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usceptibility of farmed juvenile giant grouperpinephelus lanceolatus to a newly isolated grouper iridovirusenus Ranavirus)

ao Peng a,b,1, Hongling Ma a,1, Youlu Su a, Weigeng Wen a, Juan Feng a,ixun Guo a, Lihua Qiu a,*

ey Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture, The South China Sea Fisheries

search Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, PR China

ollege of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China

Introduction

Iridoviridae, a large icosahedral enveloped virusesesent in the cytoplasm were divided into five genus:dovirus, Chloriridovirus, Lymphocystivirus, Ranavirus,

Megalocytivirus (Jancovich et al., 2012). Iridoviruses werewell known as causative agents of serious systemicdiseases among feral, cultured, and ornamental fish inthe last decade worldwide (Wang et al., 2007). Amongfamily Iridoviridae, members of genus Lymphocystivirus,Ranaviruses and Megalocytiviruses affected finfish. Bothranaviruses and megalocytiviruses cause severe systemicdisease, occur globally and affect a diversity of hosts.Ranaviruses are also significant pathogens of amphibians.In contrast, lymphocystiviruses, although widespread in

R T I C L E I N F O

icle history:

ceived 7 October 2014

ceived in revised form 27 February 2015

cepted 16 March 2015

ywords:

inephelus lanceolatus

stopathology

munohistochemistry

navirus

A B S T R A C T

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.

� 2015 Elsevier B.V. All rights reserved.

Corresponding author. Tel.: +86 20 89108308.

E-mail address: qiu902@126.com (L. Qiu).

These authors contributed equally to this paper.

Contents lists available at ScienceDirect

Veterinary Microbiology

jou r nal h o mep ag e: w ww .e ls evier . co m/lo c ate /vetm i c

p://dx.doi.org/10.1016/j.vetmic.2015.03.017

78-1135/� 2015 Elsevier B.V. All rights reserved.

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C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 271

sh, rarely cause economic loss (Whittington et al., 2010).he genus Megalocytivirus included red sea bream iridovirusSIV), infectious spleen and kidney necrosis virus (ISKNV),rbot reddish body iridovirus (TRBIV), dwarf gourami

idovirus (DGIV), Taiwan grouper iridovirus (TGIV), Seaass iridovirus (SBIV) and rock bream iridovirus (RBIV),hich caused significant mortality in multiple species ofarine and freshwater fish (Inoue et al., 1992; Kurita andakajima, 2012; Shuang et al., 2013). Histopathologicalatures of genus Megalocytiviruses were the formation of

istinctive hypertrophied cells sometimes in large numbersroughout various organs, especially spleen (Whittington

t al., 2010). The frog virus 3 (FV3), epizootic haematopoieticecrosis virus (EHNV), European catfish virus (ECV), large-outh bass virus (LMBV), Singapore grouper iridovirusGIV) and grouper iridovirus (GIV) were classified into

enus Ranaviruses, which caused severe necrosis to internalrgans of many fishes, especially in spleen and renalaematopoietic tissue (Ahne et al., 1989; Chao et al., 2002;hinchar, 2002; Langdon and Humphrey, 1987; Langdon etl., 1988, 1986; Murali et al., 2002; Pozet et al., 1992; Plumbt al., 1996, 1999; Qin et al., 2003). More and more evidenceshowed that ranavirus have become a significant cause ofisease in ectothermic animals, and that form a virological,ommercial and ecological point of view deserve additionaltudy (Chinchar, 2002).

In July, 2013, acute outbreaks of disease occurredmong E. pinephelus hybrid groupers (blotchy rock cod,pinephelus fuscoguttatus , � giant grouper, Epinephelus

nceolatus <) in Sanya, Hainan. We isolated a pathogenicidovirus from diseased hybrid groupers using MFF-1ell lines, and named here as GIV-R-SY1301. Multipleequence analysis identified that the whole nucleotideequence of MCP had four base difference between kingrouper iridovirus (KGIV) and Singapore grouper irido-irus (SGIV) (Unpublished data by Ma et al.,). During 2001nd 2009, 11 isolates of iridovirus were collected fromiant grouper (E. lanceolatus) in Tainan (Huang et al.,011) and iridovirus similar to GIV-R was found in giantrouper by epidemiological investigation in Hainan,hina. Because E. lanceolatus as the male parent of theybrid groupers has been cultivated with other groupers

widely in Southeast Asia. So, the ranavirus is also apotential threat for giant grouper aquaculture. In thepresent study, infection experiments were performed toexamine the susceptibility of E. lanceolatus to GIV-R. Bymeans of H&E and immunohistochemistry, we discussedthe histopathological changes and distribution of GIVR indifferent tissues.

2. Materials and methods

2.1. Virus

GIV-R-SY1301 was isolated from naturally diseasedEpinephelus hybrids (blotchy rock cod, E. fuscoguttatus

, � giant grouper, E. lanceolatus <) (Unpulished data byMa et al.,). GIV-R was cultured at 25 8C in MFF-1 cellsmaintained using Dulbecco’s modified Eagle’s medium(DMEM) (Invitrogen, USA) with10% (V/V) fetal bovineserum (FBS, Gibco), 100 IU ml�1penicillin G and100 mg ml�1streptomycin (Dong et al., 2008). Aftercentrifugation (12,000 � g, 10 min, 4 8C), viral culturesupernatants were subdivided into small quantities andstored at �80 8C until use. Titration of viral infectivity wasperformed using 96-well microplates seeded with MFF-1cells. After 5 days of culture, the appearance of cytopathiceffect (CPE) was evaluated to determine the 50% tissueculture infectious dose (TCID50).

2.2. Experimental design

Naive healthy E. lanceolatus (5 g, average weight) wereobtained from a local farm and maintained for acclimati-zation in culture base of South China Sea FisheriesResearch Institute, Lingshui county, Hainan Province,China. Animals were fed three times a day and theseawater was changed daily with sedimentated and sand-filtrated seawater. During the experimental period, watersalinity readings were 3.2%, temperature was between 25and 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 waschallenged by intraperitoneally with 105.5TCID50 fish�1

ig. 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

nd gill (white arrow).

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virus cell supernatant. Control group was challenged bytraperitoneally with 100 ml fish�1of DMEM medium.th groups were maintained under similar conditions

a separate tank. Fish were kept daily management andortality was monitored daily. The tissue of brain, eye,art, liver, spleen, stomach, intestines, kidney and gill oforibund fish from test group were sampled for histologi-l and immunohistochemistry analysis. When the mor-lity was stable, the control fish and surviving fish of testoup were also sampled for histological and immunohis-chemistry analysis.

. DNA extraction and PCR detection

Total genomic DNA (gDNA) was extracted from liversue using DNA extraction kit special for marineimals (Tiangen, China) following the manufacturer’sotocol. The primer-pair F (50-ATGACTTGTACAACGGGT-) and R (50-TTACAAGATAGGGAACCCCAT-30) were used

amplify the MCP of GIV-R (Huang et al., 2011). The PCRas performed in a total reaction volume of 25 mlntaining 18.3 ml of PCR-grade water, 2.5 ml of 10�action Buffer, 1 ml of dNTP mix, 1 ml of each of theimers (10 mM), 0.2 ml r-Taq DNA polymeraseOYOBO) and 1 ml of template. The PCR parametersing the primer-pair F and R were as followings: 1 cycle

94 8C for 10 min, 35 cycles of 94 8C for 40 s, 55 8C for s, and 72 8C for 90 s, followed by the final extension at 8C for 10 min.

. Histopathology

Tissues were fixed in 10% phosphate-buffered formalinr at least 24 h and dehydrated in an ethanol–xyleneries before embedding in paraffin wax. Formalin-fixed,raffin-wax-embedded (FFPE) 5 mm tissue sections werewaxed in xylene, rehydrated in an ethanol series andined with haematoxylin and eosin (H&E).

. Immunohistochemistry

Formalin-fixed, paraffin-wax-embedded (FFPE) 5 mmsue sections were dewaxed and rehydrated according

the conventional method. To unmask antigen andactivate endogenous peroxidase, deparaffinised andhydrated sections were treated with 0.01 M citrateffer (pH 6), and heated in a microwave oven for 15 min.

ides were then washed with 0.01 M PBS (NaCl 0.14 M;l 2.7 mM; Na2HPO4�12H2O 10 mM; KH2PO4 1.7 mM;

7.2) three times for 5 min. Tissue sections wereocked with 5% (w/v) bovine serum albumin (BSA) for

min, and incubated at 37 8C for 30 min. Subsequently, ml of mouse polyclonal anti-GIV-R MCP serum (1:300,ssolved by 0.01 M PBS, donated by Dr. Chuanfu Dongm the Sun Yat-sen University) was added and the

des were further incubated at 4 8C for 5 h. The tissuesere then thoroughly rinsed in PBS three times for 5 mind incubated with a secondary antibody conjugatediversal immunoenzyme polymer using GTVisonTMIII

t (Gene Tech, China) for 30 min at room temperature.

with DAB. The reaction was stopped by placing the slidesin distilled water and slides were counterstained withhematoxylin rinsed in serial graded alcohol and xylene,and mounted with mounting media. Tissues of healthyfish and diluent-only sections were used as negativecontrols.

2.6. Electron microscopy

Tissues were fixed in 2.5% glutaraldehyde in 0.1 Mphosphate buffer, pH 7.3, for 24 h. The samples werethen washed in phosphate buffer and finally post-fixedin 1% osmium tetroxide for 1 h. The fixed tissues weredehydrated in a graded series of ethanol and embeddedin Spurr’s resin. Ultrathin sections were prepared withan ultramicrotome (Leica Ultracut R, Leica Microsys-tems, Wetzlar, Germany), and subsequently double-stained with uranyl acetate and lead citrate andobserved at 80 kV with a Jeol TEM-1200EX (Akishima,Japan).

3. Results

3.1. Clinical signs and cumulative mortality

Diseased juvenile giant grouper displayed eitherlethargy or a dark coloration of the body, and sometimesascites. 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 deathfastigium was during 2–5 dpi. The cumulative mortalitywas high to 81% within 5 dpi (Fig. 2). A GIV-R specific DNAband was detected from all infected fish by PCR (Fig. 3).

3.2. Histopathology

The histopathological features of GIV-R-SY1301infected E. lanceolatus were the severe degeneration ofspleen and kidney tissues with massive pycnotic nuclei inthe hematopoietic tissue (Fig. 4C and D). Hemorrhage andinfiltration of inflammatory cells were found in gill, liver,heart, spleen and associated with tissue degeneration andnecrosis of varying severity.

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Fig. 2. Cumulative mortality of E. lanceolatus after GIV-R injection.

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Histopathological changes in spleen were the mostremarkable. Severe degeneration occurred with somesmall-size inclusion body-bearing cells (IBCs) andmassive pycnotic cell nuclei in splenic pulp. Spongiosisand disruption of ellipsoid sheaths with degeneration ofassociated cells were observed (Fig. 4C). Spleenof surviving fish showed many abnormal hypertrophiccells in splenic corpuscle (Fig. 4E). Severe degeneration ofglomerulus and tubular epithelium were observed inkidney. Pycnotic cell nuclei presented in the hematopoi-etic tissue with karyolysis of tubular epithelium. In somecase, some amorphous materials were observed indegraded tissues (Fig. 4D). Similarly, kidney of survivingfish showed masses of abnormal hypertrophic cells inrenal corpuscle (Fig. 4F). Considerable inflammatorycells infiltration around the hepatic central vein andportal area were observed in the liver, causing hepato-cyte atrophied and diminished. Central vein wallobviously thickened and partly disintegrated. The cell

ig. 3. PCR detection of infected E. lanceolatus. M. DL2000 DNA marker; B1,

2: Blank; C1, C2, C3: negative control; S1, S2, S3: diseased giant grouper.

ig. 4. E. lanceolatus. Histopathological changes in spleen, kidney and gill. (A, B) Control. (C) Spleen of diseased fish, some small-size inclusion body-bearing

ells (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

bnormal hypertrophic cells in splenic corpuscle and renal corpuscle were observed (M). (D) Kidney of diseased fish, severe degeneration of glomerulus (short

rrows) and tubular epithelium (white arrows) were noticed. Pycnotic nuclei (stars) and amorphous materials (black arrows) were observed in the

ematopoietic tissue.

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) 270–279274

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oundaries blurred and showed necrosis in local areaFig. 5C). In the heart, infiltration of inflammatory cellsaused atrophy of cardiomyocytes with swelling ofyocardial fibres, resulting in reduced staining affinity

Fig. 5D). Blood vessel in gill was expanded and fusedith tremendous erythrocyte infiltration in the sinusoid

f gill filaments. Epithelial hyperplasia, desquamation,pithelial lifting, fusion or telangiectasis was noticed inhe secondary lamellae with the breakdown of the pillarell system (Fig. 5H). In stomach, gastric gland atrophiednd partly degraded in gastric mucosa (Fig. 5G). Noistopathological change was observed in the intestines,ye and brain. Transmission electron microscopic resulthowed that many enveloped hexagonal virions mea-uring 180–200 nm were observed in cytoplasm (Fig. 6And B).

.3. Immunohistochemistry analysis

The strongest immunolabellings were obtained fromhe spleen tissues. They were accumulated in theasophilic small-size inclusion body-bearing cells andere widespread in splenic pulp (Fig. 7C). In the kidney,

ositive reactions were mainly in the renal glomerulusnd its surrounding hemopoietic tissues. But, there waso antigen labeling existed in renal tubular epithelialells (Fig. 7D). In heart, strong positive immunolabel-ngs were widespread in cardiac muscle fibers, epicar-ium and chambers of the heart (Fig. 9C and E). In thever, a few immunolabellings were observed only in theortal area of necrotic hepatocyte (Fig. 8B). Immunola-ellings in stomach were mainly in the submucosahere had rich blood vessels (Fig. 9D), secondly in theucosa (Fig. 9F), and slightly in serosa. In the intestine,

ositive reactions were visualized mostly in submucosand serosa (Fig. 8D). Immunolabellings within the gillere detected in the afferent artery and capillary vesselmen, especially in capillary vessel lumen around

artilage tissue (Fig. 7H). Weak immunolabellings werenly found in brain outer membrane (Fig. 8F). No

munolabelling was observed in eyes. Immunolabel-ngs were only observed in splenic corpuscle and renalorpuscle in surviving fish of kidney and spleen (Fig. 7End F).

4. Discussion

Grouper, the major species being maricultured in Chinaand other SE Asian countries, are high-priced and popularseafood fish. Nevertheless, iridoviruses have caused highmortality in many cultured grouper species in the lastdecades, which suggested its a severe threat to groupersaquaculture (Chua et al., 1994; Gibson-Kueh et al., 2004;Mahardika et al., 2004; Qin et al., 2003). Our infectionexperiments results showed that naive giant grouper wassusceptible to GIV-R infection with high mortality (80%).

The newly isolated ranavirus GIV-R could cause severesystemic disease to juvenile giant grouper, characterizedby degeneration and necrosis of varying severity in innerorgans, especially in spleen and kidney. This is the same asranavirus and megalocytivirus infections which alsocaused systemic infection involving multiple internalorgans with high mortalities (Williams et al., 2005). Whatis different is that there were only few small-size (about10 mm) inclusion body-bearing cells (IBCs) existed inspleen, but none in other tissues. However, otheriridoviruses diseased fishes showed systemic formationof 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 iridovirusinfection (Chen et al., 2003; He et al., 2000; Jung andOh, 2000; Shi et al., 2004; Sudthongkong et al., 2002), aswell as in grouper fishes with the iridovirus infection:brown-spotted grouper (Chua et al., 1994), Malabargrouper (Sano et al., 2002), cultured groupers (Chouet al., 1998), juvenile humpback grouper (Mahardikaet al., 2004) and hybrid grouper (Chao et al., 2004). Theenlarged cells were about 20 mm in diameter and wereobvious larger than GIV-R infected cells. The IBCs in someof iridovirus infected fish included three types: The earlystage of IBCs were hypertrophied blast-like cells thatpossessed a basophilic cytoplasm and a centrally located,enlarged nucleus containing prominent nucleoli; Themature IBCs were enlarged and usually had an entirelybasophilic cytoplasm and either a centrally or marginallylocated nucleus; The ballooning, degenerated IBCs con-tained an inclusion body with a granular appearancewithin a marginally compressed, narrow cytoplasm

Fig. 6. E. lanceolatus. Transmission electron microscopic observation of spleen. Massive virions were observed in cytoplasm (white arrows).

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.

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ontaining a pyknotic or fragmented nucleus (Mahardikat al., 2004; Sudthongkong et al., 2002). In GIV-R infectedvenile giant grouper, it seemed to have only smallature IBCs in spleen. Another prominent histopatholog-al feature of GIV-R was the formation of massive pycnotic

ell nuclei in splenic pulp and renal hematopoietic tissue.he histopathological changes in our study were mostimilar to ‘Sleepy Grouper Disease’ infected brown-spottedrouper (Chua et al., 1994).

Immunohistochemistry using mouse GIV-R MCP anti-erum showed a widespread distribution of the virus inssues. The strongest immunolabellings were obtainedom the kidney and spleen tissues. The intermediatetensity signals were observed in the heart, stomach, gill

nd liver tissues. The weakest signals were obtained frome intestine and brain. The signals were specificallycated within epithelial, endothelial, leukomonocyte and

macrophages. No signal was obtained in eyes. Immuno-labellings in spleen were accumulated in the basophilicIBCs and were widespread in splenic pulp. Many reportshave proved that iridovirus infected and proliferated in theenlarged cells or inclusion body bearing cells by IHC andISH. For example, immunohistochemical and nucleic acidsignals were labeled mainly in the enlarged cells in ISKNVinfected zebrafish and SGIV infected Malabar grouper(Huang et al., 2004; Xu et al., 2008), DNA hybridizationsignals were only obtained in basophilic enlarged cells ofspleen in TGIV infected Epinephelus hybrids (Chao et al.,2004). Moreover, many viral particles were observed ininclusion body-bearing cells of many iridovirus infectedfishes by electron microscopy (Mahardika et al., 2004;Sudthongkong et al., 2002). Necrosis and karyolysis oftubular epithelium was serious and immunolabellingswere only found in renal glomerulus and its surrounding

ig. 8. E. lanceolatus. Results of immunohistochemistry in liver, intestines and brain. The signal is observed microscopically as brown yellow staining. (A, C,

) Control. (B, D, F) Diseased fish. In liver, only a few immunolabellings were found in portal area of necrotic hepatocyte; In intestines, immunolabellings

ere detected in submucosa and serosa; In brain, only few immunolabellings were found in outer membrane of brain (black arrow).

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C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279278

mopoietic tissues, but not in tubular epithelium. Thesults were similar to the report that no signal was found

tubular epithelium examined by IHC and ISH (Cano et al.,09). This indicated that the lesions to tubular epitheliumem not to be directly virus-related. Immunolabellings inart, liver, stomach, gill and digestive tract were mainly ine sites where it had rich blood vessels. Hence, onceimary replication has taken place, virus can reach theoodstream, resulting in viraemia, as a step leading tostemic infection (Ogawa et al., 1990).On the other hand, both splenic corpuscle and renal

rpuscle of surviving fish were completely replaced bynormal hypertrophic cells (Fig. 54 and F), while nopertrophic cell was observed in the other parts of spleend kidney tissues, same case was found in juvenilempback grouper Cromileptes altivelis infected by grouper

sleepy disease iridovirus (GSDIV). Mahardika et al. (2004)had been described them as ‘abnormal IBCs’ who containeddeformed virions and had no granular masses associatedwith viral DNA and organelles (Mahardika et al., 2004).Interestingly, immunolabellings in spleen and kidney ofsurviving fish were only found in the abnormal hypertro-phic cells, which confirmed that the abnormal hypertro-phic cells contained massive viral major capsid protein,perhaps, they are deformed virions, and whether they hadviral DNA remains unknown. In surviving fish, immuno-labellings were only limited in splenic corpuscle and renalcorpuscle, it maybe because splenic corpuscle and renalcorpuscle prevented GIV-R from spreading to other sites, orsplenic corpuscle and renal corpuscle were the target siteof GIV-R infection and reproduction. The specific mecha-nism needs to further study in the future.

. 9. E. lanceolatus. Results of immunohistochemistry heart and stomach. The signal is observed microscopically as brown yellow staining. (A, B) Control.

D, E, F) Diseased fish. In heart, strong immunolabellings were found in cardiac muscle fibers, epicardium and chambers of the heart; In stomach,

munolabellings were mainly in submucosa and mucosa where had rich blood vessels (black arrows).

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. Conclusion

To sum up, GIV-R can cause fatal systemic diseases toiant grouper. It causes acute histopathological lesions inner organs. Antigens of GIV-R existed in most tissues of

iseased giant grouper, but distributed mainly in hemo-oietic tissue of spleen and kidney. Splenic corpuscle andenal corpuscle seem to have special role for GIV-R

fection.

cknowledgements

This research was supported by the key Science andechnology Program of Hainan Province under Grant No.DXM20120031 and Special Foundation of Fish Diseaserevention and Control of Guangdong Province (2012).

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