Development and application of -omics and bioinformatics approaches for a deeper understanding of infectious diseases systems INAUGURALDISSERTATION Zur Erlangung der Würde eines Doktors der Philosophie Vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät Der Universität Basel Von Pierre H. H. Schneeberger Ochlenberg (BE) und Frankreich Basel, 2017 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Jürg Utzinger, Dr. Jürg E. Frey und PD Dr. Mauro Tonolla Basel, den 13. Oktober 2015 Prof. Dr. Jörg Schibler Dekan der Philosophisch-Naturwissenschaftlichen Fakultät 2 Acknowledgements • My deepest thanks go to my four mentors, Jürg Frey, Jürg Utzinger, Christian Beuret and Joël Pothier for giving me the freedom of action as well as the required guidance to go through these amazing years. You gave me the opportunity to pursue this PhD and allowed me to bring in my own ideas – this was really a golden opportunity given to a PhD student! You were always open for discussion and there to provide support throughout the various steps of this PhD – sincere thanks for everything. • I would especially like to thank my External Referee, Dr. Mauro Tonolla, for accepting to review my work in such a short notice. • “Mes meilleurs remerciements” to all who contributed significantly within the different projects: Dr. Jean-Sebastien Reynard, Prof. Eliézer N’Goran, Dr. Brion Duffy. • Special thanks to Sören Becker, Samuel Führimann and Andreas Bühlmann for the excellent discussions and collaborations within my different projects. The three of you have, all in different ways, positively influenced me and provided me with exceptional opportunities to further expand the scope of my thesis. • An enormous thank to the people who helped me so much during my laboratory work, and this includes mainly, but is not limited to, Markus Oggenfuss, Beatrice Frey, Dr. Oliver Engler and Jasmine Portmann. • Special thanks to Geoffrey Williams who kindly corrected this document. • To all my old long-time friends from the “french” side. • A final thank goes to my parents, Christine and Georg and to my sister and brother, Séverine and Olivier who have always supported me through sometimes difficult times during my research! 3 Table of Contents Acknowledgements .................................................................................................................................... 3 Abbreviations .............................................................................................................................................. 9 List of tables .............................................................................................................................................. 10 List of figures ............................................................................................................................................. 11 Abstract ...................................................................................................................................................... 13 Résumé ...................................................................................................................................................... 16 Chapter I. Introduction ............................................................................................................................. 19 1. Infectious diseases .......................................................................................................................... 19 a. Definition ....................................................................................................................................... 19 b. Pathogens ..................................................................................................................................... 19 c. Diversity of pathogens ................................................................................................................. 21 d. Pathogenic types ......................................................................................................................... 26 e. Natural reservoirs of pathogens ................................................................................................ 27 f. Cumulative burden of coinfections ............................................................................................. 28 g. Pathogen genomics and associated challenges ..................................................................... 29 h. Pathogen identification and genetic traits ................................................................................ 29 2. Challenges in infectious diseases research ................................................................................ 30 a. Current diagnostic approaches .................................................................................................. 31 b. Culture-based diagnostics .......................................................................................................... 31 c. Microscopy .................................................................................................................................... 32 d. Immunoassays ............................................................................................................................. 33 e. Molecular-based assays ............................................................................................................. 33 3. Next-generation sequencing and implication in pathogen diagnostics ................................... 34 a. Evolution and impact of NGS technologies ............................................................................. 35 b. NGS technologies in 2015 .......................................................................................................... 36 c. NGS meta-analyses: targeted, whole-genome and -transcriptome sequencing ................ 37 4. Overarching goals of the PhD ........................................................................................................ 37 Chapter II. Development and evaluation of a bioinformatics approach for designing molecular assays for viral detection ......................................................................................................................... 40 1. Abstract ............................................................................................................................................. 41 2. Introduction ....................................................................................................................................... 42 3. Methods ............................................................................................................................................. 45 4 a. Hardware and software requirements ...................................................................................... 45 b. Input Data Used for the Workflow ............................................................................................. 46 c. Phylogenetic Analyses ................................................................................................................ 46 d. Viral Samples ............................................................................................................................... 46 e. Nucleic acid isolation ................................................................................................................... 48 f. Real-time PCR and LAMP assays ............................................................................................. 48 4. Results ............................................................................................................................................... 49 a. Workflow Concept ........................................................................................................................ 49 b. Genetic Diversity among the Tested Viruses .......................................................................... 51 c. Workflow Output ........................................................................................................................... 52 5. Discussion ......................................................................................................................................... 60 6. Supporting Information .................................................................................................................... 64 7. Acknowledgements ......................................................................................................................... 64 8. Author Contributions ........................................................................................................................ 65 9. References ........................................................................................................................................ 65 Chapter III. Biological, serological and molecular characterisation of a highly divergent strain of GLRaV-4 causing grapevine leafroll disease ....................................................................................... 72 1. Abstract ............................................................................................................................................. 73 2. Introduction ....................................................................................................................................... 73 3. Materials