Molecular determinants of IBDV pathogenesis and modulation of the host innate response Department of Virology Faculty of Medicine Katherine Louise Dulwich 30th September 2019 Supervised by Dr Andrew Broadbent and Dr Mark Fife (The Pirbright Institute) Dr Mike Skinner (Imperial College London) Submitted for the degree of Doctor of Philosophy 1 Declaration of Originality I declare the work presented in this thesis is my own, unless otherwise stated or referenced. This includes data that was generated by other sources. Katherine Dulwich Copyright Declaration ‘The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work’ 2 Abstract Viruses are known to interact with the innate immune pathways and, in some cases, strains that differ in virulence are known to interact with these pathways in different ways. This thesis aimed to directly compare IBDV strains of differing virulence to determine key interactions with the innate immune response that may contribute to disease outcome. Infection of chicken B cells with the very virulent s UK661 strain, suppressed type I IFN responses compared to both infection with the cell-adapted IBDV strain, D78 in primary bursal cells, and infection with the classical strain, F52/70 in DT40 cells. Birds infected with UK661 also had down-regulated type I IFN and pro-inflammatory responses in the bursa of Fabricius (BF), compared to infection with F52/70. No difference in the peak virus titres was detected in the BF or spleen, although UK661 reached higher titres in the caecal tonsils than F52/70. Increased type I IFN production following F52/70 infection coincided with a reduced mortality in these birds, indicating a protective role of this immune response. The UK661 VP4 protein was found to suppress IFNβ production in vitro compared to the F52/70 VP4, which instead suppressed Mx1 production, indicating that the IBDV VP4 from different strains impairs either IFN production or signalling pathways. Upon knocking out the protease function of UK661 VP4, IFNβ production remained suppressed, and multiple amino acids are likely responsible for the different phenotype between strains. This work demonstrates that UK661 and F52/70 have strain-specific differences in their interactions with the innate immune response, mediated by the VP4 protein, therefore differences in this protein between strains may contribute to virulence. This information could be useful in the development of recombinant rationally designed live attenuated IBDV vaccines, by generating a vvIBDV backbone containing a VP4 from a classical or cell-adapted strain, as a vaccine candidate. 3 Acknowledgements I would like to thank my supervisors, Andrew, Mike and Mark, for all their support over the past four years. For their helpful advice and practical guidance, I will always be grateful. I want to thank all the people I have worked alongside in the lab who have offered advice, support and assistance with experiments and made me smile. Stathis Giotis, Sarah Keep, Nicole Doyle, Helena Maier and Pip Beard have been particularly patient and supportive, offering endless motivational speeches. I would like to thank and acknowledge the support services, including the animal services and cell culture technicians, who have made the data presented in this thesis possible. Also, to thank The Pirbright Institute and BBSRC for funding my studentship, in addition to Imperial College London, The Houghton Trust and The Microbiology Society for travel grants and opportunities to network and present my work at conferences. Last, but by no means least, I would like to thank my family, friends and partner Chris, who have provided support in so many ways in the last four years. Without this support, I have no doubt this thesis would not be in existence. Thank you for not letting me give up on myself and reminding me of the curious little girl who always loved science. I would like to dedicate this thesis to the family members that started this journey with me and have been unable to finish it. “For every person that believed I couldn’t; for every person that knew I could” 4 Contents Declaration of Originality ........................................................................................................................ 2 Copyright Declaration ............................................................................................................................. 2 Abstract .................................................................................................................................................. 3 Acknowledgements ................................................................................................................................ 4 List of Figures .......................................................................................................................................... 9 List of Tables ......................................................................................................................................... 11 Abbreviations ....................................................................................................................................... 12 1. Introduction ..................................................................................................................................... 18 1.1 Birnaviruses ........................................................................................................................... 18 1.2 IBDV impact on the poultry industry ..................................................................................... 18 1.2.1 The poultry industry ...................................................................................................... 18 1.2.2 Gumboro Disease .......................................................................................................... 19 1.3 IBDV genome and structure .................................................................................................. 20 1.4 Viral proteins ......................................................................................................................... 23 1.4.1 VP1 ................................................................................................................................ 23 1.4.2 VP2 ................................................................................................................................ 24 1.4.3 VP3 ................................................................................................................................ 24 1.4.4 VP4 ................................................................................................................................ 25 1.4.5 VP5 ................................................................................................................................ 26 1.5 IBDV replication cycle ............................................................................................................ 27 1.6 IBDV Virulence ....................................................................................................................... 30 1.6.1 IBDV strains ................................................................................................................... 30 1.6.2 IBDV control .................................................................................................................. 32 1.7 Host immune responses to IBDV ........................................................................................... 36 1.7.1 Innate immune sensing of IBDV .................................................................................... 36 1.7.2 IBDV evasion and antagonism of type I IFN production ............................................... 40 1.7.3 IFN signalling and ISG production following IBDV infection ......................................... 42 1.7.4 IBDV and NF-κB signalling ............................................................................................. 43 1.7.5 Adaptive immunity during IBDV infection .................................................................... 45 1.8 IBDV host transcriptomics ..................................................................................................... 47 1.8.1 In vitro studies ............................................................................................................... 47 5 1.8.2 In vivo studies ................................................................................................................ 49 1.9 Thesis aims and objectives .................................................................................................... 53 2. Materials and Methods ................................................................................................................... 55 2.1 Cell lines and media ............................................................................................................... 55 2.1.1 DF-1 cells ....................................................................................................................... 55 2.1.2 DT40 cells .....................................................................................................................