Susceptibility of Farmed Juvenile Giant Grouper Epinephelus Lanceolatus To

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Veterinary Microbiology 177 (2015) 270–279

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Veterinary Microbiology

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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 *

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

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 , 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.

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

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 , 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

haematopoietic tissue (Ahne et al., 1989; Chao et al., 2002; serum (FBS, Gibco), 100 IU ml penicillin G and

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 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 80 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 , 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 1

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

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 tems, Wetzlar, Germany), and subsequently double-

protocol. The primer-pair F (5 -ATGACTTGTACAACGGGT- stained with uranyl acetate and lead citrate and

0 0 0

3 ) and R (5 -TTACAAGATAGGGAACCCCAT-3 ) were used observed at 80 kV with a Jeol TEM-1200EX (Akishima,

to amplify the MCP of GIV-R (Huang et al., 2011). The PCR Japan).

was performed in a total reaction volume of 25 ml

containing 18.3 ml of PCR-grade water, 2.5 ml of 10

3. Results

Reaction Buffer, 1 ml of dNTP mix, 1 ml of each of the

primers (10 mM), 0.2 ml r-Taq DNA polymerase

3.1. Clinical signs and cumulative mortality

(TOYOBO) and 1 ml of template. The PCR parameters

using the primer-pair F and R were as followings: 1 cycle

Diseased juvenile giant grouper displayed either

of 94 8C for 10 min, 35 cycles of 94 8C for 40 s, 55 8C for

lethargy or a dark coloration of the body, and sometimes

40 s, and 72 8C for 90 s, followed by the ﬁnal extension at

ascites. The infected ﬁsh showed pale gill with petechiae,

72 8C for 10 min.

congestion of spleen and liver, loose and soft muscle

(Fig. 1B). The mortalities started at 2 dpi and the death

2.4. Histopathology

fastigium was during 2–5 dpi. The cumulative mortality

was high to 81% within 5 dpi (Fig. 2). A GIV-R speciﬁc DNA

Tissues were ﬁxed in 10% phosphate-buffered formalin

band was detected from all infected ﬁsh by PCR (Fig. 3).

for at least 24 h and dehydrated in an ethanol–xylene

series before embedding in parafﬁn wax. Formalin-ﬁxed,

3.2. Histopathology

parafﬁn-wax-embedded (FFPE) 5 mm tissue sections were

dewaxed in xylene, rehydrated in an ethanol series and

The histopathological features of GIV-R-SY1301

stained with haematoxylin and eosin (H&E).

infected E. lanceolatus were the severe degeneration of

spleen and kidney tissues with massive pycnotic nuclei in

2.5. Immunohistochemistry

the hematopoietic tissue (Fig. 4C and D). Hemorrhage and

inﬁltration of inﬂammatory cells were found in gill, liver,

Formalin-ﬁxed, parafﬁn-wax-embedded (FFPE) 5 mm

heart, spleen and associated with tissue degeneration and

tissue sections were dewaxed and rehydrated according

necrosis of varying severity.

to the conventional method. To unmask antigen and

inactivate endogenous peroxidase, deparafﬁnised and

rehydrated sections were treated with 0.01 M citrate

90%

buffer (pH 6), and heated in a microwave oven for 15 min.

80%

Slides were then washed with 0.01 M PBS (NaCl 0.14 M;

tes t group

KCl 2.7 mM; Na2HPO412H2O 10 mM; KH2PO4 1.7 mM; 70%

con trol

pH 7.2) three times for 5 min. Tissue sections were 60%

blocked with 5% (w/v) bovine serum albumin (BSA) for

50%

30 min, and incubated at 37 8C for 30 min. Subsequently,

40%

50 ml of mouse polyclonal anti-GIV-R MCP serum (1:300,

dissolved by 0.01 M PBS, donated by Dr. Chuanfu Dong 30%

Cumulative mortality

from the Sun Yat-sen University) was added and the 20%

slides were further incubated at 4 8C for 5 h. The tissues

10%

were then thoroughly rinsed in PBS three times for 5 min

and incubated with a secondary antibody conjugated

TM 1 2 3456 7 8 9

universal immunoenzyme polymer using GTVison III

Days after injection

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

Tissues were thoroughly rinsed in PBS and developed Fig. 2. Cumulative mortality of E. lanceolatus after GIV-R injection.

C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279 273

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 hematopoi-

etic 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 hepato-

Fig. 3. PCR detection of infected E. lanceolatus. M. DL2000 DNA marker; B1, cyte atrophied and diminished. Central vein wall

B2: Blank; C1, C2, C3: negative control; S1, S2, S3: diseased giant grouper. 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 4. Discussion

(Fig. 5C). In the heart, inﬁltration of inﬂammatory cells

caused atrophy of cardiomyocytes with swelling of Grouper, the major species being maricultured in China

myocardial ﬁbres, resulting in reduced staining afﬁnity and other SE Asian countries, are high-priced and popular

(Fig. 5D). Blood vessel in gill was expanded and fused seafood ﬁsh. Nevertheless, iridoviruses have caused high

with tremendous erythrocyte inﬁltration in the sinusoid mortality in many cultured grouper species in the last

of gill ﬁlaments. Epithelial hyperplasia, desquamation, decades, which suggested its a severe threat to groupers

epithelial lifting, fusion or telangiectasis was noticed in aquaculture (Chua et al., 1994; Gibson-Kueh et al., 2004;

the secondary lamellae with the breakdown of the pillar Mahardika et al., 2004; Qin et al., 2003). Our infection

cell system (Fig. 5H). In stomach, gastric gland atrophied experiments results showed that naive giant grouper was

and partly degraded in gastric mucosa (Fig. 5G). No susceptible to GIV-R infection with high mortality (80%).

histopathological change was observed in the intestines, The newly isolated ranavirus GIV-R could cause severe

eye and brain. Transmission electron microscopic result systemic disease to juvenile giant grouper, characterized

showed that many enveloped hexagonal virions mea- by degeneration and necrosis of varying severity in inner

suring 180–200 nm were observed in cytoplasm (Fig. 6A organs, especially in spleen and kidney. This is the same as

and B). ranavirus and megalocytivirus infections which also

caused systemic infection involving multiple internal

3.3. Immunohistochemistry analysis organs with high mortalities (Williams et al., 2005). What

is different is that there were only few small-size (about

The strongest immunolabellings were obtained from 10 mm) inclusion body-bearing cells (IBCs) existed in

the spleen tissues. They were accumulated in the spleen, but none in other tissues. However, other

basophilic small-size inclusion body-bearing cells and iridoviruses diseased ﬁshes showed systemic formation

were widespread in splenic pulp (Fig. 7C). In the kidney, of prominent enlarged cells as inclusion body-bearing cells

positive reactions were mainly in the renal glomerulus (IBCs) in different organs, such as in large yellow croaker,

and its surrounding hemopoietic tissues. But, there was striped beakperch, angelﬁsh, farmed turbot, mandarinﬁsh,

no antigen labeling existed in renal tubular epithelial African lampeye and dwarf gourami with iridovirus

cells (Fig. 7D). In heart, strong positive immunolabel- infection (Chen et al., 2003; He et al., 2000; Jung and

lings were widespread in cardiac muscle ﬁbers, epicar- Oh, 2000; Shi et al., 2004; Sudthongkong et al., 2002), as

dium and chambers of the heart (Fig. 9C and E). In the well as in grouper ﬁshes with the iridovirus infection:

liver, a few immunolabellings were observed only in the brown-spotted grouper (Chua et al., 1994), Malabar

portal area of necrotic hepatocyte (Fig. 8B). Immunola- grouper (Sano et al., 2002), cultured groupers (Chou

bellings in stomach were mainly in the submucosa et al., 1998), juvenile humpback grouper (Mahardika

where had rich blood vessels (Fig. 9D), secondly in the et al., 2004) and hybrid grouper (Chao et al., 2004). The

mucosa (Fig. 9F), and slightly in serosa. In the intestine, enlarged cells were about 20 mm in diameter and were

positive reactions were visualized mostly in submucosa obvious larger than GIV-R infected cells. The IBCs in some

and serosa (Fig. 8D). Immunolabellings within the gill of iridovirus infected ﬁsh included three types: The early

were detected in the afferent artery and capillary vessel stage of IBCs were hypertrophied blast-like cells that

lumen, especially in capillary vessel lumen around possessed a basophilic cytoplasm and a centrally located,

cartilage tissue (Fig. 7H). Weak immunolabellings were enlarged nucleus containing prominent nucleoli; The

only found in brain outer membrane (Fig. 8F). No mature IBCs were enlarged and usually had an entirely

immunolabelling was observed in eyes. Immunolabel- basophilic cytoplasm and either a centrally or marginally

lings were only observed in splenic corpuscle and renal located nucleus; The ballooning, degenerated IBCs con-

corpuscle in surviving ﬁsh of kidney and spleen (Fig. 7E tained an inclusion body with a granular appearance

and F). 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 macrophages. No signal was obtained in eyes. Immuno-

et al., 2004; Sudthongkong et al., 2002). In GIV-R infected labellings in spleen were accumulated in the basophilic

juvenile giant grouper, it seemed to have only small IBCs and were widespread in splenic pulp. Many reports

mature IBCs in spleen. Another prominent histopatholog- have proved that iridovirus infected and proliferated in the

ical feature of GIV-R was the formation of massive pycnotic enlarged cells or inclusion body bearing cells by IHC and

cell nuclei in splenic pulp and renal hematopoietic tissue. ISH. For example, immunohistochemical and nucleic acid

The histopathological changes in our study were most signals were labeled mainly in the enlarged cells in ISKNV

similar to ‘Sleepy Grouper Disease’ infected brown-spotted infected zebraﬁsh and SGIV infected Malabar grouper

grouper (Chua et al., 1994). (Huang et al., 2004; Xu et al., 2008), DNA hybridization

Immunohistochemistry using mouse GIV-R MCP anti- signals were only obtained in basophilic enlarged cells of

serum showed a widespread distribution of the virus in spleen in TGIV infected Epinephelus hybrids (Chao et al.,

tissues. The strongest immunolabellings were obtained 2004). Moreover, many viral particles were observed in

from the kidney and spleen tissues. The intermediate inclusion body-bearing cells of many iridovirus infected

intensity signals were observed in the heart, stomach, gill ﬁshes by electron microscopy (Mahardika et al., 2004;

and liver tissues. The weakest signals were obtained from Sudthongkong et al., 2002). Necrosis and karyolysis of

the intestine and brain. The signals were speciﬁcally tubular epithelium was serious and immunolabellings

located within epithelial, endothelial, leukomonocyte and 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 sleepy disease iridovirus (GSDIV). Mahardika et al. (2004)

results were similar to the report that no signal was found had been described them as ‘abnormal IBCs’ who contained

in tubular epithelium examined by IHC and ISH (Cano et al., deformed virions and had no granular masses associated

2009). This indicated that the lesions to tubular epithelium with viral DNA and organelles (Mahardika et al., 2004).

seem not to be directly virus-related. Immunolabellings in Interestingly, immunolabellings in spleen and kidney of

heart, liver, stomach, gill and digestive tract were mainly in surviving ﬁsh were only found in the abnormal hypertro-

the sites where it had rich blood vessels. Hence, once phic cells, which conﬁrmed that the abnormal hypertro-

primary replication has taken place, virus can reach the phic cells contained massive viral major capsid protein,

bloodstream, resulting in viraemia, as a step leading to perhaps, they are deformed virions, and whether they had

systemic infection (Ogawa et al., 1990). viral DNA remains unknown. In surviving ﬁsh, immuno-

On the other hand, both splenic corpuscle and renal labellings were only limited in splenic corpuscle and renal

corpuscle of surviving ﬁsh were completely replaced by corpuscle, it maybe because splenic corpuscle and renal

abnormal hypertrophic cells (Fig. 54 and F), while no corpuscle prevented GIV-R from spreading to other sites, or

hypertrophic cell was observed in the other parts of spleen splenic corpuscle and renal corpuscle were the target site

and kidney tissues, same case was found in juvenile of GIV-R infection and reproduction. The speciﬁc mecha-

humpback grouper Cromileptes altivelis infected by grouper nism needs to further study in the future.

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

Inoue, K., Yamano, K., Maeno, Y., Nakajima, K., Matsuoka, M., Wada, Y.,

5. Conclusion

Sorimachi, M., 1992. Iridovirus infection of cultured red sea bream,

Pagrus major. Fish Pathol. (Japan).

To sum up, GIV-R can cause fatal systemic diseases to 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.

giant grouper. It causes acute histopathological lesions in

(Eds.), Family Iridoviridae. In: Virus Taxonomy: Ninth Report of the

inner organs. Antigens of GIV-R existed in most tissues of

International Committee on Taxonomy of Viruses. Elsevier Academic

diseased giant grouper, but distributed mainly in hemo- Press, San Diego, CA, pp. 193–210.

Jung, S., Oh, M., 2000. Iridovirus-like infection associated with high

poietic tissue of spleen and kidney. Splenic corpuscle and

mortalities of striped beakperch, Oplegnathus fasciatus (Temminck

renal corpuscle seem to have special role for GIV-R

et Schlegel), in southern coastal areas of the Korean peninsula. J. Fish

infection. Dis. 23, 223–226.

Kurita, J., Nakajima, K., 2012. Megalocytiviruses. Viruses 4, 521–538.

Langdon, J., Humphrey, J., 1987. Epizootic haematopoietic necrosis, a new

Acknowledgements 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

This research was supported by the key Science and

iridovirus in cultured rainbow trout, Salmo gairdneri Richardson, in

Technology Program of Hainan Province under Grant No.

Australia. J. Fish Dis. 11, 93–96.

ZDXM20120031 and Special Foundation of Fish Disease Langdon, J., Humphrey, J., Williams, L., Hyatt, A., Westbury, H., 1986. First

virus isolation from Australian ﬁsh: an iridovirus-like pathogen from

Prevention and Control of Guangdong Province (2012).

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

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