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And Herpesvirus Infecti Aus dem Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie Lehrstuhl Virologie der Ludwig-Maximilians-Universität München Vorstand: Prof. Dr. med. Oliver T. Keppler Adeno-associated virus-based heterologous replicon technology for detection and quantification of adeno- and herpesvirus infections Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Simona Langer, geb. Sigl aus Hollabrunn, Österreich 2019 Mit Genehmigung der Medizinischen Fakultät der Universität München Betreuerin: Priv. Doz. Dr. Barbara Adler Zweitgutachter: Prof. Dr. Reinhard Zeidler Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 02.08.2019 Eidesstattliche Versicherung Langer Simona Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema Adeno-associated virus-based heterologous replicon technology for detection and quantification of adeno- and herpesvirus infections selbstständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstellen einzeln nachgewiesen habe. Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde. München, 16.02.2019 Simona Langer This page intentionally left blank. Table of contents Zusammenfassung .............................................................................................................................................. v Summary ......................................................................................................................................................... vii 1. Introduction ......................................................................................................................................... 1 1.1. Microbial drug resistance .......................................................................................................................... 1 1.1.1. Viral resistance development ............................................................................................................ 2 1.1.2. Antiviral drug-resistant tests ............................................................................................................. 3 1.2. Biology of large DNA viruses and their inhibitors ...................................................................................... 5 1.2.1. Herpesviruses .................................................................................................................................... 6 1.2.2. Herpes simplex viruses ...................................................................................................................... 7 1.2.3. Human adenoviruses and their replication ..................................................................................... 15 1.3. Replicon-based reporter systems in virology .......................................................................................... 22 1.3.1. RNA virus replicon systems ............................................................................................................. 23 1.3.2. DNA virus replicon systems ............................................................................................................. 23 1.4. Adeno-associated virus as basis for a replicon vector ............................................................................. 24 1.4.1. Adeno-Associated Virus and their replication ................................................................................. 24 1.5. Objectives ................................................................................................................................................ 30 2. Material ............................................................................................................................................... 31 2.1. Devices ..................................................................................................................................................... 31 2.2. Consumables ............................................................................................................................................ 31 2.3. Reagents and biochemicals...................................................................................................................... 32 2.4. Commercial Kits ....................................................................................................................................... 33 2.5. Culture Media for bacteriology and cell culture ...................................................................................... 34 2.6. Oligonucleotides ...................................................................................................................................... 35 2.7. Enzymes for molecular biology ................................................................................................................ 36 2.8. Plasmids ................................................................................................................................................... 36 2.8.1. Plasmids that were constructed in this study ................................................................................. 37 2.9. Bacterial artificial chromosomes ............................................................................................................. 38 2.9.1. Published BACs and BACs available in the group ............................................................................ 38 2.9.2. BAC’s cloned during this study ........................................................................................................ 38 2.9.3. Viruses ............................................................................................................................................. 38 2.9.4. Recombinant viral particles ............................................................................................................. 40 3. Methods .............................................................................................................................................. 41 3.1. Propagation of recombinant DNA in E. coli ............................................................................................. 41 3.1.1. Culturing recombinant E. coli .......................................................................................................... 41 3.1.2. Preparation of electro-competent bacteria .................................................................................... 41 3.1.3. Transformation of electro-competent bacteria .............................................................................. 42 3.1.4. Isolation of DNA from bacteria ........................................................................................................ 43 3.2. Analysis and cloning of recombinant DNA ............................................................................................... 44 i 3.2.1. Determination of DNA concentration ............................................................................................. 44 3.2.2. Ethanol precipitation ....................................................................................................................... 44 3.2.3. Polymerase chain reaction (PCR) .................................................................................................... 44 3.2.4. Restriction enzyme digest ............................................................................................................... 45 3.2.5. Agarose gel electrophoresis ............................................................................................................ 45 3.2.6. Purification of DNA from agarose gel .............................................................................................. 46 3.2.7. Blunting of DNA ends ...................................................................................................................... 46 3.2.8. Ligation of DNA fragments .............................................................................................................. 46 3.2.9. DNA sequencing .............................................................................................................................. 47 3.3. Mutagenesis of BAC DNA ......................................................................................................................... 47 3.3.1. Homologous recombination of BACs .............................................................................................. 47 3.3.2. Flp/FRT recombination system ........................................................................................................ 48 3.4. Tissue culture ........................................................................................................................................... 48 3.4.1. Cultivation of mammalian cell lines ................................................................................................ 48 3.4.2. Cryopreservation of mammalian cell lines ...................................................................................... 49 3.4.3. Transfection of cultured mammalian cells .....................................................................................
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