Genetic analysis and pathogenicity of betanodavirus isolated from wild redspotted grouper Epinephelus akaara with clinical signs.

Abstract

Diseased wild redspotted grouper Epinephelus akaara were collected from Seto Inland Sea, Ehime Prefecture, in August 2002. Fish showed erratic swimming behavior and inflation of the swim bladder. The fish brains were positive for nodavirus in both RT-PCR and nested PCR. The sequence of the nested PCR product (177 nt) was closely related to that of a known betanodavirus, redspotted grouper nervous necrosis virus. When juvenile sevenband grouper E. septemfasciatus were challenged intravitreously with virus, abnormal swimming behavior and high mortality were observed. This is the first report on viral nervous necrosis in a wild population of redspotted grouper with clinical signs.

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BRIEF REPORT
Genetic analysis and pathogenicity of betanodavirus isolated
from wild redspotted grouper Epinephelus akaara
with clinical signs
Dennis K. Gomez Æ Æ Satoru Matsuoka Æ Æ
Koh-ichiro Mori Æ Æ Yasushi Okinaka Æ Æ
Se Chang Park Æ Æ Toshihiro Nakai
Received: 18 August 2008/Accepted: 9 December 2008/Published online: 8 January 2009
? Springer-Verlag 2009
Abstract
akaara were collected from Seto Inland Sea, Ehime Pre-
fecture, in August 2002. Fish showed erratic swimming
behavior and inflation of the swim bladder. The fish brains
were positive for nodavirus in both RT-PCR and nested
PCR. The sequence of the nested PCR product (177 nt)
was closely related to that of a known betanodavirus,
redspotted grouper nervous necrosis virus. When juvenile
sevenband grouper E. septemfasciatus were challenged
intravitreously with virus, abnormal swimming behavior
and high mortality were observed. This is the first report on
viral nervous necrosis in a wild population of redspotted
grouper with clinical signs.
Diseased wild redspotted grouper Epinephelus
Betanodaviruses infection, also known as viral nervous
necrosis (VNN) or viral encephalopathy and retinopathy
(VER), is considered one of the most serious viral diseases
that occur particularly during the seedling and culture
process in a variety of marine fish in Japan [14] and in
other parts of the world [15]. VNN was first reported in
hatchery-reared Japanese parrotfish Oplegnathus fasciatus
[19] and barramundi Lates calcarifer [6], and later in
other marine fishes [4, 12, 15] such as redspotted grouper
Epinephelus akaara [11]. The transmission of VNN maybe
vertical [3] or horizontal [2, 7]. The characteristics of VNN
are necrosis and vacuolation of central nervous tissues and
retina of the affected larvae and juvenile fishes showing
high mortality [4]. Nodavirus is a member of the genus
Betanodavirus of the family Nodaviridae, and it is non-
enveloped, 25 to 30 nm in diameter, spherical in shape, and
contains two segments of positive-sense single-stranded
RNA. The RNA 1 (3.1 kb) encodes the viral replicase, and
RNA 2 (1.4 kb) encodes the coat protein [1]. Genomic
classification of betanodaviruses has revealed four major
types: striped jack nervous necrosis virus (SJNNV)-type,
tiger puffer nervous necrosis virus (TPNNV)-type, barfin
flounder nervous necrosis virus (BFNNV)-type and red-
spotted grouper nervous necrosis virus (RGNNV)-type
[16]. Recently, a fifth group was suggested as turbot
nodavirus (TNV)-type from turbot Scophthalmus maximus
[10]. In the present study, moribund wild juvenile red-
spotted grouper E. akaara with clinical signs of VNN were
collected and studied. A partial nucleotide sequence of the
RNA2 coat protein gene was analyzed to elucidate the
genetic relatedness of this wild redspotted grouper isolate
to other known betanodavirus isolates.
TenmoribundwildjuvenileredspottedgrouperE.akaara
(average body weight 85 g) that showed erratic swimming
behavior and inflation of the swim bladder were obtained
D. K. Gomez
Brain Korea 21 Program for Veterinary Science,
College of Veterinary Medicine, Seoul National University,
Seoul 151-742, South Korea
S. Matsuoka
Ehime Prefectural Chuyo Fisheries Experimental Station,
Ehime 799-3125, Japan
K.-i. Mori
National Research Institute of Aquaculture,
Fisheries Research Agency, Saiki 879-2602, Japan
S. C. Park (&)
Laboratory of Aquatic Animal Medicine,
College of Veterinary Medicine, Seoul National University,
Seoul 151-742, South Korea
e-mail: parksec@snu.ac.kr
Y. Okinaka ? T. Nakai (&)
Graduate School of Biosphere Science,
Hiroshima University,
Higashi-Hiroshima 739-8528, Japan
e-mail: nakaitt@hiroshima-u.ac.jp
123
Arch Virol (2009) 154:343–346
DOI 10.1007/s00705-008-0305-5

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from Ehime Prefectural Chuyo Fisheries Experimental
Station. These wild fish were captured near the aquaculture
area in Seto Inland Sea, Japan, in August 2002. The brains
were aseptically collected and stored at -80?C until use for
virus isolation. Ten brain samples were used for virus iso-
lation using an E-11 cell line [8] previously cloned from the
SSN-1 cell line [5]. The cells were maintained at 25?C with
Leibovitz L-15 medium (Invitrogen, USA) supplemented
with 5% (V/V) fetal bovine serum, 100 IU/ml penicillin G
and 100 lg/ml streptomycin. Briefly, brain tissue homoge-
nate with nine volumes of Hank’s balanced salt solution
(HBSS; Nissui, Japan) was membrane-filtered (0.45 lm;
Millipore) and inoculated onto cellsseeded in 24-well tissue
culture plates [8]. The ten isolated samples inoculated in E-
11 cells were incubated at 25?C for 10 days, and the
supernatant of the cells of all samples showing cytopathic
effect (CPE) atthree passages were harvested and stocked at
-80?Cuntilusedforpathogenicitytesting.Titrationofvirus
infectivity in a serial 10-fold dilution of the supernatant was
performed with E-11 cells seeded in a 96-well tissue culture
plate. The virus infectivity titers (TCID50/g) were read and
determined by Reed and Muench method [17]. Total RNA
was extracted from the five representative brain samples
using an RNA extraction kit (ISOGEN; Nippon Gene,
Japan)accordingtothemanufacturer’sinstructions.Inbrief,
the tissues were homogenized with ISOGEN and shaken
withchloroform,thencentrifugedat12,0009gfor15 minat
4?C. RNA in the aqueous phase was precipitated with iso-
propanolandthendissolvedindiethylpyrocarbonate-treated
water (DEPC; Invitrogen, USA). RT-PCR and nested PCR
amplification were conducted using five oligonucleotide
primers designed from sevenband grouper nervous necrosis
virus (SGNNV) RNA2, as described previously [7]. After
reverse transcription using Reverse Transcriptase M-MLV
(TaKaRa, Japan) at 45?C for 60 min, PCR was conducted
usingExTaqpolymerase (TaKaRa, Japan)with 30cyclesof
denaturation at 94?C for 30 s, annealing at 57?C for 20 s,
and extension at 72?C for 60 s. Nested PCR was conducted
using the same protocol described above. The amplified
products were analyzed by 2% agarose gel electrophoresis
and visualized under UV irradiation after being stained with
ethidium bromide. The RNA from uninfected (negative
control) and infected [11] (positive control) redspotted
grouper larvae were used for RT-PCR and nested PCR. A
positive nested PCR product from one representative isolate
RG02Ehi was recovered from an agarose gel and was puri-
fied using the QIAquick Gel Extraction kit (Qiagen,
Germany) as described in the manufacturer’s instructions.
The purified amplification product was subjected to nucle-
otide sequence analysis according to the method described
previously [13]. As the nested PCR product included vari-
able and homologous regions commonly observed in
betanodaviruses [9], for practical reasons, the variable
region of the SGNNV RNA2 sequence (177 nt from nt no.
666–842) [9] was selected, and extra sequences were trim-
med and then realigned using the multiple alignment
algorithms in the MegAlign package Windows Version
3.12e (DNASTAR, USA) along with the known betanoda-
virussequencesobtainedfromtheGenBankdatabasetogive
a phylogenetic tree. The tree was constructed based on
neighbor-joining method (multiple alignment data using
the Jotun Hein Method in the MegAlign package). The
GenBank accession numbers of the known betanodavirus
sequences used in this study were: redspotted grouper,
RG91tok (D38636); striped jack, SJOri (D30814); barfin
flounder Verasper moseri, BF93hok (D38635); tiger puffer
Takifugu rubripes, TP93Kag (D38637); guppy Poicelia re-
ticulata, Gup (AF499774); white grouper E. aeneus,
EA040799-IL (AY284963); sevenband grouper E. septem-
fasciatus, SGWak97 (AY324870); dragon grouper E.
lanceolatus, DGNNV (AY721615); orange-spotted gro-
uper E. coioides, ECNNV (EF492143); greasy grouper E.
tauvina, ETNNV1 (AF281657); European seabass Dicen-
trarchus labrax, DL040899-IL (AY284969); white star
snapper Lutjanus stellatus, WSSNNV (AY835642); greasy
grouperE.tauvina,ETNNV2(NC_004136);greasy grouper
E. tauvina, GGNNV (AF318942); gray mullet Mugil
cephalus, MC061198-IL (AY284962) and barramundi
L. calcarifer, LC220800 (AY284973). One representative
isolate of RG02Ehi was used for experimental infections
with healthy juvenile sevenband grouper E. septemfasciatus
(average body weight 63 g), which were reared at Kamiura
Station, National Research Institute of Aquaculture, Fish-
eries Research Agency. Prior to the infection experiment,
the brains of some healthy sevenband grouper were ran-
domly examined for nodavirus by PCR assays [7], and all
were negative for nodavirus (data not shown). For the
experimental challenge of juvenile sevenband grouper, a
total of 30 fish were stocked in three 70-l aquaria at ten fish
per aquarium. The aquaria were maintained at 23–25?C for
14 days, supplied with ozonated seawater. Fish were fed
once a day with commercially prepared pellets. The
RG02Ehi (experimental treatment) and SGWak97 (positive
control) isolates 106TCID50/fish/0.03 ml/fish or HBSS
(negativecontrol)wasinjectedintoeachfishintravitreously.
During experimental infections, dead fish were removed
daily and kept at -80?C; surviving fish were collected at
14 days after challenge. Mortality of fish was observed for
2 weeks, and recovery of nodavirus from the brain of all fish
in the challenge and control groups was conducted to fulfill
Koch’s postulates.
All of the brains of the five representative fish samples
werepositivefornodavirusinbothRT-PCRandnestedPCR.
A partial coat protein gene (RNA2) sequence corresponding
to nucleotides (nt) 666–842 of one representative isolate,
RG02Ehi, was amplified by nested PCR for nucleotide
344D. K. Gomez et al.
123

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sequence analysis. A comparison of the sequences of
RG02Ehi and RG91tok (D38636) showed nearly 100%
sequence identity, with only 2 nt differences. These 2 nt
differences, at positions 736 and 775, did not affect the
encoded amino acid residues. It also showed 96.6–100%
sequence identity against other known isolates: AF499774,
AY284963, AY324870, AY721615, EF492143, AF281657,
AY284969,AY835642,NC_004136,AF318942,AY284962
and AY284973. On the other hand, RG02Ehi showed
sequence identities of 71.2% with BF93hok (D38635),
62.7% in TP93Kag (D38637) and 67.8% in SJOri
(D30814). Phylogenetic analysis confirmed that the wild
redspotted grouper isolate RG02Ehi belongs to the RGNNV
genotype (Fig. 1). In this study, we reported the isolation of
betanodavirus from the brain of wild juvenile redspotted
grouper swimming near the aquaculture area in Seto Inland
Sea. We determined the viral strain classification using
PCR-based amplification techniques with SGNNV RNA2-
specific primer sets [7], nucleotide sequencing, phylo-
genetic analysis, virus isolation and pathogenicity testing.
The isolated virus is closely related to a known betanoda-
virus, redspotted grouper nervous necrosis virus (RGNNV)
[18]. It was also confirmed that the virus isolate was
genetically distinguishable from other three major geno-
types: SJNNV, BFNNV and TPNNV, which further
elucidates the differences between these betanodavirus
strains. So far, this is the first record of VNN in the wild
marine fish population in Japan where the fish exhibited
clinical signs. Although some research facilities and rearing
ponds (Hiroshima and Tokushima Prefectural Fisheries
Experiment Stations) near the Seto Inland Sea had already
experienced and reported several outbreaks of VNN from
August to September 1990, these outbreaks had only
affected the cultured larvae and juvenile stages of red-
spotted grouper [11]. It is believed that the RGNNV
genotype is the main cause of disease in these wild popu-
lations of redspotted grouper, since this fish has been
reported to be one of its susceptible hosts [11]. We also
think that the virus isolate could be an indigenous type of
betanodavirus distributed in the Seto Inland Sea based on
the results of the present study and the past reported cases of
VNN in that area.
Sevenband grouper injected with RG02Ehi started to
show abnormal swimming behavior, loss of body balance
and poor feeding on the fourth day. Ten percent mortality
occurred on the ninth day post-injection (p.i.) and reached
60% by 2 weeks p.i. The ranges of virus titers from the dead
fish and survivors were 107.3TCID50/g to 108.6TCID50/g
and 105.2TCID50/g to 106.6TCID50/g, respectively. The
levels of pathogenicity were also similar to those of
sevenband grouper injected with virus isolate SGWak97
(positive control). These fish started to show abnormal
swimming and poor feeding behavior on the fifth day p.i.
Forty percent mortality occurred on the 9th day and reached
80% on the 14th day p.i., with virus titers ranging from 106.4
TCID50/g to 108.6TCID50/g. Twenty percent of the fish
survived, with virus titers ranging from 105.7TCID50/g to
105.9TCID50/g. Moreover, neither fish abnormality nor
virus titers (\102.05TCID50/g) was observed in the HBSS
group (negative control). It should be noted that Tanaka
et al. [18] reported the pathogenecity of nodavirus isolated
fromdiseasedsevenbandgrouper(RGNNVtype)injectedto
healthy cultured redspotted grouper induced similar
behavioral abnormalities starting at 2 days p.i. with 20%
SGWak97
RG91tok
MC061198
TP93kag
SJOri
BF93hok
Gup
ETNNV1
RG02Ehi
WSSNNV
ECNNV
GGNNV
ETNNV2
EA040799-IL
DGNNV
LC220800
DL040899-IL
100
100
100
100
77
Substitution/site
RGNNV
0.01
RGNNV
TPNNV
SJNNV
BFNNV
Fig. 1 Phylogenetic tree based
on partial nucleotide sequences
(nt 666–842) of the RNA2 coat
protein gene of RG02Ehi and
other known betanodavirus
isolates. This tree was
constructed by the neighbor-
joining method in the MegAlign
package. Numbers at branch
nodes indicate percentage
bootstrap support for the node
with 1,000 replications
Betanodavirus strains in redspotted grouper345
123

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mortality. Previous studies also reported 30% mortality in
healthy cultured redspotted grouper by experimental infec-
tion with isolated virus from the same species of grouper
[11]. Furthermore, similar clinical signs (erratic and cork-
screw swimming behavior as well as inflation of the swim
bladder) displayed by the affected fish was observed to be a
characteristic of VNN disease among cultured redspotted
grouper [4, 11] affected by the RGNNV genotype.
It has been discovered that betanodavirus isolates from
groupers that belong to the same RGNNV genotype pro-
liferate well at higher temperature in cultured cells
compared with the other three genotypes (SJNNV,
TPNNV, BFNNV), indicating that this genotypic variant
originates from warm-water fish [8]. Another finding in
groupers is that the VNN infection usually occurs in cul-
tured or hatchery-reared stages, but in the present study, it
occurs in the wild population. We speculate that these wild
redspotted grouper populations have possibly been infected
horizontally with RGNNV-type nodavirus originating from
subclinically infected or diseased cultured marine fish that
were released to the open sea by sea farming. Another
possibility is that RGNNV-type nodavirus could be trans-
mitted from other subclinically infected or diseased wild
marine fish that were there swimming around the mari-
culture area. Moreover, RGNNV-type nodavirus could also
be transmitted via environmental water and other biologi-
cal organisms that were infected in the environment.
Therefore, the present result indicates that these wild red-
spotted grouper juveniles were severely affected by
betanodavirus (RGNNV-type) infection and showed clini-
cal signs of VNN. Furthermore, these affected wild
redspotted grouper may be a persistent potential source of
RGNNV-type nodavirus to the other susceptible healthy
cultured marine fish species near the mariculture areas.
Acknowledgments
Aid Scientific Research (B) (16380132) from Ministry of Education,
Culture, Sports, Science and Technology, Japan.
This study was supported in part by a Grant-in-
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