Vol. 105: 1–8, 2013 DISEASES OF AQUATIC ORGANISMS Published July 9 doi: 10.3354/dao02594 Dis Aquat Org Megalocytivirus infection in orbiculate batfish Platax orbicularis Preeyanan Sriwanayos1, Ruth Francis-Floyd1,2, Mark F. Stidworthy3, Barbara D. Petty1,2, Karen Kelley4, Thomas B. Waltzek5,* 1Program in Fisheries and Aquatic Sciences, School of Forestry Resources and Conservation, University of Florida, Gainesville, Florida 32653, USA 2Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610, USA 3International Zoo Veterinary Group, Station House, Parkwood Street, Keighley, West Yorkshire BD21 4NQ, UK 4Interdisciplinary Center for Biotechnology Research (ICBR), Cellomics Division, Electron Microscopy and Bio-imaging Core Laboratory, University of Florida, Gainesville, Florida 32611, USA 5Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32608, USA ABSTRACT: Megalocytiviruses cause systemic disease in both marine and freshwater fishes, neg- atively impacting ornamental and food fish aquaculture. In this report, we characterize a megalo- cytivirus infection in a captive marine ornamental fish, the orbiculate batfish Platax orbicularis. Histologic examination revealed cytomegalic cells characterized by strongly basophilic granular intracytoplasmic inclusions within various organs. Transmission electron microscopy revealed icosahedral virus particles within the cytoplasm of cytomegalic cells consistent with an iridovirus infection. Analysis of the major capsid protein gene sequence confirmed that the orbiculate batfish virus is a member of the family Iridoviridae and is identical to the only other megalocytivirus reported from a marine ornamental fish, the Banggai cardinalfish Pterapogon kauderni iridovirus. KEY WORDS: Iridovirus · Megalocytivirus · Orbiculate batfish · Phylogeny Resale or republication not permitted without written consent of the publisher INTRODUCTION are large, double-stranded DNA viruses with an icosahedral capsid (Kurita & Nakajima 2012). Mem- Iridoviruses have been identified from various bers of the genus Megalocytivirus are pathogenic to species of fishes and are important problems for a wide variety of freshwater and marine fishes, and aquaculture industries (Ahne et al. 1997, Rodger et have had a negative impact on ornamental and food al. 1997, He et al. 2000, Paperna et al. 2001, Wang et fish aquaculture worldwide (Armstrong & Ferguson al. 2003, Shi et al. 2004, Wang et al. 2011). Members 1989, Anderson et al. 1993, Fraser et al. 1993, Rodger of the family Iridoviridae infect a range of poikilo- et al. 1997, Sudthongkong et al. 2002, Gibson-Kueh thermic hosts, including invertebrates, fish, amphib- et al. 2004, Jeong et al. 2008a,b, Chinchar et al. 2009, ians, and reptiles (Chinchar et al. 2009). The family is Weber et al. 2009, Kim et al. 2010, Yanong & Waltzek organized into 5 genera, Iridovirus, Chloriridovirus, 2010, Zhang et al. 2011). Megalocytiviruses induce Ranavirus, Megalocytivirus, and Lymphocystivirus; lethal systemic disease at water temperatures rang- the latter 3 genera infect fishes. Megalocytiviruses ing from 7.5 to 32°C (45.5 to 89.6°F) (Chen et al. 2003, *Corresponding author. Email: [email protected] © Inter-Research 2013 · www.int-res.com 2 Dis Aquat Org 105: 1–8, 2013 Yanong & Waltzek 2010, Wang et al. 2011, Zhang et type was recently recognized following disease al. 2011, Waltzek et al. 2012). Epizootics may result in episodes in ornamental and food fish species (Ku- up to 100% mortality under intensive aquaculture rita & Nakajima 2012), which included Banggai conditions (Fraser et al. 1993, Rodger et al. 1997, He cardinalfish iridovirus (BCIV) isolated from a mar- et al. 2000, Sudthongkong et al. 2002). These viruses ine ornamental species (Banggai cardinalfish Ptera- can spread horizontally from fish to fish by cohabi - pogon kau derni) and marbled sleepy goby iri- tation, exposure to water or equipment carrying dovirus (MSGIV) isolated from a freshwater species the virus, and ingestion of infected fish or feed items (marbled sleeper goby Oxyeleotris marmorata) cul- (He et al. 2002, Go & Whittington 2006, Yanong & tured for food in China (Weber et al. 2009, Wang Waltzek 2010). et al. 2011). The third species, turbot reddish body Megalocytivirus-infected fish may exhibit non- iridovirus (TRBIV), has primarily been associated specific clinical signs including lethargy, anorexia, with disease in a cultured Asian flatfish species, the hyperpigmentation, exophthalmos, skin lesions, un- turbot Scophthalmus maximus (Shi et al. 2004). A usual swimming behavior, severe anemia, and white fourth megalocytivirus species was recently pro- feces (Chen et al. 2003, Weber et al. 2009, Wang et al. posed, threespine stickleback iridovirus (TSIV), 2011, Waltzek et al. 2012). On post-mortem exam - from an epizootic that occurred in a Canadian col- ination, fish infected with megalocytiviruses may ex - lection of threespine stickleback Gasterosteus hibit hemorrhagic lesions, renomegaly, spleno meg - aculeatus (Waltzek et al. 2012). aly, hepa tomegaly, and coelomic distension resul ting Orbiculate batfish Platax orbicularis are tropical from hemorrhagic fluid accumulation (Chen et al. marine ornamental species belonging to the order 2003, Weber et al. 2009, Yanong & Waltzek 2010, Perciformes, family Ephippidae. They can be found Wang et al. 2011, Zhang et al. 2011, Waltzek et al. throughout the Western Indo-Pacific region (Capuli 2012). Histo pathologic examination typically reveals & Ortanez 2011). Orbiculate batfish occur in a variety cyto megalic mesenchymal cells characterized by of habitats: mangroves in shallow coastal waters, strongly basophilic or amphophilic granular intracy- other sheltered waters, deep seaweed reefs, and toplasmic inclusions observed in multiple organs open waters. They prefer water temperatures rang- including the spleen, kidney, liver, heart, brain, gills, ing from 22 to 28°C (71.7 to 82.4°F) (Capuli & Ortanez intestine, eyes, and gonads (Chen et al. 2003, Gib- 2011). Orbiculate batfish have only recently been son-Kueh et al. 2003, Weber et al. 2009, Yanong & successfully bred in captivity, and thus information Waltzek 2010, Zhang et al. 2011, Waltzek et al. 2012). regarding viral diseases in this species is limited Transmission electron microscopy (TEM) invariably (David et al. 2010). Only 2 viral infections, lympho- reveals nume rous icosahedral virus particles with a cystis and betanodavirus, have previously been capsid diameter between 120 and 200 nm in the cyto- reported in P. orbicularis (Lawler et al. 1978, David et plasm of infected cells (Chen et al. 2003, Weber et al. al. 2010). 2009, Yanong & Waltzek 2010, Wang et al. 2011, In the present study, we report a megalocytivirus Zhang et al. 2011, Kurita & Nakajima 2012, Waltzek infection in Platax orbicularis that was associated et al. 2012). with mortality in recently imported wild-caught fish Phylogenetic analyses support 4 separate species acquired by a public aquarium in Belgium. The ob - within the genus Megalocytivirus (Kurita & Naka- jective of this study was to compare clinical aspects of jima 2012, Waltzek et al. 2012). Megalocytiviruses the disease as well as microscopic and ultrastructural related to red sea bream iridovirus (RSIV) cluster features of the virus with previously described mega- into 1 of 2 genotypes and have been associated locytiviruses, including the virus associated with the with mass mortality epizootics in more than 30 only other marine ornamental epizootic reported in maricultured species in Japan, Korea, China, and Banggai cardinalfish. Southeast Asia (Inouye et al. 1992, Jung & Oh 2000, Kawakami & Nakajima 2002, Wang et al. 2003). Infectious spleen and kidney necrosis virus MATERIALS AND METHODS (ISKNV), originally isolated from mandarin fish Siniperca chuatsi raised for food in China, has also Clinical history resulted in epizootics in more than 10 species of freshwater ornamental fishes (He et al. 2000, A mortality event occurred in March 2010 among a Paperna et al. 2001, Sudthongkong et al. 2002, group of 10 orbiculate batfish quarantined at a public Yanong & Waltzek 2010). A second ISKNV geno- aquarium in Belgium. These wild-caught juvenile Sriwanayos et al.: Orbiculate batfish iridovirus 3 orbiculate batfish (average of 12 cm in total length) Transmission electron microscopy from Indonesia had been purchased by an orna - mental fish import facility in the UK and sent to a One paraffin block was selected by evaluating an quarantine facility in the UK, where they were accli- H&E-stained slide for features consistent with mega- matized and quarantined prior to delivery to the locytivirus pathology. Heart tissue was selected from aquarium in Belgium in February 2010. At the aquar- this block for TEM evaluation and sent to the Elec- ium, the batfish were maintained in a quarantine sys- tron Microscopy Laboratory (EML), Department of tem equipped with an external power filter. Water Medical Pathology and Laboratory Medicine, School quality parameters were maintained at a tempera- of Medicine, University of California at Davis. An ture of 24°C (75°F), pH 7.8, nitrite <0.3 mg l−1, nitrate area with characteristic megalocytivirus pathology in 0 to 12.5 mg l−1, salinity 32, 96% oxygen saturation, the heart was removed from the paraffin block and and undetectable total ammonia nitrogen. The orbic- placed in 100% xylene; after clearing