９愛媛大学農学部紀要（Mem. Fac. Agr., Ehime Univ.）
５２：９−１１（２００７）

Virus Detection in Diseased and Healthy Fish and Oysters
from an Indonesian Aquaculture Site: Double Infection of Viral Nervous
Necrosis Virus (VNNV) and Marine Birnavirus (MABV)
Shin-Ichi KITAMURA＊, Keisuke KANEHIRA＊, Hidetaka TAKEOKA＊ and Satoru SUZUKI＊＊
Abstract
In the south Sumatra region of Indonesia, tiger grouper, Epinephelus fuscoguttatus, and humpbuck grouper,
Cromileptes altivelis, are commercially important fish species. Recently, however, mass mortalities of the two specieshave occurred in the region and the cause of these mortalities has not been clarified. We surveyed the distribution offish viruses including viral nervous necrosis virus (VNNV), marine birnavirus (MABV) and red seabream iridovirus(RSIV) in the two grouper species and MABV in silver the lip oyster, Pinctada maxima, by PCR. VNNV was detectedfrom both healthy and diseased groupers whereas MABV was only detected in diseased groupers, suggesting that themass mortalities of the two grouper species were caused by co-infection of VNNV and MABV. PCR product of RSIV
was not observed in any fish samples. In addition, MABV was not detected in silver lip oysters from both healthy andthose exhibiting poor growth.
Key words: Tiger grouper, humpbuck grouper, lip oyster, VNNV, MABV, Indonesia

Introduction
Marine aquaculture industries including fish andshellfish species have been developed in Indonesia. In thesouth Sumatra region of Indonesia, tiger grouper,
Epinephelus fuscoguttatus, and humpbuck grouper,
Cromileptes altivelis, are commercially important fishspecies, however disease outbreaks due to unknown
causes have been encountered in these fish species,
resulting in a serious economic loss to the aquacultureindustry. In shellfish species, the silver lip oyster,
Pinctada maxima, is one of the most important cultured
shellfish species to produce pearls in the region, but
ventures in shellfish aquaculture have also faced problems
such as poor growth of the oyster due to unknown
reasons.
From the viewpoint of infectious disease, viral diseases
have often caused mass mortality of cultured fish andshellfish species. For example, viral nervous necrosis virus(VNNV) belonging to genus Betanodavirus in family
Nodaviridae (Muroga, 2001), red seabream iridovirus(RSIV) belonging to the genus Megalocytivirus in family
Iridoviridae (Inouye et al. 1992) and marine birnavirus(MABV) belonging to genus Aquabirnavirus in family
Birnaviridae (Sorimachi & Hara, 1985) were detected
Received 27 March 2007.
Accepted 22 June 2007.
＊Center for Marine Environmental Studies, Ehime University,
Bunkyo 3, Matsuyama 790‐8577, Ehime, Japan (＊＊Corresponding author)

from many diseased fish species in Asian countries. Tocontrol viral diseases, it is necessary to understand
distributions of the fish viruses in cultured fish andshellfish species as a basic research, but it has not been
fully investigated in Indonesia so far. Therefore, thedistribution of above three viruses in the tiger grouper andhumpback grouper, and silver lip oyster cultured in south
Sumatra region of Indonesia was surveyed including
examine the cause of mortalities in two grouper fish
species in this study.
Materials and Methods
Fish and shellfish
Healthy and diseased tiger grouper and humpbuck
grouper were sampled from an aquaculture facility in
Hurun Cove in south Sumatra of Indonesia in July 2003
(Fig. 1, Station 5). Diseased fish showed sluggish
behavior near the water surface and resting on the bottom,and exhibited a mortality rate of >30%. Healthy and poorgrowing silver lip oysters were collected from an oyster
farm managed by a local private company (Fig. 1, Station27).
Detection of fish viruses by PCR
Spleen and brain of fish were pooled and used forextraction of nucleic acids. In the case of shellfish,
hepatopancreas and hemocyte of the oyster wereseparately collected to detect MABV because the virus
could potentially infect both tissues in the Japanese pearl
oyster Pinctada fucata (Kitamura et al. 2000). Total
nucleic acids were extracted from the collected samples

Shin-Ichi KITAMURA, Keisuke KANEHIRA, Hidetaka TAKEOKA and Satoru SUZUKI

１０Fig.1. Map of sampling stations. ●; sampling station of tiger grouper and humpbuck grouper, ■; sampling station ofsilver lip oyster.

using the method described by Suzuki et al. (1997).
Briefly, the organ was homogenized in 4-fold volume ofTE (0.2 M Tris HCl, pH 8.0, 0.1 M EDTA), and wascentrifuged at 3,000 x g for 5 min. The 45 μl ofsupernatant was taken and 5 μl of proteinase K (1mg/ml;TaKaRa Co., Shiga, Japan) was added. The mixture wasincubated at 55 ℃ for 2 h. Nucleic acids were extractedusing phenol-chloroform. A portion of the nucleic acids
were subjected to reverse transcription (RT) for VNNVand MABV detection, described by Nishizawa et al.
(1994) and Suzuki et al. (1997), respectively; PCR wasthen performed. In order to detect RSIV, the remaining
nucleic acids were directly used for PCR by the methodof Kurita et al. (1998). PCR amplification was performed

with a DNA thermal cycler (TaKaRa Co., Shiga, Japan).
Amplified products were analyzed by agarose gelelectrophoresis, and were visualized with ethidium
bromide and UV light irradiation.
Results and Discussion
In fish samples collected from the south Sumatra regionof Indonesia, VNNV was detected from both healthy anddiseased tiger and humpbuck grouper. The detection rateof VNNV was 100% from diseased individuals of bothspecies, whereas it was <66.6% in what were designatedas healthy fish (Table 1). Since Zafran et al. (2000)
detected the same virus from diseased juvenile humpbuck

Table 1. PCR detection of three fish viruses from tiger grouper, humpbuck grouper and oyster
SpeciesCondition

Fish no.

mean B.W. (g)Tiger grouper
Tiger grouper
Humpbuck grouper
Humpbuck grouper
Silver lip (Hepatopancreas)
Silver lip (Hemocyte)
Silver lip (Hepatopancreas)
Silver lip (Hemocyte)

Healthy
Diseased
Healthy
Diseased
Healthy
Healthy
Poor growing
Poor growing

3333
10101010

41.4
52.2
28.9
36.7
49.9
−
26.9−

*Not tested

PCR positive rate (%)
VNNV

MABVRSIV33.310066.6100*NT*NTNTNT0100066.600000000NTNTNTNTVirus Detection in Diseased and Healthy Fish and Oysters from an Indonesian Aquaculture Site: Double Infection of Viral Nervous Necrosis Virus (VNNV) and Marine Birnavirus (MABV)

grouper in the Bali region of Indonesia, the virus isthought to be widely distributed in the country. In thepresent study, symptoms of a diseased fish, sluggish
behavior and resting on the bottom, was similar to thatobserved for fish affected with VNNV, suggesting that themain cause of mortality in the two grouper species isVNNV infection. RSIV was not detected from any of thegrouper samples. Since the virus has caused massmortality in many fish species, including grouper, many
Southeast Asian countries require sustained surveillance ofthe virus (Miyata et al. 1997; Sudthongkong et al. 2002;
Gibson-Kueh et al. 2004).
Interestingly, the high detection rate (>66%) of MABV
was observed in only diseased groupers, indicating thatco-infection by VNNV and MABV occurred in the fish.
Regarding the co-infection by birnavirus and the otherfish virus, from experiemental infection of Japaneseflounder (Paralichthys olivaceus). Pakingking et al.
(2003) reported that primary aquabirnavirus (ABV)
infection suppressed the secondary viral hemorrhagic
septicemia virus (VHS) infection. However, ourobservations suggest that MABV enhanced thepathogenicity of VNNV because MABV was detectedfrom only diseased individuals of grouper, despite VNNV
having been detected from both healthy and diseased fish.
To clarify the relationship between mortality and coinfection of these viruses in grouper, the experimental coinfection in consideration of the contagious turn of VNNVand MABV is necessary.
Alternatively, in the single-infection of birnavirus ingrouper (Epinephelus sp.) fry, the pathogenicity of thevirus was low in experimental infections, but it increased
when fish were exposed to a stressor such as heavy metals
and changes in salinity (Chou et al. 1999). This evidence
suggests that the virus is an opportunistic pathogen ingrouper and its pathogenicity could be enhanced byenvironmental pollution and physiological factors .
Recently, high population densities of cultured fishcoupled with environmental pollution have created ideal
conditions for infectious diseases, making it a serious
problem in Asian countries. Therefore, monitoring of
cultured fish species in Indonesia for opportunistic
pathogens such as MABV is also highly warranted.
In shellfish samples of silver lipped oysters, MABV
was not detected from any individuals (i.e. poor or regulargrowth), and therefore the cause of poor growth remains
unclear. Further examination is needed to clarify thepotential pathogenic and environmental causes of poor
growth in this commercially important species.
Acknowledgements
We thank staff of Agency for the Assessment andApplication Technology (BPPT) and National Seafarming
Development Center (BBL) for their help in fish andshellfish sampling. This work was partly supported by a１
１

Grant-in-Aid for Scientific Research (Project No.
14254004), Japanese Society for the Promotion ofSciences (JSPS), and 21st Century COE program. Dr.
Todd Miller is appreciated for his critical reading of thispaper.
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