Citation
Phylogenomic Characterization of a Novel Megalocytivirus Lineage from Archived Ornamental Fish Samples

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Title:
Phylogenomic Characterization of a Novel Megalocytivirus Lineage from Archived Ornamental Fish Samples
Creator:
Koda, Samantha A
Publisher:
University of Florida
Publication Date:
Language:
English

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Veterinary Medical Sciences
Veterinary Medicine
Committee Chair:
WALTZEK,THOMAS B
Committee Co-Chair:
FRANCIS-FLOYD,RUTH
Committee Members:
YANONG,ROY P
SUBRAMANIAM,KUTTICHANTRAN
FRASCA,SAL,JR

Subjects

Subjects / Keywords:
fish
iridovirus
megalocytivirus
saciv
trbiv
tsgiv

Notes

General Note:
The genus Megalocytivirus is the newest member of the family Iridoviridae, and as such, little is known about the genetic diversity of these globally emerging fish pathogens. Using an Illumina MiSeq sequencer, we sequenced the genomes of two megalocytiviruses (MCVs) isolated from epizootics involving South American cichlids (keyhole cichlid, Cleithracara maronii and oscar, Astronotus ocellatus) and three spot gourami Trichopodus trichopterus circulating in the ornamental fish trade in the early 1990s. Phylogenomic analyses revealed South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) possess nearly identical genomes and form a novel clade within the turbot reddish body iridovirus genotype (TRBIV Clade 2) previously reported from flatfish species reared for food in Asia (TRBIV Clade 1). The SACIV and TSGIV genomes were similar in size (111,347 and 111,591 bps, respectively), gene content (116 open reading frames for both), and %GC content (56.3 and 56.5, respectively) compared to other MCVs. However, both possess a unique truncated paralog of the major capsid protein (MCP) gene located immediately upstream of the full length parent gene. The MCP paralog likely arose through a gene duplication event and its function could be to increase antigenic diversity. Histopathological examination of archived oscar tissue sections revealed abundant cytomegalic cells characterized by basophilic granular cytoplasmic inclusions within various organs, particularly the anterior kidney, spleen and intestinal submucosa. A conventional PCR assay, designed to amplify and distinguish through Sanger sequencing all MCV genotypes, was partially validated and used to confirm the presence of SACIV DNA within archived formalin-fixed paraffin-embedded (FFPE) oscar tissues. TSGIV-infected grunt fin cells (GF) displayed cytopathic effect (e.g., cytomegaly, rounding, and refractility) as early as 96 hr post infection (pi). Ultrastructural examination revealed non-enveloped virus particles displaying hexagonal symmetry (120-144 nm) and an electron-dense core within the cytoplasm of infected GF cells, consistent with the ultrastructural morphology of a MCV. The sequencing of SACIV and TSGIV provides the first complete TRBIV Clade 2 genome sequences and expands the known host and geographic range of the TRBIV genotype to include freshwater ornamental fishes traded in North America.

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UFRGP
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5/31/2018

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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

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2017 Samantha Ayumi Koda

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To my parents who have worked hard to allow me to be able to pursue a career that I love

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4 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 wouldnt 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 ini tial 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, Dr s Thomas Waltzek, Kuttichantran Subramaniam, Ruth Franc is -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 Wil dlife and Aquatic Veterinary Disease Laboratory that have been th ere for me during my time as a m asters 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. Hi s 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

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5 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.

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6 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 In fectious 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 M egalocytiviruses .....................................................................................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 M egalocytiviruses .....................................................................................44 5 DISCUSSION .........................................................................................................................71

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7 LIST OF REFERENCES ...............................................................................................................75 BIOGRAPHICAL SKETCH .........................................................................................................84

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8 LIST OF TABLES Table page 11 Summary of megalocytiviruses used in this study .............................................................25 31 GenBank accession numbers for the full genome sequences of iridoviruses used in the 26 core gene phylogenetic analyses. ............................................................................39 32 Genome summary of the 10 megalocytivirus genomes used in the 26 and 54 core gene analyses. ....................................................................................................................41 33 Pan megalocytivirus primer set .........................................................................................42 41 Predicted open reading frames for the South American cichlid iridovirus (SACIV) genome. ..............................................................................................................................48 42 Predicted open reading frames for the three spot gourami iridovirus (TSGIV) genome. ..............................................................................................................................57

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9 LIST OF FIGURES Figure page 41 In vitro growth characteristics of TSGIV in the GF cell line.. ..........................................45 42 Transmission electron photomicrograph of a GF cell infected with three spot gourami iridovirus (TSGIV ) ..............................................................................................46 43 Microscopic examination of megalocytic cells and lesions in infected Oscar Astronus ocellatus ............................................................................................................................47 44 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 45 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 46 Phylogram illustrating the relationship of megalocytiviruses based o n the ma jor capsid protein gene ............................................................................................................69 47 Nucleotide sequence alignment of the transmembrane amino acid t ransporter protein gene ....................................................................................................................................70

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PHYLOGENOMIC CHARACTERIZATION OF A NOVEL MEGALOCYTIVIRUS LINEAGE FROM ARCHIVED ORNAMENTAL FISH SAMPLES By Samantha Ayumi Koda May 2017 Chair: Thomas Waltzek Major: Veterinary Medical Sciences The genus Megalocytivirus is the newest member of the family Iridoviridae and as such, little is known about the genetic diversity of these globally emerging fish pathogens. Using an Illumina MiSeq sequencer, we sequenced the genomes of two megalocytiviruses (MCVs) isolated from epizootics involving South American cichlids (keyhole cichlid, Cleithracara maronii and oscar, Astronotus ocellatus ) and three spot gourami Trichopodus trichopterus circulating in the ornamental fish trade in the early 1990s. Phylogenomic analyses revealed South American cichlid iridovirus (SACIV ) and three spot gourami iridovirus (TSGIV ) possess nearly identical genomes and form a novel clade within the turbot reddish body iridovirus genotype (TRBIV Clade 2) previously reported from flatfish species reared for food in Asia (TRBIV Clade 1). The SACIV and TSGIV genomes were similar in size (111,347 and 111,591 bps, respectively), gene content (116 open reading frames for both), and %GC content (56.3 and 56.5, respectively) compared to other MCVs. However, bot h possess a unique truncated paralog of the major capsid protein (MCP) gene located immediately upstream of the full length parent gene. The MCP paralog likely arose through a gene duplication event and its function could be to increase antigenic diversity Histopathological examination of archived oscar tissue sections revealed abundant cytomegalic cells characterized by basophilic granular cytoplasmic inclusions

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11 within various organs, particularly the anterior kidney, spleen and intestinal submucosa. A co nventional PCR assay, designed to amplify and distinguish through Sanger sequencing all MCV genotypes, was partially validated and used to confirm the presence of SACIV DNA within archived formalin fixed paraffinembedded (FFPE) oscar tissues. TSGIV infect ed grunt fin cells (GF) displayed cytopathic effect (e.g., cytomegaly, round ing and refracti lity ) as early as 96 hr post infection (pi). Ultrastructural examination revealed non enveloped virus particles displaying hexagonal symmetry (120 144 nm) and an e lectron dense core within the cytoplasm of infected GF cells, consistent with the ultrastructural morphology of a MCV. The sequencing of SACIV and TSGIV provides the first complete TRBIV Clade 2 genome sequences and expands the known host and geographic ra nge of the TRBIV genotype to include freshwater ornamental fishes traded in North America.

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12 CHAPTER 1 VIRUSES OF ORNAMENTAL FISH Introduction G lobal ly, aquaculture continues to be one of the fastest growing agricultural sectors with an estimated value of > $157 billion (FAO 2015). While the majority of this growth can be attributed to the production of food fish species in China, other sectors like ornamental aquaculture are important contributors to regional economies where production is conducive (e.g., Florida ornamental aquaculture industry) (Hill & Yanong 2016). More than 800 freshwater, brackish, and marine fish species and varieties are produced in Florida accounting for approximately 95% of US ornament al fish production. The most important freshwater species produced in Florida include various atheriniformes (e.g., rainbowfish), characiformes (e.g., tetras), cyprinidontiformes (e.g., livebearers and killifish) cypriniformes (e.g., danios, barbs, goldfis h, koi, rasboras, sharks), perciformes (e.g., cichlids, gourami), and siluriformes (e.g., suckermouth catfishes, Corydoras spp.). Although the value of the Florida marine ornamental aquaculture is unknown, a variety of demersal brood tending perciformes (e .g., basslets, damselfishes including clownfish, dottybacks, gobies), demersal mouthbrooding perciformes (e.g., cardinalfishes), and pouch brooding sy n gnathiformes (e.g., seahorses) are produced (Wittenrich 2007). In 2003, the farm gate value of Florida or namental fishes was approximately $47.2 million US dollars (Hill & Yanong 2016). The value of the international trade of aquacultured and wild caught ornamental fishes and invertebrates has been estimated to be approximately $300 million US dollars (Liveng ood & Chapman 2007). Finally, the approximate total revenue generated by the international ornamental fish industry including revenue generated by wholesalers, retailers, and aquarium product manufacturers is estimated to be $1 billion US dollars.

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13 Infectious agents (e.g., viruses, bacteria, fungi and water molds, unicellular and metazoan parasites) pose a significant threat to the global production of ornamental fishes (Wildgoose 2001, Noga 2010). Viruses are perhaps the least studied and among the most important pathogens negatively impacting global aquaculture production (Walker & Winton 2010). It is noteworthy that 8 out of 10 diseases reportable to the World Organization for Animal Health are viruses and 3 of these negatively impact the internati onal ornamental fish trade (i.e., spring viremia of carp, koi herpesvirus, and megalocytiviruses related to red seabream iridovirus (OIE 2016b). The lack of knowledge of ornamental fish viruses likely stems from the sheer number of species traded internati onally, a lack of interest and monies allocated toward ornamental fish virology, and the advanced laboratory equipment and tools (e.g., susceptible cell lines) needed for virological investigations. To date, only a handful of ssRNA (e.g., Rhabdoviridae ) and dsDNA (e.g., Alloherpesviridae, Poxviriidae, and Iridoviridae ) viruses have been demonstrated to induce disease in ornamental fishes. Spring viremia of carp virus (SVCV) is a negative-sense ssRNA virus within the family Rhabdoviridae that induces lethal systemic disease primarily in cyprinids (e.g., koi carp Cyprinus carpio and goldfish Carassius auratus ) and a few noncyp rinid species (Dixon 2008). SVCV was first isolated from moribund common c arp in Yugoslavia (Fijan et al. 1971) and remains a signific ant problem for European common carp aquaculture (Dixon 2008). SVCV is considered a foreign animal disease in the United States as it has only been detected from a limited number of cases in wild common carp and cultured koi carp since 2002 (Petty et al. 2016). Clinical signs of disease include lethargy, decreased respiration abnormal position in the water column, external and internal hemorrhages, and splenomegaly (Fijan 1999, OIE 2016a Petty et al. 2016 ). Direct transmission of the virus occurs through the gills, where a primary

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14 viremia is established in the branchial epithelium before spreading systemically (Ahne 1978). Spring water temperatures (12 22C) have been shown to be optimal for viral replication resulting i n natural outbreaks (Fijan 1999, McAllister 1993). Diagnostic methods useful in confirming suspected cases of SVCV disease include viral isolation, RTPCR, ELISA, virus neutralization, and immunofluorescence ( OIE 2016a Petty et al. 2016). Cyprinid herpesvirus 1 (CyHV1) is a member of the genus Cyprinivirus within the family Alloherpesviridae and is closely related to Cyprinid herpesvirus 2 (CyHV2) and Cyprinid herpesvirus 3 (CyHV3) (Waltzek et al. 2005, Hartman et al. 2016) CyHV1 infects common carp varieties including koi causing seaso nal episodes (i.e., water temperature <22C) of epidermal hyperplasia that result in unsightly mucoid to waxy growths of variable severity known as carp pox (Viadanna et al. 2017). While CyHV2 and CyHV3 are associated with lethal systemic disease in all ages of goldfish and common carp varieties, respectively, CyHV1 is rarely the cause of mortality in fish >2 mo old (Sano et al. 1991). The gross proliferative lesions are suggestive of CyHV1 disease and the presence of virus can be confirmed using immunofl uorescence, transmission electron microscopy, DNA arrays, and conventional PCR to ols (reviewed in Viadanna et al. 2017). Cyprinid herpesvirus 2 (CyHV2), known informally as herpesviral haematopoietic necrosis virus, induces lethal systemic disease in memb ers of the genus Carassius including goldfish, C. auratus (Hanson et al. 2016). CyHV2 was first reported in Japan in 1992 (Jung & Miyazaki 1995) and has since been reported globally as a result of the international ornamental goldfish trade (Hanson et al. 2016). Clinical signs of the disease include lethargy, pale gills, and mottling and enlargement of the spleen. Microscopic examination invariably reveals extensive necrosis of hematopoietic organs (e.g., anterior kidney and spleen) (Hanson et al. 2016).

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15 Morbidity and mortality can reach 100% at optimal temperatures for viral replication (i.e., 15 25C). CyHV2 has proven challenging to isolate from infected tissues; however, several PCR assays and transmission electron microscopy have been used to confirm suspected cases of CyHV2 disease (Hanson et al. 2016). Cyprinid herpesvirus 3 (CyHV3), known informally as koi herpesvirus, induces lethal systemic disease in cultured and wild varieties of common carp including koi carp (Hanson et al. 2016). The first de tection of CyHV3 was in archived koi samples from Europe in 1996 (Haenen et al. 2004). Later the disease spread wherever common carp varieties were traded including, Israel, North America, and Asia. Clinical signs of CyHV3 disease include behavioral changes (e.g., anorexia, lethargy, erratic swimming, piping ), erosions/hemorrhages of the skin and fin, and patchy white regions of the gills ( Hanson et al. 2016, Hartman et al. 2016). Outbreaks of CyHV3 disease occur at water temperatures between 17 26C with c umulative mortality reaching up to 90% (Hanson et al. 2016, Hartman et al. 2016 ). CyHV3 is difficult to isolate from infected tissues; however, several endpoint and quantitative PCR assays have been developed to rapidly confirm suspected cases of CyHV3 dis ease ( Hanson et al. 2016, Hartman et al. 2016, OIE 2016a ). Carp edema virus disease (CEVD) / koi sleepy disease (KSD) is caused by a novel fish poxvirus (Miyazaki et al. 2005, Hesami et al. 2015). The name koi sleepy disease is due to the lethargic behav ior observed in infected koi which includes hanging at the surface of the water column or laying at the bottom of the pond (Miyazaki et al. 2005, Hesami et al. 2015). CEVD/KSD was first documented in 1974 in Japanese koi, and has now been reported globally (reviewed by Hesami et al. 2015). Additional clinical signs of CEVD/KSD include hemorrhage of the skin, dermal edema, en dophthalmos, and pale gills (Miyazaki et al. 2005, Hesami et al.

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16 2015). Juvenile koi appear most susceptible with outbreaks occ urring at a water temperature between 15 25C and cumulative mortality reaching up to 100% (Miyazaki et al. 2005, Hesami et al. 2015). CEV is refractory to cell culture, and thus, a diagnosis is confirmed when common or colored carp (koi) displaying consis tent CEVD/KSD clinical signs test positive by PCR. M embers of the family Iridoviridae pose a significant threat to the international ornamental fish industry (Weber et al. 2009, Yanong & Waltzek 2010, Sriwanayos et al. 2013). Iridoviruses are large ds DNA viruses that infect a range of poikilothermic vertebrate and invertebrate hosts. They possess nucleocapsids with icosahedral symmetry that contain linear DNA genome s ranging in size from 105 350 kbp (Jancovich et al. 2012). The family is composed of five genera: Iridovirus Chloriridovirus Lymphocystivirus Ranavirus and Megalocytivirus (Jancovich et al. 2012). The genera Iridovirus and Chloriridovirus infect invertebrates such as insects and crustaceans ( Jancovich et al. 2012 ). Members of the genus Ranavirus infect fish, amphibians, and reptiles; whereas, the genera Lymphocystivirus and Megalocytivirus infect freshwater and marine fishes including species traded in the international ornamental trade. One of the most commonly encountered and easily identifiable viral diseases in the ornamental fish trade is lymphocystivirus disease virus (LCDV) (Yanong 2010). LCDVs infect a wide range of freshwater and marine fishes typically resulting in benign proliferative lesions of the skin and fins. LCD V i nfected fibroblasts of the s kin and fins (internal organ inv olvement is rare) may enlarge up to 100,000x and masses of infected fibroblasts may cluster together to form grossly visible whitish to pink masses sever al centimeters in diameter (Wolf 1988 Yanong 2010). Certain freshwater (e.g., gourami and cichlids), brackish (e.g., glassfish and scats), and marine perciformes (butterflyfishes, angelfishes, and gobies) appear predisposed to LCD V

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17 (Yanong 2010). Other commonly traded ornamental fishes belonging to the orders cypriniformes (e.g., danios, barbs, goldfish, koi, rasboras, sharks) and siluriformes (e.g., suckermouth catfishes, Corydoras spp.) are rarely if ever affected by LCD V LCD V is typically a self limiti ng disease with little or no mortality observed unless lesions obstruct respiration or feeding. Trained fish health prof essionals learn to identify LCDV grossly; however, evaluation of infected tissues by cytology / histology to confirm the clusters of enl arged cells is prudent given the gross proliferative lesions are not pathognomonic. Transmission electron microscopy and PCR are also useful options to confirm LCD V (Cano et al. 2006). The G enus Megalocytivirus The genus Megalocytivirus (MCV) i s the newest member in the family Iridoviridae MCVs possess a typical iridovirus nucleocapsid displaying icosahedral symmetry (140 200 nm in diameter) and an electron dense DNA core (Kawato et el. 2017). The MCV genome is linear dsDNA (110 112 kbp), encodes between 93135 open reading frames, and displays between 5356% GC content (Chinchar et al. 2017). MCVs are promiscuous infecting a wide range of freshwater brackish, and marine fishes from the temperate waters of British Columbia, Canada, to the tropical waters of the South China Sea ( Kurita & Nakajima 2012, Subramaniam et al. 2012, Waltzek et al. 2012 Table 1 1). A review of published literature and DNA sequences deposited into public databases revealed MCVs infect at least 125 fish species a cross 11 orders and 44 families ( Yanong & Waltzek 2016, Koda & Waltzek unpublished data ). Transmission occurs horizontally by exposure of nave fish to MCV infected fish (i.e., cohabitation), contaminated water sources, or ingestion of MCV infected prey (reviewed in Subramaniam et al. 2012). Outbreaks in hatcheries have not been reported, and thus, the risk of vertical transmission through eggs and sperm is considered low (Nakajima & Kurita, 2005). The MCV genome has been detected in multiple organs of su rvivors

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18 of experimental challenges up to 25 days pi which suggest s that viral persistence may occur (Ito et al. 2013). T ypical clinical signs associated with MCV infection s include behavioral changes (e.g., anorexia, lethargy, increas ed respiration), whit e feces gill pallor, darkened or lightened body appearance, splenomegaly, ascites, and internal/external hemorrhagic lesions (Yanong & Waltzek 201 6, Kawato et al. 2017 ). MCVs typically establish chronic infections with cumulative mortality ranging from 20 60% over a 12 month period (Kawato et al. 2017). Experimental challenge studies for each MCV genotype (i.e., ISKNV, RSIV, TRBIV) have revealed no clinical signs of disease at cooler water treatments (<20C) as compared to warmer water treatments in which up to 100% cumulative mortality has been observed (He at al. 2002, Nakajima et al. 2002, Wang et al. 2003, Oh et al. 2006, Jun et al. 2009, Subramaniam et al. 2012). Despite the lack of clinical disease at cooler temperatures PCR detection of MCV DNA at the cooler water treatments suggests fish may become infected. Histopathological examination of MCV infected fish reveals pathognomonic microscopic lesions with affected cells displaying cytomegaly and basophilic granular cytoplasmic inclusions (Gibson -Kue h et al. 2003, Weber et al. 2009, Yanong & Waltzek 201 6, Subramaniam et al. 2016). An early investigation misinterpreted these lesions as amoebae (Anderson et al. 1989) and affected cells have been described as hypertrophic, heteromorphic balloon cells, ci rcumscribed bodies, and inclusion body bearing cells (reviewed in Sudthongkong et al. 2002). Syst emic infections involving hematopoietic organs (spleen and anterior kidney), the submucosa of the gastrointestinal tract, and other organs are common ( Gibson-K ueh et al. 2003, Weber et al. 2009, Yanong & Waltzek 201 6, Subramaniam et al. 201 6). The histogenesis of affected cells remains

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19 unclear; however, affected cells may include lympho myeloid mesenchymal, endothelial, and reticuloendothelial cells (Weber et al. 2009, Subramaniam et al. 2016). The first well characterized MCV epizootic occurred in Shikoku Island, Japan in the summer of 1990 involving cultured red seabream ( Pagrus major ) (Inouye et al. 1992) and the MCV was named red seabream irido virus (RSIV ). Similar ongoing disease outbreaks have been reported as early as 1994 among Chinese freshwater farms rearing mandarin fish (Siniperca chuatsi ) and t he MCV was named Infectious spleen and kidney necrosis virus (ISKNV) to reflect the observed pathology (He et al. 2000, 2001). The first full MCV genome was reported for ISKNV and the authors suggested it was different enough from other iridoviruses to be classified into a new genus that was tentatively named the cell hypertrophy iridovirus es (He e t al. 2001). Shortly thereafter, the full genome of RSIV was reported and the authors again concluded RSIV represented a new genus in the family Iridoviridae (Kurita et al. 2002). Other authors suggested these related MCVs from tropical aquatic habitats in Asia be referred to as the tropical iridoviruses or tropiviruses (Sudthongkong et al. 2002a, b). A MCV genome distinct from ISKNV and RSIV, known as the turbot reddish body iridovirus (TRBIV), was sequenced from samples associated with farmed turbot ( Scophthalmus maximus ) epizootics in China (Shi et al. 2004). Researchers studying the microscopic and ultrastructural characteristics of MCV infections in Taiwanese hybrid grouper suggested the name megalocytivirus (Chao et al. 2004). The genus Megalocyt ivirus was accepted in 2005 by the International Committee on Taxonomy of Viruses with ISKNV as the type species and sole member of the new genus (Fauquet et al. 2005). Phylogenetic analyses based on the major capsid protein and ATPase genes have revealed the species ISKNV is composed of 3 genotypes (i.e., ISKNV, RSIV, and TRBIV) and each can

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20 be further subdivided into 2 separate well supported clades (Figure 46) (Waltzek et al. 2012, Kurita & Nakajima 2012, Go et al. 2016). The recent characterization of genetically divergent MCVs from Canadian three spine d stickleback ( Gasterosteus aculeatus ) and Australian barramundi (Lates calcarifer ) has led some authors to propose additional species in the genus (Waltzek et al. 2012, de Groof et al. 2015). Infectiou s spleen and kidney necrosis virus (ISKNV) The ISKNV genotype was first characterized from outbreaks involving farmed mandarin fish (Siniperca chuatsi ) reared for food in China (He et al. 2000, 2001, Table 1 1). The genome of ISKNV was sequenced from mandarin fish samples taken from an outbreak in 1998 and represents the only ISKNV Clade 1 genome sequenced (He et al. 2001). Related viruses have been detected in marine food fish species (Latidae, Serranidae, Sparidae) reared in Singapore in 2000, Malaysia in 2000 and 2012, Hong Kong in 2004, Taiwan from 20052008, and the Philippines in 2010 (Huang et al. 2011, Kurita & Nakajima 2012, Razak et al. 2014). In addition to freshwater and marine food fish species, the ISKNV Clade 1 MCVs have also been detected widely in freshwater ornamental fishes (Osphronemidae, Poeciliidae, Cichlidae) within Asia or exports from Asia since 1998 (Sudthongkong et al. 2002a Go et al. 2006, Rimmer et al. 2012, Whittington et al. 2010, Subramaniam et al. 2014, Go et al. 2016). Most recently, a second clade within the ISKNV genotype was described from freshwater marbled sleeper goby ( Oxyeleotris marmoratus ) cultured for food in China (Wang et al. 2011) and marine ornamental fishes (Apogonidae, Ephippidae) exported from Indonesia (Weber et al. 2009, Sriwanayos et al. 2013). Red seabream i ridovirus (RSIV) RSIV was first reported in a 1990 epizootic involving cultured red seabream (Pagrus major ) reared in the Ehime prefecture of Shikoku Islan d, Japan (Inouye et al. 1992, Table 1 1). This Ehime1 strain was the first RSIV Clade 1 genome sequenced (Kurita et al. 2002). A second

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21 RSIV Clade 1 MCV was sequenced from an isolate obtained from epizootics involving maricultured large yellow croaker ( La rimichthys crocea ) in China in 2001 (Chen et al. 2003). Since the initial RSIV outbreaks, RSIV Clade 2 MCVs have predominated involving >40 maricultured species distributed widely across East and Southeast Asia and resulting in significant economic losses (reviewed in Kurita & Nakajima 2012, and Kawato et al. 2017). The grouper sleeping disease virus described in Thailand (Sudthongkong et al. 2002a ), rock bream iridovirus in Korea (Jung & Oh 2000), and the orange spotted grouper iridovirus in China (Lu et al. 2005), and a variety of other MCVs belong to the RSIV Clade 2. The full genome for RSIV Clade 2 MCVs ha ve been determined from samples involving outbreaks in rock bream (Oplegnathus fasciatus Do et al. 2004), orange -spotted grouper (Ephinephelus coioides Lu et al. 2005), and giant seaperch (Lates calcariferi GenBank acc. no. KT781098). Neither clade of the RSIV genotype has been reported from an ornamental fish. Turbot r eddish b ody i ridovirus (TRBIV) The TRBIV genotype has been characterized from epizootics involving flatfishes (order pleuronectiformes) reared for human consumption in and around the Yellow Sea in East Asia (Kawato et al. 2017 Table 1 1). The first epizootics occurred in turbot (Scophthalmus maximus ) maricultured in Chin a (Shi et al. 2004) and then in turbot (Kim et al. 2005) and Japanese flounder (Paralichthys olivaceous ) in Korea (Do et al. 2005, Kim et al. 2005, respectively). The first TRBIV Clade 1 genome were samples associated with an epizootic in moribund turbot c ultured in China in 2004 (Shi et al. 2010). A second clade (i.e., TRBIV Clade 2) was recently proposed based on the partial MCP and ATPase sequences from material involving: 1) a 2008 outbreak in cultured rock bream fingerlings recently imported into Taiwan from Korea (Huang et al. 2011) and 2) outbreaks in freshwater ornamental fishes from the late 1980s through the early 1990s (Go et al. 2016, Chapters 25).

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22 Diagnostic Methods Red seabream iridoviral disease (RSIVD) in marine and freshwater fishes caused by the genotypes RSIV and ISKNV are reportable to the World Organization for Animal Health (OIE, 2016a ). However, it has not been decided whether similar diseases caused by TRBIV or ISKNV i n ornamental fish hosts should be considered reportable. MCVs typically induce chronic systemic infections manifesting as behavioral changes (e.g., anorexia, lethargy, increased respiration ), darkened or lightened body appearance, internal/external hemorrh agic lesions, gill pallor, splenomegaly, ascites, anemia, and white feces (Yanong & Waltzek 201 6, OIE 2016a Kawato et al. 2017). A presumptive RSIVD diagnosis is reached when one or more of the following is met: 1) fish display the aforementioned clinical signs and the observation of megalocytes on acetonefixed Giemsastained impression smears or stained tissue sections of the spleen, kidney or heart ; 2) fish display the aforementioned clinical signs and the observation of stereotypical iridoviruses vir ions within the cytoplasm of megalocytes by transmission electron microscopy; 3) virus isolation on grunt fin cells with the observation of appropriate cytopathic effect (i.e., cellular rounding and enlargement); and 4) the presence of immunofluorescent an tibody test (IFAT) positive megalocytes on acetonefixed impression smears (reviewed in OIE, 2016a). The RSIVD diagnosis is considered confirmed if 1) virus isolation with appropriate CPE i s confirmed positive by either IFAT using infected cell cultures or positive by PCR using extracted DNA from the supernatant of infected cultures; 2) positive endpoint or quantitative PCR result using extracted DNA from affected organs; and 3) presence of megalocytes showing positive IFAT results on acetonefixed impressi on smears (reviewed in OIE 2016a, Kawato et al. 2017).

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23 Megalocytivirus Mitigation Strategies Currently, there is one commercially available RSIV vaccine approved for use in Japan (Nakajima et al. 1997, 1998, OIE 2016a, Kawato et al. 2017). This formalin killed vaccine is most effective for use in red seabream, yellowtail ( Seriola quinqueradiata), greater amberjack (Seriola dumerili ), striped jack ( Pseudocaranx dentex ), Malabar grouper (Epinephelus malabaricus ), orange -spotted grouper (E coioides ), longtooth grouper (E bruneus ), and sevenband grouper (E septemfasciatus ) ( Nakajima et al. 1997, 1998, OIE 2016a, Kawato et al. 2017). However, the vaccine is not as effective in highly susceptible species of the genus Oplegnathus including rock bream (O fasciatus ) and spotted knifejaw (O punctatus ). More recently, efficacious formalin killed vaccines have been created to protect against the ISKNV (Dong et al. 2008) and TRBIV (Fan et al. 2012) genotypes. Alternative recombinant subunit vaccines and DNA v accines have also been produced for protection against the RSIV and ISKNV genotypes; however, none are commercially available (reviewed in Kawato et al. 2017). To date, no vaccine or other chemotherapeutic has been approved for use in the USA for the treatment of MCV disease (Yanong & Waltzek, 2010). Furthermore, no MCV vaccine has a proven efficacy in an ornamental species. Ornamental fish producers and wholesalers should follow proper husbandry and biosecurity practices including establishing a working r elationship with a fish health professional (reviewed in Yanong & Waltzek, 2010). Incoming fish should be quarantined and separated by origin in a separate building if possible. Each system should have separate equipment (e.g., nets, siphon hoses, buckets) that is regularly disinfected. MCV disinfection options include heat inactivation (>50 C for >30 min), elevation of pH (>11 for >30 min), UV sterilization (>10003000 uW), and chemical disinfection using sodium hypochlorite (>200 mg/L), potassi um permangan a te (>100 mg/L), or formalin (>2000 mg/L) (Nakajima & Sorimachi 1994, He et al. 2002, Kasai et al. 2002 ). The contact time for the

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24 aforementioned chemical disinfectants should be >15 min at or around 25C. Footbaths and handwashing stations should be implemented in every building and employees should be encouraged and observed for compliance. System water should be from a protected source (e.g., aquifer) versus unprotected ground water sources that may harbor infectious agents. Alternatively ground water can be sterilized (e.g., UV, ozone) prior to use. Sick fish should be quarantined and submitted for evaluation to a fish disease diagnostic laboratory. There are no appropriate treatment options for fish infected with MCVs, and thus, affected stocks should be depopulated and disposed of properly including system water and equipment. RSIV disease has not been officially been reported outside of Asia or in ornamental species. Diagnostic laboratories within the USA are encouraged to determine th e genotype of MCV cases and report any detections of RSIV disease (excluding ornamental fishes) to the appropriate authorities to ensure this foreign animal disease does not spread from Asia.

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25 Table 1 1. Summary of megalocytiviruses used in this study Host Genus species Host Common Name Host Family Marine (M) Freshwater (F) Brackish (B)a Country and Year Collected Strain Name GenB ank Accession Reference ISKNV Clade 1 Maccullochella peelii Murray cod Percichthyidae F Australia 2003 MCIV AY936203 Go et al 2006 Trichogaster lalius Dwarf gourami Osphronemidae F Singapore 2000 ISKNV DGA4/6K AB666344 Nakajima & Kurita 2005 Siniperca chuatsi Mandarin fish Percichthyidae F China 2009 ISKNV QY HQ317460 Fu et al 2011 Epinephelus lanceolatus King grouper Serranidae M/B Malaysia 2011 Sabah/RAA/2012 GGIV4 JQ253366 Razak et al 2014 Cromileptes altivelis Humpback grouper Serranidae M Malaysia 2006 Sabah/RAA/2012 HGIV65 JQ253367 Razak et al. 2014 Cromileptes altivelis Humpback grouper Serranidae M Malaysia 2006 Sabah/RAA/2012 HGIV67 JQ253369 Razak et al. 2014 Siniperca chuatsi Mandarin fish Percichthyidae F China 1998 ISKNV AF371960 (CG) He et al. 2001 Cromileptes altivelis Humpback grouper Serranidae M Malaysia 2006 Sabah/RAA/2012 HGIV69 JQ253370 Razak et al. 2014 Trichogaster lalius Dwarf gourami Osphronemidae F Japan 2000 DGIV AB109369 Sudthongkong et al. 2002a Lates calcarifer Barramundi Latidae M/B/F Taiwan 2005 GSIV/Pt/836/05 JF264350 Huang et al. 2011 Cromileptes altivelis Humpback grouper Serranidae M Malaysia 2006 Sabah/RAA/2012 HGIV73 JQ253371 Razak et al. 2014 Aplocheilichthys centralis African lampeye Poeciliidae F Japan 1998 ALIV AY285745 Sudthongkong et al. 2002a/b Lates calcarifer Barramundi Latidae M/B/F Taiwan 2005 GSIV/Pt/843/05 JF264354 Huang et al. 2011

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26 Table 1 1. Continued Host Genus species Host Common Name Host Family Marine (M) Freshwater (F) Brackish (B) a Country and Year Collected Strain Name Gen B ank Accession Reference Lates calcarifer Barramundi Latidae M/B/F Taiwan 2006 GSIV/Pt/113/06 JF264353 Huang et al. 2011 Epinephelus fuscoguttatus Brown marbled grouper Serranidae M Malaysia 2007 Sabah/RAA/2012 BMGIV46 JQ253373 Razak et al. 2014 Rhabdosargus sarba Silver seabream Sparidae M/B Taiwan 2005 SSBIV/Pt/703/05 JF264356 Huang et al. 2011 Epinephelus fuscoguttatus Brown marbled grouper Serranidae M Malaysia 2007 Sabah/RAA/2012 BMGIV48 JQ253374 Razak et al. 2014 Epinephelus lanceolatus King grouper Serranidae M/B Malaysia 2011 Sabah/RAA/2012 GGIV3 JQ253365 Razak et al. 2014 Aplocheilichthys centralis African lampeye Poeciliidae F Japan 1998 ALIV AB109368 Sudthongkong et al. 2002a/b ISKNV Clade 2 Pterapogon kauderni Banggai cardinalfish Apogonidae M SE Asia 2006 PKIV AB669096 Kurita & Nakajima 2012 Oxyeleotris marmorata Marble goby Eleotridae B/F China 2009 MSGIV HM067835 Wang et al. 2011 Platax orbicularis Orbicular batfish Ephippidae M/B Belgium 2010 OBIV KC424426 Sriwanayos et al. 2013 RSIV Clade 1 Siniperca chuatsi Mandarin fish Percichthyidae F China 2006 ISKNV XQ HQ317458 Fu et al. 2011 Siniperca chuatsi Mandarin fish Percichthyidae F China 2006 ISKNV XT HQ317457 Fu et al. 2011 Pagrus major Red seabream Sparidae M Japan 1992 RSIV Ehime 1/RS92Ehi1 AB080362 Kurita et al. 2002 Larimichthys crocea Large yellow croaker Sciaenidae M/B China 2001 LYCIV AY779031 (CG) Ao & Chen 2006

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27 Table 1 1. Continued Host Genus species Host Common Name Host Family Marine (M) Freshwater (F) Brackish (B) a Country and Year Collected Strain Name GenB ank Accession Reference RSIV Clade 2 Epinephelus lanceolatus King grouper Serranidae M/B Taiwan 2005 KGIV 05 EU847414 Wang et al. 2009 Lates calcarifer Barramundi Latidae M/B/F Taiwan 2007 BPIV 07 EU847417 Wang et al. 2009 Epinephelus lanceolatus King grouper Serranidae M/B Taiwan 2006 KGIV/Pt/96/06 JF264355 Huang et al. 2011 Oplegnathus fasciatus Rock bream Oplegnathidae M South Korea <2004 RBIV CNU 1 AY849393 Kim et al. 2007 Lateolabrax spp. Seabass Serranidae M/B/F Japan 1993 SBIV AY310917 Sudthongkong et al. 2002b Oplegnathus fasciatus Rock bream Oplegnathidae M Korea <2002 RBIV KOR TY4 AY532608 Do et al. 2005a Siniperca chuatsi Mandarin fish Percichthyidae F China 2012 ISKNV LJ2012 KC775381 Dong et al. 2013 Paralichthys olivaceus Olive flounder Paralichthyidae M Korea <2005 OFIV DQ198145 Kim & Lee unpublished Oplegnathus fasciatus Rock bream Oplegnathidae M Korea <2002 RBIV KOR TY2 AY533035 Do et al. 2005a Pagrus major Red seabream Sparidae M Korea <2005 RSIV KOR TY AY532612 Do et al. 2005a Kareius bicoloratus Stone flounder Pleuronectidae M/B/F China 2010 SFIV 724/China HQ263620 Zhao et al. unpublished Epinephelus tauvina Greasy grouper Serranidae M Thailand 1993 GSDIV AY285746 Sudthongkong et al. 2002b Oplegnathus fasciatus Rock bream Oplegnathidae M Korea 2000 RBIV KOR TY1 AY532606 (CG) Do et al. 2004 Oplegnathus fasciatus Rock bream Oplegnathidae M Korea <2004 RBIV CNU 2 AY849394 Kim et al. 2007 Lateolabrax japonicus Japanese seaperch Serranidae M/B/F Korea 2001 SBIV KOR TY AY532613 Do et al. 2005a

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28 Table 1 1. Continued Host Genus species Host Common Name Host Family Marine (M) Freshwater (F) Brackish (B) a Country and Year Collected Strain Name GenB ank Accession Reference Oplegnathus fasciatus Rock bream Oplegnathidae M Korea <2002 RBIV KOR GJ AY532609 Do et al. 2005a Sebastes schlegeli Korean rockfish Sebastidae M Korea <2002 RFIV KOR TY AY532614 Do et al. 2005a Oplegnathus fasciatus Rock bream Oplegnathidae M Korea <2002 RBIV KOR TY3 AY532607 Do et al. 2005a Leiognathus equulus Common ponyfish Leiognathidae M/B/F Taiwan 2005 CPIV 05 EU847420 Wang et al. 2009 Lates calcarifer Barramundi Latidae M/B/F Taiwan 2008 GSIV/Pt/327/08 JF264346 Huang et al. 2011 Lates calcarifer Barramundi Latidae M/B/F Taiwan <2008 GSIV K1 EU315313 Wen et al. 2008 Epinephelus lanceolatus King grouper Serranidae M/B Taiwan 2006 GGIV/Pt/36/06 JF264347 Huang et al. 2011 Seriola quinqueradiata Japanese amberjack Carangidae M China 2007 ISKNV HZhj HQ317463 Fu et al. 2011 Pagrus major Red seabream Sparidae M Japan 2005 RSIV U 6 AB461856 Shinmoto et al. 2009 Siniperca chuatsi Mandarin fish Percichthyidae F China 2006 ISKNVHT HQ317464 Fu et al. 2011 Oplegnathus fasciatus Rock bream Oplegnathidae M Korea <2002 RBIV KOR YS AY532610 Do et al. 2005a Lates calcarifer Barramundi Latidae M/B/F Taiwan 2008 BPIV 08 EU847418 Wang et al. 2009 Pagrus major Red seabream Sparidae M Japan 1994 RSIV AB109371 Sudthongkong et al. 2002b Epinephelus coioides Orange spotted grouper Serranidae M/B China 2002 OSGIV AY894343 (CG) Lu et al. 2005 TRBIV Clade 1 Scophthalmus maximus Turbot Scophthalmidae M China 2005 TRBIV I GQ273492 (CG) Shi et al. 2010

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29 Table 1 1. Continued Host Genus species Host Common Name Host Family Marine (M) Freshwater (F) Brackish (B) a Country and Year Collected Strain Name GenB ank Accession Reference Scophthalmus maximus Turbot Scophthalmidae M China 2002 TRBIV I AY590687 Shi et al. 2004 Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV EJ AY633987 Do et al. 2005b Paralichthys olivaceus Olive flounder Paralichthyidae M Korea <2007 OFLIV 1 EU276417 Jeong et al. unpublished Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV YG AY633984 Do et al. 2005b Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV SS AY633983 Do et al. 2005b Paralichthys olivaceus Olive flounder Paralichthyidae M Korea <2004 OFIV AY661546 Kim et al. unpublished Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV WD1 AY633986 Do et al. 2005b Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV PH AY633992 Do et al. 2005b Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV WD2 AY633985 Do et al. 2005b Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV DS1 AY633980 Do et al. 2005b Oplegnathus fasciatus Rock bream Oplegnathidae M Korea <2002 RBIV KOR CS AY532611 Do et al. 2005a Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV JHJ AY633991 Do et al. 2005b Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV JJ AY633988 Do et al. 2005b Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV JSY AY633989 Do et al. 2005b Scophthalmus maximus Turbot Scophthalmidae M China 2009 TIV R 603 HM596017 Zhao et al. unpublished

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30 Table 1 1. Continued Host Genus species Host Common Name Host Family Marine (M) Freshwater (F) Brackish (B) a Country and Year Collected Strain Name GenB ank Accession Reference Lateolabrax spp. Seaperch Serranidae M/B/F Korea 2000 SPIV CH 1 HM067603 Jeong & Jeong unpublished Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV MI AY633982 Do et al. 2005b Paralichthys olivaceus Olive flounder Paralichthyidae M Korea 2003 FLIV DS2 AY633981 Do et al. 2005b TRBIV Clade 2 Astronotus ocellatus Oscar Cichlidae F USA 1992 SACIV KX354221 Go et al. 2016 Pteryophyllum scalare Angelfish Cichlidae F Canada 1986 PSMV1986 KX354223 Go et al. 2016 Trichogaster lalius Dwarf gourami Osphronemidae F Australia 1988 TLMV1988 KX354222 Go et al. 2016 Astronotus ocellatus Oscar Cichlidae F USA 1992 SACIV KX354221 Go et al. 2016 Oplegnathus fasciatus Rock bream Oplegnathidae M Taiwan 2008 RBIV/Tp/45/08 JF264352 Huang et al. 2011 Trichogaster trichopterus Three spot gourami Osphronemidae F USA <1992 TSGIV N/A This study TSIV Gasterosteus aculeatus Three spined stickleback Gasterosteidae M/B/F Canada 2008 TSIV HQ857785 Waltzek et al. 2012 SDDV Lates calcarifer Barramundi Latidae M/B/F Singapore 2010 2011 C4575 NC027778 de Groof et al. 2015 Abbreviations: CG=complete genome; ISKNV = Infectious spleen and kidney necrosis virus ; RBIV= Rock bream iridovirus; RSIV= Red seabream iridovirus; SDDV= Scale drop disease virus; TRBIV= Turbot redd ish body iridovirus; TSIV=Three spine stickleback iridovirus.

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31 CHAPTER 2 PHYLOGENOMIC CHARACTERIZATION OF A NOVEL MEGALOCYTIVIRUS LINEAGE FROM ARCHIVED ORNAMENTAL FISH SAMPLES The family Iridoviridae is composed of five genera of large double stranded DNA viruses that infect arthropods (Chloriridovirus and Iridovirus ) or ectothermic vertebrates (Lymphocystivirus Ranavirus Megalocytivirus ) (Chinchar et al. 2009). Megalocytiviruses (MCVs) display the stereotypical iridovirus virion architecture including an electron dense nucleocapsid with icosahedral symmetry ranging in size from 120 200 nm in diameter (Chinchar et al. 2009). Infectious Spleen and Kidney Necrosis Virus (ISKNV) originally reported from mandarin fish Siniperca chuatsi cultured in China (He et al. 2000, 2001), represents the type species and sole member in the genus Megalocytivirus (Chinchar et al. 2009). Phylogenetic analyses based on the major capsid protein and ATPase genes have revealed 3 MCV genotypes : the red seabream irido virus (RSIV) genotype that includes strains from marine fishes in Japan, Korea, China, and SE Asia; the ISKNV g enotype that includes strains from Chinese mandarin fish and ornamental fishes cultivated in Southeas t (SE ) Asia ; and the turbot reddish body iri dovirus (TRBIV) genotype that includes strains from Asian flatfish es (Figure 4 6, Table 1 1) (Do et al. 2005, Shi et al. 2010). The MCV genotypes have each been subdivided into two separate clades. The second clade of the TRBIV genotype was recently charac terized from material derived from: 1) MCV outbreaks in freshwater ornamental fishes from the late 1980s through the early 1990s ( Go et al. 2016) and 2) a 2008 MCV outbreak involving rock bream Oplegnathus fasciatus fingerlings recently imported in to Taiwan from Korea ( Huang et al. 2011). The recent characterization of genetically divergent MCVs from three spine d stickleback Gasterosteus aculeatu s (Waltzek et al. 2012) and barramundi Lates calcarifer (de Groof et al. 2015) has led some authors to propose additional species in the genus Megalocytivirus

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32 Similar to some lymphocystiviruses and ranaviruses MCV s lack host specificity infecting a range of tropical fishes from both freshwater and marine environments (Waltzek et al. 2012, Kawato et al. 2017). Since the first suspected case of MCV infection in ram cichlids Mikrogeophagus ramirezi (Leibovitz & Riis 1980) MCVs have been detected in > 125 fish species across 11 orders and 44 families ( Yanong & Waltzek 2016, Koda & Waltzek unpublished data ). They induce lethal systemic diseases negatively impacting both food fish and ornamental aquaculture industries (Go et al. 2016, Kawato et al. 2017, Yanong & Waltzek 2016). RSIV has long been recognized as an important threat to Asian mariculture and is listed b y the World Organizat ion for Animal Health as a notifiable disease (OIE, 2016b). The use of a formalin inactivated vaccine has reduced the impact of RSIV disease on Japanese mariculture; however, its economic viability and effectiveness within ornamental aquaculture has not been established (Nakajima et al. 1997, 1998, Kawato et al. 2017). Epidemiological evidence and experimental studies suggest MCV s are transmitted horizontally through cohabitati on (He et al. 2002, Go & Whittington 2006). Megalocytivirus outbreaks are typified by high morbidity and varying degrees of mortality that can approach 100% in severe cases Experimental challeng e studies for each MCV genotype (i.e., ISKNV, RSIV, TRBIV) have revealed no clinical signs of disease a t cooler water treatments (<20C) as compared to warmer water treatments in which moribund fish were observed resulting in up to 100% cumulative mortality (He at al. 2002, Nakajima et al. 2002, Wang et al. 2003, Oh et al. 2006, Jun et al. 2009, Subramaniam et al. 2012). However, PCR detection of MCV DNA at the cooler water treatments suggests fish may become infected Affected fish exhibit nonspecific clinical signs includ ing anorexia, lethargy, increased respiration gill pallor, darkened coloration, whit e feces, and internal/external hemorrhag es (Yanong & Waltzek 2016). Although notoriously

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33 difficult to isolate in cultured cells, MCVs induce pathognomonic histopatholog ical lesions within hematopoietic organs (e.g., anterior kidney and spleen ), the submucosa of gastrointestinal tract, and others (Gibson -Kueh et al. 2003, Weber et al. 2009, Yanong & Waltzek 2016). Affected cells become cytomegalic displaying pronounced basophilic cytoplasmic inclusions. An indirect fluorescent antibody test has been developed as a confirmatory diagnostic method for RSIV and ISKNV from infected cell cultures or impression smears (OIE 2016a ). Conventional PCR assays can be used to rapidly confirm RSIV and ISKNV from infected tissues (spleen ) or cultures d isplaying MCV cytopathi c effect (i.e., enlarged, rounded, refractile cells) ( OIE 2016a ). In this study, we performed phylogenomic analyses to characteri ze a novel MCV lineage isolated from freshwater ornamental fishes (Go et al. 2016). Additionally, w e compared the in vitro growth characteristics, microscopic pathology, and ultrastructural pathology of the novel lineage to previously reported MCVs.

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34 CHAPTER 3 METHODS Archived S amples In 1991, moribund juvenile South American cichlids (oscar Astronotus ocellatus and keyhole cichlid Cleith r acara maronii ) from a commercial retail supplier in California were submitted to the Fish Health Laboratory (FHL) in Davis, CA for virological and histopathological evaluation (Go et al. 2016). Both species h ad been recently purchased from a local wholesaler/import facility and were maintained in separate 40 l display tanks prior to exhibiting lethargy, listlessness, pallor, and elevated mortality. An archived MCV isolate obtained from infected oscar tissues (Go et al. 2016), known hereafter as the South American cichlid iridovirus (SACIV ), was shipped on dry ice from the FHL to the Wildlife and Aquatic Veterinary Disease Laboratory (WAVDL) in Gainesville, FL. In addition, a second archived MCV isolate grown i n tilapia heart cells derived from moribund Florida farm raised three spot gourami Trichopodus trichopterus during epizootics in 19911992 (Fraser et al. 1993), hereafter referred to as the three spot gourami iridovirus (TSGIV), was transported on dry ice from the Bronson Animal Disease Diagnostic Laboratory in Kissimmee, FL to the WAVDL. The in vitro growth characteristics and viral genomic sequencing of the SACIV and TSGIV isolates were carried out at WAVDL Transmission electron microscopy of infected c ultured cells was performed at the University of Texas Medical Branch Electron Microscopy Laboratory (UTMB EML). The histopathological interpretation was carried out at the FHL and the Connecticut Veterinary Medical Diagnostic Laboratory in Storrs, CT. Cel l Culture and V irus E nrichment The SACIV and TSGIV isolates were inoculated onto confluent monolayers of the grunt fin (GF) cell s maintained in L15 media with 10% fetal bovine serum ( FBS ) and 1% HEPES at

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35 28C. The infected cells were monitored daily for cy topathic effect (CPE) for 14 days post inoculation Four 175 cm2 flasks of g runt fin cells displaying extensive CPE were harvested and subjected to three rounds of freez e/ thawing prior to clarifi cation of the supernatant by centrifugation at 5,520 g for 20 min at 4 C. Pelleted virus was obtained by centrifugation of the clarified supernatant at 100,000 x g for 1.25 hr at 4 C. The viral pellet was resuspended in 360 l of ATL buffer prior to extraction of viral genomic DNA (see below). Transmission E lectron M icroscopy Infected 75 cm2 flasks of GF cells displaying cytopathic effect were fixed in 15 mL of modified (2P+2G) Karnovskys fixative at room temperature for 15 minutes. After fixation, the supernatant and cells were transferred into a 15 mL conical tube and clarified at 3 000 x g for 10 minutes at 4C. The fixative and m edia was then pipetted off and the GF pellet was resuspended in 1 mL cacodylate buffer and stored at 4C until be i ng shipped to the UTMB EML Histopathology Ten separa te juvenile oscars from the 1991 outbreak were fixed in 10% neutral buffered formalin transected mid sagittal and embedded into paraffin 12 fish per block. S ections were for microscopic examination. DNA E xtraction For FFPE tissues, 50 m sections were cut from the blocks using a new microtome blade for each sample. Qiagen deparaffinization solution, ATL buffer, and proteinase K were then added to the samples and incubated at 56C overnight before extraction of DNA w as carried out using a DNA FFPE Tissue Kit (Qiagen) according to the manufacturers recommendations.

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36 DNA extraction from cell culture supernatant (SACIV) and enriched virus suspended in ATL buffer (TSGIV) was carried out using a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturers protocol for cell culture. Complete G enome Sequencing and A ssembly DNA librar ies were created using a Nextera XT DNA Kit (Illumina) for SACIV and a TruS eq Kit (Illumina) for TSGIV and sequenced using a v3 chemistry 600 cycle kit on a MiSeq platform (Illumina). De novo assembl ies of the paired end reads was performed in SPAdes 3.5.0 (Bankevich et al. 2012). The quality of the TSGIV and SACIV assembl ies w ere a ssessed by mapping the reads back to the consensus sequences in Bowtie 2 2.1.0 (Langmead & Salz berg 2012) and visually inspecting the alignment s in Tablet 1.14.10.20 (Milne et al. 2010) Genome A nnotation and P hylogenetic A nalysis Putative open reading frames (ORFs) for SACIV and TSGIV genome s were predicted usin g GeneMarkS (http://exon.biology.gatech.edu/ ) (Besemer et al. 2001) restricting the search to viral sequences. Additional criteria for annotating ORFs were : 1) larger than > 120 nucleotides 2) not overlapping with another ORF by more than 25%, 3) in the case of overlapping ORF s only the larger ORF was annotated. The gene functions were predicted based on BLAST p searches against the National Center for Biotechnology Information ( NCBI) GenBank non redundant protein sequence database and NCBI Conserved Domains Database. The 26 Iridoviridae core genes (Eaton et al. 2007) were used to construct phylogenetic trees for 47 iridoviruses including SACIV and TSGIV (Table 31). The 54 Megalocytivirus core genes were used to construct phylogenetic trees for ten MCVs including SACIV and TSGIV (Table 32). The amino acid (AA) sequence alignments for each gene were performed in MAFFT 7 using default parameters (Katoh & Standley 2013) and concatenated using Geneious 10.0.2 (https://www.geneious.com ) (Kearse et al. 2012). M aximum L ikelihood phylogenetic trees w ere

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37 constructed using IQ T REE web server (http://iqtree.cibiv.univie.ac.at/ ) (Trifinopoulos et al. 2016) using default parameters and 1000 bootstraps to determine node support PCR Detection of Megalocytiviruses To ensure the SACIV DNA was detectable within FFPE oscar tissues, a panMCV primer set was designed to amplify all MCV g enotypes (Table 3 3). The primers were designed to amplify <200 bp given DNA from FFPE tissues are typically fragmented ( Green & Sambrook 2012). The 26 iridovirus core genes were extracted from the annotated genomic sequences generated in this study for SACIV and TSGIV as well as for 8 fully sequenced MCVs available in GenBank (Table 3 1). For each gene, the AA sequences were aligned in MAFFT 7 (Katoh & Toh 2008) using default settings and the resulting alignments were imported into Geneious 10.0.2 (https://www.geneious.com, (Kearse et al. 2012)) to generate consensus sequence s with the threshold set to 100%. The consensus sequences were imported into Primer3 (http://biotools.umassmed.edu/bioapps/primer3_www.cgi ) to design pan-MCV primers with the following characteristics: conserved primer binding sites <200 bp apart with a hypervariable region in between to f acilitate MCV genotype discrimination by Sanger sequencing (Figure 4 7). Reaction volumes for the pan MCV PCR w e re 5 ed of 0.25 l of Platinum Taq DNA Polymerase (Invitrogen), 5.0 l of 10 PCR Buffer, 2.0 l of 50 mM MgCl2, 1.0 l of 10 mM dNTPs, 2.5 l of 20 M forward and reverse primers, 32.25 l of molecular grade water and 4.5 l of DNA template. An initial denaturation step of 95C for 5 min was followed by 40 cycles of a 9 4C denaturation step, a 55C annealing step, and a 72C extension step, each step run for 30 seconds and a final extension step at 72C for 5 min. PCR products were subjected to electrophoresis in 1% agarose gel Amplified products were purified using a QIAquick gel extraction kit (Qiagen). The concentration of purified DNA w as quantified fluorometrically

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38 using a Qubit 3.0 Fluorometer and dsDNA BR Assay Kit (Life Technologies). Purified DNA was sequenced in both directions on an ABI 3130 plat form (Applied Biosystems). DNA from the SACIV (TRBIV Clade 2) and TSGIV (unknown genotype) isolates were tested against the pan MCV PCR. In addition, the PCR assay was tested against DNA extracted from 1) freshly frozen tissues: splenic tissue of a moribund Florida pompano (Trichinotus carolinus ) infected with RSIV (Waltzek & Yanong, unpublished data) and hepatic tissue from a moribund ram cichlid ( Mikrogeophagus ramirezi ) infected with ISKNV (Waltzek, unpublished data) and 2) FFPE tissues: TRBIV Clade 2infected Oscar, ISKNV infected Nile tilap ia (Oreochromis niloticus ) (Subramaniam et al. 2016), and RSIV infected Florida pompano.

PAGE 39

39 Table 3 1. GenBank accession numbers for the full genome sequences of iridoviruses used in the 26 core gene phylogenetic analysi s Species name (Virus abbreviation) GenBank Acc. No. Invertebrate iridescent virus (IIV 6 ) AF303741 Armadillidium vulgare iridescent virus (IIV 31 ) HF920637 Anopheles mimivirus iridovirus (AMIV) KF938901 Invertebrate iridovirus 22 (IIV 22) HF920633 Invertebrate iridovirus 22a (IIV 22a) HF920634 Aedes taeniorhynchus iridescent virus (IIV 3) DQ643392 Invertebrate iridescent virus 30 (IIV 30) HF920636 Wiseana iridescent virus (IIV 9) GQ918152 Invertebrate iridovirus 25 (IIV 25) HF920635 European catfish virus (ECV) KT989885 European sheatfish virus (ESV) JQ724856 Testudo hermanni ranavirus (CH8/96) KP266741 Andrias davidianus ranavirus (ADRV) KC865735 Common midwife toad ranavirus (CMTV/2013/NL VB) KP056312 Pike perch iridovirus (PPIV) KX574 341 Common midwife toad ranavirus (CMTV/2008/E M) JQ231222 Frog virus 3 (FV3) AY548484 Frog virus 3 isolate SSME (SSME) KF175144 Rana grylio iridovirus (RGV) JQ654586 Soft shelled turtle iridovirus (STIV) EU627010 German gecko ranavirus (GGRV) KP266742 Bohle iridovirus (BIV) KX185156 Tiger frog virus (TFV) AF389451 Tortoise ranavirus isolate (ToRV 1) KP266743 Cod iridovirus (CoIV) KX574342 Ranavirus maximus (Rmax) KX574434 Ambystoma tigrinum stebbensi virus (ATV) AY150217 Epizootic haematopoietic necrosis virus (EHNV) FJ433873 Short finned eel ranavirus (SERV) KX353311 Doctor fish virus (DFV) Unpublished data Guppy virus 6 (GV6) Unpublished data Largemouth bass virus (LMBV) Unpublished data Grouper iridovirus (GIV) AY666015 Singapore grouper iridovirus (SGIV) AY521625 Lymphocystis disease 1 (LCDV 1) L63545 Lymphocystis disease virus isolate China (LCDV C) AY380826 Lymphocystis disease virus (LCDV Sa) PRJEB12506 Scale drop disease virus (SDDV) KR139659 Red seabream iridovirus (RSIV) BD143114 Orange spotted grouper iridovirus (OSGIV) AY894343 Giant seaperch iridovirus (GSIV K1) KT804738 Rock bream iridovirus (RBIV) AY532606

PAGE 40

40 Table 3 1. Continued Species name (Virus abbreviation) GenBank Acc. No. Infectious spleen and kidney necrosis virus (ISKNV) AF371960 Large yellow croaker iridovirus (LYCIV) AY779031 Turbot reddish body iridovirus (TRBIV) GQ273492 South American cichlid iridovirus (SACIV) This study Three spot gourami iridovirus (TSGIV) This study

PAGE 41

41 Table 3 2. Genome summary of the 10 m egalocytivirus genomes used in the 26 and 54 core gene analyses Virus (Abbreviation) Host MCV Clade Country of Origin Size (bp) No. ORFs % G+C References Scale drop disease virus (SDDV) Giant seaperch ( Lates calcarifer ) SDDV Singapore 124,244 129 36.9 Gibson Kueh, 2012 Infectious spleen and kidney necrosis virus (ISKNV) Mandarin fish (Siniperca chuatsi ) ISKNV Clade 1 China 111,362 124 54.8 He et al., 2001 Turbot reddish body iridovirus (TRBIV ) Turbot ( Scophthalmus maximus ) TRBIV Clade 1 China 110,104 115 55.0 Shi et al., 2010 South American cichlid iridovirus (SACIV ) Keyhole cichlid (Cleithracara maronii ) TRBIV Clade 2 Unknown 111,347 116 56.3 Go et al. 2016 Three spot gourami iridovirus (TSGIV ) Three spot gourami (Trichopodus trichopterus ) TRBIV Clade 2 United States 111,591 116 56.5 Fraser et al., 1993 Large yellow croaker iridovirus (LYCIV ) Large yellow croaker (Larimichthys crocea ) RSIV Clade 1 China 111,767 126 54.0 Ao &Chen, 2006 Red seabream iridovirus (RSIV ) Red seabream ( Pagrus major ) RSIV Clade 1 Japan 112,414 93 53.0 Kurita et al., 2002 Rock bream iridovirus (RBIV ) Rock bream (Oplegnathus fasciatus ) RSIV Clade 2 Korea 112,080 118 53.0 Do et al., 2004 Giant seaperch iridovirus (GSIV ) Giant seaperch ( Lates calcariferi) RSIV Clade 2 Taiwan 112,565 135 53.0 Wen & Hong, 2016 Orange spotted grouper iridovirus (OSGIV ) Orange spotted grouper (Ephinephelus coioides ) RSIV Clade 2 China 112,636 121 54.0 Lu et al., 2005

PAGE 42

42 Table 33. Pan MCV primer set targeting the transmembrane amino acid transporter protein (ORF 1L in ISKNV GenBank accession no. AF371960). The listed amp licon size includes the primers. Primer Pairs Primer Sequence (5 3) Gene of Interest Amplicon Size Reference SACIV1L F SACIV1L R CAACCCCACGTCCAAAGA ACATTGCTGGGGCATGTG Transmembrane amino acid transporter protein 173 This study

PAGE 43

43 CHAPTER 4 RESULTS C ell C ulture and Virus Enrichment Grunt fin (GF) cells displayed CPE (enlargement, rounding, refractility ) within 96 hr of being infected with the TSGIV isolate (Figure 4 1). Complete CPE was o bserved by day 10 pi in which most cells were affected but remained attached to the monolayer (Fi gure 4 1). No CPE was observed following infection of GF cells with the SACIV isolate. Transmission E lectron M icroscopy Large numbers of unenveloped, hexagonal viral particles were observed within the cytoplasm of GF cell s (Figure 4 2 A ). Vir al particles observed within the cytoplasm of the cell displayed electron dense cores, medium electron densities and electron lucent cores (Figure 4 2B ). The mean diameter ( SD) of the viral particles from opposite vertices was 144 10 nm (n = 20) and 120 7 nm (n= 20) from opposite faces. Although no enveloped virions were observed within cells or extracellularly, virions were observed within cellular blebs (Figure 4 -2). Histopathology Of the 10 fish sections examined, six displayed cytomegalic cells characterized by basophilic granular intracytoplasmic inclusions within various organs that were especially prominent in the anterior kidney spleen, and intestinal submucosa (Figure 43). In one section, approximately 75% of the lymphomyeloid cells of the anterior kidney were affected (Figure 4 3A,B ). Cytomegalic cells displaying basophilic inclusions were also noted within the pulp cavity of the teeth oropharyngeal submucosa, stomach, posterior kidney (renal interstit i um and glomeruli), branc hial lamellar capillaries, pseudobranch rete mirabile, spleen, pancreas, gonadal interstitium, coelomic cavity membrane, skeletal and cardiac muscle, cartilage, and connective tissue regardless of location O f the four oscar sections not displaying micros copic lesions

PAGE 44

44 consistent with a MCV infection one had a light epitheliocystis infection with inclusions in the branchial epithelium. Another fish section that showed a light MCV infection of megalocytic cells in the anterior kidney also displayed large gr anulomas. Complete Genome Sequencing and Assembly For SACIV, the de novo assembly of 7,721,890 paired end reads produced a contiguous consensus sequence of 111,347 bp with % G+C content of 56.3. A total of 2,421,194 reads (31.35%) aligned at a mean coverage of 4,931 reads/nucleotide. For TSGIV, the de novo assembly of 3,229,544 paired end reads produced a consensus sequence of 111,591 bp and %G+C content of 56.5. A total of 445,855 reads (13.81%) aligned at a mean coverage of 1,178 reads/nucleotide (Table 3 2). Genome A nnotation and P hylogenetic A nalysis Both genomes displayed 116 open reading frames (T able 4 1, 42). The genomes of the SACIV and TSGIV shared 99.9% nucleic acid sequence identity. Phylogenetic analyses based on the MCP gene, 26 core genes, and 54 core genes revealed SACIV and TSGIV form a well supported clade (95100% bootstrap support ) as the sister group to TRBIV (Figure s 44, 4 5, 4 6). Both SACIV and TSGIV possess a unique truncated paralog of the m ajor capsid protein gene (ORF 6L) located immediately upstream of the full length parent gen e (ORF 7L). PCR Detection of MCVs Primers against ORF 1L met the specified criteria generating a 173 bp amplicon (including primers) (Figure 47). A single band was observed for the MCV isolate DNA (SACIV and TSGIV), MCV infected tissue DNA (ISKNV and RSIV), and FFPE MCV infected tissue DNA (ISKNV, RSIV, TRBIV). Sequencing of amplicons from all samples confirmed the expected genotype including TRBIV Clade 2 for the oscar (isolate and FFPE) and TSGIV (isolate) samples.

PAGE 45

45 Figure 4 1. Microscopic examination of in vitro growth characteristics of TSGIV in the GF cell line at 28 C. (A) Control flask on day 4 post infection (pi). (B) I nfected flask on day 4 pi showing rounded, enlarged, refractile cells. (C) control flask on day 10 pi. (D) Infected flask on day 10 pi showing aggregates of rounded, enlarged, refractile cells Scale bar is 50 m.

PAGE 46

46 Figure 4 2. (A) Transmission electron photomicrograph of a GF cell infected with TSGIV displaying nonenveloped, hexagonal, virus particles (120 144 nm) having an electron dense core within the cytoplasm Smaller numbers of virus particles were also observed within the cytoplasm of cellular blebs (see arrow). Scal e bar is 1 m. (B) Higher magnification of the virus particles showing both mature virions with electron dense cores, medium electron density and electron lucent viral particles. Scale bar is 200 nm.

PAGE 47

47 Figure 4 3. Microscopic examination of megalocytic cells (arrows) a nd lesions in infected o scar Astronus ocellatus. H&E stain. (A,B) Anterior kidney. (C,D) Posterior kidney. (E,F) Intestinal submucosa. Scale bar is 50 m.

PAGE 48

48 Table 4 1 Predicted open reading fra mes for the South American cichlid iridovirus (SACIV) genome ORF Position ( nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 1L 1 1,137 379 Transmembrane amino acid transporter protein Transmembrane amino acid transporter [ TRBIV ] 0 ADE34346 2L 1,107 1,562 152 DNA dependent RNA polymerase subunit H DNA dependent RNA polymerase subunit H like protein [ ISKNV ] 1E 98 AMM04413 3R 1,688 1,945 258 Caspace recruitment domain containing protein Caspace recruitment domain containing protein [GSIV] 3E 43 AMM72786 4L 2,012 2,491 160 Hypothetical protein ORF4L [ TRBIV ] 1E 95 ADE34349 5L** 2,838 3,584 249 NLI interacting factor like phosphatase Catalytic domain of ctd like phosphatase [ TRBIV ] 1E 180 ADE34350 6L 3,723 4,043 104 Major capsid protein MCP [ KFIV ] 2E 53 AAT48718 7L* 4,223 5,584 454 Major capsid protein Major capsid protein [ RBIV ] 0 AEI85911 8L** 5,601 7,058 486 Lipid membrane protein ORF007L [ ISKNV ] 0 NP_612229 9R** 7,131 8,681 517 Hypothetical protein ORF8R [ TRBIV ] 0 ADE34353 10R 8,635 9,003 123 Hypothetical protein ORF012L [ ISKNV ] 5E 38 AMM04525 11L 9,097 9,489 131 Hypothetical protein ORF10L [ TRBIV ] 1E 93 ADE34355 12L 9,489 9,842 118 Hypothetical protein ORF011L [ ISKNV ] 8E 45 NP_612233 13R* 9,765 10,097 111 RING finger containing E3 ubiquitin ligase RING finger containing ubiquitin ligase [ TRBIV ] 3E 75 ADE34357 14R** 10,104 11,501 466 Serine/threonine protein kinase Serine/threonine protein kinase [ TRBIV ] 0 ADE34358 15R 11,756 12,727 324 Hypothetical protein ORF14R [ TRBIV ] 0 ADE34359 16R* 12,733 13,518 262 Hypothetical protein ORF15R [ TRBIV ] 3E 175 ADE34360

PAGE 49

49 Table 4 1. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 17L* 13,576 14,163 196 Hypothetical protein ORF 324R [ RSIV ] 5E 132 BAK14267 18 L 14,178 14,507 110 Hypothetical protein ORF17L [ TRBIV ] 2E 25 ADE34362 19L 14,767 14,958 64 Hypothetical protein ORF 318R [RSIV] 6E 29 BAK14265 20R** 15,024 17,870 949 DNA polymerase DNA polymerase [ TRBIV ] 0 ADE34365 21R 17,917 18,450 178 Hypothetical protein ORF024R [ GSIV ] 7E 113 AMM72671 22L* 18,434 20,008 525 Macro domain containing protein Putative phosphatase [ TRBIV ] 0 ADE34368 23R* 20,080 22,920 947 Laminin type epidermal growth factor Laminin like protein [ OFIV ] 2E 158 AAT76907 24 R** 23,029 23,967 313 Ribonucleotide reductase beta subunit Ribonucleotide reductase small chain [ TRBIV ] 0 ADE34370 25 L 24,384 24,698 105 Hypothetical protein Hypothetical protein 32HC_45 [Mycobacterium phage 32HC] 0.5628 YP_009009516 26 L* 24,727 25,050 108 Hypothetical protein ORF26L [ TRBIV ] 1E 49 ADE34371 27 L** 25,075 25,971 299 Flap endonuclease 1 DNA repair protein RAD2 [ TRBIV ] 0 ADE34372 28 L** 25,988 29,494 1169 DNA dependent RNA polymerase alpha subunit Largest subunit of the DNAdependent RNA polymerase [ TRBIV ] 0 ADE34373 29 L** 29,501 29,764 88 Transcription factor S II Elongation factor [ RSIV ] 2E 49 BAK14256

PAGE 50

50 Table 4 1. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 30 L 29,790 30,326 179 Hypothetical protein ORF037L [ ISKNV ] 2E 119 AMM04443 31 R** 30,380 30,946 189 Deoxyribonucleoside kinase Putative thymidine kinase [ ISKNV ] 3E 118 NP_612254 32 L 30,952 31,854 301 Hypothetical protein ORF32L [ TRBIV ] 0 ADE34377 33 R** 31,955 35,080 1042 DNA dependent RNA polymerase beta subunit DNA directed RNA polymerase II second largest subunit like protein [ TRBIV ] 0 ADE34378 34 L* 35,173 36,285 371 Hypothetical protein ORF34L [ TRBIV ] 0 ADE34379 35 R* 36,394 37,419 342 Hypothetical protein ORF35R [ TRBIV ] 0 ADE34380 36 L 37,471 38,820 450 Hypothetical protein ORF36L [ TRBIV ] 0 ADE34381 37 L* 38,829 40,262 478 Hypothetical protein ORF37L [ TRBIV ] 0 ADE34382 38 R 40,239 41,174 312 Hypothetical protein ORF38R [ TRBIV ] 0 ADE34383 39 L 41,167 42,315 383 Hypothetical protein ORF39L [ TRBIV ] 0 ADE34384 40 L 42,317 43,651 445 Hypothetical protein ORF40L [ TRBIV ] 0 ADE34385 41 R 43,666 44,247 194 Hypothetical protein ORF41R [ TRBIV ] 3E 117 ADE34386 42 L** 44,331 44,693 121 Erv1/Alr family protein Thiol oxidoreductase [ TRBIV] 3E 73 ADE34387 43 L 44,696 45,496 267 Hypothetical protein ORF43L [ TRBIV ] 0 ADE34388

PAGE 51

51 Table 4 1. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 44 L 45,502 46,416 305 Hypothetical protein ORF44L [ TRBIV ] 0 ADE34389 45 L* 46,410 47,093 228 Cytosine DNA methyltransferase Cytosine DNA methyltransferase [ TRBIV ] 8E 169 ADE34390 46 R* 47,253 47,516 88 Hypothetical protein ORF46R [ TRBIV ] 3E 60 ADE34391 47 R* 47,513 47,860 116 Vascular endothelial growth factor ORF47R [ TRBIV ] 7E 66 ADE34392 48 R 47,876 48,046 57 Hypothetical protein ORF049R [ISKNV] 1E 37 NP_612271 49 L* 48,106 48,534 143 Hypothetical protein ORF48L [ TRBIV ] 2E 90 ADE34393 50 L 48,765 49,265 167 Hypothetical protein ORF49L [ TRBIV ] 1E 103 ADE34394 51 R 49,267 49,482 72 Hypothetical protein ORF053R [ ISKNV ] 5E 42 NP_612275 52 L* 49,495 50,424 310 2 cysteine adaptor domain containing protein ORF 111R [ RSIV ] 0 BAK14232 53 L** 50,447 51,382 312 2 cysteine adaptor domain containing protein ORF52L [ TRBIV ] 0 ADE34397 54 L** 51,393 52,040 216 Hypothetical protein ORF53L [ TRBIV ] 7E 132 ADE34398 55 L 52,047 52,307 87 Hypothetical protein ORF057L [ ISKNV ] 2E 57 NP_612279 56 L 52,675 53,190 172 Hypothetical protein ORF55L [TRBIV] 1E 116 ADE34400 57 L** 53,261 54,064 268 Replication factor Putative replication factor [ TRBIV ] 0 ADE34401

PAGE 52

52 Table 4 1. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 58 L* 54,061 57,666 1202 Hypothetical protein ORF57L [ TRBIV ] 0 ADE34402 59 L 57,727 58,095 123 Hypothetical protein ORF_021L [ SDDV ] 6E 06 YP_009163782 60 L** 58,102 60,750 883 SNF2 family helicase SNF2 family helicase [ TRBIV ] 0 ADE34403 61L* 60,793 62,265 491 mRNA capping enzyme mRNA capping enzyme [ TRBIV ] 0 ADE34404 62 L 62,307 62,762 152 RING finger containing E3 ubiquitin ligase ORF065L [ ISKNV ] 5E 91 NP_612287 63 L 62,827 63,870 348 RING finger containing E3 ubiquitin ligase RING finger containing E3 ubiquitin ligase [ TRBIV ] 0 ADE34406 64 L 64,075 64,713 213 Hypothetical protein H ypothetical protein ORF 037R [ RSIV ] 2E 104 BAK14221 65 L 64,685 66,127 481 Hypothetical protein ORF63L [ TRBIV ] 0 ADE34408 66 L 66,183 66,839 219 Hypothetical protein Hypothetical protein ORF 029R [ RSIV ] 3E 121 BAK14219 67 L 66,954 68,564 537 Hypothetical protein ORF65L [ TRBIV ] 0 ADE34410 68 R 68,605 69,036 144 Hypothetical protein ORF66R [ TRBIV ] 3E 96 ADE34411 69 R 69,085 70,107 341 Hypothetical protein ORF67R [ TRBIV ] 0 ADE34412 70 L 70,116 70,388 91 Hypothetical protein ORF010R [RSIV] 1E 54 BAK14214 71 L** 70,390 73,560 1057 Hypothetical protein ORF69L [ TRBIV ] 0 ADE34414

PAGE 53

53 Table 4 1. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 72 R* 73,222 74,718 499 Ankyrin repeat containing protein Ankyrin repeat containing protein [ TRBIV ] 0 ADE34415 73 R* 74,715 75,179 155 Hypothetical protein ORF71R [ TRBIV ] 9E 95 ADE34416 74 R 75,334 75,975 214 US22 protein Hypothetical protein ORF 632L [ RSIV ] 2E 115 BAK14325 75 R* 75,962 76,465 168 Hypothetical protein ORF74R [ TRBIV ] 1E 101 ADE34419 76 L* 76,506 77,612 369 Hypothetical protein ORF75L [ TRBIV ] 0 ADE34420 77 R 77,484 78,020 179 Hypothetical protein ORF089R [ ISKNV ] 2E 95 AMM04489 78 L 78,058 79,413 452 Hypothetical protein ORF77L [ TRBIV ] 0 ADE34421 79 R 79,440 79,967 176 Hypothetical protein ORF78R [ TRBIV ] 1E 81 ADE34422 80 R** 79,964 80,428 155 Hypothetical protein ORF79R [ TRBIV ] 7E 111 ADE34423 81 R** 80,394 81,191 266 Ribonuclease III ORF80R [ TRBIV ] 0 ADE34424 82 L 81,188 81,679 164 SAP domain containing protein ORF095L [ GSIV ] 9E 93 AMM72718 83 R 81,652 83,223 524 Hypothetical protein ORF82R [ TRBIV ] 0 ADE34426 84 L** 83,204 84,223 340 Hypothetical protein Hypothetical protein ORF 575R [ RSIV ] 0 BAK14314 85 R 84,224 84,403 60 Hypothetical protein ORF84R [ TRBIV ] 8E 38 ADE34428

PAGE 54

54 Table 4 1. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 86 L* 84,400 85,329 310 Hypothetical protein ORF85L [ TRBIV ] 0 ADE34429 87 L 85,339 85,809 157 Hypothetical protein ORF86L [ TRBIV ] 2E 111 ADE34430 88 L* 85,860 87,020 387 Hypothetical protein ORF87L [ TRBIV ] 0 ADE34431 89 L** 87,028 87,765 246 Hypothetical protein ORF88L [ TRBIV ] 9E 171 ADE34432 90 L 87,771 88,256 162 Hypothetical protein ORF89R [ TRBIV ] 1E 115 ADE34433 91 L 88,304 88,627 108 RING finger containing E3 ubiquitin ligase RING finger domain containing E3 protein [ TRBIV ] 2E 71 ADE34434 92 L 88,680 89,264 195 Hypothetical protein ORF91L [ TRBIV ] 2E 109 ADE34435 93 L* 89,311 89,826 172 Hypothetical protein ORF92L [ TRBIV ] 4E 121 ADE34436 94 R 89,890 91,320 477 Ankyrin repeat containing protein Ankyrin repeat containing protein [ TRBIV ] 0 ADE34437 95 R 91,323 91,700 126 Suppressor of cytokine signaling Suppressor of cytokine signaling protein [ TRBIV ] 3E 77 ADE34438 96 R 91,735 92,511 259 Hypothetical protein Hypothetical protein ORF 522L [ RSIV ] 2E 178 BAK14303 97 R 92,513 92,884 124 Hypothetical protein ORF097R [ RBIV ] 4E 84 AAT71912 98 L 93,020 93,958 313 Hypothetical protein ORF97L [ TRBIV ] 0 ADE34441

PAGE 55

55 Table 4 1. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 99 L 94,103 94,705 201 Hypothetical protein ORF114L [ ISKNV ] 2E 125 AMM04510 100 L** 94,796 97,558 921 D5 family NTPase D5 family NTPase [ TRBIV ] 0 ADE34443 101 R 97,567 97,791 65 Hypothetical protein ORF100R [ TRBIV ] 8E 36 ADE34444 102 L 97,758 98,654 299 Tumor necrosis factor receptor associated factor Tumor necrosis factor type 2 receptorassociated protein [ TRBIV ] 0 ADE34445 103 R** 98,677 99,420 248 Proliferating cell nuclear antigen Proliferating cell nuclear antigen [ OSGIV ] 0 AAX82418 104 L 99,410 99,916 169 Hypothetical protein ORF119L [ ISKNV ] 2E 102 AMM04511 105 L** 99,955 102,534 860 Tyrosine kinase Tyrosine kinase [ TRBIV ] 0 ADE34449 106 R 102,643 102,945 101 Hypothetical protein Not available 107 R** 102,984 104,096 371 Immediate early protein ICP 46 Immediate early protein ICP 46 [ TRBIV ] 0 ADE34450 108 R 104,125 105,534 470 Hypothetical protein ORF107R [ TRBIV ] 0 ADE34451 109 L* 105,590 106,264 225 Hypothetical protein Early 31kDa protein [ TRBIV ] 4E 167 ADE34452 110 L 106,598 107,908 437 Ankyrin repeat containing protein Ankyrin repeat containing protein [ TRBIV ] 0 ADE34453 111 R 107,954 108,259 102 RING finger containing E3 ubiquitin ligase RING finger domain containing E3 protein [ TRBIV ] 3E 66 ADE34454

PAGE 56

56 Table 4 1. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 112 R* 108,213 108,794 194 Hypothetical protein ORF111R [ TRBIV ] 2E 120 ADE34455 113 L 108,795 109,433 213 Hypothetical protein ORF112R [ TRBIV ] 1E 122 ADE34456 114 R** 109,443 110,162 240 ATPase ATPase [ GSIV ] 0 AAL68653 115 L 110,134 110,514 127 Hypothetical protein H ypothetical protein ORF 407R [ RSIV ] 2E 85 BAK14284 116 L* 110,523 111,341 273 Ankyrin repeat containing protein Viral ankyrin repeat protein [ ISKNV ] 0 AEK52381 aSignificant hits based on NCBI BLASTp *54 core gene only **26 & 54 core gene Abbreviations: nt, nucleotides; aa, amino acids; ID, identity, GSIV= Giant seabass iridovirus; ISKNV = Infectious spleen and kidney necrosis virus ; OFIV = Olive flounder iridovirus ; OSGIV= Orange -spotted grouper iridovirus; RBIV= Rock bream iridovirus; RSIV= Red seabream iridovirus; SDDV= Scale drop disease virus; TRBIV= Turbot reddish body iridovirus.

PAGE 57

57 Table 4 2. Predicted open reading frames for the three spot gourami iridovirus (TSGIV) genome ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 1L 1 1,137 379 Transmembrane amino acid transporter protein Transmembrane amino acid transporter [ TRBIV ] 0 ADE34346 2L 1,107 1,562 152 DNA dependent RNA polymerase subunit H DNA dependent RNA polymerase subunit H like protein [ ISKNV ] 9E 92 AMM04413 3R 1,688 1,945 258 Caspace recruitment domain containing protein Caspace recruitment domain containing protein [GSIV] 3E 43 AMM72786 4L 2,012 2,491 160 Hypothetical protein ORF4L [ TRBIV ] 1E 95 ADE34349 5L ** 2,838 3,584 249 NLI interacting factor like phosphatase Catalytic domain of ctd like phosphatase [ TRBIV ] 2E 180 ADE34350 6 L 3,732 4,043 104 Major capsid protein MCP [ KFIV ] 2E 53 AAT48718 7 L ** 4,223 5,584 454 Major capsid protein Major capsid protein [ RBIV ] 0 AEI85911 8L** 5,601 7,058 486 Lipid membrane protein ORF007L [ ISKNV ] 0 NP_612229 9R* 7,131 8,681 517 Hypothetical protein ORF8R [ TRBIV ] 0 ADE34353 10 L 8,635 9,003 123 Hypothetical protein ORF012L [ ISKNV ] 5E 38 AMM04525 11 L 9,097 9,489 131 Hypothetical protein ORF10L [ TRBIV ] 1E 93 ADE34355 12L 9,489 9,842 118 Hypothetical protein ORF011L [ ISKNV ] 2E 44 NP_612233 13R* 9,765 10,097 111 RING finger containing E3 ubiquitin ligase RING finger containing ubiquitin ligase [ TRBIV ] 2E 74 ADE34357 14 R ** 10,104 11,501 466 Serine/threonine protein kinase Serine/threonine protein kinase [ TRBIV ] 0 ADE34358

PAGE 58

58 Table 4 2. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 15 R 11,756 12,727 324 Hypothetical protein ORF14R [ TRBIV ] 0 ADE34359 16R* 12,733 13,518 262 Hypothetical protein ORF15R [ TRBIV ] 8E 177 ADE34360 17 L 13,756 14,163 196 Hypothetical protein Hypothetical protein ORF 324R [ RSIV ] 6E 132 BAK14267 18L 14,178 14,504 109 Hypothetical protein ORF17L [ TRBIV ] 1E 25 ADE34362 19L 14,765 14,956 64 Hypothetical protein ORF318R [RSIV] 6E 28 BAK14265 20R** 15,022 17,868 949 DNA polymerase DNA polymerase [ TRBIV ] 0 ADE34365 21 R 17,915 18,448 178 Hypothetical protein ORF024R [ GSIV ] 7E 113 AMM72671 22 L 18,432 20,006 525 Macro domain containing protein Putative phosphatase [ TRBIV ] 0 ADE34368 23R* 20,079 22,166 696 Laminin type epidermal growth factor Laminin EGF repeat including protein [ RSIV ] 0 BAK14261 24R** 23,501 24,439 313 Ribonucleotide reductase beta subunit Ribonucleotide reductase small chain [ TRBIV ] 0 ADE34370 25 L 24,689 24,823 45 Hypothetical protein Not available Not available 26L* 24,852 25,175 108 Hypothetical protein ORF26L [ TRBIV ] 9E 46 ADE34371 27L** 25,200 26,096 299 Flap endonuclease 1 DNA repair protein RAD2 [ TRBIV ] 0 ADE34372

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59 Table 4 2. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 28L** 26,113 29,619 1169 DNA dependent RNA polymerase alpha subunit Largest subunit of the DNAdependent RNA polymerase [ TRBIV ] 0 ADE34373 29 L ** 29,626 29,829 68 Transcription factor S II Transcription elongation factor SII [ OSGIV ] 1E 43 AAX82341 30L 29,914 30,450 179 Hypothetical protein ORF037L [ ISKNV ] 3E 117 AMM04443 31 R ** 30,504 31,070 189 Deoxyribonucleoside kinase Putative thymidine kinase [ ISKNV ] 5E 116 NP_612254 32 L 31,076 31,978 301 Hypothetical protein ORF32L [ TRBIV ] 0 ADE34377 33R** 32,079 35,204 1042 DNA dependent RNA polymerase beta subunit DNA directed RNA polymerase II second largest subunit like protein [ TRBIV ] 0 ADE34378 34L* 35,297 36,409 371 Hypothetical protein ORF34L [ TRBIV ] 0 ADE34379 35R* 36,51 8 37,543 342 Hypothetical protein ORF35R [ TRBIV ] 0 ADE34380 36 L 37,595 38,944 450 Hypothetical protein ORF36L [ TRBIV ] 0 ADE34381 37L* 38,953 40,386 478 Hypothetical protein ORF37L [ TRBIV ] 0 ADE34382 38R 40,363 41,298 312 Hypothetical protein ORF38R [ TRBIV ] 0 ADE34383 39 L 41,291 42,439 383 Hypothetical protein ORF39L [ TRBIV ] 0 ADE34384

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60 Table 4 2. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 40 L 42,441 43,775 445 Hypothetical protein ORF40L [ TRBIV ] 0 ADE34385 41R 43,790 44,371 194 Hypothetical protein ORF41R [ TRBIV ] 5E 115 ADE34386 42L** 44,455 44,817 121 Erv1/Alr family protein Thiol oxidoreductase [TRBIV ] 5E 71 ADE34387 43L 44,820 45,620 267 Hypothetical protein ORF43L [ TRBIV ] 0 ADE34388 44 L 45,626 46,540 305 Hypothetical protein ORF44L [ TRBIV ] 0 ADE34389 45 L 46,534 47,217 228 Cytosine DNA methyltransferase Cytosine DNA methyltransferase [ TRBIV ] 2E 165 BAK14240 46 R 47,377 47,640 88 Hypothetical protein ORF46R [ TRBIV ] 2E 58 ADE34391 47R* 47,637 47,984 116 Vascular endothelial growth factor ORF47R [ TRBIV ] 6E 64 ADE34392 48R 48,000 48,170 57 Hypothetical protein ORF049R [ISKNV] 1E 37 NP_612271 49 L 48,230 48,658 143 Hypothetical protein ORF48L [ TRBIV ] 1E 88 ADE34393 50 L 48,889 49,389 167 Hypothetical protein ORF49L [ TRBIV ] 9E 102 ADE34394 51R 49,391 49,606 72 Hypothetical protein ORF053R [ ISKNV ] 4E 40 NP_612275 52 L 49,619 50,548 310 2 cysteine adaptor domain containing protein Hypothetical protein ORF 111R [ RSIV ] 0 BAK14232 53 L ** 50,571 51,506 312 2 cysteine adaptor domain containing protein ORF52L [ TRBIV ] 0 ADE34397

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61 Table 4 2. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 54 L ** 51,517 52,164 216 Hypothetical protein ORF53L [ TRBIV ] 6E 130 ADE34398 55 L 52,171 52,431 87 Hypothetical protein ORF057L [ ISKNV ] 1E 55 NP_612279 56 L 52,799 53,314 172 Hypothetical protein ORF55L [TRBIV] 1E 116 ADE34400 57 L ** 53,385 54,188 268 Hypothetical protein Putative replication factor [ TRBIV ] 0 ADE34401 58 L 54,185 57,910 1242 Hypothetical protein ORF57L [ TRBIV ] 0 ADE34402 59 L 57,971 58,339 123 Hypothetical protein ORF_021L [ SDDV ] 0.0005 YP_009163782 60 L ** 58,346 60,994 883 SNF2 family helicase SNF2 family helicase [ TRBIV ] 0 ADE34403 61L* 61,037 62,509 491 mRNA capping enzyme mRNA capping enzyme [ TRBIV ] 0 ADE34404 62L 62,551 63,006 152 RING finger containing E3 ubiquitin ligase ORF065L [ ISKNV ] 2E 88 NP_612287 63 L 63,071 64,114 348 RING finger containing E3 ubiquitin ligase RING finger containing E3 ubiquitin ligase [ TRBIV ] 0 ADE34406 64L 64,319 64, 957 213 Hypothetical protein H ypothetical protein ORF 037R [ RSIV ] 1E 102 BAK14221 65L 64,929 66,371 481 Hypothetical protein ORF63L [ TRBIV ] 0 ADE34408 66 L 66,427 67,083 219 Hypothetical protein Hypothetical protein ORF 029R [ RSIV ] 3E 121 BAK14219 67L 67,198 68,808 537 Hypothetical protein ORF65L [ TRBIV ] 0 ADE34410

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62 Table 4 2. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 68 R 69,849 69,280 144 Hypothetical protein ORF66R [ TRBIV ] 3E 96 ADE34411 69R 69,329 70,351 341 Hypothetical protein ORF67R [ TRBIV ] 0 ADE34412 70L 70,360 70,632 91 Hypothetical protein ORF010R [RSIV] 1E 54 BAK14214 71 L ** 70,634 73,804 1057 Hypothetical protein ORF69L [ TRBIV ] 0 ADE34414 72 R 73,625 74,962 446 Ankyrin repeat containing protein Ankyrin repeat containing protein [ TRBIV ] 0 ADE34415 73 R 74,959 75,423 155 Hypothetical protein ORF71R [ TRBIV ] 9E 95 ADE34416 74R 75,578 76,219 214 US22 protein Hypothetical protein ORF 632L [RSIV ] 2E 115 BAK14325 75 R 76,206 76,709 168 Hypothetical protein ORF74R [ TRBIV ] 1E 101 ADE34419 76 L 76,750 77,856 369 Hypothetical protein ORF75L [ TRBIV ] 0 ADE34420 77 R 77,728 78,624 179 Hypothetical protein ORF089R [ ISKNV ] 2E 95 AMM04489 78 L 78,302 79,657 452 Hypothetical protein ORF77L [ TRBIV ] 0 ADE34421 79R 79,684 80,211 176 Hypothetical protein ORF78R [ TRBIV ] 1E 81 ADE34422 80R** 80,208 80,672 155 Hypothetical protein ORF79R [ TRBIV ] 7E 111 ADE34423 81R** 80,638 81,435 266 Ribonuclease III ORF80R [ TRBIV ] 0 ADE34424

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63 Table 4 2. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 82 L 81,432 81,923 164 SAP domain containing protein ORF095L [ GSIV ] 9E 93 AMM72718 83R 81,896 83,467 524 Hypothetical protein ORF82R [ TRBIV ] 0 ADE34426 84 L ** 83,448 844,467 340 Hypothetical protein Hypothetical protein ORF 575R [ RSIV ] 0 BAK14314 85R 84,468 84,647 60 Hypothetical protein ORF84R [ TRBIV ] 8E 38 ADE34428 86L* 84,644 85,573 310 Hypothetical protein ORF85L [ TRBIV ] 0 ADE34429 87L 85,583 86,053 157 Hypothetical protein ORF86L [ TRBIV ] 2E 111 ADE34430 88L* 86,104 87,264 387 Hypothetical protein ORF87L [ TRBIV ] 0 ADE34431 89L** 87,272 88,009 246 Hypothetical protein ORF88L [ TRBIV ] 9E 171 ADE34432 90 L 88,015 88,500 162 Hypothetical protein ORF89R [ TRBIV ] 1E 115 ADE34433 91L 88,548 88,871 108 RING finger containing E3 ubiquitin ligase RING finger domain containing E3 protein [ TRBIV ] 2E 71 ADE34434 92 L 88,924 89,508 195 Hypothetical protein ORF91L [ TRBIV ] 2E 109 ADE34435 93L* 89,555 90,070 172 Hypothetical protein ORF92L [ TRBIV ] 4E 121 ADE34436 94R 90,134 91,564 477 Ankyrin repeat containing protein Ankyrin repeat containing protein [ TRBIV ] 0 ADE34437

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64 Table 4 2. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 95R 91,567 91,944 126 Suppressor of cytokine signaling Suppressor of cytokine signaling protein [ TRBIV ] 3E 77 ADE34438 96 R 91,979 92,755 259 Hypothetical protein Hypothetical protein ORF 522L [ RSIV ] 2E 178 BAK14303 97 R 92,757 93,128 124 Hypothetical protein ORF097R [ RBIV ] 4E 84 AAT71912 98L 93,264 94,202 313 Hypothetical protein ORF97L [ TRBIV ] 0 ADE34441 99L 94,347 94,949 201 Hypothetical protein ORF114L [ ISKNV ] 2E 125 AMM04510 100 L ** 95,040 97,802 921 D5 family NTPase D5 family NTPase [ TRBIV ] 0 ADE34443 101 R 97,811 98,005 65 Hypothetical protein ORF100R [ TRBIV ] 2E 36 ADE34444 102L 98,002 98,898 299 Tumor necrosis factor receptor associated factor Tumor necrosis factor type 2 receptor associated protein [ TRBIV ] 0 ADE34445 103R** 98,921 99,664 248 Proliferating cell nuclear antigen Proliferating cell nuclear antigen [ OSGIV ] 0 AAX82418 104L 99,654 100,160 169 Hypothetical protein ORF119L [ ISKNV ] 2E 102 AMM04511 105 L ** 100,199 102,778 860 Tyrosine kinase Tyrosine kinase [ TRBIV ] 0 ADE34449 106R 102,887 103,189 101 Hypothetical protein Not available Not available

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65 Table 4 2. Continued ORF Position (nt range) Product size (aa) Predicted function and conserved domain or signature Best BLAST hit a Description E value Accession no. 107R** 103,228 104,340 371 Immediate early protein ICP 46 Immediate early protein ICP 46 [ TRBIV ] 0 ADE34450 108R 104,369 105,778 470 Hypothetical protein ORF107R [ TRBIV ] 0 ADE34451 109L* 105,834 106,508 225 Hypothetical protein Early 31kDa protein [ TRBIV ] 4E 167 ADE34452 110L 106,842 108,152 437 Ankyrin repeat containing protein Ankyrin repeat containing protein [ TRBIV ] 0 ADE34453 111R 108,198 108,503 102 RING finger containing E3 ubiquitin ligase RING finger domain containing E3 protein [ TRBIV ] 3E 66 ADE34454 112R* 108,457 109,038 194 Hypothetical protein ORF111R [ TRBIV ] 2E 120 ADE34455 113L 109,039 109,677 213 Hypothetical protein ORF112R [ TRBIV ] 1E 122 ADE34456 114R** 109,687 110,406 240 ATPase ATPase [ GSIV ] 0 AAL68653 115L 110,378 110,758 127 Hypothetical protein Hypothetical protein ORF 407R [ RSIV ] 2E 85 BAK14284 116L* 110,767 111,585 273 Ankyrin repeat containing protein Viral ankyrin repeat protein [ ISKNV ] 0 AEK52381 aSignificant hits based on NCBI BL ASTp *54 core gene **26 & 54 core gene Abbreviations: nt, nucleotides; aa, amino acids; ID, identity, GSIV= Giant seabass iridovirus; ISKNV = Infectious spleen and kidney necrosis virus ; OFIV = Olive flounder iridovirus ; OSGIV= Orange -spotted grouper iridovirus; RBIV= Rock bream iridovirus; RSIV= Red seabream iridovirus; SDDV= Scale drop disease virus; TRBIV= Turbot reddish body iridovirus

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66 Figure 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 The M aximum L ikelihood tree wa s created using 1000 bootstraps in IQ T REE All nodes were supported with bootstrap support values >90 except those indicated by an asterisk (*). Branch lengths are based on the number of inferred substitutions, as indicated by the scale. See Table 4 2, 43 for a list of the 26 iridovirus core genes. See Table 3 1 for the list of included iridovirus taxa and their abbreviations.

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67 Figure 4 5. Phylogram illustrating the relationship of South American cichlid iridovirus (SACIV) and three spot gourami iridovi rus (TSGIV) to the other member of the genus Megalocytivirus based on 54 core genes. The Maximum L ikelihood tree was created using 1000 bootstraps in IQ T REE All nodes were supported with bootstrap support values >90 except those indicated by an asterisk (*). Branch lengths are based on the number of inferred substitutions, as indicated by the scale. See Table 4 2, 43 for a list of the 54 MCV core genes. See Table 3 2 for the list of included MCV taxa and their abbreviations.

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68

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69 Figure 4 6. Phylogra m (adapted from Go et al. 2016) illustrating the relationship of megalocytiviruses based on the major capsid protein gene (s ee Table 1 1 for list of all viral taxa and abbreviations). The Maximum L ikelihood tree was created using 1000 bootstraps and support values > 70 were included Branch lengths are based on the number of inferred substitutions as indicated by the scale.

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70 Figure 4 7. Nucleotide sequence alignment of Infectious spleen and kidney necrosis virus (ISKNV), red seabream iridovirus (RSIV), large yellow croaker iridovirus (LYCIV), orange spotted grouper iridovirus (OSGIV), rock bream iridovirus (RBIV), turbot reddish body iridovirus (TRBIV), South American cichlid iridovirus (SACIV), and three spot gourami iridovirus (TSGIV) for a 173 bp region of the transmembrane amino acid transporter protein gene (ORF 1L ). Highlighted regions represent the pan MCV primer (SACIV1L -F and SACIV1L -R) binding sites designed in this study.

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71 CHAPTER 5 DISCUSSION In this inve stigation, we report the first complete geno me sequences for TRBIV Clade 2 MCVs isolated from cultured South American cichlids (SACIV) and three spot gourami (TSGIV) during outbreaks in the early 1990s (Fraser et al. 1993, Go et al. 2016). Go and colleagues (2016) recently characterized TRBIV Clade 2, based on MCP and ATPase gene sequences, from even earlier outbreaks involving angelfish Pterophyllum scalare imported into Canada from Asia (Schuh and Shirley 1990) and dwarf gourami Trichogaster lalius imported into Australia from Asia (Anderson et al. 1993). It is unclear why the TRBIV C lade 2 MCVs have not been detected in the international ornamental fish trade since the 1991 SACIV outbreak in California and the 19911992 TSGIV outbreaks in Florida (Fraser et al. 1993, Go e t al 2016). The MCV genotype of more recent outbreaks in freshwater ornamental fishes have been ISKNV Clade 1 and outbreaks in marine orn amental fishes have been ISKNV C lade 2 (Weber et al. 2009, Sriwanayos et al. 2013, reviewed in Go et al. 2016). The panMCV PCR assay described here could prove to be a valuable tool in future surveillance efforts aimed at rapidly identifying the MCV genotype from isolates and fresh or fixed tissues (Figure 4 3). The most recent detection of a TRBIV Clade 2 MCV involved a high mortality event that occurred on a Taiwanese farm rearing rock bream Oplegnathus fasciatus for food (Huang et a l. 2011). Similarly, the TRBIV Clade 1 MCVs have negatively impacted turbot Scophthalmus maximus and other flatfish s pecies reared for food in China and Korea (Do et al. 2005, Shi et al. 2010). The finding of related TRBIV MCVs in both freshwater ornamental fishes and marine food fish species is not surprising given the promisc uous nature of MCVs (Yanong & Waltzek 2016, Kawato et al. 2017). Experimental challenge studies have shown that ISKNV Clade 1 MCVs derived from a freshwater fish (pearl gourami Trichogaster leeri ) can induce lethal

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72 disease in a marine food fish species rock bream ( Jeong et al. 2008). D warf gourami c an transmit the dwarf goura mi iridovirus (ISKNV Clade 1 ) by cohabitation to Murray cod, revered as a sport and food fish in Australia (Go & Whittington 2006). Finally, there is evidence that ISKNV Clade 1 has resulted in concurrent outbreaks in Nile tilapi a Oreochromis niloticus reared for food and angelfish reared for the ornamental trade on the same farm (Subramaniam et al. 2016). Given MCVs appear capable of readily crossing environmental and host boundaries, the implementation of stringent biosecurity practices should be observed when rearing highly susceptible ornamental species (e.g. angelfish and gourami) alongside susceptible food fishes (e.g., rock bream and Nile tilapia) (Jeong et al. 2008, Yanong & Wal tzek 2016, Subramaniam et al. 2016). The SACIV and TSGIV genome size, %G+C, gene number and orientation are consistent with those reported for members of the genus Megalocytivirus (Chinchar et al. 2009 ) (Tables 32) P hylogenomic analyses based on MCP gene, 26 core genes, and 54 core genes supported SACIV and TSGIV as a novel clade and sister group to the TRBIV Clade 1 MCVs ( Figure 4 4, 45, 4 6). A synapom orphy (i.e., shared derived feature) for the TRBIV Clade 2 MCVs is the presence of a truncated paralog of the major capsid protein (MCP) gene (ORF 6L) located immediately upstream of the full length parent gene (ORF 7L ; Table 41, 42). To our knowledge, this is the only report of a duplicated MCP gene in an iridovirus or the related nucleocytoplasmic large DNA viruses. The MCP paralog likely arose through a gene duplication event and if expressed its function could be to increase antigenic diversity. Gene duplication events are common mechanism microbial pathogens and parasites use to increase antigenic div ersity to evade host immune responses ( Pays et al. 1981, F erreira et al. 2004). The iridovirus major capsid protein (MCP) is the predominant structural component of the virion. The MCP is

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73 thought to be the most important protective antigen and has been the focus of several MCV vaccines (Caipang et al. 2006 Shi nmoto et al. 2010, Fu et al. 2014). Histopathological examination of archived oscar tissue sections infected with SACIV revealed stereotypical MCV microscopic lesions similar to those previously repo rted in three spot gourami infected with TSGIV ( Fraser et al. 1993). Abundant cytomegalic cells characterized by basophilic granular cytoplasmic inclusions were observed within various organs that were especially prominent in the anterior kidney, spleen, and intestinal submucosa. Although originally isolated in the tilapia heart cell line (Fraser et al. 1993), the TSGIV isolate produced the expected cytopathic effect (i.e., cellular rounding and enlargement) for MCVs (e.g., red seabream iridovirus) grown in the grunt fin (GF) cell line (Kawato et al. 2017 Figure 41). Ultrastructural examination of infected GF cells revealed nonenveloped, hexagonal, virus particles (120 144nm) having an electron dense core within the cytoplasm of infected GF cells sometim es arranged in paracrystalline arrays (Figure 4 2), consistent with previous MCV reports (Weber et al. 2009, Kawato et al 2017). In contrast to iridoviruses from related genera (e.g., ranaviruses and lymphocystiviruses), that acquire an outer envelope as they bud through the host cell plasma membrane, only unenveloped TSGIV particles were ob served within infected GF cells Furthermore, our review of previous MCV reports did not reveal a single convincing study proving MCVs acquire an outer envelope during virion morphogenesis. Interestingly, virus particles were observed with GF cellular blebs (Figure 4 2A ). T he histogenesis of the cytomegalic cells in this study likely involved cells of a mesenchymal or lymphomyeloid origin as previously described (Weber et al. 2009, Subramaniam et al. 2016). E pithelial tissue and tissue of the nervous system were not affected (Weber et al. 2009

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74 Subramaniam et al. 2016). Future research is needed to better define the cellular tropism of MCV s and the mechanism(s) by which they gain entry to and egress from hosts cells. The growth of TSGIV in GF cells (Figure 41) will permit future challenge studies to unequivocally determine the role of TRBIV Clade 2 MCVs in disease (i.e., fulfilment of Koc hs postulates). The ability to produce viral antigen will facilitate the development of serologic diagnostic assays (e.g., serum neutralization assay, Enzyme Linked Immunosorbent Assay), anti MCV antibodies, and a killed MCV vaccine. The establishment of an effective challenge model will permit the development of effective MCV mitigation strategies including testing the effect of environmental manipulation (e.g., temperature and density) and vaccination. Although a formalin inactivated vaccine has reduced the impact of RSIV disease on Japanese food fish mariculture, its economic viability and effectiveness against other MCV genotypes infecting ornamental fishes remains to be determined (Nakajima et al. 1997, 1999, Kawato et al. 2016).

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75 LIST OF REFERENCES A hne W (1978) Uptake and multiplication of spring viraemia of carp virus in carp, Cyprinus carpio L. J Fish Dis 1:265 268 Anderson IG, Prior HC, Rodwell BJ, Harris GO (1993) Iridovirus like virions in imported dwarf gourami (Colisa lal ia ) with systemic amoebiasis. Aust Vet J 70:66 67 Ao J, Chen X (2006) Identification and characterization of a novel gene encoding an RGD containing protein in large yellow croaker iridovirus. Virology 355:213 222 Bankevich A, Nurk S, Antipov D, Gurevich A A, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to singlecell sequencing. J Comput Biol 19:455 477 Baudouy A M, Danton M, Merle G (1980) Virmie printanire de la carpe: rsultants de contaminations exprimentales effectues au printemps Ann Rech Vet 11:245249 Besemer J, Lomsadze A, Borodovsky M (2001) GeneMarkS: a selftraining method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res 29:2607 2618 Caipang CM, Takano T, Hirono I, Aoki T (2006) Genetic vaccines protect red seabream, Pagrus major upon challe nge with red seabream iridovirus (RSIV). Fish Shellfish Immunol 21:130 138 Cano I, Alonso MC, Garcia Rosado E, Saint Jean SR, Castro D, Borrego JJ (2006) Detection of lymphocystis disease virus (LCDV) in asymptomatic cultured gilt head seabream ( Sparus aurata, L.) using an immunoblot technique. Vet Microbiol 113:137 141 Chao C, Chen C, Lai Y, Lin C, Huang H (2004) Histological, ultrastructural, and in situ hybridization study on enlarged cells in grouper Epinephelus hybrids infected by grouper iridivirus in Taiwan (TGIV). Dis Aquat Org 58:127 142 Chen XH, Lin KB, Wang XW (2003) Outbreaks of an iridovirus disease in maricultured large yellow croaker, Larimichthys crocea (Richardson), in China. J Fish Dis 26:615 619 Chinchar VG, Hyatt A, Miyazaki T, Williams T (2009) Family I ridoviridae : poor viral relations no longer. Curr Top Microbiol Immunol 328:123 170 Chua FHC, Ng ML, Ng KL, Loo JJ, Wee JY (1994) Investigation of outbreaks of a novel disease, 'Sleepy Grouper Disease', affecting the brown spotted grouper, Epinephelus tauvina Forskal. J Fish Dis 17:417 427

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76 Danayadol Y, Direkbusarakom S, Boonyaratpalin S, Miyazaki T, Miyata M (1996) An outbreak of iridovirus like infection in brownspotted grouper (Epinephelus malabaracus ) cultured in Thailand. AAHRI Newslett 5:6 de Groof A, Guelen L, Deijs M, van der Wal Y, Miyata M, Ng KS, van Grinsven L, Simmelink B, Biermann Y, Grisez L, van Lent J, de Ronde A, Chang SF, Schrier C, van der Hoek L (2015) A Novel Virus Causes Scale Drop Disease in Lates calcarifer PLoS Pathog 11:e1005074 Dixon PF (2008) Virus diseases of cyprinids. In: Eiras JC Segner H Wahli T, Kapoor BG (ed) Fish Diseases 1st ed. Science Publishers, Enfield, NH, USA Do JW, Cha SJ, Kim JS, An EJ, Lee NS, Choi HJ, Lee CH, Park MS, Kim JW, Ki m YC, Park JW (2005) Phylogenetic analysis of the major capsid protein gene of iridovirus isolates from cultured flounders Paralichthys olivaceus in Korea. Dis Aquat Org 64:193 200 Do JW, Moon CH, Kim HJ, Ko MS, Kim SB, Son JH, Kim JS, An EJ, Kim MK, Lee SK, Han MS, Cha SJ, Park MS, Park MA, Kim YC, Kim JW, Park JW (2004) Complete genomic DNA sequence of rock bream iridovirus. Virology 325:351 363, Dong Y, Weng S, He J, Dong C (2013) Field trial tests of FKC vaccines against RSIV genotype Megalocytivirus in cage cultured mandarin fish (Siniperca chuatsi) in an inland reservoir. Fish Shellfish Immunol 35:1598 1603 Dong C, Weng S, Shi X, Xu X, Shi N, He J (2008) Development of a mandarin fish Siniperca chuatsi fry cell line suitable for the study of infectious spleen and kidney necrosis virus (ISKNV). Virus Res 135:273 281 Eaton HE, Metcalf J, Penny E, Tcherepanov V, Upton C, Brunetti CR (2007) Comparative genomic analysis of the family Iridoviridae : re annot ating and defining the core set of iridovirus genes. Virol J 4:11 Fan T, Hu X, Wang L, Geng X, Jiang G, Yang X, Yu M (2012) Development of an inactivated iridovirus vaccine against turbot viral reddish body syndrom. J Ocean Univ China 11:65 69 FAO, 2015. FAO Global Aquaculture Production database updated to 2013 Summary information. Food and Agriculture Organization of the United Nations, Rome Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA (2005) Virus Taxonomy Classification and Nomenclatur e of Viruses: Eighth Report of the International Committee on the Taxonomy of Viruses. Academic Press, Elsevier, San Diego, CA Ferreira MU, da Silva Nunes M, Wunderlich G (2004) Antigenic diversity and immune evasion by malaria parasites. Clin Diagn Lab I mmunol 11:987 995

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84 BIOGRAPHICAL SKETCH Samantha received her BS in aquatic biology from the Universi ty of California, Santa Barbara. During her undergraduate career, she worked on the Santa Barbara Coastal Long Term Ecological Research project, as well as conducting research on larval stages of grass rockfish (Sebastes rastrelliger ). She ha s experience as an aquarist having worked in California at the Ty Warner Sea Center in Santa Barbara, Aquarium of the Pacific in Long Beach, and Sea Dwel ling Creatures in Los Angeles. She also worked for the state government as a scientific aide for the California Department of Fish and Wildlife.