GENE EXPRESSION ANALYSIS DURING WEST NILE VIRUS DISEASE, INFECTION, AND RECOVERY By MELISSA ANN BOURGEOIS, DVM A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010 1 © 2010 Melissa Ann Bourgeois 2 To my parents and my sister, for their unfailing support and unconditional love throughout the years 3 ACKNOWLEDGMENTS I thank the members of the Long lab at the University of Florida, including Maureen Long, Sally Beachboard, Katie Maldonado, Kathy Seino, and Deanne Fanta for their invaluable assistance over the entire course of this project. I also thank the members of my committee, Maureen Long, Nancy Denslow, Paul Gibbs, David Allred, David Bloom, and James Maruniak for their wonderful insight and contributions to the evolution and progression of this project. I thank the members of the UF Interdisciplinary Centers for Biotechnology and Research, Gigi Ostrow, Li Liu, and Savita Shanker for their help in accomplishing different portions of this project. And finally, I thank my wonderful family and friends for their unfailing support throughout the entire course of my graduate degree. This project was funded with a grant from the USF Centers for Biological Defense. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 12 CHAPTER 1 INTRODUCTION .................................................................................................... 14 2 LITERATURE REVIEW .......................................................................................... 18 West Nile virus ........................................................................................................ 18 Epidemiology .................................................................................................... 18 Ecology and Host Range .................................................................................. 19 Molecular Epidemiology ................................................................................... 22 Viral Structure and Life Cycle ........................................................................... 23 Experimental Models of Infection ..................................................................... 25 Clinical Signs in Horses .................................................................................... 30 Clinical Signs in Humans .................................................................................. 31 Immune Response ........................................................................................... 32 Neurological Response .................................................................................... 39 Pathology ......................................................................................................... 40 Diagnostics ....................................................................................................... 40 Treatment ......................................................................................................... 43 Vaccination ....................................................................................................... 44 High Throughput Pyrosequencing .......................................................................... 47 Microarray Analysis................................................................................................. 48 3 DEEP SEQUENCING, ANNOTATION, AND ANALYSIS OF THE EQUINE CENTRAL NERVOUS SYSTEM TRANSCRIPTOME ............................................. 51 Methodology ........................................................................................................... 51 Sample Collection ............................................................................................ 51 RNA Extraction and Quality Analysis ............................................................... 51 First Strand cDNA Synthesis and Amplification- cDNA Library Construction ... 53 Normalization of the cDNA Library ................................................................... 54 High-throughput Pyrosequencing of the Normalized cDNA Library .................. 56 Library Annotation and Analysis ....................................................................... 57 Results .................................................................................................................... 59 Nucleic Acid Quality Assessment and Normalized cDNA Library Titration ....... 59 cDNA Library Quality Assessment ................................................................... 59 Normalized cDNA Library Sequencing Results ................................................ 60 5 BLAST and GO Analysis .................................................................................. 61 Comparison of Species- NR/NT NCBI Database ............................................. 63 Novel Genes Analysis ...................................................................................... 63 Biomarker Discovery ........................................................................................ 64 Analysis Against the Human EST Database .................................................... 65 Discussion .............................................................................................................. 65 Future Work and Issues to be Addressed ........................................................ 66 Conclusions ............................................................................................................ 68 4 GENE EXPRESSION ANALYSIS OF THE CENTRAL NERVOUS SYSTEM OFHORSES DURING WNV INFECTION WITH REGARDS TO EXPOSURE, SURVIVAL, AND LOCATION ................................................................................. 83 Methodology ........................................................................................................... 83 Microarray Probe Design .................................................................................. 83 Sample Collection ............................................................................................ 83 RNA Extraction ................................................................................................. 85 cDNA Creation and Dye Labeling ..................................................................... 86 Hybridization and Scanning of Arrays .............................................................. 87 Normalization and Statistical Analysis .............................................................. 88 Gene Ontology Enrichment .............................................................................. 89 Pathway Modeling ............................................................................................ 89 Microarray Validation ........................................................................................ 90 Results .................................................................................................................... 92 Study Design .................................................................................................... 92 Array Normalization .......................................................................................... 93 Statistical Analysis ............................................................................................ 93 Gene Ontology and Pathways Analysis Overview ............................................ 95 Exposure Status ............................................................................................... 95 Immune/Survivor Status ................................................................................. 100 CNS Location ................................................................................................. 104 Analysis of Overlap Between Exposure, Survival/Immunity, and Location ..... 107 Array Validation .............................................................................................. 108 Conclusions .......................................................................................................... 121 5 CONCLUSIONS ................................................................................................... 170 APPENDIX A RNA QUALITY DATA ANALYSIS ......................................................................... 175 B LIST OF HIGHLY UPREGULATED TRANSCRIPTS RECOGNIZED BY IPA ....... 178 C LIST OF HIGHLY dOWNREGULATED TRANSCRIPTS RECOGNIZED BY IPA . 198 LIST OF REFERENCES ............................................................................................. 199 BIOGRAPHICAL SKETCH .......................................................................................... 247 6 LIST OF TABLES Table page 3-1 Experimental tissues used to create the normalized cDNA library .................... 70 3-2 Dilutions of duplex specific endonuclease used to normalize the cDNA library .. 70 3-3 Average RNA quality data for samples ............................................................... 71 3-4 Data from 454 sequencing titration run ..............................................................