An Out-Bred Animal Model of Cottontail Rabbit Papillomavirus Latent Infection
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An Out-bred Animal Model of Cottontail Rabbit Papillomavirus Latent Infection Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Jin Xi aus Sichuan, China P.R. Tübingen 2017 Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen. Tag der mündlichen Qualifikation: 26.09.2017 Dekan: Prof. Dr. Wolfgang Rosenstiel 1. Berichterstatter: Prof. Dr. Thomas Iftner 2. Berichterstatter: Prof. Dr. Friedrich Goetz CONTENT ABSTRACT ................................................................................................................. I ZUSAMMENFASSUNG ............................................................................................. III ABBREVIATIONS ....................................................................................................... V 1 INTRODUCTION ..................................................................................................... 1 1.1 Papillomavirus (PV) .............................................................................................. 1 1.2 The life cycle of PVs ............................................................................................. 3 1.2.1 Attachment, entry, uncoating and intracellular trafficking ............................................ 3 1.2.2 Viral transcription, replication, transformation and release .......................................... 3 1.3 CuHPV and Non-melanoma skin cancer (NMSC) ................................................ 5 1.3.1 Epidermodysplasia Verruciformis (EV) ........................................................................... 6 1.3.2 Organ Transplant Recipients (OTRs) .............................................................................. 7 1.3.3 UV radiation and cuHPV infection ................................................................................. 7 1.3.4 Epidermal stem cells (ESC) and cuHPV infection ............................................................ 8 1.4 PV latent infection ................................................................................................. 8 1.4.1 Clinical evidence for HPV latency .................................................................................. 9 1.4.1.1 Immunocompromised patient ............................................................................ 9 1.4.1.2 Recurrent respiratory papillomatosis (RRP) ........................................................ 9 1.4.1.3 Recurrent genital papillomas ............................................................................. 10 1.4.1.4 Latent infection of the cervix ............................................................................. 10 1.4.2 Animal models of PV latent infection ........................................................................... 10 1.4.2.1 Latency of Rabbit oral papillomavirus (ROPV) .................................................... 11 1.4.2.2 Latency of Cottontail rabbit papillomavirus (CRPV) ........................................... 11 1.4.2.3 Latency of Bovine papillomavirus 2 (BPV2) ........................................................ 12 1.4.2.4 Latency of Mastomys natalensis papillomavirus (MnPV) ................................... 12 1.4.2.5 Latency of Mus Musculus papillomavirus 1 (MmuPV1) model ........................... 12 - 1 - 1.5 Introduction to CRPV model system ................................................................... 13 1.5.1 Molecular biology of CRPV ........................................................................................... 13 1.5.1.1 Viral transcriptions ............................................................................................ 14 1.5.1.2 Viral proteins ..................................................................................................... 15 1.5.1.3 State of CRPV DNA ............................................................................................ 17 1.5.2 Immune response and regression ................................................................................ 17 1.6 Study objectives ................................................................................................. 18 2 MATERIALS AND METHODS ............................................................................... 20 2.1 Materials ............................................................................................................. 20 2.1.1 Animals and viral DNA ................................................................................................. 20 2.1.2 Equipment ................................................................................................................... 20 2.1.3 Accessories .................................................................................................................. 21 2.1.4 Chemicals .................................................................................................................... 22 2.1.5 Medical drugs .............................................................................................................. 22 2.1.6 Commercial kits ........................................................................................................... 22 2.1.7 Enzymes....................................................................................................................... 23 2.1.8 Antibodies ................................................................................................................... 23 2.1.9 Oligonucleotides .......................................................................................................... 24 2.1.10 Bacterial strains ......................................................................................................... 26 2.1.11 Eukaryotic cell lines .................................................................................................... 26 2.1.12 Cell culture Medium .................................................................................................. 26 2.1.13 Reagents and solution buffers .................................................................................... 26 2.1.14 Software .................................................................................................................... 27 2.2 Methods .............................................................................................................. 27 2.2.1 Molecular biology methods ......................................................................................... 27 - 2 - 2.2.1.1 General cloning ................................................................................................. 27 2.2.1.2 Transformation and propagation ....................................................................... 28 2.2.1.3 Mini prep and Maxi prep ................................................................................... 28 2.2.2 DNA methods .............................................................................................................. 28 2.2.2.1 DNA extraction and purification ........................................................................ 28 2.2.2.2 Normal PCR ....................................................................................................... 29 2.2.2.3 Real-time quantitative PCR (RT-qPCR) ............................................................... 29 2.2.2.4 Rolling circle amplification (RCA) ....................................................................... 30 2.2.2.5 Bisulfite-based cytosine methylation analysis .................................................... 30 2.2.2.6 DNA sequencing ................................................................................................ 30 2.2.3 RNA methods ............................................................................................................... 30 2.2.4 In vitro cell culture methods ........................................................................................ 31 2.2.4.1 Normal cell line culture ..................................................................................... 31 2.2.4.2 Ex vivo primary cell culture ................................................................................ 31 2.2.4.3 Transfection of Eukaryotic cell........................................................................... 32 2.2.4.4 Cell proliferation WST-1 assay ........................................................................... 32 2.2.4.5 Immunofluorescence staining ........................................................................... 32 2.3 Animal experiments ............................................................................................ 33 2.3.1 Ethical approval and project license application ........................................................... 33 2.3.2 Helios® Gene Gun infection .......................................................................................... 33 2.3.2.1 Precipitation of CRPV DNA to gold particles ...................................................... 33 2.3.2.2 Preparation of cartridges for Helios® Gene Gun System ..................................... 33 2.3.2.3 Delivering of CRPV DNA by Helios® Gene Gun System ........................................ 34 2.3.3 Sample collection from rabbits .................................................................................... 34 2.3.4 Drug-induced immunosuppression