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Westminsterresearch the Study of Highly Pathogenic Emerging WestminsterResearch http://www.westminster.ac.uk/westminsterresearch The study of highly pathogenic emerging zoonotic virus envelope proteins through pseudotyped virus generation Bentley, E. This is an electronic version of a PhD thesis awarded by the University of Westminster. © Miss Emma Bentley, 2017. The WestminsterResearch online digital archive at the University of Westminster aims to make the research output of the University available to a wider audience. Copyright and Moral Rights remain with the authors and/or copyright owners. Whilst further distribution of specific materials from within this archive is forbidden, you may freely distribute the URL of WestminsterResearch: ((http://westminsterresearch.wmin.ac.uk/). In case of abuse or copyright appearing without permission e-mail [email protected] THE STUDY OF HIGHLY PATHOGENIC EMERGING ZOONOTIC VIRUS ENVELOPE PROTEINS THROUGH PSEUDOTYPED VIRUS GENERATION Emma May Bentley A thesis submitted in partial fulfilment of the requirements of the University of Westminster for the degree of Doctor of Philosophy September 2017 Abstract Emerging zoonotic viruses pose an increasing threat, causing outbreaks with high rates of morbidity and mortality and frequently significant economic implications. Often, there is a lack or shortfall of effective prophylaxis and diagnostic capabilities. Research towards their development, together with improved surveillance activities are high priority activities to prepare and respond to outbreak threats. Yet handling these viruses commonly requires high containment levels. This can be circumvented by the use of replication defective pseudotyped viruses (PVs), incorporating the viral envelope protein of interest which constitutes the primary surface antigen. This permits the serological detection of neutralising antibodies without the need to handle live virus, as well as other viral entry studies. Hence, PVs are increasingly proving to be a valuable tool for emerging virus research. The aim of this study was to exploit novelties in the unique flexibility of the PV platform to allow the serological assessment of emerging viruses and evaluate technical aspects towards standardisation. Current prophylaxis provides robust protection against rabies virus, yet only confers limited protection against other lyssavirus species, which have a near 100% fatality rate. It is thought protection is afforded against isolates of phylogroup I rabies virus, yet there is limited biological data for the Arctic-like rabies virus (AL RABV) lineage which is endemic across the Middle East and Asia. Although other lyssaviruses pseudotype efficiently, titres of AL RABV PV were low. Within this study, high titre PV was produced by constructing chimeric envelope proteins, splicing the AL RABV ecto-transmembrane domain with the cytoplasmic domain of vesicular stomatitis virus. Comparisons showed this did not alter the serological profile of the AL RABV and they were effectively neutralised by vaccines and antivirals. It could therefore be concluded that they do not pose a significant public health risk. However it is recognised broadly neutralising prophylaxis needs to be developed to protect against more divergent lyssaviruses. In a further study, again utilising the flexibility to manipulate the envelope protein, PV was produced switching the five known antigenic sites of the envelope protein between a phylogroup I (rabies virus) and III (West Caucasian bat virus) isolate. Screening polyclonal sera via a neutralisation assay, the immunologically dominant sites for phylogroup I and III were identified as III and I respectively. This can act to inform future development of more broadly neutralising vaccines. The 2013-16 outbreak of Ebola virus focused global efforts towards the urgent need for effective vaccines and antivirals. To permit low containment level serology studies to assist their development, a panel of filovirus PVs were rapidly produced. Work was carried out to optimise their method of production; determining lentiviral core PV produced by transfecting HEK 293T/17 cells was most efficient. Efforts to repeat the use of chimeric envelope proteins to increase titre I proved unsuccessful. The evaluation of target cell lines permissive to infection and appropriate for neutralisation assays identified that the CHO-K1 cell line produced the clearest data. The PV neutralisation assay was subsequently applied to a range of projects to assess candidate prophylaxis and demonstrated the value of the platform to respond to emerging virus outbreaks. Given the increasing prominence in the use of PV, work was undertaken to expand their utility and methods for standardisation. An assessment of new reporter genes found a red fluorescent protein, with a nuclear localisation signal, improved the clarity of data collection and output in additional spectrum to the current repertoire. To be able to correlate the disparate readout units of fluorescent and luminescent reporters, recorded as infectious units (IFU) and relative light units (RLU) respectively, a new construct was produced to integrate and equally express two reporters from cells transduced with PV. It was determined that approximately 1260 RLU equates to 1 IFU, although future work to determine how this fluctuates between cell lines is required. Finally, alternative methods to quantify PV were evaluated, measuring the number of particles, genome copies and reverse transcriptase (RT) activity, in addition to the currently used biological titre. It was found that measures of genome copies and RT activity, in combination with biological titre provides information on the quality of PV preparations and could be used to standardise assay input. II Table of Contents ABSTRACT .......................................................................................................................................... I TABLE OF CONTENTS ..................................................................................................................... III LIST OF FIGURES ........................................................................................................................... VII LIST OF TABLES ................................................................................................................................ X ACKNOWLEDGEMENTS ................................................................................................................. XII AUTHOR’S DECLARATION ........................................................................................................... XIII LIST OF ABBREVIATIONS ............................................................................................................. XIV CHAPTER 1. INTRODUCTION ............................................................................................................ 1 1.1. EMERGING ZOONOTIC VIRUSES ................................................................................................................ 1 1.1.1. History and Processes of Zoonotic Virus Emergence.......................................................................... 1 1.1.2. Significance and Future Direction of the Emerging Virus Field ......................................................... 6 1.1.3. Lyssaviruses ........................................................................................................................................ 9 1.1.4. Filoviruses ......................................................................................................................................... 13 1.2. THE VIRAL ENVELOPE PROTEIN ............................................................................................................. 16 1.2.1. Importance and Structure of the Viral Envelope Protein ................................................................... 16 1.2.2. The Viral Envelope Protein as a Target ............................................................................................. 20 1.3. SEROLOGY ................................................................................................................................................ 21 1.3.1. Serological Investigations.................................................................................................................. 21 1.3.2. Serological Assays............................................................................................................................. 23 1.4. PSEUDOTYPED VIRUS ............................................................................................................................... 25 1.4.1. Pseudotyped Virus Definition ........................................................................................................... 25 1.4.2. History of Pseudotyped Virus ............................................................................................................ 26 1.4.3. Pseudotyped Virus Production .......................................................................................................... 32 1.4.4. Pseudotyped Virus Applications ....................................................................................................... 37 1.5. AIMS AND OBJECTIVES ............................................................................................................................ 42 CHAPTER 2. MATERIALS AND METHODS ...................................................................................... 44 2.1. MATERIALS .............................................................................................................................................
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