Molecular Identification and Genetic Characterization of Cetacean Herpesviruses and Porpoise Morbillivirus
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MOLECULAR IDENTIFICATION AND GENETIC CHARACTERIZATION OF CETACEAN HERPESVIRUSES AND PORPOISE MORBILLIVIRUS By KARA ANN SMOLAREK BENSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005 Copyright 2005 by Kara Ann Smolarek Benson I dedicate this to my best friend and husband, Brock, who has always believed in me. ACKNOWLEDGMENTS First and foremost I thank my mentor, Dr. Carlos Romero, who once told me that love is fleeting but herpes is forever. He welcomed me into his lab with very little experience and I have learned so much from him over the past few years. Without his excellent guidance, this project would not have been possible. I thank my parents, Dave and Judy Smolarek, for their continual love and support. They taught me the importance of hard work and a great education, and always believed that I would be successful in life. I would like to thank Dr. Tom Barrett for the wonderful opportunity to study porpoise morbillivirus in his laboratory at the Institute for Animal Health in England, and Dr. Romero for making the trip possible. I especially thank Dr. Ashley Banyard for helping me accomplish all the objectives of the project, and all the wonderful people at the IAH for making a Yankee feel right at home in the UK. I thank Alexa Bracht and Rebecca Woodruff who have been with me in Dr. Romero’s lab since the beginning. Their continuous friendship and encouragement have kept me sane even in the most hectic of times. I thank the newest members of the lab, Shasta McClennahan and Rebecca Grant, for their input and support over the past few months. I also thank Linda Thomas and Mia Shandell for all their help. I would like to thank my committee members, Dr. David Bloom, Dr. Donald Forrester and Dr. Charles Manire, for their guidance and expertise. iv This project would not have been possible without the assistance of the many people and organizations that sent samples, especially Dr. Ruth Ewing, Dr. Charles Manire, Dr. Jeremiah Saliki and Dr. Forrest Townsend. Big thanks go to all of them! They keep this laboratory going. I would like to thank the computer guys at CSM for solving all my computer mishaps, and although they could not retrieve any of my lost thesis information when my computer crashed, I know they gave it their all. Last but not least, I thank my sister Ali, my Floridian significant other Maura, and my husband Brock for always believing in me, even when I did not believe in myself. Finally, this work was supported by a grant from Harbor Branch Oceanographic Institution and Florida Fish and Wildlife Commission through the Marine Animal Health Program of the College of Veterinary Medicine at the University of Florida. v TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix ABSTRACT....................................................................................................................... xi CHAPTER 1 CETACEAN HERPESVIRUSES................................................................................1 Introduction...................................................................................................................1 Materials and Methods ...............................................................................................12 Sample Acquisition and DNA Extraction ...........................................................12 Nested PCR Targeting the DNA Polymerase Gene ............................................13 Cloning, Sequencing and Sequence Analysis .....................................................15 PCR Targeting the DNA Terminase Gene ..........................................................16 Results.........................................................................................................................17 Nested PCR Targeting the DNA Polymerase Gene ............................................17 PCR Targeting the DNA Terminase Gene ..........................................................38 Discussion...................................................................................................................40 2 PORPOISE MORBILLIVIRUS .................................................................................50 Introduction.................................................................................................................50 Materials and Methods ...............................................................................................56 Analysis of PMV .................................................................................................56 Reverse transcription....................................................................................56 RACE and amplification of the terminal extragenic domains .....................57 Amplification of the complete PMV N gene ...............................................58 Amplification of the C-terminus of the PMV N gene..................................60 Detection of N protein expression by Western blot .....................................60 Analysis of Stranded Cetacean Tissues for Morbillivirus...................................61 Sample acquisition, RNA extraction and reverse transcription ...................61 PCR targeting the P gene .............................................................................61 PCR targeting the β-actin gene.....................................................................62 Results.........................................................................................................................63 vi Analysis of PMV .................................................................................................63 RACE and amplification of the terminal extragenic domains .....................63 Amplification of the complete PMV N gene ...............................................65 Detection of protein expression by Western blot.........................................74 Analysis of Stranded Cetacean Tissues for Morbillivirus...................................74 Discussion...................................................................................................................74 APPENDIX A MARINE MAMMAL SAMPLES..............................................................................84 B CETACEAN TISSUE SAMPLES.............................................................................93 LIST OF REFERENCES...................................................................................................97 BIOGRAPHICAL SKETCH ...........................................................................................106 vii LIST OF TABLES Table page 1-1. Percent nucleotide identities of DNA polymerase gene fragments among members of the family Herpesviridae. .....................................................................31 1-2. Percent amino acid identities of DNA polymerase gene fragments among members of the family Herpesviridae. .....................................................................32 1-3. Percent amino acid similarities of DNA polymerase gene fragments among members of the family Herpesviridae. .....................................................................33 2-1. Percent nucleotide identities of morbillivirus N genes. ...........................................67 2-2. Percent amino acid identities of morbillivirus N genes. ..........................................67 2-3. Percent amino acid similarities of morbillivirus N genes. .......................................67 A-1. List of all samples tested for herpesvirus using the nested PCR approach..............85 B-1. List of all samples tested for morbillivirus nucleic acid using PCR targeting the phosphoprotein gene. ...............................................................................................94 viii LIST OF FIGURES Figure page 1-1. Schematic of the location of the primers used in the nested PCR targeting the herpesvirus DNA polymerase gene..........................................................................14 1-2. Gel electrophoresis of herpesvirus nested PCR products.........................................17 1-3. Wart-like lesion on the genital slit of a female dwarf sperm whale.........................18 1-4. Genital lesions of two captive Atlantic bottlenose dolphins....................................19 1-5. Blainville’s beaked whale penile lesion. ..................................................................20 1-6. Atlantic bottlenose dolphin dermatitis lesions. ........................................................21 1-7. Epithelial cells from a skin lesion of a bottlenose dolphin. .....................................22 1-8. Atlantic bottlenose dolphin with lesions on the tongue and the penis.. ...................23 1-9. Gel electrophoresis of large DNA polymerase fragments from cetacean gammaherpesviruses ................................................................................................25 1-10. Multiple alignments of the nucleotide sequences of the DNA polymerase gene fragments of cetacean herpesviruses........................................................................27 1-11. Multiple alignments