Diversity and Evolutionary Origin of the Virus Family Bunyaviridae

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Diversity and Evolutionary Origin of the Virus Family Bunyaviridae Diversity and Evolutionary Origin of the Virus Family Bunyaviridae Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Marco Marklewitz aus Hannover Bonn, 2016 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter: Prof. Dr. Christian Drosten 2. Gutachter: Prof. Dr. Bernhard Misof Tag der Promotion: 21.12.2016 Erscheinungsjahr: 2017 Danksagung Zu Beginn möchte ich mich ganz herzlich bei meinem Doktorvater Prof. Christian Drosten bedanken, dass er mir ermöglicht hat, an einem solch vielfältigen und spannenden Thema zu arbeiten. Für Fragen hatte er jederzeit ein offenes Ohr und bei auftretenden Problemen war er immer sehr hilfsbereit. Des Weiteren möchte ich mich herzlich bei meiner Prüfungskommission, bestehend aus meinem 2. Gutachter Prof. Bernhard Misof sowie Prof. Clemens Simmer und PD Dr. Lars Podsiadlowski, für ihre Zeit und Bereitschaft danken mich zu prüfen. Mein ganz besonder Dank geht an PD Dr. Sandra Junglen für ihre hervorragende und kompetente Betreuung während der Jahre meiner Doktorarbeit. Ich habe es als eine Ehre empfunden, ein Teil ihrer zu Beginn noch sehr jungen Arbeitsgruppe zu sein, danke ihr sehr für ihr Vertrauen und hoffe, sie mit meiner (zukünftigen) Arbeit stolz zu machen. Die Atmosphäre in ihrer Arbeitsgruppe ist immer sehr positiv und ermöglicht, die Arbeit mit viel Spaß zu verbinden. Insbesondere möchte ich herausstellen, dass ich ihr für die Möglichkeit besonders dankbar bin, neben meiner Doktorarbeit Feldarbeiten in Panama durchzuführen. Diese Zeit hat mein Leben auf die positivste Art und Weise nachhaltig beeinflusst. Mein großer Dank gilt auch allen Kolleginnen und Kollegen in den Virologie-Laboratorien der Augenklinik für ihre ständige Hilfs- und Diskussionsbereitschaft, ihr Zuhören bei Problemen sowie für ihre Freundschaft über all die Jahre. Es war eine sehr schöne Zeit mit vielen gemeinsamen Erlebnissen, auf die ich gerne zurückblicke. In gleicher Weise bin ich auch allen anderen Kollegen im Haupthaus der Virologie dankbar. Ich bin sehr glücklich über die Freundschaften, die während meiner Zeit in Bonn hier gewachsen sind. Meinen Eltern und Barbara möchte ich in ganz besonderer Weise danken. Der familiäre Rückhalt in jeglichen Situationen, großes Verständnis in schwierigen Zeiten sowie die Ermöglichung meines Studiums haben das Entstehen dieser Arbeit überhaupt erst ermöglicht. Die Gelegenheit möchte ich nutzen um mich von ganzem Herzen bei Euch zu bedanken. Meiner Freundin Gloria möchte ich für ihr bedingungslose Unterstützung danken. Die gemeinsame Zeit mit Dir ist für mich einfach das Größte! Meinen der Wissenschaft fernen Freunden bin ich für Ihr Verständnis im Zusammenhang mit den von außen betrachtet of seltsam wirkenden Tätigkeiten in der Forschung sehr dankbar. Das gedankliche Abschweifen von dem Alltag in der Wissenschaft hilft manchmal sehr um daraufhin mit neuer Energie weiterzumachen. Vielen Dank an Euch alle. My gratitude to national and international collaborateurs: I would like to acknowledge all coauthors and contributors for their role in the research projects my thesis is part of. Especially I would like to show my appreciation to the field workers at Taï National Park in Ivory Coast and Kibale Nationalpark in Uganda who assisted in the collection of the mosquito samples that were investigated in this thesis. It should not be underestimated how demanding fieldwork under tropical conditions can be and therefore justifies extra credit. Table of Contents 1. SUMMARY ........................................................................................................................................... 1 2. LIST OF PUBLICATIONS ........................................................................................................................ 2 3. GENERAL INTRODUCTION ................................................................................................................... 4 3.1 Insect Viruses ................................................................................................................................. 4 3.2 Arboviruses .................................................................................................................................... 4 3.3 Bunyaviruses .................................................................................................................................. 6 3.3.1 Virions, Genome and Proteins ................................................................................................ 7 3.3.2 Replication Cycle .................................................................................................................... 9 3.3.3 Immunosuppression and Antiviral Host Response .............................................................. 11 3.3.4 Taxonomic Classification ...................................................................................................... 13 3.4 Virus Diversification and Evolution ............................................................................................. 16 3.4.1 Endogenous Virus Elements and the Origin of Viruses ........................................................ 17 4. AIMS OF THE THESIS .......................................................................................................................... 19 Chapter I ............................................................................................................................................. 20 I.1 INTRODUCTION ................................................................................................................................ 21 I.2 RESULTS ............................................................................................................................................ 21 Chapter II ............................................................................................................................................. 32 II.1 INTRODUCTION ............................................................................................................................... 33 II.2 MATERIALS AND METHODS............................................................................................................. 34 II.2.1 Mosquito Collection and Species Identification ....................................................................... 34 II.2.2 Infection of Vertebrate Cells .................................................................................................... 35 II.2.3 RT-PCR Screening ..................................................................................................................... 35 II.2.4 Electron Microscopy ................................................................................................................. 36 II.2.5 Genome Sequencing ................................................................................................................ 36 II.2.6 Genome and Phylogenetic Analyses ........................................................................................ 38 II.2.7 mRNA Analyses ......................................................................................................................... 38 II.2.8 Protein Analyses ....................................................................................................................... 39 II.2.9 Nucleotide Sequence Accession Numbers ............................................................................... 40 II.3 RESULTS ........................................................................................................................................... 40 II.3.1 Detection of a Novel Clade of Mosquito-Associated Bunyaviruses ......................................... 40 II.3.2 Virus Isolation, Growth, and Morphology ................................................................................ 41 II.3.3 Genome Sequencing and Phylogenetic Analyses ..................................................................... 42 I II.3.4 Genome Organization of the Novel Bunyaviruses ................................................................... 43 II.3.5 Transcription Mechanism ......................................................................................................... 44 II.3.6 Major Structural Proteins ......................................................................................................... 46 II.4 DISCUSSION ..................................................................................................................................... 47 Chapter III ............................................................................................................................................. 56 III.1 INTRODUCTION .............................................................................................................................. 57 III.2 THE STUDY ...................................................................................................................................... 57 III.3 MATERIAL & METHODS .................................................................................................................. 63 III.3.1 Growth Kinetics ....................................................................................................................... 63 III.3.2 Amplification of GOLV Glycoprotein Precursor Gene Sequences ..........................................
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