Washington University in St. Louis Washington University Open Scholarship All Theses and Dissertations (ETDs) Summer 9-1-2014 Discovery and Characterization of Novel Polyomaviruses in Humans Erica Anne Siebrasse Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/etd Recommended Citation Siebrasse, Erica Anne, "Discovery and Characterization of Novel Polyomaviruses in Humans" (2014). All Theses and Dissertations (ETDs). 1347. https://openscholarship.wustl.edu/etd/1347 This Dissertation is brought to you for free and open access by Washington University Open Scholarship. It has been accepted for inclusion in All Theses and Dissertations (ETDs) by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biological Sciences Molecular Microbiology and Microbial Pathogenesis Dissertation Examination Committee: David Wang, Chair Jacco Boon Erika Crouch Michael S. Diamond Henry V. Huang Thaddeus S. Stappenbeck Discovery and Characterization of Novel Polyomaviruses in Humans by Erica Anne Siebrasse A dissertation presented to the Graduate School of Arts and Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2014 Saint Louis, Missouri TABLE OF CONTENTS LIST OF FIGURES AND TABLES ............................................................................... iii LIST OF ABBREVIATIONS ......................................................................................... v ACKNOWLEDGEMENTS ........................................................................................... vii ABSTRACT ....................................................................................................................... x CHAPTER 1. Introduction .............................................................................................. 1 OVERVIEW .................................................................................................................................... 2 SECTION I: DISCOVERY OF NOVEL VIRUSES ................................................................................ 2 SECTION II: THE POLYOMAVIRIDAE ............................................................................................ 10 CONCLUSIONS............................................................................................................................. 16 REFERENCES ............................................................................................................................... 18 CHAPTER 2. Identification of MW polyomavirus, a novel polyomavirus in human stool 22 ABSTRACT .................................................................................................................................. 23 INTRODUCTION ........................................................................................................................... 24 MATERIALS AND METHODS ....................................................................................................... 26 RESULTS ..................................................................................................................................... 30 DISCUSSION ................................................................................................................................ 37 ACKNOWLEDGEMENTS ............................................................................................................... 39 REFERENCES ............................................................................................................................... 40 CHAPTER 3. Determination of the cell and tissue tropism of WU and KI polyomaviruses44 ABSTRACT .................................................................................................................................. 45 INTRODUCTION ........................................................................................................................... 46 MATERIALS AND METHODS ....................................................................................................... 47 RESULTS ..................................................................................................................................... 51 DISCUSSION ................................................................................................................................ 74 ACKNOWLEDGEMENTS ............................................................................................................... 80 REFERENCES ............................................................................................................................... 81 CHAPTER 4. Progress toward a cell culture system for WU polyomavirus ............ 84 ABSTRACT .................................................................................................................................. 85 INTRODUCTION ........................................................................................................................... 86 MATERIALS AND METHODS ....................................................................................................... 89 RESULTS ..................................................................................................................................... 93 DISCUSSION .............................................................................................................................. 106 ACKNOWLEDGEMENTS ............................................................................................................. 108 REFERENCES ............................................................................................................................. 108 CHAPTER 5. Discussion ............................................................................................. 111 OVERVIEW ................................................................................................................................ 112 SECTION I: ARE THERE ADDITIONAL, NOVEL POLYOMAVIRUSES INFECTING HUMANS? ......... 112 SECTION II: DO THESE POLYOMAVIRUSES CAUSE DISEASE IN THEIR HUMAN HOSTS? ............ 119 FINAL CONCLUSIONS ................................................................................................................ 123 REFERENCES ............................................................................................................................. 124 APPENDIX I ................................................................................................................. 126 CURRICULUM VITAE ............................................................................................... 135 ii LIST OF FIGURES AND TABLES CHAPTER 1. Introduction Figure 1.1 Virus detection and discovery pipeline 7 Figure 1.2 Schematic representation of the WUPyV genome 11 Figure 1.3 The polyomavirus life cycle 12 Figure 1.4 Timeline of human polyomavirus discoveries 14 Table 1.1 Top 10 causes of death worldwide in 2011 3 Table 1.2 The human cancer viruses 5 CHAPTER 2. Identification of MW polyomavirus, a novel polyomavirus in human stool Figure 2.1 Genome organization of MWPyV 31 Figure 2.2 Phylogenetic analysis of MWPyV 35 Table 2.1 Putative proteins encoded by MWPyV (strain MA095) 32 Table 2.2 Specimens and patients testing positive for MWPyV 36 CHAPTER 3. Determination of the cell and tissue tropism of WU and KI polyomaviruses Figure 3.1 Validation of anti-KI-VP1 monoclonal antibody specificity 53 Figure 3.2 Validation of anti-WU-VP1 monoclonal antibody specificity 54 Figure 3.3 Electron micrographs of polyomavirus-like particles in a human lung 57 Figure 3.4 Nucleotide sequence of the 3’ end of the large T antigen gene from WUPyV strain Rochester-7029 58 Figure 3.5 Detection of WU-VP1 in the human respiratory tract 59 Figure 3.6 Detection of WU-VP1 in human alveolar macrophages 60 Figure 3.7 Detection of WU-VP1 in close proximity to MUC5AC-positive cells in the trachea 61 Figure 3.8 WU-VP1 detected in a BAL from a lung transplant recipient with Job’s syndrome 63 Figure 3.9 WU-VP1 detected within respiratory epithelial cells 64 Figure 3.10 WU-VP1 detected in a lung from an HIV-positive patient 66 Figure 3.11 IHC of human lung with a KI-VP1 monoclonal antibody 67 Figure 3.12 Detection of KI-VP1 in cells of hematopoietic origin in the lung 69 Figure 3.13 Detection of KI-VP1 in alveolar macrophages 70 Figure 3.14 Double IHC of the lung with monoclonal antibodies against KI-VP1 and cytokeratin at 600x 70 Figure 3.15 IHC of human spleen and lung with a KI-VP1 monoclonal antibody 73 Figure 3.16 Additional dIHC stains of human spleen 74 Table 3.1 Summary of findings 58 Table 3.2 Summary of results 75 CHAPTER 4. Progress toward a cell culture system for WU polyomavirus Figure 4.1 Establishment of a WUPyV replication system 88 Figure 4.2 Determination of the density of rWUPyV viral particles 95 Figure 4.3 Viral DNA detected in supernatants from hNECs infected with rWUPyV 97 Figure 4.4 Viral DNA detected in supernatants from hTECs infected with rWUPyV 98 iii Figure 4.5 Viral DNA detected in supernatants from hNECs infected with rWUPyV 99 Figure 4.6 Viral DNA detected in supernatants from hNECs infected with lysate passaged from a rWUPyV hNEC infection 99 Figure 4.7 Viral DNA detected in supernatants from immortal cell lines infected with rWUPyV at a MOI of ~100 101 Figure 4.8 Passage of rWUPyV-infected THP-1 cell lysates into fresh THP-1 cells