EVOLUTIONARY TRENDS IN VIRAL PATHOGENS WITHIN AND BETWEEN OUTBREAKS A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Mary E. Saha December 2017 © Copyright All rights reserved Except for previously published materials Dissertation written by Mary E Saha B.S., University of Akron, 2008 Ph.D., Kent State University, 2017 Approved by _Dr. Helen Piontkivska___________ Chair, Doctoral Dissertation Committee _Dr. Gary Koski_________________ Members, Doctoral Dissertation Committee _Dr. Christopher Woolverton_______ _Dr. Tara Smith_________________ _Dr. Walter Hoeh________________ _Dr. Gail Fraizer_________________ Accepted by __Dr. Laura Leff________________ Chair, Department of Biology __Dr. James Blank_______________ Dean, College of Arts and Sciences Table of Contents LIST OF FIGURES ......................................................................................................... V LIST OF TABLES ........................................................................................................ VII ACKNOWLEDGEMENTS ........................................................................................ VIII CHAPTER 1: INTRODUCTION .................................................................................... 1 1.1 RNA viruses .............................................................................................................. 1 1.2 Influenza A.................................................................................................................... 3 1.3 Challenges in Influenza Sampling ................................................................................ 6 1.4 Ebolavirus ................................................................................................................... 12 1.5 Immune Response against Ebolavirus ........................................................................ 17 1.6 Ebolavirus Outbreaks and Public Health .................................................................... 19 1.7 Ebolavirus Evolutionary Questions ............................................................................ 27 1.8 Research Goals............................................................................................................ 30 1.9 Overview of Subsequent Chapters .............................................................................. 33 1.10 References ................................................................................................................. 35 CHAPTER 2: SAMPLING ISSUES IN INFLUENZA A ANALYSIS: AN APPROACH TO DEALING WITH OVERSAMPLING ............................... 44 2.1 Introduction ............................................................................................................. 44 2.2 Hypotheses .............................................................................................................. 46 2.3 Methods ................................................................................................................... 46 2.4 Results ..................................................................................................................... 50 2.5 Discussion and Conclusions .................................................................................... 62 2.6 References ................................................................................................................... 71 iii CHAPTER 3: GENOME-WIDE MOLECULAR SUBSTITUTION PATTERNS IN EBOLAVIRUS .................................................................................................... 76 3.1 Introduction ............................................................................................................. 76 3.2 Hypothesis ............................................................................................................... 77 3.3 Methods ................................................................................................................... 77 3.4 Results ..................................................................................................................... 80 3.5 Discussion and Conclusions .................................................................................. 105 3.6 References ................................................................................................................. 112 CHAPTER 4: DISTRIBUTION OF POSITIVELY AND NEGATIVELY SELECTED SITES IN THE EBOLAVIRUS GENOME ............................. 115 4.1 Introduction ........................................................................................................... 115 4.2 Hypotheses ............................................................................................................ 116 4.3 Methods ................................................................................................................. 117 4.4 Results ................................................................................................................... 120 4.5 Discussion and Conclusions .................................................................................. 137 4.6 References ................................................................................................................. 146 CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS ..................................... 150 References ....................................................................................................................... 163 APPENDICES ............................................................................................................... 171 Chapter 1 ......................................................................................................................... 171 Chapter 2 ......................................................................................................................... 173 Chapter 3 ......................................................................................................................... 176 Chapter 4 ......................................................................................................................... 199 iv List of Figures Figure 1.1: Synonymous substitution rates in RNA viruses ............................................... 2 Figure 1.2: Percentages of HA Influenza A sequences available for the top ten countries (or territories) in the NCBI Influenza Database (2013). ..................................................... 8 Figure 1.3: The number of publications in PubMed for Influenza A and Ebolavirus by date .................................................................................................................................... 11 Figure 1.4: Infection Progression of Ebolavirus. .............................................................. 14 Figure 1.5: Ebolavirus life cycle and immune system effects .......................................... 17 Figure 1.6: Map of 2014 outbreak with the number of cases ........................................... 22 Figure 1.7: Map of the 2017 outbreak in the Democratic Republic of the Congo (DRC, formerly Zaire) .................................................................................................................. 24 Figure 2.1: Workflow of analysis steps. ........................................................................... 49 Figure 2.2: dN Values by Country and Year: ................................................................... 52 Figure 2.3: dS Values by Country and Year: .................................................................... 53 Figure 2.4: Nucleotide Diversity Values. ........................................................................ 57 Figure 2.5: dN Values. ...................................................................................................... 59 Figure 2.6: dS Values:....................................................................................................... 61 Figure 2.7: dN/dS values by number of sequences used and color coded by the source article................................................................................................................................. 69 Figure 3.1: Phylogenetic Tree of Ebolavirus Sequences .................................................. 89 Figure 3.2: Phylogenetic pairs mean dN. .......................................................................... 90 v Figure 3.3: Phylogenetic pairs mean dS. .......................................................................... 92 Figure 3.4: GP Between Pairs dN. .................................................................................... 94 Figure 3.5: GP Within Pairs dN. ....................................................................................... 96 Figure 3.6: Phylogenetic pairs within group values.......................................................... 98 Figure 3.7: 2014 outbreak dN-dS values for within phylogenetic pair comparisons. ... 100 Figure 3.8: Other outbreak dN-dS values for within phylogenetic pair comparisons. ... 102 Figure 3.9: GP 50% epitope dN-dS. ............................................................................... 104 Figure 4.1: Epitope Regions and Polymorphic Sites in Three Ebolavirus Genes .......... 121 Figure 4.2: Epitope Regions and Polymorphic Sites in Three Ebolavirus Genes .......... 123 Figure 4.3: Epitope Regions and Polymorphic Sites in Three Ebolavirus Genes .......... 125 Figure 4.4: Protein Structures and Polymorphic Sites in Six Ebolavirus