Novel antiviral strategies for feline coronavirus and feline calicivirus

327 Pages·2014·6.68 MB·English

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Novel antiviral strategies for feline coronavirus
and feline calicivirus

Phillip McDonagh

A thesis submitted in fulfilment of the requirements
for the degree of Doctor of Philosophy

Pathobiology Group, Faculty of Veterinary Science
The University of Sydney
2014
Declaration of authorship

Apart from the assistance stated in the acknowledgements, this thesis represents the original
work of the author. The results from this study have not been presented for any other degree
or diploma at this or any other university.

Phillip McDonagh
BVSc (hons I)
March 2014

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Dedication

For Nicki, Charlie, and Joe

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Acknowledgements
The work described within these pages was only possible with the invaluable guidance and
support of a large number of individuals. Whilst specific mention will be made of some of
these in the paragraphs below, many others have contributed in ways which they, and
perhaps I, don’t fully appreciate. So to the unnamed, I also say thank you.
I was fortunate enough to be guided in my PhD studies by my supervisor Associate
Professor Jacqui Norris and co-supervisor Associate Professor Paul Sheehy. To Jacqui,
your infectious (pun possibly intended) enthusiasm for microbiology and virology helped
inspire my interest in infectious diseases during my undergraduate studies, and it was a
privilege to be able to return to complete post-graduate training under your tutelage. I am
also eternally grateful to you for providing opportunities for me to expand my academic
horizons outside of the laboratory during the course of my PhD. To Paul, you were roped
into my PhD journey along the way, and it was an honour to work with someone with such an
obvious passion for science. As supervisors, you were both a continual source of support
and advice, allowing me plenty of scope to explore, but also making sure that I didn’t stray
too far. They say “life is what happens when you are busy making other plans”. Perhaps
that should be changed to “life is what happens when you are busy doing a PhD!” And so,
on a personal note, your kindness and understanding as the rollercoaster of life took its
twists and turns during my epic PhD journey was very much appreciated.
Thanks must also go to the staff of the McMaster building for creating an atmosphere that
was both welcoming and so conducive to research. To Denise Wigney and Veronica
Ventura in particular, thank you for keeping the lab running so smoothly. A number of
researchers, both from within the faculty and external, shared their invaluable time,
experience, not to mention their expensive equipment, which allowed me to successfully
undertake the studies reported herein. I am particularly grateful to Karen Matthews (The
University of Sydney) for providing what may be the most painless introduction to PCR ever
That things “just worked” is a testament to your skill and experience in this field. Thanks also
go to Dr Adrian Smith and Steven Allen (Centenary Institute of Cancer Medicine and Cell
Biology), to Dr Louise Cole (Bosch Advanced Microscopy Facility), and to Dr Donna Lai
(Bosch Molecular Biology Facility) for your expert advice and assistance. The feline
calicivirus studies would not have been possible without the assistance of Professor James
Gilkerson and Ms Natalie Job (The University of Melbourne) in providing the reference strain
FCV F9 in addition to field isolates from Victoria, and Anne Fawcett for collecting FCV
samples from cats in Sydney. Thanks also go to Mark Kelman of Virbac Australia for
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donating recombinant feline interferon omega for these studies, and to Dr Chris Grant of
Custom Monoclonals International for the donation of a range of antibodies for testing.
To my fellow PhD students, thank you for your companionship, support, and advice over the
years – it has been a pleasure to work with like-minded people. I still however stand by my
decision not to join in the “Carcassonne” tournaments lest my PhD candidature go even
longer.
Thanks go to the Australian Companion Animal Health Foundation (Australian Veterinary
Association) and the Dr William Richards Award in Veterinary Pathology (The University of
Sydney) for providing financial support for these studies.
Quite simply none of this work would have been possible without my family. To my parents
and brothers, thank you for your unwavering support over the years, not only through this
PhD, but through every step of my life. To Mum, Dad, and Bill, thanks in particular for letting
me turn your lives upside down just a little as Nicki, Charlie, and I took up residence in your
homes for those final few “months” of lab work that dragged on for longer than anyone
wanted. To my late brother Dave, I wish you were here to see the completed thesis. I
imagine you would have skimmed rather than read it, but I know you would have been proud,
and that means a lot.
The biggest thanks go my wife and sons – I cannot hope to express in these words what it is
that you bring to my life. Nicki, you literally travelled to the other side of the world so I could
indulge my intellectual curiosity, and that is something I will never forget. Along the way you
have managed to keep me (relatively) sane, providing support and encouragement, all the
while shouldering considerably more than your fair share of the household burden in raising
two young children with a dad, sometimes physically, and often mentally, in absentia. In the
almost embarrassingly long time I have spent in university as a student I have been fortunate
to learn a lot, however to Charlie and Joe, in the blink of an eye you have taught me the most
important lesson of all. Thank you.
Oh, and boys, as we draw this thesis to a close, it is now officially time for the study to be
renamed the Lego room! Let’s play…

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List of publications and conference
proceedings
Refereed publications
McDonagh, P, Sheehy, P.A., Norris, J.M. (2011) In vitro inhibition of feline coronavirus
replication by small interfering RNAs. Veterinary Microbiology. 150 (3-4): 220-229

Conference presentations
Antiviral activity of small interfering RNAs on feline coronavirus replication in vitro. Australian
Society for Microbiology Annual Scientific Meeting. Hobart, 2011

Publications not arising from this thesis
Worthing, K., Wigney, D.I., Dhand, N.K., Fawcett, A., McDonagh, P., Malik, R., Norris, J.M.
(2012) Risk factors for feline infectious peritonitis in Australian cats. Journal of Feline
Medicine and Surgery. 14(6): 405-412.

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Summary
Both feline coronavirus (FCoV) and feline calicivirus (FCV) are common infections in
domestic cats, and are an important cause of morbidity and mortality in this species. Whilst
most FCoV infections are asymptomatic, or result in mild self-limiting gastrointestinal
disease, infection with virulent mutant FCoV biotypes can result in the development of feline
infectious peritonitis (FIP), an invariably fatal immune mediated disease for which there is
currently no effective therapy. Similarly, whilst many FCV infections result in only mild self-
limiting oro-respiratory disease, more severe disease manifestations, such as the newly
recognised FCV-associated virulent systemic disease, can occur with a significant impact on
the health and wellbeing of affected cats. As with FCoV, a lack of effective antiviral agents
limits treatment of FCV-associated disease to supportive therapy. The aim of the studies
described in this thesis was to begin to address this therapeutic shortfall by identifying
effective antiviral therapeutics for the treatment of these two important feline viruses.
Chapter 1 reviews the pertinent literature on the inherent difficulties associated with antiviral
chemotherapy across all animal species, the different classes of antiviral therapeutics, with a
particular emphasis on nucleic acid based therapies, and the process of antiviral drug
development. The chapter culminates in a systematic review of the two viruses which are
the focus of this work in terms of their importance in feline medicine, aspects of their physical
structure and biology relevant to therapeutics, and places into context the unmet need for
effective and safe treatments.
Chapter 2 describes some of the general methods used throughout the studies in this thesis,
including general cell and viral culture methods, molecular biology methods, and imaging
methods.
Veterinary practice, and in particular feline therapeutics, has a long history of therapeutic
‘trial and error’ in which drugs with proposed or theoretical benefit have been trialled in
patients, with variable success. Advances in available in vitro methods, higher expectations
of the profession and the public, industry support for the companion animal sector, and the
concerns regarding ‘off label’ use of drugs has seen a shift towards a more rigorous and
structured approach to drug development and testing. In light of this, Chapter 3 describes
the development of cytopathic effect inhibition assays for screening compounds for antiviral
efficacy against FCoV and FCV. Two different assay formats were optimised and tested for
each virus, a resazurin-based assay which detects viable cells through their reduction of the
substrate resazurin to fluorescent resorufin, and a sulforhodamine B-based assay which
provides a measure of cell biomass, and thus an indirect indication of cell viability. Both
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assay formats demonstrated excellent performance for FCoV and FCV, and based on the
calculated Z’-factors would be suitable in their current form for high throughput screening.
Although the SRB-based assay resulted in slightly higher Z’-factors for both viruses, the
resazurin-based assay was selected for subsequent screening due to significant practical
advantages, including low cost and assay simplicity. The development of this economical,
robust, and reliable screening assay opens up avenues for small and large scale compound
screening, allowing a physiologically relevant assessment of the efficacy of potential antiviral
compounds.
Chapter 4 describes the use of the optimised resazurin-based CPE inhibition assay to screen
a focused panel of nineteen compounds for antiviral activity against FCoV. Compounds
were selected for inclusion in the panel based on published demonstration of antiviral effects
against coronaviruses or other RNA viruses. Three compounds, chloroquine, mefloquine,
and hexamethylene amiloride were demonstrated to markedly inhibit CPE (> 75% inhibition)
in the screening assay and were confirmed to be potent antiviral agents at low micromolar
concentrations in orthogonal confirmatory assays. Preliminary investigation into the
mechanism of action of the compounds demonstrated chloroquine and hexamethylene
amiloride were effective only when present in the early stages of viral replication, while
mefloquine remained effective when added as late as 5 h post infection suggesting a
different mechanism of action. As replication of virulent biotypes of FCoV is a triggering and
perpetuating factor in the pathogenesis of FIP, the successful clinical application of these
results would likely provide a critical missing piece of the therapeutic puzzle. Given two of
the compounds identified, mefloquine and chloroquine, are commonly available antimalarial
compounds with a long history of prophylactic and therapeutic use in humans, if
demonstrated effective in vivo these treatments should be rapidly accessible and eminently
affordable, avoiding some the practical barriers seen with the introduction of a therapeutic
based on a new molecular entity.
RNA interference (RNAi) provides a promising new approach to antiviral therapy.
Preliminary studies on the efficacy of this approach against FCoV, using synthetic small
interfering RNAs (siRNAs), are presented in Chapter 5. All of the eight designed siRNAs had
some inhibitory effect on FCoV replication, two of which were highly effective, resulting in >
95% reduction in extracellular viral titre. Further characterisation of these demonstrated
them to be effective at low nanomolar concentrations, when used in combination, and when
used against high viral challenge. Serial passage of virus through siRNA treated cells
highlighted a weakness of the RNAi-based approach, with antiviral resistance rapidly
emerging; however combination therapy with three siRNAs was able to considerably delay
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this. A structural siRNA variant, Dicer-substrate siRNA, was tested and shown to provide
similar or better efficacy, depending on the target, over canonical siRNAs targeted at the
same motif. In addition to demonstrating the efficacy of an antiviral RNAi approach for
inhibiting FCoV, the results of this study also informs its potential therapeutic application, with
combinatorial therapy with a minimum of three siRNAs needed to minimise the development
of viral resistance when used in vivo. The successful therapeutic application of these results
for FIP however, will require an appropriate and affordable systemic delivery system. Based
on the results reported, the benefits of this potent and specific antiviral approach should
encourage further research to enable the translation of the results into a clinical setting for
further evaluation.
Chapter 6 reports the identification and characterisation of a small molecule antiviral effective
against feline calicivirus. Antiviral screening of the compound panel used in Chapter 4 with
the optimised resazurin-based CPE inhibition assay identified only a single compound,
mefloquine, displaying marked inhibitory effects against FCV. Orthogonal testing with virus
yield reduction assays and plaque reduction assays confirmed the antiviral effects at low
micromolar concentrations. Mefloquine, a commonly available antimalarial, was shown to be
effective against a panel of recent Australian FCV isolates, with greater potency
demonstrated against these field isolates than the reference strain. The seven field isolates
tested were taken from cats with a range of typical FCV-associated clinical disease and from
two geographically distinct regions, and thus represent an unbiased sample of circulating
viruses. Demonstration of efficacy against a broad range of circulating viruses is important
when considering the therapeutic application of mefloquine against a virus as genetically
diverse as FCV, as treatment will only be clinically useful if effective against relevant field
viruses. Combination treatment with mefloquine and recombinant feline interferon omega
demonstrated additive effects and may be a clinically useful approach. Based on these data,
consideration should be given to in vivo trials of mefloquine in cats with severe FCV-
associated disease. Given the paucity of antiviral treatments available for caliciviruses in
other species, the identification of a well characterised pharmaceutical with antiviral
properties against feline calicivirus should prompt further investigation into its use against
other related viruses.
The final experimental chapter (Chapter 7) reports on the use of RNAi against FCV. Despite
the small length and highly variable nature of the FCV genome, four short highly-conserved
regions were identified as suitable target regions for siRNA design. Three of the eight
siRNAs designed demonstrated a marked antiviral effect with a greater than 99% reduction
in extracellular viral titre. Titration of these effective siRNAs demonstrated a clear
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