Indiana University-Purdue University Indianapolis (IUPUI) / Enteric viruses are among the most common infectious human viruses worldwide, causing an estimated 10-15 million infections per year in the United States. Among enteric viruses, Coxsackievirus is commonly isolated and can lead to the development of meningitis, encephalitis, pancreatitis, and hepatitis. Furthermore, Coxsackievirus B3 is the primary cause of viral myocarditis and can lead to pleurodynia, with nearly 40,000 symptomatic cases reported in the United States each year. The enteroviral ssRNA genome contains a 5’ untranslated region (5’UTR) which consists of two structural components, the cloverleaf and the internal ribosome entry site (IRES), both shown to be integral to viral success. Additionally, the viral genome encodes four structural VP proteins as well as 11 non-structural proteins. Polymorphisms found within the CVB3 population have been linked to viral virulence. Here, we compare two CVB3 Nancy variants to elucidate the downstream effects observed in response to mutations found in the CVB3 genome. Implementing our novel oral inoculation model, we aimed to determine the impact mutations found in the 5’UTR and VP regions exert on viral pathogenesis. We also aimed to delineate the in vitro effects of the observed mutations. We investigated the role mutations found in the structural regions played in virus host cell attachment, in vitro cell viability, and replication. Our work has further confirmed the relevance and impact of mutations found in the VP region of the CVB3 genome.

Contamination of drinking water sources, such as groundwater, by pathogens (protozoa, bacteria and viruses) is of major concern globally. Due to their small size, mobility and high infectivity, enteric viruses have been a focus of groundwater research. However, the behaviour of enteric viruses in aquifer media is still poorly understood, which is partially attributable to the lack of reliable surrogates for these viruses. In the study reported in this thesis, a new type of surrogate was characterised and validated for its use in studying virus fate and transport in groundwater. The surrogates developed were composed of 70 nm carboxylated silica nanoparticles, labelled with dsDNA tags for sensitive detection, and coated with selected proteins to mimic the physico-chemical characteristics (size, charge, density) of two enteric viruses, human rotavirus and adenovirus, frequently found in faecal-contaminated groundwater. The selected enteric viruses and a commonly used virus surrogate, the MS2 bacteriophage, were purified and characterised in terms of size, surface charge, hydrophobicity and aggregation. For validation, the characteristics, the adsorption, degradation and transport of the surface-modified nanoparticles and the viruses were investigated in laboratory studies and compared. The characterisation of the viruses and particles revealed that the modified silica nanoparticles resemble the size and negative surface charge of the rotavirus and adenovirus. In general, the nanoparticles were found to be less hydrophobic than the enteric viruses, thus presumably less interactive with hydrophobic media. In contrast, the MS2 bacteriophage was smaller in size than the enteric viruses studied and considerably more hydrophobic implying stronger interactions with hydrophobic media. The surface-modified nanoparticles were found to be more stable and remained more monodispersed over time than the purified enteric viruses. In laboratory studies using simulated groundwater, the DNA-labelled nanoparticles were more stable over time than the rotavirus, the adenovirus or a plasmid DNA on its own. Interestingly, the study revealed that rotavirus was more persistent than the adenovirus over time in terms of degradation and aggregation, however, day light considerably enhanced rotavirus degradation. The adsorption studies revealed strong interactions between the enteric viruses and natural aquifer media (gravel and sand), whereas most of the surface-modified nanoparticles adsorbed weakly to these media. Only the casein-coated nanoparticles adsorbed strongly to the sand. The MS2 adsorbed to the gravel strongly, but weakly to the sand implying different interactions. The studies on virus and nanoparticle adsorption to hydrophobic-coated and non-modified Ottawa sand supported the results of characterisation. Column studies investigating the transport of the viruses and the nanoparticles in gravel and sand showed that even though gravel had high adsorption capacity in the adsorption tests, all viruses and nanoparticles travelled though the gravel columns with little retention, probably due to insufficient interaction time. This highlights the vulnerability of gravel aquifers to virus contamination. Experiments using sand columns showed great differences in the transport of the particles. Results suggested that the recovery of the DNA-labelled nanoparticles was similar to the recovery of the adenovirus, however, their transport pattern was different. The glycoprotein-, the protein A- and the AMBP-coated nanoparticles mimicked the transport pattern and low recovery of the rotavirus. In contrast, the streptavidin- and casein-coated nanoparticles were not recovered, emphasising the great importance of surface structure in particle transport. The results of this study demonstrated the usefulness of protein-coated silica nanoparticles as virus surrogates in groundwater studies. Surface-modified nanoparticles are able to mimic the surface characteristics of viruses. The glycoprotein-, protein A- and AMBP-coated particles were found to be suitable surrogates for rotavirus, whereas the DNA-labelled nanoparticles resembled adenovirus behaviour in hydrophilic media. Using particles with different material, size and protein-coating other pathogens can be modelled as well. Furthermore, these particles are expected to besafe to humans and the environment, thus can be used in a great variety of experiments in environmental research.

To protect public health, drinking water systems are monitored for indicator organisms that correlate with fecal contamination and suggest the presence of human pathogens. Total coliforms, fecal coliforms, and E. coli are the most commonly used indicator organisms. These bacteria generally colocate with fecal pollution, but some limitations exist. In particular, the ability of indicator bacteria to predict the presence of enteric viruses is questionable because of distinct transport and survival characteristics of bacteria and viruses. Although viral indicators of enteric viruses have been proposed, none have been implemented into the current regulatory framework. In this thesis, the correlation of bacteria and viruses in drinking water sources and treatment systems is reviewed, and the potential of Torque Teno virus (TTV) to qualify as an indicator virus is discussed. TTV is unique among enteric viruses as it infects approximately 80% of healthy individuals worldwide, is transmitted by the fecal-oral route, causes no observable illness, and lacks seasonal fluctuations.

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.

Réutiliser les eaux usées en irrigation agricole aide à répondre aux besoins croissants en eau, réduit leur décharge dans les eaux conventionnelles et participe à la fertilisation des sols. Les eaux usées d'origine domestique contiennent des virus entériques de l'homme responsables d'épidémies transmises par voies hydrique et alimentaire. Leur transmission aérienne avec maladie à la clé a été mise en évidence dans d'autres contextes, mais il existe peu d’études sur le devenir de virus déposés à la surface du sol ou de végétaux. Aussi cette thèse de Doctorat avait-elle pour objectifs d'évaluer et décrire (i) l'aérosolisation des virus préalablement apportés au sol par irrigation avec des eaux usées et (ii) leur inactivation dans l'atmosphère.Pour répondre à ces objectifs, des suivis expérimentaux ont été réalisés en conditions semi-contrôlées pour l'aérosolisation (virus déposés in situ sur des placettes de sol couvertes par des tunnels) et en conditions contrôlées pour l'inactivation (virus en réacteur atmosphérique de laboratoire). La souche MC0 du mengovirus murin a été utilisée pour l'ensemble des expérimentations. Elle a été multipliée sur des cellules BGM. La teneur en ARN viral a été suivie par RT-qPCR et, pour l'étude de l'inactivation, les virus infectieux ont été simultanément dénombrés par comptage de plages de lyse sur cellules BGM. Ces suivis ont été couplés au suivi des conditions environnementales (rayonnement global, température, humidité relative de l'air, teneur en O3, auxquels s'ajoutent l'humidité et la température de surface du sol pour l'étude de l'aérosolisation). Ces travaux ont nécessité de concevoir un nouveau réacteur atmosphérique, d'évaluer les performances de biocollecteurs (Impingers et filtres), et d’améliorer le dénombrement des virus infectieux.Un modèle a été proposé pour décrire l'aérosolisation d'un ou plusieurs pools de virus aérosolisables, chacun étant caractérisé par sa taille et un coefficient cinétique d'aérosolisation. Nous l'avons utilisé pour générer des expériences numériques reproduisant la variabilité des mesures réelles, et pour ajuster à ces expériences numériques des simulations portant soit sur l'aérosolisation cumulée soit sur l'aérosolisation « instantanée ». Les ajustements sur l'aérosolisation instantanée donnent des estimations plus précises du coefficient cinétique d'aérosolisation ; il n'en va pas forcément de même pour l'estimation de la quantité de virus aérosolisables. Un modèle de dépendance du coefficient d'inactivation à l'humidité relative de l'air a été proposé.Eu égard à l'aérosolisation des virus à partir du sol, les Impingers donnent des aérosolisations estimées plus élevées que les membranes en raison de différences de piégeage et/ou d'extraction. Toutefois, ils ne piègeraient qu'environ 77 % des virus et relargueraient des virus piégés avec un coefficient de réaérosolisation de 0.11. Ces imperfections aboutissent à des estimations des quantités de virus aérosolisés environ 2 fois moins élevées qu'en réalité ; à l'inverse, elles n'affectent pas les estimations des coefficients cinétiques d'aérosolisation. Sans tenir compte de ce biais, entre 1 et 10% des virus apportés ont été aérosolisés. À notre connaissance, c'est la première mise en évidence d'un tel phénomène. On distingue un pool de virus aérosolisés quasi-instantanément (environ 1/3 des virus aérosolisés) d'un pool de virus aérosolisés de manière cinétique. Pour ce dernier pool, la constante cinétique d'aérosolisation varie entre 0.007 et 0.21 h-1 : 90 % des virus du pool cinétique sont aérosolisés au bout de respectivement 13 j et 11 h dans nos conditions. La taille du pool cinétique est bien prédite à partir de la vitesse du vent, de la température de surface du sol et de la nature de l'eau d'irrigation. / Wastewater reuse for agricultural irrigation allows coping increasing water requirements, reduces wastewater discharge in conventional water bodies, and contributes to soil fertilization. Wastewaters of domestic origin are contaminated with human enteric viruses responsible for waterborne and foodborne outbreaks. Air transmission of viruses that leads to diseases has been noted in other contexts, but nothing is known about the fate of viruses brought at the surface of the soil. The aims of this PhD thesis were to assess and describe the aerosolization of viruses previously brought to the soil surface by wastewater irrigation, and their inactivation in the atmosphere.To fulfil these objectives, experiences were performed in semi-controlled conditions for aerosolization (viruses brought in situ on soil small plots covered by wind tunnels) and in controlled conditions for inactivation (viruses in laboratory atmospheric reactor). The MC0 murine mengovirus strain was used for all these experiences. It was propagated on BGM cells. The viral RNA content was monitored by RT-qPCR; for inactivation studies, infectious viruses were simultaneously enumerated by plaque assay on BGM cells. Variations in the total or infectious virus numbers were analyzed with regard to variations in global radiation, air temperature and relative humidity, O3 concentration, as well as soil surface moisture and temperature. This work required to design a new laboratory atmospheric reactor, to assess the performance of air biocollectors (impingers and membranes), and to optimize method for infectious virus enumeration.A model has been proposed to describe the aerosolization of one or some pools of viruses, each of these pools being characterized by its size and a kinetic coefficient of aerosolization. We used it to generate numerical experiences having the same variability as actual measurements, and to fit simulations of either cumulative or "instantaneous" aerosolized virus quantities to these numerical experiences. Fitting simulations to "instantaneous" aerosolized virus quantities leads to more precise estimates of the kinetic coefficient of aerosolization; it didn't lead to better estimates of the total amount of viruses that could be aerosolized. A relationship between the kinetic coefficient of virus inactivation and air relative moisture has also been proposed.Dealing with virus aerosolization from the soil, the amount of aerosolized viruses estimated from impinger data were higher than those estimated from membrane data, because of differences in trapping and/or latter extraction. Impingers would have trapped about 77% of air virus and virus re-aerosolization from Impingers would have concerned about 11% of the trapped viruses every hour. These limits would have resulted in estimates of the total amount of viruses that could be aerosolized about 2 times lower than in reality; conversely, they do not affect the estimates of the kinetic coefficient of aerosolization. Regardless of this bias, between 1 and 10% of viruses supplied to the soil were aerosolized. To our knowledge, this is the first evidence of such a phenomenon. We distinguish a pool of viruses that could be aerosolized nearly instantaneously (about 1/3 of aerosolized viruses) from a pool of viruses that would be aerosolized kinetically. For this last pool, the kinetic aerosolization coefficient varied between 0.007 and 0.21 h-1: 90% of the viruses belonging to the kinetic pool would be aerosolized after 13 days or 11 hours, respectively. The total amount of viruses belonging to the kinetic pool is correctly predicted by a model accounting simultaneously for the wind, the soil surface temperature and the type of irrigation water.

Made available in DSpace on 2016-12-23T14:04:26Z (GMT). No. of bitstreams: 1 Susanne Mariani Loss.pdf: 2049956 bytes, checksum: 69145238536f7bbbe30c59f18c5e2885 (MD5) Previous issue date: 2012-12-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A Ilha das Caieiras está localizada em uma das regiões mais carentes no município de Vitória/ES, a Grande São Pedro. Esta região é densamente povoada e é conhecida como um bolsão de pobreza da capital, com a população sobrevivendo, principalmente, da coleta e comercialização de frutos do mar do manguezal localizado em seu entorno. Estudos realizados anteriormente na região mostraram que a água deste estuário está contaminada com microrganismos patogênicos provenientes, principalmente, de esgotos domésticos lançados sem prévio tratamento. O objetivo deste trabalho foi avaliar a presença de vírus entéricos (adenovírus AdV, rotavírus RV e norovírus GII NoV) e indicadores bacterianos de poluição fecal (coliformes termotolerantes e enterococos) em amostras de água de quatro pontos deste importante estuário da Ilha das Caieiras. O monitoramento ocorreu de janeiro de 2011 a julho de 2012 (19 meses) e utilizou-se as técnicas moleculares PCR Qualitativa e PCR em Tempo Real (qPCR), para detecção de vírus entéricos, e membrana filtrante, para análise de bactérias, além de avaliação de parâmetros físico-químicos da água. Os resultados das análises microbiológicas da água nos pontos estudados (P1, P2, P3 e P4) demonstraram a presença de coliformes termotolerantes, com médias geométricas de 1,66x102, 1,31x102, 2,29x103 e 3,13x102 UFC / 100 mL de água, respectivamente. Para enterococos, as médias encontradas foram 6,30x101 UFC / 100 mL em P1, 5,56x101 UFC / 100 mL em P2, 1,89x103 UFC / 100 mL em P3 e 1,62x103 UFC / 100 mL em P4. Os vírus entéricos foram detectados nos quatro pontos de monitoramento pelas duas técnicas moleculares utilizadas, com valores máximos de 2,00x103, 5,24x104 e 1,50x104 CG / 100 mL para AdV, RV e NoV, respectivamente, quantificados por qPCR. A frequências de detecção de vírus nas amostras de água do estuário variou de 21 27% para AdV, 42 53% para RV e 10 42% para NoV GII. Neste estudo foi possível verificar que o estuário da Ilha das Caieiras apresenta-se contaminado devido, possivelmente, aos lançamentos de esgoto in natura e/ou de lixiviados de áreas rurais. A ação antropogênica sobre este manguezal é evidente e preocupante, pois se reflete na qualidade ambiental deste meio, na saúde da população e na economia da região / The Ilha das Caieiras is located in one of the poorest regions in Vitória / ES, the Grande São Pedro. This region is densely populated and is known as a pocket of poverty in the capital, with a population surviving mainly on the collection and marketing of seafood mangrove located in their surroundings. Previous studies have shown that the region of this estuary water is contaminated with pathogenic microorganisms from mainly domestic sewage released without prior treatment. The aim of this study was to evaluate the presence of enteric viruses (adenovirus - AdV, rotavirus - RV and norovirus GII - NoV) and bacterial indicators of fecal pollution (fecal coliform and enterococci) in water samples from four sites of this important Ilha das Caieiras estuary. Monitoring occurred from January 2011 to July 2012 (19 months) and used molecular techniques Qualitative PCR and Real Time PCR (qPCR) for detection of enteric viruses, and membrane filter for analysis of bacteria, and assessment of physico-chemical parameters of the water. The microbiological analysis of water studied sites (P1, P2, P3 and P4) showed the presence of fecal coliform, with geometric means of 1.66 x102, 1.31 x102, 2.29 x103 and 3.13 x102 CFU / 100 mL water, respectively. For enterococci, the averages were 6.30 x101 CFU / 100 mL in P1, 5.56 x101 CFU / 100 mL at P2, 1.89 x103 CFU / 100 mL at P3 and 1.62 x103 CFU / 100 mL at P4. Enteric viruses were detected in the four monitoring sites by both molecular techniques used, with maximum values of 2,00x103, 5,24x104 and 1,50x104 GC / 100 mL for AdV, NoV and RV, respectively, quantified by qPCR. The frequencies of virus detection in samples of estuarine water ranged from 21 - 27% for AdV, 42 - 53% for RV and 10 - 42% for NoV GII. In this study we found that the estuary of the Ilha das Caieiras presents contaminated, possibly due to sewage releases fresh and / or leachate from rural areas. The anthropogenic mangrove on this is clear and worrisome because environmental quality is reflected in this environment, the health of the population and the economy of the region

Les entérovirus (EV) sont des Picornavirus (virus nus à génome ARN positif), caractérisés par une grande diversité génétique et antigénique (116 types classés en 4 espèces taxonomiques EV-A à D) et une évolution rapide. Les infections humaines sont très fréquentes, hautement contagieuses à partir des selles et épidémiques. La plupart des infections sont asymptomatiques ou bénignes ; elles peuvent être graves voire mortelles, en particulier chez les jeunes enfants. La poliomyélite, modèle d’infection à EV, est en voie d’éradication grâce aux programmes de vaccination et de surveillance sous l’égide de l’OMS. La détection de poliovirus sauvages dans des pays déclarés exempts de polio depuis plusieurs années et l’émergence récente de plusieurs EV non poliomyélitiques (EV-A71, EV-D68) associés à des manifestations cliniques sévères dans plusieurs régions du monde montrent l’importance de surveiller la circulation des EV dans la population humaine. Le but de la thèse était de rechercher et caractériser les EV dans les eaux usées de l’agglomération de Clermont-Ferrand et de comparer les données à celles de la surveillance clinique pour avoir une image plus complète de la circulation virale dans la population générale. Une méthode de concentration virale à partir des eaux usées prélevées en entrée (eaux usées brutes) et sortie (eaux usées traitées) de station d’épuration a été mise au point, permettant la détection moléculaire des EV et de 6 autres virus entériques humains. La présence de génomes viraux a été détectée dans tous les échantillons d’octobre 2014 à octobre 2015, avec une médiane de 6 virus différents en entrée de station et de 4 virus en sortie. L’analyse phylogénétique des séquences d’EV et des virus des hépatites A et E présents dans les eaux usées et les prélèvements cliniques des patients hospitalisés au CHU de Clermont-Ferrand pendant la même période, a validé l’approche mise en place pour surveiller la circulation communautaire d’un virus entérique. La diversité des EV présents dans les eaux usées brutes a été analysée par séquençage d’amplicons avec une technique haut débit Illumina (metabarcoding). Les résultats montrent la présence d’une grande diversité d’EV et la circulation silencieuse de 25 types (notamment 9 EV-C, dont des séquences de poliovirus 1 vaccinal) dans la population générale. L’analyse phylogénétique des variants intra-typiques a mis en évidence plusieurs profils épidémiques parmi les principaux types ayant circulé pendant la période d’étude. Les données obtenues montrent la faisabilité et la sensibilité de la stratégie développée pour détecter et caractériser les EV présents dans les eaux usées. Ils permettent de discuter la place de la surveillance environnementale dans la surveillance des infections à EV non polio (études épidémiologiques, prévention des épidémies, alertes sanitaires). Surveiller conjointement les virus entériques dans l’environnement et chez les patients permet une meilleure compréhension de leur prévalence. Cette approche globale de la circulation virale et de l’écologie de la santé représente un engagement important de la part des laboratoires et nécessitera une intégration dans des réseaux structurés de collaboration nationales et internationales dépassant la seule surveillance des EV. / Enterovirus (EV) are Picornaviruses (non-enveloped, positive-sense RNA viruses), characterized by a large genetic and antigenic diversity (116 types classified within 4 taxonomic species EV-A to D) and rapid evolution. Human infections are frequent, highly contagious from stools and occur as outbreaks. The infections are mainly asymptomatic or benign but severe or fatal cases can be reported in young children. Poliomyelitis is the model EV infection. Combined with clinical and virological surveillance, mass vaccination is closer than ever to achieve the WHO program of the Global Polio Eradication Initiative. However, the detection of wild type polioviruses in polio-free countries and the recent worldwide emergence of non-polio enteroviruses (EV-A71, EV-D68) associated with severe clinical manifestations underscore the importance of surveilling EV circulation in the general population. The aim of the PhD thesis was the detection and identification of EV strains in wastewater treated in the sewage treatment plant at Clermont-Ferrand (France). The viral data were compared with those reported through clinical surveillance to obtain a comprehensive picture of the viral circulation in the local population. A method was developed to concentrate viruses from raw and treated wastewater and molecular assays were used to detect EVs and 6 other human enteric viruses. The viral genomes were detected in all samples from October 2014 to October 2015, with a median of 6 and 4 different viruses in raw and treated wastewater respectively. Phylogenetic analysis of viral sequences (EV, hepatitis A and E viruses) determined in wastewater and reported in patients during the sampling period, showed the efficiency of the method for surveilling enteric viruses in the community. The EV diversity in raw wastewater was analyzed by sequencing of amplicons with the Illumina high throughput technology (metabarcoding). The analysis revealed a large viral diversity and the silent circulation of 25 types not detected from hospital data (in particular 9 EV-C, of which sequences of vaccine poliovirus 1). The phylogenetic analyses of intra-typic variants showed different epidemic patterns in the predominant EV types circulating over the study period. The data demonstrate the feasibility and sensitivity of the strategy developed for the detection and characterization of EV in wastewater and provide a future prospect for the implementation of environmental surveillance of non-polio EV infections in epidemiological studies, epidemic prevention, and for health alert. Combining the surveillance of enteric viruses in the environment and in the clinical setting allows a better understanding of their prevalence. This global approach of virus circulation and ecological health represents an important investment for laboratories, which will require integration in national and international collaboration networks beyond the scope of enterovirus surveillance.

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Les eaux usées traitées d'origine domestique présentent généralement une charge non négligeable en virus entériques pathogènes de l'Homme, malgré leur traitement. Afin de pouvoir à terme comparer les risques sanitaires associés à différents modes de gestion de ces eaux usées, nous avons entrepris un travail sur (i) la prévision des quantités de virus entériques excrétés à partir des données épidémiologiques relatives aux gastroentérites aiguës, (ii) le devenir environnemental de ces virus lorsque les eaux usées sont rejetées en rivière, et (iii) le devenir de ces virus lorsqu'ils sont apportées au sol par irrigation. L'étude a porté sur un bassin de collecte des eaux usées de 240 000 habitants à proximité de Clermont-Ferrand, sur les rivières Artière et Allier et la nappe alluviale de l'Allier potentiellement contaminées par les rejets d'eau usée, et sur un sol du périmètre d'irrigation réutilisant ces eaux. Les concentrations en divers virus ont été suivies sur la même période dans les eaux usées brutes et traitées, dans les eaux douces de surface et souterraine. Nous avons proposé une méthode d'estimation du nombre journalier de nouveaux cas de gastroentérites aiguës d'étiologie virale en 2015-2016 à partir de données épidémiologiques et avons combiné ces estimations à un modèle d'excrétion virale pour évaluer les quantités de virus entériques arrivant à la station d'épuration. Le devenir des virus a été modélisé en tenant compte d'un abattement en station d'épuration et de dilutions-mélanges en rivières. Le devenir des virus apportés au sol par les eaux usées traitées réutilisées en irrigation agricole a été étudié sur un sol bien représenté dans le périmètre en utilisant un virus modèle. Ce devenir a été décrit par un modèle combinant transfert, immobilisation réversible et élimination, et en distinguant eau mobile et eau immobile comme virus libres et virus adsorbés sur des colloïdes en suspension. La méthode permettant de passer de l'épidémiologie à une excrétion de virus nous a permis de bien simuler les arrivées de virus à la station d'épuration avec un pic hivernal et l'impact prépondérant des norovirus GII sur les cas de gastroentérites virales. La simulation de l'abattement en station d'épuration et des phénomènes de dilutions-mélanges en rivière permet de simuler correctement la charge virale en aval du rejet d'eaux usées, mais leur devenir ultérieur reste mal caractérisé. Apporté au sol, le virus modèle était progressivement éliminé ou immobilisé de façon irréversible avec un abattement journalier de 0.38 log10. La fraction réversiblement immobilisée pouvait être estimée par une isotherme de Freundlich. L'ajout de Mg2+ a favorisé l'immobilisation du virus comme son adsorption sur des colloïdes dispersés dans l'eau mobile. Alors que les eaux usées stérilisées n'avaient pas d'effet majeur sur l'immobilisation du virus par rapport à une solution artificielle de sol en raison d'effets antagonistes des composés organiques et des cations minéraux, l'eau souterraine riche en Mg2+ favorisait l'immobilisation des virus. Un volet plante réalisé en marge de ce travail a montré l'impact d'irrigations sur les contaminations de surface et après internalisation via les racines. Complétée et améliorée, notre étude pourrait être couplée à une évaluation quantitative des risques viraux. / Urban treated wastewaters may be heavily contaminated by human pathogenic enteric viruses that cause acute gastroenteritis, despite their treatment. In order to compare health risks of different wastewater management scenarios, we investigated (i) how to predict virus shedding from epidemiological data on acute gastroenteritis, (ii) the environmental fate of these viruses when wastewaters are discharged into rivers, and (iii) the fate of these viruses when they are brought to the soil by irrigation. Our study focused on a wastewater collection basin of 240,000 inhabitants near Clermont-Ferrand, on the Artière and Allier Rivers and the Allier alluvial groundwater potentially contaminated by wastewater discharges, and on a soil in the irrigation perimeter reusing these wastewaters. Concentrations of various viruses were monitored over the same period in raw and treated wastewaters, as well as in surface and underground freshwaters. We proposed a method based on epidemiological data to estimate the daily number of new cases of acute gastroenteritis of viral etiology in 2015-2016; and we combined these estimates with a viral shedding model to estimate the quantities of enteric viruses arriving at the treatment plant. The fate of viruses has been simulated by taking into account the removal of viruses in the treatment plant and dilution-mixing in rivers. The fate of viruses brought to the soil by treated wastewater reused in agricultural irrigation was studied on a well-represented soil in the perimeter using a surrogate virus. Its fate has been described by a model combining transfer, reversible immobilization and removal; the model distinguished between mobile and immobile waters, as well as between free viruses and viruses adsorbed on colloids in suspension. The method for switching from epidemiology to virus shedding allowed us to accurately simulate virus inflows to the treatment plant, including a winter peak and the prominent role of norovirus GII in viral gastroenteritis cases. The simulation of virus removal in the treatment plant and subsequent dilution-mixing phenomena in rivers allow correctly simulating the viral load in the river downstream of the wastewater discharge, but their subsequent fate remains poorly characterized. When brought to the soil, the surrogate virus was progressively removed or irreversibly immobilized, according to a 0.38 log10 daily removal. The reversibly immobilized fraction could be estimated by a Freundlich isotherm. The addition of Mg2+ favored the immobilization of viruses, as well as their adsorption on colloids dispersed in mobile water. While sterilized wastewater had no major effect on virus immobilization compared to artificial soil solution due to the antagonistic effects of their organic compounds and mineral cations, groundwater rich in Mg2+ favored immobilization of viruses. An additional work, complementary to this PhD, showed the impact of irrigations on vegetable surface and internalized contaminations. After improvement, our study could be coupled with a quantitative viral risks assessment.Key words: enteric virus, sewage, discharge, reuse, irrigation, environmental fate, scenarios, assessment,