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Molecular Characterization and Pathogenicity Studies of Canadian Infectious Laryngotracheitis Virus Isolates

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Molecular Characterization and Pathogenicity Studies of Canadian Infectious Laryngotracheitis Virus Isolates University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2021-02-02 Molecular characterization and pathogenicity studies of Canadian infectious laryngotracheitis virus isolates Perez Contreras, Ana Paulina Perez Contreras, A. P. (2021). Molecular characterization and pathogenicity studies of Canadian infectious laryngotracheitis virus isolates (Unpublished master's thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/113067 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Molecular characterization and pathogenicity studies of Canadian infectious laryngotracheitis virus isolates by Ana Paulina Perez Contreras A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIRMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN VETERINARY MEDICAL SCIENCES CALGARY, ALBERTA FEBRUARY, 2021 ©Ana Paulina Perez Contreras 2021 ABSTRACT The extensive use of live-attenuated vaccines to control the upper respiratory tract viral infection in chicken known as infectious laryngotracheitis (ILT), has been associated with a surge in vaccine related ILT outbreaks. It is documented that these ILT outbreaks are due to the regaining of virulence of the vaccine viruses due to multiple bird to bird passages following vaccination. These vaccine-originated infectious laryngotracheitis virus (ILTV) isolates are known as vaccine revertants. An additional concern is that the multiple live attenuated vaccine ILTV and wild-type ILTV can recombine, resulting in ILTV strains with higher pathogenicity. To date, little is known about the molecular nature of the Canadian ILTV. The objectives of the present thesis work are to, 1) molecularly characterize the ILTV associated with ILT outbreaks in poultry flocks in Canada using a whole genome sequence approach and 2) study the pathogenicity of representative ILTV isolates in vivo. In achieving objective 1, It was found that most of the ILTV isolates of Canadian origin used in this study were genetically related to chicken embryo origin (CEO) live attenuated vaccine ILTV strains. Evidence of recombination involving commonly used live attenuated ILT vaccines was also detected in an ILTV isolate belonging to the British Columbia province. A second recombination event was found this time involving an ILTV isolate belonging to Alberta. This Alberta ILTV strain acted as a parental strain along with another live attenuated ILT vaccine strain to give rise to an ILTV strain previously isolated in United states (US) territory. In objective 2, the pathogenicity of two wild- type and one CEO vaccine revertant ILTV isolates was compared, by infecting specific pathogen free chickens along with age matched mock infected controls. We also used naïve contact chickens in order to determine the transmission potential of these ILTV isolates. It was found that the tested ii CEO vaccine revertant ILTV isolate can induce not only severe disease but also to transmit more efficiently than the wild-type ILTV isolates used for this study. Keywords: Poultry, infectious laryngotracheitis virus, whole genome sequencing, recombination, vaccine revertant, pathogenicity, transmission iii PREFACE The studies described in this thesis were performed at the Department of Ecosystem and Public health, Faculty of Veterinary Medicine, University of Calgary, Alberta, Canada. The in vivo investigations and laboratory analyses were carried out by me, Ana Paulina Perez Contreras, from January 2019 to January 2021 under the supervision of Dr. M. Faizal Abdul-Careem. Provost Chantale propagated and isolated ILTV from three samples originated from Quebec. Mohamed Sarjoon Abdul-Cader supported me with virus propagation. Catalina Barboza Solis supported me with virus propagation, virus titration and animal experiments. Shahnas Mohamed Najimudeen supported me with processing of swab samples and animal experiments. Mohamed S. H. Hassan supported me with animal experiments. The thesis contains the materials already published or to be submitted for publication elsewhere, which are listed below. Published Manuscripts Contreras, P.A.; Van der Meer, F.; Checkley, S.; Joseph, T.; King, R.; Ravi, M.; Peters, D.; Fonseca, K.; Gagnon, C. A.; Provost, C.; Ojkic, D.; Abdul-Careem, M. F., Analysis of Whole- Genome Sequences of Infectious laryngotracheitis Virus Isolates from Poultry Flocks in Canada: Evidence of Recombination. Viruses, 2020. 12(1302). Manuscript to be published Contreras, P. A.; Barboza-Solis, C.; Najimudeen, M. S.; Checkley, S. Van der Meer, F.; Abdul- Careem, M. F.; Joseph, T.; King, R.; Ravi, M.; Peters, D.; Fonseca, K.; Gagnon, C. A.; Ojkic, D. Pathogenic and transmission potential of chicken embryo origin (CEO) vaccine revertant infectious laryngotracheitis virus. To be submitted to Viruses. iv AKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor, Dr. M. Faizal Abdul- Careem, for giving me the opportunity to further develop my skills by allowing me to conduct my thesis research as a member in his lab, under his guidance. I am also thankful to him for providing me with tools necessary to accomplish this milestone, for his enormous patience and continuous mentorship, by which I have learned the importance of critical thinking. Most importantly, for bestowing me with his trust and setting a prime example of what hard work can accomplish. I would like to thank the University of Calgary and the Faculty of Veterinary Medicine for welcoming me into their excellent graduate program and community, which has been my home, away from home for the past two years. I am indebted to my supervisory committee members Dr. Frank van der Meer and Dr. Kevin Fonseca, for their input on this project which helped shape it to the outcome that it is today. I would like to thank the funding agencies, Egg farmers of Alberta and Alberta Agriculture and Forestry for providing the funding necessary to carry out this work. I would also like to acknowledge the Agri Food Laboratories, from Alberta Agriculture and Forestry (Edmonton, AB), the Animal Health Center (Abbotsford, BC), and the Laboratoire de Diagnostic Moléculaire, the University of Montreal (QC), for providing me with the ILTV positive clinical samples or sequences. I would like to thank all the people whose assistance was a milestone in the completion of this project, Mohammed Sarjoon Abdul-Cader for helping me and sharing his knowledge on cell culture, virus propagation and nucleic acid extraction. Upasama de Silva Senapathi for introducing me to quantitative polymerase chain reaction assay and other techniques which I used extensively v throughout my thesis research. I thank Victor Palomino-Tapia for sharing his knowledge on chicken primary cell culture and the use of embryonated eggs for viral propagation. I am thankful to Mohamed S.H. Hassan for his assistance on animal experiments. I also thank Catalina Barboza- Solis for helping with the processing of the original ILTV samples, with cell culture, viral propagation, and titration, as well as with animal experiments. I also thank Shahnas Mohamed Najimudeen for helping me with the nucleic acid extraction process and animal experiments. I am thankful to Mohammad Mostafa Nazari Zanjani for sharing his knowledge and his assistance in the aspects of my research involving bioinformatics. I whole heartedly appreciate the invaluable assistance I received from my lab members. To them and to Sabrina Buharideen, I wish to thank for their support and most of all, for their unvaluable friendship throughout this period. I wish to acknowledge Lilian Oribhabo and Bukola Ali, from the Prion/Virology Animal Facility (PVF) for their assistance on animal care and animal facility management. I would like to express my gratitude to Paul Gajda for his assistance on microscopy and digital imaging for cell culture. I would also like to thank Dr. Grace Kwong for her guidance on statistical analyses of this thesis research data. I wish to show my gratitude to Chantale Provost and Carl Gagnon for handling all the sequencing of the ILTV isolates used in this thesis. Finally, I wish to show my gratitude to Adrian Sacher and my family, whose love, and unconditional support has encouraged me to fulfill this milestone. vi DEDICATION I dedicate this work to my beloved parents Frida, and Pablo. vii TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii PREFACE ...................................................................................................................................... iv AKNOWLEDGEMENTS................................................................................................................v DEDICATION .............................................................................................................................. vii TABLE OF CONTENTS ............................................................................................................. viii LIST OF TABLES ......................................................................................................................
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