Developing Antiviral Platforms And Assessing Interferon Against Kyasanur Forest Disease Virus by

175 Pages·2015·8.72 MB·English

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.

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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!

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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 DOSE (TCID50) DETERMINATION ....................................................................... 50
2.3. VIRUS QUANTIFICATION FOR KFDV AND VSV-GFP ..................................................... 51
X