ucalgary_2016_mohamedabdulcader_mohamedsarjoon.pdf

UNIVERSITY OF CALGARY
Cytosine-guanosine deoxynucleotides (CpG DNA)-mediated Antiviral Response Against Avian
Influenza Virus Infection
by
M Sarjoon Abdul-Cader
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
GRADUATE PROGRAM IN VETERINARY MEDICAL SCIENCES
CALGARY, ALBERTA
SEPTEMBER, 2016
M Sarjoon Abdul-Cader 2016

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ABSTRACT
Cytosine-guanosine deoxynucleotides (CpG DNA) can be used for the stimulation of toll-like
receptor (TLR)21 signaling pathway in avian species, that ultimately leads to upregulation of
gene transcription for pro-inflammatory molecules including nitric oxide (NO) and recruitment
of innate immune cells. These innate immune mediators, play various roles in the innate immune
system against viruses that infect mammals and avian species. The objective of the study was to
determine the antiviral effect of NO, produced in response to in ovo delivery of CpG DNA,
against H4N6 low pathogenic avian influenza virus (LPAIV) infection in the avian respiratory
system. First, we observed that when CpG DNA is delivered at embryo day (ED)18 in ovo and
subsequently challenged with H4N6 LPAIV at ED19 pre-hatch and day 1 post-hatch, CpG DNA
reduces H4N6 LPAIV replication in the respiratory tract. Second, we observed that CpG DNA-
mediated H4N6 LPAIV inhibition was attributable to NO production.Third, we observed that
the antiviral response elicited by in ovo CpG DNA delivery is also associated with macrophage
recruitment in the lungs. Finally, we showed that NO originated from macrophages is capable of
eliciting antiviral response against H4N6 LPAIV infection in a dose-dependent manner.
Altogether, CpG DNA-mediated antiviral response against H4N6 LPAIV infection is attributable
to increased macrophage numbers in the lungs and elevated NO production originated from
macrophages. The study provides insights into the mechanisms of CpG DNA-mediated antiviral
response, particularly against H4N6 LPAIV infection in avian species.
Key words:In ovo, avian influenza virus, CpG DNA, nitric oxide, TLR21

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PREFACE
The studies illustrated in this thesis were performed at the Department of Ecosystem and Public
Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada. The
work described in this thesis was carried out by myself, M Sarjoon Abdul-Cader, from
September 2014 to September 2016 under the supervision of Dr. Faizal Careem. The technical
and analytical supports in the immunofluorescent assay to quantify macrophages were obtained
from Aruna Amarasinghe. Hanaa Ahmed-Hassan supported me in CpG DNA uptake
experiments and the pertinent data analysis. The thesis contains the materials already published
or submitted for publication elsewhere, which are listed below.
Chapter one of this thesis contains materials published in:
Abdul-Cader MS, Amarasinghe A, Abdul-Careem MF (2016) Activation of toll-like
receptor signaling pathways leading to nitric oxide-mediated antiviral responses. Arch
Virol. Aug;161(8):2075-86
Chapter two of this thesis contains materials published in:
Abdul-Cader MS, Ahmed-Hassan H, Amarasinghe A, Nagy E, Sharif S, Abdul-Careem
MF (2016) CpG DNA-mediated antiviral response against avian influenza virus infection
is attributable to macrophage recruitment and NO production. Virology (submited)

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ACKNOWLEDGEMENTS
All the thanks and praises belong to Almighty Allah. I would like to thank the University of
Calgary, Faculty of Veterinary Medicine for providing me the opportunity to conduct studies for
my MSc degree.
Foremost, I wish to thank my supervisor, Dr. Faizal Careem for giving me the opportunity to
work under his excellent supervision with his continuous guidance and support throughout my
studies. Not only his motivation and enthusiasm, but also his direct involvement in animal
experiments with me, inspired me to think critically and to work hard. His timely comments and
constructive advice on experiments were invaluable for my future research career. His immense
guidance on scientific writings and presentations were precious, helped me to develop my
writing and presentation skills and overall confidence. I could not have made it through this
challenging experience without his continuous support and motivation.
I would like to acknowledge the rest of the supervisory committee members, Dr. Hermann
Schäetzl, Dr. Karen Liljebjelke and Dr. Kevin Fonseca for their time commitments and for
providing suggestions and directions to solve research related problems that facilitated timely
completion of my masters thesis. I am also thankful to Dr. Carla S Coffin for agreeing to be an
external examiner for my thesis examination.
I am very grateful to my past lab member, Simrika Thapa for training me in many of the
techniques described in this thesis. My success would not have been possible without her
guidance and support. I also want to thank current lab members, Aruna Amarasinghe, Hanaa
Ahmed-Hassan and Victor Palomino for their company and their support in many of my
experiments. Aruna Amarasinghe is a long-time friend of mine and I can not forget his support

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whenever I needed, particularly with maintaining cell cultures during weekends and also with
animal infections and sampling. In addition, he helped me with the immunofluorescent assays
and related data analysis. Hanaa Ahmed-Hassan helped me with CpG DNA uptake experiment in
chicken macrophage cell line, taking images and data analysis. Hanaa Ahmed-Hassan not only
helped with my experiments, but also kept on inspiring and motivating me. Her helpful and
friendly nature with my family made our journey in this new environment very pleasurable. I
also thank all of the previous summer students, Bakhtawar, Yuwei, John, Nasrif, Sadiya, and
Godsent, for sharing nice moments and for their help, particularly with cell culture works.
I also would like to thank Sampson Law, Angelo Bianchi, Li Lu and Su Shim of the
Prion/virology animal facility at Foothill campus, University of Calgary for the monitoring and
management of experimental animals. I am thankful to Dr. Greg Muench for training me in
animal handling procedures. I am also greatly appreciating all the assistance from the members
of the Czub, van Marle, Van der Meer, Schäetzl, Glich, and Coffin labs. I also wish to thank the
Grewal lab for providing access to spectrophotometer for NO measurment. Also, thanks go to
Paul Gajda for providing me necessary training for operating the fluorescent microscope. I wish
to acknowledge the weekly meetings of the "Virology Group" for providing an opportunity to
present the progress of my work and constructive comments that improved the quality of my
work and expanded my scientific knowledge.
Last but not least, I am very grateful to my parents, wife, and children, who are always besides
me in every ups and downs of my life, making this journey successful. Their continuous love and
support are the strengths and inspirations for the success in my studies sofar.