Fish

Pathology,33(1),17-23,1998.3

Polymerase Chain Reaction(PCR)Amplification

of DNA

of

Red Sea Bream Iridovirus(RSIV)

Jun Kurita*1,
KazuhiroNakajima*1,Ikuo Hirono*2and TakashiAoki*2
*1NationalResearchInstitute
ofAquaculture,Fisheries
Agency,Nansei,Mie 516-0193Japan.
*2Department of
Aquatic Biosciences
,Tokyo University
ofFisheries,
Kohnan,Minato-ku,Tokyo 108-8477Japan.
(Received November

20,1997)

The polymerasechainreaction(PCR)wasusedtoamplifyDNA oftheredseabream iridovirus(RSIV).Four

oligonucleotide
primersetsbasedon theATPase gene,DNA polymerasegene,and a PstI-restriction
fragmentof
RSIV genomic DNA were synthesized.PCR productsoftheexpectedsizewere amplifiedwitheachprimerset
after30 cyclesusingtemplateDNA which was extracted
from thesupernatant
of tissue-cultured
GF cellsinfected
withRSIV. These amplifiedproductswere shown tobe specific
forthegenomic DNA ofRSIV by Southernblot
hybridization.In addition,
PCR productswere obtainedfrom the DNAs of the spleenand blood of red sea
bream,Pagrus ma/or,artificially
infectedwithRSIV. Furthermore,thePCR productswere detectedfrom the
tissues
of culturedmarinefishnaturally
infected
with RSIV and from thesupernatant
oftissue-cultured
GF cells
infectedwithRSIV isolated
from culturedmarinefish. However, PCR productswere notobtainedat3 months
post-challenge
from thespleensof redseabream infectedby intraperitoneal
inoculation
of RSIV. None of the
PCR productswere obtainedfrom thesupernatant
of tissue-cultured
FHM cellsinfectedwithfrogvirus3 or from
thelymphocystistissue
of Japaneseflounder,
Paralichthys
olivaceus,
infectedwithfishlymphocystis
diseasevirus.
Key

words:red

sea bream

iridovims, RSIV,

red sea bream,

Pagrus

major,

PCR,

rapid diagnosis

reductase(RNRS)
An epizooticcausedby a red sea bream iridovirus smallsubunitof theribonucleotide
(RSIV)was reportedto be the leadingcausesof high
gene of RSIV. Subsequently,
PCR successfully
am
mortalities
in cultured
red sea bream(Pagrus major)in
plified
uirus-specific
nucleicacidswitha primersetbased
southwestJapan(lnouyeetal.,1992).Biological
and
on theRNRS gene. We alsofound thatPCR amplifi
cationusingprimersetsbasedon genomic genesofRSIV
physico-chemical
properties
of the viruswere reported
(Nakajimaetal.,1994)andrapidmethod fordiagnosis
provideda rapid,simple,and sensitive
method of diag
ofthediseaseusingan immunofluorescence(IF)test
with
nosis.
amonoclonal antibodywas alsodeveloped(Nakajima
etal.,1995). However, a highlysensitive
method is
Materials and Methods
desirable
fordetecting
thevirusinfishattheearlystage
of thedisease. The polymerasechainreaction(PCR)
Virus
isolates
and
cells
issignificantly
more sensitive
than currently
used im
The
Ehime-1
strain(Nakajima
et al.,1995)of
RSIV
munologicalassayssuchastheenzyme-linkedimmuno
was
used
in this study.
The
virus
was
propagated
in
sorbentassay(Stoeckl
etal.1989,van der Ryst etal.,
the Grunt
fin(GF)cell
line at 25
in basal
medium
1996,Poieszetal.1997). PCR has alreadybeen used
eagle(BME)(Flow
Laboratories)supplemented
with
todetectfish-pathogenic
virus,
epizootic
haematopoietic
10%(V/V)fetal
bovine
serum(Bio
Whittaker),10%(V/
necrosisvirus(EHNV)and Bohle iridovirus
which be
V) ,human
serum(ICN
Biomedicals)and
100mg/ml
long tothe Iridoviridae(Gold
etal.,1995),as well as
spreptomycin
and
100
IU/ml
penicillin.
severalof the fishvirusessuch as infectious
haema
topoietic
necrosisvirus(IHNV), channelcatfishvirus
Viruspurification
(CCV), infectious
pancreatic
necrosis
virus(IPNV)and
The tissueculturesupernatant
from GF cellscultured
striped
jack nervousnecrosisvirus(SJNNV)(Arakawa
withRSIV was collected,
pooledand centrifuged
at3,200
.,1990,Boyle etal.,1991,Lopez-Lastra
et al
et al.,1994,
rpm for5 min toprecipitate
celldebris. The superna
Nishizawaetal.,1994).Oshima etal.(1996)cloneda
tantwas then successively
filtered
throughthedecreas-

J.Kurita,K. Nakajima,I.Hironoand T. Aoki

18

ing

size

order

The filtrate
XL-90

8.0,3.0,0.8

The

volume

resultant

of TNE

EDTA,

0.45m

dient

was
24h

and

of

obtained

80

until

The

with

TNE

of

chloride.

was

The

gra

at 30,000

by

Artificial

rpm

harvested

buffer
were

2h

RSIV

with

centrifugation.

frozen

and

DNA

sequence

at

ml

the

and

viral

deter

genomic
m

DNA
A

virus

genomic

following
was

DNA

extracted

RSIV

virions

(0.01

from

the virions.

were

soaked

and

30min

by

followed

and

against

digested

binant
coli

plasmids
strain

plasmid

enzyme

were

used

XL1-Blue

methylated
DNAs

Some

were
et

were

prepared

al.,1989).

used

overnight.

Sau3A

with

Sph

I.

DNA

inserted

into

fragments

of

an alkaline

Sequitherm

the

multi

the recom

to Escherichira

is capable

Double-stranded
the

I, Sac

fragments

and

to transform

for
and

extracted

I, Pst

pGEM3zf(+),

by

at 55

The

with

were

buffer

extraction

MRF'which

DNA.

(Sambrook
DNAs

each

site of the plasmid

TE

pH8.0)containing

digested
with

the

of RSIV

this, pelleted
with

proteinase

buffer
was

partially

with

cloning

1 mg/ml

DNA

digested

achieve

incubated
EDTA,

by

DNA

phenol-chloroform

TE

genomic

constructed

genomic
To

and

Tris-HCl,0.001

dialysis

was

Initially,

0.5%(W/V)SDS

viral

library

procedure.

were

cycle

of

cloning

recombinant
lysis method
template
sequencing

red

of

RSIV.
running

of

sea

inoculated

pernatant

kept

library
of

infection

Juvenile
a

with

genomic

nucleotide

kit(Air Brawn). The electrophoresedfragments were

sequenced in an automated DNA sequencer(Li-Cor
Model 4000). The determined nucleotide sequences
were analyzed using the GENETYX-MAC(SDC,
soft
ware)computer grogram.

a continuous

rotor

band

pellets

for

in a small
NaCl,0.015

top

cesium

extraction.

Construction of
of

on

within

rpm

soaked

Tris,0.15M

a SW-28

virus

virion
DNA

ination

in

washed

The

then

layered

at 4.

syringe

was

buffer(0.04

centrifuged

rotor

25,000

pellet

pH7.4)and

membranes.

in a SW-40Ti

10-35%(W/V)gradient

for

and

centrifuged

ultracentrifuge(Beckman)at

at 4.

of

was

red

sea

bream

bream(mean

body

intraperitoneally

of the

GF

The
sea

cell
fish

water

held

0.1

containing
in a 50l

maintained

ml

27.3
of

the

g)
su

104.0TCID50/
tank

supplied

at 25.

Preparation of samples PCR

for
Samples of the spleensof red seabream artificially
infected
withRSIV and thoseofuninfected
fish,aswell
as spleensof culturedmarinefish(1sample from each
culturedmarine fish),
which were diagnosedas natu
rallyinfectedwith RSIV by an IF test,were used for
DNA extractions
and theseDNAs wereusedastemplates
forthe PCR. The DNA extractedfrom lymphocystis
tissue
ofJapaneseflounder
infected
withfishlymphocystis
diseasevirus(FLDV)was alsousedfortemplateDNA.
These DNAs were extractedusingPuregeneDNA ex
traction
kit(Gentra
System Inc.). TemplateDNA from
bloodof a moribund red seabream was preparedusinga
Takarasingle-tube
PCR kit(TakaraShuzo). Template
DNAs from thesupernatant
ofGF cellcultures
infected
with RSIV and the supernatant
of FHM cellculturein
fectedwithFV3 were preparedusingInstageneMatrix
(Bio-RadLaboratories).These preparations
were per
formed accordingto supplier's
protocol. DNAs ex
tractedfrom the spleensof variouskinds of cultured
marine fish(Table1)with diseasecaused by natural

Table 1. PCR samples from tissuesof naturally

infectedculturedmarine fishand
cultures of the virus infected cells

RSIV
weight

with

culture
were

with

PCR

RSIV

infection and from

cultured with RSIV

amplification

the supernatant of GF

of red sea bream

cells

isolated from various cultured ma

rine fish were also used as templates in this study.

PCR primersand amplification

ofviralgenomes
A totalof fouroligonucleotide
primersetswere syn
thesizedfor use in thePCR assays. Two primersets,
No.1(1-F and 1-R)and No.2(2-F and 2-R),were de
signed from the nucleotidesequence of the 959-bp
Pst I restriction
fragment(DDBJ accessionnumber:
AB006954)of RSIV genomic DNA. The primersets

Table

2.

iridovirus DNA

19

of No.3(3-F and 3-R) and No.4(4-F and 4-R)were

based on nucleotidesequences of theputativeDNA poly
merase gene(DDBJ accession number:AB007366)and
adenosine triphosphatase(ATPase)gene(DDBJ
acces
sion number:AB007367)cloned
from RSIV, respec
tively. These primer sequences and the expected
lengths of the amplified fragments are shown in Table
1. For nested PCR, the primer setsof No.5(1-F and
2-R)and No.6(2-F and 1-R)were used(Table 2).

Primer

list

CTGCAGTTGCCGCTCAAACACTCTGGCTCATCTATGTCATCGTAGTCGTCCATTCCGCTGCCCCCATC
primer

1-F

GTCAAGCAGTGTAGGCGGTGGAGTAACATTATCGGTGTCTGTTGGCAGCTCACATGAGACACCTACAC
AAGGCTGACTGTCAGATGAGATGCGGCTGGCGTGGCATGTGACGGTCTGCACAGGGTGAGGTTTCAGC
TTGATGACAGACAAGATGGTACCGTCATACAGCACCACTCCATGCTTCAGGACTTCACTGCTGTTGCG
GCCTACATGGACCACCTCGCCATGTACAACATGCTCCGCCAAGAGGCTGTTGCTGTCGCTTGACCAAA
primer
2-F
CAATCTTCACATCGGTCTCTCGAGGTACCCCGCAGCTGAGGGTGGTCGTCTGGTTGTCGATTTCCAGG
TTATAGAAGGTGGTGGCGTGAGTACACGCCACAGTCAGCAACAGAAGAAGTAGCAGGGTCGCCATTGC
TCATGTAGCTATGATTCACAGTAGTCACCTATGACATGAGGATATTCAAAATTTTTATACAAGTAAAA
GATGTTCACTGTGCTTGAGATAGGAGATGTGTTGGTGCTAGTGTCGCGTGACGACATACGTGTGATGT
primer
1-R
ACAACCGTGACCCGGGACAATCTGTGGCGTTAACTAATACCACCAGAGGGTGTGTCACGGTTACCGCG
GTGCCTGACAACGCGGATTATGAGACATTGCGCAGGTGTCTTATAGTCGCAGCCCAGATTTTCGGTTC
ACGGCTGCGTGTGCCTATTACACTGATCCACCCAGACATGTCACAAGATGGATTTGTCAATTACCAGG
ACATGGGCACGCACATTGGCTTCTGCCCTCACTACTTTAACGCAGCGCAAATGCCGGTGGTTAACTAC
primer
2-R
GCCGCAAGAGAACTGATCTCATCCGGGGGTCGCCCTCAACCAATGACAATCATCGTGTATGTGTCTGC
ACTGCAG
Fig.1.

The nucleotide sequences of DNA fragments digested with Pst I cloned from red sea bream iridovirus(RSIV). The
nucleotidesequences of the forward and reverse primers of primer set 1 are underlined and those of primer set 2 are double
underlined.

J. Kurita, K. Nakajima, I. Hirono and T. Aoki

20

PCR

amplification

Reaction
mM

of

mixes

contained

DNA

polymerase

with

a commercially

Shuzo)

20

incubated

for

cycler

(Takara

thermal

an

1 min

30

cycles

in
MP)

period

and

of

buffer

ExTaq

supplied

ExTaq

kit

(Takara

The

an

200

U of

template.

cycler

at 58,

extension

PCR

Takara
of

primer,

1.25

Mg2+

volume

were

by

mM

available

a small

at 94,

of each

triphosphate,
in

with

min

1 mM

deoxynucleotide

mixture

automatic

thermal

programmed

1 min

min

for

at 72,

at

72

0.5

followed

after

the

last

cycle.

Nested

PCR

Nested
spleens
The

of
first

set

sea

with

same

then

0.8%

were

agarose-TBE
acid,

0.002

DNA

and

products

per-

conditions

PCR

by

were

amplification.

electrophoresis

(0.089

EDTA)

The

product

Hybond

using

an

viral

DNA,

in

nylon

the

gel

membrane

probes
ECL

0.089

DNA

N+
with

in

Tris-borate,

gels.

PCR

hybridized

themselves

primer

were

products

to
and

the

the

challenge.
with

Reaction

analyzed

each

transferred

(Amersham)

from

post

performed

for

buffer

cell

were

6.

amplified

Boric

GF

No.

DNA

months

amplifications

described

products

amplify
3

second

set

those

of PCR

PCR

to
at

were

the

primer

as

Analysis

used

bream

amplifications

5 and

formed

were

red

PCR

No.

the

amplification
PCRs

of

each

PCR

detection

system

(Amersham).

Results

959-bp

cloned

from

structed
to

Pst
the

two

amplify

(Fig.

1).

(570

or

after

of the
was

30

using

in

cycles

the

DNA

same

primer

the

bp

from

2).

size

(563bp

fragment

obtained

from

the

ture

(data

not

shown).

that

all

cells

3,

supernatant

or

3 and

band

(Fig.

Fig.

PCR
DNA

tissue

2.

Agarose
from

2).

by

template

uninfected

primer

in length)

amplified

4 with

of

with

DNA

or 568bp

not

the

infected

single

sets

was

size
from

using

2)

fragment

molecular

amplification

(Fig.

con-

1 and

amplified

GF

2,

959

expected
were

primer

1,

We

bp

of PCR

sets

library.
sets

of

using

randomly

(primer

length)

molecular

the

was

primers

tissue-cultured

amplified

However,

564

2 respectively

expected
also

or

bp

of

1 and

PCR

fragments

564

supernatant

sets

bp

fragment

genomic

of

DNA

bp

RSIV

RSIV

pairs
570

I-digested

cul-

gel electrophoresis
supernatant

with

RSIV

rose

gel;

using
Panel

blot

analysis

Southern

blot

products

themselves

1, 2, 3,

and

tissue-cultured

4,

of
which
GF

PCR
were
cells

product

DNAs

amplified
infected

using
from

with

RSIV,

primer

C, D,

and

hybridizations

Lane

set.
E are
with

using

products

GF

cells

Panel

infected

A is an

aga-

autoradiographs
the

of

probes

of PCR

primer

set

1, 2,

1, , DNA/Hind

III

molecular

3 and

4,
size

showed
marker;

probes

each
B,

respectively.
A Southern

of amplification

of tissue-cultured

primer

supernatant
hybridized

set
of

DNA;

lane
and

2, RSIV

lanes

genomic

4, 5, 6, and

supernatant

of tissue-cultured

RSIV

primer

using

set

DNA;
7 are PCR
GF

1, 2, 3, and

cells
4,

Lane

3, GF

products
infected
respectively.

cell
from
with

PCR

amplification

of red sea bream

with viral genomic DNA, however, they did not hybridize with the DNA extracted from GF cells (Fig. 2).
A PCR product was not obtainedfrom the lymphocystis
tissue of Japanese flounder infected with FLDV or the
supernatant of tissue-cultured FHM cells infected with
FV3 (Fig. 3).
A single DNA fragment with the same molecular size
was amplified not only from the spleens of red sea bream
experimentally infected with RSIV (Fig. 4) but also from
the blood of moribund red sea bream experimentally infected with RSIV (data not shown) using various combinations of primers. On the other hand, no PCR product was obtained from the spleens of uninfected red sea
bream or from those of the fish at 3 months post-challenge (Fig. 4). Expected molecular size DNA fragment
of RSIV DNA was also not detected from the spleen of
red sea bream at 3 months post-challenge using the nested
PCR method (data not shown).
A fragment of the appropriate size was amplified using each of the primer sets 1, 2, 3, and 4, with template
DNAs obtained from the spleens of several cultured
marine fish (red sea bream, Japanese parrot fish, striped
jack, sea bass, yellowtail, amberjack, and bluefin tuna)
(Fig. 5). All four primer sets yielded the same PCR
products from the supernatant of tissue-culture d GF cells
infected with virus isolated from cultured red sea bream,
Japanese parrot fish, striped jack, sea bass, and bluefin
tuna.

iridovirus

DNA

21

Discussion
A Pst I-digested fragment of 959 bp in length was
cloned from RSIV genomic DNA and sequenced.
No
significantly homologous sequences were found in
EMBL and GenBank.
We designed two primer sets
(primer sets 1 and 2) based on this nucleotide sequence
for amplification of RSIV using the PCR method.
We
also constructed two primer sets (nos. 3 and 4) that were,
respectively, the ORF of ATPase gene and the ORF of
the DNA polymerase gene cloned from RSIV genomic
DNA (Table 2). The primer sets 1, 2 and 4 could am-

D
Fig.

Fig.

3.

Amplification
ing

PCR

2-4),

of DNA
with

primer

10) and
Hind

four
set

primer

III

DNA
infected

cells

amplified

DNA

phocystis
FLDV;
the
with

with

with

tissue
lanes

supernatant
FV3.

4,

of
7,

primer

marker;

RSIV; 1
the

Japanese
10,

and

of tissue-cultured

8-

, DNA/

2, 5, 8 and

3, 6,

extracted
flounder

13,

(lanes

11,

of tissue-cultured
anes

DNA

us-

3 (lanes

Lane 1
lanes

FV3

set 1
set

11-13).

the supernatant
with

and

Primer

5-7),

size

FLDV

sets.

4 (lanes

molecular

GF

primer

2 (lanes
set

amplified

of RSIV,

amplified
FHM

9 and
from

infected
DNA
cells

12,

4.

Detection

of RSIV

artificially

infected

using

primer

each

agarose

gels

products
Lane

genomic
with

GF

lanes

3, 4,

cells
and

with

6, 7, and

8, amplified

with

artificially

infected

and

sea

bream

13,

the
with

5, amplified

non-infected

12,

PCR

amplification

A,

B, C, and

sea

red

of

1, 2, 3, and

molecular

from

lym-

infected

sets
III

infected

red

red sea

by

Panels

primer

DNA

tured

from

electrophoresis

1, DNA/Hind

2, amplified

RSIV

sets.

after

using

DNA

bream

size

supernatant
RSIV
DNA

(negative

DNA

from

sea

bream;

4, respectively.
markers;

amplified

DNA

from

at 3 months

post

infection.

and
the

lane

of tissue-cul-

the

control);
spleens

control);

the

D show

amplification

(positive
from

bream

spleens
lanes
spleens

of
lanes

of dead
9, 10,

11,

of

red

J. Kurita, K. Nakajima, I. Ffirono and T. Aoki

22

D
Fig.

5.

Detection
after

of RSIV

naturally

electrophoresis

of

respectively.
cially

Lane

infected

control);
shown
lane

3-9,

5,

striped

jack

Lanes

infected

with

(positive
fishes

control);

lane

cultured

lane
11-17,

12, no
fishes.

15, striped

jack

sea
6,

infection
Lane
(Ehime);

from

lane

DNA

(negative

lane

from

16, sea

non-infected
with

lane

Lane

10,

the

Lane

(Ishikawa);
bass

4, Japanese

(Ehime);

13-17,

lane

3,
bream

bream

culture
parrot

strain

viruses

14, Japanese

(negative

locations
fish

17, albacore

lane

GF

isolated

are

(Ehime),

molecular

Ehime-1

parrot

4,

artifi-

(Ehime),
III

gels
and

of tissue-cultured

11, RSIV

lane

sea

agarose
2,

sea

yellowtail

DNA/Hind

supernatant
Lane

Fish

7,

1,

I. red
red

RSIV.
lane

D show

sets

Lane

(Ehime);

control).

sea bream

plify a fragment of the expected size from the supernatant of tissue-cultured GF cells infected with RSIV, and
from spleens of cultured marine fish infected with RSIV.
Primer set 3 amplified a common fragment of the same
molecular size from marine fish described above and, in
addition, yielded one or two fragments of a lower molecular size from red sea bream, striped jack, sea bass,
yellow tail, and amberjack. However, the primer sets
did not amplify the same molecular size fragment from
the supernatant of tissue-cultured cells infected with FV3
and lymphocystis tissue of Japanese flounder infected
with FLDV, which belong to Iridoviridae (Fig. 3).

2,

sources.

B, C, and
primer

spleens.

(Ehime);

bass

different

13, red

the

(Kochi).

A,

using

infected

bream

sea

albacore

amplified

strains

from

control);

lane
9,

Panels

products

naturally

3, red

(Ehime);

RSIV

RSIV.

DNA

RSIV

Lane

(Ehime);

with

amplified

cultured

in parentheses.

markers.

lane

with

lanes

amberjack

ent

1-9,

infected
amplification

8,
size

cells

(positive
from

fish

differ(Ehime);

(Kochi).

Recently, Mao et al. (1997) demonstrated that nine

iridoviruses from fish, reptile, and amphibians were more
similar to FV3 of the genus Ranavirus.
A common
primer based on the conserved major capsid protein of
iridoviruses was developed by Mao et al. (1997) for
amplification of the virus-specific nucleic acid from
iridoviruses infecting fish, reptiles, and amphibians.
Ohshima et al. (1996) cloned a part of RNRS gene of
RSIV and also succeeded in RSIV detection from naturally infected red sea bream by PCR using the primers
based on this gene sequence.
We also developed the
four primer sets and could detect the RSIV by PCR us-

Fish

Pathology,33(1),17-23,1998.3

Polymerase Chain Reaction(PCR)Amplification

of DNA

of

Red Sea Bream Iridovirus(RSIV)

Jun Kurita*1,
KazuhiroNakajima*1,Ikuo Hirono*2and TakashiAoki*2
*1NationalResearchInstitute
ofAquaculture,Fisheries
Agency,Nansei,Mie 516-0193Japan.
*2Department of
Aquatic Biosciences
,Tokyo University
ofFisheries,
Kohnan,Minato-ku,Tokyo 108-8477Japan.
(Received November

20,1997)

The polymerasechainreaction(PCR)wasusedtoamplifyDNA oftheredseabream iridovirus(RSIV).Four

oligonucleotide
primersetsbasedon theATPase gene,DNA polymerasegene,and a PstI-restriction
fragmentof
RSIV genomic DNA were synthesized.PCR productsoftheexpectedsizewere amplifiedwitheachprimerset
after30 cyclesusingtemplateDNA which was extracted
from thesupernatant
of tissue-cultured
GF cellsinfected
withRSIV. These amplifiedproductswere shown tobe specific
forthegenomic DNA ofRSIV by Southernblot
hybridization.In addition,
PCR productswere obtainedfrom the DNAs of the spleenand blood of red sea
bream,Pagrus ma/or,artificially
infectedwithRSIV. Furthermore,thePCR productswere detectedfrom the
tissues
of culturedmarinefishnaturally
infected
with RSIV and from thesupernatant
oftissue-cultured
GF cells
infectedwithRSIV isolated
from culturedmarinefish. However, PCR productswere notobtainedat3 months
post-challenge
from thespleensof redseabream infectedby intraperitoneal
inoculation
of RSIV. None of the
PCR productswere obtainedfrom thesupernatant
of tissue-cultured
FHM cellsinfectedwithfrogvirus3 or from
thelymphocystistissue
of Japaneseflounder,
Paralichthys
olivaceus,
infectedwithfishlymphocystis
diseasevirus.
Key

words:red

sea bream

iridovims, RSIV,

red sea bream,

Pagrus

major,

PCR,

rapid diagnosis

reductase(RNRS)
An epizooticcausedby a red sea bream iridovirus smallsubunitof theribonucleotide
(RSIV)was reportedto be the leadingcausesof high
gene of RSIV. Subsequently,
PCR successfully
am
mortalities
in cultured
red sea bream(Pagrus major)in
plified
uirus-specific
nucleicacidswitha primersetbased
southwestJapan(lnouyeetal.,1992).Biological
and
on theRNRS gene. We alsofound thatPCR amplifi
cationusingprimersetsbasedon genomic genesofRSIV
physico-chemical
properties
of the viruswere reported
(Nakajimaetal.,1994)andrapidmethod fordiagnosis
provideda rapid,simple,and sensitive
method of diag
ofthediseaseusingan immunofluorescence(IF)test
with
nosis.
amonoclonal antibodywas alsodeveloped(Nakajima
etal.,1995). However, a highlysensitive
method is
Materials and Methods
desirable
fordetecting
thevirusinfishattheearlystage
of thedisease. The polymerasechainreaction(PCR)
Virus
isolates
and
cells
issignificantly
more sensitive
than currently
used im
The
Ehime-1
strain(Nakajima
et al.,1995)of
RSIV
munologicalassayssuchastheenzyme-linkedimmuno
was
used
in this study.
The
virus
was
propagated
in
sorbentassay(Stoeckl
etal.1989,van der Ryst etal.,
the Grunt
fin(GF)cell
line at 25
in basal
medium
1996,Poieszetal.1997). PCR has alreadybeen used
eagle(BME)(Flow
Laboratories)supplemented
with
todetectfish-pathogenic
virus,
epizootic
haematopoietic
10%(V/V)fetal
bovine
serum(Bio
Whittaker),10%(V/
necrosisvirus(EHNV)and Bohle iridovirus
which be
V) ,human
serum(ICN
Biomedicals)and
100mg/ml
long tothe Iridoviridae(Gold
etal.,1995),as well as
spreptomycin
and
100
IU/ml
penicillin.
severalof the fishvirusessuch as infectious
haema
topoietic
necrosisvirus(IHNV), channelcatfishvirus
Viruspurification
(CCV), infectious
pancreatic
necrosis
virus(IPNV)and
The tissueculturesupernatant
from GF cellscultured
striped
jack nervousnecrosisvirus(SJNNV)(Arakawa
withRSIV was collected,
pooledand centrifuged
at3,200
.,1990,Boyle etal.,1991,Lopez-Lastra
et al
et al.,1994,
rpm for5 min toprecipitate
celldebris. The superna
Nishizawaetal.,1994).Oshima etal.(1996)cloneda
tantwas then successively
filtered
throughthedecreas-