Real Time PCR-Based Infectivity Assay and Characterization of Cell Surface Receptors for Turkey Hemorrhagic Enteritis Virus Hassan M. Mahsoub Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biomedical and Veterinary Sciences F. William Pierson, Chair Roger J. Avery Kurt L. Zimmerman Lijuan Yuan Dec 9, 2015 Blacksburg, Virginia Keywords: Hemorrhagic Enteritis, THEV, Siadenovirus, titration, receptor Copyright 2015, Hassan M. Mahsoub Real Time PCR-Based Infectivity Assay and Characterization of Cell Surface Receptors for Turkey Hemorrhagic Enteritis Virus Hassan M. Mahsoub ABSTRACT Turkey hemorrhagic enteritis virus (THEV) is responsible for the hemorrhagic enteritis (HE) disease in commercial turkeys through infections by its virulent strains. HE is an acute condition characterized by depression, immunosuppression, bloody droppings, intestinal hemorrhage, and death. THEV (also known as turkey adenovirus 3) is an official member of the family Adenoviridae, genus Siadenovirus, species Turkey siadenovirus A. Two main types of live vaccines are currently used for the protection of turkeys against HE; a crude splenic vaccine propagated in live turkeys, and a cell culture-based vaccine generated in RP19 cells. The only laboratory-adapted tests for assessing the titers of these vaccines are agar gel immunodiffusion test and cell culture endpoint dilution, respectively. The assays suffer from low sensitivity, inaccuracy, and time consumption. A SYBR Green-based real time PCR assay for determining the genomic titer of THEV through the quantification of its hexon gene was developed. The assay was applied as a quality control for the titration of splenic vaccines and was found useful in distinguishing the differences in virus titer among many vaccine batches. Additionally, using the qPCR assay along with a cell culture system, a novel infectivity assay was developed for the titration of THEV, as an alternative for the endpoint dilution assay. Applying the assay on nine batches of commercial HE cell culture vaccines, high variations in infectious virus titers were detected. The new method is rapid, sensitive, and very accurate. A strong correlation was found between the genomic titer and qPCR infectious titer in HE cell culture vaccines. Moreover, the qPCR infectivity assay proved as an instrumental research tool. It was used to measure the effect of several treatments of RP19 cells on virus infection. The main target cell type for THEV infection and replication is B-lymphocytes, which are represented in vitro by the B lymphoblastoid, RP19 cells. The cellular surface components used by the virus to gain entry into cells are unknown. As an adenovirus, we hypothesized that THEV uses two different molecules on RP19 cells for the attachment and internalization. A recent study has shown that the synthesized THEV fiber knob domain binds to sialyllactose, based on a glycan array analysis. In our studies, the treatment of RP19 cells with neuraminidases and lectins resulted in high reduction of virus entry, which provide a strong evidence of the utilization of cell surface sialic acids as attachment receptor for THEV. Destruction of surface carbohydrates and proteins on RP19 cells also reduced virus entry, indicating that these components are part of the THEV receptor. Using virus overlay protein blot assay, THEV was found to specifically bind to two RP19 surface membrane proteins, most likely, representing primary and secondary receptors for virus entry. Further studies are required to identify these proteins and verify their role in THEV endocytosis in host cells. Dedication This dissertation is dedicated to My wife Marwa Elsenderissy My sons Mohamed and Hamza My daughter Khadija My beloved uncle Hassan Mahsoub & My grandfather Mohamed E. Shoaib iii Acknowledgements First and foremost, I praise ALLAH (God) for guiding me throughout my work on this dissertation until I achieved it and for blessing me with the many people who helped me and encouraged me to accomplish this work in the best form it could be. I would like to thank my supervisor, Dr. Pierson. He provided me with exceptional guidance throughout my dissertation work. Dr. Pierson has always been very supportive of me individually or when I have gone before my PhD advising committee. He knows very well how to make his students self-confident, self-sufficient, and creative researchers. He appreciates my strengths and works with me to improve my weaknesses. Dr. Pierson has never put unnecessary pressure on my shoulders and always shows appreciation for my work. He understands that trial and error is an important part of professional development. When I reached the end of my knowledge and experience, he has always put me back on the right path. As an emeritus professor here at VA Tech once said to me “if your advisor does not consider you as his son, you will find it hard to succeed”. This statement is positively applied to Dr. Pierson. A lot of thanks go to my committee members, Dr. Avery, Dr. Zimmerman, and Dr. Yuan. I could have never imagined having an advisory committee better than this. Every time I went to a committee meeting, I had concerns about the quality and quantity of the lab work I did. However, they always surprised me with their appreciation of what I have accomplished. From my first committee meeting till the day of my dissertation defense, their criticism has been always constructive and friendly, and their words have been always encouraging. I do appreciate all kind of support and advising they have provided me throughout my PhD studies and dissertation writing. I feel obliged to thank Becky Jones, our BMVS program coordinator, for her extraordinary effort and advices which kept my visa paperwork in a good shape, although not part of her responsibilities. Thanks again for keeping me safe and keeping my assistantship uninterrupted during the last three years. I am also thankful to Cyndi Booth for her administrative roles. Special thanks are to Dr. Nathan Beach, a previous member in our lab, for his great help during my first year in the lab and afterwards. I thank him also for teaching me lab techniques and troubleshooting, and for continuous encouragement. I would like also to thank Dr. Sriranganathan for his friendly scientific and non- scientific discussions, and Nancy Tenpenny and Paige Smith for their efforts to make my stay at CMMID as comfortable as possible. I would like to express my appreciation to all the friendly and lovely faces I have met in CMMID and the College of Veterinary Medicine (faculty, staff, and students). You have truly made my life very pleasant and I really enjoyed the good times we spent together. I would like also to say thank you to Dr. Nicholas Evans for keeping our research uninterrupted during the last few years through his efficient management of our laboratory financial and organizational matters. Also, he has never hesitated to give help in explaining things that I did not understand and reviewing my abstracts, presentations, and other stuff, when asked to do so. iv I am so grateful to Kay Carlson, a previous lab manager at CMMID, for her tremendous effort in reviewing and proofreading the majority of my dissertation. I am thankful to many friends in and out of Blacksburg: Dr. Mohamed Seleem, Mostafa Ali, Abdullah Awaysheh, Tariq Abuhamdia, Michael Tomaszewicz, and many others. You have been great friends. I would like also to thank my best friends in Egypt: Mahmoud Hafez, Hassan Eltaweell, and Fahmy Abdelaziz. My first four years in the graduate school at Virginia Tech was supported by a scholarship from the Egyptian Ministry of Higher Education and Scientific Research. I gratefully acknowledge this support. Thanks also to the staff of the Egyptian Cultural and Educational Bureau in Washington, DC, for facilitating my paperwork until I finished my degree. I am exceptionally thankful to my father Mostafa Mahsoub, my mother Kawthar Shoaib, my brother Mohamed, my sisters Wesam and Reham and their husbands Mohamed Elian and Mohamed Motawea, my parents-in-law Taiseer Eltahawy and Salaheldin Elsenderissy, and my sisters-in-law Maiada, Mai, Mona, and Mariam. Thanks to those family members and all friends in Egypt who remembered me in their prayers all the time and wished me success in my PhD and future profession. Finally, I will never be able to find the suitable words to express how grateful I am to my wife Marwa, who kept my life in the past seven years the easiest one possible. She has exerted a tremendous effort in taking care of me and our three. God bless you my wife and reward you for all what you have done and are still doing for us. I love you. My gorgeous kids (Mohamed, Hamza, and Khadija), your smiles were a major reason that I have survived my long-term stress until finished my PhD work. May Allah bless you all! v Table of Contents Abstract ii Acknowledgments iv Table of Contents vi List of Figures viii List of Tables xi List of Abbreviations xii Chapter 1: Introduction 1 1.1. Background 1 1.2. Previous status of THEV research 1 1.3. Scope of the problem 3 1.4. Overall research approach 3 1.5. Hypotheses 5 1.6. References 6 Chapter 2: Review of Literature 2.1. Current taxonomy and nomenclature of adenoviruses 10 2.2. Section I: Turkey hemorrhagic enteritis virus and other siadenoviral 11 infections 2.3. Section II: Virus titration methods and infectivity assays 39 2.4. Section III: Adenovirus cellular receptors 59 2.5. References 70 Chapter 3: Development and Applications of Real-Time PCR for Quantification of Turkey Hemorrhagic Enteritis Virus Genomes (THEV): Genomic Titration of HE Live Vaccines and Assessment of Virus Replication in Cell Culture Systems 85 3.1.