O objetivo deste estudo foi avaliar a qualidade virológica da água de reúso produzida em uma das estações de tratamento de esgoto da cidade de São Paulo. Para tanto, foram coletadas concomitantemente 177 amostras de esgoto tratado (100L) e bruto (15L) e os vírus concentrados utilizando método Viradel-ultracentrifugação. Em seguida as amostras foram tratadas com Vertrel XF e os ácidos nucléicos extraídos para a detecção de adenovírus (HAdV), rotavírus (RV-A), norovírus e vírus da hepatite A (VHA). A detecção por PCR e/ou RT-PCR evidenciou RV-A (G1-G5), VHA e HAdV incluindo os da espécie F tanto no esgoto bruto quanto no tratado, no entanto norovírus não foram detectados em ambos os efluentes. A infectividade de RV-A e HAdV foi avaliada por cultivo celular e os rotavírus RV-A foram também quantificados por reação de imunoperoxidase direta. PCR em tempo real foi padronizada para quantificação de vírus não cultiváveis ou de difícil cultivo como os VHA. Com base nos resultados obtidos foi verificada a ocorrência e a distribuição anual de cada vírus nas águas de reúso. / The aim of the study was to evaluate the virological quality from one Sewage Treatment Plant in the state of São Paulo. From January/2005 to November/2006, 177 samples (15L) of raw sewage were collected at the entrance and another 177 (100L) at the end of treatment, twice a week. Viruses were concentrated by filtration through positively charged microporous filters, followed by ultracentrifugation. Virus concentrates were treated by using Vertrel-XF and the viral genomes extracted for detection of adenovirus (HAdV), rotavirus (RV-A), norovirus and hepatitis A virus (HAV). PCR and RT-PCR revealed RV-A (G1-G5), HAV and HAdV, including the enteric ones (species F) in sewage and treated wastewater samples. Norovirus was not detected in any samples. The infectivity of HAdV and RV-A was assayed by inoculating onto suitable cell line. Immunoperoxidase assay was used to calculate the rotavirus FFU/L in the samples. Real time-PCR was standardized for enumeration of non-cultivable virus. The occurrence and annual distribution of each virus in reuse water were analyzed.

Os esgotos domésticos contêm uma alta quantidade de bactérias e outros organismos patogênicos e não patogênicos que são descarregados em corpos hídricos. A contaminação da água, pela presença de bactérias e vírus patogênicos, traz conseqüências indesejáveis, principalmente de saúde pública. O objetivo deste trabalho foi avaliar a possibilidade de emprego da análise de colifagos, como indicador viral. Para isso, amostras de esgoto bruto, efluente tratado por reator UASB e efluente de lodo ativado, da Estação de Tratamento de Esgotos São João Navegantes, Porto Alegre, foram coletadas durante o período de junho/2006 a junho/2007. As análises físicas, químicas e microbiológicas, DBO5, turbidez, sólidos totais, quantificação de coliformes totais, fecais e de colifagos foram realizadas em 99 amostras, conforme APHA, 2005. Na determinação da presença de adenovírus e enterovírus foram testadas 36 amostras, utilizando-se a técnica de concentração por filtração-eluição e ultracentrifugação. Posteriormente, purificação e avaliação da infectividade. Por fim, as amostras foram submetidas a teste de nested PCR e RT-PCR, a fim de verificar a presença de adenovírus e enterovírus, respectivamente. Nas 36 amostras testadas, a presença de enterovírus foi confirmada em apenas 5,56% das amostras e, adenovírus, em 16,67% das amostras. Não foram encontradas correlações significativas entre colifagos e bactérias coliformes totais e fecais, bem como entre colifagos e os parâmetros físicos e químicos. Desta independência encontrada entre vírus e bactérias se destaca a necessidade de monitoramento conjunto de ambos os microrganismos. Verificou-se uma similaridade em relação à sazonalidade referente ao comportamento de colifagos e o descrito na literatura referente a vírus entéricos. Observou-se, também, a baixa eficiência do tratamento para remoção de microrganismos, ressaltando-se a importância de realização de um tratamento terciário para assegurar a qualidade microbiológica do efluente. As relações entre colifagos e vírus entéricos não foram possíveis de serem analisadas, devido aos dados inconclusivos obtidos no presente trabalho. / Domestic sewage contains a high amount of bacteria and other pathogens and non pathogens that are discharged into water bodies. Contamination of water by the presence of pathogenic bacteria and viruses, brings undesirable consequences, especially for public health. The objective of this work was to evaluate the possibility of using analysis of coliphages as viral indicator. Samples of raw sewage, treated effluent by UASB reactor and by activated sludge, from Wastewater Treatment Systems São João Navegantes, Porto Alegre, were collected during the period of june/2006 to june/2007. Physical, chemical and microbiological analyzes, namely BOD5, turbidity, total solids, quantification of total coliforms, fecal coliforms and coliphages were performed in 99 samples, following the techniques described in APHA 2005. The presence of enteric viruses was tested on 36 samples. The technique of concentration by filtration-elution and ultra-centrifugation was used for this purpose. Subsequently, purification and infectivity test was performed. The samples were tested to nested PCR and RT-PCR to verify the presence of adenovirus and enterovirus, respectively. In 36 samples tested, the presence of enteroviruses was confirmed in only 5.56% of the samples, and adenovirus, in 16.67% of the samples. No significant correlation was found among coliform bacteria and coliphages, and also between the presence of coliphages and physical and chemical parameters. The independence found between viruses and bacteria indicates the need for joint monitoring of both microrganisms. The behavior of coliphages was observed to follow the path described in the literature about enteric viruses in relation to seasonality. There was observed a low efficiency in terms of microorganisms removal, emphasizing the need for a tertiary treatment to ensure the microbiological quality of the effluents tested. Relations between coliphages and enteric viruses were not able to be analyzed, due to inconclusive data obtained in this work.

Os esgotos domésticos contêm uma alta quantidade de bactérias e outros organismos patogênicos e não patogênicos que são descarregados em corpos hídricos. A contaminação da água, pela presença de bactérias e vírus patogênicos, traz conseqüências indesejáveis, principalmente de saúde pública. O objetivo deste trabalho foi avaliar a possibilidade de emprego da análise de colifagos, como indicador viral. Para isso, amostras de esgoto bruto, efluente tratado por reator UASB e efluente de lodo ativado, da Estação de Tratamento de Esgotos São João Navegantes, Porto Alegre, foram coletadas durante o período de junho/2006 a junho/2007. As análises físicas, químicas e microbiológicas, DBO5, turbidez, sólidos totais, quantificação de coliformes totais, fecais e de colifagos foram realizadas em 99 amostras, conforme APHA, 2005. Na determinação da presença de adenovírus e enterovírus foram testadas 36 amostras, utilizando-se a técnica de concentração por filtração-eluição e ultracentrifugação. Posteriormente, purificação e avaliação da infectividade. Por fim, as amostras foram submetidas a teste de nested PCR e RT-PCR, a fim de verificar a presença de adenovírus e enterovírus, respectivamente. Nas 36 amostras testadas, a presença de enterovírus foi confirmada em apenas 5,56% das amostras e, adenovírus, em 16,67% das amostras. Não foram encontradas correlações significativas entre colifagos e bactérias coliformes totais e fecais, bem como entre colifagos e os parâmetros físicos e químicos. Desta independência encontrada entre vírus e bactérias se destaca a necessidade de monitoramento conjunto de ambos os microrganismos. Verificou-se uma similaridade em relação à sazonalidade referente ao comportamento de colifagos e o descrito na literatura referente a vírus entéricos. Observou-se, também, a baixa eficiência do tratamento para remoção de microrganismos, ressaltando-se a importância de realização de um tratamento terciário para assegurar a qualidade microbiológica do efluente. As relações entre colifagos e vírus entéricos não foram possíveis de serem analisadas, devido aos dados inconclusivos obtidos no presente trabalho. / Domestic sewage contains a high amount of bacteria and other pathogens and non pathogens that are discharged into water bodies. Contamination of water by the presence of pathogenic bacteria and viruses, brings undesirable consequences, especially for public health. The objective of this work was to evaluate the possibility of using analysis of coliphages as viral indicator. Samples of raw sewage, treated effluent by UASB reactor and by activated sludge, from Wastewater Treatment Systems São João Navegantes, Porto Alegre, were collected during the period of june/2006 to june/2007. Physical, chemical and microbiological analyzes, namely BOD5, turbidity, total solids, quantification of total coliforms, fecal coliforms and coliphages were performed in 99 samples, following the techniques described in APHA 2005. The presence of enteric viruses was tested on 36 samples. The technique of concentration by filtration-elution and ultra-centrifugation was used for this purpose. Subsequently, purification and infectivity test was performed. The samples were tested to nested PCR and RT-PCR to verify the presence of adenovirus and enterovirus, respectively. In 36 samples tested, the presence of enteroviruses was confirmed in only 5.56% of the samples, and adenovirus, in 16.67% of the samples. No significant correlation was found among coliform bacteria and coliphages, and also between the presence of coliphages and physical and chemical parameters. The independence found between viruses and bacteria indicates the need for joint monitoring of both microrganisms. The behavior of coliphages was observed to follow the path described in the literature about enteric viruses in relation to seasonality. There was observed a low efficiency in terms of microorganisms removal, emphasizing the need for a tertiary treatment to ensure the microbiological quality of the effluents tested. Relations between coliphages and enteric viruses were not able to be analyzed, due to inconclusive data obtained in this work.

Os esgotos domésticos contêm uma alta quantidade de bactérias e outros organismos patogênicos e não patogênicos que são descarregados em corpos hídricos. A contaminação da água, pela presença de bactérias e vírus patogênicos, traz conseqüências indesejáveis, principalmente de saúde pública. O objetivo deste trabalho foi avaliar a possibilidade de emprego da análise de colifagos, como indicador viral. Para isso, amostras de esgoto bruto, efluente tratado por reator UASB e efluente de lodo ativado, da Estação de Tratamento de Esgotos São João Navegantes, Porto Alegre, foram coletadas durante o período de junho/2006 a junho/2007. As análises físicas, químicas e microbiológicas, DBO5, turbidez, sólidos totais, quantificação de coliformes totais, fecais e de colifagos foram realizadas em 99 amostras, conforme APHA, 2005. Na determinação da presença de adenovírus e enterovírus foram testadas 36 amostras, utilizando-se a técnica de concentração por filtração-eluição e ultracentrifugação. Posteriormente, purificação e avaliação da infectividade. Por fim, as amostras foram submetidas a teste de nested PCR e RT-PCR, a fim de verificar a presença de adenovírus e enterovírus, respectivamente. Nas 36 amostras testadas, a presença de enterovírus foi confirmada em apenas 5,56% das amostras e, adenovírus, em 16,67% das amostras. Não foram encontradas correlações significativas entre colifagos e bactérias coliformes totais e fecais, bem como entre colifagos e os parâmetros físicos e químicos. Desta independência encontrada entre vírus e bactérias se destaca a necessidade de monitoramento conjunto de ambos os microrganismos. Verificou-se uma similaridade em relação à sazonalidade referente ao comportamento de colifagos e o descrito na literatura referente a vírus entéricos. Observou-se, também, a baixa eficiência do tratamento para remoção de microrganismos, ressaltando-se a importância de realização de um tratamento terciário para assegurar a qualidade microbiológica do efluente. As relações entre colifagos e vírus entéricos não foram possíveis de serem analisadas, devido aos dados inconclusivos obtidos no presente trabalho. / Domestic sewage contains a high amount of bacteria and other pathogens and non pathogens that are discharged into water bodies. Contamination of water by the presence of pathogenic bacteria and viruses, brings undesirable consequences, especially for public health. The objective of this work was to evaluate the possibility of using analysis of coliphages as viral indicator. Samples of raw sewage, treated effluent by UASB reactor and by activated sludge, from Wastewater Treatment Systems São João Navegantes, Porto Alegre, were collected during the period of june/2006 to june/2007. Physical, chemical and microbiological analyzes, namely BOD5, turbidity, total solids, quantification of total coliforms, fecal coliforms and coliphages were performed in 99 samples, following the techniques described in APHA 2005. The presence of enteric viruses was tested on 36 samples. The technique of concentration by filtration-elution and ultra-centrifugation was used for this purpose. Subsequently, purification and infectivity test was performed. The samples were tested to nested PCR and RT-PCR to verify the presence of adenovirus and enterovirus, respectively. In 36 samples tested, the presence of enteroviruses was confirmed in only 5.56% of the samples, and adenovirus, in 16.67% of the samples. No significant correlation was found among coliform bacteria and coliphages, and also between the presence of coliphages and physical and chemical parameters. The independence found between viruses and bacteria indicates the need for joint monitoring of both microrganisms. The behavior of coliphages was observed to follow the path described in the literature about enteric viruses in relation to seasonality. There was observed a low efficiency in terms of microorganisms removal, emphasizing the need for a tertiary treatment to ensure the microbiological quality of the effluents tested. Relations between coliphages and enteric viruses were not able to be analyzed, due to inconclusive data obtained in this work.

Les virus entériques sont l'une des principales causes d'épidémies de gastroentérites chez l'Homme. Ces virus sont essentiellement excrétés dans les selles et sont donc observés en grande quantité au sein des eaux usées. L'abattement viral est très variable en fonction des types de traitements mis en place dans les stations d'épurations (STEP). Malgré ces traitements, la grande majorité des STEP rejettent d'importantes quantités de virus entériques dans l'environnement. L'objectif de ce travail a été d'apporter des éléments de réponse concernant la circulation des virus entériques humains dans un environnement hydrique urbain. Pour cela, des méthodes d'analyse ont été mises au point afin de permettre la détection d'un large panel de virus entériques dans différentes matrices hydriques susceptibles d'être étudiées dans l'environnement. Trois contrôles ont été développés afin de vérifier le bon déroulement de cette méthode et donc de valider les résultats obtenus. Une campagne d'échantillonnage a été réalisée au niveau d'un tronçon de la Seine impacté par les affluents naturels et des effluents de STEP. La prise en compte des débits au niveau des différents points de prélèvement a permis de calculer les flux viraux qui correspondent à la quantité de virus circulant en un point au cours d'une unité de temps. Cette approche a permis de démontrer la contribution majeure des effluents de STEP dans la contamination virale du milieu récepteur. Parmi l'ensemble des virus entériques recherchés, les adénovirus, les norovirus, les astrovirus et les rotavirus présentaient les charges virales les plus importantes dans les effluents de STEP et dans les eaux de surface. Leurs quantités au sein de ces matrices d'eau étaient les plus importantes durant la période hivernale ce qui était cohérent avec les données épidémiologiques. Un lien étroit entre l'état sanitaire de la population humaine reliée au réseau d'assainissement et la concentration en virus entériques des effluents de STEP et des eaux de surface a pu être établi. Sur ces mêmes prélèvements, l'étude de la diversité des astrovirus et des norovirus de génogroupes I et II a été réalisée par pyroséquençage. Cette approche a permis d'établir un profil des différents génotypes circulant dans les effluents de STEP. Au regard des données épidémiologiques recueillies sur la même période par le Centre National de Référence des virus entériques, un grand nombre de génotypes communs ont pu être observés entre ces deux jeux de données. Ce constat met en évidence le caractère informatif de l'analyse des effluents de STEP qui fournit un profil relativement pertinent des virus entériques circulant dans l'environnement mais aussi dans la population humaine. La présence de virus entériques présente dans les eaux de surface, ressources utilisées pour la potabilisation, est un réel problème de santé publique. C'est pourquoi la dernière partie de ce travail s'intéresse à évaluer leur persistance dans l'eau de surface et l'eau destinée à la consommation humaine, mais aussi face à des traitements de désinfection, fréquemment utilisés dans les usines de potabilisation et parfois en sortie de STEP. L'absence de modèle cellulaire pour la plupart de ces virus nous a conduit à développer une approche basée sur l'emploi d'agents intercalants permettant d'évaluer l'intégrité de la capside des particules virales détectées par PCR en temps réel. L'utilisation de ces agents intercalants s'est avérée pertinente à partir du moment où les conditions conduisent à une dégradation des capsides virales. Par conséquent, leur emploi sur des échantillons d'eau potabilisée a permis une meilleure estimation du risque sanitaire associé à la consommation de l'eau produite. En conclusion, ce travail propose des méthodes d'analyse qui ont permis de mettre en exergue les relations existantes entre la circulation des virus entériques dans l'environnement et l'état sanitaire de la population humaine / Enteric viruses are a major cause of gastroenteritis outbreaks in humans. These viruses are mainly excreted in the faeces and are therefore observed in large amounts in wastewater. Depending on the types of treatments, virus removal from effluent may vary from one wastewater treatment plants (WWTP) to another. Despite these treatments, most of the WWTP releases significant quantities of enteric viruses in the environment. The aim of this study was to better understand the circulation of human enteric viruses in urban rivers. For this purpose methods have been developed to enable the detection of a broad panel of enteric viruses in various water matrices that may be collected from the environment. Since there is important inhibition observed in this type of water samples, three controls were developed to verify the success of this method, from the sample concentration to the PCR quantification, and thus to validate the results. A sampling campaign was carried out at the Seine River section (Paris area) affected by its natural tributaries and WWTP effluents. The river and effluent flows at the different sampling points were used to calculate the viral flows as the amount of virus circulating at a point during a time unit. This approach has demonstrated the major implication WWTP effluents in the viral contamination of the Seine River. Among all the targeted enteric viruses, adenoviruses, norovirus, astrovirus and rotavirus showed the most significant viral loads in WWTP effluents and consequently in river waters. Their quantities in WWTP effluents and surface water were the most important during the winter period which was consistent with the epidemiological data from the French Reference Center for enteric viruses. A close connection between the health status of the Parisian population and enteric virus concentration in WWTP effluents and in the Seine River could be established. The study of the diversity of astrovirus and norovirus of genogroups I and II was carried out by pyrosequencing on WWTP effluent and Seine river samples to develop a profile of the different genotypes circulating in the environment. Many common genotypes were observed between the effluent dataset and the epidemiological data collected over the same period by the French Reference Center. These results highlight the complementarity of the two datasets, and the fact that WWTP effluents provide a relevant profile of enteric viruses circulating in the environment but also in the human population. The amount of enteric viruses present in drinking water resources such as surface waters is a real public health problem. Therefore, it is important to evaluate viral persistence in surface waters during tertiary treatment and in drinking water, but also the viral persistence after usual disinfection treatments such chlorination and ultraviolet treatment. The absence of cell model for culture of most enteric viruses, has led us to develop a method using intercalating dyes to assess the capsid integrity of the viral particles quantified by real time PCR. The use of these intercalating agents was relevant when the environmental conditions and disinfection treatments caused a degradation of the viral capsid. Consequently, their use for the analysis of water samples from drinking water plants allowed to prevent an overestimation of the risk of transmission to humans by the consumption of tap water. In conclusion, this work provides development of analysis methods highlighting the relationships between the circulation of infectious enteric viruses in the environment and the health status of the human population

Tesis por compendio / [ES] El objetivo inicial de esta tesis era detectar y rastrear virus entéricos en diferentes matrices acuáticas mediante la combinación de protocolos moleculares y metagenómicos. Se establecieron como objetivos principales el desarrollo de procedimientos para la concentración de virus en muestras de aguas residuales, el análisis de virus indicadores de contaminación fecal y la caracterización del viroma de estas muestras. En cuanto al desarrollo de procedimientos para la concentración de virus entéricos emergentes en muestras de aguas residuales, el protocolo de floculación con hidróxido de aluminio, utilizado habitualmente en el grupo para la concentración de otros virus entéricos, resultó muy eficaz para el virus de la Hepatitis E, permitiendo su detección en muestras de aguas de entrada y salida de estaciones depuradoras de aguas residuales (EDAR). Por lo que respecta al seguimiento de la prevalencia de otros virus entéricos e indicadores virales en muestras de entrada y salida de EDARs, esta tesis proporciona información cuantitativa sobre la presencia del indicador crAssphage y otros virus entéricos de cápside intacta en aguas de diferentes EDARs valencianas. Además, los resultados de la correlación indican que crAssphage podría no ser un indicador óptimo de la presencia de virus entéricos infecciosos en las aguas residuales tratadas. En relación a la caracterización del viroma de las muestras de agua de las EDARs analizadas, en la presente tesis se describe un procedimiento de referencia que permite la detección y caracterización de las poblaciones virales en las muestras de aguas residuales recogidas a la entrada y salida de la planta depuradora. También se refleja el sesgo existente en los perfiles del viroma que se obtienen según las librerías de secuenciación que se empleen. En este sentido, esta investigación arroja luz sobre la diversidad de las comunidades virales en influentes y efluentes de aguas residuales, proporcionando información valiosa también en términos de indicadores fecales virales. Con la llegada de la pandemia de COVID-19 a principios de 2020, el SARS-CoV-pasó a ser el protagonista de la segunda parte de la tesis. En este aspecto, se marcaron como objetivos implementar un sistema de monitorización de SARS-CoV-2 en aguas residuales y desarrollar y optimizar métodos moleculares rápidos para inferir la infectividad del SARS-CoV-2. Los resultados obtenidos han demostrado que la aplicación de la epidemiología basada en aguas residuales (WBE) es eficiente para estimar la presencia de COVID-19 en comunidades y puede servir de herramienta para la salud pública como alerta temprana ante situaciones pandémicas. Asimismo, esta tesis incluye el primer estudio publicado en España que realizó un análisis metagenómico de la diversidad del SARS-CoV-2 presente en las aguas residuales en las tres primeras oleadas epidemiológicas, cuyos resultados confirmaron el potencial de la secuenciación masiva de aguas residuales para detectar nuevas mutaciones y linajes del SARS-CoV-2. Además, también se han comparado y optimizado los protocolos de concentración, extracción y detección de ácidos nucleicos de coronavirus a partir de muestras de aguas residuales, superficiales y de mar. Así, este trabajo amplía el conocimiento sobre los procedimientos analíticos para la detección del SARS-CoV-2 en aguas residuales favoreciendo la implementación global del COVID-19 WBE. Finalmente, se ha implementado un protocolo de RT-qPCR de viabilidad basado en el cloruro de platino para evitar la amplificación del ARN del SARS-CoV-2 no infeccioso. Además, los resultados de esta tesis apoyan la idea de que el SARS-CoV-2 presente en las aguas residuales no es infeccioso. En general, en el marco de esta tesis doctoral se ha desarrollado una herramienta analítica rápida basada en la RT-qPCR de viabilidad para inferir la infectividad del SARS-CoV-2 con potencial aplicación en la evaluación de riesgos, la prevención y el control en los programas de salud / [CA] L'objectiu inicial d'esta tesi era detectar i rastrejar virus entèrics en diferents matrius aquàtiques mitjançant la combinació de protocols moleculars i metagenòmics. Es van establir com a objectius específics el desenvolupament de procediments per a la concentració de virus en mostres d'aigües residuals, l'anàlisi de virus indicadors de contaminació fecal i la caracterització del viroma d'estes mostres. Pel que fa al desenvolupament de procediments per a la concentració de virus entèrics emergents en mostres d'aigües residuals, el protocol de floculació amb hidròxid d'alumini, utilitzat habitualment al grup per a la concentració d'altres virus entèrics, va resultar molt eficaç per al virus de l'hepatitis E, permetent-ne la detecció en mostres d'aigües d'entrada i de sortida d'estacions depuradores d'aigües residuals (EDAR). Pel que fa al seguiment de la prevalença d'altres virus entèrics i indicadors virals en mostres d'entrada i de sortida d'EDARs, esta tesi proporciona informació quantitativa sobre la presència de l'indicador crAssphage i altres virus entèrics de càpside intacta en aigües de diferents EDARs valencianes. A més, els resultats de la correlació indiquen que el bacteriòfag crAssphage podria no ser un indicador òptim de la presència de virus entèrics infecciosos a les aigües residuals tractades. Pel que fa a la caracterització del viroma de les mostres d'aigua de les EDARs analitzades, a la present tesi es descriu un procediment de referència que permet la detecció i caracterització de les poblacions virals a les mostres d'aigües residuals recollides a l'entrada i sortida de la planta depuradora. També es reflecteix el biaix existent als perfils del viroma que s'obtenen segons les llibreries de seqüenciació que s'utilitzen. En este sentit, esta investigació aporta coneixement sobre la diversitat de les comunitats virals en influents i efluents d'aigües residuals, proporcionant informació valuosa també en termes d'indicadors fecals virals. Amb l'arribada de la pandèmia de COVID-19 a principis del 2020, el SARS-CoV-va passar a ser el protagonista de la segona part de la tesi. En este aspecte, es van establir com a objectius implementar un sistema de monitorització de SARS-CoV-2 en aigües residuals i desenvolupar i optimitzar mètodes moleculars ràpids per inferir la infectivitat del SARS-CoV-2. Els resultats obtinguts han demostrat que l'aplicació de l'epidemiologia basada en aigües residuals (WBE) és eficient per estimar la presència de COVID-19 a les comunitats i pot servir d'eina per a la salut pública com a alerta primerenca davant de situacions pandèmiques. Així mateix, esta tesi inclou el primer estudi publicat a Espanya que va realitzar una anàlisi metagenòmica de la diversitat del SARS-CoV-2 present a les aigües residuals a les tres primeres onades epidemiològiques, els resultats del qual van confirmar el potencial de la seqüenciació massiva d'aigües residuals per a detectar noves mutacions i llinatges del SARS-CoV-2. A més, també s'han comparat i optimitzat els protocols de concentració, extracció i detecció d'àcids nucleics de coronavirus a partir de mostres d'aigües residuals, superficials i de mar. Així, este treball amplia el coneixement sobre els procediments analítics per a la detecció del SARS-CoV-2 en aigües residuals afavorint la implementació global del COVID-19 WBE. Finalment, s'ha implementat un protocol de RT-qPCR de viabilitat basat en el clorur de platí per evitar l'amplificació de l'ARN del SARS-CoV-2 no infecciós. A més, els resultats d'esta tesi donen suport a la idea que el SARS-CoV-2 present a les aigües residuals no és infecciós. En general, en el marc d'esta tesi doctoral s'ha desenvolupat una eina analítica ràpida basada en la RT-qPCR de viabilitat per inferir la infectivitat del SARS-CoV-2 amb potencial aplicació en l'avaluació de riscos, la prevenció i el control en els programes de Salut Pública. / [EN] The initial aim of this thesis was to detect and monitor the presence of enteric viruses in different aquatic matrices using both molecular and metagenomic protocols. The specific objectives were the development of procedures for the concentration of viruses in wastewater samples, the analysis of viruses indicative of faecal contamination and the characterisation of the virome in these samples. Regarding the development of procedures for the concentration of emerging enteric viruses in wastewater samples, the flocculation protocol with aluminium hydroxide, commonly used in the group for the concentration of other enteric viruses, proved to be very effective for Hepatitis E virus, allowing its detection in influent and effluent water samples from wastewater treatment plants (WWTPs). Regarding the monitoring of the prevalence of other enteric viruses and viral indicators in incoming and outgoing samples from WWTPs, this thesis provides quantitative information on the presence of the indicator crAssphage and other enteric viruses with intact capsid in water from different Valencian WWTPs. Moreover, the correlation results indicate that crAssphage may not be an optimal indicator of the presence of infectious enteric viruses in treated wastewater. In relation to the virome characterisation of the WWTP water samples analysed, the present thesis describes a reference procedure that allows the detection and characterisation of viral populations in wastewater. It also reflects the existing bias in the virome profiles obtained depending on the sequencing libraries used. In this sense, this research sheds light on the diversity of viral communities in influent and effluent wastewater, providing valuable information also in terms of faecal viral indicators. With the advent of the COVID-19 pandemic in the early 2020s, SARS-CoV-2 became the focus of the second part of the thesis. In this aspect, the objectives were to implement a monitoring system for SARS-CoV-2 in wastewater and to develop and optimise rapid molecular methods to infer SARS-CoV-2 infectivity. The results obtained have demonstrated that the application of wastewater-based epidemiology (WBE) is efficient for estimating the presence of COVID-19 in communities and can serve as a Public Health tool for early warning of pandemic situations. Furthermore, this thesis includes the first study published in Spain that performed a metagenomic analysis of the SARS-CoV-2 diversity present in wastewater in the first three epidemiological waves, the results of which confirmed the potential of mass sequencing of wastewater to detect new SARS-CoV-2 mutations and lineages. In addition, protocols for the concentration, extraction and detection of coronavirus nucleic acids from sewage, surface and seawater samples have also been compared and optimised. Thus, this work expands the knowledge on analytical procedures for the detection of SARS-CoV-2 in wastewater favouring the global implementation of the COVID-19 WBE. Finally, a viability RT-qPCR protocol based on platinum chloride has been implemented to avoid amplification of non-infectious SARS-CoV-2 RNA. Furthermore, the results of this thesis support the idea that SARS-CoV-2 present in wastewater is not infectious. Overall, in the framework of this PhD thesis, a rapid analytical tool based on feasibility RT-qPCR has been developed to infer the infectivity of SARS-CoV-2 with potential application in risk assessment, prevention and control in Public Health programmes. / This thesis has been funded by an "Ayudas para contratos predoctorales para la formación de doctores 2018” grant, awarded by the Ministry of Science and Innovation. / Cuevas Ferrando, E. (2022). Detection and tracking of emerging viruses of public health interest in waters through molecular and metagenomic procedures [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/190899 / Compendio

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Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-13T17:29:42Z No. of bitstreams: 1 andressasilvinoferreiraassis.pdf: 3364177 bytes, checksum: ff9c20080e11e36f28daf805d56ecc7e (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-13T19:33:58Z (GMT) No. of bitstreams: 1 andressasilvinoferreiraassis.pdf: 3364177 bytes, checksum: ff9c20080e11e36f28daf805d56ecc7e (MD5) / Made available in DSpace on 2017-03-13T19:33:58Z (GMT). No. of bitstreams: 1 andressasilvinoferreiraassis.pdf: 3364177 bytes, checksum: ff9c20080e11e36f28daf805d56ecc7e (MD5) Previous issue date: 2016-12-19 / O tratamento de esgoto pode ser insuficiente para a completa eliminação de vírus entéricos, tais como adenovírus humanos (HAdV) e rotavírus do grupo A (RVA). Portanto, o retorno do lodo e do efluente tratado ao ambiente pode representar riscos à saúde pública. Este estudo foi conduzido para otimizar um protocolo de floculação orgânica para recuperação viral a partir de lodo de esgoto e efluente tratado, bem como realizar um monitoramento de HAdV e RVA na estação de tratamento de esgoto (ETE) de Juiz de Fora, MG. Nos estudos de otimização, foram propostas adaptações no protocolo de floculação com leite desnatado para lodo e efluente tratado, com modificações no tempo de agitação da amostra, na concentração final de leite desnatado e/ou na etapa de centrifugação. No estudo de monitoramento, esgoto bruto (P1), esgoto primário (P2), lodo (P3) e efluente tratado (P4) foram coletados bimensalmente em 2014 (durante as épocas seca e chuvosa), totalizando 96 amostras (simples e compostas). As cargas virais foram determinadas por PCR quantitativo e o bacteriófago PP7 foi usado como controle interno. Amostras de HAdV e RVA foram submetidas ao sequenciamento e a viabilidade das partículas de HAdV foi avaliada em amostras de P4. Os parâmetros físico-químicos e a contagem de coliformes termotolerantes (CT) foram determinados em cada ponto. Nos estudos de otimização, foram selecionadas duas condições que apresentaram as maiores taxas de recuperação viral no lodo (menor tempo de agitação e maior concentração de leite desnatado) e no efluente tratado (sem primeira etapa de centrifugação e com maior concentração de leite desnatado). Ambas provaram ser ferramentas úteis para pesquisa viral em amostras de campo, inclusive para a pesquisa de vírus gigantes. No monitoramento, o HAdV foi detectado em 85,4% (82/96) dos concentrados, com cargas virais variando de 3,27 x 102 a 2,42 x 106 cópias do genoma por mililitro (cg/mL), ao longo do ano. A presença de RVA foi observada em 52,1% (50/96) dos concentrados (1,38 x 103 a 3,65 x 105 cg/mL), com maior detecção na época seca. A carga viral não foi influenciada pelo tipo de amostra, sendo detectada tanto em amostras simples, quanto em amostras compostas. Todas as amostras de HAdV sequenciadas pertenciam à espécie F tipo 41 e as amostras de RVA pertenciam ao genótipo I1. O tratamento de esgoto reduziu a quantidade de matéria orgânica e sólidos, bem como a contagem de CT e as cargas virais. No entanto, a presença de HAdV e RVA foi observada mesmo após o tratamento, inclusive em amostras de efluente tratado consideradas adequadas pelas legislações atuais, com detecção de partículas infecciosas de HAdV. Foram observadas correlações positivas entre a carga viral e a demanda bioquímica de oxigênio, os sólidos sedimentáveis e sólidos suspensos totais. Os dois protocolos otimizados neste estudo podem ser facilmente adequados para uso em laboratório de rotina, podendo impulsionar o monitoramento viral nos subprodutos gerados na ETE. A carga viral detectada na ETE salienta a disseminação ambiental de RVA e HAdV e aponta o potencial do HAdV como um marcador viral de contaminação em ambientes aquáticos. / Sewage treatment may be insufficient for the complete elimination of enteric viruses such as human adenoviruses (HAdV) and group A rotaviruses (RVA). Thus, the return of sewage sludge and treated effluent to the environment poses concerns potential for public health. This study was conducted to optimize an organic flocculation procedure for viral recovery from sludge and treated effluent, and carry out a surveillance of HAdV and RVA in a wastewater treatment plant (WWTP) at Juiz de Fora, MG. In optimization studies, conditions were proposed for sludge and treated effluent with changes in the stirring time, in the final concentration of skimmed-milk and/or in the centrifugation step. In surveillance study, raw sewage (P1), primary sewage (P2), sludge (P3) and treated effluent (P4) were collected bimonthly in 2014 (during the dry and the rainy season), totaling 96 samples (simple and composite). Quantitative PCR determined viral loads and PP7 bacteriophage was used as internal control. HAdV and RVA strains were selected for sequencing, and the HAdV viability was evaluated in P4 samples. Physicochemical parameters and thermotolerant coliforms (TC) counting were determined at each point. After the optimization studies, two conditions were selected: the ones that showed the highest viral recovery rates in sludge (lower stirring time and higher concentration of skimmed-milk) and treated effluent (without the first centrifugation step and with a higher concentration of skimmed-milk). These conditions proved to be a useful tool for viral search in the field samples, including for the research of giant virus. In surveillance study, HAdV was detected in 85.4% (82/96) of the concentrated, with viral loads ranging from 3.27 x 102 to 2.42 x 106 genome copies per milliliter (gc/mL), throughout the year. RVA presence was observed in 52.1% (50/96) of the samples (1.38 x 103 to 3.65 x 105 gc/mL) with detection greater in the dry season. Viral load was not influenced by the type of sample being detected both in single samples, as in composite samples. All the sequenced HAdV strains belonged to species F type 41, and RVA strains belonged to genotype I1. Sewage treatment reduced the content of organic matter and solids, as well as the TC counts and the viral loads. However, the presence of HAdV and RVA was observed after treatment, even in samples considered adequate by current laws with detection of infectious HAdV particles. Positive correlations were observed between viral load and biochemical oxygen demand, sedimented solids and total suspended solids. Two optimized protocols in this study are easily suitable for routine laboratory use and can boost viral monitoring in by-products generated in the WWTP. Viral load detected in WWTP stress the environmental dissemination of HAdV and RVA and addressed the potential of HAdV as a virological marker of contamination in aquatic environments.

MSc (Microbiology) / Department of Microbiology