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Cowpox Virus Virulence Determinants

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Cowpox Virus Virulence Determinants Aus dem Institut für Virologie des Fachbereichs Veterinärmedizin der Freien Universität Berlin COWPOX VIRUS VIRULENCE DETERMINANTS Inaugural-Dissertation zur Erlangung des Grades eines Doktors der Veterinärmedizin an der Freien Universität Berlin vorgelegt von Aistė Tamošiūnaitė Tierärztin aus Kaunas, Litauen Berlin 2017 Journal-Nr.: 3934 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Dekan: Univ.-Prof. Dr. Jürgen Zentek Erster Gutachter: Univ.-Prof. Dr. Nikolaus Osterrieder Zweiter Gutachter: Univ.-Prof. Dr. Robert Klopfleisch Dritter Gutachter: PD Dr. Sandra Blome Deskriptoren (nach CAB-Thesaurus): cowpox virus; DNA; plasmids; restriction fragment lenght polymorphism; immunohistochemistry; polymerase chain reaction; electrophoresis; antibiotics; antibodies Tag der Promotion: 20.07.2017 Bibliografische Information der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über <http://dnb.ddb.de> abrufbar. ISBN: 978-3-86387-832-0 Zugl.: Berlin, Freie Univ., Diss., 2017 Dissertation, Freie Universität Berlin D 188 Dieses Werk ist urheberrechtlich geschützt. Alle Rechte, auch die der Übersetzung, des Nachdruckes und der Vervielfältigung des Buches, oder Teilen daraus, vorbehalten. Kein Teil des Werkes darf ohne schriftliche Genehmigung des Verlages in irgendeiner Form reproduziert oder unter Verwendung elektronischer Systeme verarbeitet, vervielfältigt oder verbreitet werden. Die Wiedergabe von Gebrauchsnamen, Warenbezeichnungen, usw. in diesem Werk berechtigt auch ohne besondere Kennzeichnung nicht zu der Annahme, dass solche Namen im Sinne der Warenzeichen- und Markenschutz-Gesetzgebung als frei zu betrachten wären und daher von jedermann benutzt werden dürfen. This document is protected by copyright law. No part of this document may be reproduced in any form by any means without prior written authorization of the publisher. Alle Rechte vorbehalten | all rights reserved © Mensch und Buch Verlag 2017 Choriner Str. 85 - 10119 Berlin [email protected] – www.menschundbuch.de Parts of this thesis are based on following manuscript: Tamošiūnaitė, A., D. Hoffmann, A. Franke, J. Schluckebier, K. Tauscher, B. K. Tischer, M. Beer, R. Klopfleisch, N. Osterrieder. 2016. Histopathological and Immunohistochemical Studies of Cowpox Virus Replication in a Three-Dimensional Skin Model. Journal of Comparative Pathology. 155(1):55-61. __________________________________________________________________________Table of Contents V Table of Contents List of Figures ....................................................................................................................... VII List of Tables ........................................................................................................................ VIII List of Abbreviations .............................................................................................................. IX 1. Introduction ..................................................................................................................... 13 1.1. Poxviruses ............................................................................................................. 13 1.1.1. Classification of poxviruses .......................................................................... 13 1.1.2. Historic hallmarks ......................................................................................... 13 1.1.3. Structure of the poxvirus genome ................................................................. 14 1.1.4. Poxvirus virion forms .................................................................................... 15 1.1.5. The poxvirus replication cycle ...................................................................... 15 1.1.6. Immunomodulatory proteins of OPV ............................................................ 17 1.2. Cowpox virus ........................................................................................................ 18 1.2.1. Clinical presentation ...................................................................................... 20 1.2.2. Variety of cowpox ......................................................................................... 21 1.3. Bacterial artificial chromosomes (BACs) ............................................................. 22 1.4. Project Introduction .............................................................................................. 22 1.5. Specific aims of the project .................................................................................. 23 2. Materials and Methods ................................................................................................... 24 2.1. Materials ............................................................................................................... 24 2.1.1. Chemicals, consumables and equipment ....................................................... 24 2.1.2. Enzymes and markers .................................................................................... 27 2.1.3. Kits ................................................................................................................ 27 2.1.4. Buffers, media and solutions ......................................................................... 28 2.1.5. Antibiotics ..................................................................................................... 29 2.1.6. Antibodies ..................................................................................................... 29 2.1.7. Bacteria, cells and viruses ............................................................................. 30 2.1.8. Bacterial artificial chromosomes (BACs), plasmids and primers ................. 31 2.2. Methods ................................................................................................................ 38 2.2.1. Cell culture. ................................................................................................... 38 2.2.2. Virologic al methods...................................................................................... 39 2.2.3. Molecula r biolog y techniques ....................................................................... 40 2.2.4. Anima l Studie s.............................................................................................. 48 3. Results .............................................................................................................................. 50 3.1. Generation of the knock-in mutants ..................................................................... 50 3.1.1. Sequence analysis of genes of interest .......................................................... 50 3.1.2. Transfer constructs ........................................................................................ 51 __________________________________________________________________________VI Table of Contents 3.1.3. Recombination .............................................................................................. 53 3.1.4. Reconstitution and TK recovery of gain of function mutants ....................... 54 3.2. Generation of the chimeric viruses ....................................................................... 54 3.3. Phenion® full-thickness 3D skin model as possible replacement for animal exper iments........................................................................................................... 60 3.3.1. Histopathological studies of the Uninfected and CPXV-Infected the Phenion® full-thickness skin model Skin Model ......................................... 60 3.3.2. Analysis of Cell Cycle and Gene Expression in the Infected 3D Skin Culture Model................................................................................................ 61 3.4. Virus gr owth ki netics............................................................................................ 63 3.5. In vivo experiments .............................................................................................. 64 4. Discussion ......................................................................................................................... 67 4.1. Generation of recombinant BR, with putative RatPox09 virulence markers inserted.................................................................................................................. 67 4.2. Generation of chimeric viruses. ............................................................................ 70 4.3. Cowpox virus replication in a three-dimensional skin model .............................. 70 4.4. Animal experiments .............................................................................................. 71 5. Outlook ............................................................................................................................. 73 Summary ................................................................................................................................. 74 Zusammenfassung.................................................................................................................. 75 References ............................................................................................................................... 77 List of publications ................................................................................................................. 89 Acknowledgements................................................................................................................
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