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

PHYLOGENOMIC CHARACTERIZATION OF A NOVEL MEGALOCYTIVIRUS LINEAGE FROM ARCHIVED ORNAMENTAL FISH SAMPLES

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

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

ACKNOWLEDGMENTS

On a daily basis, I continue to be inspired and reminded by those I love, to pursue my dreams. I attribute my passion for aquatic animals to my parents who have always been huge supporters of zoos and aquariums all my life. I wouldn’t have been able to get this far without the support and guidance from my family, friends, and the many teachers that have inspired me to ultimately pursue a career in fish health. Throughout my schooling I have had the pleasure of learning from some of the most passionate teachers and I would like to thank Mr.Schmitz for inspiring my initial interest in animal sciences, and Drs. Dan Reed and Scott Cooper for their mentorship roles during my undergraduate career. I have had the most amazing opportunities that have led me to my current success and I am very grateful for all the educational and hands on experience that I have gained at Asahi Koi Shop, Ty Warner Sea Center, California

Department of Fish and Wildlife, Aquarium of the Pacific, and Sea Dwelling Creatures.

I would like to thank my committee members, Drs. Thomas Waltzek, Kuttichantran

Subramaniam, Ruth Francis-Floyd, Roy Yanong, and Salvatore Frasca, for their expertise, guidance, and time with my research project and degree. I would also like to thank the Bronson

Animal Disease Diagnostic Laboratory for providing one of the viral isolates for my study, Dr.

Joseph Groff for providing FFPE material, histology slides, and guidance on my pathological findings, and Patrick Thompson for training me on all the laboratory procedures for my research project.

Lastly, I would like to thank all of the members of the Wildlife and Aquatic Veterinary

Disease Laboratory that have been there for me during my time as a master’s student. I would especially like to thank Dr. Waltzek, who has been one of the most supportive, dedicated, and passionate advisors I have ever met. His love for aquatic animal science has been a tremendous positive influence on me, and has led to many lab retreats and group activities that have been

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.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

ABSTRACT ...... 10

CHAPTER

1 VIRUSES OF ORNAMENTAL FISH ...... 12

Introduction...... 12 The Genus Megalocytivirus ...... 17 Infectious spleen and kidney necrosis virus (ISKNV) ...... 20 Red seabream iridovirus (RSIV) ...... 20 Turbot reddish body iridovirus (TRBIV) ...... 21 Diagnostic Methods ...... 22 Megalocytivirus Mitigation Strategies ...... 22

2 PHYLOGENOMIC CHARACTERIZATION OF A NOVEL MEGALOCYTIVIRUS LINEAGE FROM ARCHIVED ORNAMENTAL FISH SAMPLES ...... 31

3 METHODS ...... 34

Archived Samples ...... 34 Cell Culture and Virus Enrichment ...... 34 Transmission Electron Microscopy ...... 35 Histopathology ...... 35 DNA Extraction ...... 35 Complete Genome Sequencing and Assembly ...... 36 Genome Annotation and Phylogenetic Analysis ...... 36 PCR Detection of Megalocytiviruses ...... 37

4 RESULTS ...... 43

Cell Culture and Virus Enrichment ...... 43 Transmission Electron Microscopy ...... 43 Histopathology ...... 43 Complete Genome Sequencing and Assembly ...... 44 Genome Annotation and Phylogenetic Analysis ...... 44 PCR Detection of Megalocytiviruses ...... 44

5 DISCUSSION ...... 71

LIST OF REFERENCES ...... 75

BIOGRAPHICAL SKETCH ...... 84

LIST OF TABLES

Table page

1-1 Summary of megalocytiviruses used in this study ...... 25

3-1 GenBank accession numbers for the full genome sequences of iridoviruses used in the 26 core gene phylogenetic analyses...... 39

3-2 Genome summary of the 10 megalocytivirus genomes used in the 26 and 54 core gene analyses...... 41

3-3 Pan-megalocytivirus primer set ...... 42

4-1 Predicted open reading frames for the South American cichlid iridovirus (SACIV) genome...... 48

4-2 Predicted open reading frames for the three spot gourami iridovirus (TSGIV) genome...... 57

LIST OF FIGURES

Figure page 4-1 In vitro growth characteristics of TSGIV in the GF cell line...... 45

4-2 Transmission electron photomicrograph of a GF cell infected with three spot gourami iridovirus (TSGIV) ...... 46

4-3 Microscopic examination of megalocytic cells and lesions in infected Oscar Astronus ocellatus...... 47

4-4 Phylogram illustrating the relationship of South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) to the other members of the family Iridoviridae based on 26 core genes ...... 66

4-5 Phylogram illustrating the relationship of South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) to the other member of the genus Megalocytivirus based on 54 core genes...... 67

4-6 Phylogram illustrating the relationship of megalocytiviruses based on the major capsid protein gene ...... 69

4-7 Nucleotide sequence alignment of the transmembrane amino acid transporter protein gene ...... 70

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

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, 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.

CHAPTER 1 VIRUSES OF ORNAMENTAL FISH

Introduction

Globally, 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 ornamental 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, goldfish, 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 syngnathiformes (e.g., seahorses) are produced

(Wittenrich 2007). In 2003, the farm-gate value of Florida ornamental 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 (Livengood & 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.

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 international 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 internationally, 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 non-cyprinid species (Dixon 2008).

SVCV was first isolated from moribund common carp in Yugoslavia (Fijan et al. 1971) and remains a significant 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

viremia is established in the branchial epithelium before spreading systemically (Ahne 1978).

Spring water temperatures (12 - 22°C) have been shown to be optimal for viral replication resulting in natural outbreaks (Fijan 1999, McAllister 1993). Diagnostic methods useful in confirming suspected cases of SVCV disease include viral isolation, RT-PCR, 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 seasonal episodes (i.e., water temperature <22°C) 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 immunofluorescence, transmission electron microscopy, DNA arrays, and conventional PCR tools (reviewed in Viadanna et al. 2017).

Cyprinid herpesvirus 2 (CyHV2), known informally as herpesviral haematopoietic necrosis virus, induces lethal systemic disease in members 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).

Morbidity and mortality can reach 100% at optimal temperatures for viral replication (i.e., 15-

25°C). 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 detection 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 17-26°C with cumulative 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 disease (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 behavior 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, endophthalmos, and pale gills (Miyazaki et al. 2005, Hesami et al.

2015). Juvenile koi appear most susceptible with outbreaks occurring at a water temperature between 15-25°C 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 consistent CEVD/KSD clinical signs test positive by PCR.

Members 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 dsDNA viruses that infect a range of poikilothermic vertebrate and invertebrate hosts. They possess nucleocapsids with icosahedral symmetry that contain linear

DNA genomes 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. LCDV-infected fibroblasts of the skin and fins (internal organ involvement is rare) may enlarge up to 100,000x and masses of infected fibroblasts may cluster together to form grossly visible whitish to pink masses several 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 LCDV

(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 LCDV. LCDV is typically a self-limiting disease with little or no mortality observed unless lesions obstruct respiration or feeding. Trained fish health professionals learn to identify LCDV grossly; however, evaluation of infected tissues by cytology / histology to confirm the clusters of enlarged cells is prudent given the gross proliferative lesions are not pathognomonic. Transmission electron microscopy and PCR are also useful options to confirm LCDV (Cano et al. 2006).

The Genus Megalocytivirus

The genus Megalocytivirus (MCV) is 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 93-135 open reading frames, and displays between 53-

56% 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 across 11 orders and 44 families (Yanong & Waltzek 2016,

Koda & Waltzek unpublished data). Transmission occurs horizontally by exposure of naïve 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 survivors

of experimental challenges up to 25 days pi which suggests that viral persistence may occur (Ito et al. 2013).

Typical clinical signs associated with MCV infections include behavioral changes (e.g., anorexia, lethargy, increased respiration), white feces, gill pallor, darkened or lightened body appearance, splenomegaly, ascites, and internal/external hemorrhagic lesions (Yanong &

Waltzek 2016, Kawato et al. 2017). MCVs typically establish chronic infections with cumulative mortality ranging from 20-60% over a 1-2 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 (<20ºC) 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-Kueh et al. 2003, Weber et al. 2009, Yanong & Waltzek 2016, 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, circumscribed bodies, and inclusion body-bearing cells (reviewed in Sudthongkong et al. 2002). Systemic infections involving hematopoietic organs (spleen and anterior kidney), the submucosa of the gastrointestinal tract, and other organs are common (Gibson-Kueh et al. 2003, Weber et al. 2009,

Yanong & Waltzek 2016, Subramaniam et al. 2016). The histogenesis of affected cells remains

unclear; however, affected cells may include lymphomyeloid, 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 iridovirus (RSIV). Similar ongoing disease outbreaks have been

reported as early as 1994 among Chinese freshwater farms rearing mandarin fish (Siniperca

chuatsi) and the 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 iridoviruses (He et 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 Megalocytivirus 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

be further subdivided into 2 separate well supported clades (Figure 4-6) (Waltzek et al. 2012,

Kurita & Nakajima 2012, Go et al. 2016). The recent characterization of genetically divergent

MCVs from Canadian three-spined 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).

Infectious 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 2005-2008, 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 iridovirus (RSIV)

RSIV was first reported in a 1990 epizootic involving cultured red seabream (Pagrus major) reared in the Ehime prefecture of Shikoku Island, Japan (Inouye et al. 1992, Table 1-1).

This Ehime-1 strain was the first RSIV Clade 1 genome sequenced (Kurita et al. 2002). A second

RSIV Clade 1 MCV was sequenced from an isolate obtained from epizootics involving maricultured large yellow croaker (Larimichthys 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 have 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 reddish body iridovirus (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 China (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 cultured 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 2-5).

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 in 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 hemorrhagic lesions, gill pallor, splenomegaly, ascites, anemia, and white feces (Yanong & Waltzek 2016, 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 acetone-fixed Giemsa-stained 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 virions 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 antibody test (IFAT) positive megalocytes on acetone-fixed impression smears (reviewed in OIE,

2016a). The RSIVD diagnosis is considered confirmed if 1) virus isolation with appropriate CPE is 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 acetone-fixed impression smears (reviewed in OIE 2016a, Kawato et al.

2017).

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 vaccines 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 relationship 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 (>1000-3000 μW•sec/cm2 uW), and chemical disinfection using sodium hypochlorite (>200 mg/L), potassium permanganate (>100 mg/L), or formalin (>2000 mg/L)

(Nakajima & Sorimachi 1994, He et al. 2002, Kasai et al. 2002). The contact time for the

aforementioned chemical disinfectants should be >15 min at or around 25°C. 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 the 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.

Table 1-1. Summary of megalocytiviruses used in this study

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

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

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

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

Table 1-1. Continued Host Marine (M) Host Genus Country and GenBank Common Host Family Freshwater (F) Strain Name Reference species Year Collected Accession Name Brackish (B)a 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 Jeong et al. olivaceus Olive flounder Paralichthyidae M Korea <2007 OFLIV-1 EU276417 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 Kim et al. olivaceus Olive flounder Paralichthyidae M Korea <2004 OFIV AY661546 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 Zhao et al. maximus Turbot Scophthalmidae M China 2009 TIV R-603 HM596017 unpublished

Table 1-1. Continued Host Marine (M) Host Genus Country and GenBank Common Host Family Freshwater (F) Strain Name Reference species Year Collected Accession Name Brackish (B)a Lateolabrax Jeong & Jeong spp. Seaperch Serranidae M/B/F Korea 2000 SPIV CH-1 HM067603 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 Huang et al. fasciatus Rock bream Oplegnathidae M Taiwan 2008 RBIV/Tp/45/08 JF264352 2011 Trichogaster Three spot trichopterus gourami Osphronemidae F USA <1992 TSGIV N/A This study TSIV Gasterosteus Three-spined Waltzek et al. aculeatus stickleback Gasterosteidae M/B/F Canada 2008 TSIV HQ857785 2012 SDDV Singapore 2010- de Groof et al. Lates calcarifer Barramundi Latidae M/B/F 2011 C4575 NC027778 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 reddish body iridovirus; TSIV=Threespine stickleback iridovirus.

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 iridovirus (RSIV) genotype that includes strains from marine fishes in Japan, Korea, China, and SE Asia; the ISKNV genotype that includes strains from Chinese mandarin fish and ornamental fishes cultivated in Southeast (SE) Asia; and the

turbot reddish body iridovirus (TRBIV) genotype that includes strains from Asian flatfishes

(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

characterized 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 into Taiwan from

Korea (Huang et al. 2011). The recent characterization of genetically divergent MCVs from

three-spined stickleback Gasterosteus aculeatus (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.

Similar to some lymphocystiviruses and ranaviruses, MCVs 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 by the

World Organization 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 MCVs are transmitted

horizontally through cohabitation (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 challenge studies for each MCV genotype (i.e., ISKNV,

RSIV, TRBIV) have revealed no clinical signs of disease at cooler water treatments (<20ºC) 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 non-specific

clinical signs including anorexia, lethargy, increased respiration, gill pallor, darkened coloration,

white feces, and internal/external hemorrhages (Yanong & Waltzek 2016). Although notoriously

difficult to isolate in cultured cells, MCVs induce pathognomonic histopathological 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 displaying MCV cytopathic effect (i.e., enlarged, rounded, refractile cells) (OIE 2016a).

In this study, we performed phylogenomic analyses to characterize a novel MCV lineage isolated from freshwater ornamental fishes (Go et al. 2016). Additionally, we compared the in vitro growth characteristics, microscopic pathology, and ultrastructural pathology of the novel lineage to previously reported MCVs.

CHAPTER 3 METHODS

Archived Samples

In 1991, moribund juvenile South American cichlids (oscar Astronotus ocellatus and keyhole cichlid Cleithracara 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 had 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 in tilapia heart cells derived from moribund Florida farm-raised three spot gourami Trichopodus trichopterus during epizootics in 1991-1992 (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 cultured 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.

Cell Culture and Virus Enrichment

The SACIV and TSGIV isolates were inoculated onto confluent monolayers of the grunt fin (GF) cells maintained in L15 media with 10% fetal bovine serum (FBS) and 1% HEPES at

28°C. The infected cells were monitored daily for cytopathic effect (CPE) for 14 days post- inoculation.

Four 175 cm2 flasks of grunt fin cells displaying extensive CPE were harvested and

subjected to three rounds of freeze/thawing prior to clarification of the supernatant by centrifugation at 5,520 × g for 20 min at 4º C. Pelleted virus was obtained by centrifugation of the clarified supernatant at 100,000 x g for 1.25 hr at 4ºC. The viral pellet was resuspended in

360 µl of ATL buffer prior to extraction of viral genomic DNA (see below).

Transmission Electron Microscopy

Infected 75 cm2 flasks of GF cells displaying cytopathic effect were fixed in 15 mL of

modified (2P+2G) Karnovsky’s fixative at room temperature for 15 minutes. After fixation, the

supernatant and cells were transferred into a 15 mL conical tube and clarified at 3000 x g for 10 minutes at 4°C. The fixative and media was then pipetted off and the GF pellet was resuspended in 1 mL cacodylate buffer and stored at 4°C until being shipped to the UTMB-EML.

Histopathology

Ten separate juvenile oscars from the 1991 outbreak were fixed in 10% neutral buffered

formalin, transected mid-sagittal, and embedded into paraffin 1-2 fish per block. Sections were

cut at 5 μm and stained routinely with hematoxylin and eosin (H&E) for microscopic

examination.

DNA Extraction

For FFPE tissues, 50 µm 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 56ºC overnight before extraction of DNA was carried out

using a DNA FFPE Tissue Kit (Qiagen) according to the manufacturer’s recommendations.

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 manufacturer’s protocol for cell culture.

Complete Genome Sequencing and Assembly

DNA libraries were created using a Nextera XT DNA Kit (Illumina) for SACIV and a

TruSeq Kit (Illumina) for TSGIV and sequenced using a v3 chemistry 600 cycle kit on a MiSeq

platform (Illumina). De novo assemblies of the paired end reads was performed in SPAdes 3.5.0

(Bankevich et al. 2012). The quality of the TSGIV and SACIV assemblies were assessed by

mapping the reads back to the consensus sequences in Bowtie 2 2.1.0 (Langmead & Salzberg

2012) and visually inspecting the alignments in Tablet 1.14.10.20 (Milne et al. 2010).

Genome Annotation and Phylogenetic Analysis

Putative open reading frames (ORFs) for SACIV and TSGIV genomes were predicted

using 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 ORFs, only the larger ORF was annotated. The gene functions were predicted based

on BLASTp 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 3-1). The 54 Megalocytivirus core genes

were used to construct phylogenetic trees for ten MCVs including SACIV and TSGIV (Table 3-

2). 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). Maximum Likelihood phylogenetic trees were

constructed using IQ-TREE 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 pan-MCV primer set was designed to amplify all MCV genotypes (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 sequences 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 facilitate MCV genotype discrimination by Sanger sequencing (Figure 4-7).

Reaction volumes for the pan-MCV PCR were 50 μl and consisted of 0.25 µl of Platinum

Taq DNA Polymerase (Invitrogen), 5.0 µl of 10× PCR Buffer, 2.0 µl of 50 mM MgCl2, 1.0 µl of

10 mM dNTPs, 2.5 µl of 20 µM forward and reverse primers, 32.25 µl of molecular grade water, and 4.5 µl of DNA template. An initial denaturation step of 95°C for 5 min was followed by 40 cycles of a 94°C denaturation step, a 55°C annealing step, and a 72°C extension step, each step run for 30 seconds, and a final extension step at 72°C 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 was quantified fluorometrically

using a Qubit® 3.0 Fluorometer and dsDNA BR Assay Kit (Life Technologies). Purified DNA was sequenced in both directions on an ABI 3130 platform (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 2-infected Oscar, ISKNV-infected Nile tilapia

(Oreochromis niloticus) (Subramaniam et al. 2016), and RSIV-infected Florida pompano.

Table 3-1. GenBank accession numbers for the full genome sequences of iridoviruses used in the 26 core gene phylogenetic analysis 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) KX574341 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

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

Table 3-2. Genome summary of the 10 megalocytivirus genomes used in the 26 and 54 core gene analyses

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

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

CHAPTER 4 RESULTS

Cell Culture 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 observed by day 10 pi in which most cells were affected but remained attached to the monolayer (Figure 4-1). No CPE was observed following infection of GF cells with the SACIV isolate.

Transmission Electron Microscopy

Large numbers of unenveloped, hexagonal viral particles were observed within the cytoplasm of GF cells (Figure 4-2A). Viral 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 4-3). 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 interstitium and glomeruli), branchial lamellar capillaries, pseudobranch, rete mirabile, spleen, pancreas, gonadal interstitium, coelomic cavity membrane, skeletal and cardiac muscle, cartilage, and connective tissue regardless of location. Of the four oscar sections not displaying microscopic lesions

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 granulomas.

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 Annotation and Phylogenetic Analysis

Both genomes displayed 116 open reading frames (Table 4-1, 4-2). 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 (95-100% bootstrap support) as the sister group to TRBIV (Figures 4-4, 4-5, 4-

6). Both SACIV and TSGIV possess a unique truncated paralog of the major capsid protein gene

(ORF 6L) located immediately upstream of the full length parent gene (ORF 7L).

PCR Detection of MCVs

Primers against ORF 1L met the specified criteria generating a 173 bp amplicon

(including primers) (Figure 4-7). 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.

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) Infected 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 µm.

Figure 4-2. (A) Transmission electron photomicrograph of a GF cell infected with TSGIV displaying non-enveloped, 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). Scale bar is 1 μm. (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.

Figure 4-3. Microscopic examination of megalocytic cells (arrows) and lesions in infected oscar Astronus ocellatus. H&E stain. (A,B) Anterior kidney. (C,D) Posterior kidney. (E,F) Intestinal submucosa. Scale bar is 50 µm.

Table 4-1. Predicted open reading frames for the South American cichlid iridovirus (SACIV) genome. ORF Position Product Predicted function and Best BLAST hita (nt range) size (aa) conserved domain or signature Description E-value Accession no. 1L* 1-1,137 379 Transmembrane amino acid Transmembrane amino 0 ADE34346 transporter protein acid transporter [TRBIV] 2L 1,107-1,562 152 DNA dependent RNA DNA dependent RNA 1E-98 AMM04413 polymerase subunit H polymerase subunit H- like protein [ISKNV] 3R 1,688-1,945 258 Caspace recruitment domain Caspace recruitment 3E-43 AMM72786 containing protein domain containing protein [GSIV] 4L 2,012-2,491 160 Hypothetical protein ORF4L [TRBIV] 1E-95 ADE34349 5L** 2,838-3,584 249 NLI interacting factor-like Catalytic domain of ctd- 1E-180 ADE34350 phosphatase like phosphatase [TRBIV] 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 0 AEI85911 [RBIV] 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 RING-finger-containing 3E-75 ADE34357 ubiquitin ligase ubiquitin ligase [TRBIV] 14R** 10,104- 466 Serine/threonine protein Serine/threonine protein 0 ADE34358 11,501 kinase kinase [TRBIV] 15R 11,756- 324 Hypothetical protein ORF14R [TRBIV] 0 ADE34359 12,727 16R* 12,733- 262 Hypothetical protein ORF15R [TRBIV] 3E-175 ADE34360 13,518

Table 4-1. Continued ORF Position Product Predicted function and Best BLAST hita (nt range) size (aa) conserved domain or signature Description E-value Accession no. 17L* 13,576- 196 Hypothetical protein ORF324R [RSIV] 5E-132 BAK14267 14,163 18L 14,178- 110 Hypothetical protein ORF17L [TRBIV] 2E-25 ADE34362 14,507 19L 14,767- 64 Hypothetical protein ORF 318R [RSIV] 6E-29 BAK14265 14,958 20R** 15,024- 949 DNA polymerase DNA polymerase 0 ADE34365 17,870 [TRBIV] 21R 17,917- 178 Hypothetical protein ORF024R [GSIV] 7E-113 AMM72671 18,450 22L* 18,434- 525 Macro domain containing Putative phosphatase 0 ADE34368 20,008 protein [TRBIV] 23R* 20,080- 947 Laminin-type epidermal Laminin-like protein 2E-158 AAT76907 22,920 growth factor [OFIV] 24R** 23,029- 313 Ribonucleotide reductase beta Ribonucleotide reductase 0 ADE34370 23,967 subunit small chain [TRBIV] 25L 24,384- 105 Hypothetical protein Hypothetical protein 0.5628 YP_009009516 24,698 32HC_45 [Mycobacterium phage 32HC] 26L* 24,727- 108 Hypothetical protein ORF26L [TRBIV] 1E-49 ADE34371 25,050 27L** 25,075- 299 Flap endonuclease-1 DNA repair protein 0 ADE34372 25,971 RAD2 [TRBIV] 28L** 25,988- 1169 DNA dependent RNA Largest subunit of the 0 ADE34373 29,494 polymerase alpha subunit DNA-dependent RNA polymerase [TRBIV] 29L** 29,501- 88 Transcription factor S-II Elongation factor [RSIV] 2E-49 BAK14256 29,764

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

Table 4-1. Continued ORF Position Product Predicted function and Best BLAST hita (nt range) size (aa) conserved domain or signature Description E-value Accession no. 44L 45,502- 305 Hypothetical protein ORF44L [TRBIV] 0 ADE34389 46,416 45L* 46,410- 228 Cytosine DNA Cytosine DNA 8E-169 ADE34390 47,093 methyltransferase methyltransferase [TRBIV] 46R* 47,253- 88 Hypothetical protein ORF46R [TRBIV] 3E-60 ADE34391 47,516 47R* 47,513- 116 Vascular endothelial growth ORF47R [TRBIV] 7E-66 ADE34392 47,860 factor 48R 47,876- 57 Hypothetical protein ORF049R [ISKNV] 1E-37 NP_612271 48,046 49L* 48,106- 143 Hypothetical protein ORF48L [TRBIV] 2E-90 ADE34393 48,534 50L 48,765- 167 Hypothetical protein ORF49L [TRBIV] 1E-103 ADE34394 49,265 51R 49,267- 72 Hypothetical protein ORF053R [ISKNV] 5E-42 NP_612275 49,482 52L* 49,495- 310 2-cysteine adaptor domain ORF111R [RSIV] 0 BAK14232 50,424 containing protein 53L** 50,447- 312 2-cysteine adaptor domain ORF52L [TRBIV] 0 ADE34397 51,382 containing protein 54L** 51,393- 216 Hypothetical protein ORF53L [TRBIV] 7E-132 ADE34398 52,040 55L 52,047- 87 Hypothetical protein ORF057L [ISKNV] 2E-57 NP_612279 52,307 56L 52,675- 172 Hypothetical protein ORF55L [TRBIV] 1E-116 ADE34400 53,190 57L** 53,261- 268 Replication factor Putative replication 0 ADE34401 54,064 factor [TRBIV]

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

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

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

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

Table 4-1. Continued ORF Position Product Predicted function and Best BLAST hita (nt range) size (aa) conserved domain or signature Description E-value Accession no. 112R* 108,213- 194 Hypothetical protein ORF111R [TRBIV] 2E-120 ADE34455 108,794 113L 108,795- 213 Hypothetical protein ORF112R [TRBIV] 1E-122 ADE34456 109,433 114R** 109,443- 240 ATPase ATPase [GSIV] 0 AAL68653 110,162 115L 110,134- 127 Hypothetical protein Hypothetical protein 2E-85 BAK14284 110,514 ORF 407R [RSIV] 116L* 110,523- 273 Ankyrin repeat containing Viral ankyrin repeat 0 AEK52381 111,341 protein protein [ISKNV] 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.

Table 4-2. Predicted open reading frames for the three spot gourami iridovirus (TSGIV) genome ORF Position Product size Predicted function and conserved Best BLAST hita (nt range) (aa) domain or signature Description E-value Accession no. 1L* 1-1,137 379 Transmembrane amino acid Transmembrane 0 ADE34346 transporter protein amino acid transporter [TRBIV] 2L 1,107-1,562 152 DNA dependent RNA polymerase DNA dependent 9E-92 AMM04413 subunit H RNA polymerase subunit H-like protein [ISKNV] 3R 1,688-1,945 258 Caspace recruitment domain- Caspace recruitment 3E-43 AMM72786 containing protein domain-containing protein [GSIV] 4L 2,012-2,491 160 Hypothetical protein ORF4L [TRBIV] 1E-95 ADE34349 5L** 2,838-3,584 249 NLI interacting factor-like Catalytic domain of 2E-180 ADE34350 phosphatase ctd-like phosphatase [TRBIV] 6L 3,732-4,043 104 Major capsid protein MCP [KFIV] 2E-53 AAT48718 7L** 4,223-5,584 454 Major capsid protein Major capsid protein 0 AEI85911 [RBIV] 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 10L 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] 2E-44 NP_612233

13R* 9,765-10,097 111 RING-finger-containing E3 ubiquitin RING-finger- 2E-74 ADE34357 ligase containing ubiquitin ligase [TRBIV] 14R** 10,104- 466 Serine/threonine protein kinase Serine/threonine 0 ADE34358 11,501 protein kinase [TRBIV]

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

Table 4-2. Continued ORF Position Product size Predicted function and conserved Best BLAST hita (nt range) (aa) domain or signature Description E-value Accession no. 28L** 26,113- 1169 DNA dependent RNA polymerase Largest subunit of 0 ADE34373 29,619 alpha subunit the DNA-dependent RNA polymerase [TRBIV] 29L** 29,626- 68 Transcription factor S-II Transcription 1E-43 AAX82341 29,829 elongation factor SII [OSGIV] 30L 29,914- 179 Hypothetical protein ORF037L [ISKNV] 3E-117 AMM04443 30,450 31R** 30,504- 189 Deoxyribonucleoside kinase Putative thymidine 5E-116 NP_612254 31,070 kinase [ISKNV] 32L 31,076- 301 Hypothetical protein ORF32L [TRBIV] 0 ADE34377 31,978 33R** 32,079- 1042 DNA dependent RNA polymerase DNA-directed RNA 0 ADE34378 35,204 beta subunit polymerase II second largest subunit-like protein [TRBIV] 34L* 35,297- 371 Hypothetical protein ORF34L [TRBIV] 0 ADE34379 36,409 35R* 36,518- 342 Hypothetical protein ORF35R [TRBIV] 0 ADE34380 37,543 36L 37,595- 450 Hypothetical protein ORF36L [TRBIV] 0 ADE34381 38,944 37L* 38,953- 478 Hypothetical protein ORF37L [TRBIV] 0 ADE34382 40,386 38R 40,363- 312 Hypothetical protein ORF38R [TRBIV] 0 ADE34383 41,298 39L 41,291- 383 Hypothetical protein ORF39L [TRBIV] 0 ADE34384 42,439

Table 4-2. Continued ORF Position Product size Predicted function and conserved Best BLAST hita (nt range) (aa) domain or signature Description E-value Accession no. 40L 42,441- 445 Hypothetical protein ORF40L [TRBIV] 0 ADE34385 43,775 41R 43,790- 194 Hypothetical protein ORF41R [TRBIV] 5E-115 ADE34386 44,371 42L** 44,455- 121 Erv1/Alr family protein Thiol oxidoreductase 5E-71 ADE34387 44,817 [TRBIV]

43L 44,820- 267 Hypothetical protein ORF43L [TRBIV] 0 ADE34388 45,620 44L 45,626- 305 Hypothetical protein ORF44L [TRBIV] 0 ADE34389 46,540 45L* 46,534- 228 Cytosine DNA methyltransferase Cytosine DNA 2E-165 BAK14240 47,217 methyltransferase [TRBIV] 46R* 47,377- 88 Hypothetical protein ORF46R [TRBIV] 2E-58 ADE34391 47,640 47R* 47,637- 116 Vascular endothelial growth factor ORF47R [TRBIV] 6E-64 ADE34392 47,984 48R 48,000- 57 Hypothetical protein ORF049R [ISKNV] 1E-37 NP_612271 48,170 49L* 48,230- 143 Hypothetical protein ORF48L [TRBIV] 1E-88 ADE34393 48,658 50L 48,889- 167 Hypothetical protein ORF49L [TRBIV] 9E-102 ADE34394 49,389 51R 49,391- 72 Hypothetical protein ORF053R [ISKNV] 4E-40 NP_612275 49,606 52L* 49,619- 310 2-cysteine adaptor domain containing Hypothetical protein 0 BAK14232 50,548 protein ORF 111R [RSIV] 53L** 50,571- 312 2-cysteine adaptor domain containing ORF52L [TRBIV] 0 ADE34397 51,506 protein

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

Table 4-2. Continued ORF Position Product size Predicted function and conserved Best BLAST hita (nt range) (aa) domain or signature Description E-value Accession no. 68R 69,849- 144 Hypothetical protein ORF66R [TRBIV] 3E-96 ADE34411 69,280 69R 69,329- 341 Hypothetical protein ORF67R [TRBIV] 0 ADE34412 70,351 70L 70,360- 91 Hypothetical protein ORF010R [RSIV] 1E-54 BAK14214 70,632 71L** 70,634- 1057 Hypothetical protein ORF69L [TRBIV] 0 ADE34414 73,804 72R* 73,625- 446 Ankyrin repeat containing protein Ankyrin repeat- 0 ADE34415 74,962 containing protein [TRBIV] 73R* 74,959- 155 Hypothetical protein ORF71R [TRBIV] 9E-95 ADE34416 75,423 74R 75,578- 214 US22 protein Hypothetical protein 2E-115 BAK14325 76,219 ORF 632L [RSIV]

75R* 76,206- 168 Hypothetical protein ORF74R [TRBIV] 1E-101 ADE34419 76,709 76L* 76,750- 369 Hypothetical protein ORF75L [TRBIV] 0 ADE34420 77,856 77R 77,728- 179 Hypothetical protein ORF089R [ISKNV] 2E-95 AMM04489 78,624 78L 78,302- 452 Hypothetical protein ORF77L [TRBIV] 0 ADE34421 79,657 79R 79,684- 176 Hypothetical protein ORF78R [TRBIV] 1E-81 ADE34422 80,211 80R** 80,208- 155 Hypothetical protein ORF79R [TRBIV] 7E-111 ADE34423 80,672 81R** 80,638- 266 Ribonuclease III ORF80R [TRBIV] 0 ADE34424 81,435

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

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

Table 4-2. Continued ORF Position Product size Predicted function and conserved Best BLAST hita (nt range) (aa) domain or signature Description E-value Accession no. 107R** 103,228- 371 Immediate early protein ICP-46 Immediate early 0 ADE34450 104,340 protein ICP-46 [TRBIV] 108R 104,369- 470 Hypothetical protein ORF107R [TRBIV] 0 ADE34451 105,778 109L* 105,834- 225 Hypothetical protein Early 31kDa-protein 4E-167 ADE34452 106,508 [TRBIV] 110L 106,842- 437 Ankyrin repeat containing protein Ankyrin repeat- 0 ADE34453 108,152 containing protein [TRBIV] 111R 108,198- 102 RING-finger-containing E3 ubiquitin RING-finger 3E-66 ADE34454 108,503 ligase domain-containing E3 protein [TRBIV] 112R* 108,457- 194 Hypothetical protein ORF111R [TRBIV] 2E-120 ADE34455 109,038 113L 109,039- 213 Hypothetical protein ORF112R [TRBIV] 1E-122 ADE34456 109,677 114R** 109,687- 240 ATPase ATPase [GSIV] 0 AAL68653 110,406 115L 110,378- 127 Hypothetical protein Hypothetical protein 2E-85 BAK14284 110,758 ORF 407R [RSIV] 116L* 110,767- 273 Ankyrin repeat containing protein Viral ankyrin repeat 0 AEK52381 111,585 protein [ISKNV] aSignificant hits based on NCBI BLASTp *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

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 Maximum Likelihood tree was created using 1000 bootstraps in IQ-TREE. 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, 4-3 for a list of the 26 iridovirus core genes. See Table 3-1 for the list of included iridovirus taxa and their abbreviations.

Figure 4-5. Phylogram illustrating the relationship of South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) to the other member of the genus Megalocytivirus based on 54 core genes. The Maximum Likelihood tree was created using 1000 bootstraps in IQ-TREE. 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, 4-3 for a list of the 54 MCV core genes. See Table 3-2 for the list of included MCV taxa and their abbreviations.

Figure 4-6. Phylogram (adapted from Go et al. 2016) illustrating the relationship of megalocytiviruses based on the major capsid protein gene (see Table 1-1 for list of all viral taxa and abbreviations). The Maximum Likelihood 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.

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 (ORF1L). Highlighted regions represent the pan-MCV primer (SACIV1L-F and SACIV1L-R) binding sites designed in this study.

CHAPTER 5 DISCUSSION

In this investigation, we report the first complete genome 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 Clade 2 MCVs have not been detected in the international ornamental fish trade since the 1991 SACIV outbreak in California and the 1991-1992 TSGIV outbreaks in Florida (Fraser et al. 1993, Go et al. 2016).

The MCV genotype of more recent outbreaks in freshwater ornamental fishes have been ISKNV

Clade 1 and outbreaks in marine ornamental fishes have been ISKNV Clade 2 (Weber et al.

2009, Sriwanayos et al. 2013, reviewed in Go et al. 2016). The pan-MCV 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 al.

2011). Similarly, the TRBIV Clade 1 MCVs have negatively impacted turbot Scophthalmus maximus and other flatfish species 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 promiscuous 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

disease in a marine food fish species rock bream (Jeong et al. 2008). Dwarf gourami can transmit

the dwarf gourami 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 tilapia 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 & Waltzek 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 3-

2) Phylogenomic 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,

4-5, 4-6). A synapomorphy (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 4-1, 4-2). 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

diversity to evade host immune responses (Pays et al. 1981, Ferreira et al. 2004). The iridovirus

major capsid protein (MCP) is the predominant structural component of the virion. The MCP is

thought to be the most important protective antigen and has been the focus of several MCV

vaccines (Caipang et al. 2006, Shinmoto 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 reported 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 4-1).

Ultrastructural examination of infected GF cells revealed non-enveloped, hexagonal, virus

particles (120-144nm) having an electron-dense core within the cytoplasm of infected GF cells, sometimes 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 observed 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). The 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).

Epithelial tissue and tissue of the nervous system were not affected (Weber et al. 2009,

Subramaniam et al. 2016). Future research is needed to better define the cellular tropism of

MCVs and the mechanism(s) by which they gain entry to and egress from hosts cells.

The growth of TSGIV in GF cells (Figure 4-1) will permit future challenge studies to unequivocally determine the role of TRBIV Clade 2 MCVs in disease (i.e., fulfilment of Koch’s

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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BIOGRAPHICAL SKETCH

Samantha received her BS in aquatic biology from the University 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 has 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 Dwelling

Creatures in Los Angeles. She also worked for the state government as a scientific aide for the

California Department of Fish and Wildlife.