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Alphavirus and flavivirus infection of Ixodes tick cell lines: an insight into tick antiviral immunity Claudia Rückert Submitted for the degree of Doctor of Philosophy The University of Edinburgh 2014 Table of contents DECLARATION ........................................................................................................ 5 ACKNOWLEDGEMENTS ....................................................................................... 6 ABBREVIATIONS .................................................................................................... 8 ABSTRACT .............................................................................................................. 11 1. INTRODUCTION ............................................................................................... 13 1.1 INTRODUCTION .................................................................................... 14 1.2 ARBOVIRUSES ..................................................................................... 14 1.2.1 Alphaviruses ...................................................................................................... 18 1.2.2 Flaviviruses ........................................................................................................ 25 1.2.3 Orbiviruses ......................................................................................................... 27 1.2.4 Vectors of arboviruses ....................................................................................... 28 1.3 TICKS AND TICK-BORNE DISEASES ......................................................... 31 1.3.1 Ticks as vectors of pathogens ........................................................................... 33 1.3.2 Tick cell lines ..................................................................................................... 35 1.4 ARTHROPOD ANTIVIRAL IMMUNITY.......................................................... 39 1.4.1 RNA interference ............................................................................................... 41 1.4.2 Antiviral responses triggered by innate immunity signalling pathways ............. 46 1.4.3 Other antiviral defences of arthropods .............................................................. 50 1.5 AIM OF THE PROJECT ............................................................................ 52 1.5.1 Objectives: ......................................................................................................... 52 2. MATERIALS AND METHODS ........................................................................ 53 2.1 CELL CULTURE .................................................................................... 55 2.1.1 Tick cell culture .................................................................................................. 55 2.1.2 Mammalian cell culture ...................................................................................... 55 2.1.3 Mosquito cell culture .......................................................................................... 56 2.2 VIRUS PROPAGATION ........................................................................... 56 2.2.1 SFV reporter viruses .......................................................................................... 56 2.2.2 LGTV ................................................................................................................. 59 2.2.3 Determination of virus titre by plaque assay ..................................................... 60 1 2.3 INFECTION OF TICK CELLS ..................................................................... 61 2.3.1 Infection with SFV .............................................................................................. 61 2.3.2 Infection with LGTV ........................................................................................... 62 2.4 INFECTION OF MOSQUITO CELLS ............................................................ 62 2.5 FLUORESCENCE MICROSCOPY .............................................................. 62 2.6 ELECTRON MICROSCOPY ...................................................................... 63 2.7 LUCIFERASE ASSAY .............................................................................. 63 2.8 FLOW CYTOMETRY ............................................................................... 64 2.8.1 Flow cytometry experiments carried out at Roslin (Chapters 3, 4) ................... 64 2.8.2 Flow cytometry experiments carried out at Pirbright (Chapter 5) ...................... 64 2.9 RNA EXTRACTION ................................................................................ 67 2.10 CDNA SYNTHESIS BY REVERSE TRANSCRIPTION .................................... 67 2.10.1 Reverse transcription using Super Script III (Roslin)......................................... 67 2.10.2 Reverse transcription using high capacity cDNA synthesis kit (Pirbright) ......... 68 2.11 POLYMERASE CHAIN REACTION (PCR) ................................................... 69 2.12 QUANTITATIVE REAL TIME PCR (QPCR) ............................................... 71 2.13 IDENTIFICATION OF GENE ORTHOLOGUES IN THE I. SCAPULARIS GENOME .. 72 2.13.1 Basic Local Alignment Search Tool ................................................................... 72 2.13.2 Alignment of sequences .................................................................................... 74 2.13.3 Phylogeny of I. scapularis STAT ....................................................................... 74 2.14 KNOCKDOWN OF GENE EXPRESSION IN TICK CELLS ................................. 75 2.15 KNOCKDOWN OF GENE EXPRESSION IN MOSQUITO CELLS......................... 78 2.16 KNOCKDOWN VALIDATIONS ................................................................... 79 2.17 INHIBITORS OF STAT PHOSPHORYLATION .............................................. 79 2.18 WESTERN BLOT ................................................................................... 79 2.18.1 Sample preparation ........................................................................................... 79 2.18.2 Sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS-PAGE) . 80 2.18.3 Transfer of proteins to nitrocellulose membranes ............................................. 81 2.18.4 Immunolabelling................................................................................................. 82 2.19 CLONING OF IXVAGO ............................................................................ 83 2.20 IMMUNOFLUORESCENCE STAINING ......................................................... 84 2.21 HEAT-INACTIVATION OF E. COLI ............................................................. 84 2.22 MODELLING OF PROTEIN STRUCTURE ..................................................... 85 2.23 SIGNAL PEPTIDE PREDICTION ................................................................ 85 2 2.24 STATISTICAL ANALYSIS ......................................................................... 85 2.24.1 Knockdown experiments ................................................................................... 85 2.24.2 Timecourse experiments ................................................................................... 86 3. CHARACTERISATION OF SFV AND LGTV INFECTION OF THREE IXODID TICK CELL LINES ................................................................................. 87 3.1 INTRODUCTION .................................................................................... 88 3.1.1 Objectives .......................................................................................................... 89 3.1.2 Materials and Methods ...................................................................................... 89 3.2 RESULTS ............................................................................................. 89 3.2.1 Initial experiments .............................................................................................. 89 3.2.2 Visualisation of virus replication in tick cells ...................................................... 90 3.2.3 Establishing experimental conditions for IRE11 and ISE18 cells ...................... 96 3.2.4 Kinetics of SFV non-structural gene expression in ISE18 and IRE11 ............... 98 3.2.5 Kinetics of SFV production in ISE18 and IRE11 ............................................. 108 3.2.6 Transfection of expression plasmids into tick cells .......................................... 109 3.2.7 Selection of the I. scapularis cell line IDE8 for further study ........................... 111 3.2.8 LGTV infection of IDE8 tick cells ..................................................................... 115 3.2.9