Development of a cell culture-based method for detecting infectious tick-borne encephalitis virus (TBEV) in milk products.

Tick-borne encephalitis outbreaks have been reported in Europe after consumption of raw milk products from infected animals. While molecular methods are commonly used in viral foodborne outbreak investigations due to their sensitivity, specificity and rapidity, there are very few methods to detect infectious tick-borne encephalitis virus (TBEV) in milk products for routine use/analyses. To address this gap, we developed a cell culture-based method to detect infectious TBEV in artificially contaminated raw goat milk and raw goat cheese, and evaluated the sensitivity of TBEV infectivity assays. Raw goat milk samples were spiked with TBEV to achieve inoculation levels ranging from 106 to 100 TCID50/mL, and Faisselle and Tomme cheese samples were spiked so their TBEV concentrations ranged from 9.28 × 105 to 9.28 × 101 TCID50 per 2.5g. To detect infectious TBEV, Vero cells were infected by raw goat milk. For cheese samples, after homogenisation and membrane filtration, Vero cells were infected with samples adsorbed on the filter (method A) or with samples eluted from the filter (method B). After 5 days, cytopathic effects (CPEs) were observed and TBEV replication in Vero cells was confirmed by an increase in the number of genome copies/mL that were detected in cell supernatant. Infected Vero cells exhibited CPEs for both milk and cheese samples. Infectious TBEV was detected to 103 TCID50/mL in raw milk samples and to 9.28 × 101 TCID50 from Faisselle samples using both methods A and B. For Tomme samples, method A was able to detect TBEV to 9.28 × 102 TCID50/2.5g and method B to 9.28 × 103 TCID50/2.5g. The number of positive samples detected was slightly higher with method A than with method B. To conclude, this qualitative cell culture-based method can detect infectious TBEV artificially inoculated into raw milk and cheese; it should be further evaluated during foodborne outbreak investigations to detect infectious TBEV from naturally contaminated milk and cheese.

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.

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.

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.

Evaluation of methods for elution of HEV particles in naturally contaminated sausage, figatellu and pig liver.

Foodborne transmission of HEV is a growing public health concern in industrialised countries, where the disease is mainly autochthonous, caused by zoonotic HEV of either genotype 3 or 4. Foodstuffs containing pig's liver were suspected on several occasions to be the cause of autochthonous cases of HEV infection, while the transmission was associated with animal contact and the ingestion of raw or uncooked meat, especially liver. In assessing the risk related to the presence of HEV in food, detection methods were previously developed but HEV detection rates seem to vary with the type of samples and methods. As foodstuff containing pig liver can be contaminated with HEV internally, an efficient virus extraction procedure is required. The aim of this study was to evaluate six methods for their efficiency in releasing HEV viral particles from figatelli, pig liver sausages and liver samples previously tested positive for the presence of HEV. The ratio weight to volume of elution buffer (1:5) and the FastPrep®-24 homogeniser showed to significantly improve the quantity of HEV genomes released per gram of figatelli and pig liver sausages. To our knowledge, this study is the first to evaluate several methods for elution of HEV particles from naturally contaminated pig liver products, and may be extended for quantifying other viral genomes from food of animal origin.

Comparison of three extraction methods to detect noroviruses in dairy products.

Noroviruses (NoV) are currently the most common cause of viral foodborne diseases and RT-qPCR is widely used for their detection in food because of its sensitivity, specificity and rapidity. The ISO/TS (15216-1, 15216-2) procedures for detecting NoV and HAV in high-risk food categories such as shellfish, bottled water and vegetables were published in 2013. Milk products are less implicated in foodborne viral outbreaks but they can be contaminated with fruit added to these products or by the food handler. Thus, the development of sensitive and reliable techniques for the detection of NoV in dairy products is needed to ensure the safety of these products. The aim of this study was to develop a RT-qPCR based method for the detection of NoV in milk products. Three methods were tested to recover NoV from artificially contaminated milk and cottage cheese. The selected method was based on the use of proteinase K and the recovery efficiencies ranged from 54.87% to 98.87% for NoV GI, 61.16%-96.50% for NoV GII. Murine norovirus and mengovirus were used as process controls and their recovery efficiencies were respectively 60.59% and 79.23%. The described method could be applied for detecting NoV in milk products for routine diagnosis needs.

Hepatitis A virus subgenotyping based on RT-qPCR assays.

BACKGROUND: The hepatitis A virus (HAV) is the most frequent cause of viral hepatitis worldwide and is recognized as one of the most widespread foodborne pathogens. HAV genotypes and subtypes differ in their geographic distribution and the incidence of HAV infection varies considerably among countries, and is particularly high in areas with poor sanitation and hygiene. Phylogenetic analyses are traditionally used in clinical microbiology for tracing the geographic origin of HAV strains. In food microbiology, this approach is complicated by the low contamination levels of food samples. To date, real-time reverse-transcription PCR has been one of the most promising detection methods due to its sensitivity, specificity and ability to deliver quantitative data in food samples, but it does not provide HAV subtyping information. RESULTS: Six subtype-specific RT-qPCR assays were developed for human HAV. The limit of detection of HAV was 50 genome copies/assay for subtype IIB, 500 genome copies assay for IA, IB, IIA and IIIB and 5000 genome copies/assay for IIIA. The specificity of the assays was evaluated by testing reference isolates and in vitro HAV RNA transcripts. No significant cross reactivity was observed. Subtyping results concordant with sequencing analysis were obtained from 34/35 clinical samples. Co-infection with a minor strain of a different subtype was suggested in 5 cases and a recombinant event in one case. CONCLUSIONS: These RT-qPCR assays may be particularly useful for accurately tracing HAV in low-level contaminated samples such as food matrices but also to allow co-infection identification in human samples.

Transcytosis of HTLV-1 across a tight human epithelial barrier and infection of subepithelial dendritic cells.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to blood transfusion and sexual transmission, HTLV-1 is transmitted mainly through prolonged breastfeeding, and such infection represents a major risk for the development of adult T-cell leukemia/lymphoma. Although HTLV-1-infected lymphocytes can be retrieved from maternal milk, the mechanisms of HTLV-1 transmission through the digestive tract remain unknown. In the present study, we assessed HTLV-1 transport across the epithelial barrier using an in vitro model. Our results show that the integrity of the epithelial barrier was maintained during coculture with HTLV-1-infected lymphocytes, because neither morphological nor functional alterations of the cell monolayer were observed. Enterocytes were not susceptible to HTLV-1 infection, but free infectious HTLV-1 virions could cross the epithelial barrier via a transcytosis mechanism. Such virions were able to infect productively human dendritic cells located beneath the epithelial barrier. Our data indicate that HTLV-1 crosses the tight epithelial barrier without disruption or infection of the epithelium to further infect target cells such as dendritic cells. The present study provides the first data pertaining to the mode of HTLV-1 transport across a tight epithelial barrier, as can occur during mother-to-child HTLV-1 transmission during breastfeeding.

A Useful Method to Provide Infectious and Cultivable In Vitro Naked Viral Particles of Hepatitis A Virus.

Hepatitis A virus (HAV) is an enteric virus mainly transmitted by the faecal-oral route. Belonging to the Picornaviridae family, HAV was first described as small naked particles, like all viruses of this family. However, for about a decade, it was demonstrated that HAV particles can exist surrounded by a lipid bilayer. This type of particle, called enveloped HAV (eHAV), acquires its lipid bilayer by hijacking a part of cell membranes during the virion egress in the last steps of the viral cycle. In vitro culture systems produce mainly eHAV, and so, to date, most of the studies on HAV have been carried out using this type of viral particle. In this study, a method based on lipid bilayer removal by chemical delipidation is proposed for the production of naked HAV particles. The resulting naked HAV particles conserve their infectivity and are therefore fully cultivable in vitro. By using this method, naked HAV particles can easily be produced in vitro and can be useful to perform further studies such as inactivation processes for the food industry, as HAV is a main concern for food safety.

Discrimination of infectious hepatitis A virus and rotavirus by combining dyes and surfactants with RT-qPCR.

BACKGROUND: Human enteric viruses are major agents of foodborne diseases. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, real-time reverse transcriptase PCR is now widely used for the detection of RNA viruses in food samples. However this approach detects viral nucleic acids of both infectious and non infectious viruses, which limits the impact of conclusions with regard to public health concern. The aim of the study was to develop a method to discriminate between infectious and non-infectious particles of hepatitis A virus (HAV) and two strains of rotavirus (RV) following thermal inactivation by using intercalating dyes combined with RT-qPCR. RESULTS: Once the binding of propidium monoazide (PMA) or ethidium monoazide (EMA) was shown to be effective on the viral ssRNA of HAV and dsRNA of two strains of RV (SA11 and Wa), their use in conjunction with three surfactants (IGEPAL CA-630, Tween 20, Triton X-100) prior to RT-qPCR assays was evaluated to quantify the infectious particles remaining following heat treatment. The most promising conditions were EMA (20 µM) and IGEPAL CA-630 (0.5%) for HAV, EMA (20 µM) for RV (WA) and PMA (50 µM) for RV (SA11). The effectiveness of the pre-treatment RT-qPCR developed for each virus was evaluated with three RT-qPCR assays (A, B, C) during thermal inactivation kinetics (at 37°C, 68 C, 72°C, 80°C) through comparison with data obtained by RT-qPCR and by infectious titration in cell culture. At 37°C, the quantity of virus (RV, HAV) remained constant regardless of the method used. The genomic titers following heat treatment at 68°C to 80°C became similar to the infectious titers only when a pre-treatment RT-qPCR was used. Moreover, the most effective decrease was obtained by RT-qPCR assay A or B for HAV and RT-qPCR assay B or C for RV. CONCLUSIONS: We concluded that effectiveness of the pre-treatment RT-qPCR is influenced by the viral target and by the choice of the RT-qPCR assay. Currently, it would be appropriate to further develop this approach under specific conditions of inactivation for the identification of infectious viruses in food and environmental samples.

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.

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.

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.

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.

Comparison of two extraction methods for the detection of hepatitis A virus in semi-dried tomatoes and murine norovirus as a process control by duplex RT-qPCR.

Enteric viruses are important agents of foodborne diseases. Due to their low infectious doses and low concentrations in food samples, an efficient and rapid virus concentration method is required for routine control. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, reverse transcription quantitative real-time PCR (RT-qPCR) is now widely used for the detection of RNA viruses in food samples. One of the general requirements for viral diagnosis concerns the use of a process control to monitor the efficiency of viral particle concentration, nucleic acid extraction and the presence of potential inhibitors of the RT-PCR reaction. Recent epidemiological studies have linked hepatitis A outbreaks to the consumption of semi-dried tomatoes (SDT) in Australia, the Netherlands and France. In this study, the virus concentration reference method proposed by the CEN/TC275/WG6/TAG4 working group for samples of soft fruit and salad vegetables was compared to a method including an ultracentrifugation step to recover hepatitis A virus (HAV) in SDT. Murine norovirus (MNV-1) was used as a process control and detected simultaneously with HAV in a one-step duplex RT-qPCR in both procedures. The LOD of HAV was 10 PFU and 1 PFU of HAV/25 g of SDT in the presence or absence of MNV-1 respectively, whatever the method used. We conclude that both methods achieved an identical limit of detection and that the MNV-1 offers a very reliable and simple way to monitor the quality of the extraction procedures and the presence of RT-qPCR inhibitors.