Time-resolved cryo-EM has already provided ground-breaking discoveries in various fields, including structural biology, biochemistry, and drug development. Compared to traditional structural biology methods where mostly stabilized conformations are reconstructed, the main advantage of time-resolved cryo-EM is its ability to capture dynamic processes in biological samples at near-atomic resolution, which allows for studying biological structures as they change and interact in real-time. In this project, I focused on the expression and purification of the individual proteins of two dynamic molecular complexes – Staphylococcus aureus (S. aureus) DNA gyrase and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) replication-transcription complex – and attempted to assemble them into their functional forms for cryo-EM imaging.  Both of these complexes are interesting drug targets as they play an essential role in nucleic acid replication. The function of DNA gyrase is to modulate DNA supercoiling, facilitate DNA replication, and resolve intertwined DNA molecules. The replication-transcription complex of SARS-CoV-2 comprises, among other proteins, the RNA-dependent RNA polymerase, which, together with non-structural proteins 7 and 8, is responsible for the replication of the viral genome. There are still many questions about the underlying mechanisms of these key processes, and time-resolved cryo-EM studies will provide valuable information to advance our understanding of them. Here I present expression and purification protocols for S. aureus DNA gyrase subunits A and B and SARS-CoV-2 non-structural proteins 7, 8 and 12. DNA gyrase subunits A and B were expressed in Escherichia coli (E. coli) and purified in several steps, including affinity chromatography (His-Trap), ion exchange chromatography (IEX) and size exclusion chromatography (SEC). Despite many challenges with gyrase A precipitation, I obtained enough of both subunits for the intended cryo-EM. Different strategies to assemble them into a functional tetramer were tested but did not result in the expected outcome. The gained knowledge about the behaviour of the subunits in solution will serve as a basis for further optimization of the protocols before the assembly of the complex can be attempted again. Non-structural proteins 7 and 8 were expressed in E. coli as a polyprotein and successfully purified using His-Trap and SEC. I obtained a great amount of the polyprotein and established a protocol for its cleavage. Nsp12 was expressed using the baculovirus-insect cell expression system. The immunofluorescence assay data showed that the tested lipofection protocol works, and nsp12 is being produced in sufficient quantities. This result provides a solid base for further experiments to establish a purification method and assemble the nsp12-nsp7-nsp8 complex for cryo-EM imaging.

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The pandemic that struck the entire world 2020 caused by the SARS-CoV-2 (COVID-19) virus, will have an enormous interest for statistical and economical analytics for a long time. While the pandemic of 2020 is not the first that struck the entire world, it is the first pandemic in history where the data were gathered to this extent. Most countries have collected and shared its numbers of cases, tests and deaths related to the COVID-19 virus using different storage methods and different data types. Gaining quality data from the COVID-19 pandemic is a problem most countries had during the pandemic, since it is constantly changing not only for the current situation but also because past values have been altered when additional information has surfaced. The importance of having the latest data available for government officials to make an informed decision, leads to the usage of Business Intelligence tools and techniques for data gathering and aggregation being one way of solving the problem. One of the mostly used software to perform Business Intelligence is the Microsoft develop Power BI, designed to be a powerful visualizing and analysing tool, that could gather all data related to the COVID-19 pandemic into one application. The pandemic caused not only millions of deaths, but it also caused one of the largest drops on the stock market since the Great Recession of 2007. To determine if the deaths or other reasons directly caused the drop, the study modelled the volatility from index funds using Generalized Autoregressive Conditional Heteroscedasticity. One question often asked when talking of the COVID-19 virus, is how deadly the virus is. Analysing the effect the pandemic had on the mortality rate is one way of determining how the pandemic not only affected the mortality rate but also how deadly the virus is. The analysis of the mortality rate was preformed using Seasonal Artificial Neural Network. Forecasting deaths from the pandemic using the Seasonal Artificial Neural Network on the COVID-19 daily deaths data.

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

Effectively treating infectious diseases often requires a multi-step approach to target different components involved in disease pathogenesis. Similarly, the COVID-19 pandemic has become a global health crisis that requires a comprehensive understanding of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection to develop effective therapeutics. One potential strategy to instill greater immune protection against COVID-19 is boosting the innate immune system. This boosting, termed trained immunity, employs immune system modulators to train innate immune cells to produce an enhanced, non-specific immune response upon reactivation following exposure to pathogens, a process that has been studied in the context of and clinical studies prior to the COVID-19 pandemic. Evaluation of the underlying pathways that are essential to inducing protective trained immunity will provide insight into identifying potential therapeutic targets that may alleviate the COVID-19 crisis. Here we review multiple immune training agents, including Bacillus Calmette-Guérin (BCG), β-glucan, and lipopolysaccharide (LPS), and the two most popular cell types involved in trained immunity, monocytes and natural killer (NK) cells, and compare the signaling pathways involved in innate immunity. Additionally, we discuss COVID-19 trained immunity clinical trials, emphasizing the potential of trained immunity to fight SARS-CoV-2 infection. Understanding the mechanisms by which training agents activate innate immune cells to reprogram immune responses may prove beneficial in developing preventive and therapeutic targets against COVID-19.

This study compared the immunogenicity of inactivated SARS-CoV-2 vaccines between people living with HIV (PLWH) and HIV-negative individuals. We recruited 120 PLWH and 53 HIV-negative individuals aged 18-59 years who had received an inactivated SARS-CoV-2 vaccine in two Chinese cities between April and June 2021. Blood samples were tested for immunogenicity of the inactivated SARS-CoV-2 vaccines. The prevalence and severity of adverse events associated with SARS-CoV-2 vaccines were similar between PLWH and HIV-negative individuals. The seropositivity of neutralizing activity against authentic SARS-CoV-2, of the total amount of antibody (total antibody) and of S-IgG were 71.3%, 81.9%, and 92.6%, respectively, among fully vaccinated PLWH. Among all participants, PLWH had lower neutralizing activity, total antibody, S-IgG, and T-cell-specific immune response levels, compared to HIV-negative individuals, after controlling for types of vaccine, time interval between first and second dose, time after receiving the second dose, and sociodemographic factors. PLWH with a longer interval since HIV diagnosis, who received their second dose 15-28 days prior to study commencement, and who had an interval of ≥21 days between first and second dose had higher neutralizing activity levels. The immunogenicity of the inactivated SARS-CoV-2 vaccines was lower among PLWH as compared to HIV-negative individuals. Vaccination guideline specific for PLWH should be developed.

Discussion of the hematologic complications of vaccination for severe acute respiratory syndrome coronavirus-2 (COVID-19) has primarily focused on the development of vaccine-associated immune thrombosis with thrombocytopenia (VITT). Other hematologic complications are uncommon. We report the case of a patient who developed immunoglobulin G (IgG)-mediated autoimmune hemolytic anemia (AIHA) after the Moderna COVID-19 messenger ribonucleic acid (mRNA) vaccine.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

Nous sommes toujours aux prises avec la pandémie de SARS-CoV-2 plus de deux ans après son début. Le virus a depuis accumulé de nombreuses mutations qui ont mené à différentes souches virales au long de la pandémie. Plusieurs de ces mutations sont récurrentes: il y a un excès de mutations C > U dans les génomes viraux, certains codons sont fréquemment mutés vers différents acides aminés et certaines mutations sont convergentes, c’est-à-dire que la même substitution est apparue de manière indépendante sur différentes lignées. Dans ce mémoire, nous avons identifiés différentes manières par lesquelles le SARS-CoV-2 évolue à travers l’étude de ces mutations récurrentes et évaluons leur impact sur l’évasion immunitaire. Premièrement, nous avons déterminés que les mutations C > U sont responsable de l’introduction et du retrait préférentiel d’acides aminés spécifiques dans les épitopes viraux. Nous avons déterminé la significativité statistique de ces patrons de mutation à l’aide de simulations génomiques virales. Deuxièmement, nous avons participé à la surveillance des variants au Québec durant la deuxième vague de la pandémie, qui s’est déroulée d’août 2020 à mars 2021. C’était une période intéressante pour la diversité virale, puisque les restrictions de déplacement ont créé de multiples poches de variants locaux en compétition les uns avec les autres qui partagent des mutations convergentes. Notamment, nous reportons que les lignées B.1.160 et B.1.1.176 comptaient pour 50% des échantillons séquencés au sommet de la deuxième vague dans la province. Finalement, nous avons analysé les patrons mutationnels intra-hôte qui sont apparus de novo dans le contexte d’infections au SARS-CoV-2 de longue durée chez des patients atteints de cancers hématologiques. Une de ces patientes est une patiente québécoise infectée par B.1.160 et dans laquelle nous avons identifié la présence d’un réservoir viral. Nous avons également trouvé des éléments probants montrant différentes quasiespèces virales avec des propriétés d’évasion immunitaire. Nos résultats permettent de mieux comprendre les différentes manières dont les pressions sélectives façonnent l’évolution virale. / We are still living in the SARS-CoV-2 pandemic over two years after its start. The virus has since accumulated many mutations that have led to different viral strains throughout the pandemic. Several of these mutations are recurrent: there is an excess of C > U substitutions in viral genomes, some codons are frequently mutated to different amino acids, and some mutations are convergent, meaning that the same substitution has occurred independently on different lineages. In this thesis, we identified different ways in which SARS-CoV-2 evolves through these recurrent mutations and assess their impact on immune escape. First, we determined that C > U mutations drive the preferential introduction and removal of specific amino acids in viral epitopes. Using genetic simulations, we determined the statistical significance of these patterns. Second, we participated in the surveillance of variants in Quebec during the second wave of the pandemic that went from the end of August 2020 to the end of March 2021. This was an interesting period of viral diversity owing to imposed travel restrictions that created competition between multiple pockets of local strains that share convergent mutations. Notably, we found that lineages B.1.160 and B.1.1.176 account for 50% of samples sequenced at the height of the second wave in the province. Finally, we analyzed intra-host mutational patterns that arose de novo in the context of long-term infections of patients with hematological cancers, one of which was from Québec and infected by B.1.160. We have identified a pattern consistent with the presence of a viral reservoir in this patient. We have also found evidence of different viral quasispecies with immune escape properties. These results shed light on different ways in which selective pressures shape the evolution of SARS-CoV-2.

Background: To investigate whether vaccination against SARS-CoV-2 is associated with the onset of retinal vascular occlusive disease (RVOD). Methods: In this multicentre study, data from patients with central and branch retinal vein occlusion (CRVO and BRVO), central and branch retinal artery occlusion (CRAO and BRAO), and anterior ischaemic optic neuropathy (AION) were retrospectively collected during a 2-month index period (1 June–31 July 2021) according to a defined protocol. The relation to any previous vaccination was documented for the consecutive case series. Numbers of RVOD and COVID-19 vaccination were investigated in a case-by-case analysis. A case– control study using age- and sex-matched controls from the general population (study participants from the Gutenberg Health Study) and an adjusted conditional logistic regression analysis was conducted. Results: Four hundred and twenty-one subjects presenting during the index period (61 days) were enrolled: one hundred and twenty-one patients with CRVO, seventy-five with BRVO, fifty-six with CRAO, sixty-five with BRAO, and one hundred and four with AION. Three hundred and thirty-two (78.9%) patients had been vaccinated before the onset of RVOD. The vaccines given were BNT162b2/BioNTech/Pfizer (n = 221), followed by ChadOx1/AstraZeneca (n = 57), mRNA- 1273/Moderna (n = 21), and Ad26.COV2.S/Johnson & Johnson (n = 11; unknown n = 22). Our case–control analysis integrating population-based data from the GHS yielded no evidence of an increased risk after COVID-19 vaccination (OR = 0.93; 95% CI: 0.60–1.45, p = 0.75) in connection with a vaccination within a 4-week window. Conclusions: To date, there has been no evidence of any association between SARS-CoV-2 vaccination and a higher RVOD risk.