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Journal of
Journal of Invertebrate Pathology 97 (2008) 197202

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,*
KRF Zoonotic Disease Priority Research Institute, Seoul National University, Seoul 151-74, Republic of Korea
Faculty of Marine Technology, Chonnam National University, Yeosu 550-749, Republic of Korea
Brain Korea 21 Program for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea
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


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

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

0022-2011/$ - see front matter  2007 Elsevier Inc. All rights reserved.
198 D.K. Gomez et al. / Journal of Invertebrate Pathology 97 (2008) 197202

Viral nervous necrosis was rst reported in larvae and 40 (5 species) and 78 (18 species) samples were randomly
juveniles of hatchery-reared Japanese parrotsh Oplegna- collected from Hwanghae (October 2005) and Namhae
thus fasciatus (Yoshikoshi and Inoue, 1990). The same viral (OctoberNovember 2005) areas, respectively (Table 1).
disease has also been observed in barramundi Lates calca- The sample species of marine invertebrates targeted for
rifer (Glazebrook et al., 1990) and later on in other marine sampling included shrimp, crab and shellsh. The organs
shes (Mori et al., 1992; Muroga, 2001; Munday et al., were aseptically collected, frozen and then stored at
2002). Recent studies also demonstrated that a certain pop- 80 C until used.
ulation of apparently healthy wild marine sh carried bet-
anodaviruses, and suggested that these wild sh can be a 2.2. RNA extraction
persistent potential source of virus for cultured sh (Barker
et al., 2002; Gomez et al., 2004; Baeck et al., 2007). In Total RNA was extracted from organ tissues by TRI-
Korea, there were reported incidences of VNN, which zol Reagent (Invitrogen, California, USA) using RNA
cause mortality on several cultured sh. In this study, we extraction kit, according to manufacturers instruction.
examined apparently healthy wild marine invertebrates by In brief, the brain or hepatopancreas tissues were
polymerase chain reaction (PCR)-based techniques and homogenized with 900 ll TRIzol reagent and incubated
phylogenetically analyzed in order to know whether the for 5 min at room temperature. Two-hundred microliters
invertebrates population in the coastal areas might be con- of chloroform were added to the suspension, shaken and
taminated and could be an inoculum source of genetically incubated for 23 min at room temperature, and then
closely related betanodaviruses that is transmitted horizon- centrifuged at 12,000g for 15 min at 4 C. The RNA in
tally to cultured shes in the Korean Peninsula. 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
2. Materials and methods 12,000g for another 10 min at 4 C. The pellet obtained
after centrifugation was washed with 1 ml of 75% etha-
2.1. Invertebrate samples nol and centrifuged at 7500g for 5 min at 4 C. The
RNA pellet was air-dried and dissolved in 50 ll of
Samples (n = 118) from wild populations of apparently diethylpyrocarbonate-treated water (DEPC) (Biosesang,
healthy marine invertebrates (21 species) were collected Seoul, Korea).
near the mariculture area of Korea. All invertebrates were
caught using double line system shing gear by demersal 2.3. PCR amplication
trawling survey of the grounds o the west (Hwanghae)
and south (Namhae) coasts of Korea (Fig. 1). A total of 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-
Fig. 1. Map of Korean Peninsula, location of the sampling sites: (A) fer. The gel band of the target size was recovered from
Hwanghae and (B) Namhae. (d) indicating trawling areas used to collect agarose gels and puried using the power gel extraction
wild marine invertebrates from October to November 2005. kit (DyneBioInc, Gyeonggi-do, Korea) following the
D.K. Gomez et al. / Journal of Invertebrate Pathology 97 (2008) 197202 199

Table 1
Betanodaviruses detected by nested PCR in apparently healthy wild marine invertebrates
Invertebrates species Sourcesa No of positive sample
No. of invertebrates examined
Swimming crab (Charybdis japonica) H 0/5
Spiny lobster (Ibacus ciliatus) H 0/3
Beka squid (Loligo beak) H 0/7
Swimming crab (Ovalipes punctatus) H 0/20
Swimming crab (Portunus trituberculatus) H 0/5
Divaricated nut clam (Acila divaricata) N 0/5
Snapping shrimp (Alpheus japonicus) N 0/2
Goby shrimp (Alpheus rapax) N 0/1
Monkey crab (Carcinoplax longimana) N 0/11
Sea crab (Carcinoplax vestitus) N 0/5
Charybdid crab (Charybdis bimaculata) N 1/9b
Swimming crab (Charybdis japonica) N 0/5
Sand shrimp (Crangon hakodatei) N 0/4
Flatnose shrimp (Latreutes planirostris) N 0/1
Mediterranean mussel (Mytilus galloprovincialis) N 2/2c
Southern humpback shrimp (Pandalus hypsinotus) N 1/8d
Smooth shell shrimp (Parapenaeopsis tenellus) N 0/1
Three spots swimming crab (Portunus sanguinolentus) N 0/2
Swimming crab (Portunus trituberculatus) N 0/1
Cuttlesh (Sepia ocinalis) N 0/5
Bonnet whelk (Siphonalia cassidariaeformis) N 0/11
Big head shrimp (Solenocera melantho) N 0/1
Mantis shrimp (Squilla oratoria) N 0/4
Total 4/118
Rate (%) 3.38
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.
MM05WIKr1 and MM05WIKr2.

manufacturers instruction. The puried amplication 3. Results

products were sequenced using BigDyeTM terminator
cycle sequencing kit (Macrogen Genomic Division, 3.1. PCR amplication
Seoul, South Korea). Electrophoresis of sequencing reac-
tions was completed using Automatic Sequencer 3730xl Detection of betanodavirus by nested PCR were 4/118
DNA analyzer (Applied Biosystems, California, USA). (3.38%) and the results are summarized in Table 1. From
As the nested PCR products include variable regions the apparently healthy wild marine invertebrates collected,
and homologous regions commonly observed in betan- 40 samples in Hwanghae were negative for nodavirus in
odaviruses (Iwamoto et al., 2004), variable regions both RT-PCR and nested PCR tests. Four of 78 samples
sequences were selected and used for phylogenetic anal- collected in Namhae were positive for nodavirus in nested
ysis. The variable region of RGNNV RNA2 (177 nt PCR. Positive nested PCR results were obtained from the
from nt no. 666 to 842) (Iwamoto et al., 2004) was brain of Charybdid crab Charybdis bimaculata, hepatopan-
aligned with the betanodavirus sequences obtained in creas of Southern humpback shrimp Pandalus hypsinotus
this study as well as those retrieved from the GeneBank and Mediterranean mussel Mytilus galloprovincialis.
database. Extra sequences were trimmed by comparing
with the 177 nt RGNNV RNA2 sequence. All nucleo- 3.2. Sequence and phylogenetic tree analysis
tide sequences and deduced amino acid sequences were
aligned using the multiple alignment algorithm in the Partial coat protein gene (RNA2) nucleotide sequences
MegAlign package (Windows version 3.12e; DNASTAR, of the four strains detected were determined and designated
Wisconsin, USA) to give a phylogenetic tree. The Gen- a code (Table 1). The four Korean nodavirus strains
Bank accession numbers of the known betanodavirus showed 100% nucleotide similarity with RGNNV
sequences used in this phylogenetic analysis were striped (D38636), 79.7% with BFNNV (D38635), 71.2% with
jack (D30814), barn ounder (D38635), redspotted TPNNV (D38637) and 67.8% with SJNNV (D30814)
grouper (D38636) and tiger puer (D38637). (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
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 and shellsh) contaminated with nodavirus in the water or
SHS05Wikr1, CC05WIKr1, MM05WIKr1 and environment also attributes VNN infection in cultured
MM05WIKr2 were 100% similar to each other (Table 2). marine shes in Korea.
The sequences of the four nested PCR products obtained In this study, nPCR yielded four positive results consist-
from the four Korean nodavirus strains were identical ing of three dierent species of marine invertebrates
and showed only two nucleotide dierences at the same (shrimp, crab and mussel). The results showed that approx-
positions (nt no. 677 and 827) between the RGNNV-type. imately 3.38% of the invertebrate samples collected in the
These two nucleotide dierences did not aect the encoded southern coastal area of Korea were positive for nodavirus.
amino acid residue (Fig. 2). Phylogenetic tree was con- Four nodavirus strains detected were sequenced and phylo-
structed based on RNA2 coat protein gene. The host range genetic analysis revealed that all strains were identical and
of Korean nodavirus strains includes three species of wild belongs to the RGNNV genotype. To eliminate the pres-
marine invertebrates; the phylogenetic analysis revealed ence of PCR product contamination, four positive PCR
that all Korean strains belong to the RGNNV genotype samples were tested using E-11 cell lines (Iwamoto et al.,
(Fig. 2). 2000) for virus isolation. There were only two viruses iso-
lated (data not shown). It may be due to the presence of
4. Discussion low virus titer in carrier invertebrates and a low virus detec-
tion limits in E-11 cell lines. Usually, the samples that were
Nodaviruses were already known to infect insects, fresh- negative for nodavirus on RT-PCR, but were conrmed
water and marine shes (Ball and Johnson, 1998; Hedge positive on nested PCR, suggest that these marine inverte-
et al., 2003; Munday et al., 2002). The rst description of brates have been latently infected with betanodaviruses.
VNN on the southern coast of Korea was reported appar- Almost all of the subclinically infected wild marine inverte-
ently in 1998 in cage-cultured groupers Epinephelus septem- brates detected and identied showed no gross pathology
fasciatus, although in this case, the causative agent was not suggesting that virus might have been present at low con-
identied (Sohn et al., 1998). There were also incidences of centrations in the aected tissues of invertebrates that can
nodavirus disease outbreaks from 1999 to 2000 in four be classied as asymptomatic carriers. The lack of any clin-
hatcheries culturing red drum Sciaenops ocellatus larvae ical symptoms and obvious pathology is typical of viral
located at Yochon and Yosu (Namhae) areas of the south- infections reported from wild invertebrates. Most infec-
ern coast of Korea (Oh et al., 2002). However, we have tions are presumably latent and pathology does not mani-
long suspected that other biological organisms (crustacean 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 201

from acute infections; thus, it is dicult to nd such infec- previously occurred (Muroga, 2001; Gomez et al., 2004)
tions among wild population. The data of positive nested have transmitted nodavirus horizontally to the inverte-
PCR results indicate that nodavirus is not only widespread brates population in Korea because of the closer distance
in apparently healthy cultured and wild marine sh between the two countries. This might explain the preva-
(Gomez et al., 2004; Baeck et al., 2007) but also in appar- lence of nodavirus among the wild invertebrates in Korean
ently healthy wild marine invertebrates. So far, this is the Peninsula.
rst reported case of apparently healthy wild marine inver- It is dicult to draw any clear conclusions about the
tebrates to be subclinically infected with betanodavirus in importance of betanodaviruses in wild invertebrate, but
(Namhae) southern coastal area of Korea. as the present study shows, it cannot be excluded that
In the present study, crustacean samples such as P. hyp- invertebrate may also constitute an inoculum source for
sinotus Southern humpback shrimp and C. bimaculata betanodavirus infection and cause mortality to susceptible
Charybdid crab were subclinically infected with nodavirus sh species cultured in Korea. In order to demonstrate
in the hepatopancreas and in the brain of the samples, these speculations, however, further intensive molecular
respectively. The presence of virus in crustacean diseases epidemiological, pathological and virological analyses will
has previously been reported (Bonami, 1980; Mari, 1987). be required.
Gomez et al. (2006) have detected nodavirus from the brain
of the spiny lobster Pamulirus versicolor, a marine inverte- Acknowledgments
brate imported from Japan in one of the commercial
aquarium in Korea. In Taiwan, Chi et al. (2000, 2003) This study was supported by the Korea Research Foun-
reported the detection of nodavirus in other live food dation Grant (KRF-005-E00076) and the Research Insti-
organisms, such as crustaceans including brine shrimp nau- tute for Veterinary Science.
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- Arcier, J.M., Herman, F., Lightner, D.V., Redman, R.M., Mari, J.,
bergii with white tail disease (WTD) have also been Bonami, J.R., 1999. A viral disease associated with mortalities in
infected with a virus in the hepatopancreas, and the causa- hatchery-reared postlarvae of the giant freshwater prawn Macrobrach-
tive pathogen has been identied as the M. rosenbergii ium rosenbergii. Dis. Aquat. Org. 38, 177181.
Arimoto, M., Mushiake, K., Mizuta, Y., Nakai, T., Muroga, K.,
nodavirus (MrNV) (Arcier et al., 1999). In the present
Furusawa, I., 1992. Detection of striped jack nervous necrosis virus
study, the sample tested was consistent with the report of (SJNNV) by enzyme-linked immunosorbent assay (ELISA). Fish
Arcier et al. (1999), that nodavirus was also positive in Pathol. 27 (4), 191195.
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M. galloprovincialis Mediterranean mussel, nodavirus was Ball, L.A. (Eds.), The Insect Viruses. Plenum Press, New York, pp.
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