PHYLOGENOMIC CHARACTERIZATION OF A NOVEL MEGALOCYTIVIRUS LINEAGE FROM ARCHIVED ORNAMENTAL FISH SAMPLES By SAMANTHA AYUMI KODA 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 2017 © 2017 Samantha Ayumi Koda To my parents, who have worked hard to allow me to be able to pursue a career that I love ACKNOWLEDGMENTS On a daily basis, I continue to be inspired and reminded by those I love, to pursue my dreams. I attribute my passion for aquatic animals to my parents who have always been huge supporters of zoos and aquariums all my life. I wouldn’t have been able to get this far without the support and guidance from my family, friends, and the many teachers that have inspired me to ultimately pursue a career in fish health. Throughout my schooling I have had the pleasure of learning from some of the most passionate teachers and I would like to thank Mr.Schmitz for inspiring my initial interest in animal sciences, and Drs. Dan Reed and Scott Cooper for their mentorship roles during my undergraduate career. I have had the most amazing opportunities that have led me to my current success and I am very grateful for all the educational and hands on experience that I have gained at Asahi Koi Shop, Ty Warner Sea Center, California Department of Fish and Wildlife, Aquarium of the Pacific, and Sea Dwelling Creatures. I would like to thank my committee members, Drs. Thomas Waltzek, Kuttichantran Subramaniam, Ruth Francis-Floyd, Roy Yanong, and Salvatore Frasca, for their expertise, guidance, and time with my research project and degree. I would also like to thank the Bronson Animal Disease Diagnostic Laboratory for providing one of the viral isolates for my study, Dr. Joseph Groff for providing FFPE material, histology slides, and guidance on my pathological findings, and Patrick Thompson for training me on all the laboratory procedures for my research project. Lastly, I would like to thank all of the members of the Wildlife and Aquatic Veterinary Disease Laboratory that have been there for me during my time as a master’s student. I would especially like to thank Dr. Waltzek, who has been one of the most supportive, dedicated, and passionate advisors I have ever met. His love for aquatic animal science has been a tremendous positive influence on me, and has led to many lab retreats and group activities that have been 4 some of the highlights of my graduate career. To everyone in WAVDL, your countless stories, words of encouragement, and daily adventures made my time as a graduate student more joyous than I could have ever imagined. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................................10 CHAPTER 1 VIRUSES OF ORNAMENTAL FISH ...................................................................................12 Introduction.............................................................................................................................12 The Genus Megalocytivirus ....................................................................................................17 Infectious spleen and kidney necrosis virus (ISKNV) ....................................................20 Red seabream iridovirus (RSIV) .....................................................................................20 Turbot reddish body iridovirus (TRBIV) ........................................................................21 Diagnostic Methods .........................................................................................................22 Megalocytivirus Mitigation Strategies ............................................................................22 2 PHYLOGENOMIC CHARACTERIZATION OF A NOVEL MEGALOCYTIVIRUS LINEAGE FROM ARCHIVED ORNAMENTAL FISH SAMPLES ...................................31 3 METHODS .............................................................................................................................34 Archived Samples ...................................................................................................................34 Cell Culture and Virus Enrichment ........................................................................................34 Transmission Electron Microscopy ........................................................................................35 Histopathology ........................................................................................................................35 DNA Extraction ......................................................................................................................35 Complete Genome Sequencing and Assembly .......................................................................36 Genome Annotation and Phylogenetic Analysis ....................................................................36 PCR Detection of Megalocytiviruses .....................................................................................37 4 RESULTS ...............................................................................................................................43 Cell Culture and Virus Enrichment ........................................................................................43 Transmission Electron Microscopy ........................................................................................43 Histopathology ........................................................................................................................43 Complete Genome Sequencing and Assembly .......................................................................44 Genome Annotation and Phylogenetic Analysis ....................................................................44 PCR Detection of Megalocytiviruses .....................................................................................44 5 DISCUSSION .........................................................................................................................71 6 LIST OF REFERENCES ...............................................................................................................75 BIOGRAPHICAL SKETCH .........................................................................................................84 7 LIST OF TABLES Table page 1-1 Summary of megalocytiviruses used in this study .............................................................25 3-1 GenBank accession numbers for the full genome sequences of iridoviruses used in the 26 core gene phylogenetic analyses. ............................................................................39 3-2 Genome summary of the 10 megalocytivirus genomes used in the 26 and 54 core gene analyses. ....................................................................................................................41 3-3 Pan-megalocytivirus primer set .........................................................................................42 4-1 Predicted open reading frames for the South American cichlid iridovirus (SACIV) genome. ..............................................................................................................................48 4-2 Predicted open reading frames for the three spot gourami iridovirus (TSGIV) genome. ..............................................................................................................................57 8 LIST OF FIGURES Figure page 4-1 In vitro growth characteristics of TSGIV in the GF cell line.. ..........................................45 4-2 Transmission electron photomicrograph of a GF cell infected with three spot gourami iridovirus (TSGIV) ..............................................................................................46 4-3 Microscopic examination of megalocytic cells and lesions in infected Oscar Astronus ocellatus. ............................................................................................................................47 4-4 Phylogram illustrating the relationship of South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) to the other members of the family Iridoviridae based on 26 core genes .......................................................................66 4-5 Phylogram illustrating the relationship of South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) to the other member of the genus Megalocytivirus based on 54 core genes. ................................................................67 4-6 Phylogram illustrating the relationship of megalocytiviruses based on the major capsid protein gene ............................................................................................................69 4-7 Nucleotide sequence alignment of the transmembrane amino acid transporter protein gene ....................................................................................................................................70 9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of