Evaluating models of Influenza A virus Infection Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy By Elsa Gayle Zekeng BSc March 2019 1 AUTHOR’S DECLARATION Apart from the help and advice acknowledged, this thesis represents the unaided work of the author ................................... Elsa Gayle Zekeng, March 2019 This research was carried out in the Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool 2 ACKNOWLEDGEMENTS Over the course of my PhD, there are many people who provided me with mental and emotional support without whom I will not be able to get to this point today. It has been a great pleasure to complete my PhD at the University of Liverpool and more importantly, one of the greatest opportunities to do this in the Hiscox laboratory. First and foremost, I will like to thank God for giving me this opportunity and seeing me through my PhD. To my parents, Dr. & Dr. Mrs. Zekeng; the best gift a parent can give their child is education, you gave me that and so much more. For that, I will like to thank you from the bottom of my heart. For the sacrifices, you have made to give me this opportunity, they didn’t go unnoticed, I hope I’ve made you proud and I am able to pay you back some day. To my sister, Dr. Cherryl Zekeng, thank you for listening to me, for supporting me and pushing me to my full potential. To my other siblings, Miss Rita Tatah and Mr. Leopold Zekeng; thank you for the moral and emotional support. To Miss Zoey Leonore Awah and Mr. Liam Zuriel Awah, thank you for putting a smile on my face from the day you were born. To my supervisor, Prof. Julian Hiscox, thank you for giving me the opportunity to work with you, it has been my pleasure working with your group. Thank you to the whole of the Hiscox respiratory viruses’ group. Thank you to our collaborators in Public Health England; Prof. Miles Carroll, Dr. Anthony Marriott and Dr. Catherine Whittaker. Thank you to Professor Paul Digard at the Roslin Institute, University of Edinburgh for all the scientific support. Thank 3 you to Prof. James Stewart for your scientific support. Particular thanks to Dr. Isabel Garcia, Dr. Stuart Armstrong, Dr. Dong Xia, Dr. Nathifa Moyo, Dr. Diane Munday, Dr. Olivier Touzelet, Dr. Simon Clegg and Ms. Jill Hudson for all their scientific support during my PhD. A heartfelt thank you to all my friends in the IC2 in the infection Biology department for their friendship and scientific support over this time. To my friend, Miss Kabila Gana, thank you very much for your love, mental and emotional support and your continuous encouragement and prayers, I will forever be grateful. To Dr. Irene Afreh-Mensah and Miss Sharon Ngang, thank you for your emotional, mental and physical support. To Mr. Jamaal Brathwaite, thank you very much for your continuous support and love over the last year, it was indeed a very trying year, but your love and support carried and encouraged me through. Last but certainly not the least, a very special thanks to Dr. Sarah Tade. For the constant love, encouragement, prayers and support during the last five years, I could not have done this without her. 4 ABSTRACT High throughput proteomics and transcriptomics has provided a platform to further understand viral – host interaction. This provides a window into the host proteome and transcriptome with and without infection. This leads to identification of potential biomarkers, understanding IAV pathogenesis and also drawing a comparison of how hosts respond to viral infection. This thesis used two independent high throughput approaches to explore the proteome and transcriptome of samples from hosts (in vitro and in vivo) infected with influenza A virus (IAV) compared to samples from hosts (in vitro and in vivo) non-infected with IAV. The independent high throughput approaches used were; proteomics on a Q-Exactive platform and transcriptomics on a MinION sequencer. These approaches were used to further understand IAV infection in these different hosts and secondly to explore and search further for a potential biomarker for the diagnosis of IAV and potential drug targets. To our knowledge, this is the first study that has used high throughput approaches to analyses samples from different hosts. This allowed for comparison across hosts but also provides vast amounts of data that are reliable and consistent. A549 cells that were mock-infected and IAV- infected were subjected to the Q-Exactive platform and MinION sequencing. This provided insight into the in vitro host-viral interaction on a cellular level. For the first time, showed the potential of MinION sequencing as a method for understanding the viral-host interaction of IAV infected cells and identifying potential biomarkers. This highlighted transcripts such as NUP54, RBM42, HPGD, GCLC, ANPEP, AKAP13, RACGAP1, CREB1, MAN2B1 and PRKCI. These transcripts were 5 identified by bioinformatic analysis as host factors that play a crucial role in replication of IAV in the host. The corresponding proteins to these transcripts were also identified by proteomics. To better understand IAV in hosts, in vivo, Non-human primates (NHP) were infected with IAV and the broncho-alveolar fluid (BALF) was collected and compared to BALF from naïve NHP. After analysis on the Q-Exactive platform, the results obtained drew parallels on a cellular level to that observed in vitro models. Proteins such as; DDX58, EIF3A, HSP90AA1, MAPK1, MX1 and STAT1 involved in the “replication of IAV” were highlighted. In addition to the cellular changes, the NHP studies provided insight into an immune response similar to that observed in humans following IAV infection. This provided an added dimension in understanding IAV infection. Finally, nasopharyngeal aspirates (NAs) from humans IAV- infected and IAV non-infected from three different cohorts (Alder Hey Children’s hospital (AHCH), Liverpool, Great Ormond Street Hospital (GOSH), London and Institute Pasteur Dakar (IPD)) were analysed on the Q-Exactive platform. This provided a full circle loop to compare if the changes observed in vitro model – A549 cells and in vivo model-NHP were relatable back to humans. Proteins identified in vitro and in vivo studies were concordant with proteins identified in the human NAs. These proteins include; COPA, STAT1, TUBB and HSPB1. Additionally, three proteins were identified in human NAs across all three cohorts; BPIFA1/SPLUNC1, Lactotransferrin and Fibrinogen A, B and G. These proteins play crucial roles in elucidating IAV infection in the host. This study presents the first time these proteins have been highlighted using label-free Mass spectrometry in human NAs across three cohorts from different geographical locations. 6 This thesis illustrates how proteomic analysis of IAV-infected samples compared to non-infected samples can be used to identify markers that may serve as potential diagnostic indicators for IAV infection. 7 TABLE OF CONTENTS ABBREVIATIONS ....................................................................................... 15 CHAPTER 1: ............................................................................................... 19 INTRODUCTION ......................................................................................... 19 1.1 GENERAL INTRODUCTION ....................................................................... 20 1.2 INTRODUCTION TO THE CLASSIFICATION OF FAMILIES ............................... .22 1.2.1 The Orthomyxoviridae family ....................................................... 22 1.3 ANTIGENIC DRIFT, ANTIGENIC SHIFT, AND RECOMBINATION ........................ 28 1.3.1 Antigenic drift – mutation ............................................................. 28 1.3.2 Antigenic shift – reassortment ...................................................... 29 1.3.3 Recombination ............................................................................. 31 1.4 INFLUENZA EPIDEMICS AND PANDEMICS ................................................... 33 1.4.1 Epidemics .................................................................................... 33 1.4.2 Pandemics ................................................................................... 34 1.5 INFLUENZA VIRION STRUCTURE, GENOME ORGANISATION, AND PROTEINS ... 37 1.5.1 Virion structure ............................................................................. 37 1.5.2 Genome structure, organisation, and viral proteins ..................... 39 1.6 INFLUENZA A VIRUS REPLICATION CYCLE ................................................. 45 1.6.1 Virus attachment and entry to host cell surface receptors ........... 45 1.6.2 Replicating their genome ............................................................. 47 1.6.3 vRNPs leaving the nucleus .......................................................... 48 1.6.4 Assembly and release .................................................................. 49 1.7 VIRAL PATHOGENESIS AND HOST IMMUNE RESPONSE................................ 51 1.7.1 Host immune response ................................................................ 51 8 1.7.2 Virus factors that influence viral pathogenesis ............................. 54 1.8 TREATMENT AND VACCINES .................................................................... 58 1.9 QUANTITATIVE PROTEOMIC TECHNIQUES USED TO CHARACTERISE VIRAL- HOST INTERACTIONS.................................................................................... 60 1.9.1