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Marek’s Disease Virus Pathogenesis and Latency Gillian Hunter M Phil The University of Edinburgh 2012 i DECLARATION I declare that all work included in this thesis has been composed by myself. Contributions to the work of this thesis by colleagues are fully acknowledged. No part of this work has been or will be submitted for any other degree or qualification. Gillian Hunter January 28, 2011 Centre for Infectious Diseases The Roslin Institute Royal (Dick) School of Veterinary Studies Summerhall Tower Block University of Edinburgh Edinburgh EH9 1QH ii ABSTRACT Marek’s Disease virus (MDV) is a highly contagious, widespread and persistent neoplastic herpesvirus causing extensive lymphoblastic tumours in chickens. The virus is shed in feather dust and spread through inhalation. Vaccines are available to protect against the effects of MDV but not replication of the virus and subsequent contamination of the environment leading to flock exposure. Increased virulence in strains of MDV has been identified and currently available vaccines may not offer protection from the disease. Disease outbreaks result in economic losses as well as welfare issues. To break this cycle better methods of controlling MDV preventing both tumourogenesis and shedding of infectious virus must be developed. Targeting specific MDV genes key to maintaining latency and viral replication using siRNA could potentially be used as a control strategy. At the present time, many of the unique genes in MDV are largely uncharacterised. 15 uncharacterised open reading frames (ORFs) were screened for expression in a MDV latent infection model in a non-producer MDV transformed chicken lymphoblast cell line, RPL-1. Of these uncharacterised ORFs LORF1, LORF3, LORF11, LORF12, ANTISENSE, US2, MLTI, RLORF11, RLORF12, 23kDa and RLORF6 were expressed during latency. To investigate the effect of post-transcriptional knockdown of these ORF products two 25-mer siRNA oligonucleotides were designed for each gene, transfected into RPL-1 cells and analyzed using growth rate as an indicator of changed phenotype over a period of 120 hours post-transfection. RPL-1 cells transfected with a nonsense siRNA oligonucleotide were used as the control group. No significant changes in transfected cell growth over the controls were identified in LORF3, ANTISENSE, RLORF12, LORF1, LORF11 or MLTI. Increased RPL-1 cell growth was observed (adjusted p-value of 0.0094) in one of the two siRNA oligonucleotides specific for RLORF6 at 72 hours post-transfection. RLORF6 was further characterised using confocal microscopy techniques and was found to be localized in the nucleus but not the nucleolus of chicken embryo fibroblasts and chicken lymphoblastic cells. iii ACKNOWLEDGEMENTS I would firstly like to thank the British Biotechnology and Scientific Research Council (BBSRC) and Aviagen for their mentoring, support and funding of this project. Thanks are also due to Professor Tony Nash, Dr. Bob Dalziel, Dr. Bernadette Dutia, Mr. Ian Bennet, Mrs. Yvonne Ligertwood and Dr. Jeanette Webb at the University of Edinburgh Centre for Infectious Disease, Prof. Helen Sang at the Roslin Institute and Dr. Barry Thorp, Dr. Bill Stanley and Dr. Chris Morrow at Aviagen for all their patience, help and advice. A special thanks should also go to Dr. Ola Ali Hassanin at the Division of Pathway Medicine, University of Edinburgh for her friendship and assistance with plasmids, to Dr. Venugopal Nair at the Institute for Animal Health, Compton, UK for all of his assistance with everything and to Mrs. Trudi Gillespie at the University of Edinburgh Impact Imaging Facility for all her assistance with the confocal microscopy. I would also like to thank all my colleagues in the department who were a constant source of help and advice. Finally, I would like to thank my family and particularly my husband Rory, and sons Alex and Ian for their love, support, encouragement and loads of patience throughout the project. iv TABLE OF CONTENTS Declaration ii Abstract iii Acknowledgements iv Contents v List of Figures viii List of Tables ix Abbreviations x Chapter 1: Introduction 1.1 Herpesviridae………………………………………………………….. 1 1.1.1 Taxonomic Classification………...………………………..………2 1.1.2 Virus Particle Structure…………………………………..…..…... 6 1.1.3 Genome Arrangement..……………………………..……………..8 1.1.4 Herpesvirus Life Cycle……………………………..…………….. 14 1.1.5 Herpesvirus MicroRNAs………...…………………..………….... 22 1.2 Marek’s Disease Virus (Gallid Herpesvirus 2)……………………..... 25 1.2.1 History………………………………………………..……………26 1.2.2 Taxonomy & Strain Variation……………………………….….... 26 1.2.3 Genome Structure & Gene Function………………………...…….28 1.2.4 Life Cycle & Biology …………………………………………..…30 1.2.5 MDV Tumourigenesis…………………………………..………... 34 1.2.6 Diagnosis of MDV….…………………………………..…………40 1.2.7 Control of MDV-Vaccination & Viral Vectors.…………..….…... 42 1.2.8 Control of MDV-Resistance………………………………..…….. 47 1.2.9 Introduction to siRNA Methods for Gene Silencing in Chickens……………………………………………………..…….50 1.3 Investigation of Selected Uncharacterised MDV Genes……………..52 1.3.1 The ‘Meq Loci’ Genes………………………………………….....54 1.3.2 Repeat Long Regions of the Genome………………………..…… 55 1.3.3 Other Regions of the Genome…………………………………..…58 1.4 Project Aims………………………………………………….…...……. 61 Chapter 2: Materials and Methods 2.1 Molecular Techniques…………………………………………………. 63 2.1.1 Restriction Endonuclease Digestion…………………………….. 63 2.1.2 DNA Isolation & Purification…………………………………....63 2.1.3 RNA Isolation & Purification………………………………….... 63 2.1.4 DNaseI Treatment of RNA Samples…………………………..…64 2.1.5 Synthesis of first strand cDNA from RNA……………………....65 2.1.6 Concentration of DNA Samples……………………………….... 65 2.1.7 Purification of RNA by Phenol: Chloroform Extraction……..…. 66 2.1.8 Polymerase Chain Reaction (PCR)……………………………....67 v 2.1.9 Reverse Transcriptase Quantitative PCR (RTqPCR).………..…. 68 2.1.10 Normalization of cDNA Samples……………………………….. 71 2.1.11 Purification of PCR Products………………………………….....71 2.1.12 Agarose Gel Electrophoresis……………………………………..71 2.1.13 DNA Extraction from Agarose Gels…………………………..…72 2.1.14 Quantitation of Nucleic Acid by Spectrophotometry………….... 73 2.1.15 Sequencing of DNA…………………………………………..….73 2.2 Bacterial Techniques…………………………………………………... 74 2.2.1 Bacterial Culture……………………………………………….... 74 2.2.2 Transformation of Chemically Competent E. coli…………….....74 2.2.3 Plasmid DNA Isolation from Bacteria (Small-Scale)…………....75 2.2.4 Plasmid DNA Isolation from Bacteria (Large-Scale)…………....75 2.2.5 Marek’s Disease Virus Bacterial Artificial Chromosome Preparation…………………………………………………….… 75 2.2.6 Preparation of Bacterial Stocks for Long Term Storage………....77 2.3 Protein Blots………………………………………………...………... 77 2.3.1 Protein Electrophoresis……………………………………..…… 77 2.3.2 Transfer of Protein to Nitrocellulose Membranes……………..... 79 2.3.3 Immunological Detection of Protein Blots…………………….... 80 2.4 Tissue Culture and Virus Growth Techniques……………..……… 81 2.4.1 Growth of Established Cell Lines…………………………..……81 2.4.2 Preparation of Cell Lines for Liquid Nitrogen Storage……......... 81 2.4.3 Growing Cell Lines from Frozen Liquid Nitrogen Stocks…….... 82 2.4.4 Counting Cells Using Haemocytometer……………………….... 82 2.4.5 Transformed Chicken Lymphoblastic Cell Lines……………..…83 2.4.6 Chick Embryo Fibroblasts………………………………………. 84 2.5 In vitro Experiments………………………………………..………... 85 2.5.1 Transfection of CEFs Using Lipofectamine…………………..… 85 2.5.2 Transfection of CEFs Using Electroporation……………..……...86 2.5.3 Transfection of RPL-1 and Rb1b T cells Using Electroporation...87 2.6 Dual-Reporter Luciferase Assays……………………………………89 2.7 Confocal Laser Microscopy…………………………………………. 91 2.7.1 Suspension Cell Lines…………………………………………....91 2.7.2 Adherent Cell Lines……………………………………………... 92 2.8 Statistical Analysis…………………………………………………… 93 2.9 Recipes………………………………………………………………... 93 2.9.1 Commonly Used Solutions………..…………………………….. 93 2.9.2 Protein Electrophoresis…………………………………….......... 94 2.9.3 Nucleic Acid Electrophoresis…………………………………….95 2.9.4 Bacterial Media………………………..…………………………96 2.9.5 Tissue Culture Media…………………………………………….96 vi Chapter 3: siRNA Studies of Selected MDV Genes Results 3.1 Project Objectives…………………………………………………… 97 3.2 Selection of Genes for Study………………………………………... 97 3.3 PCR Screening for Expression in Latency………………………… 102 3.4 Sequencing of Genes of Interest in MDV JM Strain……………… 120 3.5 Knockdown of Viral Gene Expression Using siRNA……………… 124 3.6 Summary……………………………………………………………... 142 Discussion 3.7 Overview and Introduction………………………………………….144 3.8 Selection of Genes for Study………………………………………... 145 3.9 PCR Screening for Expression in Latency………………………… 148 3.10 Sequencing of Genes of Interest in JM Strain