Medizinische Hochschule Hannover Institut für Virologie/ Helmholtz Zentrum für Infektionsforschung Detection of murine herpesvirus 68 by the innate immune system and studies on Mus musculus rhadinovirus 1 in its natural host INAUGURALDISSERTATION zur Erlangung des Grades einer Doktorin der Naturwissenschaften - Doctor rerum naturalium - (Dr. rer. nat.) vorgelegt von Sripriya Murthy aus Bangalore Hannover 2016 Angenommen vom Senat der Medizinischen Hochschule Hannover am 16.09.2016 Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover Präsident: Prof. Dr. med. Christopher Baum Betreuer: Prof. Dr. rer. nat. Melanie M. Brinkmann Kobetreuer: Prof. André Bleich, PhD 1. Gutachter: Prof. Dr. rer. nat. Melanie M. Brinkmann 2. Gutachter: Prof. André Bleich, PhD 3. Gutachter: Prof. Dr. rer. nat. Beate Sodeik Tag der mündlichen Prüfung vor der Prüfungskomission: 16.09.2016 Prof. Dr. rer. nat. Christine Falk Prof. Dr. rer. nat. Melanie M. Brinkmann Prof. André Bleich, PhD Prof. Dr. rer. nat. Beate Sodeik Table of contents 1 Introduction ............................................................................................................ 1 1.1 Herpesviridae .................................................................................................... 1 1.1.1 Betaherpesvirinae ......................................................................................... 2 1.1.2 Gammaherpesvirinae .................................................................................... 4 1.2 Host defense ..................................................................................................... 9 1.2.1 Pattern recognition receptors ...................................................................... 12 1.2.2 UNC93B and TLR trafficking ....................................................................... 20 1.2.3 TLR-mediated detection and control of MHV68 ........................................... 24 1.3 Aims of the study ............................................................................................. 28 2 Materials and Methods ......................................................................................... 29 2.1 Buffers and solutions ....................................................................................... 29 2.2 Commercially available kits .............................................................................. 30 2.3 Cloning of plasmid controls .............................................................................. 30 2.3.1 Bacteria ....................................................................................................... 31 2.3.2 Determination of copy number to assess PCR sensitivity ............................ 33 2.4 Extraction of DNA from tissue samples ............................................................ 33 2.5 PCR analysis for the detection of MmusRHV1, MCMV1, MCMV2 and MHV68 .................................................................................................... 34 2.5.1 Nested PCR to detect the presence of MmusRHV1, MCMV1 and MCMV2 ................................................................................................ 34 2.5.2 Non-invasive blood PCR to screen for the presence of MmusRHV1 ................................................................................................. 34 2.5.3 Limiting dilution nested PCR for the detection of MHV68 ORF50 ........................................................................................................ 37 2.6 Cell culture ...................................................................................................... 38 2.6.1 Primary cells – preparation of dendritic cells with Flt3 ligand and GM-CSF ............................................................................................... 38 2.7 Viruses ............................................................................................................ 39 2.8 Cell sorting ...................................................................................................... 40 2.8.1 Fluorescence-activated cell sorting (FACS) ................................................. 40 2.8.2 Magnetic-activated cell sorting (MACS) ....................................................... 40 2.9 Stimulation of MmusRHV1 reactivation ............................................................ 42 2.10 Stimulation of TLR with viruses and TLR ligands for TNFα and IFNα measurement by enzyme-linked immunosorbent assay (ELISA) ............ 42 2.11 ELISA .............................................................................................................. 43 2.11.1 TNFα ELISA ............................................................................................. 44 2.11.2 IFNα ELISA ............................................................................................... 45 2.12 Mice ................................................................................................................. 45 2.13 Genotyping of mouse strains used in the study of MHV68 infection both in vitro and in vivo ................................................................................... 49 2.14 In vivo infection ................................................................................................ 51 2.15 Perfusion ......................................................................................................... 51 2.16 Determination of viral titers in organs by tissue culture infective dose (TCID50) .................................................................................... 52 2.17 Ex vivo reactivation assay................................................................................ 53 2.18 Statistical analyses .......................................................................................... 54 3 Results .................................................................................................................. 55 3.1 Attempt to establish a novel autologous mouse model for herpesvirus infection ........................................................................................ 55 3.1.1 Colony screen verified presence of MmusRHV1 and MCMV2 in wild mouse colonies ............................................................................... 56 3.1.2 Efforts to isolate MmusRHV1 in an in vitro setting ....................................... 57 3.1.3 Maintenance of wild mouse colony as a resource of MmusRHV1 at the HZI ................................................................................................... 59 3.1.4 Establishment of a non-invasive blood PCR screen for MmusRHV1 ........... 63 3.1.5 Co-housing strategy and design to study natural transmission of MmusRHV1 ................................................................................................. 65 3.2 The role of Toll-like receptors in the detection of MHV68 ................................. 69 3.2.1 Identification of cell types responsible for the production of type I interferons upon MHV68 infection in vitro .................................................. 69 3.2.2 Characterization of the role of TLR expressed by pDC for sensing MHV68 in vitro ............................................................................................ 72 3.3 Characterization of the role of TLR in sensing MHV68 infection in vivo............ 78 3.3.1 Role of TLR in the detection of MHV68 after intranasal infection ................. 80 3.3.2 Role of TLR in the detection of MHV68 after intraperitoneal infection .......... 88 3.3.3 Role of TLR in the detection of MHV68 after intravenous infection .............. 93 3.4 Role of TLR in the establishment of latency and reactivation of MHV68 after intravenous infection ................................................................. 103 3.4.1 The role of TLR in the establishment of latency ......................................... 105 3.4.2 The role of TLR in reactivation of MHV68 .................................................. 108 4 Discussion .......................................................................................................... 114 4.1 Establishment of an autologous model to study gammaherpesvirus infections ...................................................................................................... 114 4.1.1 Isolation of MmusRHV1............................................................................. 114 4.1.2 Characterization of the natural transmission of MmusRHV1 in Mus musculus ........................................................................................... 116 4.2 Characterization of co-infections and its effect on herpesvirus latency in naturally infected mouse models ............................................................... 120 4.3 Role of UNC93B and specifically TLR7 and 9 in the detection of MHV68 in vitro ............................................................................................... 124 4.4 Role of TLR during MHV68 infection in vivo upon different routes of infection ..................................................................................................... 127 4.4.1 Intranasal infection .................................................................................... 127 4.4.2 Intraperitoneal infection ............................................................................. 131 4.4.3 Intravenous infection ................................................................................. 132 4.5 Role of TLR and gender for establishment of MHV68