identified at the time the infection probably occurred; HEV RNA was also detected in a private water source that was connected to the public water network. The water flowing from the taps was quite turbid during the break. The private water supply containing HEV RNA was the likely source of the contamination. Private water supplies not disconnected from the public network are still frequent in rural areas, where they may contribute to public water pollution.

Evaluation of a Proteinase K-Based Extraction Method to Detect Hepatitis A Virus, Hepatitis E Virus and Norovirus in Artificially Contaminated Dairy Products.

Human norovirus and hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) are leading causes of foodborne disease worldwide. Among the various food products, different types of dairy products can be implicated in viral foodborne outbreaks and contamination can occur at different stages, such as preparation, contact with contaminated equipment or via other foods. The aim of this study was to characterise a proteinase K method adapted from the ISO 15216 method for the detection of HAV, HEV and norovirus in artificially contaminated dairy products, based on the recent international standard of ISO 16140-4. Results showed that the recovery yields obtained from pure RNA in dairy products ranged from 5.76% to 76.40% for HAV, from 35.09% to 100.00% for HEV, from 25.09% to 100.00% for norovirus GI and from 47.83% to 100.00% for norovirus GII. The process control MNV-1 was detected in all RNA extracts, with recovery yields between 36.83% and 100.00%. The limit of detection (LOD) of the method was between 184 and 642 genome copies/mL (or/g) for the LOD50 and 802 and 2800 genome copies/mL or/g for the LOD95 according to the virus analysed. This method proved to be suitable for detecting viruses in dairy products for routine diagnostic needs.

First Expert Elicitation of Knowledge on Possible Drivers of Observed Increasing Human Cases of Tick-Borne Encephalitis in Europe.

Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over the past years in Europe as well as the emergence of the disease in new areas. To better understand this phenomenon, we investigated the drivers of TBE emergence and increase in incidence in humans through an expert knowledge elicitation. We listed 59 possible drivers grouped in eight domains and elicited forty European experts to: (i) allocate a score per driver, (ii) weight this score within each domain, and (iii) weight the different domains and attribute an uncertainty level per domain. An overall weighted score per driver was calculated, and drivers with comparable scores were grouped into three terminal nodes using a regression tree analysis. The drivers with the highest scores were: (i) changes in human behavior/activities; (ii) changes in eating habits or consumer demand; (iii) changes in the landscape; (iv) influence of humidity on the survival and transmission of the pathogen; (v) difficulty to control reservoir(s) and/or vector(s); (vi) influence of temperature on virus survival and transmission; (vii) number of wildlife compartments/groups acting as reservoirs or amplifying hosts; (viii) increase of autochthonous wild mammals; and (ix) number of tick species vectors and their distribution. Our results support researchers in prioritizing studies targeting the most relevant drivers of emergence and increasing TBE incidence.

Development of an Extraction Method to Detect Hepatitis A Virus, Hepatitis E Virus, and Noroviruses in Fish Products.

Viruses are a leading cause of foodborne disease worldwide. Hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) and human norovirus are recognized as the main viruses of public health concern in food hygiene. ISO 15216 approved procedures are not validated for detection of HAV and human norovirus in foodstuffs, such as fishes, leading to an inability to ensure the safety of these products. This study aimed to provide a rapid and sensitive method for detecting these targets in fish products. An existing method that includes proteinase K treatment was selected for further validation using artificially contaminated fish products, according to the recent international standard ISO 16140-4. Recovery efficiencies in pure RNA extracts of viruses ranged from 0.2% to 66.2% for HAV, 4.0% to 100.0% for HEV, 2.2% to 100.0% for norovirus GI, and 0.2% to 12.5% for norovirus GII. LOD50 values were between 144 and 8.4 × 104 genome copies/g for HAV and HEV, and 104 and 2.0 × 103 copies/g for norovirus GI and norovirus GII, respectively. LOD95 values were between 3.2 × 103 and 3.6 × 105 genome copies/g for HAV and HEV, and between 8.8 × 103 and 4.4 × 104 genome copies/g for norovirus GI and norovirus GII, respectively. The method developed here was successfully validated in various fish products and can be applied for routine diagnostic needs.

Risk of Monkeypox virus (MPXV) transmission through the handling and consumption of food.

Monkeypox (MPX) is a zoonotic infectious disease caused by Monkeypox virus (MPXV), an enveloped DNA virus belonging to the Poxviridae family and the Orthopoxvirus genus. Since early May 2022, a growing number of human cases of Monkeypox have been reported in non-endemic countries, with no history of contact with animals imported from endemic and enzootic areas, or travel to an area where the virus usually circulated before May 2022. This qualitative risk assessment aimed to investigate the probability that MPXV transmission occurs through food during its handling and consumption. The risk assessment used "top-down" (based on epidemiological data) and "bottom-up" (following the agent through the food chain to assess the risk of foodborne transmission to human) approaches, which were combined. The "top-down" approach first concluded that bushmeat was the only food suspected as a source of contamination in recorded cases of MPXV, by contact or ingestion. The "bottom-up" approach then evaluated the chain of events required for a human to become ill after handling or consuming food. This approach involves several conditions: (i) the food must be contaminated with MPXV (naturally, by an infected handler or after contact with a contaminated surface); (ii) the food must contain viable virus when it reaches the handler or consumer; (iii) the person must be exposed to the virus and; (iv) the person must be infected after exposure. Throughout the risk assessment, some data gaps were identified and highlighted. The conclusions of the top-down and bottom-up approaches are consistent and suggest that the risk of transmission of MPXV through food is hypothetical and that such an occurrence was never reported. In case of contamination, cooking (e.g., 12 min at 70°C) could be considered effective in inactivating Poxviridae in foods. Recommendations for risk management are proposed. To our knowledge, this is the first risk assessment performed on foodborne transmission of MPXV.

Database of SARS-CoV-2 and coronaviruses kinetics relevant for assessing persistence in food processing plants.

SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), a virus causing severe acute respiratory disease in humans, emerged in late 2019. This respiratory virus can spread via aerosols, fomites, contaminated hands or surfaces as for other coronaviruses. Studying their persistence under different environmental conditions represents a key step for better understanding the virus transmission. This work aimed to present a reproducible procedure for collecting data of stability and inactivation kinetics from the scientific literature. The aim was to identify data useful for characterizing the persistence of viruses in the food production plants. As a result, a large dataset related to persistence on matrices or in liquid media under different environmental conditions is presented. This procedure, combining bibliographic survey, data digitalization techniques and predictive microbiological modelling, identified 65 research articles providing 455 coronaviruses kinetics. A ranking step as well as a technical validation with a Gage Repeatability & Reproducibility process were performed to check the quality of the kinetics. All data were deposited in public repositories for future uses by other researchers.

Method for detecting norovirus, hepatitis A and hepatitis E viruses in tap and bottled drinking water.

Viruses are a leading cause of foodborne disease worldwide. Human norovirus and hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) are recognised to be the main viruses of importance to public health. The ISO 15216 procedure describes molecular methods for detecting HAV and norovirus in bottled water by using an electropositive filter to concentrate viruses. The aim of this study was to validate the Zeta Plus 1MDS membrane (1MDS) for detecting enteric viruses from tap and bottled water using the recent international standard ISO/DIS/16140-4:2018, which describes the protocol for validating methods for microbiology in the food chain. Method with direct lysis of viruses from the 1MDS filter, and RNA extraction was used for detecting noroviruses, HAV and HEV from different tap and bottled drinking water. By taking into account virus's inoculation levels above the LOD, the recovery rates of noroviruses and HAV obtained from pure RNA extracts ranged from 2.50% to 14.31% and for HEV from 27.87% to 53.54% according to the water samples analysed. The virus recovery rates did not differ according to the operator or drinking water analysed but did according to the virus inoculated. The LOD95 values were respectively 50 genome copies/mL for HAV and 2.8 genome copies/mL for HEV, 420 genome copies/mL for norovirus GI and 134 genome copies/mL of water sample for norovirus GII. LOQs were determined for HAV and HEV by the total error approach and were 15.8 genome copies/mL for HAV and 2.8 genome copies/mL of water sample for HEV. The described method could be used for detecting viruses from tap and bottled water for routine diagnosis needs.

A One-Health Approach to Investigating an Outbreak of Alimentary Tick-Borne Encephalitis in a Non-endemic Area in France (Ain, Eastern France): A Longitudinal Serological Study in Livestock, Detection in Ticks, and the First Tick-Borne Encephalitis Virus Isolation and Molecular Characterisation.

Tick-borne encephalitis virus' (TBEV) geographic range and the human incidence are increasing throughout Europe, putting a number of non-endemic regions and countries at risk of outbreaks. In spring 2020, there was an outbreak of tick-born encephalitis (TBE) in Ain, Eastern France, where the virus had never been detected before. All patients but one had consumed traditional unpasteurised raw goat cheese from a local producer. We conducted an investigation in the suspected farm using an integrative One Health approach. Our methodology included (i) the detection of virus in cheese and milk products, (ii) serological testing of all animals in the suspected farm and surrounding farms, (iii) an analysis of the landscape and localisation of wooded area, (iv) the capture of questing ticks and small mammals for virus detection and estimating enzootic hazard, and (v) virus isolation and genome sequencing. This approach allowed us to confirm the alimentary origin of the TBE outbreak and witness in real-time the seroconversion of recently exposed individuals and excretion of virus in goat milk. In addition, we identified a wooded focus area where and around which there is a risk of TBEV exposure. We provide the first TBEV isolate responsible for the first alimentary-transmitted TBE in France, obtained its full-length genome sequence, and found that it belongs to the European subtype of TBEV. TBEV is now a notifiable human disease in France, which should facilitate surveillance of its incidence and distribution throughout France.

Method for tick-borne encephalitis virus detection in raw milk products.

The transmission of tick-borne encephalitis virus (TBEV) through food is rare, but can occur through the consumption of raw milk products from animals infected by tick bites. In 2020, France faced a TBEV outbreak linked to the consumption of unpasteurized goat cheese. The aim of this study was to develop and characterize a molecular method for the detection of TBEV in raw milk products based on the recent international standard PR ISO/DIS 16140-4. The TBEV recovery rates varied with the inoculation level and settings. The LOD50 and LOD95 of TBEV were 6.40 × 103 genome copies per g or per mL and 2.84 × 104 genome copies per g or per mL, respectively. The percentages of RT-qPCR inhibitions were lower than 75% and the murine norovirus (MNV-1), used as process control, was detected in all samples with a recovery rate higher than 1%, as recommended in ISO 15216. We conclude that the described method is appropriate to detect TBEV in raw milk products for routine diagnosis, and to assess potential health risks.

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. Our results were finally validated in nasopharyngeal swabs from persons who tested positive for COVID-19 and in wastewater samples positive for SARS-CoV-2 RNA. We established a rapid viability RT-qPCR that selectively detects potentially infectious SARS-CoV-2 particles in complex matrices. In particular, the confirmed positivity of nasopharyngeal swabs following the viability procedure suggests their potential infectivity, while the complete prevention of amplification in wastewater indicated either non-infectious particles or free RNA. The viability RT-qPCR approach provides a more accurate ascertainment of the infectious viruses detection and it may complement analyses to foster risk-based investigations for the prevention and control of new or re-occurring outbreaks with a broad application spectrum.

Detection of norovirus, hepatitis A and hepatitis E viruses in multicomponent foodstuffs.

Among the enteric viruses implicated in foodborne outbreaks, the human norovirus and hepatitis viruses A and E (HAV and HEV) represent a serious public health concern. International standard ISO 15216 proposes methods for detecting HAV and norovirus (genogroups I and II) RNA from soft fruit, leaf, stem and bulb vegetables, bottled water or food surfaces. These methods had not previously been validated for detecting the targeted viruses in other foodstuffs such as multicomponent foods, nor for detecting other viruses in foodstuffs. The aim of this study was to characterise a method derived from the vegetable method described in ISO 15216 to detect HAV, HEV and norovirus in artificially-contaminated multicomponent foodstuffs according to the recent international standard ISO 16140-4. Results showed that the mean recovery rates for all settings did not differ according to the operator. The mean extraction yields ranged from 0.35% to 40.44% for HAV, 5.19% to 100% for HEV, 0.10% to 40.61% for norovirus GI and 0.88% to 69.16% for norovirus GII. The LOD95 was 102 genome copies/g for HAV, HEV and norovirus GII and 103 genome copies/g for norovirus GI. The LOQ was 2.90 × 104, 1.40 × 103, 1.60 × 104 and 1.30 × 104 genome copies/g for HAV, HEV, norovirus GI and norovirus GII respectively. The MNV-1 process control was detected in 120 out of 128 RNA extracts analysed and was recovered with an efficiency of between 3.83% and 50.22%. The mean inhibition rates of quantitative real-time RT-PCR reaction ranged from 3.25% to 28.70% and varied significantly with the type of food matrix. The described method could be used to detect viruses in composite food products for routine diagnosis needs.

Modeling the Inactivation of Viruses from the Coronaviridae Family in Response to Temperature and Relative Humidity in Suspensions or on Surfaces.

Temperature and relative humidity are major factors determining virus inactivation in the environment. This article reviews inactivation data regarding coronaviruses on surfaces and in liquids from published studies and develops secondary models to predict coronaviruses inactivation as a function of temperature and relative humidity. A total of 102 D values (i.e., the time to obtain a log10 reduction of virus infectivity), including values for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were collected from 26 published studies. The values obtained from the different coronaviruses and studies were found to be generally consistent. Five different models were fitted to the global data set of D values. The most appropriate model considered temperature and relative humidity. A spreadsheet predicting the inactivation of coronaviruses and the associated uncertainty is presented and can be used to predict virus inactivation for untested temperatures, time points, or any coronavirus strains belonging to Alphacoronavirus and Betacoronavirus genera.IMPORTANCE The prediction of the persistence of SARS-CoV-2 on fomites is essential in investigating the importance of contact transmission. This study collects available information on inactivation kinetics of coronaviruses in both solid and liquid fomites and creates a mathematical model for the impact of temperature and relative humidity on virus persistence. The predictions of the model can support more robust decision-making and could be useful in various public health contexts. A calculator for the natural clearance of SARS-CoV-2 depending on temperature and relative humidity could be a valuable operational tool for public authorities.

Evaluation of three different filters and two methods for recovering viruses from drinking water.

Among the enteric viruses implicated in waterborne outbreaks, human norovirus and hepatitis A virus (HAV) are a serious public health issue. Most foodborne viruses are difficult or currently unlikely to cultivate. Because of the lack of a cell culture method, real-time reverse transcriptase PCR is commonly used for the detection of norovirus in foodstuffs and environmental samples. Due to low infectious doses in humans and low virus concentration in water sample, filter adsorption methods were used for concentrating viruses from water. The ISO (Anonymous, ISO 15216-1, 2017) describes standardized molecular methods for detecting HAV and norovirus in bottled water. This method includes a two-step procedure: concentrating the virus using a microporous electropositive filter (47 mm diameter, 0.45 µm pore size) then molecular detection. The Zetapor filter, which had a charged membrane with a pore size of 0.45 µm, was commonly used in the past to concentrate viruses from water or from salad leaves following virus elution. But, unfortunately, the Zetapor filter is no longer marketed and it is therefore necessary to assess an alternative filter. The aim of this study was to compare the ability of two electropositive filters with a pore size of 0.45 µm or 0.22 µm and one uncharged filter (0.45 µm) to recover norovirus and HAV from two different types of drinking water (bottled water and tap water) with the adsorption-elution method proposed by ISO (Anonymous, ISO 15216-1, 2017) (method A) and with direct viral extraction using filters (method B). The mean extraction yields for norovirus and HAV calculated with RNA extracts ranged from 0.2 % - 4.81 % with method A and from 5.05 % - 53.58 % with method B, and did not differ significantly between the two types of drinking water tested. For method B, the mean extraction yields for HAV and norovirus were evaluated according to results from the three filters used. The recovery rate of HAV and norovirus ranged between 3.47 % and 62.41 % with the 0.45 µm electropositive filter and were higher than the other filters. The 0.45 µm electropositive filter could be used to concentrate viruses for routine viral monitoring of drinking water for researchers who want to adopt the method in their lab routine.

Virological analyses in collective catering outbreaks in France between 2012 and 2017.

Enteric viruses cause the majority of foodborne illnesses and common symptoms of many foodborne illnesses include vomiting, diarrhea, abdominal pain, and fever. Among the enteric viruses, human Norovirus (NoV) and hepatitis virus (HAV and HEV) are the main viruses suspected to cause foodborne outbreaks and represent a serious public health. The study presents survey tools of viruses in a wide variety of foodstuffs and results obtained during 56 foodborne outbreaks investigation in France between 2012 and 2017. 246 suspected foods were examined for the presence of four human enteric viruses (NoV GI and NoV GII, HAV or HEV) either using methods described in the EN ISO 15216-1 or in house methods. All viral analysis of food samples were performed with the implementation of process control and an external amplification controls. Eighteen of 56 foodborne outbreaks investigated included at least one positive food sample (16/18 NoV, 1/18 HAV and 1/18 HEV). The genomic levels of four viruses detected ranged from < 102 to 107 genome copies per g or per L. This study showed the interest to develop methods for the extraction of viruses in different foodstuffs to increase the possibility to identify the association between viral illness and food consumption.

Development of an extraction method to detect enteric viruses in dressed vegetables.

Food-borne viral infections are caused mainly by noroviruses (NoV) and the hepatitis A virus (HAV), which respectively cause gastroenteritis and hepatitis. Various foods have been implicated in viral outbreaks, including vegetables that are consumed in a variety of forms, often with salad dressing. NF EN ISO procedures (15216-1:2017) propose standard methods for quantifying NoV and HAV in high-risk food categories, such as vegetables, based on viral elution and PEG concentration methods, but these methods are not suitable for composite meals like salads dressed with oily, fatty or emulsified food ingredients. The development of sensitive and reliable techniques for the detection of viruses in these products is therefore needed to ensure the safety of these products. The aim of this study was to develop an RT-qPCR based method for the detection and quantification of NoV and HAV in various vegetables with different dressings. Three methods for recovering NoV and HAV from artificially contaminated dressed vegetables were evaluated. The selected method was based on the use of Trizol reagent and, according to the type of dressing, the limit of detection ranged from 104 to 106 genome copies/g for NoV and from 102 to 103 PFU/g for HAV. The described method can be applied for detecting NoV and HAV in food containing salad dressing for routine diagnosis needs.