Application of metagenomic sequencing to characterization of the virome in bovine respiratory disease Submitted to the College of Graduate and Postdoctoral Studies of the University of Saskatchewan in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Veterinary Pathology University of Saskatchewan Saskatoon By MAODONG ZHANG © Copyright Maodong Zhang, September 2020. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis/dissertation in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis/dissertation. Requests for permission to copy or to make other uses of materials in this thesis in whole or part should be addressed to: Head of Department of Veterinary Pathology University of Saskatchewan Saskatoon, Saskatchewan S7N 5B4 Canada OR Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan S7N 5C9 Canada i ABSTRACT Bovine respiratory disease (BRD) is the most concerning disease in the cattle industry worldwide due to the enormous economic losses it causes, and concerns regarding antimicrobial resistance that is emerging due to use of massive amounts of antimicrobial agents to control the disease. The concept of primary environmental, management or viral factors with ensuing secondary bacterial infections is widely accepted, however, morbidity and mortality of feedlot cattle due to BRD are not reduced despite viral vaccination and antimicrobial usage. This indicates that the etiology and pathogenesis of BRD are not yet fully understood, and that all of the infectious agents and environmental causal factors may not have been identified. The advent of high throughput sequencing (HTS) using Illumina MiSeq and Oxford Nanopore sequencing technologies has revolutionized the genomics field, particularly microorganism identification, antimicrobial resistance prediction and microbiome analysis. The main objective of my study was to use HTS to identify unconventional viruses (those viruses are not included in BRD vaccine and no available diagnostic assays) and characterize their association with BRD. We first characterized and compared the upper and lower respiratory tract viromes of Canadian feedlot cattle with or without BRD through metagenomic sequencing on the Illumina MiSeq platform. The presence of influenza D virus (IDV), bovine rhinitis A virus (BRAV), bovine rhinitis B virus (BRBV), bovine coronavirus (BCV) and bovine respiratory syncytial virus (BRSV) was associated with BRD. Agreement between identification of these viruses in nasal swabs and tracheal washes was generally weak, indicating that sampling location may affect detection of infection. We next characterized the virome of bovine pneumonia lungs collected in western Canada using metagenomic sequencing on the Illumina MiSeq, and combined this information with bacterial ii culture and targeted real-time PCR results from the same samples to determine the relationships between the microorganisms detected and different types of pneumonia defined by histopathological examination. Detection of Histophilus somni and Pasteurella multocida was associated with suppurative bronchopneumonia (SBP) and concurrent bronchopneumonia and bronchointerstitial pneumonia (BP&BIP), respectively. None of these viruses, however, was significantly associated with a particular type of pneumonia. Unconventional viruses such as IDV and BRBV were detected, although sparsely, consistent with our previous findings in upper respiratory tract samples. In the third chapter, we used IDV as a representative BRD-associated virus to examine the feasibility of using metagenomic sequencing for detection of viruses in clinical bovine respiratory samples. We compared results of long-read sequencing on the Oxford Nanopore GridION platform and previously generated Illumina MiSeq data to the results of an IDV-specific qPCR. We concluded that both MiSeq and Nanopore sequencing were capable of detecting IDV in clinical specimens with a range of Cq values. In the last chapter, we applied Nanopore metagenomic sequencing to characterize the viromes of cattle upon arrival at nine feedlots in Western Canada, and related the findings to health outcomes of these cattle to determine if the composition of the virome of individual animals could be used to predict the likelihood of their development of BRD. No relationship was found between BRD development and the number of viruses detected, the presence of any specific individual virus, or combination of viruses. In summary, results of these studies demonstrate the diversity of viruses in bovine respiratory tracts, and highlight the need for further research into prevention and control of BRD development in the context of mixed infections. Meanwhile, our results also demonstrate the potential of iii metagenomic sequencing on the Illumina MiSeq and Oxford Nanopore platforms for detection of viruses in clinical samples from naturally infected animals with a wide range of viral loads. iv ACKNOWLEDGEMENTS First and foremost, I want to thank my fantastic and excellent supervisors Dr. Yanyun Huang and Dr. Janet Hill. How blessed I am to have these two supervisors in my life to pursue my PhD. They are always happy to talk to me, help me out of issues and provide me with great opportunities and freedom to do my research. Their thinking ways inspire me a lot. I really appreciate what they have done and will carry on doing for me. Thanks so much! Second, I want to thank my committee members: Dr. Dale Godson, Dr. Matthew Links, Dr. Nathan Erikson, Dr. Bruce Wobeser and Dr. Elemir Simko (former graduate chair). They are always ready to help and provide insightful comments. To Dr. Trevor Alexander, thanks for providing those valuable samples and launching amazing cooperation. I want to thank Hill lab members: Salahuddin, Pashupati, Scott, Shakya, Mckayla, Laura, Sarah, Elina and Aline for their critical comments in our group meetings, which is one of the most enjoyable moments. I also thank them for their presentations in our journal clubs, which greatly extends my knowledge from virome to broader microbiome. A great thank to Champika Fernando for her help and encouragement. I thank Anju Tumber, Arnie Berg, all the pathologists, the technicians, staff and faculty in PDS and WCVM for their support. I also want to thank my parents, brother, sister, my wife and lovely son for their love and support. I love you all! v Table of Contents PERMISSION TO USE ................................................................................................................................. i ABSTRACT .................................................................................................................................................. ii ACKNOWLEDGEMENTS .......................................................................................................................... v LIST OF TABLES ....................................................................................................................................... ix LIST OF FIGURES ...................................................................................................................................... x CHAPTER 1. Literature review .................................................................................................................... 1 1.1. Introduction of bovine respiratory disease (BRD) and its economic impact ..................................... 1 1.2. Pathogenesis and etiology of BRD .................................................................................................... 2 1.3. Pathogens associated with BRD ........................................................................................................ 3 1.3.1 Bovine respiratory syncytial virus (BRSV) ................................................................................. 3 1.3.2 Bovine parainfluenza virus 3 (BPIV3) ......................................................................................... 4 1.3.3 Bovine coronavirus (BCV) .......................................................................................................... 5 1.3.4 Bovine viral diarrhea virus (BVDV) ............................................................................................ 7 1.3.5 Bovine herpesvirus 1 (BHV1) ...................................................................................................... 8 1.3.6 Influenza D virus (IDV) ............................................................................................................... 9 1.3.7 Bacterial pathogens .................................................................................................................... 10 1.4. Current