ROLE OF VIRAL GENETICS IN DENGUE EPIDEMIOLOGICAL FITNESS GAYATHRI MANOKARAN B.Science (Hons), Monash University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2014 Acknowledgements I would like to express my heartfelt gratitude to Associate Professor Ooi Eng Eong for his patient guidance, invaluable advice and encouragement throughout my research. His continued support and mentorship significantly aided the progress of the project. I would also like to thank Professor Mariano Garcia-Blanco for his scientific guidance and kind encouragement. To Professor Duane Gubler: A big thank you for having the hindsight of collecting the clinical isolates used in this study and the generosity to allow me to work with them. My sincere appreciation is also extended to the rest of my thesis advisory committee, Professor David Virshup, Assistant Professor Manoj Krishnan and Professor Naoki Yamamoto for the stimulating discussions and critical suggestions during my research. Special thanks to Esteban Finol, Eugenia Ong, Justin Bahl, Dr Alex Ward, Dr Katell Bidet, Tan Hwee Cheng and Summer Zhang for providing technical assistance and support in my research. And not forgetting colleagues from Duke-NUS who have made this a very conducive environment for scientific research. Lastly, I would like to thank my mum, dad, sister and dog for their love, encouragement and support all these years. This work would not have been possible without them. “The more I study science, the more I believe in God” – Albert Einstein. With that, I’ll like to extend a final thank you to God for His continued presence throughout these grueling PhD years. i Table of Contents Acknowledgements i Table of contents iii Summary ix List of tables x List of figures xi List of abbreviations xv List of publications xxi Chapter 1: An Introduction to the role of viral genetics in dengue epidemiological fitness. 1.1 1.2 Dengue Virus 1.1.1 Virus structure and genome organization 1.1.2 Lifecycle of dengue virus 1.1.3 Virus Transmission 1.1.4 Clinical presentation and progression 10 1.1.5 Prevention and control of dengue 13 1.1.5.1 Vector control programs 13 1.1.5.2 Vaccines in clinical development 15 Disease Pathogenesis 19 1.2.1 Factors contributing to dengue pathogenesis 19 iii 1.2.1.1 Role of secondary infection in severe dengue pathogenesis 1.2.1.2 19 Viral virulence contributes to pathogenesis 1.2.2 Immune responses to dengue infection 1.2.2.1 Host innate immune response against dengue 1.2.2.2 Subversion of innate immune responses by dengue 1.2.2.2.1 Role of dengue coding regions 24 28 30 32 33 1.2.2.2.2 Role of dengue noncoding regions – sfRNA 36 1.2.2.2.2.1 Inhibition of cellular RNA decay 40 1.2.2.2.2.2 Perturbing the RNAi pathway 40 1.2.2.2.2.3 Interference with IFN-mediated innate immune responses 1.3 Dengue epidemiology 41 44 1.3.1 Dengue is an emerging disease 44 1.3.2 Problems caused by dengue as an emerging disease 47 1.3.3 Interplay of host and viral factors in dengue epidemics 49 iv 1.4 1.3.3.1 Serotype switch linked to dengue epidemics 49 1.3.3.2 Virus genetics linked to recent epidemics 52 What is the molecular basis for dengue epidemics? – Gaps in Knowledge 58 Chapter 2: Materials and Methods 2.1 Cells 61 2.2 Viruses 62 2.3 Plaque assay 62 2.4 Infections 63 2.5 Sequence and structure analysis 64 2.6 Quantification of RNA 65 2.7 Northern hybridization 67 2.8 siRNAs 69 2.9 Western blot 69 2.10 Constructs 70 2.11 In vitro transcription 70 2.12 Electroporation and dual-luciferase assay 71 v 2.13 sfRNA transfections 72 2.14 RNA-protein interactions 73 2.14.1 SILAC-qMS 73 2.14.2 RNA-immunoprecipitations 74 2.15 Co-immunoprecipitation and immunoblot analysis 75 2.16 Statistical analysis 76 Chapter 3: Results 3.1 Subgenomic flavivirus RNA is a critical determinant of the epidemiological fitness of Dengue-2 78 3.1.1 Comparative analysis of the 1994 Puerto Rican DENV-2 isolates 79 3.1.2 Viral isolates from the PR-2B clade produce less viral RNA and infectious particles at 24 hours p.i but higher relative amounts of sfRNA 3.1.3 Increased sfRNA:gRNA in Nicaraguan DENV-2 3.2 87 95 PR-2B DENV-2 replication is associated with reduced expression of IFN-β relative to PR-1 DENV-2. 101 3.2.1 sfRNA suppresses type I interferon expression for greater replication rates in the later stages of infection 102 vi NONO 0.859 0.950 1.064 -0.068 0.526 1.772 1.712 1.748 0.802 0.000 1.970 1.710 1.543 0.793 0.016 DAZAP1 0.826 0.813 1.150 -0.124 0.526 1.603 1.180 1.037 0.324 0.224 1.901 1.487 0.863 0.429 0.331 CAND1 0.952 1.029 0.968 -0.026 0.528 0.900 0.915 0.905 -0.141 0.003 0.893 0.891 0.916 -0.152 0.007 TMEM109 0.959 0.876 1.054 -0.058 0.529 0.668 0.672 0.716 -0.545 0.003 0.586 0.679 0.596 -0.692 0.009 RPS6KA3 1.021 0.968 0.973 -0.019 0.530 1.039 0.986 0.735 -0.137 0.472 0.961 1.000 0.720 -0.177 0.358 COPB1 1.032 0.948 0.964 -0.028 0.532 0.850 0.990 0.730 -0.234 0.206 0.859 0.962 0.762 -0.222 0.149 XRN1 0.466 1.198 0.947 -0.307 0.532 0.518 1.469 0.803 -0.237 0.641 1.085 1.300 0.935 0.133 0.435 RNH1 1.055 0.935 1.176 0.072 0.533 0.643 0.621 0.799 -0.550 0.040 0.668 0.719 0.674 -0.543 0.004 LRPPRC 0.836 1.228 1.332 0.150 0.543 0.921 1.478 1.668 0.395 0.270 0.962 1.298 1.275 0.223 0.251 CCDC124 0.719 1.041 1.028 -0.126 0.545 0.794 0.633 0.753 -0.468 0.042 1.024 0.989 0.651 -0.200 0.441 RRP9 0.741 0.741 1.249 -0.181 0.546 1.277 1.297 1.324 0.378 0.002 1.695 1.517 1.410 0.620 0.015 1.031 1.035 0.976 0.020 0.549 0.953 0.966 0.970 -0.055 0.019 0.871 0.919 0.940 -0.137 0.052 EIF5A;EIF 5AL1 261 ACTR3 0.905 1.012 1.009 -0.038 0.549 1.091 1.153 1.065 0.141 0.053 1.125 1.051 1.019 0.090 0.167 0.976 1.078 0.764 -0.106 0.549 1.101 1.397 0.998 0.206 0.290 1.202 1.304 1.262 0.328 0.011 OB1A 1.046 0.973 0.891 -0.047 0.553 0.882 0.855 1.140 -0.073 0.637 0.837 0.836 1.057 -0.145 0.325 SMC2 0.949 0.946 1.042 -0.032 0.553 1.297 1.271 1.151 0.308 0.029 1.383 1.382 1.224 0.409 0.020 CTSL2 1.004 0.862 1.028 -0.056 0.554 0.880 0.679 0.697 -0.421 0.071 0.680 0.643 0.622 -0.626 0.004 ADAR 1.045 1.192 0.932 0.072 0.555 1.302 1.260 2.025 0.577 0.120 1.175 1.113 2.074 0.480 0.237 GAPDH 1.008 0.995 0.980 -0.008 0.557 0.922 0.901 0.856 -0.165 0.035 0.912 0.890 0.871 -0.167 0.013 TRNT1 1.024 0.921 0.992 -0.032 0.558 0.745 0.855 0.901 -0.267 0.082 0.738 0.861 0.864 -0.289 0.062 SEC23A 0.979 0.997 1.007 -0.008 0.559 1.037 1.024 0.930 -0.006 0.912 0.922 0.955 0.908 -0.107 0.038 PHYH 0.964 1.240 0.989 0.080 0.559 0.941 1.049 0.870 -0.073 0.448 0.883 1.345 0.869 0.015 0.948 GNG12 1.043 0.907 1.343 0.115 0.560 0.858 0.861 0.611 -0.382 0.146 0.751 0.965 0.469 -0.518 0.231 RBM4B;R BM4 MOB1B;M 262 FAM98B 1.019 1.104 0.964 0.039 0.561 0.965 1.004 0.915 -0.058 0.278 0.878 0.978 0.967 -0.089 0.212 HEATR5A 1.207 0.785 0.787 -0.141 0.565 0.732 0.408 0.429 -0.988 0.067 0.467 0.479 0.632 -0.940 0.021 LSM2 1.101 1.016 0.966 0.037 0.566 0.538 0.604 0.559 -0.821 0.004 0.487 0.580 0.554 -0.892 0.007 CROCC 0.963 1.023 1.094 0.036 0.569 1.019 0.939 0.944 -0.049 0.328 0.938 0.915 0.852 -0.151 0.069 USP9X 1.052 0.936 0.941 -0.037 0.574 0.731 0.706 0.642 -0.532 0.011 0.755 0.788 0.722 -0.407 0.008 KATNAL2 0.976 1.054 0.876 -0.050 0.583 0.459 0.637 0.507 -0.919 0.022 0.431 0.593 0.572 -0.925 0.024 SUB1 1.001 0.954 1.195 0.063 0.588 0.595 0.721 1.337 -0.268 0.527 0.620 0.713 0.921 -0.432 0.123 FTSJ1 1.083 0.969 1.013 0.029 0.594 0.706 0.791 0.814 -0.379 0.026 0.654 0.749 0.805 -0.448 0.036 HBS1L 0.986 1.116 0.983 0.038 0.594 1.080 1.065 0.993 0.064 0.233 1.064 0.984 0.996 0.020 0.622 USMG5 0.745 1.250 1.682 0.216 0.594 0.942 1.047 0.874 -0.072 0.443 0.897 0.815 0.393 -0.600 0.252 RPL14 0.989 1.031 0.926 -0.028 0.598 1.961 1.733 1.650 0.829 0.008 1.722 1.652 1.497 0.697 0.007 SNX9 1.078 0.899 0.931 -0.049 0.602 1.118 1.025 1.018 0.074 0.230 1.150 1.086 1.100 0.153 0.026 PRIC285 1.450 0.642 0.657 -0.236 0.603 0.946 0.916 0.353 -0.571 0.346 0.688 1.509 0.624 -0.208 0.657 263 RPL10;RP L10L 1.311 1.127 0.852 0.111 0.605 1.515 0.814 1.114 0.153 0.614 1.106 0.859 1.425 0.146 0.561 GEMIN5 0.828 1.109 0.935 -0.073 0.610 0.737 0.977 0.814 -0.257 0.164 0.838 0.889 0.815 -0.240 0.023 PPP2R1A 0.869 1.164 1.183 0.086 0.610 0.730 0.799 0.842 -0.342 0.029 0.842 0.737 0.707 -0.396 0.035 NEMF 1.308 0.795 1.285 0.139 0.614 0.717 0.320 0.823 -0.802 0.200 0.511 0.474 0.622 -0.910 0.016 NAA38 1.145 0.918 1.067 0.055 0.616 0.509 0.596 0.669 -0.767 0.022 0.457 0.652 0.564 -0.857 0.029 PTBP1 0.998 1.050 0.987 0.016 0.617 0.925 1.108 1.213 0.105 0.457 0.975 1.040 1.093 0.049 0.410 PKN2 0.985 0.920 1.039 -0.029 0.621 0.537 0.500 0.624 -0.859 0.012 0.486 0.553 0.563 -0.909 0.005 RPRD1B 1.015 0.909 1.017 -0.031 0.625 1.868 1.707 2.001 0.891 0.005 1.821 1.655 1.919 0.844 0.005 SHMT1 1.410 0.906 0.986 0.111 0.628 1.921 0.760 1.116 0.235 0.607 1.312 0.861 1.090 0.100 0.627 K1 1.062 1.016 0.800 -0.071 0.634 1.041 1.012 0.911 -0.020 0.764 1.133 1.164 1.051 0.157 0.071 CRAT 1.209 0.974 0.961 0.059 0.634 1.217 1.091 0.920 0.096 0.498 0.979 1.154 0.946 0.032 0.753 TNIK;MIN 264 ANKHD1 0.940 1.101 1.043 0.037 0.635 0.941 0.884 0.904 -0.137 0.036 0.864 0.931 0.984 -0.113 0.174 PTPN11 0.946 1.012 1.150 0.046 0.636 0.852 0.699 1.153 -0.181 0.479 0.800 0.695 0.950 -0.307 0.143 CCT7 1.009 0.990 1.012 0.005 0.640 1.246 1.280 1.258 0.335 0.001 1.230 1.272 1.205 0.305 0.006 PRIM2 0.845 0.937 1.110 -0.063 0.640 1.028 1.224 1.178 0.189 0.132 1.195 1.232 1.054 0.211 0.092 CRYZ 0.967 1.094 0.829 -0.063 0.641 1.055 1.091 0.933 0.034 0.666 1.133 1.121 1.072 0.148 0.025 PSPC1 0.879 0.925 1.101 -0.053 0.643 1.535 1.715 1.889 0.771 0.012 1.966 1.767 1.723 0.861 0.005 FAM136A 1.055 0.957 0.934 -0.029 0.648 0.974 0.985 1.143 0.044 0.617 0.954 0.953 1.046 -0.024 0.643 EEF2 0.985 1.042 1.001 0.013 0.655 0.856 0.779 0.786 -0.310 0.019 0.956 0.861 0.801 -0.200 0.114 GTPBP1 0.961 0.960 1.040 -0.020 0.656 0.637 0.531 1.583 -0.300 0.600 0.537 0.539 1.091 -0.554 0.245 TRMT11 0.864 1.045 1.012 -0.043 0.661 0.784 0.801 0.919 -0.265 0.066 0.863 0.850 1.003 -0.147 0.193 MRPL46 1.013 1.666 0.816 0.154 0.664 2.369 3.373 0.890 0.943 0.243 1.806 1.716 0.593 0.293 0.632 EPRS 1.021 1.002 0.946 -0.016 0.673 1.043 0.989 1.024 0.027 0.357 0.954 0.913 1.054 -0.041 0.569 SRP54 1.040 1.074 0.947 0.027 0.673 1.008 0.994 1.154 0.070 0.419 0.928 0.906 1.131 -0.024 0.832 265 PDIA3 0.928 1.064 0.954 -0.029 0.675 1.016 1.039 1.005 0.028 0.178 1.059 1.015 1.033 0.050 0.106 C7orf50 1.178 0.732 0.949 -0.096 0.676 1.277 1.621 2.676 0.823 0.120 0.983 2.324 2.487 0.835 0.192 ACP1 1.066 0.958 0.917 -0.031 0.678 0.816 0.777 0.828 -0.310 0.008 0.794 0.797 0.861 -0.292 0.016 GFPT1 0.937 1.015 1.144 0.040 0.679 1.040 0.934 1.055 0.012 0.847 1.058 0.911 0.864 -0.088 0.421 LRPAP1 0.989 1.108 0.967 0.028 0.688 0.494 0.555 0.479 -0.976 0.004 0.523 0.522 0.484 -0.973 0.001 WASF1 1.149 1.316 0.809 0.097 0.690 0.941 1.808 1.324 0.391 0.288 0.864 1.311 1.249 0.167 0.472 COPE 1.033 1.117 0.931 0.035 0.694 1.040 1.018 0.931 -0.007 0.898 0.974 0.904 0.844 -0.143 0.140 TPR 1.280 0.919 0.976 0.066 0.696 0.654 0.812 0.772 -0.429 0.045 0.681 0.742 0.839 -0.412 0.042 TXLNA 0.856 0.966 1.097 -0.046 0.697 0.823 0.727 1.644 -0.008 0.984 0.993 0.896 1.307 0.073 0.699 NXA2P2 0.937 1.046 1.080 0.028 0.697 0.625 0.626 0.653 -0.656 0.001 0.698 0.595 0.597 -0.671 0.012 MYH9 0.967 0.986 1.113 0.028 0.699 0.947 1.152 0.938 0.011 0.923 1.083 1.152 0.892 0.051 0.689 CLPB 1.140 1.056 0.908 0.043 0.700 1.299 1.316 0.892 0.203 0.386 1.181 1.073 0.812 0.014 0.940 ANXA2;A 266 MAP1LC3 B2;MAP1L C3B 0.912 1.123 0.885 -0.047 0.703 0.752 0.758 0.631 -0.492 0.030 0.678 0.602 0.656 -0.634 0.006 GGH 1.007 0.969 1.060 0.016 0.705 0.856 0.884 0.852 -0.212 0.006 0.831 0.813 0.777 -0.309 0.008 RBM42 0.721 0.929 1.223 -0.096 0.705 1.206 1.255 1.211 0.291 0.004 1.607 1.398 1.065 0.419 0.138 PTK2 1.120 0.904 0.899 -0.046 0.705 1.693 1.448 0.956 0.409 0.238 1.406 1.369 1.217 0.409 0.024 MLLT4 0.754 0.782 1.333 -0.116 0.705 0.128 0.461 0.776 -1.481 0.195 0.168 0.762 0.620 -1.219 0.216 CCDC25 1.243 0.738 0.894 -0.096 0.706 0.863 0.747 0.554 -0.495 0.119 0.727 0.931 0.651 -0.394 0.123 FTSJ3 0.908 1.157 0.841 -0.060 0.709 2.531 2.528 1.923 1.207 0.012 2.729 2.694 2.264 1.352 0.004 0.889 0.918 1.540 0.110 0.711 2.339 2.592 2.143 1.233 0.004 2.372 2.726 1.311 1.028 0.087 PDIA3 0.971 1.062 1.002 0.016 0.717 1.056 1.098 0.820 -0.025 0.868 1.037 1.059 0.802 -0.061 0.682 EDC4 0.901 1.311 0.661 -0.118 0.719 0.327 0.903 0.131 -1.564 0.192 0.321 0.679 0.170 -1.583 0.111 MAGED2 0.974 1.077 0.891 -0.033 0.721 0.946 0.960 0.811 -0.147 0.199 0.977 1.007 0.881 -0.069 0.360 RAB3GAP 267 CHMP5 0.962 1.042 1.028 0.014 0.725 0.952 0.967 0.980 -0.049 0.057 0.904 0.956 0.870 -0.137 0.073 SNRPD2 0.983 0.981 1.021 -0.008 0.726 1.430 1.586 1.858 0.692 0.024 1.349 1.486 1.679 0.583 0.024 SBDS 1.013 1.027 0.923 -0.019 0.727 1.074 1.012 0.981 0.031 0.505 1.106 0.970 0.913 -0.010 0.917 TRPT1 1.106 1.083 0.901 0.037 0.733 1.206 1.079 0.673 -0.064 0.827 1.043 0.959 0.677 -0.188 0.428 GIGYF2 1.091 0.954 0.898 -0.032 0.736 0.612 0.493 0.684 -0.758 0.032 0.569 0.576 0.638 -0.753 0.005 C2 0.848 1.205 1.104 0.058 0.739 0.870 1.676 0.918 0.140 0.689 0.987 1.724 0.826 0.164 0.660 PPIB 1.032 1.007 0.978 0.008 0.746 0.890 0.966 0.970 -0.087 0.166 0.831 0.918 0.903 -0.179 0.056 BRIX1 0.722 1.019 1.162 -0.076 0.748 1.540 1.777 1.101 0.530 0.122 2.070 1.784 0.885 0.570 0.271 SNUPN 0.908 0.836 1.177 -0.054 0.750 0.763 0.931 0.688 -0.344 0.115 0.934 0.947 0.887 -0.117 0.055 HGD 0.981 1.054 0.990 0.012 0.753 0.985 1.015 0.948 -0.026 0.456 1.064 0.922 0.916 -0.051 0.544 PICALM 0.999 1.089 0.958 0.020 0.753 1.106 1.093 0.925 0.054 0.587 1.045 1.030 1.078 0.071 0.067 1.167 1.025 0.905 0.038 0.754 0.974 0.998 0.901 -0.064 0.287 0.822 0.855 0.898 -0.221 0.026 ERC1;ER UBE2D3; 268 UBE2D2 PSMD11 0.876 1.056 1.190 0.046 0.755 1.096 1.263 1.637 0.393 0.146 1.173 1.042 1.231 0.196 0.111 UGDH 1.021 1.007 0.951 -0.011 0.760 0.991 1.106 0.973 0.031 0.646 0.910 1.103 1.032 0.017 0.851 SF3B3 1.035 0.965 1.024 0.011 0.765 1.625 1.648 1.772 0.749 0.003 1.635 1.700 1.704 0.748 0.001 SMC1A 0.676 1.303 1.449 0.117 0.766 0.808 0.896 1.042 -0.135 0.332 1.068 0.646 0.760 -0.311 0.283 PUS3 0.954 1.213 0.751 -0.067 0.768 0.721 0.802 3.624 0.356 0.683 0.838 0.637 4.802 0.452 0.669 SLK 0.930 0.979 1.181 0.035 0.770 1.636 1.477 1.715 0.684 0.009 1.544 1.444 1.343 0.528 0.012 EPS8L2 0.904 1.132 1.044 0.032 0.771 0.718 1.031 1.774 0.131 0.762 0.993 0.914 1.316 0.085 0.646 GSPT1 1.042 0.935 1.069 0.020 0.772 0.699 0.553 1.330 -0.320 0.487 0.688 0.686 1.025 -0.349 0.211 0.901 1.014 1.046 -0.022 0.774 0.969 0.908 0.773 -0.186 0.195 0.957 0.797 0.706 -0.297 0.145 LSM6 1.045 1.000 0.976 0.009 0.775 0.549 0.589 0.530 -0.849 0.003 0.464 0.544 0.529 -0.968 0.005 ATP6V1B 269 RAB39A;R AB6A;RA B6B 0.969 1.021 1.029 0.009 0.777 1.000 1.211 1.121 0.147 0.209 1.022 1.170 0.860 0.013 0.926 CDC5L 1.187 0.665 1.072 -0.080 0.784 0.849 0.948 1.865 0.195 0.638 0.879 1.893 1.672 0.492 0.288 PUS7 1.008 0.889 1.064 -0.023 0.791 0.689 0.730 0.683 -0.513 0.003 0.709 0.908 0.759 -0.345 0.084 NAA15 0.982 1.004 1.025 0.005 0.793 1.207 1.095 1.010 0.139 0.203 1.197 1.026 0.960 0.080 0.487 HSPA6 1.059 0.901 1.004 -0.020 0.795 0.724 0.796 0.763 -0.395 0.010 0.639 0.850 0.813 -0.393 0.092 BUD31 0.984 0.968 1.084 0.015 0.795 0.568 0.605 0.584 -0.772 0.001 0.561 0.597 0.501 -0.859 0.007 DIS3 0.975 1.194 0.768 -0.054 0.797 1.238 0.905 0.884 -0.005 0.980 1.503 0.700 1.275 0.141 0.715 SRSF1 1.248 0.910 0.780 -0.058 0.799 2.727 2.961 3.439 1.598 0.004 2.411 3.366 4.173 1.694 0.018 GANAB 1.092 0.893 0.975 -0.024 0.800 0.440 0.529 0.490 -1.044 0.005 0.387 0.632 0.531 -0.981 0.042 0.855 1.208 1.054 0.041 0.806 1.059 1.384 1.113 0.235 0.186 0.980 1.206 1.041 0.099 0.381 TUBB8;R P11631M21.2 270 CAD 1.149 0.934 0.869 -0.033 0.808 0.759 0.683 0.696 -0.490 0.009 0.844 0.817 0.828 -0.269 0.003 LLGL2 1.119 0.808 1.022 -0.038 0.811 0.743 0.504 0.575 -0.739 0.046 0.752 0.631 0.625 -0.585 0.021 STAU1 0.999 1.057 0.966 0.010 0.817 0.807 1.623 1.241 0.234 0.510 0.959 1.407 1.056 0.170 0.413 ARSL 0.898 1.075 1.088 0.024 0.817 1.035 1.136 1.180 0.157 0.107 1.062 1.077 0.987 0.058 0.277 TROVE2 1.066 0.868 1.029 -0.023 0.823 0.882 0.955 0.992 -0.087 0.226 0.830 1.072 0.924 -0.095 0.470 SEC24B 0.775 1.091 1.088 -0.041 0.828 1.187 1.017 0.810 -0.011 0.952 1.551 1.118 0.746 0.124 0.724 EHD4 0.962 0.992 1.073 0.011 0.830 1.946 0.927 2.000 0.617 0.231 2.189 0.959 1.838 0.649 0.215 TRMT112 1.061 0.969 0.948 -0.012 0.831 0.630 0.750 0.785 -0.477 0.039 0.532 0.782 0.737 -0.568 0.081 LMNA 1.271 1.191 0.745 0.058 0.833 1.870 1.738 1.113 0.618 0.118 1.419 1.377 1.338 0.462 0.003 OGFR 0.895 1.349 0.912 0.046 0.834 1.456 1.780 1.781 0.735 0.017 1.643 1.548 1.799 0.731 0.007 PARD6B 1.088 0.928 1.024 0.016 0.837 1.255 1.035 1.477 0.313 0.169 1.001 1.033 0.961 -0.003 0.922 PPP6R3 1.195 0.765 0.999 -0.044 0.837 0.540 0.509 1.067 -0.590 0.228 0.697 0.667 0.884 -0.428 0.077 HARS2;H 271 UXT 1.147 1.267 0.765 0.051 0.839 1.417 1.784 0.766 0.318 0.475 1.241 1.323 0.823 0.145 0.569 TMEM62 1.280 0.907 0.778 -0.048 0.841 1.901 0.841 0.876 0.162 0.713 1.592 0.962 1.004 0.206 0.469 PKP2 0.695 1.125 1.490 0.073 0.841 1.086 1.465 1.747 0.492 0.133 1.313 1.306 1.366 0.409 0.002 SEPHS1 1.184 0.947 0.939 0.025 0.843 1.267 1.352 1.162 0.331 0.035 1.082 1.543 1.213 0.340 0.153 HTATIP2 1.103 0.721 1.430 0.062 0.849 0.769 0.859 1.206 -0.109 0.633 0.688 0.725 0.745 -0.476 0.005 OSTF1 1.028 0.988 0.975 -0.005 0.854 0.779 0.941 0.831 -0.238 0.097 0.667 0.842 0.724 -0.433 0.048 CCNH 0.870 1.252 0.848 -0.038 0.854 0.948 1.657 0.905 0.169 0.608 1.119 1.264 1.177 0.245 0.041 CSE1L 1.034 0.985 0.991 0.004 0.858 1.000 1.078 1.071 0.069 0.183 1.050 1.095 1.086 0.107 0.029 KIF21A 1.118 0.909 0.948 -0.018 0.859 0.458 0.443 0.311 -1.329 0.018 0.536 0.516 0.361 -1.108 0.026 COPS3 0.866 0.999 1.109 -0.020 0.865 0.573 0.742 0.674 -0.601 0.031 0.597 0.758 0.751 -0.519 0.044 NXF1 1.020 1.057 0.946 0.009 0.866 0.589 0.986 1.138 -0.199 0.562 0.817 0.908 1.201 -0.055 0.770 MYL6 0.862 1.022 1.197 0.026 0.869 0.773 1.039 0.832 -0.194 0.269 0.811 1.015 0.683 -0.277 0.236 41884 0.852 0.861 1.267 -0.035 0.869 1.761 1.777 2.237 0.936 0.014 1.945 2.388 1.751 1.008 0.017 272 USP19 0.894 0.996 1.088 -0.015 0.871 1.656 1.955 1.389 0.723 0.037 1.488 1.995 1.357 0.670 0.057 ARPC2 0.856 1.128 0.995 -0.019 0.883 1.153 1.160 1.014 0.146 0.146 1.181 1.054 1.071 0.138 0.114 FUBP1 1.056 1.022 0.943 0.008 0.885 0.937 0.989 0.887 -0.094 0.174 0.852 0.960 0.894 -0.150 0.094 LSM4 0.987 1.002 1.015 0.002 0.887 0.545 0.619 0.652 -0.728 0.011 0.514 0.634 0.662 -0.737 0.022 ARL1 1.122 1.009 0.910 0.014 0.887 0.909 0.983 0.871 -0.121 0.143 0.771 1.024 1.009 -0.109 0.497 MICU1 1.145 0.953 0.883 -0.018 0.888 1.405 0.855 0.940 0.058 0.816 1.200 0.918 1.078 0.083 0.537 BAIAP2L1 0.905 1.392 0.725 -0.043 0.890 0.859 1.614 0.484 -0.192 0.739 0.847 1.144 0.620 -0.245 0.438 UGP2 1.020 0.955 1.016 -0.005 0.891 0.622 0.646 0.615 -0.672 0.001 0.643 0.677 0.553 -0.685 0.016 IKBKAP 1.076 0.862 1.045 -0.016 0.891 0.996 1.012 0.876 -0.060 0.458 0.771 1.650 0.896 0.063 0.868 OXSR1 0.964 1.049 0.977 -0.006 0.892 0.830 0.711 0.818 -0.351 0.039 0.863 0.833 0.837 -0.245 0.004 EIF3J 0.908 1.032 1.093 0.012 0.896 0.504 0.867 1.634 -0.162 0.772 0.753 0.894 1.205 -0.101 0.662 VPS33B 0.987 1.188 0.813 -0.023 0.897 0.332 0.324 1.608 -0.845 0.385 0.328 0.266 1.984 -0.843 0.456 CACYBP 0.997 1.143 0.904 0.014 0.899 0.877 0.812 0.961 -0.182 0.122 0.908 0.875 0.882 -0.171 0.009 273 IQGAP2 0.942 1.014 1.063 0.007 0.902 0.389 0.776 0.665 -0.772 0.125 0.563 0.791 0.643 -0.601 0.052 DTD1 1.259 0.900 0.840 -0.024 0.908 1.243 1.117 0.685 -0.024 0.935 0.876 0.869 0.732 -0.281 0.079 DNASE2 0.900 1.046 1.045 -0.008 0.923 0.810 0.920 0.916 -0.183 0.093 0.771 0.837 0.782 -0.329 0.012 CCT5 0.978 0.973 1.043 -0.003 0.928 1.364 1.297 1.260 0.386 0.007 1.336 1.287 1.208 0.351 0.014 THUMPD1 1.054 0.928 1.034 0.006 0.931 0.807 0.840 0.916 -0.229 0.052 0.729 0.839 0.924 -0.274 0.110 1;TENC1 0.738 1.230 1.057 -0.020 0.935 0.758 1.011 0.965 -0.145 0.376 1.001 0.928 0.928 -0.072 0.187 RAB21 1.252 0.896 0.866 -0.014 0.941 1.691 1.176 1.106 0.379 0.185 1.361 1.243 1.244 0.358 0.015 TBCE 0.879 1.081 1.069 0.008 0.943 0.926 1.088 0.992 -0.001 0.994 0.649 0.956 0.888 -0.287 0.236 HSPA14 1.001 1.092 0.925 0.005 0.944 0.741 0.634 0.582 -0.624 0.026 0.686 0.695 0.644 -0.567 0.003 CSNK1A1 1.038 1.077 0.906 0.006 0.946 1.438 1.298 1.043 0.320 0.144 1.296 1.326 1.121 0.315 0.053 PA2G4 1.015 0.983 1.000 -0.001 0.950 1.176 1.025 1.133 0.150 0.127 1.152 1.004 1.114 0.122 0.178 CYFIP2 1.088 0.857 1.091 0.008 0.951 1.120 0.947 1.191 0.112 0.373 1.134 1.108 0.917 0.068 0.556 TNS3;TNS 274 SRSF2;SF RS2 0.988 1.046 0.964 -0.002 0.953 1.184 1.364 1.362 0.379 0.030 1.187 1.273 1.214 0.292 0.010 GTF2F1 1.140 0.674 1.356 0.020 0.954 0.735 0.627 0.977 -0.384 0.176 0.598 0.672 0.887 -0.496 0.098 SPATS2 0.930 1.051 1.018 -0.003 0.964 1.202 1.134 1.023 0.160 0.144 1.148 1.025 0.930 0.043 0.670 OVCA2 1.216 0.926 0.875 -0.007 0.965 0.959 0.877 0.936 -0.115 0.095 0.705 0.845 0.944 -0.277 0.153 FGA 1.212 0.832 0.976 -0.008 0.965 1.062 1.183 1.001 0.110 0.259 1.004 1.060 0.892 -0.026 0.761 ZNFX1 1.161 0.980 0.889 0.005 0.966 0.500 0.586 0.572 -0.859 0.007 0.525 0.571 0.531 -0.884 0.002 CYCS 0.960 1.041 0.997 -0.002 0.966 0.939 0.951 0.959 -0.075 0.014 0.908 0.818 0.919 -0.184 0.075 NRD1 1.073 0.802 1.147 -0.007 0.970 0.371 0.487 0.522 -1.135 0.017 0.323 0.612 0.482 -1.130 0.052 WASL 0.985 1.074 0.949 0.002 0.971 0.841 0.955 0.836 -0.192 0.092 0.826 0.938 0.859 -0.196 0.069 AB13 1.105 0.915 0.982 -0.003 0.971 0.584 0.638 0.727 -0.628 0.021 0.577 0.664 0.769 -0.587 0.039 ELAVL1 1.155 0.929 0.923 -0.004 0.972 0.955 0.875 0.857 -0.161 0.079 0.886 0.866 0.802 -0.233 0.033 RAB8B;R 275 HNRNPUL 1.056 0.935 1.009 -0.002 0.972 1.080 1.103 1.081 0.122 0.006 1.109 1.054 1.091 0.117 0.033 MPG 1.145 0.818 1.079 0.005 0.977 1.573 1.560 1.933 0.749 0.018 1.358 1.769 1.697 0.676 0.029 ILKAP 1.216 0.936 0.871 -0.005 0.978 2.703 1.999 1.976 1.139 0.016 1.611 1.758 2.021 0.839 0.013 CSK 0.729 0.986 1.373 -0.006 0.983 1.068 0.956 1.383 0.166 0.404 1.220 0.887 1.005 0.040 0.791 OPA1 0.920 1.132 0.956 -0.002 0.985 0.996 1.126 1.011 0.060 0.393 0.984 0.850 0.924 -0.124 0.181 HK1 1.125 0.858 1.041 0.002 0.985 1.465 0.840 0.808 -0.003 0.992 1.120 1.024 0.881 0.005 0.966 SMG8 1.176 1.110 0.771 0.003 0.988 2.377 2.297 0.852 0.739 0.267 1.933 2.687 1.150 0.859 0.137 SEC24D 0.912 0.989 1.111 0.001 0.989 1.070 1.006 1.145 0.101 0.202 1.105 1.047 1.016 0.077 0.160 DECR1 1.109 1.159 0.773 -0.003 0.989 1.204 1.106 1.114 0.190 0.040 1.158 1.137 1.366 0.282 0.078 1orf57 0.987 1.001 1.013 0.000 0.992 0.797 0.808 0.849 -0.290 0.009 0.821 0.777 0.738 -0.362 0.015 TOMM34 1.401 0.973 0.733 0.000 1.000 1.990 1.615 1.231 0.662 0.081 1.224 1.626 1.205 0.420 0.096 NTPCR;C 276 [...]... the globe has led to increasing viral genetic diversity, some of which appears to be associated with greater epidemic potential The underlying mechanisms of viral fitness in an epidemiological setting, however, remain poorly defined We conducted in vitro studies and identified a determinant of fitness in a foreign dominant (PR-2B) DENV-2 clade, which emerged during the 1994 epidemic in Puerto Rico and... RNA of dengue- 2 virus binds tripartite motif 25 protein to inhibit interferon expression providing a mechanism for epidemiological fitness Previous Publications Manokaran, G., Lin, Y.N., Soh, M.L., Lim, E.A., Lim, C.W., and Tan, B.H (2008) Detection of porcine circovirus type 2 in pigs imported from Indonesia Vet Microbiol 132, 165-170 xxi CHAPTER 1: An Introduction to the role of viral genetics in dengue. .. IFIT Interferon induced proteins with tetratricopeptide repeats IFITM IFN Interferon IFNα Interferon alpha IFNβ Interferon beta IFNγ Interferon gamma IFNAR IFN-α/β receptor IgG Immunoglobulin G IgM Immunoglobulin M IKK IκB kinase IL Interleukin IRF Interferon-regulatory factor ISG Interferon stimulated genes ISRE IFN-stimulated response elements JAK xvi Interferon-induced transmembrane protein Janus kinase... showing the distribution of select RNA binding proteins 3.19 119 Validation of TRIM25 binding by RNA-immunoprecipitation (RIP) 121 3.20 RNA-immunoprecipitation of MAVS 122 3.21 Immunoprecipitation of TRIM25 upon viral infection 124 3.22 Immunoprecipitation of RIG-I after transfection of cells with sfRNA 4.1 126 Proposed model depicting how sfRNA inhibits RIG-I signaling for amplified and sustained... a RNA-binding protein that binds better to PR-2B sfRNA than PR-1 sfRNA 117 3.4.2 PR-2B 3’UTR binds to TRIM25 more stably than PR-1, preventing its de-ubiquitylation 123 Chapter 4: Discussion 4.1 Summary of findings 4.2 128 sfRNA-mediated de-ubiquitylation of TRIM25 leads to increased DENV epidemiological fitness 4.3 131 Role of 3’ noncoding RNA – Key to DENV pathogenesis? 136 vii 4.3.1 Amount of sfRNA... acidification of the late endosomes triggers trimerization of the E protein, inducing fusion of viral and cell endosomal membranes, thus allowing the viral nucleocapsid to uncoat (Kuhn et al., 2002; Li et al., 2008; van der Schaar et al., 2008) The DENV genome is then released into cells and translated Once the viral replication complex is synthesized by viral and co-opted host proteins, RNA synthesis begins in. .. Sequence of sfRNA produced 138 4.4 Experimental limitations and alternative explanations 139 4.5 Future directions 141 4.5.1 Role of 3’UTR in DENV epidemiological fitness – further investigation warranted 141 4.5.2 Deeper insights on the contribution of sfRNA to viral pathogenesis 4.6 Conclusion 142 143 References 145 Appendix 167 viii Summary The staggeringly large number of dengue virus (DENV) infections... DENV-2 produced increased levels of subgenomic relative to genomic RNA during replication The subgenomic RNA bound and inhibited deubiquitylation of tripartite motif 25 (TRIM25), which is needed to activate its E3 ligase activity critical for sustained and amplified RIG-I induced type-I interferon expression Our findings demonstrate a unique viral RNA-host protein interaction to evade the innate immune... for increased epidemiological fitness ix List of Tables Table Title 1.1 Candidate dengue vaccines 15 2.1 List of primers used for qRT-PCR 66 3.1 Amino acid and nucleotide substitutions present in at least 25 %of the PR-2B viruses x Page Number 86 List of Figures Figure Title 1.1 Page Number Flaviviruses and medically important viruses within the genus 1.2 2 Global evidence consensus and burden of dengue. .. consensus and burden of dengue in 2010 (A) National and subnational evidence on dengue Red indicates complete presence through to green indicating complete absence (B) Cartogram of annual number of infections for all ages as a proportion of national or sub-national (China) geographical area There are four serotypes of DENV (DENV-1, DENV-2, DENV-3, DENV4) that are immunologically distinct but antigenically . underlying mechanisms of viral fitness in an epidemiological setting, however, remain poorly defined. We conducted in vitro studies and identified a determinant of fitness in a foreign dominant. against dengue 30 1.2.2.2 Subversion of innate immune responses by dengue 32 1.2.2.2.1 Role of dengue coding regions 33 1.2.2.2.2 Role of dengue noncoding regions – sfRNA 36 1.2.2.2.2.1 Inhibition. Table of contents iii Summary ix List of tables x List of figures xi List of abbreviations xv List of publications xxi Chapter 1: An Introduction to the role of viral genetics in dengue epidemiological