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Veterinary Microbiology 177 (2015) 270–279 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic Susceptibility of farmed juvenile giant grouper Epinephelus lanceolatus to a newly isolated grouper iridovirus (genus Ranavirus) Chao Peng a,b,1, Hongling Ma a,1, Youlu Su a, Weigeng Wen a, Juan Feng a, Zhixun Guo a, Lihua Qiu a,* 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 College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China b A R T I C L E I N F O A B S T R A C T Article history: Received 7 October 2014 Received in revised form 27 February 2015 Accepted 16 March 2015 A ranavirus was isolated from the diseased farmed groupers (Grouper iridovirus in genus Ranavirus, GIV-R), Epinephelus hybrids (blotchy rock cod, Epinephelus fuscoguttatus , giant grouper, Epinephelus lanceolatus <), in Sanya, Hainan, in July 2013. In this study, 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 infection. Histologically, severe degeneration with massive pycnotic nuclei in spleen and kidney tissues was observed, and some small-size inclusion body-bearing cells (IBCs) existed in spleen. Hemorrhage and infiltration of inflammatory cells were presented in gill, liver and heart along with tissue degeneration and necrosis of varying severity. The results 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. Keywords: Epinephelus lanceolatus Histopathology Immunohistochemistry Ranavirus 1. Introduction Iridoviridae, a large icosahedral enveloped viruses present in the cytoplasm were divided into five genus: Iridovirus, Chloriridovirus, Lymphocystivirus, Ranavirus, * Corresponding author. Tel.: +86 20 89108308. E-mail address: [email protected] (L. Qiu). 1 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. Megalocytivirus (Jancovich et al., 2012). Iridoviruses were well known as causative agents of serious systemic diseases among feral, cultured, and ornamental fish in the last decade worldwide (Wang et al., 2007). Among 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. Ranaviruses are also significant pathogens of amphibians. In contrast, lymphocystiviruses, although widespread in C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 fish, rarely cause economic loss (Whittington et al., 2010). The genus Megalocytivirus included red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis virus (ISKNV), turbot reddish body iridovirus (TRBIV), dwarf gourami iridovirus (DGIV), Taiwan grouper iridovirus (TGIV), Sea bass iridovirus (SBIV) and rock bream iridovirus (RBIV), which caused significant mortality in multiple species of marine and freshwater fish (Inoue et al., 1992; Kurita and Nakajima, 2012; Shuang et al., 2013). Histopathological features of genus Megalocytiviruses were the formation of distinctive hypertrophied cells sometimes in large numbers throughout various organs, especially spleen (Whittington et al., 2010). The frog virus 3 (FV3), epizootic haematopoietic necrosis virus (EHNV), European catfish virus (ECV), largemouth bass virus (LMBV), Singapore grouper iridovirus (SGIV) and grouper iridovirus (GIV) were classified into genus Ranaviruses, which caused severe necrosis to internal organs of many fishes, especially in spleen and renal haematopoietic tissue (Ahne et al., 1989; Chao et al., 2002; Chinchar, 2002; Langdon and Humphrey, 1987; Langdon et al., 1988, 1986; Murali et al., 2002; Pozet et al., 1992; Plumb et al., 1996, 1999; Qin et al., 2003). More and more evidences showed that ranavirus have become a significant cause of disease in ectothermic animals, and that form a virological, commercial and ecological point of view deserve additional study (Chinchar, 2002). In July, 2013, acute outbreaks of disease occurred among E. pinephelus hybrid groupers (blotchy rock cod, Epinephelus fuscoguttatus , giant grouper, Epinephelus lanceolatus <) in Sanya, Hainan. We isolated a pathogenic iridovirus from diseased hybrid groupers using MFF-1 cell lines, and named here as GIV-R-SY1301. Multiple sequence analysis identified that the whole nucleotide sequence of MCP had four base difference between king grouper iridovirus (KGIV) and Singapore grouper iridovirus (SGIV) (Unpublished data by Ma et al.,). During 2001 and 2009, 11 isolates of iridovirus were collected from giant grouper (E. lanceolatus) in Tainan (Huang et al., 2011) and iridovirus similar to GIV-R was found in giant grouper by epidemiological investigation in Hainan, China. Because E. lanceolatus as the male parent of the hybrid groupers has been cultivated with other groupers 271 widely in Southeast Asia. So, the ranavirus is also a potential threat for giant grouper aquaculture. In the present study, infection experiments were performed to examine the susceptibility of E. lanceolatus to GIV-R. By means of H&E and immunohistochemistry, we discussed the histopathological changes and distribution of GIVR in different tissues. 2. Materials and methods 2.1. Virus GIV-R-SY1301 was isolated from naturally diseased Epinephelus hybrids (blotchy rock cod, E. fuscoguttatus , giant grouper, E. lanceolatus <) (Unpulished data by Ma et al.,). GIV-R was cultured at 25 8C in MFF-1 cells maintained using Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, USA) with10% (V/V) fetal bovine serum (FBS, Gibco), 100 IU ml1penicillin G and 100 mg ml1streptomycin (Dong et al., 2008). After centrifugation (12,000 g, 10 min, 4 8C), viral culture supernatants were subdivided into small quantities and stored at 80 8C until use. Titration of viral infectivity was performed using 96-well microplates seeded with MFF-1 cells. After 5 days of culture, the appearance of cytopathic effect (CPE) was evaluated to determine the 50% tissue culture infectious dose (TCID50). 2.2. Experimental design Naive healthy E. lanceolatus (5 g, average weight) were obtained from a local farm and maintained for acclimatization in culture base of South China Sea Fisheries Research Institute, Lingshui county, Hainan Province, China. Animals were fed three times a day and the seawater was changed daily with sedimentated and sandfiltrated seawater. During the experimental period, water salinity readings were 3.2%, temperature was between 25 and 28 8C and water was kept continuous aeration. Fishes were distributed into two groups: test group (n = 36) and control group (n = 30). Test group was challenged by intraperitoneally with 105.5TCID50 fish1 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). C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 of virus cell supernatant. Control group was challenged by intraperitoneally with 100 ml fish1of DMEM medium. Both groups were maintained under similar conditions in a separate tank. Fish were kept daily management and mortality was monitored daily. The tissue of brain, eye, heart, liver, spleen, stomach, intestines, kidney and gill of moribund fish from test group were sampled for histological and immunohistochemistry analysis. When the mortality was stable, the control fish and surviving fish of test group were also sampled for histological and immunohistochemistry analysis. 2.3. DNA extraction and PCR detection Total genomic DNA (gDNA) was extracted from liver tissue using DNA extraction kit special for marine animals (Tiangen, China) following the manufacturer’s protocol. The primer-pair F (50 -ATGACTTGTACAACGGGT30 ) and R (50 -TTACAAGATAGGGAACCCCAT-30 ) were used to amplify the MCP of GIV-R (Huang et al., 2011). The PCR was performed in a total reaction volume of 25 ml containing 18.3 ml of PCR-grade water, 2.5 ml of 10 Reaction Buffer, 1 ml of dNTP mix, 1 ml of each of the primers (10 mM), 0.2 ml r-Taq DNA polymerase (TOYOBO) and 1 ml of template. The PCR parameters using the primer-pair F and R were as followings: 1 cycle of 94 8C for 10 min, 35 cycles of 94 8C for 40 s, 55 8C for 40 s, and 72 8C for 90 s, followed by the final extension at 72 8C for 10 min. 2.4. Histopathology Tissues were fixed in 10% phosphate-buffered formalin for at least 24 h and dehydrated in an ethanol–xylene series before embedding in paraffin wax. Formalin-fixed, paraffin-wax-embedded (FFPE) 5 mm tissue sections were dewaxed in xylene, rehydrated in an ethanol series and stained with haematoxylin and eosin (H&E). 2.5. Immunohistochemistry Formalin-fixed, paraffin-wax-embedded (FFPE) 5 mm tissue sections were dewaxed and rehydrated according to the conventional method. To unmask antigen and inactivate endogenous peroxidase, deparaffinised and rehydrated sections were treated with 0.01 M citrate buffer (pH 6), and heated in a microwave oven for 15 min. Slides were then washed with 0.01 M PBS (NaCl 0.14 M; KCl 2.7 mM; Na2HPO412H2O 10 mM; KH2PO4 1.7 mM; pH 7.2) three times for 5 min. Tissue sections were blocked with 5% (w/v) bovine serum albumin (BSA) for 30 min, and incubated at 37 8C for 30 min. Subsequently, 50 ml of mouse polyclonal anti-GIV-R MCP serum (1:300, dissolved by 0.01 M PBS, donated by Dr. Chuanfu Dong from the Sun Yat-sen University) was added and the slides were further incubated at 4 8C for 5 h. The tissues were then thoroughly rinsed in PBS three times for 5 min and incubated with a secondary antibody conjugated universal immunoenzyme polymer using GTVisonTMIII kit (Gene Tech, China) for 30 min at room temperature. Tissues were thoroughly rinsed in PBS and developed with DAB. The reaction was stopped by placing the slides in distilled water and slides were counterstained with hematoxylin rinsed in serial graded alcohol and xylene, and mounted with mounting media. Tissues of healthy fish and diluent-only sections were used as negative controls. 2.6. Electron microscopy Tissues were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 24 h. The samples were then washed in phosphate buffer and finally post-fixed in 1% osmium tetroxide for 1 h. The fixed tissues were dehydrated in a graded series of ethanol and embedded in Spurr’s resin. Ultrathin sections were prepared with an ultramicrotome (Leica Ultracut R, Leica Microsystems, Wetzlar, Germany), and subsequently doublestained with uranyl acetate and lead citrate and observed at 80 kV with a Jeol TEM-1200EX (Akishima, Japan). 3. Results 3.1. Clinical signs and cumulative mortality Diseased juvenile giant grouper displayed either lethargy or a dark coloration of the body, and sometimes ascites. The infected fish showed pale gill with petechiae, congestion of spleen and liver, loose and soft muscle (Fig. 1B). The mortalities started at 2 dpi and the death fastigium was during 2–5 dpi. The cumulative mortality was high to 81% within 5 dpi (Fig. 2). A GIV-R specific DNA band was detected from all infected fish by PCR (Fig. 3). 3.2. Histopathology The histopathological features of GIV-R-SY1301 infected E. lanceolatus were the severe degeneration of spleen and kidney tissues with massive pycnotic nuclei in the hematopoietic tissue (Fig. 4C and D). Hemorrhage and infiltration of inflammatory cells were found in gill, liver, heart, spleen and associated with tissue degeneration and necrosis of varying severity. 90% 80% Cumulative mortality 272 test group 70% control 60% 50% 40% 30% 20% 10% 0% 1 2 3 4 5 6 Days after injection 7 8 9 Fig. 2. Cumulative mortality of E. lanceolatus after GIV-R injection. C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 Fig. 3. PCR detection of infected E. lanceolatus. M. DL2000 DNA marker; B1, B2: Blank; C1, C2, C3: negative control; S1, S2, S3: diseased giant grouper. 273 Histopathological changes in spleen were the most remarkable. Severe degeneration occurred with some small-size inclusion body-bearing cells (IBCs) and massive pycnotic cell nuclei in splenic pulp. Spongiosis and disruption of ellipsoid sheaths with degeneration of associated cells were observed (Fig. 4C). Spleen of surviving fish showed many abnormal hypertrophic cells in splenic corpuscle (Fig. 4E). Severe degeneration of glomerulus and tubular epithelium were observed in kidney. Pycnotic cell nuclei presented in the hematopoietic tissue with karyolysis of tubular epithelium. In some case, some amorphous materials were observed in degraded tissues (Fig. 4D). Similarly, kidney of surviving fish showed masses of abnormal hypertrophic cells in renal corpuscle (Fig. 4F). Considerable inflammatory cells infiltration around the hepatic central vein and portal area were observed in the liver, causing hepatocyte atrophied and diminished. Central vein wall obviously thickened and partly disintegrated. The cell Fig. 4. E. lanceolatus. Histopathological changes in spleen, kidney and gill. (A, B) Control. (C) Spleen of diseased fish, some small-size inclusion body-bearing cells (IBCs) (white arrows) and massive pycnotic cell nuclei in splenic pulp (black arrows) were observed. (E, F) Spleen and kidney of surviving fish, masses of abnormal hypertrophic cells in splenic corpuscle and renal corpuscle were observed (M). (D) Kidney of diseased fish, severe degeneration of glomerulus (short arrows) and tubular epithelium (white arrows) were noticed. Pycnotic nuclei (stars) and amorphous materials (black arrows) were observed in the hematopoietic tissue. 274 C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 Fig. 5. E. lanceolatus. Histopathological changes in liver, heart, stomach and intestines. (A, B, E, F) Control. (C) Liver of infected fish, Infiltration of inflammatory cells and macrophages in portal area casued hepatocyte atrophy (white arrows); venous blood wall became thick and ruptured (black arrow). (D) Heart of infected fish, infiltration of inflammatory cells (star), atrophy of cardiomyocytes (black arrows), and swell of myocardial fibres resulting in reduced staining affinity (white arrows) were seen. (G) Stomach of infected fish, gastric gland atrophied and partly degraded in gastric mucosa (black arrow). (H) Gill of diseased fish, blood vessel in gill was expanded associated with tremendous erythrocyte infiltration in the sinusoid of gill filaments (star); Epithelial hyperplasia, desquamation (black arrow), epithelial lifting (short arrow), or telangiectasis (white arrow) were observed in the secondary lamellae. C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 275 Fig. 6. E. lanceolatus. Transmission electron microscopic observation of spleen. Massive virions were observed in cytoplasm (white arrows). boundaries blurred and showed necrosis in local area (Fig. 5C). In the heart, infiltration of inflammatory cells caused atrophy of cardiomyocytes with swelling of myocardial fibres, resulting in reduced staining affinity (Fig. 5D). Blood vessel in gill was expanded and fused with tremendous erythrocyte infiltration in the sinusoid of gill filaments. Epithelial hyperplasia, desquamation, epithelial lifting, fusion or telangiectasis was noticed in the secondary lamellae with the breakdown of the pillar cell system (Fig. 5H). In stomach, gastric gland atrophied and partly degraded in gastric mucosa (Fig. 5G). No histopathological change was observed in the intestines, eye and brain. Transmission electron microscopic result showed that many enveloped hexagonal virions measuring 180–200 nm were observed in cytoplasm (Fig. 6A and B). 3.3. Immunohistochemistry analysis The strongest immunolabellings were obtained from the spleen tissues. They were accumulated in the basophilic small-size inclusion body-bearing cells and were widespread in splenic pulp (Fig. 7C). In the kidney, positive reactions were mainly in the renal glomerulus and its surrounding hemopoietic tissues. But, there was no antigen labeling existed in renal tubular epithelial cells (Fig. 7D). In heart, strong positive immunolabellings were widespread in cardiac muscle fibers, epicardium and chambers of the heart (Fig. 9C and E). In the liver, a few immunolabellings were observed only in the portal area of necrotic hepatocyte (Fig. 8B). Immunolabellings in stomach were mainly in the submucosa where had rich blood vessels (Fig. 9D), secondly in the mucosa (Fig. 9F), and slightly in serosa. In the intestine, positive reactions were visualized mostly in submucosa and serosa (Fig. 8D). Immunolabellings within the gill were detected in the afferent artery and capillary vessel lumen, especially in capillary vessel lumen around cartilage tissue (Fig. 7H). Weak immunolabellings were only found in brain outer membrane (Fig. 8F). No immunolabelling was observed in eyes. Immunolabellings were only observed in splenic corpuscle and renal corpuscle in surviving fish of kidney and spleen (Fig. 7E and F). 4. Discussion Grouper, the major species being maricultured in China and other SE Asian countries, are high-priced and popular seafood fish. Nevertheless, iridoviruses have caused high mortality in many cultured grouper species in the last decades, which suggested its a severe threat to groupers aquaculture (Chua et al., 1994; Gibson-Kueh et al., 2004; Mahardika et al., 2004; Qin et al., 2003). Our infection experiments results showed that naive giant grouper was susceptible to GIV-R infection with high mortality (80%). The newly isolated ranavirus GIV-R could cause severe systemic disease to juvenile giant grouper, characterized by degeneration and necrosis of varying severity in inner organs, especially in spleen and kidney. This is the same as ranavirus and megalocytivirus infections which also caused systemic infection involving multiple internal organs with high mortalities (Williams et al., 2005). What is different is that there were only few small-size (about 10 mm) inclusion body-bearing cells (IBCs) existed in spleen, but none in other tissues. However, other iridoviruses diseased fishes showed systemic formation of prominent enlarged cells as inclusion body-bearing cells (IBCs) in different organs, such as in large yellow croaker, striped beakperch, angelfish, farmed turbot, mandarinfish, African lampeye and dwarf gourami with iridovirus infection (Chen et al., 2003; He et al., 2000; Jung and Oh, 2000; Shi et al., 2004; Sudthongkong et al., 2002), as well as in grouper fishes with the iridovirus infection: brown-spotted grouper (Chua et al., 1994), Malabar grouper (Sano et al., 2002), cultured groupers (Chou et al., 1998), juvenile humpback grouper (Mahardika et al., 2004) and hybrid grouper (Chao et al., 2004). The enlarged cells were about 20 mm in diameter and were obvious larger than GIV-R infected cells. The IBCs in some of iridovirus infected fish included three types: The early stage of IBCs were hypertrophied blast-like cells that possessed a basophilic cytoplasm and a centrally located, enlarged nucleus containing prominent nucleoli; The mature IBCs were enlarged and usually had an entirely basophilic cytoplasm and either a centrally or marginally located nucleus; The ballooning, degenerated IBCs contained an inclusion body with a granular appearance within a marginally compressed, narrow cytoplasm 276 C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 Fig. 7. E. lanceolatus. Results of immunohistochemistry in spleen, kidney and gill. The signal is observed microscopically as brown yellow staining. (A, B, G) Control. (C, D, H) Diseased fish. Strong immunolabellings were accumulated in the basophilic small-size inclusion body-bearing cells and were widespread in splenic pulp. In kidney, strong immunolabellings were observed in renal corpuscle and its surrounding hemopoietic tissues. In gill, immunolabellings were detected mainly around the afferent artery and capillary vessel lumen, especially in capillary vessel lumen around cartilage tissue (black arrow). (E, F) Surviving fish of kidney and spleen. Immunolabellings were only observed in splenic corpuscle and renal corpuscle. C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 277 Fig. 8. E. lanceolatus. Results of immunohistochemistry in liver, intestines and brain. The signal is observed microscopically as brown yellow staining. (A, C, E) Control. (B, D, F) Diseased fish. In liver, only a few immunolabellings were found in portal area of necrotic hepatocyte; In intestines, immunolabellings were detected in submucosa and serosa; In brain, only few immunolabellings were found in outer membrane of brain (black arrow). containing a pyknotic or fragmented nucleus (Mahardika et al., 2004; Sudthongkong et al., 2002). In GIV-R infected juvenile giant grouper, it seemed to have only small mature IBCs in spleen. Another prominent histopathological feature of GIV-R was the formation of massive pycnotic cell nuclei in splenic pulp and renal hematopoietic tissue. The histopathological changes in our study were most similar to ‘Sleepy Grouper Disease’ infected brown-spotted grouper (Chua et al., 1994). Immunohistochemistry using mouse GIV-R MCP antiserum showed a widespread distribution of the virus in tissues. The strongest immunolabellings were obtained from the kidney and spleen tissues. The intermediate intensity signals were observed in the heart, stomach, gill and liver tissues. The weakest signals were obtained from the intestine and brain. The signals were specifically located within epithelial, endothelial, leukomonocyte and macrophages. No signal was obtained in eyes. Immunolabellings in spleen were accumulated in the basophilic IBCs and were widespread in splenic pulp. Many reports have proved that iridovirus infected and proliferated in the enlarged cells or inclusion body bearing cells by IHC and ISH. For example, immunohistochemical and nucleic acid signals were labeled mainly in the enlarged cells in ISKNV infected zebrafish and SGIV infected Malabar grouper (Huang et al., 2004; Xu et al., 2008), DNA hybridization signals were only obtained in basophilic enlarged cells of spleen in TGIV infected Epinephelus hybrids (Chao et al., 2004). Moreover, many viral particles were observed in inclusion body-bearing cells of many iridovirus infected fishes by electron microscopy (Mahardika et al., 2004; Sudthongkong et al., 2002). Necrosis and karyolysis of tubular epithelium was serious and immunolabellings were only found in renal glomerulus and its surrounding 278 C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 Fig. 9. E. lanceolatus. Results of immunohistochemistry heart and stomach. The signal is observed microscopically as brown yellow staining. (A, B) Control. (C, D, E, F) Diseased fish. In heart, strong immunolabellings were found in cardiac muscle fibers, epicardium and chambers of the heart; In stomach, immunolabellings were mainly in submucosa and mucosa where had rich blood vessels (black arrows). hemopoietic tissues, but not in tubular epithelium. The results were similar to the report that no signal was found in tubular epithelium examined by IHC and ISH (Cano et al., 2009). This indicated that the lesions to tubular epithelium seem not to be directly virus-related. Immunolabellings in heart, liver, stomach, gill and digestive tract were mainly in the sites where it had rich blood vessels. Hence, once primary replication has taken place, virus can reach the bloodstream, resulting in viraemia, as a step leading to systemic infection (Ogawa et al., 1990). On the other hand, both splenic corpuscle and renal corpuscle of surviving fish were completely replaced by abnormal hypertrophic cells (Fig. 54 and F), while no hypertrophic cell was observed in the other parts of spleen and kidney tissues, same case was found in juvenile humpback grouper Cromileptes altivelis infected by grouper sleepy disease iridovirus (GSDIV). Mahardika et al. (2004) had been described them as ‘abnormal IBCs’ who contained deformed virions and had no granular masses associated with viral DNA and organelles (Mahardika et al., 2004). Interestingly, immunolabellings in spleen and kidney of surviving fish were only found in the abnormal hypertrophic cells, which confirmed that the abnormal hypertrophic cells contained massive viral major capsid protein, perhaps, they are deformed virions, and whether they had viral DNA remains unknown. In surviving fish, immunolabellings were only limited in splenic corpuscle and renal corpuscle, it maybe because splenic corpuscle and renal corpuscle prevented GIV-R from spreading to other sites, or splenic corpuscle and renal corpuscle were the target site of GIV-R infection and reproduction. The specific mechanism needs to further study in the future. C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 5. Conclusion To sum up, GIV-R can cause fatal systemic diseases to giant grouper. It causes acute histopathological lesions in inner organs. Antigens of GIV-R existed in most tissues of diseased giant grouper, but distributed mainly in hemopoietic tissue of spleen and kidney. Splenic corpuscle and renal corpuscle seem to have special role for GIV-R infection. Acknowledgements This research was supported by the key Science and Technology Program of Hainan Province under Grant No. ZDXM20120031 and Special Foundation of Fish Disease Prevention and Control of Guangdong Province (2012). References Ahne, W., Schlotfeldt, H., Thomsen, I., 1989. Fish viruses: isolation of an icosahedral cytoplasmic deoxyribovirus from sheatfish (Silurus glanis). J. Vet. Med. Series B 36, 333–336. Cano, I., Ferro, P., Alonso, M.C., Sarasquete, C., Garcia-Rosado, E., Borrego, J.J., Castro, D., 2009. Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L. J. Fish Dis. 32, 143–150. Chao, C.-B., Chen, C.-Y., Lai, Y.-Y., Lin, C.-S., Huang, H.-T., 2004. Histological, ultrastructural, and in situ hybridization study on enlarged cells in grouper Epinephelus hybrids infected by grouper iridovirus in Taiwan (TGIV). Dis. Aquat. Org. 58, 127–142. Chao, C., Yang, S., Tsai, H., Chen, C., Lin, C., Huang, H., 2002. A nested PCR for the detection of grouper iridovirus in Taiwan (TGIV) in cultured hybrid grouper, giant seaperch, and largemouth bass. J. Aquat. Anim. Health 14, 104–113. Chen, X.H., Lin, K.B., Wang, X.W., 2003. Outbreaks of an iridovirus disease in maricultured large yellow croaker, Larimichthys crocea (Richardson), in China. J. Fish Dis. 26, 615–619. Chinchar, V., 2002. Ranaviruses (family Iridoviridae): emerging coldblooded killers. Arch. Virol. 147, 447–470. Chou, H.Y., Hsu, C.C., Peng, T.Y., 1998. Isolation and characterization of a pathogenic iridovirus from cultured grouper (Epinephelus sp.) in Taiwan. Fish Pathol. (Japan). Chua, F., Ng, M., Ng, K., Loo, J., Wee, J., 1994. Investigation of outbreaks of a novel disease,‘Sleepy Grouper Disease’, affecting the brown-spotted grouper, Epinephelus tauvina Forskal. J. Fish Dis. 17, 417–427. Dong, C., Weng, S., Shi, X., Xu, X., Shi, N., He, J., 2008. Development of a mandarin fish Siniperca chuatsi fry cell line suitable for the study of infectious spleen and kidney necrosis virus (ISKNV). Virus Res. 135, 273–281. Gibson-Kueh, S., Ngoh-Lim, G., Netto, P., Kurita, J., Nakajima, K., >Ng, M., 2004. A systemic iridoviral disease in mullet, Mugil cephalus L., and tiger grouper, Epinephelus fuscoguttatus Forsskal: a first report and study. J. Fish Dis. 27, 693–699. He, J., Wang, S., Zeng, K., Huang, Z., Chan, S.M., 2000. Systemic disease caused by an iridovirus-like agent in cultured mandarinfish, Siniperca chuatsi (Basilewsky), in China. J. Fish Dis. 23, 219–222. Huang, C., Zhang, X., Gin, K.Y.H., Qin, Q.W., 2004. In situ hybridization of a marine fish virus, Singapore grouper iridovirus with a nucleic acid probe of major capsid protein. J. Virol. Methods 117, 123–128. Huang, S.-M., Tu, C., Tseng, C.-H., Huang, C.-C., Chou, C.-C., Kuo, H.-C., Chang, S.-K., 2011. Genetic analysis of fish iridoviruses isolated in Taiwan during 2001–2009. Arch. Virol. 156, 1505–1515. 279 Inoue, K., Yamano, K., Maeno, Y., Nakajima, K., Matsuoka, M., Wada, Y., Sorimachi, M., 1992. Iridovirus infection of cultured red sea bream, Pagrus major. Fish Pathol. (Japan). Jancovich, J.K., Chinchar, V.G., Hyatt, A., Miyazaki, T., Williams, T., Zhang, Q.Y., 2012. In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Family Iridoviridae. In: Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San Diego, CA, pp. 193–210. Jung, S., Oh, M., 2000. Iridovirus-like infection associated with high mortalities of striped beakperch, Oplegnathus fasciatus (Temminck et Schlegel), in southern coastal areas of the Korean peninsula. J. Fish Dis. 23, 223–226. Kurita, J., Nakajima, K., 2012. Megalocytiviruses. Viruses 4, 521–538. Langdon, J., Humphrey, J., 1987. Epizootic haematopoietic necrosis, a new viral disease in redfin perch, Perca fluviatilis L., in Australia. J. Fish Dis. 10, 289–297. Langdon, J., Humphrey, J., Williams, L., 1988. Outbreaks of an EHNV-like iridovirus in cultured rainbow trout, Salmo gairdneri Richardson, in Australia. J. Fish Dis. 11, 93–96. Langdon, J., Humphrey, J., Williams, L., Hyatt, A., Westbury, H., 1986. First virus isolation from Australian fish: an iridovirus-like pathogen from redfin perch, Perca fluviatilis L. J. Fish Dis. 9, 263–268. Mahardika, K., Yamamoto, A., Miyazaki, T., 2004. Susceptibility of juvenile humpback grouper Cromileptes altivelis to grouper sleepy disease iridovirus (GSDIV). Dis. Aquat. Org. 59, 1–9. Murali, S., Wu, M.F., Guo, I.C., Chen, S.C., Yang, H.W., Chang, C.Y., 2002. Molecular characterization and pathogenicity of a grouper iridovirus (GIV) isolated from yellow grouper, Epinephelus awoara (Temminck &Schlegel). J. Fish Dis. 25, 91–100. Ogawa, M., Ahne, W., Fischer-Scherl, T., Hoffmann, R.W., Schlotfeldt, H.J., 1990. Pathomorphological alterations in sheatfish fry Silurus glanis experimentally infected with iridovirus-like agent. Dis. Aquat. Org. 9, 187–191. Plumb, J.A., Grizzle, J.M., Young, H.E., Noyes, A.D., Lamprecht, S., 1996. An iridovirus isolated from wild largemouth bass. J. Aquat. Anim. Health 8, 265–270. Plumb, J.A., Noyes, A.D., Graziano, S., Wang, J., Mao, J., Chinchar, V.G., 1999. Isolation and identification of viruses from adult largemouth bass during a 1997-1998 survey in the southeastern United States. J. Aquat. Anim. Health 11, 391–399. Pozet, F., Morand, M., Moussa, A., Torhy, C., De Kinkelin, P., 1992. Isolation and preliminary characterization of a pathogenic icosahedral deoxyribovirus from the catfish Ictalurus melas. Dis. Aquat. Org. 14, 35–42. Qin, Q., Chang, S., Ngoh-Lim, G., Gibson-Kueh, S., Shi, C., Lam, T., 2003. Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina. Dis. Aquat. Org. 53, 1–9. Sano, M., Minagawa, M., Nakajima, K., 2002. Multiplication of red sea bream iridovirus (RSIV) in the experimentally infected grouper Epinephelus malabaricus. Fish Pathol. (Japan). Shi, C.-Y., Wang, Y.-G., Yang, S.-L., Huang, J., Wang, Q.-Y., 2004. The first report of an iridovirus-like agent infection in farmed turbot, Scophthalmus maximus, in China. Aquaculture 236, 11–25. Shuang, F., Luo, Y., Xiong, X., Weng, S., Li, Y., He, J., 2013. Virions proteins of an RSIV-type megalocytivirus from spotted knifejaw Oplegnathus punctatus (SKIV-ZJ07). Virology 437, 27–37. Sudthongkong, C., Miyata, M., Miyazaki, T., 2002. Iridovirus disease in two ornamental tropical freshwater fishes: African lampeye and dwarf gourami. Dis. Aquat. Org. 48, 163–173. Wang, Y., Lü, L., Weng, S., Huang, J., Chan, S.-M., He, J., 2007. Molecular epidemiology and phylogenetic analysis of a marine fish infectious spleen and kidney necrosis virus-like (ISKNV-like) virus. Arch. Virol. 152, 763–773. Whittington, R., Becker, J., Dennis, M., 2010. Iridovirus infections in finfish–critical review with emphasis on ranaviruses. J. Fish Dis. 33, 95–122. Williams, T., Barbosa-Solomieu, V., Chinchar, V.G., 2005. A decade of advances in iridovirus research. Adv. Virus Res. 65, 174. Xu, X., Zhang, L., Weng, S., Huang, Z., Lu, J., Lan, D., Zhong, X., Yu, X., Xu, A., He, J., 2008. A zebrafish model of infectious spleen and kidney necrosis virus (ISKNV) infection. Virology 376, 1–12.