Toll-Like Receptor Ligands as Antiviral Agents against Avian Influenza Viruses

169 Pages·2017·1.31 MB·English

Toll-Like Receptor Ligands as Antiviral Agents against Avian Influenza
Viruses

by

Neda Barjesteh

A Thesis
presented to
The University of Guelph

In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
in
Pathobiology

Guelph, Ontario, Canada

© Neda Barjesteh, January, 2017
ABSTRACT

Toll-Like Receptor Ligands as Antiviral Agents against Avian Influenza Viruses

Neda Barjesteh Advisor:
University of Guelph, 2017 Professor Shayan Sharif

Avian influenza viruses (AIV) are a significant threat to the poultry industry and public health.
Recent outbreaks of AIV have provoked notable concerns about measures available for control of
AIV. In conjunction with the research efforts focused on developing effective vaccines, there is a
need to explore novel approaches to modulate host innate responses, as part of a strategy for rapid
response against AIV outbreaks. The research presented here evaluated the antiviral activities of
Toll-like receptor (TLR) ligands against AIV infection and assessed the underlying mechanisms
of these activities. Initially, the potential ability of TLR ligands as antiviral agents against AIV
was investigated using in vitro and in ovo models. Our results confirmed that TLR ligands were
able to reduce AIV replication in chicken cells. However, different levels of antiviral activities
were observed among TLR ligands. CpG ODN1826 caused the highest reduction in AIV
replication in chicken macrophages and embryonated chicken eggs. Furthermore, results of the
present research showed that TLR ligands were able to induce antiviral responses in chicken cells,
as demonstrated by an increase in the expression of proinflammatory cytokines, type I interferons
and some interferon-stimulated genes. Subsequently, several TLR ligands were examined in vivo
to determine whether TLR ligands can reduce viral shedding from infected chickens. Our results
demonstrated that all TLR ligand treatments reduced AIV shedding, with the CpG ODN1826 being
the most efficacious to reduce oral virus shedding, whereas lipopolysaccharide (LPS) from
Escherichia coli (E. coli) 026:B6 resulted in the largest reduction in cloacal virus shedding. To
further explore the role of TLR ligands in inducing antiviral responses in the chicken respiratory
system, tracheal organ cultures (TOC) were used to investigate localized innate responses to TLR
ligands. The antiviral activity of TLR ligands in TOCs was demonstrated by induced expression
of type I interferons (IFNs) and interferon-stimulated genes (ISGs) in TOCs and the reduction of
AIV replication in treated TOCs.
Overall, the results in this thesis identified candidate TLR ligands for control of AIV infection
in chickens and also provided a better understanding of innate responses in chickens that
contribute to protection against AIV.
Acknowledgements
I would like to extend my sincere gratitude to my advisor, Dr. Shayan Sharif for his support,
encouragement, and continuous guidance. It would be unlikely to count all the ways that he has helped me
in my career. I am so grateful for everything that I have learned from him during my Ph.D. program.
I would like to thank Drs. Bonnie Mallard, Éva Nagy, and Faizal Careem for guiding me on the right path
and their advice through my Ph.D. program. I will always be thankful to them.
I would also like to thank Drs. Byram Bridle and Maricarmen García for serving in my Ph.D. defense exam
and their insightful and constructive comments.
I would like to acknowledge my labmates in the Sharif lab, both past, and present, including
Jennifer Brisbin, Michael St Paul, Tamiru Alkie, Bahram Shojadoost, Douglas Hodgins, Khaled
Taha Abd Abdelaziz, Leah Read, Alexander Bekele-Yitbarek, Ravi Kulkarni, Jegarubee
Bavananthasiv and Jake Astill for their kind assistance, inspiration, motivation and friendship.
I would like to thank my fellow graduate students, research technicians and all of my friends and
colleagues in the Department of Pathobiology, Ontario Veterinary College for providing friendly and
innovative environment and helping me during the course of my Ph.D. program.
I would also like to acknowledge all of the staff and faculty in the Department of Pathobiology, Ontario
Veterinary College for their help and support during the course of my Ph.D. program.
I appreciatively acknowledge the financial support from National Sciences and Engineering
Research Council of Canada, The Ontario Ministry of Agriculture, Food and Rural Affairs.

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I would like to dedicate this thesis to my family, my beloved
husband, Mehdi, and my inspiring and devoted parents, and
affectionate sisters, Laleh, Leila and Vida for their constant
support and unconditional love.

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Table of Contents
List of Tables ................................................................................................................................. xi
List of figures ................................................................................................................................ xii
List of abbreviations .................................................................................................................... xiv
Declaration of work performed .................................................................................................... xix
Chapter 1 ......................................................................................................................................... 1
Introduction ..................................................................................................................................... 1
1.1. Background information .......................................................................................................... 1
1.2. Literature review ...................................................................................................................... 3
1.2.1. Influenza virus ...................................................................................................................... 3
1.2.2. Host range and susceptibility to AIV in avian species ......................................................... 4
1.2.3. Structure of influenza virus A ............................................................................................... 5
1.2.4. Virus life cycle ...................................................................................................................... 6
1.2.5. Innate responses .................................................................................................................... 8
1.2.6. Sensing microbes ................................................................................................................ 11
1.2.7. Sensing influenza A virus ................................................................................................... 18
1.2.8. The vertebrate interferon response ...................................................................................... 20
1.2.9. Natural killer cells ............................................................................................................... 22
1.2.10. Adaptive immune system responses against influenza virus ............................................ 22
1.2.11. Applications of Toll-like receptor ligands ........................................................................ 26
Chapter 2 ....................................................................................................................................... 31
TLR ligands induce antiviral responses in chicken macrophages ................................................ 31
2.1. Abstract .................................................................................................................................. 32
2.2. Introduction ............................................................................................................................ 33
2.3. Materials and methods ........................................................................................................... 35
2.3.1. Avian influenza virus .......................................................................................................... 35
2.3.2. Cell culture .......................................................................................................................... 35
2.3.3. Avian influenza virus infection of cells .............................................................................. 35
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2.3.4. TLR ligands ........................................................................................................................ 36
2.3.5. Macrophage treatment with TLR ligands and cell infection with avian influenza virus .... 36
2.3.6. Gene expression in chicken macrophages stimulated with TLR ligands ........................... 37
2.3.7. RNA extraction and cDNA synthesis ................................................................................. 38
2.3.8. Real-time PCR .................................................................................................................... 38
2.3.9. Flow cytometry ................................................................................................................... 39
2.4. Statistical analysis .................................................................................................................. 39
2.5. Results .................................................................................................................................... 39
2.5.1. Low pathogenic avian influenza virus replicates in chicken macrophages ........................ 39
2.5.2. TLR ligands induce NO production in chicken macrophages ............................................ 40
2.5.3. Pam3CSK4, LPS and CpG ODNs decrease viral replication in chicken macrophages ..... 41
2.5.4. Induction of gene expression in macrophages by TLR ligands .......................................... 42
2.5.5. TLR 2, 4 and 21 ligands up-regulate macrophage activation markers ............................... 43
2.6. Discussion .............................................................................................................................. 44
2.7. Acknowledgements ................................................................................................................ 48
Chapter 3 ....................................................................................................................................... 57
Induction of antiviral responses against avian influenza virus in embryonated chicken eggs with
TLR ligands .................................................................................................................................. 57
3.1. Abstract .................................................................................................................................. 58
3.2. Introduction ............................................................................................................................ 59
3.3. Materials and methods ........................................................................................................... 61
3.3.1. Avian influenza virus .......................................................................................................... 61
3.3.2. TLR ligands ........................................................................................................................ 61
3.3.3. Embryonated egg treatment with TLR ligands and infection with avian influenza virus .. 61
3.3.4. Gene expression of the chorioallantoic membrane stimulated with TLR ligands .............. 62
3.3.5. RNA extraction and cDNA synthesis ................................................................................. 63
3.3.6. Real-time PCR .................................................................................................................... 63
3.4. Statistical analysis .................................................................................................................. 63
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3.5. Results .................................................................................................................................... 64
3.5.1. The chorioallantoic membrane expresses TLRs ................................................................. 64
3.5.2. TLR2/1, 4, 7 and 21 ligands decrease viral replication in embryonated eggs .................... 64
3.5.3. Induction of gene expression in the CAM by TLR ligands ................................................ 65
3.6. Discussion .............................................................................................................................. 67
3.7. Acknowledgements ................................................................................................................ 72
Chapter 4 ....................................................................................................................................... 80
Reduction of avian influenza virus shedding by administration of Toll-like receptor ligands to
chickens......................................................................................................................................... 80
4.1. Abstract .................................................................................................................................. 81
4.2. Introduction ............................................................................................................................ 82
4.3. Materials and methods ........................................................................................................... 83
4.3.1. Chickens .............................................................................................................................. 83
4.3.2. Avian influenza virus .......................................................................................................... 84
4.3.3. TLR ligands ........................................................................................................................ 84
4.3.4. Experimental design............................................................................................................ 84
4.3.5. RNA extraction, cDNA synthesis and real-time PCR ........................................................ 85
4.4. Statistical analysis .................................................................................................................. 85
4.5. Results .................................................................................................................................... 86
4.5.1. TLR ligands administered intranasally and intramuscularly significantly reduced the oral
shedding of H4N6 AIV ................................................................................................................. 86
4.5.2. TLR ligands administered intranasally and intramuscularly significantly reduced cloacal
shedding of H4N6 AIV ................................................................................................................. 86
4.5.3. TLR2/1, 4 and 21 ligands administered intranasally induce antiviral responses in trachea 87
4.5.4. TLR2/1, 4 and 21 ligands administered intranasally induce antiviral responses in cecal
tonsils ............................................................................................................................................ 88
4.6. Discussion .............................................................................................................................. 89
4.7. Acknowledgements ................................................................................................................ 92
Chapter 5 ....................................................................................................................................... 99
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Local innate responses to TLR ligands in the chicken trachea ................................................... 100
5.1. Abstract ................................................................................................................................ 101
5.2. Introduction .......................................................................................................................... 102
5.3. Materials and methods ......................................................................................................... 105
5.3.1. Tracheal organ culture (TOC) ........................................................................................... 105
5.3.2. Avian influenza virus (AIV) ............................................................................................. 106
5.3.3. Stimulation of TOC with TLR Ligands ............................................................................ 106
5.3.4. cDNA synthesis and real-time PCR .................................................................................. 107
5.3.5. Stimulation of chicken macrophages with tracheal supernatants ..................................... 107
5.4. Statistical analysis ................................................................................................................ 108
5.5. Results .................................................................................................................................. 109
5.5.1. TLR ligands induce the expression of innate response genes in chicken tracheal organ
cultures ........................................................................................................................................ 109
5.5.2. Supernatants from chicken tracheas treated with TLR ligands activate chicken
macrophages ............................................................................................................................... 111
5.5.3. Antiviral compounds produced by stimulated TOCs are able to limit AIV replication in
chicken macrophages .................................................................................................................. 111
5.6. Discussion ............................................................................................................................ 112
5.7. Acknowledgments................................................................................................................ 116
Chapter 6 ..................................................................................................................................... 121
6.1. General discussion ............................................................................................................... 122
References ................................................................................................................................... 131

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