The Antiviral Properties of Melaleuca alternifolia concentrate

270 Pages·2016·8.55 MB·English

The Antiviral Properties of Melaleuca alternifolia
concentrate (MAC) against West Nile virus

Adriana C. Pliego Zamora
BBiology (Hons) Universidad de las Américas Puebla

School of Medical Science
Griffith Health
Griffith University

Submitted in fulfilment of the requirements of the degree of
Doctor of Philosophy

March 2015
Abstract
Many Flaviviruses are mosquito-borne viruses that can causes disease in humans and
animals including Dengue virus (DENV), West Nile virus (WNV), yellow fever virus
(YFV), Japanese encephalitis virus (JEV) and tick-borne encephalitis virus (TBEV).
These viruses are highly pathogenic to humans causing extensive morbidity and
mortality and most importantly, their recent re-emergence and global spread is a current
growing public health issue. Currently, DENV alone causes an approximate 50 million
infection cases annually and WNV infections have been reported in the five continents.
Despite the existence of licensed vaccines for YFV, JEV and TBEV, a major challenge
remains given there are no specific and effective antiviral therapies against flaviviral
infections.
Considering the homologies between flaviviral replication strategies, it may be possible
to identify broad-spectrum compounds with both prophylactic and therapeutic activity
against different flavivirus strains. Because the steps involved in compound discovery
to their clinical use are protracted and expensive, studies should include testing existing
clinically approved drugs as antivirals. Melaleuca alternifolia extracts are good
candidates to be explored for novel uses as they have been extensively studied and their
composition, chemistry, safety and toxicity are well documented. Melaleluca
alternifolia concentrate (MAC) is obtained from the further purification of Tea Tree Oil
(TTO) and is a new product exhibiting promising antimicrobial and anti-cancer
activities.
Oil and Aqueous (Aq.MAC) formulations of MAC were tested against the flaviviral
particles and infection of Vero cells with the non-pathogenic Australian strain of WNV,
Kunjin (KUNV) or the pathogenic American strain New York 99 (NY99). Both
formulations of MAC exhibited virucidal activity and diminished the infectivity of
KUNV and NY99 WNV particles in vitro by 1 log PFU. However, only the Aq.MAC
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formulation showed a dose-dependent and potent antiviral activity against infection
with both WNV strains in Vero cells, as it decreased the viral induced cytopathic effect
by ~25% and suppressed the replication of WNV, depicted by a 100-fold reduction in
viral titres and ~50% reduction in the number of cells expressing the non-structural viral
protein 1 (NS1).
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The antiviral mechanism of MAC against WNV in vitro was examined, and several
hypotheses were tested including affecting intra-cellular pH, induction of apoptosis and
cell cycle arrest in the G0/G1 phase. It was found that Aq.MAC, at the concentrations
used in this study, did not alter the endosomal pH nor did it induce apoptosis, but it was
capable of arresting cell growth in the G0/G1 cell cycle phase. Studies comparing the
cell growth arrest and antiviral properties of Aq.MAC versus Lovastatin (LOV)
concluded that although cell cycle arrest in the G0/G1 phase by itself is not sufficient to
exert an antiviral effect, Aq.MAC is a potent post-infection antiviral agent with stronger
antiviral properties than LOV. In addition, these studies indicated that the antiviral
activity of Aq.MAC must be cell mediated, and as such it is unlikely for WNV to
become resistant to this treatment.
Murine studies aimed to optimize Aq.MAC treatment against WNV for its future
clinical use, revealed that Aq.MAC treatment administered at different times pre- or
post-infection did not exhibit a significant antiviral effect against KUNV in weanling
wild type (WT) mice or against NY99 in adult WT mice. Only in adult IFN-deficient
mice was Aq.MAC capable of significantly delaying onset of WNV (KUNV)
encephalitis symptoms and reducing the viral titres in the brain by 1000-fold.
Additionally, Aq.MAC showed a moderate immunostimulatory effect in macrophages
in the blood, neutrophils in the spleen and a potent suppressive effect on dendritic cells
in the peritoneal cavity.
Overall, the potent antiviral properties of MAC against WNV in vitro suggest that by
reducing viral replication, the viral loads would decrease to a manageable level so that
the host immune system could potentially clear the infection thus attenuating the disease
pathogenesis. In particular, the results from the IFN-deficient mice support that by
inhibiting viral replication, MAC could reduce the initial acute viremia such that the
immune system might clear the infection. In addition, the virucidal activity of MAC
could also help to reduce the viral titres, preventing viral spread, circumventing any
cytotoxic or negative effects to the cell, and making the virion more vulnerable to
clearance by the immune system. Moreover, these results provide solid evidence for the
potential of MAC to be developed as an anti-flaviviral agent and merits further
investigation in its precise molecular mechanism of action, as well as to optimize the
delivery of MAC in murine models.
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Statement of originality
"This work has not previously been submitted for a degree or diploma in any university.
To the best of my knowledge and belief, the thesis contains no material previously
published or written by another person except where due reference is made in the thesis
itself."

Signed _______________________
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Acknowledgments
I would like to acknowledge my two supervisors Prof. Steve Ralph and Prof. Alex
Khromykh for their guidance and support throughout this project. Also, thank you to
Prof. Khromykh for providing access to his laboratory where most of this work was
done. I would also like to sincerely thank Dr. Judy Edmonds for her valued help, advice
and time, as well as to thank Prof. Nigel McMillan for his support.
From Griffith University I would like to thank Dr. Koichi Ito for his support and
friendship; Dr. Jelena Vider for her assistance, support, advice and chocolates; Dr.
Amanda Clark for her help and support; Dr. Daniel Clarke for his assistance and the
moments of laughter; Mariel Familiar Lopez for her sincere friendship, support and
great moments, and Dr. Beatrice Philip, Reem Alhulais and Rhys Pritchard for the good
moments and laughter.
From the University of Queensland I would like to thank Dr. Lucy Young for her
support, help and cupcakes; Dr. Penny Rudd for her support and friendship; Dr.
Ezequiel Balmori for his advice, support and the moments of laughter; and Nick Owens,
Shessy Torres, Dr. Justin Roby and Dr. Setoh Yin Xiang for the good moments and
laughter.
Thank you to my friends from Australia, México or wherever they may be for their
support and true friendship.
Last but never least, I would like to express my deepest gratitude to my parents, sisters
and brothers as they have always been very supportive and encouraging despite the
distance. Finally, I will always be infinitely thankful with Jaime; my dear companion of
adventures, for his enormous patience with me, lovely support and encouraging words
and hugs at all times.

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List of publications during PhD
Adriana Pliego Zamora, Judith H. Edmonds, Maxwell J Reynolds, Alexander A.
Khromykh, Stephen J. Ralph. “The in vitro and in vivo antiviral properties of Melaleuca
alternifolia concentrate against West Nile virus infection”. Journal of Antimicrobial
Chemotherapy (5.4) Submitted

Adriana Pliego Zamora, Judith H. Edmonds, Maxwell J Reynolds, Alexander A.
Khromykh, Stephen J. Ralph. “Melaleuca alternifolia concentrate delays West Nile
virus pathogenesis in IRF-3/-7 mice but not in wild type mice, and suppresses dendritic
cells”. In preparation.

Maxwell J Reynolds, Stephen J. Ralph, Adriana Pliego Zamora. “Pre-clinical and Phase
I studies of Melaleuca alternifolia concentrate (MAC): antiviral activity against West
Nile virus in vitro, and safety and pharmacodynamics in humans. In preparation.

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Table of contents
Chapter 1 Literature review ............................................................................................ 2
1.1 Flaviviruses ............................................................................................................. 3
1.1.1 Dengue ............................................................................................................. 5
1.1.2 West Nile virus ............................................................................................... 10
1.2 Candidate antiviral compounds against Flaviviruses ............................................ 26
1.2.1 Antiviral therapies against flaviviral components .......................................... 26
1.2.2 Host cell factors as targets for antiviral therapies .......................................... 30
1.3 Traditional and complementary medicines ........................................................... 34
1.3.1 Plant-derived antiviral compounds ................................................................ 35
1.3.2 Essential oils ................................................................................................... 45
1.4 Melaleluca alternifolia Concentrate ...................................................................... 46
1.4.1 Extraction process .......................................................................................... 46
1.4.2 Composition of MAC ..................................................................................... 47
1.4.3 Toxicity of MAC and TTO ............................................................................ 49
1.4.4 Formulation and administration of MAC ....................................................... 50
1.4.5 Biological activity of MAC ............................................................................ 50
1.4.6 Clinical trials: Melaleuca oil as an antiviral treatment .................................. 54
1.5 Aims ...................................................................................................................... 56
Chapter 2 Materials and Methods ................................................................................. 58
2.1 Cell culture ............................................................................................................ 59
2.1.1 Maintenance of cell lines................................................................................ 59
2.1.2 Cell plating ..................................................................................................... 60
2.2 Virus Stocks .......................................................................................................... 60
2.2.1 KUNV: FLSDX cloning ................................................................................ 61
2.2.2 NY99 .............................................................................................................. 64
2.2.3 Plaque Assay .................................................................................................. 65
2.3 Viral cytopathic effect .......................................................................................... 66
2.3.1 Cytopathic effect (CPE) of KUNV ................................................................ 66
2.4 Compound preparation and solubilisation, working solutions and cytotoxicity .. 69
2.4.1 Oil MAC, Terpinen-4-ol, Limonene and Aromadendrene working solutions 69
2.4.2 Serial dilutions of test compounds ................................................................. 69
2.4.3 Endotoxin levels of Aq.MAC......................................................................... 69
2.4.4 Cytotoxicity .................................................................................................... 70
2.5 Antiviral properties of MAC ................................................................................. 71
2.5.1 Antiviral activity of Oil MAC and Aq.MAC on the CPE caused by KUNV
infection ................................................................................................................... 71
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2.5.2 Antiviral activity of Aq.MAC, Terpinen-4-ol and TTO on the CPE caused by
KUNV infection ...................................................................................................... 72
2.5.3 Virucidal assay ............................................................................................... 73
2.5.4 Viral growth kinetics ...................................................................................... 73
2.5.5 NS1 expression ............................................................................................... 74
2.6 The antiviral mechanism of MAC ........................................................................ 75
2.6.1 Acridine orange (AO) intracellular pH measurements .................................. 75
2.6.2 Apoptosis detection by Annexin V and PI staining ....................................... 76
2.6.3 Cell cycle analysis .......................................................................................... 76
2.6.4 Cell cycle analysis, NS1 expression and viral growth kinetics ...................... 76
2.6.5 Cell growth kinetics ....................................................................................... 77
2.7 Antiviral activity of MAC and immune response against WNV in murine models
.................................................................................................................................... 79
2.7.1 KUNV infection and MAC treatment of mice ............................................... 79
2.7.2 NY99 infection of adult C57BL/6 WT mice and MAC treatment ................. 85
2.8 Processing of mouse tissues .................................................................................. 88
2.8.1 ELISA for detecting anti- KUNV or -NY99 antibodies in infected mouse sera
................................................................................................................................. 88
2.8.2 Tissue Culture Infectious Dose 50 ................................................................. 89
2.8.3 Processing of mouse tissues ........................................................................... 91
2.8.4 Flow cytometry analysis of mouse tissues ..................................................... 93
2.9 Statistical analysis ................................................................................................. 97
Chapter 3 The in vitro antiviral activity of MAC against West Nile virus ................... 98
3.1 Introduction ........................................................................................................... 99
3.2 Results ................................................................................................................. 101
3.2.1 West Nile virus stocks: KUNV and NY99................................................... 101
3.2.2 Cytopathic effect of KUNV in Vero and HeLa cells ................................... 104
3.2.3 MAC cytotoxicity and tolerance for Vero cells ........................................... 106
3.2.4 Preliminary studies of MAC treatment against KUNV ............................... 109
3.2.5 Oil and Aq.MAC exhibit virucidal activity and diminish the infectivity of
WNV particles in vitro .......................................................................................... 116
3.2.6 Aq.MAC reduces the cytopathic effect of KUNV infection in Vero cells .. 119
3.2.7 Aq.MAC treatment suppresses WNV replication in Vero cells .................. 120
3.2.8 Aq.MAC treatment reduces the percentage of WNV NS1-positive cells .... 124
3.3 Discussion ........................................................................................................... 131
Chapter 4 Studies to identify the in vitro mechanism of action of MAC against West
Nile virus ...................................................................................................................... 136
4.1 Introduction ......................................................................................................... 137
4.2 Results ................................................................................................................. 139
4.2.1 Aq.MAC treatment does not alter the endosomal pH .................................. 139
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4.2.2 Aq.MAC treatment does not induces apoptosis ........................................... 142
4.2.3 Aq.MAC treatment arrests cell growth in the G0/G1 cell cycle phase ........ 144
4.2.4 Cell growth arrest in G0/G1 phase by Aq.MAC is not responsible for
inhibiting WNV production .................................................................................. 148
4.3 Discussion ........................................................................................................... 157
Chapter 5 The antiviral and immunostimulatory activity of MAC treatment against
West Nile virus infection in vivo .................................................................................. 162
5.1 Introduction ......................................................................................................... 163
5.2 Optimization of KUNV infection in C57BL/6 weanling mice ........................... 165
5.3 Aq.MAC toxicity in weanling C57BL/6 mice .................................................... 167
5.4 Determination of the antiviral activity of Aq.MAC treatment against KUNV
infection in weanling C57BL/6 mice ........................................................................ 169
5.4.1 Aq.MAC treatment at twenty four hours post KUNV-infection .................. 170
5.4.2 Aq.MAC treatment at the time of KUNV infection ..................................... 171
5.4.3 Aq.MAC as a prophylactic treatment before KUNV infection. ................... 173
5.4.4 Aq.MAC treatment at the same time as infection and with increased doses
from day 5 to 10 post KUNV-infection ................................................................ 174
5.4.5 Aq.MAC treatment with increased dosing from day 5 to 10 post KUNV-
infection ................................................................................................................. 176
5.5 Determination of the antiviral properties of Aq.MAC treatment against KUNV
infection in C57BL/6 IRF 3 -/-/ 7-/- Knock Out mice .............................................. 177
5.6 Body weight loss as a risk factor for developing symptoms of WNV encephalitis
.................................................................................................................................. 181
5.7 Determination of the antiviral properties of Aq.MAC treatment against NY99
infection in adult C57BL/6 wild type mice .............................................................. 183
5.7.1 Infection with 100 PFU of NY99 and treatment with Aq.MAC treatment at
the same time ......................................................................................................... 183
5.7.2 Aq.MAC as a prophylactic treatment before infection with 1,000 PFU of
NY99 ..................................................................................................................... 195
5.8 Discussion ........................................................................................................... 199
Chapter 6 Concluding remarks and future directions ................................................. 206
6.1 The in vitro antiviral activity of MAC against West Nile virus ......................... 207
6.2 The antiviral mechanism of action of MAC against West Nile virus ................. 208
6.3 The antiviral efficacy and immunomodulatory effect of MAC treatment in West
Nile virus infection murine models .......................................................................... 214
References.................................................................................................................... 218
Appendix ..................................................................................................................... 245
Appendix I ................................................................................................................ 246
Flow cytometry analysis of KUNV NS1-positive cells ........................................ 246
Flow cytometry analysis of NY99 NS1-positive cells .......................................... 247
Appendix II ............................................................................................................... 248
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Appendix III .............................................................................................................. 250
Flow cytometry analysis of CD11chi/CD11bhi dendritic cells (DC) from adult
C57BL/6 WT mock-infected and Aq.MAC treated mice ..................................... 250
Flow cytometry analysis of CD11chi/CD11bhi dendritic cells (DC) from adult
C57BL/6 WT NY99-infected and Aq.MAC treated mice .................................... 251
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