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Investigation of Dengue Virus Envelope Gene Quasispecies Variation in Patient Samples: Implications for Virus Virulence and Disease Pathogenesis

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Investigation of Dengue Virus Envelope Gene Quasispecies Variation in Patient Samples: Implications for Virus Virulence and Disease Pathogenesis Hannah Love Investigation of dengue virus envelope gene quasispecies variation in patient samples: implications for virus virulence and disease pathogenesis Cranfield Health PhD 2011 Supervisors: Prof. David Cullen (Cranfield University) Dr. Jane Burton (Health Protection Agency) Dr. Kevin Richards (Health Protection Agency) This thesis is submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy September 2011 © Cranfield University, 2011. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder. ABSTRACT i Abstract Due to the error-prone nature of RNA virus replication, each dengue virus (DV) exists as a quasispecies within the host. To investigate the hypothesis that DV quasispecies populations affect disease severity, serum samples were obtained from dengue patients hospitalised in Ragama, Sri Lanka. From the patient sera, DV envelope glycoprotein (E) genes were amplified by high-fidelity RT-PCR, cloned, and multiple clones per sample sequenced to identify mutations within the quasispecies population. A mean quasispecies diversity of 0.018% was observed, consistent with reported error rates for viral RNA polymerases (0.01%; Smith et al., 1997). However, previous studies reported 8.9 to 21.1-fold greater mean diversities (0.16% to 0.38%; Craig et al., 2003; Lin et al., 2004; Wang et al., 2002a). This discrepancy was shown to result from the lower fidelity of the RT-PCR enzymes used by these groups for viral RNA amplification. Previous studies should therefore be re-examined to account for the high number of mutations introduced by the amplification process. Nonsynonymous mutation locations were modelled to the crystal structure of DV E, identifying those with the potential to affect virulence due to their proximity to important structural features. No correlation was observed between the extent of quasispecies variation and disease severity. However, genome-defective quasispecies variants, and variants with surface accessible amino acid substitutions, or those proximal to the fusion peptide, proposed receptor binding sites, or other E oligomers, were observed predominantly in patients with severe dengue. Recombinant virus-like particles were produced for nine quasispecies variants, and the effects of the mutations on protein function assessed. Altered heparin binding abilities were demonstrated for four of the nine variants, indicative of differing cell attachment capabilities. Further work is required to assess differences in antibody binding, replication efficiency, virion and oligomer assembly, low pH-induced conformational changes required for fusion, and transmissibility of variants. ii ACKNOWLEDGEMENTS iii Acknowledgements This research project would not have been possible without the support of many people. Firstly I would like to thank my supervisors Prof. David Cullen, Dr. Jane Burton and Dr. Kevin Richards for their guidance and support during this project. I am grateful to Major Mark Bailey of the Royal Army Medical Corps for allowing me to use the dengue samples and clinical data from his fever study, and for providing medical expertise in the early stages of this project regarding the dengue patient disease severity classifications. Also thanks to his colleagues Dr. Ranjan Premaratna and Prof. Janaka de Silva from the Department of Medicine at the University of Kelaniya in Sri Lanka. I would also like to express my thanks to my colleagues at HPA CEPR, in particular the Diagnostic Support and C20 Project groups, for giving me the time and invaluable support to complete this project. Special thanks go to Dr. Richard Vipond, Dr. Daniel Bailey and Dr. Brian Dove for their expert advice and troubleshooting over the last four years; Dr. Stuart Dowall for his comments on Chapter 6, Howard Tolley for electron microscopy, Dr. Jane Osborne for help with phylogenetic analysis, Steve Kidd for initial guidance with ELISAs, and also to Mike Stubbington for cloning and sequencing advice. A huge thank you is due to my friends, for their support and encouragement, but mostly to my fellow PhD students at HPA CEPR, Ruth, Debs, Leanne, Simon, H and Lotte, for sharing the pain and making it a bit more bearable. Finally the biggest thanks go to my family, for putting up with me and learning when it’s best not to ask how it’s going; to Jenny, for sending the care package that made me smile; and especially to Simon, for his love, patience and understanding, and for looking after me. iv LIST OF CONTENTS v List of contents ABSTRACT ...................................................................................................................... I ACKNOWLEDGEMENTS ................................................................................................ III LIST OF CONTENTS ........................................................................................................ V LIST OF FIGURES ............................................................................................................ X LIST OF TABLES ........................................................................................................... XIV NOTATION ................................................................................................................ XVII CHAPTER 1. INTRODUCTION...................................................................................... 1 1.1 THESIS OVERVIEW ..................................................................................................................... 2 1.2 DENGUE .................................................................................................................................... 3 1.2.1 Aetiology of dengue ......................................................................................................... 3 1.2.2 Symptoms of dengue......................................................................................................... 6 1.2.3 Clinical and laboratory diagnosis of dengue .................................................................... 6 1.3 DENGUE VIRUSES .................................................................................................................... 12 1.3.1 Dengue virus structure and genome organisation ........................................................... 12 1.3.2 Dengue virus replication ................................................................................................ 13 1.3.3 Dengue virus binding to target host cells ........................................................................ 15 1.4 DETERMINANTS OF VIRAL REPLICATION AND DISEASE PATHOGENESIS ....................................... 17 1.4.1 Antibody-dependent enhancement of dengue virus infection ............................................ 17 1.4.2 Dengue virus sequence variation and virulence .............................................................. 18 1.4.3 Dengue in Sri Lanka ...................................................................................................... 25 1.4.4 Recombination in dengue viruses ................................................................................... 26 1.4.5 Dengue virus as a quasispecies ...................................................................................... 27 1.5 FLAVIVIRUS ENVELOPE PROTEINS AND THEIR ROLE IN VIRULENCE ............................................. 30 1.5.1 Flavivirus envelope protein structure ............................................................................. 30 1.5.2 Flavivirus envelope gene mutations and altered pathogenesis ......................................... 33 1.6 HYPOTHESIS, AIMS AND OBJECTIVES ........................................................................................ 37 1.6.1 Hypothesis ..................................................................................................................... 37 1.6.2 Aims and objectives ....................................................................................................... 37 CHAPTER 2. MATERIALS AND METHODS ................................................................. 40 2.1 GENERAL INFORMATION .......................................................................................................... 41 2.1.1 Chemicals and reagents ................................................................................................. 41 2.1.2 Oligonucleotides ............................................................................................................ 41 2.1.3 Flavivirus RNA used as a positive control template for RT-PCR ..................................... 41 2.1.4 Dengue patient material ................................................................................................. 41 2.1.5 Bacterial strains and culture media ................................................................................ 42 2.1.6 Insect cell lines and culture medium ............................................................................... 42 2.1.7 Insect viruses and virus transfer vectors ......................................................................... 43 2.2 IN SILICO TECHNIQUES ............................................................................................................. 44 2.2.1 Acquisition of published Flavivirus sequence data .......................................................... 44 2.2.2 Sequence analysis for primer design..............................................................................
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