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Molecular Epidemiology of Dengue Viruses from Complete Genome Sequences Molecular Epidemiology of Dengue Viruses from Complete Genome Sequences Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Swee Hoe Ong aus Malaysia Basel, 2010 Original document stored on the publication server of the University of Basel edoc.unibas.ch This work is licenced under the agreement „Attribution Non-Commercial No Derivatives – 2.5 Switzerland“. The complete text may be viewed here: creativecommons.org/licenses/by-nc-nd/2.5/ch/deed.en Attribution-Noncommercial-No Derivative Works 2.5 Switzerland You are free: to Share — to copy, distribute and transmit the work Under the following conditions: Attribution. You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Noncommercial. You may not use this work for commercial purposes. No Derivative Works. You may not alter, transform, or build upon this work. • For any reuse or distribution, you must make clear to others the license terms of this work. The best way to do this is with a link to this web page. • Any of the above conditions can be waived if you get permission from the copyright holder. • Nothing in this license impairs or restricts the author's moral rights. Your fair dealing and other rights are in no way affected by the above. This is a human-readable summary of the Legal Code (the full license) available in German: http://creativecommons.org/licenses/by-nc-nd/2.5/ch/legalcode.de Disclaimer: The Commons Deed is not a license. It is simply a handy reference for understanding the Legal Code (the full license) — it is a human-readable expression of some of its key terms. Think of it as the user-friendly interface to the Legal Code beneath. This Deed itself has no legal value, and its contents do not appear in the actual license. Creative Commons is not a law firm and does not provide legal services. Distributing of, displaying of, or linking to this Commons Deed does not create an attorney-client relationship. Quelle: http://creativecommons.org/licenses/by-nc-nd/2.5/ch/deed.en Datum: 3.4.2009 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Hernn Prof. Dr. Marcel Tanner, Herrn Prof. Dr. Hans-Peter Beck und Dr. Mark J. Schreiber. Basel, den 28. Mai 2009. Prof. Dr. Eberhard Parlow Dekan Basel, 2010 Table of Contents Table of Contents................................................................................................i Acknowledgements............................................................................................v Summary...........................................................................................................vi Zusammenfassung .......................................................................................... vii List of Original Publications ............................................................................. iix List of Abbreviations...........................................................................................x Preface............................................................................................................. xii 1. Literature Review.........................................................................................1 1.1 Dengue the Disease.................................................................................1 1.1.1 Etymology..........................................................................................1 1.1.2 Symptoms..........................................................................................1 1.1.3 Classification .....................................................................................4 1.1.4 Brief History of Dengue and DHF......................................................5 1.1.5 Vectors and Transmission Cycles.....................................................6 1.1.6 Geographical Distribution ..................................................................8 1.1.7 Factors Influencing Transmission......................................................9 1.1.8 Economic impact ............................................................................ 11 1.2 Dengue the Virus .................................................................................. 12 1.2.1 Taxonomy....................................................................................... 12 1.2.2 Virion Morphology........................................................................... 13 1.2.3 Genomic Organization.................................................................... 14 1.2.4 Genetic Diversity ............................................................................ 17 1.2.5 Role of virus genetics and evolution .............................................. 23 1.3 References............................................................................................ 24 2. Development of a Dengue Virus Genome Database............................. 32 2.1 Abstract ................................................................................................. 33 2.2 Introduction............................................................................................ 33 2.3 Material and Methods............................................................................ 34 2.3.1 Complete genome sequencing of DENV ....................................... 34 i 2.3.2 Inclusion criteria of public domain DENV sequences .................... 36 2.3.3 DENV genome sequence annotation............................................. 36 2.3.4 DENV subtype classification .......................................................... 37 2.3.5 Technical specifications of DENV genome database .................... 38 2.4 Results and Discussion......................................................................... 39 2.4.1 Complete genome sequencing of DENV ....................................... 39 2.4.2 DENV genome sequence annotation............................................. 40 2.4.3 DENV genome database Web application..................................... 42 2.4.4 State of the DENV genome database ............................................ 47 2.5 Conclusion............................................................................................. 50 2.6 Acknowledgements ............................................................................... 50 2.7 Author Contributions ............................................................................. 51 2.8 References............................................................................................ 51 3. Periodic re-emergence of endemic strains with strong epidemic potential—A proposed explanation for the 2004 Indonesian dengue epidemic......................................................................................................... 53 3.1 Abstract ................................................................................................. 54 3.2 Introduction............................................................................................ 54 3.3 Material and Methods............................................................................ 56 3.3.1 Virus sample collection and preparation ........................................ 56 3.3.2 Virus propagation, RNA extraction and virus typing via RT-PCR .. 56 3.3.3 Primer design ................................................................................. 57 3.3.4 Viral cDNA amplification, sequencing, assembly and annotation.. 57 3.3.5 Strain nomenclature ....................................................................... 58 3.3.6 Phylogenetic analysis of the DENV genomes................................ 58 3.3.7 Clade-specific mutations of the DENV-3 isolates .......................... 59 3.3.8 Site-specific selection pressures.................................................... 59 3.4 Results .................................................................................................. 61 3.4.1 Genome sequencing of dengue isolates........................................ 61 3.4.2 Phylogeny of the DENV-1, DENV-2 and DENV-4 isolates ............ 61 3.4.3 Diversity and phylogeny of the DENV-3 isolates............................ 65 3.4.4 Clade-specific mutations of the DENV-3 isolates .......................... 68 3.5 Discussion ............................................................................................. 71 ii 3.6 Acknowledgements ............................................................................... 74 3.7 Author Contributions ............................................................................. 75 3.8 References............................................................................................ 75 4. Genomic Epidemiology of a Dengue Virus Epidemic in Urban Singapore ...................................................................................................... 78 4.1 Abstract ................................................................................................. 79 4.2 Introduction............................................................................................ 79 4.3 Material and Methods............................................................................ 80 4.3.1 Collection of viral samples.............................................................. 80 4.3.2 Molecular Analysis.......................................................................... 81 4.3.3 Evolutionary analysis.....................................................................
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