Production of a novel Affimer based biosensor for the detection of porcine reproductive and respiratory syndrome virus nucleocapsid protein. Zoe Jackson Submitted in accordance with the requirements for the degree of Doctor of Philosophy University of Leeds Faculty of Biological Sciences School of Molecular and Cellular Biology November 2017 The candidate confirms that the work submitted is her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from this thesis may be published without proper acknowledgement. © 2017 The University of Leeds and Zoe Jackson Acknowledgments I would first like to thank my primary supervisor Professor Adrian Whitehouse who saw potential in a shy and nervous Masters student and encouraged me to challenge myself with the undertaking of a PhD. His encouragement, guidance and support have allowed me to progress into a confident scientist. I would also like to thank my co-supervisor Dr Darren Tomlinson for his invaluable support during the dark times of Affimer screening and for our useful brainstorming sessions featuring my excellent drawings. A huge thank you to the Whitehouse and Hewitt group members past and present without whom I would not have managed to complete year one let alone year five of this PhD. The happy working environment, the cake and the laughs have been a welcome side offering in addition to plenty of scientific advice. In particular I would like to mention Dr Sophie Schumann, Dr David Hughes and Dr Brian Jackson. I must also take this opportunity to thank the Wellcome Trust for providing the funding for me to carry out this research and for providing a PhD scheme which allowed me to assist in the design of my project and the opportunity to experience such exciting science. I undertook a placement at BBI Solutions, Cardiff, and would like to thank them for providing me with the training and support for lateral flow development. Finally I would like to thank my family (the one I already had and the one I gained recently) initially for their encouragement, belief and also for listening to me at times of despair. But secondly for their help in babysitting duties which have been life-saving at the end. Last but not least, I would like to say a huge thank you to my husband Brian (again) for his unwavering support and who has read this entire thesis multiple times, offering his thoughts and suggestions, as well as listening to me when I was worrying at all hours about finishing with a new baby. And so that leaves Hugo, the little boy who arrived so close to the end. Thank you for keeping me awake most nights, giving the best cuddles after a hard day’s work and for being the best son anyone could wish for. Although you were not here at the start, the completion of this thesis is for you as well as me and I hope you will be proud. i Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important infection with no current point of care (POC) diagnostic available. PRRSV causes reproductive and respiratory illness in swine with the recent emergence of highly pathogenic strains. This highlights the need for measures to control the spread of this infection to be taken more seriously in order to reduce the economic impact of this virus. Current diagnostics for PRRSV are laboratory based and inherently these tests are expensive and are not rapid enough for the adequate management of outbreaks of the virus and implementation of biosecurity measures. This study presents a novel lateral flow device (LFD), using Affimer binding proteins to detect the nucleocapsid protein of PRRSV within a clinical sample to provide a cheap, rapid and reliable diagnostic for this infection in clinical samples. Affimer reagents were raised against the nucleocapsid proteins from two strains of PRRSV, a high pathogenic and a low pathogenic strain. Affimers that were able to distinguish between the two were taken forward for assessment in lateral flow. The Affimers were able to bind to the nitrocellulose membrane component of the device and were stable once dehydrated. The Affimers were able to migrate through the membrane via capillary action when rehydrated and can detect the viral protein at a test line within a clinical sample, swine serum. This study provides the basis for further investigations in to the applications of Affimer reagents in lateral flow devices able to detect other viral infections as well as medically important diseases such as cancer. In addition to their use in diagnostics, this study proposes the use of Affimers raised against the nucleocapsid protein of PRRSV as molecular tools for the further investigation into the role of this protein in the viral lifecycle as well as their potential as anti-viral therapeutics to address the lack of these medicines against this virus. ii Contents Acknowledgements……………………………………………………………………………………………..i Abstract………………………………………………………………………………………………………………ii Contents…………………………………………………………………………………………………………….iii List of tables……………………………………………………………………………………………………….ix List of figures………………………………………………………………………………………………………x Abbreviations…………………………………………………………………………………………………..xiii 1 Introduction .......................................................................................................... 2 1.1 Infectious diseases of livestock ............................................................................. 2 1.2 Virus classification ................................................................................................. 3 1.2.1 Baltimore classification of viruses ................................................................ 3 1.3. Nidovirales ............................................................................................................. 3 1.3.1. Introduction to the Nidovirus order ............................................................. 3 1.3.2. Nidovirus hosts ............................................................................................. 4 1.3.3. Nidovirus genome......................................................................................... 5 1.3.4. Nidovirus virion architecture ........................................................................ 6 1.3.5. Members of the Nidovirus order .................................................................. 7 1.3.5.1. Coronaviridae .......................................................................................... 7 1.3.5.2. Toroviridae .............................................................................................. 7 1.3.5.3. Mesoniviridae ......................................................................................... 8 1.3.5.4. Arteriviridae ............................................................................................ 8 1.3.6. Arteriviruses ................................................................................................. 8 1.3.6.1. Genome organisation ............................................................................. 9 1.3.6.2. Arterivirus lifecycle ............................................................................... 11 1.3.6.3. Arterivirus virion ................................................................................... 14 1.3.6.4. Arteriviral proteins ................................................................................ 16 1.3.6.4.1. Polyprotein1ab .............................................................................. 16 1.3.6.4.2. RdRp .............................................................................................. 18 1.3.6.4.3. Nucleocapsid protein .................................................................... 19 1.3.6.4.4. Glycoproteins ................................................................................ 21 1.3.6.4.5. M protein ...................................................................................... 23 1.3.6.4.6. E protein ........................................................................................ 24 1.3.6.5. Arteriviral pathogenesis ........................................................................ 24 iii 1.3.6.5.1. Arteriviral innate immune response ............................................. 25 1.3.6.5.2. Humoral immune response ........................................................... 25 1.3.6.5.3. Cell-mediated immune response .................................................. 25 1.4. Porcine reproductive and respiratory syndrome virus (PRRSV) ......................... 25 1.4.1. PRRSV clinical presentation and pathogenicity .......................................... 26 1.4.1.1. Evasion of the immune response ......................................................... 27 1.4.1.2. Modulation of cellular signalling pathways .......................................... 27 1.4.1.3. Modulation of other host cytokines ..................................................... 30 1.4.1.4. Hijacking of host miRNAs ...................................................................... 30 1.4.1.5. Modulation of T-cell response .............................................................. 31 1.4.1.6. Delayed detection of virus .................................................................... 32 1.4.1.7. Antibody dependent enhancement ...................................................... 32 1.4.1.8. Clinical manifestation of PRRSV ............................................................ 33