The Pennsylvania State University The Graduate School Eberly College of Science THE MOLECULAR EVOLUTION AND PHYLOGEOGRAPHY OF DENGUE VIRUSES IN ASIA A Dissertation in Biology by Maia Anita Rabaa © 2012 Maia A. Rabaa Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2012 The dissertation of Maia A. Rabaa was reviewed and approved* by the following: Andrew F. Read Professor of Biology and Entomology Chair of Committee Edward C. Holmes Professor of Biology Dissertation Advisor Isabella M. Cattadori Assistant Professor of Biology Timothy Reluga Assistant Professor of Mathematics and Biology Douglas R. Cavener Professor and Head of Biology *Signatures are on file in the Graduate School ABSTRACT The global burden of dengue and its impact on the health and economics of tropical countries is vast and continues to grow. Outbreaks of severe dengue disease are increasingly common in regions where dengue was thought to be rare only a decade ago. Our knowledge of the epidemiological and evolutionary processes by which dengue viruses (DENV) invade new populations and become established as persistent viral lineages has been limited by a lack of data relating virus to host and vector populations, as well as the immune landscapes in which they spread over time. Virus lineages enter new populations regularly, but become established only occasionally and are generally detected long after establishment has occurred. The introduction of novel viral lineages has been shown to perturb the immune landscape in endemic environments, often causing massive epidemics and long-term changes in clinical manifestations within the host population. The use of large sets of spatially- and temporally- related DENV genome and envelope gene (E) sequences to investigate these lineage establishment events allows us to trace the timing and routes of viral movement between host populations, including the evolutionary dynamics of the viral lineages themselves. Determining whether trends in viral introduction and establishment can be distinguished within DENV phylogenies, and what these trends may be, is integral to understanding and controlling the spread of disease at local, regional, and global scales. In this thesis, I explore the spatio-temporal dynamics of DENV dispersal to identify the routes by which dengue invades new populations, characterize trends in viral migration, and identify the factors that affect the speed and scope of DENV migration at various scales and in heterogeneous transmission environments. In hyperendemic settings, I determined that DENV iii diversity is maintained in densely populated urban areas, frequently spreading through urban and suburban populations and into rural areas, and following a predictable pattern of human movement that suggests a limited role for the vector in the rapid dispersal of an emerging lineage across the population. Once arriving in rural areas, transmission is remarkably spatially and temporally focal. Where climate conditions are suitable, these populations may show persistence of viral populations over multiple seasons, but the likelihood of lineage fade-out here is increased as a result of lower population densities and seasonal bottlenecks in DENV populations. Hyperendemic urban areas further seed more distant populations at rates that appear to be dependent upon transmission intensities in the donor population and the connectedness of the human host populations, while the establishment of viral populations may show a greater dependence upon the immune landscape and the climate into which the virus is introduced. This is also true at the largest spatial and temporal scales, where I identify three geographic regions in tropical Asia that act as sources of global DENV diversity and suggest that immune- mediated competition allows for the DENV populations in these areas to be maintained largely in isolation of one another. These analyses are the first to indicate a primary role for South Asia in the evolution and maintenance of global DENV diversity. Not only does South Asia act as a primary source of DENV, but it also appears to be the most frequent source of novel DENV diversity (i.e., new lineages) for populations outside of Asia. Thus, characterizing the diversity of DENV in this region and the processes by which it is maintained and dispersed into new populations is integral to identifying the factors responsible for their emergence, persistence, and expansion into new landscapes across the globe. iv TABLE OF CONTENTS LIST OF FIGURES .................................................................................................... v LIST OF TABLES ...................................................................................................... vi ACKNOWLEDGEMENTS .......................................................................................... vii Chapter 1 Introduction ........................................................................................... 1 Ecology and origins of DENV ............................................................................. 1 Molecular biology of DENV ................................................................................. 5 Immune response to DENV infection .................................................................. 11 Global dengue epidemiology and ecology .......................................................... 17 Evolution of DENV .............................................................................................. 23 Spatial dynamics and phylogeography of dengue .............................................. 29 Phylogenetic and phylogeographic analysis of DENV ........................................ 32 Aims of this thesis ............................................................................................... 38 Chapter 2 Frequent in-migration and highly focal transmission of dengue viruses among children in Kamphaeng Phet, Thailand ................................ 40 Abstract ............................................................................................................... 40 Introduction ......................................................................................................... 41 Results and Discussion ...................................................................................... 43 Materials and Methods ....................................................................................... 57 Chapter 3 Phylogeography of recently emerged DENV-2 in Viet Nam .............. 65 Abstract ............................................................................................................... 65 Introduction ......................................................................................................... 66 Results ................................................................................................................ 68 Discussion .......................................................................................................... 76 Materials and Methods ....................................................................................... 77 Supplementary Figures ....................................................................................... 88 v Chapter 4 Dengue virus in sub-tropical northern and central Viet Nam: population immunity and climate shape patterns of viral invasion and maintenance ............................................................................................................. 92 Abstract ............................................................................................................... 92 Introduction ......................................................................................................... 94 Results ................................................................................................................ 97 Discussion…………………………………………………………………………….110 Materials and Methods………………………………………………………………115 Supplementary Tables………………………………………………………………121 Chapter 5 Modern global dengue diversity: out of Asia…………………………122 Abstract……………………………………………………………………………….122 Introduction…………………………………………………………………………...123 Results and Discussion……………………………………………………………..126 Conclusions…………………………………………………………………………..139 Materials and Methods……………………………………………………………...140 Supplementary Tables………………………………………………………………145 Chapter 6 Discussion…………………………………………………………………..150 REFERENCES…………………………………………………………………………….157 Appendix A The emergence of rotavirus G12 and the prevalence of enteric viruses in hospitalized pediatric diarrheal patients in southern Vietnam…….185 Appendix B The evolutionary consequences of blood-stage vaccination on the rodent malaria Plasmodium chabaudi…….…….…….…….…….…….…….…….194 vi LIST OF FIGURES Figure 1-1: Phylogenetic relationships among dengue viruses. ................................ 4 Figure 1-2: Dengue virus genome and virion structures. ........................................... 5 Figure 1-3: Model of antibody-mediated enhancement of viral infection. .................. 15 Figure 1-4: Global distribution of DENV. .................................................................... 25 Figure 2-1: Kamphaeng Phet Provincial Hospital data of all reported and PCR confirmed dengue cases from Muang District, Kamphaeng Phet during 2004 to 2007. ..... 43 Figure 2-2: The timing of isolation of DENV lineages circulating in each of five participating sub-districts of Kamphaeng Phet, Thailand during 2004 – 2007 .... 45 Figure 2-3: ML tree