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DETECTION and MOLECULAR CHARACTERIZATION of MANATEE PAPILLOMAVIRUS in CUTANEOUS LESIONS of the FLORIDA MANATEE (Trichechus Manatus Latirostris)

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DETECTION and MOLECULAR CHARACTERIZATION of MANATEE PAPILLOMAVIRUS in CUTANEOUS LESIONS of the FLORIDA MANATEE (Trichechus Manatus Latirostris) DETECTION AND MOLECULAR CHARACTERIZATION OF MANATEE PAPILLOMAVIRUS IN CUTANEOUS LESIONS OF THE FLORIDA MANATEE (Trichechus manatus latirostris) By REBECCA ANN WOODRUFF 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 Rebecca Ann Woodruff This document is dedicated to the graduate students of the University of Florida. ACKNOWLEDGMENT I would like to extend my greatest appreciation and thanks to my mentor, Dr. Carlos Romero, for allowing me to join his laboratory and enjoy many opportunities that I would not have been able to elsewhere. I would also like to thank my committee members, Dr. Peter McGuire and Dr. Ayalew Mergia, for their time, assistance, and suggestions. I would like to thank Dr. Ellis Greiner for first introducing me to the pathobiology graduate program at the University of Florida. I extend great thanks to Bob Bonde, not only for obtaining all of the manatee samples used in this study, but also for providing his expertise and knowledge of manatees. This project was funded by the Florida Fish and Wildlife Commission through the Marine Mammal Animal Health Program of the College of Veterinary Medicine at the University of Florida, and would not have been possible without the U. S. Geological Survey (USGS) Department of the Interior, contributions of collaborators affiliated with numerous zoological parks, and those involved with manatee capture release studies. I would also like to thank my fellow lab-mates, Alexa Bracht, Kara Smolarek-Benson, Shasta McClenahan, and Rebecca Grant, for their much valued friendships. I am greatly appreciative of my best friend, David Kottke, for supporting me unconditionally and following me to pursue my dreams, even if it meant putting his own aside. I would also like to thank my family for their constant encouragement during these past two years. iv TABLE OF CONTENTS page ACKNOWLEDGMENT.................................................................................................... iv LIST OF TABLES........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix ABSTRACT....................................................................................................................... xi CHAPTER 1 INTRODUCTION ........................................................................................................1 Classification of Papillomaviruses ...............................................................................1 Papillomavirus Genome Organization and Characterization .......................................2 Papillomavirus Replication...........................................................................................4 Papillomavirus Transmission........................................................................................5 Papillomavirus Pathogenesis ........................................................................................6 Diagnosis of Papillomavirus Infection .........................................................................8 Treatment and Prevention of Papillomavirus Infection................................................9 Human Papillomaviruses............................................................................................10 Non-Human Papillomaviruses....................................................................................11 Marine Mammal Papillomaviruses.............................................................................13 The Florida Manatee (Trichechus manatus latirostris)...............................................13 2 MATERIALS AND METHODS ...............................................................................16 Clinical Data ...............................................................................................................16 Source of Samples ......................................................................................................16 DNA Extraction from Papillomatous Lesions............................................................17 General Methods of PCR............................................................................................18 Taq Polymerase Reactions ..................................................................................18 Accuprime™ Taq DNA Polymerase Reactions ..................................................19 Expand High Fidelity Plus Reactions..................................................................19 PCR Targeting the L1 458-bp Fragment ....................................................................20 PCR Targeting the ∆L1 TmlPV 458-bp Fragment.....................................................20 PCR Targeting the TmlPV L1-E1 Region..................................................................20 PCR Targeting the Complete TmlPV E6 Gene ..........................................................21 PCR Targeting the Complete TmlPV E7 Gene ..........................................................21 v PCR Targeting the TmlPV L1-L2 Region..................................................................21 PCR Targeting the Complete TmlPV L1 Gene ..........................................................22 PCR Targeting the Complete TmlPV L2 Gene ..........................................................22 Gel Electrophoresis.....................................................................................................22 General Cloning of PCR Products..............................................................................23 Cloning into pCR 2.1® TOPO® T/A Vector ........................................................23 Cloning into P-Target™ Mammalian Expression Vector ...................................24 Cloning into pcDNA ™3.1 Directional TOPO® Expression Vector .................24 Analyzing Recombinants............................................................................................25 Sub-cloning of Purified Recombinants.......................................................................27 Sub-Cloning into pcDNA 3.1/Zeo+ Expression Vector......................................27 L1 Gene Sub-Cloning into pFastBac™1 Vector.................................................29 L1 Gene Sub-Cloning into pBlueBac 4.5............................................................30 Sequencing of PCR-amplified and Cloned Products..................................................30 Transfection of Insect Cell Cultures...........................................................................31 Culturing Insect Cells..........................................................................................31 Transformation of MAX Efficiency® DH10Bac™ Competent E. coli..............32 Transfection of Insect Cells with Bacmid DNA Recombinants..........................33 Harvest of Recombinant Baculovirus Stocks......................................................34 Reverse Transcription PCR (RT-PCR) of Infected Cell Cultures.......................34 Generation of Recombinant Baculovirus ............................................................35 Transfection of Mammalian Cells ..............................................................................36 Culturing African Green Monkey Kidney (COS-7) Cells...................................36 Electroporation of COS-7 Cells with Recombinants for Immunofluorescence Assays ..............................................................................................................37 Culturing Florida Manatee Respiratory Epithelial Cells.....................................38 Transfection of TmlRE Cells with DNA Recombinants for Immunofluorescence Assays ...........................................................................39 3 RESULTS...................................................................................................................44 PCR Results................................................................................................................44 PCR Targeting the Papillomavirus L1 458-bp Fragment....................................44 PCR Targeting the ∆L1 TmlPV Fragment..........................................................45 PCR Targeting the TmlPV L1-E1 Region...........................................................45 PCR Targeting the Complete TmlPV E6 Gene...................................................45 PCR Targeting the Complete TmlPV E7 Gene...................................................45 PCR Targeting the TmlPV L1-L2 Region...........................................................46 PCR Targeting the Complete TmlPV L1 Gene...................................................46 PCR Targeting the Complete TmlPV L2 Gene ..........................................................46 Sequencing Results and Genetic Analyses.................................................................46 TmlPV L1 458-bp Fragments..............................................................................46 L1-E1 TmlPV Region..........................................................................................48 TmlPV E6 Gene ..................................................................................................49 TmlPV E7 Gene ..................................................................................................49 TmlPV L1-L2 Region..........................................................................................50
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