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Evaluation of Diagnostic Methods for Vector-Borne Viral Infections and Development of Tools for the Study of Yellow Fever Infections

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Evaluation of Diagnostic Methods for Vector-Borne Viral Infections and Development of Tools for the Study of Yellow Fever Infections Evaluation of diagnostic methods for vector-borne viral infections and development of tools for the study of yellow fever infections Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. Nat.) submitted to the Department of Biology, Chemistry and Pharmacy of the Freie Universität of Berlin by Camille Escadafal from Châtenay-Malabry (France) 2014 The presented Ph.D. thesis was conducted from September 2011 to May 2014 at Robert Koch Institute, Berlin, Germany under the supervision of Prof. Dr. Matthias Niedrig. 1st reviewer: Prof. Dr. Matthias Niedrig Robert Koch Institute, Berlin 2nd reviewer: Prof. Dr. Rupert Mutzel Freie Universität Berlin Date of defense: 18th July 2014 DECLARATION OF AUTHORSHIP I certify that the work presented here is, to the best of my knowledge and belief, original and the result of my own investigations, except as acknowledged. The present work has not been submitted, either in part or completely, for a degree at this or any other University. Date : ______________ Signature: _______________ I II ACKNOWLEDGEMENTS It is a pleasure for me to thank the persons who have supported me during the achievement of this PhD thesis: My supervisor at the Robert Koch Institute, Professor Matthias Niedrig, for offering me such a great working experience and learning opportunity. At his side, I have learned a lot in the field of virology but also about human relationships. I also thank him for his kindness, support and liberty of initiative he gave me throughout all this work. My supervisor at the Freie Universität, Professor Rupert Mützel, for listening and advising me and facilitating every stage of the process. Nina Stock, for her availability, her support, her joyful energy and her friendship. Pranav Patel, for his help and for sharing knowledge and fructifying ideas. Oliver Donoso-Mantke, for being the greatest coordinator I ever had! Pauline Prüger, for her constant dedication and for her communicative thirst of knowledge. Dia Roy Chowdhury and Ravish Paliwal, for their words and laughs of solidarity throughout our collective adventure. Nadine Litzba, for sharing her knowledge in science and her wisdom in life. Peter Hagerdorn, for introducing me to the fascinating world of ticks! Regina Schädler, for being such a great support since my first day in Berlin. Andrea Sanchini and Sergejs Nikisins, for providing an improved version of the EUPHEM fellow in Berlin and a fun multicultural work environment! Marion Mielke, for sweeping away bad moods each morning. Anette Teichmann, for her joyful assistance. My parents, Richard and Colette, for supporting me in all my choices. My husband, Omar Latsouck, for his every day comfort, love and indulgence. III ABSTRACT Vector-borne diseases represent over 17% of all infectious diseases in humans causing one billion deaths each year. Public health efforts to strengthen vector-borne disease detection, surveillance and control have been intensified and laboratories play a key role in this process. Since diagnostic methods for vector-borne infections are often not standardized, it is essential to evaluate the quality of diagnostic methods currently in use in laboratories by organizing external quality assurance (EQA) programs. The first chapter of this dissertation presents the organization and analysis of five EQA studies on the serological and/or molecular detection of hantavirus, yellow fever virus, Rift Valley fever virus and Crimean-Congo hemorrhagic fever virus. The analysis of all study results enabled to identify important weak points in the diagnostic procedures performed by the different participating laboratories and propose recommendations for the future development of EQAs. The next chapters of the dissertation focus on the study and the development of diagnostic tools for yellow fever virus (YFV) infections. The second chapter describes the development of an isothermal molecular method for YFV detection using recombinase polymerase amplification (RPA). The YFV RPA assay proved to be a specific and sensitive detection method during testing with a low detection limit and rapid processing time by using a portable instrument. Therefore the RPA method can provide an affordable alternative to current PCR-based technologies allowing an early and accurate molecular diagnosis of YFV infections in low-resource remote endemic areas. A second diagnostic approach for YFV infections was investigated by producing antibodies for the specific detection of the non-structural 1 (NS1) protein which is already used as an early diagnostic marker for dengue infections. Production and characterization of three polyclonal anti-NS1 sera from guinea pig was performed and allowed specific detection of the YFV NS1 in immunofluorescence and Western blot assays. Even though the development of an ELISA assay could not be brought to success within the frame of this study, preliminary results confirm the NS1 protein as a promising diagnostic marker for early detection of YFV infections and provide a basis for the further development of a quantitative assay for YFV NS1 detection. The third and last chapter of this dissertation describes the development of tools for the study of the pathogenesis of YFV infections in live cell imaging microscopy. For that purpose, fluorescent labeling of viral proteins of the 17D vaccine strain was performed by using the tetracysteine (TC-) tag technology (FlAsH). The project had been initiated at the Robert Koch IV Institute where four full-length YFV-17D plasmids with the TC-tag in the envelope or the capsid (C) protein had been constructed. In this work, the potential use of the four YFV-17D constructs as models for the study of YFV pathogenesis was investigated. One construct with the TC-tag in the C protein lead to the production of live infectious virus particles and showed similar growth characteristics compared to the parental 17D-virus. However, further experiments revealed that the TC-tagged virus was not able to produce fluorescence after staining with the FlAsH-reagent. In summary, the various results of this PhD work are a significant contribution to the development of international EQA programs for diagnostic methods of vector-borne viral infections and to the enlargement of the methodological spectrum for the investigation and diagnostics of yellow fever virus. V ZUSAMMENFASSUNG Vektorübertragene Krankheiten verursachen über 17% aller Infektionskrankheiten im Menschen und dadurch eine Milliarde Todesfälle pro Jahr. Bestrebungen des Gesundheitswesens zur Erkennung, Überwachung und Kontrolle vektorübetragener Krankheiten sind kontinuierlich verstärkt worden und mikrobiologische Labore spielen dabei eine Schlüsselrolle. Da diagnostische Methoden zur Detektion vektorübertragener Krankheiten häufig nicht standardisiert sind, ist es von groẞer Bedeutung die Qualität der derzeitig in den Laboren verwendeten Methoden durch die Durchführung externer Qualitätskontrollen (EQA) zu evaluieren. Das erste Kapitel dieser Dissertation beschreibt die Organisation und Analyse von fünf EQA Studien zur serologischen und/oder molekularbiologischen Detektion von Hantaviren, Gelbfieberviren, Rift Valley Fieber- und Krim-Kongo Hämorrhagisches Fieberviren. Die Analyse aller Untersuchungsergebnisse zeigt Schwachpunkte in den diagnostischen Prozessen der teilnehmenden Labore auf und liefert Verbesserungsvorschläge für die zukünftige Entwicklung von EQAs. Die weiteren Kapitel dieser Arbeit konzentrieren sich auf die Untersuchung und Entwicklung diagnostischer Methoden zum Nachweis von Gelbfieberviren (GFV). Das zweite Kapitel beschreibt die Entwicklung einer molekularen, isothermalen Methode zur GFV Detektion mittels recombinase polymerase amplification (RPA). Hierbei erwies sich der GFV RPA Assay als spezifische und sensitive Nachweismethode mit einem niedrigen Detektionslimit und kurzer Bearbeitungszeit, die in einem tragbaren Gerät durchgeführt werden kann. Die RPA Methode bietet daher eine erschwingliche Alternative zu gegenwärtigen PCR-basierten Technologien, die eine frühe und genaue Diagnose von GFV Infektionen besonders in unterversorgten und entlegenen Endemiegebieten ermöglicht. Ein zweiter diagnostischer Ansatz zum Nachweis von GFV Infektionen wurde durch die Produktion von Antikörpern zur spezifischen Detektion des NS1-Proteins untersucht. Das NS1-Protein wird bereits als früher diagnostischer Marker bei Infektionen mit dem Denguevirus verwendet. Insgesamt wurden drei polyklonale anti-NS1 Seren in Meerschweinchen hergestellt und charakterisiert, die eine spezifische Detektion des GFV NS1-Proteins im Immunfluoreszenztest und Western Blot ermöglichen. Obwohl die Entwicklung eines ELISA-Tests im Rahmen dieser Arbeit nicht erfolgreich abgeschlossen werden konnte, bestätigen die vorläufigen Ergebnisse das diagnostische Potential des NS1- Proteins für eine frühe Detektion von GFV-Infektionen und liefern eine Grundlage für die weitere Entwicklung eines quantitativen Tests zum Nachweis des GFV NS1-Proteins. VI Das dritte und letzte Kapitel dieser Dissertation beschreibt die Entwicklung eines Tools zur Untersuchung der GFV-Pathogenese mittels live cell imaging-Mikroskopie. Dazu wurden virale Proteine des GFV Impfstammes 17D unter Anwendung der Tetracystein (TC)- Technologie fluoreszenzmarkiert. Das Projekt wurde zuvor am Robert Koch-Institut initiiert, wo vier GFV-17D Volllängeklone hergestellt wurden, die den TC-Marker im Hüll- oder Capsid (C)-Protein tragen. In dieser Arbeit wurde die Möglichkeit untersucht, diese vier GFV-17D Konstrukte
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