Veterinary Microbiology 177 (2015) 270–279 Contents lists available at ScienceDirect Veterinary Microbiology jou rnal homepage: www.elsevier.com/locate/vetmic Susceptibility of farmed juvenile giant grouper Epinephelus lanceolatus to a newly isolated grouper iridovirus (genus Ranavirus) a,b,1 a,1 a a a Chao Peng , Hongling Ma , Youlu Su , Weigeng Wen , Juan Feng , a a, Zhixun Guo , Lihua Qiu * a Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture, The South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, PR China b College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China A R T I C L E I N F O A B S T R A C T A ranavirus was isolated from the diseased farmed groupers (Grouper iridovirus in genus Article history: Received 7 October 2014 Ranavirus, GIV-R), Epinephelus hybrids (blotchy rock cod, Epinephelus fuscoguttatus Received in revised form 27 February 2015 , Â giant grouper, Epinephelus lanceolatus <), in Sanya, Hainan, in July 2013. In this study, Accepted 16 March 2015 susceptibility of farmed juvenile giant grouper E. lanceolatus to GIV-R was determined by intraperitoneally injection. The cumulative mortality reached to 81% at 5 day post Keywords: infection. Histologically, severe degeneration with massive pycnotic nuclei in spleen and Epinephelus lanceolatus kidney tissues was observed, and some small-size inclusion body-bearing cells (IBCs) Histopathology existed in spleen. Hemorrhage and infiltration of inflammatory cells were presented in gill, Immunohistochemistry liver and heart along with tissue degeneration and necrosis of varying severity. The results Ranavirus of immunohistochemistry analysis showed that the strongest immunolabellings were obtained from the kidney and spleen tissues, while intermediate intensity signals were observed in the heart, stomach, gill and liver tissues, and the weakest signals were obtained from the intestine and brain, but no signal was obtained in eyes. Electron microscopy revealed that spleen of moribund fish contained many viral particles in cytoplasm. Interestingly, in surviving fish, abnormal hypertrophic cells were observed in both splenic corpuscle and renal corpuscle, while no hypertrophic cell was observed in the other parts of spleen and kidney tissues. Moreover, immunolabellings only stained the hypertrophic cells in splenic corpuscle and renal corpuscle. This indicated that splenic corpuscle and renal corpuscle play an important role in GIV-R infection and replication. ß 2015 Elsevier B.V. All rights reserved. 1. Introduction Megalocytivirus (Jancovich et al., 2012). Iridoviruses were well known as causative agents of serious systemic Iridoviridae, a large icosahedral enveloped viruses diseases among feral, cultured, and ornamental fish in present in the cytoplasm were divided into five genus: the last decade worldwide (Wang et al., 2007). Among Iridovirus, Chloriridovirus, Lymphocystivirus, Ranavirus, family Iridoviridae, members of genus Lymphocystivirus, Ranaviruses and Megalocytiviruses affected finfish. Both ranaviruses and megalocytiviruses cause severe systemic disease, occur globally and affect a diversity of hosts. * Corresponding author. Tel.: +86 20 89108308. Ranaviruses are also significant pathogens of amphibians. E-mail address: [email protected] (L. Qiu). 1 In contrast, lymphocystiviruses, although widespread in These authors contributed equally to this paper. http://dx.doi.org/10.1016/j.vetmic.2015.03.017 0378-1135/ß 2015 Elsevier B.V. All rights reserved. C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 271 fish, rarely cause economic loss (Whittington et al., 2010). widely in Southeast Asia. So, the ranavirus is also a The genus Megalocytivirus included red sea bream iridovirus potential threat for giant grouper aquaculture. In the (RSIV), infectious spleen and kidney necrosis virus (ISKNV), present study, infection experiments were performed to turbot reddish body iridovirus (TRBIV), dwarf gourami examine the susceptibility of E. lanceolatus to GIV-R. By iridovirus (DGIV), Taiwan grouper iridovirus (TGIV), Sea means of H&E and immunohistochemistry, we discussed bass iridovirus (SBIV) and rock bream iridovirus (RBIV), the histopathological changes and distribution of GIVR in which caused significant mortality in multiple species of different tissues. marine and freshwater fish (Inoue et al., 1992; Kurita and Nakajima, 2012; Shuang et al., 2013). Histopathological 2. Materials and methods features of genus Megalocytiviruses were the formation of distinctive hypertrophied cells sometimes in large numbers 2.1. Virus throughout various organs, especially spleen (Whittington et al., 2010). The frog virus 3 (FV3), epizootic haematopoietic GIV-R-SY1301 was isolated from naturally diseased necrosis virus (EHNV), European catfish virus (ECV), large- Epinephelus hybrids (blotchy rock cod, E. fuscoguttatus mouth bass virus (LMBV), Singapore grouper iridovirus , Â giant grouper, E. lanceolatus <) (Unpulished data by (SGIV) and grouper iridovirus (GIV) were classified into Ma et al.,). GIV-R was cultured at 25 8C in MFF-1 cells genus Ranaviruses, which caused severe necrosis to internal maintained using Dulbecco’s modified Eagle’s medium organs of many fishes, especially in spleen and renal (DMEM) (Invitrogen, USA) with10% (V/V) fetal bovine À1 haematopoietic tissue (Ahne et al., 1989; Chao et al., 2002; serum (FBS, Gibco), 100 IU ml penicillin G and À1 Chinchar, 2002; Langdon and Humphrey, 1987; Langdon et 100 mg ml streptomycin (Dong et al., 2008). After al., 1988, 1986; Murali et al., 2002; Pozet et al., 1992; Plumb centrifugation (12,000 Â g, 10 min, 4 8C), viral culture et al., 1996, 1999; Qin et al., 2003). More and more evidences supernatants were subdivided into small quantities and showed that ranavirus have become a significant cause of stored at À80 8C until use. Titration of viral infectivity was disease in ectothermic animals, and that form a virological, performed using 96-well microplates seeded with MFF-1 commercial and ecological point of view deserve additional cells. After 5 days of culture, the appearance of cytopathic study (Chinchar, 2002). effect (CPE) was evaluated to determine the 50% tissue In July, 2013, acute outbreaks of disease occurred culture infectious dose (TCID50). among E. pinephelus hybrid groupers (blotchy rock cod, Epinephelus fuscoguttatus , Â giant grouper, Epinephelus 2.2. Experimental design lanceolatus <) in Sanya, Hainan. We isolated a pathogenic iridovirus from diseased hybrid groupers using MFF-1 Naive healthy E. lanceolatus (5 g, average weight) were cell lines, and named here as GIV-R-SY1301. Multiple obtained from a local farm and maintained for acclimati- sequence analysis identified that the whole nucleotide zation in culture base of South China Sea Fisheries sequence of MCP had four base difference between king Research Institute, Lingshui county, Hainan Province, grouper iridovirus (KGIV) and Singapore grouper irido- China. Animals were fed three times a day and the virus (SGIV) (Unpublished data by Ma et al.,). During 2001 seawater was changed daily with sedimentated and sand- and 2009, 11 isolates of iridovirus were collected from filtrated seawater. During the experimental period, water giant grouper (E. lanceolatus) in Tainan (Huang et al., salinity readings were 3.2%, temperature was between 25 2011) and iridovirus similar to GIV-R was found in giant and 28 8C and water was kept continuous aeration. grouper by epidemiological investigation in Hainan, Fishes were distributed into two groups: test group China. Because E. lanceolatus as the male parent of the (n = 36) and control group (n = 30). Test group was 5.5 À1 hybrid groupers has been cultivated with other groupers challenged by intraperitoneally with 10 TCID50 fish Fig. 1. Clinical signs of GIV-R-infected E. lanceolatus. A. Control fish. B. GIV-R infected fish showed soft muscle (black arrow) and congestion of spleen, liver and gill (white arrow). 272 C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 of virus cell supernatant. Control group was challenged by with DAB. The reaction was stopped by placing the slides À1 intraperitoneally with 100 ml fish of DMEM medium. in distilled water and slides were counterstained with Both groups were maintained under similar conditions hematoxylin rinsed in serial graded alcohol and xylene, in a separate tank. Fish were kept daily management and and mounted with mounting media. Tissues of healthy mortality was monitored daily. The tissue of brain, eye, fish and diluent-only sections were used as negative heart, liver, spleen, stomach, intestines, kidney and gill of controls. moribund fish from test group were sampled for histologi- cal and immunohistochemistry analysis. When the mor- 2.6. Electron microscopy tality was stable, the control fish and surviving fish of test group were also sampled for histological and immunohis- Tissues were fixed in 2.5% glutaraldehyde in 0.1 M tochemistry analysis. phosphate buffer, pH 7.3, for 24 h. The samples were then washed in phosphate buffer and finally post-fixed 2.3. DNA extraction and PCR detection in 1% osmium tetroxide for 1 h. The fixed tissues were dehydrated in a graded series of ethanol and embedded Total genomic DNA (gDNA) was extracted from liver in Spurr’s resin. Ultrathin sections were prepared with tissue using DNA extraction kit special for marine an ultramicrotome (Leica Ultracut R, Leica Microsys- animals (Tiangen, China) following the manufacturer’s