Die COVID-19 Pandemie ist die bisher verheerendste Pandemie des 21. Jahrhunderts. Durch die Einführung neuer mRNA-basierter Impfstoffe sowie der hohen Rate natürlicher Infektionen konnte die weltweite SARS-CoV-2-Immunität gesteigert werden. Trotz aller Erfolge zur Eindämmung der Pandemie kann eine Infektion auch heute noch zu schweren Verläufen und Tod führen. Eine adäquate COVID-19-Therapie ist folglich auf potente Virostatika angewiesen. Eine durch Umgehung zeitaufwändiger klinischer Studien schnell verfügbare Alternative zu neu entwickelten Arzneimitteln ist die Anwendung etablierter Medikamente. Wir isolierten und charakterisierten ein von einem Patienten stammendes SARS-CoV-2-Virus. Dieses Virusisolat wurde bisher in elf Publikationen verwendet. Mittels quantitativer Echtzeit-Polymerasekettenreaktion untersuchten wir eine Substanzbibliothek mit mehr als 300 neuen und bereits zugelassenen Wirkstoffen auf ihre Wirksamkeit gegen SARS-CoV-2. Dabei konnten wir zeigen, dass der selektive Serotonin-Wiederaufnahmehemmer Fluoxetin die SARS-CoV-2-Replikation ab einer Dosis von 0,8 μg/ml signifikant inhibiert, einer bei der Behandlung von Depressionen häufig angewandten Dosierung. Der EC50-Wert lag bei 387 ng/ml. Die Behandlung mit Fluoxetin resultierte in einer reduzierten Zahl an Virusprotein-produzierenden Zellen, was darauf hindeutet, dass es die virale Reinfektion und/oder Proteinexpression inhibiert. Fluoxetin ist ein racemisches Gemisch, wobei das (S)-Enantiomer der potentere Serotonin-Wiederaufnahmehemmer ist. Wir konnten zeigen, dass beide Enantiomere einen vergleichbaren antiviralen Effekt gegen SARS-CoV-2 aufweisen, wodurch das (R)-Enantiomer bei virologischer Indikation gegebenenfalls präferiert werden sollte. Fluoxetin hat keinen Einfluss auf die Replikation des Tollwut-Virus und des Humanen Respiratorischen Synzytial-Virus, was auf eine Virusspezifität hindeutet. Weitere aus der Bibliothek stammende signifikante Inhibitoren der SARS-CoV-2-Replikation sind die am Institut für Organische Chemie Würzburg entwickelten Substanzen AKS 232 und AKS 128. Neben der medikamentösen Therapie ist die akkurate Bestimmung neutralisierender Antikörper gegen SARS-CoV-2 zur Quantifizierung des bestehenden (Re-) Infektionsschutzes sowie zur Planung zukünftiger Impfstrategien von großer Bedeutung. Im Rahmen dieser Arbeit entwickelten wir unter Verwendung der quantitativen Echtzeit-Polymerasekettenreaktion erfolgreich ein zuverlässiges Testverfahren zur Detektion neutralisierender anti-SARS-CoV-2 Antikörper. / The COVID-19 pandemic is the most fatal pandemic of the 21st century. The introduction of new mRNA-based vaccines and the high rate of natural infections have increased global SARS-CoV-2 immunity. Despite the successes in containing the pandemic, infection can still lead to severe courses and death. COVID-19 treatment is therefore dependent on potent antivirals. The development of new antivirals is a prolonged process. By bypassing time-consuming clinical trials, repurposing established medication offers a quickly available alternative to newly developed drugs. We isolated and characterized a patient-derived SARS-CoV-2 virus. This virus isolate has been used in 11 publications so far. Using quantitative real-time polymerase chain reaction, we investigated a compound library with more than 300 new and already approved substances against SARS-CoV-2. We showed that the selective serotonin reuptake inhibitor Fluoxetine significantly inhibits SARS-CoV-2 replication at a concentration of 0.8 µg/ml, a dosage frequently used in the treatment of depression. The EC50 was determined with 387 ng/ml. Treatment with Fluoxetine resulted in a decrease in viral protein-expressing cells, indicating that it inhibits viral reinfection and/or protein expression. Fluoxetine is a racemate with the (S)-enantiomer being the dominant serotonin reuptake inhibitor. We have shown that both stereoisomers have a comparable antiviral effect against SARS-CoV-2, suggesting the (R)-enantiomer might be the preferred option for virological indications. Fluoxetine suppressed neither Rabies virus nor human Respiratory Syncytial Virus replication, indicating it is virus specific. Other significant inhibitors of SARS-CoV-2 replication originating in the compound library are AKS 232 and AKS 128, developed with the Institute of Organic Chemistry Würzburg. In addition to drug therapy, the accurate determination of neutralising antibodies against SARS-CoV-2 is of great importance for quantifying the existing (re)infection protection and for planning future vaccination strategies. In this work, we successfully developed a reliable test procedure for the detection of neutralising anti-SARS-CoV-2 antibodies using the quantitative real-time polymerase chain reaction.

By reviewing pre-existing academic literature, research, and data (both international and domestic), this report examines information from a variety of sources to contextualize the threat of COVID-19 against the negative consequences of COVID-19 non-pharmaceutical intervention (NPI) response measures. The qualitative and quantitative data in this report highlights costs associated with COVID-19 response measures relative to the threat of COVID-19 and has been collected to inform a Multiple Accounts Cost-Benefit Analysis (CBA). This report emphasizes the costs associated with NPIs as they relate to physical and mental health, as well as human rights and economic concerns. Overall, a review of available evidence did find a relationship between COVID-19 NPI response measure implementation and negative outcomes. In fact, it remains unclear if NPIs are proportionate or even effective against the risk posed by COVID-19. How NPIs might be optimized (i.e., to reduce the negative effects of their implementation) remains unclear as mild to severe NPI implementation can yield similar outcomes. Following the above analysis, this report provides recommendations to the Government of BC to ensure that COVID-19 response measures are optimized and proposes that a Multiple Accounts CBA of NPI implementation be completed. / Graduate

The complex molecular machinery of the cell controls its response to various signals and environmental conditions. A natural approach to study these molecular mechanisms and cellular processes is with protein interaction networks. Due to the complexity of these networks, sophisticated computational techniques are required to extract biological insights from them. In this thesis, I develop and apply network-based algorithms for three different challenges. 1. I develop a novel, highly-scalable algorithm for network-based label prediction methods that enables the integration of functional annotations and interaction networks across many species in order to predict the functions of genes in newly-sequenced bacteria. 2. To overcome the limitations of experimental approaches to find human proteins and processes that are hijacked by SARS-CoV-2, I adapt network propagation approaches for predicting human interactors of the virus. 3. Large-scale experimental techniques to screen chemicals for toxicity have tested their effects on many individual proteins. I integrate human protein-protein interactions with this data to gain insights into the molecular networks those chemicals affect. For each of these research problems, I perform comprehensive evaluations and downstream analyses to demonstrate both the accuracy of our approaches and their utility in obtaining a broader understanding of the molecular systems in question. / Doctor of Philosophy / The functions of all living cells are governed by complex networks of molecular interactions. A major goal of systems biology is to understand the components of this machinery and how they regulate each other to control the cell's response to various conditions and signals. Advances in experimental techniques to understand these systems over the past couple of decades have led to an explosion of data that probe various aspects of a cell such as genome sequencing, which reads the DNA blueprint, gene expression, which measures the amount of each gene's products in the cell, and the interactions between those products (i.e., proteins). To extract biological insights from these datasets, increasingly sophisticated computational methods are required. A powerful approach is to model the datasets as networks where the individual molecules are the nodes and the interactions between them are the edges. In this thesis, I develop and apply network-based algorithms to utilize molecular systems data for three related problems: (i) predicting the functions of genes in bacterial species, (ii) predicting human proteins and processes that are hijacked by the SARS-CoV-2 virus, and (iii) suggesting cellular signaling pathways affected by exposure to a chemical. Developments such as those presented in these three projects are critical to obtaining a broader understanding of the functions of genes in the cell. Therefore, I make the methods and results for each project easily accessible to aid other researchers in their efforts.

Pandemics are periodic events characterized by rapid and widespread transmission of infectious disease affecting a significant proportion of the population over a large geographical area. Zoonotic strains of influenza viruses and coronaviruses have both caused pandemics in the recent past. Although vaccination is the often the most effective way to prevent infection or serious outcomes of infection, vaccine development, production, distribution, and deployment are all time- consuming and logistically challenges processes. Alternative readily deployed approaches must be quickly executed to mitigate the toll of future pandemics, especially during the early phases. The work described in this thesis describes some of these approaches. Firstly, I describe the process by which I performed genome-wide CRISPR-Cas9 knockout screens using SARS-CoV-2 variants of concern to discover crucial host factors as targets for broad-acting antivirals. I found that all variants rely on similar host pathways to replicate in the glial cell line used for the screen. I identified BCL-xL, a regulator of apoptosis, as a potential target for a broad- acting antiviral. I show that chemical inhibition of BCL-xL results in accelerated cell death in infected cells in vitro, but improved clinical signs and disease mortality of SARS-CoV-2 in our murine infection model. Secondly, I describe a unique mechanism for cooperative antiviral combination therapy. I demonstrate that chemical inhibition of neuraminidase by oseltamivir improved immune effector cell activation by hemagglutinin stalk-binding antibodies. Combination therapy of oseltamivir and stalk-binding antibodies also improved clinical signs and disease mortality of influenza in our murine infection model compared to monotherapy in both prophylactic and therapeutic contexts. Finally, I show that non-pharmaceutical public health interventions used to restrict the spread of COVID-19 were also effective against several other infectious diseases. I used an interrupted time- series analysis on Ontario public health administrative data during the early COVID-19 pandemic period and found a drastic and sustained decline in outpatient visits for diseases that are typically caused by viruses that transmit by droplet or aerosol. The three projects described in this thesis outlines broadly-protective and distinct strategies to curb the spread of novel respiratory viruses. These new tools may be leveraged to improve the response and to mitigate the burden of future pandemics. / Thesis / Doctor of Philosophy (PhD) / Most pandemics in recent history have been caused by viruses that infect the respiratory tract. Vaccination is often the best way to prevent the spread of these pandemic viruses, but making these vaccines takes time. Vaccines also work less well in the very young, the elderly, and those with a compromised immune system. These people are often also the most vulnerable to severe disease. My work describes three novel approaches to help combat the next pandemic, especially during the early phases when vaccines are still being developed, or for the segments of the population that respond poorly to vaccination. These include discovering and using new drugs that work against a wide range of viruses, using combinations of previously-discovered antiviral drugs, and using non-pharmaceutical methods such as physical distancing and wearing masks.

Yes / Since December 2019, a new type of coronavirus called novel coronavirus (2019-nCoV, or COVID-19) was identified in Wuhan, China and on March 11, 2020, the World Health Organization (WHO) has declared the novel coronavirus (COVID-19) outbreak a global pandemic. With more than 101,797,158 confirmed cases, resulting in 3,451,354 deaths as of May 21, 2021, the world faces an unprecedented economic, social, and health impact. The clinical spectrum of COVID-19 has a wide range of manifestations, ranging from an asymptomatic state or mild respiratory symptoms to severe viral pneumonia and acute respiratory distress syndrome. Several diagnostic methods are currently available for detecting the coronavirus in clinical, research, and public health laboratories. Some tests detect the infection directly by detecting the viral RNA using real time reverse transcriptase polymerase chain reaction (RT-PCR) and other tests detect the infection indirectly by detecting the host antibodies. Additional techniques are using medical imaging diagnostic tools such as X-ray and computed tomography (CT). Various approaches have been employed in the development of COVID-19 therapies. Some of these approaches use drug repurposing (eg Remdesivir and Dexamethasone) and combinational therapy (eg Lopinavir/Ritonavir), whilst others aim to develop anti-viral vaccines (eg mRNA and antibody). Additionally, health experts integrate data sharing, provide with guidelines and advice to minimize the effects of the pandemic. These guidelines include wearing masks, avoiding direct contact with infectious people, respiratory and personal hygiene.

COVID-19 Patientinnen und Patienten haben ein hohes thrombotisches Risiko. Die Sicherheit und Wirksamkeit verschiedener Antikoagulationsschemata bei COVID-19 Patientinnen und Patienten sind unklar. Acht RCTs mit 5580 Patientinnen und Patienten wurden identifiziert, wovon zwei RCTs Antikoagulation in halbtherapeutischer und sechs RCTs Antikoagulation in therapeutischer Dosierung mit der Standard Thromboembolieprophylaxe verglichen haben. Die halbtherapeutische Antikoagulation kann wenig oder gar keinen Einfluss auf thrombotische Ereignisse oder Todesfälle haben (RR 1,03, 95% KI 0,86-1,24), kann aber schwere Blutungen (RR 1,48, 95% KI 0,53-4,15) bei mittelschweren bis schweren COVID-19 Patientinnen und Patienten verstärken. Therapeutische Antikoagulation kann thrombotische Ereignisse oder den Tod bei Patientinnen und Patienten mit mittelschwerem COVID-19 (RR 0,64, 95% KI 0,38-1,07) verringern, kann aber bei Patientinnen und Patienten mit schwerer Erkrankung (RR 0,98, 95% KI 0,86-1,12) wenig oder keine Wirkung haben. Das Risiko schwerer Blutungen kann unabhängig vom Schweregrad der Erkrankung zunehmen (RR 1,78, 95% KI 1,15-2,74). Die Evidenzsicherheit ist immer noch gering. Mäßig betroffene COVID-19 Patientinnen und Patienten können von einer therapeutischen Antikoagulation profitieren, jedoch ist das Blutungsrisiko erhöht. / COVID-19 patients have a high risk of thrombotic disease. The safety and efficacy of various anticoagulation regimens in COVID-19 patients remains unclear. Eight RCTs with 5580 patients were identified, with two comparing anticoagulation in intermediate doses and six comparing therapeutic doses to standard thromboembolism prophylaxis. Intermediate anticoagulation may have little or no effect on thrombotic events or deaths (RR 1.03, 95% CI 0.86-1.24), but may increase major bleeding (RR 1.48, 95% CI 0.53-4.15) in moderate to severe COVID-19 patients. Therapeutic anticoagulation may reduce thrombotic events or death in patients with moderate COVID-19 (RR 0.64, 95% CI 0.38-1.07), but may have little or no effect in patients with severe disease (RR 0.98, 95% CI 0.86-1.12). The risk of major bleeding may increase regardless of the severity of the disease (RR 1.78, 95% CI 1.15-2.74). The certainty of evidence is still low. Moderately affected COVID-19 patients may benefit from therapeutic anticoagulation, but the risk of bleeding is increased.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25203号 / 医科博第159号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 濵﨑 洋子, 教授 中川 一路, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

–1 programmed ribosomal frameshifting (–1PRF) is a fundamental recoding process that makes alternative reading frames accessible during translation. Especially RNA viruses rely on this mechanism to regulate gene expression for example while main- taining the correct stoichiometry of replicative enzymes and structural proteins. –1PRF is at the heart of viral replication and it has been shown that perturbations of –1PRF efficiency can have dramatic effects on viral fitness. In the present work, two viral –1PRF elements were studied: severe acute respi- ratory syndrome coronavirus 2 (SARS-CoV-2) ORF1 a/b and human immunodeficiency virus 1 (HIV-1) gag-pol. In a mutational study of the SARS-CoV-2 –1PRF element it was confirmed that it functions in its pseudoknot conformation. Additionally, through mutagenesis as well as targeting by antisense oligonucleotides, it was shown that the structure of the RNA directly correlates to –1PRF efficiency. Next, protein interactors of both –1PRF elements were captured revealing over 100 interactors for SARS-CoV-2 and almost 50 for HIV-1. Through this, a set of 18 core interactors of the unrelated –1PRF elements of SARS-CoV-2 and HIV-1 was identified. Among those, the two proteins HNRNPH1 and ZNF346 were shown to reduce –1PRF efficiency significantly for both sites. Among the interactors of SARS-CoV or HIV-1 alone, the strongest effect was mediated by ZAP-S for SARS-CoV-2 and SRBD1 for HIV-1. Finally, it was shown that overexpression of ZAP-S reduced the replication of SARS- CoV-2 by 95% in collaboration with the group of Prof. Dr. Čičin-Šain. In addition, using pseudotyped HIV-1 particles, overexpression of SRBD1 reduced RTase levels and by extension viral titers by 90%. / Das –1 programmierte ribosomale Frameshifting (–1PRF) ist ein grundlegender Umkodierungsprozess, der alternative Leseraster während der Translation zugänglich macht. Insbesondere RNA-Viren sind auf diesen Mechanismus angewiesen, um beispielsweise die Genexpression zu regulieren und gleichzeitig die korrekte Stöchiometrie der replizierenden Enzyme und Strukturproteine aufrechtzuerhalten. Das –1PRF ist das Herzstück der viralen Replikation, und es hat sich gezeigt, dass Störungen der –1PRF-Effizienz dramatische Auswirkungen auf die virale Fitness haben können. In der vorliegenden Arbeit wurden zwei virale –1PRF-Elemente untersucht: das Coronavirus 2 des schweren akuten Respirationssyndroms (SARS-CoV-2) ORF1 a/b und das humane Immunschwächevirus 1 (HIV-1) gag-pol. In einer Mutationsstudie des SARS-CoV-2 –1PRF-Elements wurde bestätigt, dass es in seiner Pseudoknotenkonformation funktioniert. Darüber hinaus wurde durch Mutagenese sowie durch Targeting mit Antisense-Oligonukleotiden gezeigt, dass die Struktur der RNA direkt mit der –1PRF-Effizienz korreliert. Als Nächstes wurden die Protein-Interaktoren beider –1PRF-Elemente erfasst, wobei über 100 Interaktoren für SARS-CoV-2 und fast 50 für HIV-1 gefunden wurden. Auf diese Weise wurde eine Gruppe von 18 Kerninteraktoren der nicht miteinander verbundenen –1PRF-Elemente von SARS-CoV-2 und HIV-1 identifiziert. Unter diesen konnten die beiden Proteine HNRNPH1 und ZNF346 die –1PRF-Effizienz an beiden Stellen deutlich verringern. Unter den Interaktoren von SARS-CoV oder HIV-1 allein wurde die stärkste Wirkung durch ZAP-S für SARS-CoV-2 und SRBD1 für HIV-1vermittelt. Schließlich wurde in Zusammenarbeit mit der Gruppe von Prof. Dr. Čičin-Šain gezeigt, dass die Überexpression von ZAP-S die Replikation von SARS-CoV-2 um 95% reduziert. Darüber hinaus reduzierte die Überexpression von SRBD1 unter Verwendung von pseudotypisierten HIV-1-Partikeln die RTase-Werte und damit auch die Virustiter um 90%.

The aim of this diploma thesis was to describe information about SARS-CoV-2 virus and coronavirus disease (COVID-19), to evaluate the course of the disease in the Home with a special regime in Břevnice, in which the epidemic took place at the beginning of the first wave of COVID-19 pandemic in spring 2020. Another goal was to perform an antibody analysis and evaluate the obtained data. I divided the diploma thesis into three main parts: theoretical, experimental and discussion. In the theoretical part, I worked with the literature and described information related not only to COVID-19, but also to other serious infections caused by human coronaviruses. In the experimental part, which I performed in the immunological laboratory of the Department of Joint Laboratories at Havlíčkův Brod Hospital, I dealt with the issue of the clinical course of the disease, the severity of the disease and possible consequences after the infection. I also dealt with methods for the determination of antibodies, the principles of which are described in Chapter 4.5. The diagnostic methods and procedures used are described in Chapter 5.3 Laboratory Assays. From the obtained data, I prepared graphs and tables and processed data on the clinical course of the infection in the Břevnice Home, both for the clients and the...

Se validó y evaluó un método de RT-PCR en tiempo real usando cebadores y sondas específicas para los genes RdRP de SARS-CoV-2 y GAPDH de humanos; este último fue usado como control endógeno. Se evaluó la especificidad y sensibilidad; además, se evaluó otros parámetros como la robustez, la repetibilidad, reproducibilidad, comparabilidad y el límite de detección. La sensibilidad, especificidad, los valores predictivos positivo y negativo, la robustez, comparabilidad y la repetibilidad-reproducibilidad de la prueba de RT-PCR en tiempo real dúplex fue de 100%, con un límite de detección de 100 copias/μL, de acuerdo con los criterios de aceptación establecidos para validación del protocolo. Esta prueba estandarizada es una buena alternativa para el diagnóstico de COVID-19; además, la prueba fue aplicada de manera exitosa en personas sospechosas de la enfermedad permitiendo controlar el número de falsos negativos.