SIADENOVIRUSES: A COMPARATIVE MOLECULAR AND PHYLOGENETIC STUDY Ph.D. Thesis Endre Kovács Supervisor: Prof. Dr. Mária Benkı Biological Studies Doctorate School Faculty of Science, Eötvös Loránd University Head: Prof. Dr. Anna Erdei Doctorate Program: Theoretical and Evolutionary Biology Head: Prof. Dr. Eörs Szathmáry Veterinary Medical Research Institute Hungarian Academy of Sciences Budapest 2011 "There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved." Charles Darwin 2 CONTENTS 1 INTRODUCTION ....................................................................................................................................... 5 1.1 HISTORY AND SIGNIFICANCE OF ADENOVIRUS RESEARCH ..................................................................... 5 1.2 MORPHOLOGY ...................................................................................................................................... 6 1.3 TAXONOMY AND GENOME ORGANIZATION ........................................................................................... 8 1.3.1 Mastadenovirus............................................................................................................................. 11 1.3.2 Aviadenovirus................................................................................................................................ 11 1.3.3 Atadenovirus ................................................................................................................................. 12 1.3.4 Siadenovirus.................................................................................................................................. 12 1.3.5 Ichtadenovirus............................................................................................................................... 13 1.4 BASIC BIOLOGY .................................................................................................................................. 13 1.5 ADENOVIRUS INFECTION IN BIRDS OF PREY AND IN SONGBIRDS .......................................................... 14 1.6 HISTORY OF SIADENOVIRUS ................................................................................................................. 15 1.7 OBJECTIVES ........................................................................................................................................ 17 2 MATERIALS AND METHODS.............................................................................................................. 19 2.1 SAMPLE ORIGINS ................................................................................................................................. 19 2.1.1 Raptor adenovirus......................................................................................................................... 19 2.1.2 Great tit adenovirus ...................................................................................................................... 20 2.2 DNA EXTRACTION .............................................................................................................................. 21 2.3 NON -SPECIFIC WHOLE GENOME AMPLIFICATION ................................................................................. 21 2.4 PRIMER DESIGN AND PRIMER TYPES .................................................................................................... 22 2.5 PCR.................................................................................................................................................... 24 2.6 MOLECULAR CLONING ........................................................................................................................ 24 2.7 POLY -A TAILING ................................................................................................................................. 25 2.8 SEQUENCING ....................................................................................................................................... 26 2.9 SEQUENCE ANALYSIS .......................................................................................................................... 27 2.9.1 Staden............................................................................................................................................ 27 2.9.2 Artemis .......................................................................................................................................... 28 2.9.3 Similarity Plotting ......................................................................................................................... 28 2.10 PHYLOGENETIC ANALYSIS .................................................................................................................. 28 2.10.1 Nucleotide and protein alignments........................................................................................... 28 2.10.2 Maximum likelihood ................................................................................................................. 28 2.10.3 Bayesian inference.................................................................................................................... 28 3 RESULTS................................................................................................................................................... 30 3.1 RAPTOR ADENOVIRUS 1 ...................................................................................................................... 30 3.1.1 General features............................................................................................................................ 30 3.1.2 Sialidase........................................................................................................................................ 33 3.1.3 ORF4 and hydrophobic protein .................................................................................................... 33 3.1.4 Regions "E3" and "E4" ................................................................................................................. 34 3.1.5 Similarity plotting.......................................................................................................................... 34 3.2 GREAT TIT ADENOVIRUS 1 .................................................................................................................. 35 3.2.1 Genome features............................................................................................................................ 35 3.3 PHYLOGENY OF SIADENOVIRUS ........................................................................................................... 37 4 DISCUSSION............................................................................................................................................. 40 4.1 CURRENT STATE OF SIADENOVIRUS ..................................................................................................... 40 4.2 COMPARATIVE GENOME ANALYSIS ..................................................................................................... 42 4.2.1 Divergent adenovirus genomes ..................................................................................................... 42 4.2.2 Siadenovirus genomes................................................................................................................... 43 4.3 PHYLOGENY ....................................................................................................................................... 46 5 NEW SCIENTIFIC RESULTS ................................................................................................................ 50 6 SUMMARY................................................................................................................................................ 51 7 SUMMARY IN HUNGARIAN (ÖSSZEFOGLALÁS) .......................................................................... 52 8 ABBREVIATIONS.................................................................................................................................... 53 3 9 ACKNOWLEDGEMENTS ...................................................................................................................... 55 10 REFERENCES .......................................................................................................................................... 56 11 PUBLICATIONS....................................................................................................................................... 71 11.1 RELATED PUBLICATIONS ..................................................................................................................... 71 11.2 PRESENTATIONS AT INTERNATIONAL CONFERENCES........................................................................... 71 11.3 UNRELATED PUBLICATIONS ................................................................................................................ 71 4 1 INTRODUCTION 1.1 HISTORY AND SIGNIFICANCE OF ADENOVIRUS RESEARCH Adenoviruses have been known since the middle of the twentieth century. Rowe et al. in 1953 cultured surgically removed human pharyngeal tonsils, which led to the observation of spontaneous cellular degeneration. At about the same time, a virus with similar cell-damaging effects was isolated from the tracheal