Developing Antiviral Platforms And Assessing Interferon Against Kyasanur Forest Disease Virus by Bradley William Michael Cook A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements of the degree of Doctor of Philosophy Department of Microbiology, University of Manitoba Winnipeg, Manitoba, Canada Copyright © 2015 by Bradley William Michael Cook Abstract Kyasanur Forest disease virus (KFDV) of the Flaviviridae virus family has caused seasonal infections and periodic outbreaks in Karnataka, India. First identified in 1957, KFDV annually infects 400-500 people and has a fatality rate of 3-5%; there are no approved antivirals and the existing licensed vaccine’s effectiveness appears to be questionable. Many tools for KFDV research are limited and this work sought to develop methods for analysing antivirals, including interferon (IFN)-α/β species. The BHK-21 (ATCC) cell line allowed for high virus propagation and distinguishable cytopathic effects (CPE) for determining antiviral effectiveness. The additional tool of a reverse genetics system expressing a full-length cDNA KFDV genome with a GFP reporter failed to propagate, despite numerous GFP genome-insertion strategies. The clinically approved IFN-α2a or IFN-α2b has had variable success at combatting flavivirus diseases in people, especially in the immuno-compromised. The continued passaging of KFDV- infected cells with repeated IFN-α2a treatment did not eliminate KFDV and had little effect on infectious particle production. IFN-α species, αWA and αΚ were more effective than IFN-α2a and α2b at reducing KFDV; however dose ranges indicated that while low concentrations could limit CPE, higher concentrations were needed to inhibit virion release. Avoidance of IFN-α/β through Jak/STAT signalling repression was attributed to the NS5 protein, specifically the RdRp domain based on data obtained with luciferase and vesicular stomatitis virus (VSV) recovery assays. However, the mechanism appears to act subsequently to STAT1/2 activation without NS5 binding to any Jak/STAT components. A non-infectious, replicative system serving as a platform for antiviral drug testing against KFDV in a high throughput manner could only provide II luciferase signals when the NS proteins capable of driving replication, were supplied in cis (subgenomic) but not in trans (antigenome). To conclude, IFN-α species such as IFN-αWA may be better suited than the licensed IFN-α2a for treatment of KFDV infections; however, IFN effects appear to be subdued in vitro due to the actions of the NS5 protein. While IFN may not be a successful antiviral against KFDV, the work in this thesis provides a foundation for evaluating other potential anti-KFDV therapeutics. III List Of Abbreviations AHFV-Alkhurma hemorrhagic fever virus BHK-Baby Hamster Kidney C-Capsid protein CAT-chloramphenicol-acetyl-transferase CC-Cytotoxicity concentration cDNA-Complementary Deoxyribonucleic acid CL-2-Containment level 2 CL-4-Containment level 4 CM-Convoluted membranes CPE-Cytopathic effect DENV-Dengue virus DLR-Dual luciferase reporter DMEM-Dulbecco’s minimal essential medium DNA-Deoxyribonucleic acid E-Envelope protein E. coli-Escherichia coli EDTA-ethylene-diamine-tetra-acetic acid EMCV-Encephalomyocarditis Virus EMEM-Eagle’s minimal essential medium ER-Endoplasmic reticulum IV FACS-Fluorescence-associated cell sorting FBS-Fetal bovine serum GFP-Green fluorescent protein GTP-Guanosine tri-phosphate HA-Hemagglutinin HCV-Hepatitis C virus HEK 293T-Human embryonic kidney 293 T-antigen HIV-Human immunodeficiency virus HRP-Horse-radish peroxidase HRV-Human rhinovirus hScrib-Human scribble IC-Inhibitory concentration IFN-Interferon IFNAR1-Interferon alpha receptor 1 IRES-Internal ribosome entry sequence IRF-9-Interferon regulatory factor – 9 ISG-Interferon stimulated genes ISGF3-Interferon-stimulated growth factor 3 ISRE-Interferon-stimulated response element Jak-Janus kinase JEV-Japanese encephalitis virus Kb-Kilo base V kDa-Kilo Dalton KFDV-Kyasanur Forest disease virus LGTV-Langat virus M-Membrane protein Mda5-Melanoma differentiation-association gene 5 MDCK-Madin-Darby canine kidney MEM-Minimal essential medium MOI-Multiplicity of infection MTase-Methyl-transferase NML-National Microbiology Laboratory NS-Non-Structural proteins NTPase-Nucleoside tri-phosphatase PBS-Phosphate-buffered saline PC-Paracrystalline membranes PCR-Polymerase chain reaction PKR-Protein kinase R PMSF-Phenyl-methane-sulfonyl-fluoride prM-Pre-Membrane protein PS-Porcine stable kidney RC-Replication complex RdRp-RNA-dependent RNA polymerase RGS-Reverse genetics system VI RIG-I-Retinoic acid-inducible gene I RLU-Raw light units RNA-Ribonucleic Acid RT-PCR-Reverse transcription polymerase chain reaction SDS-Sodium dodecyl sulfate SDS-PAGE-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SeAP-Secreted alkaline phosphatase STAT-Signal transducer and activator of transcription TBE serocomplex-Tick-borne encephalitis serocomplex TBEV-Tick-borne encephalitis virus TCID-Tissue culture infectious dose TRIM-Tripartite motif UTR-Untranslated Region VLP-Virus-like particle VP-Vesicle packets VSV-Vesicular stomatitis virus VSV-GFP-Vesicular stomatitis virus-green fluorescent protein WNV-West Nile virus YFV-Yellow fever virus VII Acknowledgements I would like to thank Dr. Steven Theriault and Dr. Deborah Court for the opportunity to follow the research endeavors that I wanted and for their unwavering support throughout this process. Thank you to my remaining committee members Dr. Darwyn Kobasa and Dr. Teresa de Kievit for their candor, which undoubtedly, shaped my project and helped to improve myself as a scientist. I would like to thank the external examiner Dr. Michael Holbrook for his constructive criticisms, which undeniably helped to shape this thesis. Thank you to our technician Mr. Todd Cutts, we may not always see “eye to eye” but the success of this project can be attributed to your technical expertise, advice and support. Thank you to Mr. Aidan Nikiforuk for help with many aspects of the project, but most importantly for aiding in the dreaded minigenome/subgenomic clone systems. I hope we never have to talk about that project ever again! I would also like to extend thanks to all of the former members of the ABRP group who have supported me throughput this process, specifically Mr. Jay Krishnan and Mrs. Diane Gordon-Pappis. Special thank you to the CL-4 support staff: Mrs. Lisa Fernando, Mr. Allen Grolla, Mr. Shane Jones and Mr. Jason Gren, for their fantastic CL-4 training and technical expertise. A big thank you for the rest of Special Pathogens program, I cannot thank you all enough for the support and the guidance that I have received over the years!! I would finally like to thank everyone at the National Microbiology Laboratory, especially the DNA core and Proteomics departments and the department of Microbiology at the University of Manitoba. VIII Dedication My work is dedicated to my lord and savior Jesus Christ and to the loving memories of my grandmother, mother and sister. I would also like to dedicate this work to my wife My Kim Tran, her endless support and encouragement really drove this degree. This work is also dedicated to the rest of my close friends/brothers in team Showtime! IX ABSTRACT ................................................................................................................................................ II LIST OF ABBREVIATIONS .................................................................................................................. IV ACKNOWLEDGEMENTS .................................................................................................................. VIII DEDICATION .......................................................................................................................................... IX LIST OF TABLES. ................................................................................................................................ XIII LIST OF FIGURES. .............................................................................................................................. XIII LIST OF COPYRIGHTED MATERIALS. ........................................................................................ XIV CHAPTER 1. THESIS INTRODUCTION .............................................................................................15 1.1. INTRODUCTION TO KYASANUR FOREST DISEASE VIRUS (KFDV) .................................. 17 1.2. DEVELOPING KYASANUR FOREST DISEASE VIRUS THAT EXPRESSES A GREEN FLUORESCENT PROTEIN DURING REPLICATION ..................................................................... 23 1.3. INTRODUCTION TO INTERFERON AND INTERFERON AS A TREATMENT FOR VIRAL DISEASE .................................................................................................................................... 29 1.4. FLAVIVIRUSES AND THEIR ABILITY TO HALT CELLULAR INTERFERON RESPONSES .... 36 1.5. ANTIGENOME AND SUBGENOMIC CLONE SYSTEMS FOR KFDV .................................... 41 1.6. HYPOTHESIS, RESEARCH GOALS AND OBJECTIVES ........................................................ 48 CHAPTER 2. MATERIAL AND METHODS ........................................................................................49 2.1. CELLS, VIRUSES AND INTERFERON .................................................................................. 49 2.2. APPROPRIATE CELL LINES FOR KFDV PROPAGATION AND 50% TISSUE CULTURE INFECTIOUS