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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.

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.

Hepatitis A virus (HAV) is one of the most common agents causing acute liver disease worldwide. HAV has been increasingly reported as the cause of foodborne disease outbreaks. The standard method currently available for detection of the genome of HAV in vulnerable foodstuffs is by RT-qPCR (ISO 15216). Despite its usefulness in the investigation of foodborne viruses, the use of RT-qPCR in food virology has been shown to overestimate the quantity of infectious virus or to highly underestimate the effect of the treatment on virus inactivation. The gold standard methods currently used for evaluating the efficacy of inactivation treatments on the adapted strain of HAV (HM175/18f) are either the plaque assay or the end-point dilution assay (TCID ). However, both assays are labor-intensive and time-consuming. The aim of this study was to evaluate the use of the xCELLigence real-time cell analysis (RTCA) system for detecting the infectivity of the adapted strain of HAV. Kinetics of cell impedance showed that HAV induced a decrease in cell index (CI) correlated with the onset of HAV-induced cell death. In addition, the time to which the HAV-induced CI drop occurred was dependent on the viral concentration. An inverse linear relation could be established over a range of 5 log between the concentration of HAV and the time to reach 50% of CI decrease (TCI ), showing that the RTCA assay could be used as a titration method for HAV. In addition, the RTCA-based assay could be performed in less than 6 days instead of 12 to 14 days with the gold standard methods. Therefore, the RTCA-based titration method is a powerful and suitable tool for high-throughput screening of anti-viral treatments. Its usefulness in HAV inactivation studies will improve the assessment of viral risk in food virology, as controlling transmission of viruses through their removal from foodstuffs is also an important challenge in reducing the burden of viral foodborne illnesses.

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.

Human T-cell Lymphotropic Virus type 1 (HTLV-1) is a human retrovirus that infects at least 5–10 million people worldwide, and is the etiological agent of a lymphoproliferative malignancy; Adult T-cell Leukemia/Lymphoma (ATLL); and a chronic neuromyelopathy, HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), as well as other inflammatory diseases such as infective dermatitis and uveitis. Besides sexual intercourse and intravenous transmission, HTLV-1 can also be transmitted from infected mother to child during prolonged breastfeeding. Some characteristics that are linked to mother-to-child transmission (MTCT) of HTLV-1, such as the role of proviral load, antibody titer of the infected mother, and duration of breastfeeding, have been elucidated; however, most of the mechanisms underlying HTLV-1 transmission during breast feeding remain largely unknown, such as the sites of infection and cellular targets as well as the role of milk factors. The present review focuses on the latest findings and current opinions and perspectives on MTCT of HTLV-1.

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.

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.

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.

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.Measurement(s)Decimal reduction timeTechnology Type(s)Modelling of kineticsFactor Type(s)Temperature • Relative humidity • Genus • StrainSample Characteristic - OrganismCoronaviridae