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Download (7Mb) University of Warwick institutional repository: http://go.warwick.ac.uk/wrap A Thesis Submitted for the Degree of PhD at the University of Warwick http://go.warwick.ac.uk/wrap/63027 This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. Characterisation of host-range determinants in Chandipura virus by Emily Louise Stock Thesis Submitted for the degree of Doctor of Philosophy The University of Warwick School of Life Sciences June 2014 Contents List of tables vi List of figures vii Acknowledgements ix Declaration x Abbreviations xi Summary xii Chapter 1: Introduction 1 1.1. Emerging viruses 2 1.2. Chandipura virus (CHPV) classification 5 1.2.1. Order Mononegavirales 5 1.2.2. Family Rhabdoviridae 7 1.2.3. Genus Vesiculovirus 9 1.3. CHPV structure and lifecycle 13 1.3.1. CHPV structure 13 1.3.2. Genome organisation 14 1.3.3. Replication cycle 15 1.3.4. Attachment and fusion mediated by the G protein 21 1.3.5. Transport of viral nucleocapsids through the 24 endocytic pathway 1.3.6. Replication complex proteins 24 1.3.6.1. Nucleocapsid (N) protein 24 1.3.6.2. Phosphoprotein (P) 25 1.3.6.3. Large (L) protein 26 1.3.7. Matrix (M) protein 29 1.3.8. Leader RNA 32 1.4. Host range (hr) and temperature-dependent host range (tdCE) 32 mutants 1.5. Vesiculovirus disease: infection of the central nervous system 38 1.5.1. Causative agents of viral encephalitis 38 1.5.2. Viral encephalitis in India 43 1.5.3. Human epidemics of Chandipura virus encephalitis 43 1.5.4. Human clinical studies of CHPV infection 48 1.5.5 Non-human isolations of CHPV 48 1.6. Experimental CHPV infection and disease 49 1.6.1. CHPV infection in cultured cells 49 1.6.2. Mouse model of CHPV pathogenesis 50 1.7. Aims 52 Chapter 2: Materials and Methods 54 2.1. Cell lines and culture 55 2.1.1. Cell lines 55 i 2.1.2. Maintenance of cells in culture 56 2.1.3. CHPV minigenome rescue 57 2.1.4. Rescue of infectious recombinant viruses 57 2.1.5. Generation of CHPV-G glycoprotein pseudotyped 58 lentivirus 2.1.6. Cryopreservation of cell lines 58 2.2. Viruses and virus growth 59 2.2.1. Viruses 59 2.2.2. Growth of CHPV stocks 59 2.2.3. Determination of virus titre by plaque assay 59 2.2.4. Microplaque assay 60 2.2.5. Virus growth curves 61 2.2.6. Semi-purifying virus 61 2.3. Mice 61 2.3.1. In vivo infection with CHPV 61 2.3.2. Titration of CHPV 62 2.3.3. Timecourse of infection with CHPV 62 2.3.4. Homogenising tissue 63 2.3.5. Sectioning of fresh brain tissue 63 2.3.6. Deparaffinisation and rehydration of paraffin- 64 embedded sections 2.3.7. Heat-induced antigen retrieval 64 2.4. Organotypic mouse cerebellar slice cultures 64 2.4.1. Preparation and culture of cerebellar slice cultures 64 2.4.2. Viral infection of cerebellar slice cultures 64 2.5. Bacterial culture 65 2.5.1. Bacterial culture 65 2.5.2. Preparation of competent E.coli DH5α cells 65 2.5.3. Transformation of competent cells and culture of 66 transformants 2.5.4. Preparation and recovery of glycerol stocks of bacteria 66 2.6. DNA techniques 67 2.6.1. Plasmid DNA purification from transformed E.coli 67 2.6.2. Polymerase Chain Reaction (PCR) 67 2.6.3. Quickchange site-directed mutagenesis 68 2.6.4. Agarose gel electrophoresis 69 2.6.5. Extraction of DNA from agarose gels 69 2.6.6. Restriction digestion 69 2.6.7. Ligation 70 2.6.8. DNA sequencing 70 70 2.7. RNA techniques 2.7.1. Viral RNA extraction 70 2.7.2. Reverse transcription 70 2.8. Protein techniques 71 ii 2.8.1. Antibodies 71 2.8.2. Immunofluorescence 72 2.8.3. Immunohistochemistry 72 2.8.4. Whole cell lysates 73 2.8.5. Immunoprecipitation of Flag fusion proteins using 73 ANTI-FLAG® M2 magnetic beads 2.8.6. SDS-PAGE 73 2.8.7. Coomassie staining and destaining 74 2.8.8. Western blotting 74 2.8.9. Plaque reduction assay 75 Chapter 3: The host range tdCE phenotype of Chandipura virus is 76 determined by mutations in the polymerase gene 3.1 Introduction 77 3.2 Identifying CHPV gene(s) associated with tdCE mutant phenotype 78 3.2.1 Characteristics of CHPV tdCE mutants 78 3.2.1.1. Growth of tdCE mutant viruses in a range of 80 cultured cells 3.2.2 Sequencing wild-type CHPV and tdCE mutant genomes 83 3.2.3 Generation of recombinant viruses containing the 86 identified point mutations 3.2.4 Recovery of recombinant viruses containing the point 90 mutations associated with host range 3.3 Discussion 94 Chapter 4: The construction and characterisation of tagged wild type 98 CHPV and tdCE mutant replication complex proteins 4.1 Introduction 99 4.2. Generation of recombinant viruses containing tagged proteins 101 4.2.1. Recombinant CHPV encoding eGFP in the variable region 101 upstream of domain VI of L protein could not be recovered 4.2.2. Construction of Flag tagged virus proteins 102 4.2.2.1. Construction of pT7L Flag plasmid 103 4.2.2.2. The L/Flag protein is functional in minigenome 105 assays 4.2.2.3. Insertion of c-terminal flag tag to L in wild type 106 and tdCE mutant pT7CV using overlapping PCR 4.2.3. Construction of pT7CV-PRFP containing the tdCE 108 mutations with and without L/Flag 4.2.4. Recovery and growth of P/RFP, L/Flag and dual 112 recombinant viruses 4.2.4.1. Titres of the recovered recombinant viruses 112 4.2.4.2. Single-cycle growth analysis of P/RFP and L/Flag 113 recombinant viruses 4.2.4.3. Fluorescence of P/RFP recombinant viruses 114 4.2.4.4. L/Flag could not be detected in infected cells by 116 immunofluorescence 4.2.5. The P/RFP protein can be detected in infected cells by 116 Western blot iii 4.2.6. The Flag-tagged CHPV L protein is stable 117 4.3. The location of CHPV tdCE mutant replication complexes in 118 permissive and non-permissive conditions 4.4. Identification of cellular factors associated with CHPV L protein in 121 mammalian and avian cells 4.4.1. Introduction and strategy 121 4.4.2. Determination of cellular binding partners of the CHPV L 121 protein 4.5 Discussion 136 Chapter 5: The pathogenesis of Chandipura virus-induced encephalitis 144 5.1 Introduction 145 5.2 Ex vivo investigation of the cell tropism of CHPV 148 5.2.1 rCVeGFP-infection of cerebellar slice cultures 148 5.2.2 Elucidating the identity of rCVeGFP-positive cells using brain 153 cell marker proteins 5.3 Establishing an in vivo model of CHPV-induced encephalitis 165 5.3.1. Dose-dependent induction of encephalitis in mice by CHPV 165 5.3.1.1. Titration of CHPV in mice 165 5.3.1.2. Virus was detected in the brains (but not lungs or 169 liver) of infected mice 5.3.2. Progression of wild-type CHPV infection in vivo 171 5.3.2.1. Weight change and clinical assessment 171 5.3.2.2. Virus was detected in the mouse brain 24 hours post- 174 infection with wild-type CHPV 5.3.2.3. Pathological examination of brains from mice infected 176 with wild-type CHPV showed pronounced damage to the cerebellum 5.3.3. The dose of recombinant wild-type CHPV required to cause 180 encephalitis 5.3.3.1. A ten-fold higher dose of rCHPV (500 pfu) was 180 required to induce encephalitis compared with wild-type CHPV strain I653514 5.3.4. Progression of recombinant wild-type CHPV infection in vivo 183 5.3.4.1. Weight change and clinical assessment 183 5.3.4.2. Virus was detected at high titre in the mouse brain 3 186 days post-infection with rCHPV 5.3.4.3 Pathological examination of brains from mice infected 189 with rCHPV 5.3.5. The dose of a GFP-tagged CHPV (CVeGFP) required to cause 190 encephalitis 5.3.5.1. A dose of 1000 pfu of CVeGFP was required to induce 191 encephalitis 5.3.6. Progression of CVeGFP infection in vivo 192 5.3.6.1. Weight change and clinical assessment 192 5.3.6.2. There was a two day delay in the onset of CVeGFP- 194 induced encephalitis compared with rCHPV 5.3.6.3. Virus was detected at high titre in the mouse brain four 195 days post-infection with CVeGFP 5.3.6.4. Significantly lower levels of virus was present in the 198 iv brains of CVeGFP-infected mice compared to rCHPV-infected mice at days 3 and 4 post-inoculation 5.3.6.5. rCVeGFP passaged in the mouse brain contains functional 198 GFP 5.3.6.6. Pathological examination of brains from mice infected with 199 rCVeGFP 5.3.7. Brain cell types infected with rCVeGFP following 202 intraperitoneal inoculation of mice with the virus 5.4 The effect of tdCE point mutations on the pathogenesis of infection 206 5.5 Discussion 208 Chapter 6: Conclusions and future questions 217 References 227 Appendix 1 244 Appendix 2 247 v List of tables Table 1.1.
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