A NON MOUSE-ADAPTED DENGUE VIRUS STRAIN AS A NEW MODEL OF SEVERE DENGUE INFECTION IN AG129 MICE TAN KAI XIN GRACE B.SC (HONS) A THESIS SUBMITTED FOR THE DEGREE OF MASTERS OF SCIENCE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor, Dr Sylvie Alonso for her guidance and support throughout my master candidature I thank her for all the advices she has given and the trust she has in me to carry out my project independently Also, I am thankful for the many opportunities she has given me to publish various pieces of work with her None of this would have been possible without her expertise, patience and encouragement I sincerely thank Jowin, for his relentless help and support throughout this period of time To Annabelle, Emily, Weixin, Wenwei: for the advices they have given me in various aspects of my work and being there for me whenever I need them and to the rest of the lab members: for making the lab environment full of life and an enjoyable place to work in I would also like to thank my parents for all the support they have given me all these while and their understanding for the times when I have to devote my time to my work Last but not least, I would like to thank my husband, Andrew, for every single thing he has done for me I wouldn’t have been able to achieve all these without his help and kind understanding i Publications PUBLICATIONS Journals: Tan GK, Ng JKW, Trasti SL, Schul W, Yip G and Alonso S (2010) A non mouseadapted dengue virus strain as a new model of severe dengue infection in AG129 mice PLoS Negl Trop Dis 4(4): e672 Tan GK and Alonso S (2009) Pathogenesis and prevention of dengue virus infection: state-of-the-art Curr Opin Infect Dis (Invited review) 22(3):302-8 Sim AC, Lin W, Tan GK, Sim MS, Chow VT, Alonso S (2008) Induction of neutralizing antibodies against dengue virus type upon mucosal administration of a recombinant Lactococcus lactis strain expressing envelope domain III antigen Vaccine 26(9): 1145-54 ii Contents CONTENTS ACKNOWLEDGEMENTS i PUBLICATIONS .ii CONTENTS iii SUMMARY vii TABLES AND FIGURES ix ABBREVIATIONS xi INTRODUCTION 1.1 DENGUE VIRUS .1 1.1.1 CLASSIFICATION 1.1.2 DENGUE VIRUS PROTEINS 1.1.2.1 CAPSID (C) PROTEIN .1 1.1.2.2 MEMBRANE (M), PRE-M (PRM) AND ENVELOPE (E) PROTEINS 1.1.2.3 NS1 PROTEIN 1.1.2.4 NS3 PROTEIN 1.1.2.5 NS5 PROTEIN – THE VIRAL RNA-DEPENDENT RNA POLYMERASE (RDRP) 1.1.2.6 OTHER NON-STRUCTURAL PROTEINS .5 1.2 TRANSMISSION CYCLE OF DENGUE VIRUS 1.3 DENGUE VIRUS REPLICATION IN HUMANS 1.4 THE SPREAD AND THREAT OF DENGUE VIRUS 10 1.5 DENGUE INDUCED DISEASE 10 1.5.1 CLASSIFICATION 10 1.5.2 CLINICAL MANIFESTATIONS OF DHF/DSS 10 iii Contents 1.6 DENGUE PATHOGENESIS 14 1.6.1 IMMUNE RESPONSE TO DENGUE VIRUS INFECTION 14 1.6.1.1 PROTECTIVE ROLE OF IMMUNE RESPONSE AGAINST DENGUE VIRUS INFECTION 14 1.6.1.2 IMMUNOPATHOGENESIS OF DENGUE VIRUS INFECTION: THE ENHANCEMENT HYPOTHESIS OF DHF/DSS PATHOGENESIS 15 1.6.1.3 NON-ENHANCING MECHANISMS AS RISK FACTORS FOR DHF/DSS: AUTOIMMUNE AND COMPLEMENT-MEDIATED PATHOGENESIS 20 1.6.2 HOST GENETIC FACTORS .22 1.6.3 VIRUS VIRULENCE .23 1.7 DENGUE PREVENTION 25 1.7.1 VECTOR CONTROL 25 1.7.2 DENGUE VIRUS VACCINES 26 1.7.2.1 LIVE ATTENUATED VACCINE (LAV) 28 1.7.2.2 CHIMERIC VIRUS VACCINE 29 1.7.2.3 SUBUNIT DENGUE VACCINE 30 1.8 MODELS FOR DENGUE VIRUS INFECTION 33 1.8.1 NONHUMAN PRIMATE ANIMAL MODEL .33 1.8.2 ESTABLISHED MOUSE MODELS 34 1.8.2.1 MOUSE-ADAPTATION OF DENGUE VIRUS 34 1.8.2.2 WILDTYPE MOUSE STRAINS 35 1.8.2.3 MOUSE-HUMAN CHIMERAS 36 1.8.2.4 KNOCKOUT MOUSE STRAINS 38 AIM 41 MATERIALS AND METHODS 42 iv Contents 3.1 VIRUS STRAIN AND GROWTH CONDITIONS 42 3.2 VIRUS QUANTITATION 42 3.3 MICE INFECTION 43 3.4 ENZYME-LINKED IMMUNOADSORBENT ASSAY (ELISA) 43 3.5 PLAQUE REDUCTION NEUTRALIZATION TEST (PRNT) .44 3.6 VIRUS QUANTIFICATION IN INFECTED MICE .45 3.7 HISTOPATHOLOGY .46 3.8 VASCULAR LEAKAGE ASSESSMENT 46 3.9 CYTOKINE DETECTION 47 3.10 HAEMATOLOGY .47 3.11 STATISTICAL ANALYSIS 47 RESULTS PART I 48 INTRAPERITONEAL INFECTION OF D2Y98P IN AG129 48 4.1 SURVIVAL RATE IN D2Y98P INFECTED AG129 48 4.2 VIREMIA PROFILE IN D2Y98P INFECTED AG129 50 4.3 SPECIFIC ANTIBODY TITRES AND PRNT50 LEVELS AGAINST D2Y98P 52 4.4 TISSUE TROPISM AND KINETIC OF VIRUS REPLICATION IN D2Y98P-INFECTED AG129 55 4.5 HISTOLOGICAL EXAMINATION OF ORGANS IN D2Y98P-INFECTED AG129 57 4.6 VASCULAR LEAKAGE IN D2Y98P-INFECTED AG129 .61 4.7 CYTOKINE EXPRESSION LEVELS IN D2Y98P-INFECTED AG129 63 4.8 HAEMATOLOGY IN D2Y98P-INFECTED AG129 65 RESULTS PART II .67 SUBCUTANEOUS INFECTION OF D2Y98P IN AG129 67 v Contents 5.1 COMPARATIVE ANALYSIS OF SURVIVAL RATE AND MEAN VIRUS TITRES IN IP VS SC D2Y98P-INFECTED AG129 .67 5.2 HISTOPATHOLOGICAL CHANGES AND VASCULAR LEAKAGE IN SC INFECTED AG129 71 5.3 DOSE-DEPENDENT SURVIVAL RATE IN D2Y98P SC INFECTED AG129 74 5.4 SPECIFIC ANTIBODY TITRES AND PRNT50 LEVELS AGAINST D2Y98P IN SC INFECTED AG129 .77 5.5 TISSUE TROPISM AND KINETIC OF VIRUS REPLICATION IN AG129 INFECTED SC WITH 10 PFU OF D2Y98P VIRUS 78 5.6 HISTOPATHOLOGY, VASCULAR PERMEABILITY AND CYTOKINE EXPRESSION LEVELS IN 10 PFU D2Y98P SC INFECTED AG129 83 5.7 HAEMATOLOGICAL PARAMETERS IN D2Y98P SC INFECTED AG129 88 DISCUSSION 90 6.1 MOUSE MODELS FOR DENGUE INFECTION .90 6.2 THE AG129 MOUSE MODEL FOR DENGUE INFECTION 91 6.3 INFECTION OF D2Y98P IN AG129 MICE 92 6.3.1 IP ROUTE OF D2Y98P INFECTION IN AG129 MICE 93 6.3.2 SC ROUTE OF D2Y98P INFECTION IN AG129 MICE 98 6.4 CLINICAL RELEVANCE TO DENGUE DISEASE IN HUMANS 102 CONCLUSION 104 FUTURE WORK 105 REFERENCES 107 10 APPENDIX .127 vi Summary SUMMARY The spread of dengue (DEN) worldwide combined with an increased severity of the DEN-associated clinical outcomes have made this mosquito-borne virus of great global public health importance Progress in understanding DEN pathogenesis and in developing effective treatments has been hampered by the lack of a suitable small animal model Most of the DEN clinical isolates and cell culture-passaged DENV strains reported so far, either require the need for host adaptation, inoculation with a high dose and/or intravenous (iv.) administration to elicit a virulent phenotype in mice, which results at best in a productive infection with none, few or irrelevant disease manifestations, and with mice dying at the peak of viremia Here we describe a non mouse-adapted DEN2 virus strain (D2Y98P) that is highly infectious in AG129 mice (lacking interferon-α/β and -γ receptors) upon intraperitoneal (ip.) and subcutaneous (sc.) administration Infection with a high dose (107 PFU) of D2Y98P (via the ip and sc route) induced a cytokine storm, massive organ damage, and severe vascular leakage, leading to hemorrhage and rapid death of the animals at the peak of viremia In contrast, very interestingly and uniquely, ip infection with a low dose of D2Y98P (104 PFU) led to asymptomatic viral dissemination and replication in relevant organs, followed by non-paralytic death of the animals few days after virus clearance, similar to the disease kinetic described in DEN-infected patients Tissue damage and increased vascular permeability but no hemorrhage, were observed only at moribund state of infected animals, suggesting intact vascular integrity, a cardinal feature of DEN shock syndrome Interestingly, whereas high pro-inflammatory (TNF-α, IL-6 and IFN-γ) cytokine levels were detected at the peak of viremia – during which the extent of tissue damage and vii Summary vascular leakage was minimal; basal production of these cytokines was instead measured in the animals at moribund state, where tissue damage and vascular leakage were more severe This observation suggests that other cytokines or other inflammation-independent mechanisms may be responsible for the vascular leakage phenomenon observed in the infected animals at moribund state Strikingly, infection with 104 PFU of D2Y98P by the sc route instead, induced clinical manifestations and disease which closely resembled that of the high dose model, indicating that the sc route of infection is more potent than the ip route at triggering a virulent phenotype of DEN infection in AG129 Altogether, infection with the D2Y98P strain thus offers the opportunity to further decipher some of the aspects of DEN pathogenesis and provides a new platform for drug and vaccine testing viii Tables and Figures TABLES AND FIGURES Figure 1.1: The intracellular life cycle of DENV .……………………………9 Figure 1.2: Classification and clinical manifestations in humans infected with DENV … ………………………………………………………12 Figure 1.3: A diagrammatic view of the antibody dependent enhancement (ADE) phenomenon of DENV infection ……………………………… 17 Figure 1.4: Schematic representation of the immunopathogenesis of severe DEN disease mediated by cross reactive anti-DEN antibodies and T cells ……………………………………………………………….……….19 Table 1.1: Proposed mechanisms involving the host immune system in mediating severe DEN disease in humans …………………………………… 21 Table 1.2: Characteristics of an ideal DEN vaccine ………….……………… 27 Figure 1.5: Summary of candidate DEN vaccines currently in development .…32 Figure 4.1: Survival rate in AG129 mice infected with a dose range of D2Y98P virus …………………………………………………………………49 Figure 4.2: Body weight changes in D2Y98P-infected AG129 ……………… 49 Figure 4.3: Viremia profile in D2Y98P-infected AG129 ……………………….51 Figure 4.4: Specific antibody titres against D2Y98P ………………………… 53 Figure 4.5: Neutralizing antibody titres specific against D2Y98P …………… 54 Figure 4.6: Gross pathological changes in D2Y98P-infected AG129 ………… 56 Figure 4.7: Quantification of virus titres in the liver, spleen and brain of D2Y98Pinfected AG129 …………………………………………………… 56 Figure 4.8: Histopathology of D2Y98P-infected AG129 ……………………….59 Figure 4.9: Serum levels of aspartate (AST) and alanine (ALT) transaminases 60 Figure 4.10: Vascular leakage in D2Y98P-infected AG129 …………………… 62 Figure 4.11 Pro-inflammatory cytokine expression in D2Y98P-infected AG129 ……………………………………………………………………… 64 Table 4.1: Haematology in D2Y98P-infected AG129 …………………………66 ix Chapter 10: Appendix 139 Chapter 10: Appendix 140 Chapter 10: Appendix 141 Chapter 10: Appendix 142 Chapter 10: Appendix 143 Chapter 10: Appendix 144 Chapter 10: Appendix 145 Chapter 10: Appendix 146 Chapter 10: Appendix 147 Chapter 10: Appendix 148 Chapter 10: Appendix 149 Chapter 10: Appendix 150 Chapter 10: Appendix 151 Chapter 10: Appendix 152 Chapter 10: Appendix 153 ... has also been implicated in DEN disease; hence elevations in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (Burke et al., 1988), - indicative of liver damage - are... levels of aspartate (AST) and alanine (ALT) transaminases ……………………………………………………………………… 85 Figure 5.13: Comparative analysis of vascular leakage and albumin concentration in D2Y98P sc infected AG129. .. levels of aspartate (AST) and alanine (ALT) transaminases 60 Figure 4.10: Vascular leakage in D2Y98P-infected AG129 …………………… 62 Figure 4.11 Pro-inflammatory cytokine expression in D2Y98P-infected AG129