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

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.Les principaux virus entériques à l’origine de toxi-infections alimentaires collectives sont les norovirus génogroupes I et II (NoV GI, NoV GII) et le virus de l’hépatite A (VHA) responsables respectivement de gastro-entérites et d’hépatites. Ces virus sont transmissibles par la voie féco-orale directe ou via l’ingestion d’eaux ou d’aliments consommés crus ou peu cuits (coquillages, fruits et légumes). Le niveau de contamination virale des aliments souvent faible nécessite d’utiliser des méthodes de détection très sensibles. La plupart des virus entériques étant non cultivable, ces méthodes reposent sur la détection / quantification des génomes viraux par RT-qPCR ce qui ne permet pas de déterminer l’infectiosité des virus et limite l’appréciation du risque viral en santé publique. Les travaux de thèse visaient à proposer des méthodes moléculaires pour la détection, la quantification et le typage des virus entériques, à évaluer l’apport de nouvelles technologies moléculaires (comme la Digital RT-PCR (RT-dPCR) et la RT-PCR à haut débit) dans le cadre du diagnostic viral dans les aliments et enfin à développer des traitements précédant les réactions de RT-qPCR pour détecter des génomes issus de particules virales infectieuses. Une nouvelle technique d’extraction du VHA à partir de la laitue a été développée et évaluée équivalente à la technique de référence décrite dans les spécifications techniques publiées en 2013 (ISO/TS 15216-1 et 15216-2). Pour favoriser les études phylogénétiques dans le domaine alimentaire, 6 modèles moléculaires de RT-qPCR spécifiques des 6 sous-types humains du VHA (IA, IB, IIA, IIB, IIIA, IIIB) ont été développés et évalués pour le génotypage d’échantillons cliniques contaminés par le VHA. Ils peuvent être utiles pour tracer les sous-types du VHA dans des échantillons faiblement contaminés comme des matrices alimentaires, mais aussi permettre l'identification de co-infection de l'homme ou de souches de VHA recombinantes. La RT-dPCR en nanofluidique a été comparée à la RT-qPCR pour la quantification des génomes de NoV GI, NoV GII et VHA en présence de 2 contrôles de process (mengovirus et norovirus murin) dans des échantillons de laitues et d’eau embouteillée artificiellement contaminés. Un contrôle externe d’amplification a permis d’évaluer et de comparer l’inhibition des réactions de RT-qPCR et RT-dPCR. Les rendements d’extraction viraux se sont révélés significativement plus élevés après RT-dPCR qu’après RT-qPCR pour les NoV GI et mengovirus dans l'eau et pour les NoV GII et VHA dans les échantillons de laitue. De plus, les essais de RT-dPCR se sont avérés être plus tolérants à la présence de substances inhibitrices issues de laitues. La technologie qPCR en nanofluidique a également été utilisée afin de proposer une « puce » capable de détecter 20 virus entériques. Des limites de détection similaires ont été obtenues avec la qPCR et la dPCR. La qPCR nanofluidique a été trouvé moins sensible d’environ 1 à 3 log10 (du fait des faibles volumes (~nanolitre) d’échantillons analysés). Des prétraitements à base de monoazide +/- détergent à réaliser avant la RT-qPCR pour la détection de virus infectieux (VHA, rotavirus) ont été développés et évalués en réalisant des cinétiques d’inactivations thermiques. [...] Suite et fin du résumé dans la thèse

Foodborne enteric viruses : detecting, typing, infectivity and new technologies

Les principaux virus entériques à l’origine de toxi-infections alimentaires collectives sont les norovirus génogroupes I et II (NoV GI, NoV GII) et le virus de l’hépatite A (VHA) responsables respectivement de gastro-entérites et d’hépatites. Ces virus sont transmissibles par la voie féco-orale directe ou via l’ingestion d’eaux ou d’aliments consommés crus ou peu cuits (coquillages, fruits et légumes). Le niveau de contamination virale des aliments souvent faible nécessite d’utiliser des méthodes de détection très sensibles. La plupart des virus entériques étant non cultivable, ces méthodes reposent sur la détection / quantification des génomes viraux par RT-qPCR ce qui ne permet pas de déterminer l’infectiosité des virus et limite l’appréciation du risque viral en santé publique. Les travaux de thèse visaient à proposer des méthodes moléculaires pour la détection, la quantification et le typage des virus entériques, à évaluer l’apport de nouvelles technologies moléculaires (comme la Digital RT-PCR (RT-dPCR) et la RT-PCR à haut débit) dans le cadre du diagnostic viral dans les aliments et enfin à développer des traitements précédant les réactions de RT-qPCR pour détecter des génomes issus de particules virales infectieuses. Une nouvelle technique d’extraction du VHA à partir de la laitue a été développée et évaluée équivalente à la technique de référence décrite dans les spécifications techniques publiées en 2013 (ISO/TS 15216-1 et 15216-2). Pour favoriser les études phylogénétiques dans le domaine alimentaire, 6 modèles moléculaires de RT-qPCR spécifiques des 6 sous-types humains du VHA (IA, IB, IIA, IIB, IIIA, IIIB) ont été développés et évalués pour le génotypage d’échantillons cliniques contaminés par le VHA. Ils peuvent être utiles pour tracer les sous-types du VHA dans des échantillons faiblement contaminés comme des matrices alimentaires, mais aussi permettre l'identification de co-infection de l'homme ou de souches de VHA recombinantes. La RT-dPCR en nanofluidique a été comparée à la RT-qPCR pour la quantification des génomes de NoV GI, NoV GII et VHA en présence de 2 contrôles de process (mengovirus et norovirus murin) dans des échantillons de laitues et d’eau embouteillée artificiellement contaminés. Un contrôle externe d’amplification a permis d’évaluer et de comparer l’inhibition des réactions de RT-qPCR et RT-dPCR. Les rendements d’extraction viraux se sont révélés significativement plus élevés après RT-dPCR qu’après RT-qPCR pour les NoV GI et mengovirus dans l'eau et pour les NoV GII et VHA dans les échantillons de laitue. De plus, les essais de RT-dPCR se sont avérés être plus tolérants à la présence de substances inhibitrices issues de laitues. La technologie qPCR en nanofluidique a également été utilisée afin de proposer une « puce » capable de détecter 20 virus entériques. Des limites de détection similaires ont été obtenues avec la qPCR et la dPCR. La qPCR nanofluidique a été trouvé moins sensible d’environ 1 à 3 log10 (du fait des faibles volumes (~nanolitre) d’échantillons analysés). Des prétraitements à base de monoazide +/- détergent à réaliser avant la RT-qPCR pour la détection de virus infectieux (VHA, rotavirus) ont été développés et évalués en réalisant des cinétiques d’inactivations thermiques. [...] Suite et fin du résumé dans la thèse.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

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System.

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

Discrimination of infectious and heat-treated norovirus by combining platinum compounds and real-time RT-PCR

International audienceHuman noroviruses (NoV) are major agents of foodborne outbreaks. Because of the lack of a standardized cell culture method, real-time reverse transcriptase PCR is now commonly used for the detection of NoV in foodstuffs and environmental samples. However, this approach detects the viral nucleic acids of both infectious and non-infectious viruses and needs to be optimized to predict infectivity for public health risk assessment. The aim of this study was to develop a viability PCR method to discriminate between native and heat-treated virus, for both NoV and its surrogate, murine norovirus (MNV).To this end, screening of viability markers (monoazide dyes, platinum and palladium compounds) was performed on viral RNA, native virus or heat-treated virus, and incubation conditions were optimized with PtCl4, the most efficient viability marker. Multiple MNV molecular models were designed: no impact of amplicon length was observed on inactivated MNV genomic titer; but the 5′NTR, ORF1 and 3′UTR regions resulted in higher reductions than central genomic regions. The optimal viability PCR conditions developed (incubation with 2.5 mM PtCl4 in PBS for 10 min at 5 °C) were finally applied to MNV by performing heat inactivation studies and to native and heat-treated NoV clinical strains. The viability PCR discriminated efficiently between native and heat-inactivated MNV at 72 °C and 80 °C, and efficiently reduced the genomic titer of heat-treated NoV strains.This viability PCR method could be useful to study heat inactivation kinetics of NoV and MNV. It could also be evaluated for the identification of infectious enteric viruses in foodstuffs and environmental samples

GenBank accession number for viral sequences used to obtain recombinant plasmids.

<p>GenBank accession number for viral sequences used to obtain recombinant plasmids.</p

Screening and viral quantification in clinical samples (stools and viral supernatants from cell culture) by RT-qPCR and novel nanofuidic approaches (RT-qPCR and RT-dPCR).

<p>Samples were firstly screened by RT-PCR array and then quantified by RT-dPCR. Absolute viral quantification (by RT-dPCR) was compared to relative quantification (by RT-qPCR).</p

Primers and probes used in this study.

<p>(F: Forward; R: Reverse; P: Probe)</p

Comparison of RT-qPCR, RT-dPCR and RT-PCR array assays.

<p>Characteristics of standard curves based on the RT-qPCR assays and limit of detection (LOD) of viral targets by RT-qPCR, by RT-dPCR and RT-PCR array assays. The differences between relative quantification (by RT-qPCR) and absolute quantification (by RT-dPCR) were indicated.</p