魚病研究 Fish Pathology, 33 (4), 201-206, 1998. 10

Isolation and Characterization of a Pathogenic Iridovirus from Cultured Grouper (Epinephelus sp.) in Taiwan

Hsin-Yiu Chou*, Chung-Che Hsu and Tsui-Yi Peng

Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan 202, ROC

(Received March 18, 1998)

A new epizootic viral disease has threatened cultured grouper (Epinephelus sp.) in Taiwan since 1995.

Icosahedral viral particles with a diameter of 230 •} 10 nm were observed in the spleen of moribund fish. Cell

rounding, a cytopathic effect (CPE), was induced in KRE cell line by the virus isolated from diseased

groupers. Virus infectivities declined rapidly during serial passages in the KRE cell line. The susceptibility of

the virus to chloroform and ether treatments indicates that the virus may be enveloped. When stained with

acridine orange, the inclusions in the KRE cells exhibited a greenish fluorescence, and this together with the

results of IUDR treatment suggest that the viral genome of the virus is double-stranded DNA. Based on its

general properties and viral morphology, the name Grouper Iridovirus of Taiwan (TGIV) was proposed. When

healthy juvenile groupers were experimentally infected by intraperitoneal injection with TGIV, cumulative

mortalities reached 100% within 11 days, while no grouper died in the control groups. Virus similar in morphol

ogy to that observed in spontaneously diseased fish was then re-isolated from the experimentally infected

groupers. These results suggest that this virus may be the causative agent of the epizootic disease that now

afflicts cultured grouper in Taiwan.

Key words: iridovirus, grouper, pathogenicity, Taiwan

Fisheries production is important to the economy and food supply of Taiwan. In recent years, high-priced marine species such as sea bass (Lateolabrax spp.),

grouper (Epinephelus spp.) and red sea bream (Pagrus major) have shown good profitability, which has encouraged the development of marine aquaculture industry in this island. But since 1992, red sea bream culture in

Taiwan's Pon-Fu Island has been plagued by outbreaks of a new disease. Based on the evidence from electron microscopy, these outbreaks are conjectured to be caused by an iridovirus-like infection (Chou et al., 1994; M. C. Tung, pers. comm.). The viral agent seems to be highly contagious to fishes; several kinds of cultured marine fishes other than red sea bream, such as grouper and sea bass were also observed with an iridovirus-like infection. Farmers have reported serious damages to

production of marine fishes in Taiwan apparently due to the iridovirus infection.

In 1995, an acute disease which caused up to 60%

mortality among farmed groupers was reported from southern Taiwan. Diseased fish swam in circles and

were anemic. During the course of viral examination, a cytopathic effect (CPE) was observed. In this study,

we describe our electron microscope observations of diseased grouper and the general characteristics of the

isolated viral agent. As well as attempting to study the

pathogenicity of the virus and electron microscopic observations of the experimentally infected groupers were made. The isolate is subsequently referred to as

grouper iridovirus of Taiwan (TGIV).

Materials and Methods

Cell culture

Leibovitz's L-15 medium (Flow Laboratories) supple

mented with fetal bovine serum (FBS) and antibiotics

(50 IU/ml penicillin, 50 ƒÊg/ml streptomycin, 1.75 ƒÊg/

ml fungizone) was used to culture KRE cells derived

from hybrid groupers (Fernandez et al., 1993). For

routine passage and virus titration, 10% and 2% FBS

was added to the medium, respectively. The cells were

incubated at 25•Ž.

* Correspondence: Hsin-Yiu Chou , Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan 202, ROC

Tel: 886-2-24622192 Ext.5214; Fax: 886-2-24634176

Email: hychou@ntou66.ntou.edu.tw

202 H. Y. Chou, C. C. Hsu and T. Y Peng

Grouper

Diseased groupers (5 -8 cm in total length) were

collected from a culture farm at Chieh-Ting area in

Kao-Hsiung Prefecture in southern Taiwan. The

healthy groupers were verified to be virus-free by virus

isolation before being subjected to pathogenicity

tests. All of the groupers were maintained in 25-28•Ž

aquaria with aeration and fed commercially obtained

artificial food twice daily.

Transmission electron microscopy

Organs were removed from each diseased grouper and

immediately prefixed in 2.5% glutaraldehyde in 0.1M

cold phosphate buffer solution (PBS, pH 7.4) for 2 h at

4•Ž. Subsequently, samples were washed several

times with cold PBS, and then postfixed in 1% osmium

tetroxide for 3 h at 4•Ž. The samples were dehydrated

and embedded in Spurr's resin. Thick and ultrathin

sections were prepared on Richert-Jung Ultracut E

Ultrotome. Thick sections were stained with toluidine

blue and observed under a light microscope. Where

abnormal cell changes were found, ultrathin sections

were prepared and stained with uranyl acetate and lead

citrate. The sections were observed with a HITACHI

H-600 transmission electron microscope.

Virus isolation

Samples of kidney, spleen, heart and liver from each

diseased grouper were pooled, weighed and ground with

a chilled mortar and pestle. L-15 medium containing

no FBS (L-15-0) was added to homogenates in the ratio

of 9 : 1. After being centrifuged at 2,000•~ g (Sigma

2K15 rotor 12141) for 10 min, the supernatant fluid was

filtered through a 0.45ƒÊm membrane. One of two dif

ferent quantities (50ƒÊl or 150ƒÊl) of the filtrates were

inoculated into 24-well plates containing confluent

monolayers of KRE cells prepared 24 h previously.

These were incubated at 25•Ž until a CPE was observed.

Stability of serial passages

For studying the passage stability of TGIV, the

primary isolated virus suspension was collected and

inoculated into KRE cells in a 25 cm<SUP>2<SUP> flask at a multi

plicity of infection (MOI) of 0.1•`0.01. The virus was

then allowed to multiply for 7 days at 25•Ž. The cul

ture medium, along with the adherent cells removed by

scraping, was pooled, and then frozen and thawed three

times. The processed product was treated as the first

passage, and the second to fifth passages were prepared

by repeating the procedures described above. Virus in

fectivity of each passage was measured by TCID50 assay and calculated by the method of Reed and Muench

(1938).

General properties of TGIV

Organic solvents sensitivity: The treatment of TGIV

with ether and chloroform was used for envelope

detection. L-15-0 was used in place of organic solvents

to serve as control (Andrewes and Horstmann, 1949;

Feldman and Wang, 1961).

Stability at pH 3.0 and pH 11.0: Stability at pH 3.0

and 11.0 were determined by adding 0.1 ml virus sus

pension to 0.9 ml L-15-0 adjusted to pH 3.0 or 11.0,

holding for 4 h at 4•Ž, then measuring virus infectivity

in KRE cells. Virus was added to L-15 medium at pH

7.2 as a control.

Nucleic acid inhibition test: The nucleic acid type

of the virus was presumptively determined by growing

it in the presence of the DNA inhibitor 5-iodo-2-

deoxyuridine (IUDR). L-15 medium alone, or L-15

medium containing either 100 or 200 ƒÊg IUDR was

added to monolayer cultures of KRE cells prepared in 9

6-well plates. Virus dilutions from 1 to 10-11 in 10-fold

dilutions were then inoculated into the cells and

allowed to react at 25•Ž for 4 h. After 4 h, the culture

media containing virus and IUDR were replaced with

L-15 medium, and all the plates were incubated at 25•Ž

and examined for CPE. An RNA virus (IPNV) grown

in CHSE-214 cells served as negative control.

Acridine orange stain: Monolayer cultures of KRE

cells were grown on round cover glasses 12 mm in di

ameter and inoculated with TGIV at MOI = 0.1•`0.01.

Controls and infected cells were incubated for 24 h at

25•Ž and fixed in Carnoy's solution (1 : 6 : 3 glacial

acetic acid -- absolute ethanol-- chloroform) for 10

min. Subsequently, samples were dip washed through

70% ethanol, 50% ethanol, 1% acetic acid solution and

distilled water. The cells on the cover glasses were

then immersed in a citrate-phosphate buffer solution (pH

3.8) for 5 min, which was followed by a 3 min staining

with a buffered 0.01% acridine-orange solution. The

samples were again immersed in buffer for 3 min and

examined for fluorescence with an Olympus fluores

cence microscope (Argot and Malsberger, 1972).

Pathogenicity test An infection trial was performed by intraperitoneal

injection. After food had been withheld for 24 h, two replicates of 15 groupers (1.9 g average weight, 4.9 cm average length) were injected with virus solution at a

Iridovirus from cultured grouper in Taiwan 203

concentration of 103.3 TCID50/0.1 ml/fish. Groupers in-

jected with medium served as control. Water tempera-

ture and salinity were 25-28•Ž and 25-30 ppt, respec-

tively, throughout the experiment. The mortality was

observed daily and the moribund fish were collected and

examined by transmission electron microscopy and

virus re-isolation.

Results

Electron microscopyThe spontaneously diseased grouper sampled from the

Chieh-Ting area of Kao-Hsiung were off their feed andunderweight, and appeared lethargic (Fig. 1). Abnor-

mal hypertrophied cells were observed in the thicksections of the spleen from the diseased groupers (Fig.

2). The ultrathin sections of the spleen from spontane-

ously diseased groupers revealed numerous icosahedral

virions (Figs. 3A and B). The virus particles were 230

•} 10 nm in diameter (n = 30), and had an inner electron-

dense central core of indefinite structure, approximately

120 •} 10 nm in diameter. Many partially disrupted par-

ticles were also present.

Virus isolationVirus was successfully isolated from the visceral

organs of diseased groupers. CPE in the form of cellrounding was induced in KRE cells 3 days after being

inoculated with filtrate from diseased groupers (Fig. 4).The entire cell sheet was lysed within 7 days.

Stability of serial passages

Virus titers of the original isolate and the further pas-sages in KRE cells are shown in Table 1. A virus titer

A

B

Fig. 1. External appearance of spontaneously diseased grou-

per which were sampled from Chieh-Ting area of Kao-Hsiung. Emaciated fish are off their feed and lethar-

gic.

Fig. 2. Giant cells (arrows) in the spleen of spontaneously

diseased grouper. Thick section, toluidine blue

stain, •~ 1200.

Fig. 3. Transmission electron micrograph of the spleen from

spontaneously diseased grouper. (A) Viral particles

in the cyoplasm of a giant cell. Nu: nucleus. (B)

High magnification of viral particles. Scale bars = 5

ƒÊm in A, 500 nm in B.

204 H. Y. Chou, C. C. Hsu and T. Y Peng

A

B

of 106.0 TCID50/ml was attained on the first passage, but

this declined rapidly to 101.9 TCID50/ml through five

passages. Serial passages of the virus in KRE cellsresulted in a rapid decrease in infectivity.

General properties of TGIVGeneral properties of TGIV are summarized in Table

2. The susceptibility of TGIV to ether and chloroform

indicates that the virus may be enveloped. TGIV was

partially inactivated by exposure to pH 3 but treatmentat pH 11 showed no effect on viral infectivity. Thedeoxyuridine analogue IUDR was effective in blocking

A B

C

viral replication. Replication of the reference RNAvirus, IPNV, was not affected by the presence of IUDR

in the medium. When infected cells were stained withacridine orange, the cytoplasm contained inclusions that

exhibited a greenish fluorescence (Fig. 5).

Pathogenicity test

By experimental infection, TGIV was shown to behighly pathogenic to grouper, with cumulative mortali-

ties reaching 100% in 11 days, while no grouper died inthe control groups (Fig. 6). No clinical signs weredetected in any of the experimental fish, but large

aggregations of icosahedral virions were found in the

Fig. 4. Cytopathic effect (CPE) in KRE cells 3 days after

inoculating filtrate from diseased groupers. (A)

Uninfected KRE cells, (B) infected KRE cells.

Table 1. Virus titers of TGIV during serial passages in the

KRE cell line

Table 2. General properties of TGIV

N/A: not avalible

Fig. 5. Acridine-orange stained KRE cells. (A) Uninfected

KRE cells. (B, C) TGIV-infected KRE cells (12 h

after infection). Inclusions (arrows) having yellow-

greenish fluoresence in cytoplasm were observed.

Iridovirus from cultured grouper in Taiwan 205

cytoplasm of the spleen cells of these artificially infected

groupers when they were examined under transmissionelectron microscopy (Fig. 7). No differences in virionmorphology between spontaneously diseased and

experimentally infected groupers were recognized. Inaddition, marked CPE characterized by cell rounding

was induced when KRE cells were inoculated withfiltrates prepared from either pooled spleens or livers of

experimentally infected grouper.

Discussion

The prevalence of iridoviruses in both wild and cul-tured fish populations has been well documented around

the world. These include lymphocystis virus (Wolf etal., 1966), erythrocytic necrosis virus (Appy et al., 1976),

cod iridovirus (Jensen et al., 1979), goldfish iridovirus

(Berry et al., 1983), carp gill necrosis iridovirus(Shchelkunov and Shchelkunova, 1984), redfin perchiridovirus (Langdon et al., 1986, 1988; Langdon and

Humphrey, 1987) and sheatfish iridovirus (Ahne et al.,1991). In Singapore, an iridovirus has been implicated

in a novel disease, "Sleepy Grouper Disease" in brown-spotted grouper Epinephelus tauvina (Chua et al., 1994).

In Japan, iridoviral disease has been occurred with 2060% mortalities among cultured marine fish such as

red sea bream (Pagrus major), yellowtail (Seriola

quinquerdiata), sea bass (Lateolabrax sp.) and Japanese

parrot fish (Oplegnathus fasciatus) since 1990. Thedisease is characterized by bathophilic enlarged cells in

A

B

the spleen, heart, kidney, liver and gill. Hexagonalvirions, measuring 200 — 240nm in diameter, were foundin the cytoplasm of infected cells. The causative viruswas tentatively designated as red sea bream iridovirus

(RSIV) (Inouye et al., 1992; Nakajima and Sorimachi,1994).

Similarly, outbreaks of an acute, highly contagiousdisease of marine fishes which has often caused highmortality and significant economic loss have occurredin Taiwan since 1992. In this study, we isolated a viralagent from diseased groupers and, based on its general

properties and viral morphology, we have tentativelyclassified it as an iridovirus and propose the name Grou-

per Iridovirus of Taiwan (TGIV). Because TGIV and

Fig. 6. Cumulative mortality (%) of grouper (Epinephelus sp.)experimentally infected by intraperitoneal injectionwith TGIV (103.3 TCID50/0.1 ml /fish). Groupersinjected with medium only served as control. Datashown are the mean of 2 replicates.

Fig. 7. Transmission electron micrograph of spleen from

grouper experimentally infected with TGIV. (A)

Aggregation of viral particles in the cytoplasm. Nu:

nucleus. (B) High magnification of viral particles

shows their resemblance to those found in spontane-

ously affected fish (cf Fig 3B). Scale bar = 2ƒÊm in

A, 1 ƒÊm in B.

206 H. Y. Chou, C. C. Hsu and T. Y Peng

RSIV are similar in morphology and histopathology and their epidemiological circumstances are also similar, we suspect that these two viral agents might in fact be closely related. Clearly, further comparison of TGIV with RSIV will be required. The results of the patho

genicity test that the isolated virus caused high mortality in healthy groupers and the similar virus was reisolated from them suggested that TGIV may possibly be the causative agent for the epizootic disease that now afflicts cultured grouper in Taiwan. The experimentally infected groupers, however, failed to exhibit any clinical signs. The reason for this seems to have been that TGIV was directly injected into the fish, which

probably caused unusually rapid and acute responses accompanied by high mortality.

Informal pilot studies in which TGIV was used to infect healthy silver bream (Sparus sarba) and black sea bream (Acanthopagrus latus) suggest that the virus may also constitute a threat to other marine fishes, and inasmuch as prevention and quarantine are essential parts of any strategy intended to combat the disease, a rapid method of diagnosis is urgently needed. However, to classify TGIV more definitively as well as to develop a

quick diagnostic method, purification of TGIV will be necessary. Unfortunately, TGIV quantity and quality declined rapidly during serial passages in KRE cells (See Table 1). Similar findings have also been reported for RSIV (Nakajima and Sorimachi, 1994). This instability of TGIV in KRE cells is a barrier to the purification of the virus. Further studies need to be performed to more effectively culture the agent and to increase virus yield in the purification process, as well as determine the DNA structure and other specific characteristics of this virus.

Acknowledgements

The work was supported by the Council of Agriculture under grant No. 85-AST-1.13-FID-06(II). We

would like to express our sincere thanks to Dr. M. Yoshimizu, Hakkaido University, for his kind provision

of the KRE cell line. We are also thankful to Mr. N.-H. Yu for his assistance in sampling; and to Mr. P.-L. Yeh

and Mr. H.-L. Yeh for providing the fish.

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