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Journal of
INVERTEBRATE
PATHOLOGY
Journal of Invertebrate Pathology 97 (2008) 197202
www.elsevier.com/locate/yjipa

Molecular detection of betanodaviruses from apparently

healthy wild marine invertebrates
Dennis K. Gomez a, Gun Wook Baeck b, Ji Hyung Kim c,d
, Casiano H. Choresca Jr. c,d
,
Se Chang Park a,c,d,*
a
KRF Zoonotic Disease Priority Research Institute, Seoul National University, Seoul 151-74, Republic of Korea
b
Faculty of Marine Technology, Chonnam National University, Yeosu 550-749, Republic of Korea
c
Brain Korea 21 Program for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea
d
Laboratory of Aquatic Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea

Received 3 January 2007; accepted 31 October 2007

Available online 7 November 2007

Abstract

One hundred eighteen samples (21 species) of wild marine invertebrates were collected from western and southern coastal area of Kor-
ean Peninsula. Four of 78 (18 species) samples collected at Namhae (South) area were positive for nodavirus in nested PCR test. Of the
40 samples (5 species) collected at Hwanghae (West) areas, all samples were negative for nodavirus in both RT-PCR and nested PCR
tests. Positive nested PCR results were obtained from the following species: Charybdis bimaculata Charybdid crab; Pandalus hypsinotus
Southern humpback shrimp and Mytilus galloprovincialis Mediterranean mussel. Phylogenetic analysis based on the partial nucleotide
sequence (177 bases) of the RNA2 coat protein gene showed that the four strains were highly homologous (100%) and closely related to
that of the known betanodaviruses, redspotted grouper nervous necrosis virus (RGNNV). These results indicate that nodavirus is present
from wild marine invertebrates in the southern coastal areas of Korean Peninsula. These subclinically infected marine invertebrates may
constitute an inoculum source for betanodavirus infection and cause mortality in cultured shes in Korea.
 2007 Elsevier Inc. All rights reserved.

Keywords: Betanodaviruses; PCR detection; Wild invertebrates; Subclinical infection; Phylogenetic analysis; Viral nervous necrosis

1. Introduction show abnormal swimming behavior. The spread of VNN

Betanodavirus (Nodaviridae) infection also known as
viral nervous necrosis (VNN) causes encephalopathy and
retinopathy (VER) in a variety of cultured marine shes
(more than 30 species) in most parts of the world. This dis-
ease particularly occurs during the seedling period and the
culture process (Munday et al., 2002; OIE, 2003). Necrosis
and the vacuolation of central nervous tissues (brain and
spinal cord) and eye retina are the most characteristic
lesions of VNN. The aected larvae and juvenile shes
*
Corresponding author. Address: Laboratory of Aquatic Animal
Medicine, College of Veterinary Medicine, Seoul National University,
Seoul 151-742, Republic of Korea. Fax: +82 2 880 1213.
E-mail address: parksec@snu.ac.kr (S.C. Park).
among populations of cultured marine sh may be attribut-
able to either vertical (Arimoto et al., 1992; Breuil et al.,
2002; Kim et al., 2006; Tsuchihashi et al., 2002; Watanabe
et al., 2000) or horizontal transmission (Castric et al., 2001;
Le Breton et al., 1997). The virus contains two segments of
positive-sense single-stranded RNA. The RNA1 (3.1 kb)
and RNA2 (1.4 kb) of SJNNV encode 100 kDa (presum-
ably RNA-dependent RNA polymerase) and a major coat
protein of 42 kDa, respectively (Mori et al., 1992). Based
on partial nucleotide sequence of coat protein gene, betan-
odaviruses are divided into four genotypes: striped jack
nervous necrosis virus (SJNNV)-type, tiger puer nervous
necrosis virus (TPNNV)-type, barn ounder nervous
necrosis virus (BFNNV)-type, and redspotted grouper ner-
vous necrosis (RGNNV)-type (Nishizawa et al., 1997).

0022-2011/$ - see front matter  2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.jip.2007.10.012
198 D.K. Gomez et al. / Journal of Invertebrate Pathology 97 (2008) 197202
Viral nervous necrosis was rst reported in larvae and
juveniles of hatchery-reared Japanese parrotsh Oplegna-
thus fasciatus (Yoshikoshi and Inoue, 1990). The same viral
disease has also been observed in barramundi Lates calca-
rifer (Glazebrook et al., 1990) and later on in other marine
shes (Mori et al., 1992; Muroga, 2001; Munday et al.,
2002). Recent studies also demonstrated that a certain pop-
ulation of apparently healthy wild marine sh carried bet-
anodaviruses, and suggested that these wild sh can be a
persistent potential source of virus for cultured sh (Barker
et al., 2002; Gomez et al., 2004; Baeck et al., 2007). In
Korea, there were reported incidences of VNN, which
cause mortality on several cultured sh. In this study, we
examined apparently healthy wild marine invertebrates by
polymerase chain reaction (PCR)-based techniques and
phylogenetically analyzed in order to know whether the
invertebrates population in the coastal areas might be con-
taminated and could be an inoculum source of genetically
closely related betanodaviruses that is transmitted horizon-
tally to cultured shes in the Korean Peninsula.
2. Materials and methods
2.1. Invertebrate samples
Samples (n = 118) from wild populations of apparently
healthy marine invertebrates (21 species) were collected
near the mariculture area of Korea. All invertebrates were
caught using double line system shing gear by demersal
trawling survey of the grounds o the west (Hwanghae)
and south (Namhae) coasts of Korea (Fig. 1). A total of
Fig. 1. Map of Korean Peninsula, location of the sampling sites: (A)
Hwanghae and (B) Namhae. (d) indicating trawling areas used to collect
wild marine invertebrates from October to November 2005.
40 (5 species) and 78 (18 species) samples were randomly
collected from Hwanghae (October 2005) and Namhae
(OctoberNovember 2005) areas, respectively (Table 1).
The sample species of marine invertebrates targeted for
sampling included shrimp, crab and shellsh. The organs
were aseptically collected, frozen and then stored at
80 C until used.
2.2. RNA extraction
Total RNA was extracted from organ tissues by TRI-
zol Reagent (Invitrogen, California, USA) using RNA
extraction kit, according to manufacturers instruction.
In brief, the brain or hepatopancreas tissues were
homogenized with 900 ll TRIzol reagent and incubated
for 5 min at room temperature. Two-hundred microliters
of chloroform were added to the suspension, shaken and
incubated for 23 min at room temperature, and then
centrifuged at 12,000g for 15 min at 4 C. The RNA in
the aqueous upper phase was precipitated by adding
500 ll of isopropanol; the mixture was incubated for
10 min at room temperature and then centrifuged at
12,000g for another 10 min at 4 C. The pellet obtained
after centrifugation was washed with 1 ml of 75% etha-
nol and centrifuged at 7500g for 5 min at 4 C. The
RNA pellet was air-dried and dissolved in 50 ll of
diethylpyrocarbonate-treated water (DEPC) (Biosesang,
Seoul, Korea).
2.3. PCR amplication
The PCR amplication was conducted using primers
BNV-RT, BNV-UR1, BNV-UF1 for reverse transcrip-
tion-polymerase chain reaction (RT-PCR), and BNV-
UR2, BNV-UF2 for nested polymerase chain reaction
(nPCR). These primers target regions 570 and 420 bp of
the RGNNV RNA2, as previously described (Gomez
et al., 2004). After reverse transcription using Reverse
Transcriptase SuperScript II (Invitrogen, California,
USA) at 45 C for 60 min, PCR was conducted using Ex
Taq polymerase (TaKaRa, Shiga, Japan) with 30 cycles
of denaturation at 94 C for 30 s, annealing at 57 C for
20 s, and a nal extension at 72 C for 60 s. Nested PCR
was conducted using the same protocol described above.
The PCR products were analyzed by 2% agarose gel elec-
trophoresis. The RNA from uninfected (negative control)
and infected (positive control) redspotted grouper Epi-
nephelus akaara larvae was used for RT-PCR and nested
PCR, respectively.
2.4. Sequence and analysis of sequence data
Four positive nPCR products were electrophoresed in
2% agarose gel in 0.5 TrisacetateEDTA (TAE) buf-
fer. The gel band of the target size was recovered from
agarose gels and puried using the power gel extraction
kit (DyneBioInc, Gyeonggi-do, Korea) following the
 D.K. Gomez et al. / Journal of Invertebrate Pathology 97 (2008) 197202
Table 1
Betanodaviruses detected by nested PCR in apparently healthy wild marine invertebrates
Invertebrates species
Swimming crab (Charybdis japonica)
Spiny lobster (Ibacus ciliatus)
Beka squid (Loligo beak)
Swimming crab (Ovalipes punctatus)
Swimming crab (Portunus trituberculatus)
Divaricated nut clam (Acila divaricata)
Snapping shrimp (Alpheus japonicus)
Goby shrimp (Alpheus rapax)
Monkey crab (Carcinoplax longimana)
Sea crab (Carcinoplax vestitus)
Charybdid crab (Charybdis bimaculata)
Swimming crab (Charybdis japonica)
Sand shrimp (Crangon hakodatei)
Flatnose shrimp (Latreutes planirostris)
Mediterranean mussel (Mytilus galloprovincialis)
Southern humpback shrimp (Pandalus hypsinotus)
Smooth shell shrimp (Parapenaeopsis tenellus)
Three spots swimming crab (Portunus sanguinolentus)
Swimming crab (Portunus trituberculatus)
Cuttlesh (Sepia ocinalis)
Bonnet whelk (Siphonalia cassidariaeformis)
Big head shrimp (Solenocera melantho)
Mantis shrimp (Squilla oratoria)
Total
Rate (%)
a
H, wild marine invertebrates from Hwanghae (West Sea) area and N, wild marine invertebrates from Namhae (South Sea) area. Designated code of
four Korean nodavirus strain sequenced for the phylogenetic analysis.
b
CC05WIKr1.
c
MM05WIKr1 and MM05WIKr2.
d
SHS05WIKr1.
manufacturers instruction. The puried amplication
products were sequenced using BigDyeTM terminator
cycle sequencing kit (Macrogen Genomic Division,
Seoul, South Korea). Electrophoresis of sequencing reac-
tions was completed using Automatic Sequencer 3730xl
DNA analyzer (Applied Biosystems, California, USA).
As the nested PCR products include variable regions
and homologous regions commonly observed in betan-
odaviruses (Iwamoto et al., 2004), variable regions
sequences were selected and used for phylogenetic anal-
ysis. The variable region of RGNNV RNA2 (177 nt
from nt no. 666 to 842) (Iwamoto et al., 2004) was
aligned with the betanodavirus sequences obtained in
this study as well as those retrieved from the GeneBank
database. Extra sequences were trimmed by comparing
with the 177 nt RGNNV RNA2 sequence. All nucleo-
tide sequences and deduced amino acid sequences were
aligned using the multiple alignment algorithm in the
MegAlign package (Windows version 3.12e; DNASTAR,
Wisconsin, USA) to give a phylogenetic tree. The Gen-
Bank accession numbers of the known betanodavirus
sequences used in this phylogenetic analysis were striped
jack (D30814), barn ounder (D38635), redspotted
grouper (D38636) and tiger puer (D38637).
199
Sourcesa No of positive sample
No. of invertebrates examined
H 0/5
H 0/3
H 0/7
H 0/20
H 0/5
N 0/5
N 0/2
N 0/1
N 0/11
N 0/5
N 1/9b
N 0/5
N 0/4
N 0/1
N 2/2c
N 1/8d
N 0/1
N 0/2
N 0/1
N 0/5
N 0/11
N 0/1
N 0/4
4/118
3.38
3. Results
3.1. PCR amplication
Detection of betanodavirus by nested PCR were 4/118
(3.38%) and the results are summarized in Table 1. From
the apparently healthy wild marine invertebrates collected,
40 samples in Hwanghae were negative for nodavirus in
both RT-PCR and nested PCR tests. Four of 78 samples
collected in Namhae were positive for nodavirus in nested
PCR. Positive nested PCR results were obtained from the
brain of Charybdid crab Charybdis bimaculata, hepatopan-
creas of Southern humpback shrimp Pandalus hypsinotus
and Mediterranean mussel Mytilus galloprovincialis.
3.2. Sequence and phylogenetic tree analysis
Partial coat protein gene (RNA2) nucleotide sequences
of the four strains detected were determined and designated
a code (Table 1). The four Korean nodavirus strains
showed 100% nucleotide similarity with RGNNV
(D38636), 79.7% with BFNNV (D38635), 71.2% with
TPNNV (D38637) and 67.8% with SJNNV (D30814)
(Table 2). The identities of the sequences from nucleotides
200 D.K. Gomez et al. / Journal of Invertebrate Pathology 97 (2008) 197202

Table 2
Nucleotide (nt 666842) percent similarity and divergence among betanodavirus strainsa
1 2 3 4 5 6 7 8
BFNNV 1 64.4 72.9 79.7 79.7 79.7 79.7 79.7
SJNNV 2 48.0 71.2 67.8 67.8 67.8 67.8 67.8
TPNNV 3 33.7 36.3 71.2 71.2 71.2 71.2 71.2
RGNNV 4 23.8 42.0 36.3 100.0 100.0 100.0 100.0
SHS05WIKr1 5 23.8 42.0 36.3 0.0 100.0 100.0 100.0
CC05WIKr1 6 23.8 42.0 36.3 0.0 0.0 100.0 100.0
MM05WIKr1 7 23.8 42.0 36.3 0.0 0.0 0.0 100.0
MM05WIKr2 8 23.8 42.0 36.3 0.0 0.0 0.0 0.0
a
Determined by using the Jotun Hein Method MegAlign package (Windows version 3.12e; DNASTAR, Madison, WI). Nucleotide similarities and
divergence are presented in the upper right half and the lower left half, respectively. The nucleotide sequences of four genotypes were obtained from
GenBank and their accession numbers are as follows: BFNNV (D38635), SJNNV (D30814), TPNNV (D38637) and RGNNV (D38636).

666 to 842 of the RNA2 coat protein gene among
SHS05Wikr1, CC05WIKr1, MM05WIKr1 and
MM05WIKr2 were 100% similar to each other (Table 2).
The sequences of the four nested PCR products obtained
from the four Korean nodavirus strains were identical
and showed only two nucleotide dierences at the same
positions (nt no. 677 and 827) between the RGNNV-type.
These two nucleotide dierences did not aect the encoded
amino acid residue (Fig. 2). Phylogenetic tree was con-
structed based on RNA2 coat protein gene. The host range
of Korean nodavirus strains includes three species of wild
marine invertebrates; the phylogenetic analysis revealed
that all Korean strains belong to the RGNNV genotype
(Fig. 2).
4. Discussion
Nodaviruses were already known to infect insects, fresh-
water and marine shes (Ball and Johnson, 1998; Hedge
et al., 2003; Munday et al., 2002). The rst description of
VNN on the southern coast of Korea was reported appar-
ently in 1998 in cage-cultured groupers Epinephelus septem-
fasciatus, although in this case, the causative agent was not
identied (Sohn et al., 1998). There were also incidences of
nodavirus disease outbreaks from 1999 to 2000 in four
hatcheries culturing red drum Sciaenops ocellatus larvae
located at Yochon and Yosu (Namhae) areas of the south-
ern coast of Korea (Oh et al., 2002). However, we have
long suspected that other biological organisms (crustacean
and shellsh) contaminated with nodavirus in the water or
environment also attributes VNN infection in cultured
marine shes in Korea.
In this study, nPCR yielded four positive results consist-
ing of three dierent species of marine invertebrates
(shrimp, crab and mussel). The results showed that approx-
imately 3.38% of the invertebrate samples collected in the
southern coastal area of Korea were positive for nodavirus.
Four nodavirus strains detected were sequenced and phylo-
genetic analysis revealed that all strains were identical and
belongs to the RGNNV genotype. To eliminate the pres-
ence of PCR product contamination, four positive PCR
samples were tested using E-11 cell lines (Iwamoto et al.,
2000) for virus isolation. There were only two viruses iso-
lated (data not shown). It may be due to the presence of
low virus titer in carrier invertebrates and a low virus detec-
tion limits in E-11 cell lines. Usually, the samples that were
negative for nodavirus on RT-PCR, but were conrmed
positive on nested PCR, suggest that these marine inverte-
brates have been latently infected with betanodaviruses.
Almost all of the subclinically infected wild marine inverte-
brates detected and identied showed no gross pathology
suggesting that virus might have been present at low con-
centrations in the aected tissues of invertebrates that can
be classied as asymptomatic carriers. The lack of any clin-
ical symptoms and obvious pathology is typical of viral
infections reported from wild invertebrates. Most infec-
tions are presumably latent and pathology does not mani-
fest itself. Invertebrates will quickly die when suering

Fig. 2. Multiple alignment of deduced amino acid sequences of RNA2 coat protein gene (aa 224280) of four major sh genotypes and the Korean
invertebrate betanodavirus strains. GenBank accession numbers of four major genotypes used in the above are as follows: BFNNV (D38635), SJNNV
(D30814), TPNNV (D38637) and RGNNV (D38636).
 D.K. Gomez et al. / Journal of Invertebrate Pathology 97 (2008) 197202
from acute infections; thus, it is dicult to nd such infec-
tions among wild population. The data of positive nested
PCR results indicate that nodavirus is not only widespread
in apparently healthy cultured and wild marine sh
(Gomez et al., 2004; Baeck et al., 2007) but also in appar-
ently healthy wild marine invertebrates. So far, this is the
rst reported case of apparently healthy wild marine inver-
tebrates to be subclinically infected with betanodavirus in
(Namhae) southern coastal area of Korea.
In the present study, crustacean samples such as P. hyp-
sinotus Southern humpback shrimp and C. bimaculata
Charybdid crab were subclinically infected with nodavirus
in the hepatopancreas and in the brain of the samples,
respectively. The presence of virus in crustacean diseases
has previously been reported (Bonami, 1980; Mari, 1987).
Gomez et al. (2006) have detected nodavirus from the brain
of the spiny lobster Pamulirus versicolor, a marine inverte-
brate imported from Japan in one of the commercial
aquarium in Korea. In Taiwan, Chi et al. (2000, 2003)
reported the detection of nodavirus in other live food
organisms, such as crustaceans including brine shrimp nau-
plii Artemia sp., the copepod Tigriopus japonicus, and the
shrimp Acetesinte medius. It has also been reported from
Taiwan, China and French West Indies (Sri Widada
et al., 2003), that freshwater shrimp Macrobrachium rosen-
bergii with white tail disease (WTD) have also been
infected with a virus in the hepatopancreas, and the causa-
tive pathogen has been identied as the M. rosenbergii
nodavirus (MrNV) (Arcier et al., 1999). In the present
study, the sample tested was consistent with the report of
Arcier et al. (1999), that nodavirus was also positive in
the hepatopancreas of the shrimp. For mollusk sample
M. galloprovincialis Mediterranean mussel, nodavirus was
also subclinically infected with nodavirus in the hepatopan-
creas. It was also observed in Japan that marine inverte-
brates such as mollusks, Japanese common squid
Todarodes pacicus used as feeds for aquaculture were sub-
clinically infected by betanodaviruses in the brain (D.K.
Gomez, personal communication). This was the rst report
of nodavirus in the aected mollusk species in Japan.
What is the implication for the presence of nodavirus
from apparently healthy wild marine invertebrates? Detec-
tion of betanodavirus from these wild marine invertebrates
populations may indicate a horizontal transmission from
either diseased or subclinically infected cultured or wild
marine shes from the mariculture areas that were released
to the open sea via sea-ranching or contact with other sub-
clinically infected or diseased wild marine sh that has been
there swimming around or near the mariculture area.
Gomez et al. (2004) have reported high frequencies (67%)
of betanodaviruses detection by nPCR method from
apparently healthy cultured and wild marine sh near the
mariculture areas in Japan. Baeck et al. (2007) also
detected about 7.2% betanodaviruses by nPCR from wild
marine sh in the southern coastal area of Korea. Another
possibility that some highly migratory sh from Japan
such as Japanese jack mackerel in which VNN has
201
previously occurred (Muroga, 2001; Gomez et al., 2004)
have transmitted nodavirus horizontally to the inverte-
brates population in Korea because of the closer distance
between the two countries. This might explain the preva-
lence of nodavirus among the wild invertebrates in Korean
Peninsula.
It is dicult to draw any clear conclusions about the
importance of betanodaviruses in wild invertebrate, but
as the present study shows, it cannot be excluded that
invertebrate may also constitute an inoculum source for
betanodavirus infection and cause mortality to susceptible
sh species cultured in Korea. In order to demonstrate
these speculations, however, further intensive molecular
epidemiological, pathological and virological analyses will
be required.
Acknowledgments
This study was supported by the Korea Research Foun-
dation Grant (KRF-005-E00076) and the Research Insti-
tute for Veterinary Science.
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