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Fish & Shellfish Immunology 17 (2004) 1e11
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In vivo and in vitro analysis of the resistance against viral
haemorrhagic septicaemia virus in Japanese flounder
(Paralichthys olivaceus) precedingly infected with
aquabirnavirus
Rolando Pakingking Jra, Yasushi Okinakaa, Koh-Ichiro Morib,
Misao Arimotob, Kiyokuni Murogaa,c, Toshihiro Nakaia,)
a
Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima 739e8528, Japan
b
Kamiura Station, Japan Sea-Farming Association, Oita, 879e2602, Japan
c
Graduate School of Agricultural Science, Tohoku University, Sendai, 981e8555, Japan
Received 4 July 2003; accepted 15 October 2003
Abstract
The resistance of Japanese flounder (Paralichthys olivaceus Temminck et Schlegel) against a viral haemorrhagic
septicaemia virus (VHSV) challenge induced by a preceding non-lethal aquabirnavirus (ABV) challenge was investigated
through experimental dual-infections with different intervals between the two challenges. The non-specific protection
conferred by the primary ABV infection against the secondary VHSV infection commenced at Day 3 and persisted up to
Day 14 but vanished at Day 21 post-ABV challenge. The in vitro assay using HINAE (hirame natural embryo) cells
demonstrated anti-VHSV activity in the serum of ABV-challenged flounder from Day 1 to Day 14 but not at Day 21
post-ABV challenge. A high expression of a Mx gene, a molecular marker of type I interferon(s) (IFN) occurred in the
head kidneys of ABV-challenged flounder from Day 1 to Day 7. These results suggest that the non-specific protection
against the secondary VHSV infection in flounder was due to IFN(s) induced by the primary ABV infection.
2003 Elsevier Ltd. All rights reserved.
Keywords: Interferon; Mx protein; Dual-infection; Viral haemorrhagic septicaemia virus; Aquabirnavirus; Paralichthys olivaceus
1. Introduction
Japanese flounder Paralichthys olivaceus (Temminck et Schlegel) is an important fish species in
aquaculture in Japan. However, occurrence of infectious diseases, particularly of bacterial and viral
aetiologies is a major impediment besetting production [1]. Recently, viral haemorrhagic septicaemia (VHS)
has been reported as a new disease problem in flounder culture in Japan [2].
) Corresponding author. Tel.: +81-824-247947; fax: +81-824-227059.
E-mail address: [email protected] (T. Nakai).
1050-4648/$ - see front matter 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2003.10.005
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R. Pakingking et al. / Fish & Shellfish Immunology 17 (2004) 1e11
In a surveillance made in 1999e2000 on virus distribution among wild Japanese flounder populations
collected in several coastal areas of Japan, aquabirnavirus (ABV), serologically identified as yellowtail
ascites virus (YTAV=YAV) [3], was frequently isolated (ca 40%) in addition to viral haemorrhagic
septicaemia virus (VHSV) (6.6%) [4]. The high virulence of VHSV isolates in flounder was revealed by
experimental infection while ABV isolates failed to induce pathology to young flounder. In a successive
survey on the virus distribution, ABV was isolated from 15% of wild flounder but at a very low rate (1.5%)
from cultured ones [5]. To investigate the role of ABV in infections with other pathogens in flounder, we
sequentially conducted experiments with VHSV and two pathogenic bacteria (Edwarsiella tarda and
Streptococcus iniae) as the secondary pathogens. It was found that the primary infection of flounder with
ABV slightly reduced resistance against these bacterial infections, but apparently increased resistance
against VHSV infection [6].
Increased knowledge about the non-specific defense mechanisms of Japanese flounder against viruses
should help explain the phenomenon we previously demonstrated through the dual-infection experiment. In
the previous study, we speculated that the non-specific protection induced by the preceding ABV infection was
due to interferon (IFN), which is an important first line of defense against viral infections in vertebrates [7]. In
salmonids, induction of the type I IFN-like activity has been experimentally demonstrated in vivo and in vitro,
either after virus infection or exposure to the dsRNA polyinosinic polycytidylic acid ( poly I:C) [8e10].
In the present study, a more detailed investigation on the onset and duration of the resistance in
Japanese flounder conferred by the preceding ABV infection against subsequent VHSV infection was
conducted. We analyzed the mechanisms of this resistance by correlating the high survival rates of fish
dually infected with ABV and VHSV with an in vitro antiviral activity against VHSV in the sera of ABVinfected flounder collected at different time intervals. Additionally, we monitored the level of Mx gene
expression in the kidney of ABV-infected fish because recent studies have implicated the usefulness of Mx
protein as a putative molecular marker of the type I IFN production in fish [11e13], coupled by the fact
that the Japanese flounder Mx (JFMx) cDNA has already been cloned [14].
2. Materials and methods
2.1. Viral strains
2.1.1. Aquabirnavirus
A strain (FBV) of ABV originally isolated from Japanese flounder [15] was used as the primary agent in
the dual-infection experiment. ABV was propagated in rainbow trout gonad-2 (RTG-2) cells maintained at
20 (C in Eagle’s minimum essential medium (MEM, Nissui) supplemented with 10% (V/V) fetal bovine
serum (=MEM10), 150 IU ml ÿ1 penicillin G and 50 mg ml ÿ1 kanamycin sulfate at a multiplicity of
infection (MOI) of 0.01 TCID50 cell ÿ1. When the cytopathic effect (CPE) was extensive, the supernatant
was obtained by centrifugation and stocked at ÿ80 (C until used. The titer (TCID50 ml ÿ1) of the virus
stock was determined by end point dilution [16] in 96-well plates seeded with RTG-2 cells.
2.1.2. VHSV
The Obama25 strain of VHSV [4] at the 3rd passage from the original isolation was used as the
secondary agent in the dual-infection experiment. VHSV was propagated (MOI: 0.01 TCID50 cell ÿ1) in
fathead minnow (FHM) cells maintained in MEM10 supplemented with antibiotics at 20 (C. The harvested
supernatant was processed and stocked as described above.
2.2. Experimental fish
Healthy Japanese flounder produced and reared at the Kamiura Station of Japan Sea-Farming Association
(JASFA) were used in this study. Average body weight of fish was 18 g for the dual-infection experiment and
R. Pakingking et al. / Fish & Shellfish Immunology 17 (2004) 1e11
3
25 g for assays of IFN-like activity and Mx gene expression. Fish were acclimatized in the laboratory for
10 days and maintained in a tank supplied with sand-filtered and flow-through seawater at 20 (C. Ten fish
were randomly sampled to ascertain the absence of ABV and VHSV by isolation using the above cell lines.
2.3. Dual-infection
The dual-infection experiment was carried out following a modified protocol adapted from our previous
study [6]. The primary infection with ABV (106.9 TCID50 fishÿ1) via intramuscular (IM) injection was
conducted on 160 flounder that were consequently divided into eight subgroups at a density of 20 fish in
a 100 l aquarium. The same number of fish (control group) were injected with MEM10 and also divided into
eight subgroups (20 fish per aquarium). Twenty extra fish injected with the same dose of ABV were placed
in another 100 l aquarium and five fish were randomly sampled for virus titration at Days 3, 7, 14, and 21
post-ABV challenge. ABV titration was done by the method described in the next section.
At Day 3 post-ABV challenge, two subgroups from the ABV-injected group were randomly chosen; fish
in one subgroup were IM injected with VHSV (107.1 TCID50 fishÿ1) (=ABV+VHSV) while the other were
given MEM10 (=ABV+MEM10). Similarly, two subgroups from the control group were randomly chosen
and injected with the same dose of VHSV (=MEM10+VHSV) or MEM10 (=MEM10+MEM10). These
four subgroups of fish were collectively designated as Day 3-challenged group. The challenge of fish with
the same dose of VHSV or MEM10 obtained from each of the ABV-injected and control group were also
done at Days 7, 14, and 21 post-ABV or -MEM10 injection and designated as Day 7, 14, and 21-challenged
groups, respectively. After the injection, flounder were returned to their respective aquaria and maintained
in the same conditions as before. Fish mortalities were recorded and dead fish were removed from the
aquaria daily. Experiments were terminated 14 days after VHSV inoculation.
2.4. Quantification of ABV and VHSV titers
VHSV and ABV infectious titers in the head kidneys of all dead and surviving dual-infected fish were
quantified following the method published previously [6]. Briefly, head kidneys of dead and surviving
flounder were aseptically excised, homogenized in nine volumes of Hanks’ balanced salt solution,
centrifuged, and the supernatants were filtered using a 0.45-mm filter. VHSV titration was carried out in 96well plates seeded with FHM cells after the sample homogenate was treated with a diluted rabbit antiserum
(1:100) against YTAV to neutralize ABV infectivity. Head kidneys of dead fish solely challenged with
VHSV were titrated in the same cell line without YTAV-neutralizing process. Since RTG-2 cell line in our
laboratory is not highly susceptible to flounder isolates of VHSV [17], the filtered homogenates were
directly titrated in RTG-2 cells to quantify ABV titers. Virus multiplication in the FHM or RTG-2 cells,
that produced CPE, was confirmed by an indirect fluorescent antibody technique using a diluted rabbit
antiserum against VHSV (1:100) or YTAV (1:500) [6].
2.5. Assays of anti-VHSV activity in the sera and Mx gene expression in the head kidneys
of ABV-infected fish
2.5.1. Sample preparation
Twenty-five flounder were IM injected with 106.9 TCID50 fishÿ1of ABV and maintained at 20 (C in a 100
l aquarium. Protocols for blood collection, processing and storage (ÿ80 (C) of serum samples were
conducted following standard procedures [18]. Random collections of blood and head kidneys from five
individual fish per sampling were conducted at Days 1, 3, 7, 14 and 21 post-ABV challenge. Ten fish
injected with MEM10 were randomly sampled (5 fish samplingÿ1) at Days 1 and 3 post-injection to serve as
negative control. Immediately after the blood collection, the head kidney of each fish was aseptically taken
and frozen at ÿ80 (C until assayed for Mx gene expression.
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R. Pakingking et al. / Fish & Shellfish Immunology 17 (2004) 1e11
2.5.2. Measurement of IFN-like activity
Measurement of IFN-like activity in the serum of individual flounder with ABV infection was conducted
following a cell protection assay adapted from Renault et al. [19]. Before measurements of IFN-like activity
(except for the negative control), serum samples initially diluted at 1:10 in Leiboritz’s L-15 medium
(Gibco-BRL, USA) supplemented with 2% FBS were treated with a rabbit anti-YTAV (1:100) for 1 h to
inactivate ABV infectivity in the samples, and then serially diluted 2-fold up to 1:640 using the same
medium. Because flounder sera showed high cytotoxicity at lower dilutions to RTG-2 and FHM cells, the
assay for IFN-like activity was carried out on cell monolayers of Japanese flounder natural embryo
(HINAE) cells [20] grown at 20 (C in L-15 medium with 10% FBS, 100 IU ml ÿ1 penicillin G, and 100 mg
ml ÿ1 streptomycin. VHSV and ABV used in this assay were propagated (MOI 0.01) in HINAE cells and
the titer of the stocked virus at ÿ80 (C was determined in the same cells. HINAE cells (105 cells 100 ml ÿ1)
were seeded on to 96-well plates and allowed to grow at 20 (C until they attained confluence, usually less
than 48 h. Prior to the assay, the old medium was removed and 100 ml of each dilution of the serum sample
was transferred into 4 wells. Some wells were left untreated to serve as controls. After treating cells for
16e18 h at 20 (C, the cell monolayers were washed once with a fresh medium and replaced by 200 ml
well ÿ1of L-15 medium containing 10% FBS and VHSV with a MOI of 0.06. Incubation was then carried
out at 20 (C for 72 h until VHSV-induced CPE was clearly seen in the control wells. The cells were
subsequently fixed and stained with 1% crystal violet in 10% formalin, washed and air-dried, and the
degree of cell protection was assessed by measuring the absorbance of the dried stained cell monolayers at
600 nm using an ELISA reader (MPR-A4, Tosoh, Japan). The IFN-like activity units per ml (IFLU ml ÿ1)
were expressed as the reciprocal dilution of the serum that provided a cell layer with 50% of the dye
absorbance of the uninfected control cell layer [19].
2.5.3. Assay for Mx gene expression
Total RNA was extracted from the head kidney tissues of flounder using ISOGEN (Nippon Gene, Japan)
according to the manufacturer’s instructions and dissolved in diethylpyrocarbonate-treated water. Equal
amounts of total RNA extracted from each of the five fish sampled at the same time were pooled and used for
a reverse transcription polymerase chain reaction (RTePCR). To obtain 1st strand cDNA as a PCR
template, reverse transcription was performed at 45 (C for 60 min using a 10 ml reaction mixture which
contained 3 mg of the pooled flounder RNA, 50 ng random hexamer primers (Invitrogen, USA), 0.5 mM
dNTP, 10 mM dithiothreitol, 2 ml of 5! first strand buffer (Invitrogen, USA), 200 U Superscript II
(Invitrogen, USA), and 8 U RNase inhibitor (TOYOBO, Japan). The reaction was terminated at 72 (C for 10
min. For amplification of Mx cDNA, a 25 ml reaction mixture which contained 1 ml of the 1st strand cDNA,
0.4 mM Mx gene specific primers (JFMx-F, 5#-TATGAGGAGAAGGTGCTGCCCTGCAT-3# and
JFMx-R, 5#-TTCAAGGCCTCTGTGGTTGCTATGTC-3#), 0.1 mM dNTP, 2.5 ml of 10! Storage buffer
B (Promega, USA), and 1.25 U Taq DNA polymerase (Promega, USA) was used. PCR was performed with
30 cycles of denaturation at 94 (C for 30 s, annealing at 52 (C for 20 s, and extension at 72 (C for 60 s. These
primers were designed to amplify a 557-bp fragment based on the Japanese flounder Mx cDNA sequence
reported [14]. Japanese flounder b-actin gene-specific primers (JFbAct-F, 5#-GACAGAAGGACAGCTACGTG-3# and JFbAct-R, 5#-CATGTAGTCAGTCAGATCTC-3#) were also synthesized to amplify
a 426-bp fragment as internal control. The PCR products amplified by the same protocol as used in the PCR
for Mx gene were separated on 1% agarose gel and stained with ethidium bromide.
2.6. Statistical analyses
Differences in cumulative mortalities between groups of fish infected single or dually were compared
using the c2test at 5% confidence level. Additionally, statistical differences among treatments in the assay
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for IFN-like activity were determined using One-way ANOVA at 5% level of significance. The least
significant difference was used to further determine the significant differences among treatments.
3. Results
3.1. Onset and duration of the resistance to secondary VHSV infection in flounder after primary
ABV infection
The non-specific protection occurred at Day 3 (mortality rate: 0%) and persisted up to Day 7 (10%) and
Day 14 (45%) post-ABV challenge (Fig. 1AeC). The cumulative mortalities of these three dual-infected
groups were significantly lower than those of their corresponding control groups (infected with VHSV
without preceding ABV challenge). However, the mortality (90%) of the Day 21 challenged group was not
statistically different from that of the control (100%) (Fig. 1D). Dead fish solely injected with VHSV
exhibited behavioral changes like anorexia and lethargy, and external signs like dark coloration of the
body, distended abdomen and haemorrhagic fins. When these fish samples were necropsied, the presence of
ascitic fluid in the peritoneal cavity, swollen kidney and spleen, and haemorrhage in the muscle and/or
Cumulative mortality (%)
100
100
A
80
80
60
60
40
40
20
20
B
0
0
0
100
3
6
9
12
0
15
100
C
80
80
60
60
40
40
20
20
0
3
6
9
12
15
3
6
9
12
15
D
0
0
3
6
9
12
15
0
Days after VHSV challenge
Fig. 1. Cumulative mortalities in groups (each n=20) of Japanese flounder with primary ABV infection followed by secondary VHSV
infection at (A) Day 3, (B) Day 7, (C) Day 14, and (D) Day 21 post-ABV challenge. B: ABV+VHSV (test group); C:
MEM10+VHSV ( positive control); ¤: ABV+MEM10(negative control).
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R. Pakingking et al. / Fish & Shellfish Immunology 17 (2004) 1e11
viscera were observed. The same observations were noted in dead fish dually infected with ABV and VHSV
with a few exceptions in the Day 7-challenged group. In contrast, surviving fish with the dual infections did
not show any pathological signs of VHS. Additionally, no mortality or abnormal condition was noted in
groups of mock-infected fish or in fish injected with ABV alone.
3.2. ABV and VHSV infectious titers
The kinetics of ABV replication in flounder sampled at different time points is shown in Fig. 2. Fish
examined at Days 3, 7, 14, and 21 post ABV-challenge were all positive for the virus with infectious titers
ranging 103.8e106.8, 107.3e108.3, 104.6e107.8, and 103.1e106.2 TCID50 gÿ1, respectively.
Fig. 3A shows the re-isolation rates of ABV and VHSV and their corresponding mean infectious titers in
the head kidneys of live fish in the dual-infected groups. The titers of ABV gradually decreased from Day
3-challenged group (103.6e107.3 TCID50 gÿ1) to Day 21-challenged group (103.1 TCID50 gÿ1). VHSV
infectious titers were detected only in 4 out of 11 fish in the Day 14-challenged group, ranging from 103.1e
106.0 TCID50 gÿ1. In the dead fish, as shown in Fig. 3B, ABV titers decreased from Day 7 (105.3e
107.5 TCID50 gÿ1) to Day 21 (103.2e104.0 TCID50 gÿ1), similar to the changes in survivors. On the other
hand, VHSV was re-isolated from all dead fish in the Day 7, 14, and 21 challenged groups with virus titers
ranging from 103.6e105.5 (Day 7), 105.0e107.8 (Day 14) and 107.0e109.5 (Day 21) TCID50 gÿ1, showing an
increasing trend.
All fish injected with VHSV after preceding MEM10injection ( positive control) died (Fig. 1AeD) and
the virus was recovered from 100% (n=80) of dead fish with high virus infectious titers (mean: 1010.1
TCID50 gÿ1).
3.3. Kinetics of IFN-like activity in the serum of ABV-infected flounder
The CPE in HINAE cells by VHSV was evident within a few days after inoculation. ABV infection in
flounder induced the synthesis of an IFN-like substance(s) in their sera as supported by the results shown in
Fig. 4. The sera obtained from flounder 1 day after ABV infection already showed antiviral activity with the
maximum activity at 3 days post-ABV challenge. However, from Day 7 onward, the anti-VHSV activity
started to decline so that the sera obtained from flounder at Day 21 post-ABV challenge had low antiviral
activity similar to those in the control fish.
Log10 TCID50 g-1
10
8
6
4
2
0
3
7
14
21
Days post-ABV challenge
Fig. 2. ABV titers in the head kidneys of Japanese flounder collected at Days 3, 7, 14, and 21 post-challenge. Data represent the
mean G SD (n=5) of each group.
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R. Pakingking et al. / Fish & Shellfish Immunology 17 (2004) 1e11
100
A. Survivors
10
80
8
60
6
40
4
20
Log10 TCID50 g-1
2
0
0
ABV
VHSV
Day 3 (n=20)
12
10
ABV
VHSV
Day 7 (n=18)
ABV
VHSV
Day 14(n=11)
ABV
VHSV
Day 21(n=2)
100
B. Dead
80
8
Reisolation rate (%)
12
60
6
40
4
20
2
0
0
ABV
VHSV
Day 3(n=0)
ABV
VHSV
Day 7 (n=2)
ABV
VHSV
Day 14(n=9)
ABV
VHSV
Day 21(n=18)
Post-ABV challenge
ÿ1
Fig. 3. Virus titers in log10 TCID50 g (bar) and reisolation rates (small circle) of ABV and VHSV in the head kidneys of surviving (A)
and dead (B) dual-infected Japanese flounder. Fish (n=20 each) were challenged with VHSV at Days 3, 7, 14, and 21 post-ABV
inoculation and observed for 14 days. Data are given as mean virus titer of each group with standard deviation. n: number of fish
examined.
3.4. Mx gene expression in the head kidneys of ABV-infected flounder
As shown in Fig. 5, JFMx gene expression level was higher in the head kidneys of ABV-infected fish at
Day 1 post-infection compared with that of the control. The expression level was apparently maintained
until Day 3 and was slightly reduced at Day 7 post-infection. At Days 14 and 21, JFMx gene expression was
further reduced though the level was still higher than that of the control. RTePCR amplification of b-actin
mRNA, which is transcribed constitutively irrespective of cellular physical conditions, gave similar
amounts of product throughout the treatments.
4. Discussion
The present study was undertaken to define the clinical impact of dual viral infections with ABV and
VHSV in Japanese flounder. In particular, the onset and duration of the heterologous protection conferred
by the infection with a less pathogenic ABV against the infection with a highly virulent VHSV were
documented. One of the prominent highlights of the current study is the finding that the on-going ABV
infection in flounder could induce the secretion of a potent IFN-like substance that could protect fish from
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IFN-like activity (IFLU ml-1)
3500
c
3000
2500
2000
b
1500
b
ab
1000
500
a
a
0
control
1
3
7
14
21
Days post-ABV challenge
Fig. 4. IFN-like activity in the serum of Japanese flounder with ABV infection. HINAE cells were treated with serially diluted serum
samples for 16e18 h before inoculated with VHSV (MOI: 0.06) and the dye absorbance of the cells was measured at 72 h post
infection. Data given as mean G SD (n=5) for each group are expressed in IFN-like units per ml (IFLU ml ÿ1) based on the reciprocal
dilution of the serum that provided a cell layer with 50% of the dye absorbance of the uninfected control cell layer. Column bars with
the same superscripts are not statistically different at P=0.05.
the lethal effects of the secondary VHSV infection. This finding could be expected considering the fact that
double stranded RNA viruses and DNA vaccines [21e23] are inducers of type I IFN(s) in fish [9]. However,
the production of the IFN-like substance and its activity must be influenced by a number of factors
including host fish species, kinds of primary inducer viruses or secondary target viruses, and water
temperature [9,19,24,25]. Protection in fish and fish cell lines by IFN-like activity against several viruses
including infectious haematopoietic necrosis virus (IHNV) [8,10], VHSV [26], infectious pancreatic necrosis
virus (IPNV) [13,19,27], and infectious salmon anemia virus (ISAV) [28] have been extensively studied in
salmonids both in vitro and in vivo, and one study in sea bass [29].
The results of our dual-infection experiments revealed that the non-specific protection conferred by the
primary ABV infection against the secondary VHSV infection in flounder commenced at Day 3 and
persisted up to Day 14, but vanished at Day 21 post-ABV challenge. These results clearly show that the
1
2
3
4
5
6
JFMx
E -actin
Fig. 5. Time course of JFMx gene expression in the head kidneys of ABV-infected Japanese flounder. The upper gel shows the 557-bp
RTePCR product specific to JFMx mRNA. b-actin gene expression (426-bp PCR fragment) was also detected as internal control and
was shown in the lower gel. Lane 1: control (MEM10-injected fish); Lanes 2e6: ABV-injected fish sampled at Day 1 (Lane 2), Day 3
(Lane 3), Day 7 (Lane 4), Day 14 (Lane 5), and Day 21 (Lane 6) post-infection.
R. Pakingking et al. / Fish & Shellfish Immunology 17 (2004) 1e11
9
time interval between the preceding ABV infection and the succeeding VHSV infection is very critical in
determining the extent of disease progression attributed to the latter agent. In accordance with our previous
study [6], our current data show that protection conferred by ABV against VHSV appeared to be greatest
within 7 days following ABV exposure, and the resistance disappeared at Day 21 post-challenge. This in
vivo resistance was supported by the absence of VHSV in surviving dual-infected fish shown in Fig. 3 except
for the presence of VHSV in some survivors of Day 14-challenged group.
The period of the resistant state conferred in flounder by prior infection with ABV directly correlated
with the kinetics of the anti-VHSV activity in the sera of fish tested by the in vitro assay. The anti-viral
activity was already detected in the sera of flounder at 1-day post-ABV challenge, and the maximal antiviral
activity was obtained at Day 3 and gradually declined thereafter. The higher survival rates of dual-infected
flounder in Day 3- and 7-challenged groups could be explained in the context of the non-specific protection
directly influenced by the quantity of the IFN-like substance that acts on various target cells against VHSV.
Japanese flounder IFN has not been purified or cloned yet, thus we were not able to detect IFN directly
from virally stimulated fish and could not sequentially analyze how it acts and converts the cell into an
antiviral state. Because ABV has a predilection for the hematopoietic tissues of the kidney of flounder [4,6],
the entry of this virus in the kidney cells of flounder probably induces the synthesis of IFN(s)-like proteins
that prevent the intracellular invasion of VHSV or triggers a cascade of events leading to the upregulation
of other cytokines possessing antiviral activity.
Mx proteins are GTPases specifically induced by the type I IFNs in vertebrates and prevent the growth
of certain classes of viruses in vivo and in vitro [30]. Aside from Japanese flounder [14], Mx cDNA has
already been cloned from Atlantic salmon Salmo salar L. [31], rainbow trout Oncorhynchus mykiss [32], and
Atlantic halibut Hippoglossus hippoglossus [33]. Whereas expression of a Mx protein in transfected chinook
salmon embryo cells (CHSE-214) demonstrated no antiviral activity against IHNV in vitro [32], HINAE
cells expressing a JFMx cDNA has been reported to slightly inhibit the replication of hirame rhabdovirus
(HIRRV) and VHSV [34]. In the present study, monitoring of the IFN-like substance in the sera of fish was
done by studying the expression of JFMx gene in the head kidneys of ABV-infected flounder collected at
different time points by RTePCR. The kinetics of JFMx gene expression in the head kidneys of ABVinfected flounder may explain the appearance and duration of antiviral activity in the fish, suggesting that
Mx protein in Japanese flounder inhibits the multiplication of VHSV in the host. Alternatively, Mx gene
upregulation might be an indicator for the type I IFN production that has a major contribution to the
antiviral activity.
In conclusion, this study clearly defined the essential role of ABV in limiting the proliferation of VHSV
in flounder in ABV-VHSV dual-infection. It documented the onset and duration of the non-specific
protection induced by the preceding ABV infection in flounder against the lethal effects of the succeeding
VHSV infection. Additionally, it highlighted the ability of a less pathogenic strain of ABV to induce the
synthesis of a potent IFN-like substance that possesses antiviral activity against VHSV. With the
emergence of new viruses in economically important fish, either cultured or feral, efficacious vaccines are
required. The ‘‘immunoadjuvant potential’’ of ABV in the formulation of vaccines remains for further
investigation.
Acknowledgements
The authors thank Dr M. Yoshimizu of Hokkaido University for providing the HINAE cell line and
some staff of JASFA for their kind assistance during the experiment. This study was financially supported
by JASFA and the Ministry of Education, Science, Sport and Culture of Japan (Grant-in-Aid: No.
14206024).
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