Virus entériques transmissibles par voie alimentaire : détection, typage, pouvoir infectieux et nouvelles technologies

Abstract : The main enteric viruses that cause foodborne outbreaks are noroviruses genogroupe I and II (NoV GI and NoV GII) and hepatitis A virus (HAV), respectively responsible for gastroenteritis and hepatitis. They are mainly transmitted via the faecal-oral route either by person-to-person contact or by ingestion of contaminated water, raw and undercooked food, particularly shellfish, soft fruits and vegetables. Viral contamination level is often low and requires sensitive methods of detection. As most enteric viruses are not cultivable, these methods are based on viral genome detection and quantification by real time RT-PCR. Such an approach provides no information regarding virus infectivity and therefore limits viral risk assessment in public health. These thesis works aim to propose molecular methods for enteric viruses detection, quantification and typing, also to evaluate new molecular technologies contribution (as Digital PCR and PCR high throughput) for food viral diagnosis and finally to develop treatments combined with RT-qPCR to only detect genomes from infectious viral particles. A new HAV extraction from lettuce method was developed and assessed as similar to the reference method which is described in the technical specifications published in 2013 (ISO/TS 15216-1; ISO/TS 15216-2). In order to facilitate phylogenetic analysis in food microbiology, six subtype-specific RT-qPCR assays for human HAV (HAV IA, IB, IIA, IIB, IIIA, IIIB) were developed and evaluated by testing HAV contaminated clinical samples genotyping. These assays may be particularly useful for accurately tracing HAV in low-level contaminated samples such as food matrices and moreover, to allow co-infection identification in human samples and/or HAV recombinant strains. Nanofluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for NoV GI, NoV GII, and HAV genomes quantification, in presence of two process controls (mengovirus and murine norovirus) in artificially contaminated bottled water and lettuce samples. External amplification control allowed evaluating and comparing RT-qPCR and RT-dPCR assays inhibitions. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. Nanofluidic PCR Array (PCR Array) has also been used in order to propose an array able to simultaneously detect 20 enteric viruses. Similar detection limits were obtained with qPCR and dPCR but PCR Array was found less sensitive of 1 to 3 log10 (due to the weak volumes (nanolitre) of analyzed samples). Pretreatments based on the use of monoazides +/- surfactant and to do before RT-qPCR were developed for discriminating between infectious and non-infectious particles of HAV and rotavirus. They have been evaluated with thermal inactivation kinetic curves. Last and final summary in the thesis.
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Coralie Coudray-Meunier. Virus entériques transmissibles par voie alimentaire : détection, typage, pouvoir infectieux et nouvelles technologies. Médecine humaine et pathologie. AgroParisTech, 2014. Français. ⟨NNT : 2014AGPT0055⟩. ⟨tel-01166044⟩

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