Molecular comparison of iridoviruses isolated from marine fish cultured in Korea and imported from China

Aquaculture 255 (2006) 105 – 116 www.elsevier.com/locate/aqua-online

Molecular comparison of iridoviruses isolated from marine fish cultured in Korea and imported from China Joon Bum Jeong a , Ho Yeoul Kim a , Ki Hong Kim a , Joon-Ki Chung a , Jack L. Komisar b , Hyun Do Jeong a,⁎ a b

Department of Aquatic Life Medicine, Pukyong National University, 599-1 Dae Yeon Dong, Nam Ku, Busan 608-737, South Korea Department of Immunology, Walter Reed Army Institute of Research, 503 Robert Grant Ave. Silver Spring, MD 20910-7500, USA Received 7 July 2004; received in revised form 1 October 2005; accepted 5 December 2005

Abstract We have identified three iridovirus isolates, Sachun and Namhae from diseased fish, and CH-1 from infected imported fish. In order to determine whether these iridovirus isolates are distinguishable from other fish iridoviruses reported as Ranavirus, nucleotide sequence analysis of the K2 region was carried out. This 5905 bp K2 region located between DPOL and RNRS gene includes the 4436 bp K1 region analyzed in our laboratory previously and its flanking regions. The sequences of this region in various iridovirus isolates from fish differed from one another (percent identity = 72∼92) by nucleotide substitutions, insertions, and deletions. Most of the various insertions and deletions, with lengths of up to 163 bp, appeared in clusters in the repeating sequences in both the open reading frames (ORFs) and intergenic regions in K2 region. High homologies, N 71%, were demonstrated in the nucleotide sequences of the ORFs in the K2 regions of iridovirus strains including the infectious spleen and kidney necrosis virus (ISKNV), notably N 91% between isolates Sachun and Namhae isolated in Korea. Thus, these iridoviruses isolated from marine fish might be considered to be members of the tropical iridovirus group, which includes the red sea bream (Pagrus major) iridovirus (RSIV) Ehime-1 and is different from the genera Ranavirus, Lymphocystivirus, and Iridovirus in the family Iridoviridae. Identification of these iridovirus isolates was based on the different sizes of the resulting amplicons in discriminative PCR with primers derived from the K2 region, which contained different lengths of insertion (or deletion) depending upon the isolates. We also have described the possibility of asymptomatic iridovirus infection in fish. © 2005 Elsevier B.V. All rights reserved. Keywords: Iridovirus; Genomic variation; DNA homology; Gene amplification; Repetitive element

1. Introduction Members of the family Iridoviridae are an emerging group of viral pathogens that threaten the aquaculture industry, causing great economic losses throughout the ⁎ Corresponding author. Tel.: +82 51 620 6143; fax: +82 51 628 7430. E-mail address: [email protected] (H.D. Jeong). 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.12.015

world. The International Committee on Taxonomy of Viruses (ICTV) currently describes four genera within the family Iridoviridae, namely Ranavirus, Lymphocystivirus, Iridovirus, and Chloriridovirus (Langdon et al., 1986; Rodger et al., 1997; Sudthongkong et al., 2002). After the reports of three iridoviruses, the epizootic hematopoietic necrosis virus (EHNV), the European sheatfish (Silurus glanis) iridovirus (ESV), and the European catfish (Ictalurus melas) iridovirus (ECV)

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(Langdon, 1989; Ahne et al., 1989; Pozet et al., 1992) related to the frogvirus 3 (FV3), the type species of the genus Ranavirus (Hedrick et al., 1992), other types of iridovirus infections, the chromide cichlid (Etroplus maculatus) iridovirus, the red sea bream (Pagrus major) iridovirus (RSIV), the “sleepy grouper disease” virus, the fresh water angelfish (Pterophyllum scalare) iridovirus, and the mandarin fish (Siniperca chuati) infectious spleen and kidney necrosis virus (ISKNV) were described (Armstrong and Ferguson, 1989; Inouye et al., 1992; Chua et al., 1994; Rodger et al., 1997; He et al., 2001). The relationships among these iridoviruses are currently being investigated. Iridoviruses have many interesting biological and molecular biological features. The genomes of these viruses are circularly permuted and terminally redundant, the only eukaryotic viruses with this feature (Delius et al., 1984; Darai et al., 1983, 1985; He et al., 2001). To date, the complete genome sequences of three vertebrate iridoviruses that are infectious in fish, lymphocystis disease virus-1 (LCDV-1), RSIV, and ISKNV, have been determined. The genome of LCDV-1 (Tidona and Darai, 1997) is 102,653 bp long, encoding 195 potential proteins, the genome of RSIV Ehime-1 strain (GenBank accession number: BD143114) is 112,414 bp long, and the genome of ISKNV (He et al., 2001) is 111,362 bp long, encoding 124 potential proteins. One of the reported iridoviruses, RSIV, is a piscine iridovirus and causes an acute and highly contagious disease (Inouye et al., 1992). Since 1990, outbreaks of this viral infection, designated red sea bream iridoviral disease (RSIVD), have caused severe economic losses, primarily during the summer, in Asian countries including Korea (Sohn et al., 2000; Jung and Oh, 2000), Japan (Inouye et al., 1992), Thailand (Danayadol et al., 1996; Sudthongkong et al., 2002), Taiwan (Chua et al., 1998) and China (He et al., 2001). For the detection of this virus, polymerase chain reaction (PCR) analysis with primer sets based on the nucleotide sequences of the various genomic regions, such as the DNA polymerase (DPOL) gene, the adenosine triphosphatase (ATPase) gene, the ribonucleotide reductase small subunit (RNRS) gene, and the Pst I restriction fragment (Kurita et al., 1998; Oshima et al., 1998), are being used. Recently, we have isolated three different types of RSIVs, two from sea perch (Lateolabrax sp.) and rock bream (Oplegnathus fasciatus) of Korea and one from sea perch (Lateolabrax sp.) imported from China (Jeong et al., 2003, 2004), and identified as Namhae, Sachun, and CH1, respectively, based on the variations of the nucleotide sequences in the four genomic regions mentioned above (Kurita et al., 1998; Oshima et al., 1998). In these three

isolates, Namhae appeared to be the same as RSIV Ehime1, at least with respect to the nucleotide sequences in the regions that were analyzed. Additionally, we also have cloned and sequenced the 3′ flanking region of the DPOL gene of Namhae using cassette ligation-mediated PCR (Jeong et al., 2003). This sequence, called the K1 region (GenBank accession number: AF506370), was 4436 bp long and possessed two open reading frames (named ORF-1 and ORF-2) oriented in opposite directions, and long intergenic regions, as well as the Pst I restriction fragment of Kurita et al. (1998). Thus, there remains a need for a genetic comparison of this genomic region so as to be able to characterize and discriminate these iridovirus variants more precisely. The aim of the present study was to determine genomic and deduced amino acid sequences for the K1 region and its flanking regions in the RSIVs Sachun, Namhae, and CH-1, in order to distinguish these viruses from each other and from another related iridovirus. The results indicated that the nucleotide sequences of each of the different iridoviruses can be used to discriminate and classify these iridoviruses and pave the way for more precise understanding of the genomic structure of iridoviruses. 2. Materials and methods 2.1. Virus In previous studies (Jeong et al., 2003), we have reported finding two representative RSIVs of Korea, Namhae and Sachun, in sea perch (Lateolabrax sp.) and rock bream (O. fasciatus). Both fishes were weighing 100 g and suffering from typical RSIV infections in September of 1999 and 2000 from the aquatic farms in Korea. We also have obtained RSIV CH-1 from the sea perch (Lateolabrax sp.) of 5–7 g body weight, by screening for the presence of RSIV using PCR with the primers of Kurita et al. (1998). These fish were imported as a live fry in the spring of 2000 from China and healthy externally. Other cultured fishes obtained from 1998 to 2003 from the aquatic farms of the South Sea of Korea, 2 sea perch (Lateolabrax sp.), 3 rock bream (O. fasciatus), 2 yellow tail (Seriola quinqueradiata), 1 black porgy (Acanthopagrus schlegeli) and 1 red sea bream (P. major) showing symptoms of RSIV infection with enlarged spleen cells (Inouye et al., 1992), were also used for the analysis of iridovirus infection. 2.2. Isolation of viral nucleic acids For DNA isolation, samples of 20 mg of spleen from infected fish were homogenized for 10 min in 355 μl of

J.B. Jeong et al. / Aquaculture 255 (2006) 105–116

TE buffer (100 mM Tris–HCl, 10 mM EDTA) using motor for pellet pestle (Sigma-Aldrich, Co., Ltd.) and centrifuged at 8000 g for 10 min. Supernatants were treated with 40 μl of 10% SDS and 5 μl of 20 mg/ml proteinase K (Roche, Mannheim, Germany) for 1 h at 37 °C. After three extractions with phenol-chloroform, the DNA was precipitated with ethanol in the presence of 0.3 M sodium acetate, redissolved in 50 μl TE buffer, and stored at − 80 °C until use. 2.3. Primers for PCR An oligonucleotide primer set that has been used for the diagnosis of RSIV infection, 4F/4R, was synthesized (Bioneer Co., Taejon, Korea) (Table 1) based on the nucleotide sequence of the DPOL gene (Kurita et al., 1998), (GenBank accession numbers: AB007366). For the cloning of the genomic region described in this study, two other primers, P1F as the sense primer and P3R as the antisense primer, were designed from the nucleotide sequences of the 3' end region of DPOL gene and the 5' end region of the RNRS gene. Other related primers (Table 1) were derived from the nucleotide sequence of the K1 region of Jeong et al. (2003). 2.4. PCR PCR amplification was carried out in a 50 μl reaction mixture containing 0.5 μl (200 ng for CH-1, 20 ng for Sachun and Namhae) of the viral nucleic acids extracted from the spleen, 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% w/v gelatin, 0.5% Tween-20, 200 μM of each dNTP, 1 μM of each primer, 1.25 U AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT, USA) in a Perkin-Elmer 2400 thermal cycler. After 2 min of predenaturation at 94 °C, the mixtures were

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incubated for 35 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, followed by an extension period at 72 °C for 7 min. The results of amplification were analyzed by 1% agarose gel electrophoresis. 2.5. DNA sequencing The PCR products were purified by agarose gel electrophoresis using a Prep-A-Gene DNA Purification System (Bio-Rad Laboratories, Hercules, CA, USA) and cloned into the TOPO-TA vector following the instructions of the manufacturer (Invitrogen Co., Carlsbad, CA, USA). To avoid errors due to the PCR process, PCR was performed three times with the same primers and each PCR product was cloned and sequenced using the Big Dye Terminator Cycle DNA Sequencing Kit (ABI PRISM, PE Applied Biosystems, Foster City, CA, USA) and an automatic sequencer. Nucleotide sequences and the deduced amino acid sequences were compared based upon a gene alignment using the BioEdit program (Version 5.0.9. Department of Microbiology, North Carolina State University, Raleigh, NC, USA). Based on the alignment, a phylogenetic tree was constructed using the Clustal W program (European Molecular Biology Laboratory, Heidelberg, Germany) and generated by the MEGA2 program (Version 2.1. Department of Biology, Arizona State University, Tempe, AZ, USA). 3. Results 3.1. Sequencing of the K2 region in RSIV isolates Infection of iridovirus Sachun in rock bream of Korea and CH-1 in sea perch imported from China was determined by PCR with the 4F/4R primer pair of Kurita

Table 1 PCR primers used for the cloning and differentiation of the iridovirus isolates Object

Genomic region

Primer

Direction

Oligonucleotide sequence (5′ to 3′ direction)

Expected size of amplicons⁎

Detection of iridovirus

DPOL gene P1

Cloning of K2 region

P2

Cloning of K2 region

P3

Differentiation

Va

Differentiation

Vb

Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense

CGGGGGCAATGACGACTACA CCGCCTGTGCCTTTTCTGGA GACCAGCTACTTGTTAC CTATGCACGTGCTCATG GTGACAATCTCATATCG AGCCATTTGTGTATCTC GTTACTCCACCGCCTAC TCTGTCCAATGGCGTAC AAGGTAGACCACATCCA CACTAACACACTACGAT GGGTCTTGCATGGTTGC TCTGTCCAATGGCGTAC

567 bp

Cloning of K2 region

4F 4R P1F P1R P2F P2R P3F P3R V1F V1R V2F P3R

⁎: based on the nucleotide sequence of Namhae isolate.

1820 bp 1095 bp 3008 bp 780 bp 341 bp

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Fig. 1. Amplification of DNA of RSIVs using PCR with P3F/P3R primer set. Lane 1, DNA of RSIV Sachun; Lane 2, DNA of RSIV Namhae; Lane 3, DNA of RSIV CH-1; M, 1 kb DNA ladder.

et al. (1998) (data not shown). Another PCR analysis with the P3F/P3R primer pair (Table 1) was also performed to determine the nucleotide sequence of the K1 region (Jeong et al., 2003) and its flanking regions in the genomic DNA of both iridovirus strains. Interestingly, Sachun and CH-1 produced amplicons different from the expected sizes as expected from the corresponding sequence of the RSIV Ehime-1 (Fig. 1). However, Namhae, which had the same nucleotide sequences in the four genomic regions, the RNRS gene, the ATPase gene, the DPOL gene and the Pst I restriction fragment, as those of the Ehime-1 strain (Jeong et al., 2003) produced an amplicon of the expected size. This result suggested that there would be variations in the genomic DNA of different iridovirus isolates. In order to detect any differences in the nucleotide sequences in the entire K1 region and its flanking regions (designated as K2 region in this study) of

different iridovirus isolates, the amplicons produced in three different PCR, P1 (P1F/P1R primer pair), P2 (P2F/ P2R primer pair), and P3 (P3F/P3R primer pair), with template DNA from the different isolates were cloned and sequenced (Table 1, Fig. 2). It was found that the total lengths of the K2 region for the Sachun, Namhae, and CH-1 isolates were 5905, 5630, and 5169 bp long, respectively, all of them different from the 4996 bp length of the K2 region of ISKNV found in mandarin fish (He et al., 2001), (GenBank accession number: AF371960) (Fig. 3). Nucleotide sequences of the K2 regions of the Sachun, Namhae, and CH-1 isolates have been deposited in GenBank (GenBank accession numbers: AY628698, AF506370, and AY628699, respectively). Alignment of the sequences revealed the presence of many nucleotide substitutions and insertions (or deletions) of different lengths in the K2 region in four iridovirus isolates (Fig. 3), ranging in length from 1 to 163 bp, in the regions of the repeating sequences. Insertions (or deletions) were found in both the ORFs and the intergenic sequence of the K2 region, occurring in clusters in three different isolates, commonly between base positions 1195-1391 of ORF-1, 4695-4864 of ORF-2, and 5580-5742 of the intergenic region flanking the 3′ end of ORF-2, with these numbers referring to the base positions of the Sachun strain (Fig. 3). There was no frame shifting in ORFs of K2 region of four iridoviruses and an intergenic region flanking the 3′ end ORF-2 contained many repeating sequences and showed extensive genomic changes in the iridovirus isolates. However, we have also found insertion in the region of ORF-2 of Sachun (from 3979 to 4119) and Namhae (from 3857 to 3997), in the absence of the repeating sequences found in the other iridovirus isolates. As expected, Namhae demonstrated 99.8%

DPOL gene

RNRS gene ORF1 (1701bp)

ORF2 (3135bp)

K2 region of RSIV Sachun (5,905 bp) K1 region (4,525 bp) P2 (1,092bp) P1 (1,942bp) Va (888bp)

-500

0

P3 (3,163bp) Vb(506bp)

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 5905 +500 +1000

Fig. 2. Schematic illustration of the physical map of the K2 genomic region of the Sachun isolate of RSIVs. The K2 and K1 genomic region of the RSIV Sachun isolate is shown as a thick black box and a gray box, respectively. The thick arrows represent non-overlapping ORFs with respect to their size, position and orientation. The target regions of PCR amplification for cloning (P1, P2, and P3) and discrimination (Va and Vb) of RSIVs are shown as narrow arrows.

J.B. Jeong et al. / Aquaculture 255 (2006) 105–116

109

P 1F S N C I

gaccagctacttgttac ................. ................. .................

-101 -101 -101 -101

S N C I

ggccggctacgggcctgtgtgcagcaaggtatatgcggcacacttacagttggcgtacgtgcacaaacaattaatgagacgcatgacgcccaccgtataa .........t............................................................................t............. .........t.............................g.....g...........................c.........c...a............ ...............c.......................g.............................g...c.........c......tg......g.

-1 -1 -1 -1

K2 region from here

DPOL gene

S N C I

cccaatgccacatgcccgacgtgccgtcgtgtcaagtacgattattgagacactttatacccgctatataaaaacaatggcagactcatcaccaaca--..................--------...............---------...............................................----.---..--------..--------.......gtac....---------...........t......................g..c.........---------.------------------...c.c.gt-cga..---------..................................g............aca

97 80 67 65

S N C I

gccgttccacacgccg---atgtcgctccgacagacgtgccaaagaaagaggccacactgtcgtgcaattctgtcaaagagcaactgaaggttgcacttg ...........tc...---.c.....c...g..................................................................... ........g.....t.---.c.....c..ag..................................................................... ..............t.ctg.....c.a..ag....t................t...c..........................................

194 177 164 165

S N C I

gacaggactgccggctatctggcagtgtgctgcatgaggccatcgcacacatcaaggaggtttacatgactgacctgtgcacacgcatcataggtgcact ......................................................................c............................. ...........a....c.................c..........................g...................................... .............a..c.................c.......................t................................g........

294 277 264 265

S N C I

ccagtacaataacagcaaaactgtgacagatcgcttactcaaggtgattggcgtcactgtatggcccgaggtggacgtgcctgtatatcacatcaagctg ..............................c..........................c.......................................... .....................c............c.g.................g.....g......c.........................t...... t........c.....t..................c.g........................................................t......

394 377 364 365

S N C I

ggcacaatcatcaggatgcttaaagagcggatgccgggtgttcggatacagcaacaggtgcgcaaaacattacagcaccacctgtgcgcatatctccatg ......................................................................................t..c........c. ....................c........c.....a..c..c..c........g...............c............a...............c. ........................c....a.....a...........................g.......g..........................c.

494 477 464 465

S N C I

tcatgggcatcctcctgggccgctatgtgcagtcaacgggcataggtctcatcaaggtagaccacatccagcatgtgttgcgcaccaaccgcatcgaccc .................................................................................................... ...................t.................t.....t........t.................a............................. ..................................c.t...............................................................

V1F 594 577 564 565

M2 S N C I

tattgcactgggttgaagtatgtaggcaactggctattgtatacgtcaaagtcagcattgctgtacgtacagaaaacaatgtgatcaaaagcacccgggt ...............................................................................................g.... ......g.....c.a.t......g...................ac.........t........a............tg...........g.....g.... .........................................c..c.g............a...a.............g.................g....

694 677 664 665

S N C I

gctgtattacatatgcacgtacactcgacatggccacatgaacggcatcctcaatcgggtagttgtacacacccgtggatatggacgggaacgcgatggt .......g..............g...........................a............c.................................... .......g..............g...............................................g............................. .......g..............g.............................................g.g.......................a.....

794 777 764 765

S N C I

gcgcacaccatttgcctgggccacatgcaatgactggatataacaactggtcaatacacgcttatcggcctgggtgggtcgctgtcctgcattaataata ...............t.................................................................................... ...tg.c........t.......................g..............c........g..c...aca.....a..agca..ctt.c.c.gt... ....g.c........t..........................g...........c..g.....g..c..tgc......a..agca..cct...c.gt...

894 877 864 865

S N C I

ggaccaactgtgtggatgacataagtcgctggcagccgatagccgcctgtgatttttgcctccccaaacccaatcccaccaagggtcctgcactccctct .............................................a........................tg............................ ..g.....a...............................g..............................................tga.......... ..g.....a.............................................................t...a............tc...........

994 977 964 965

S N C I

taagttcgcgacctgccactctgtgaatacggccatcgacaccgccaccacccagccctacggtgtttgctgcgttaactatggcatctactcttaaaga .g......g.......................................................a................c......c........... .g......g.g..c....tct..........a.............t......a......gt......g..c............................. .g..c...g.g.......tc..a.......tc...........a........g......c...g...g.......................c........

1094 1077 1064 1065

S N C I

ggttatatcatccaacacaacactcacattagtctgtctatggggctctatgtcgtcatcgaaatgaacgtgtcgagctggcgccggtgctggagcaggc ..............................g.................------...............a........c..............t..t... .....................................................................ac....c..a..a.at...t..c.c...... ....................................c....t.t........ct......a........a........c.....t..---...c..t...

1194 1171 1152 1162

S N C I

attggtcgcggtgccggtaccggagatggtggtcgcgctggcattggtggcggcgctggagctggcaccggagatggtggtcgcgctggcattggtggcg .c------......t..c....----------------------..c....a..a.c..................................--------------------------------------------------------------------.....tg.t..c.......c..tt.tg...t------------------------------------------------------------------------------------------------------------

1294 1235 1183 1162

S N C I

gcgctggagctggcaccggagatggtggtcgcgctggcaccggaactggtaccggcaccggagatggtggtcgcgctggcgccggaactggagctggcgc ---------------............................t....-------------------------------------------------... ---------------............................t.. -------------------------------------------------... ---------------------------------.c..ag.t..tg..c.------------------------------------------------... -----------------------------------...g.t..------------------------------------------------------tag

1394 1272 1202 1173

Fig. 3. Comparison of the nucleotide sequences of the K2 regions of RSIVs. Identical residues and gaps are represented as dots and dashes, respectively. Start and stop codons for ORF-1 and ORF-2 are designated M1/M2 and T1/T2, respectively. Primers used in PCR for the cloning and differentiation of RSIVs are in black and white boxes, respectively. S, RSIV Sachun; N, RSIV Namhae; C, RSIV CH-1; I, ISKNV.

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J.B. Jeong et al. / Aquaculture 255 (2006) 105–116

V1R S N C I

tatctgaggtaatattacttcatcgtagtgtgttagtgtgtcatagtcgataggcattacacccccaatgggtgaattaaaccgctgcaatgcatttgca ......................................c............................................................. ....................g..............................g..............g................................. ...........---------..........................gt...........t.................................g......

1494 1372 1302 1264

S N C I

agctgtccagtggcagacatcaatgtgttaggagcaagagggtatccattggtacctggggtgtatgaaacctccatttgtacaccatccattgtatcta ......................................t..............g............................t........a........ ......................g.........g.....t..............g..c........................................... ........g..........................g..t.....g.....t.....c........a..........................c.cc....

1594 1472 1402 1364

S N C I

tgtcagcccatgtgtatgtgttgtggttgtgtcgagatttgagcacaatgccattacgtgtggcatatacatgcaacatgacacctggtgatacgggccc .....................................................................................c.............. ....g...............g..........c....t...............g.cg.............................g..g.c...t..t.. ..........c....................c.................a...................................g..g.....c..t..

1694 1572 1502 1464

S N C I

caatggcaacggcacatttacataatatcggaacgatgatgccattccggtgtctacgtctgtgttggagtgcttgtcaatgcaagtgacaatctcatat ....................................................................c............................... ............t..............g.....t....................a............................................. .................................g...........g.......................................................

S N C I

cgaccgtggtaaaacatgagcacgtgcatagcgccattggcctgaccaccatcctttatgtgtaccagctgccgaagacgatcaagctgtagtgcagaca .........................................t...............................................c.......... ...................................................g......................g..............c.a........ ......c.........................a..................g......................g..g...........c..........

1894 1772 1702 1664

S N C I

catacacgatgattgtcattggttgagggcgacccccggatgagatgagttctctcgcggcatagttaaccaccggcatttgcgctgcattaaagtagtg .......................................................t.....g..........................g........... ......................c................................g.................g.......................... .......a..............c...................................a..............t..........................

1994 1872 1802 1764

S N C I

agggcagaagccaatgtgtgtgcccatgtcctggtaattgacaaatccatcttgtgacatgtctgggtggatcagtgtaataggcacacgcagccgcgaa ..................c.............................................................................t... .........................g.......................................................g.....g...........c .......................................................................................g...........c

2094 1972 1902 1864

S N C I

ccaaaaatctgggctgcgactataagacacctgcgcaatgtctcataatccacattgtcaggcaccgcggtaactgtgacacaccctctggtggtattat ..g................................................g.g....................c........................g ..g...............................................tg.g.....g........c..g..c.....g......t............ ...................................................c.g..............c..g..c.....g......tg...........

2194 2072 2002 1964

S N C I

ttaacgccacagattgtcccgggtcacggttgtacatcacacgtatgtcgtcacgcgatactagcaccaacacatctcctatctcaagcacagtgaacat ..........................................................c......................................... ..........g.c...c........g.............tg...........g.....c..............g....................a..... ..........g.c...c........g..............g.......t.........c..............g..........................

S N C I

cttttacttgtataaaaattttgaatatcctcatgtcatatgtgactactgtaaatcatacctacatgagcaatggcaaccctgctatttcttctgttgc ........................................g...........g.......g................g.........c............ ..................................c.....ca.............g.........c...........g..tt.....c....g....... .........................................a....c....g...a........gcg..c.......g..t......c.....g......

2394 2272 2202 2164

S N C I

tgactgtggcgtatagtcatgccaccaccttctataacctggaaatcggcaaccagacgacaaccctcagctgcggggtacctcgagagactgatgtgaa ............g..c...c............................a............c.............................g........ ..........t.gc---..c............................a............c.......................g.............. ..........a.....c..c..........................t.a............c......................................

2494 2372 2299 2264

S N C I

gattgtttggtcaagcgacagcaacagcctcttggcggagcatgttgtacatggcgaggtggtccatgtaggccgcaacagcagtgaagtcctgaagcat .................................................................................................... ....c.......g..................................................g....................c..........g.... ..........g...................g.....a..........................a....................c...........a...

2594 2472 2399 2364

S N C I

ggagtggtgctgtatgacggtaccatcttgtctgtcatcaagctgaaacctcaccctgtgcagaccgtcacatgccacgccagccgcatctcatctgaca .................................................................................................... .................t..a...............................t...............g..g.................t.......... .................t........tg....................................g...................................

2694 2572 2499 2464

S N C I

gtcagccatgtgtaggtgtctcatgtgagctgccaacagacaccgatgatgttactccaccgcctacactgcttgacgatgggggcagtggaatggacga .......t.......................................a........................................c........... .......t..............g.................................................................c........... .......t........c.................g..............c...........a..........................c...........

S N C I

ctacgat---atagacgagccagagtgtttgagcggcaactgcagtgactgcataaatgtggtgagatacacaaatggctctttggagtgtctggacgat .......gac.....t...................................t............................................t... ....t..gac....t.t.t..g........................c....t............................................t... .......gac.....t...................................tga..........................................t..-

2891 2772 2699 2663

S N C I

aagatgtgtgtccccggtgaaatgccctacaatatgctccagtgctaccaatcaaatgtcacatgccactgtgacaatgggctatgtagagtgagctacg ...c................g................................g..........................a.................t. c...................g..........g.......g........................................a......c.t........t. -----...............g................................g..............t...........a......c....a.....t.

2991 2872 2799 2758

P 2F 1794 1672 1602 1564

P1R

M1 2294 2172 2102 2064

T1

P3F 2794 2672 2599 2564

P 2R

Fig. 3 (continued).

J.B. Jeong et al. / Aquaculture 255 (2006) 105–116

111

S N C I

atgggaccgatgtgtgctgtccagttggctactatggcgacccaatgtcatgccaccaatgcccatgtccagaagacggtccgtgtgaaatacgtatggg ..........c..............c........c........g..t........a....................t.....a..........a...... ........a.c..............c........c........g.ct........a................g.t.ta....a.........ta..c... .....g..c.c.............ac........c........g..t........a....................t............g.c.a......

3091 2972 2899 2858

S N C I

tcgcctggtatgtacaagctgtccaccgggacacataggagacacatgcaaccgctgtctggatggatattatcaggccgaagatggtgtgtgccgtgaa ...........................................................................a...a....c............... ...............c.t.........................g.....g..a.a......tc..........a.a.a.cgt..............c... .............t................t.....t............g..a........a...........a.a...cgt..............g...

3191 3072 2999 2958

S N C I

tgtcagtgtccattcgataggccgtgtatactggagaatggc---acagcaacctgtttatgccccatcggacataccggacgaaggtgcgaggtgtgtg ...............a....c.......c.........c...---..g........cg........................a................. ...g.c.........a....ct..............g.c.a.gat..g.tg.....cg.......a..................tat..t...a...... ...gca.....g...a..g.c........gtc.........t---...at.gt....g....t..ag.t...t..g.t..c.c.ca...t..t..a...c

3288 3169 3099 3055

S N C I

aatcaggctacttttggacaggacaacaatgtacacgctgcccgtgtgatggacaatgtgcactgcaacatgacggacaggtgatctgctttgggcgtgt .........................c.......t.....................c..................................a.c..t.... ...ac.........c....g........t....g..c............................................c..........cact.... ..atg........c.........g.gt.t....gc........a..c..............g........ca.t..c..a...g....ta.gcaat..ag

3388 3269 3199 3155

S N C I

ggattgtgtaggagaaggcccatgccatgtagacatgactaacaacaatagc---atatattgtgtggactgcccatacggctcaagcgggcaatactgt .......................................c..t..g......---............................................. ta.......g..tcg............c...........c...g.....------.....c...............cg....g...cg........t... cc.a...cg.....g...a..c...tt.a.ga..tat..cg..g.......tggtg..c.......c.........act....tcgt....ag......c

3485 3366 3293 3255

S N C I

gaaatgtgtgacgttggcttctttcgtgttgtagatggatgcaggccatgtccatgccctaacaatggagcatgtagacaggctggcaataacatcatat ..............c.............g.tc.a...................g.....c........g............................... ...c........acc..........tc.a..gca...a........a.....ag.....c........g..g.......g.................... ...ca.....cacc......t..c.a....tc...........ta........g......c.......gc.g.........a....ag.g..........

3585 3466 3393 3355

S N C I

gtacaggctgccctatcaataccacaaagaacaacttgtgcgaagagtgcctcgatgggtcatttggcgacccaagtggcttactgggccctgttcgccc ...................................................................t.....g........g..a.............. ....g.a....a.................g.t...gca.................g.................c........g.gc.............. ....g........................g.....gca...................................t........g.ga..............

3685 3566 3493 3455

S N C I

atgcaggcgctgccactgttcaggcaacattgacgacaatccagtaggacagtgtaaccctaagactggcgaatgcctgcgctgcctgcataataccgat g..t........t................c.........c...............c.....g....g..t.....ta....................... g.............t........................c..............cg.t...g..............t....................... g..............a.......................c.................t...g......................................

3785 3666 3593 3555

S N C I

ggattcttctgtgacaggtgcgccaccagctattatggcaacgccctgagttctgaccctactaacaagtgcaaaccatgtgtttgcagtgggcgcggct .........................ggg.a...............................---....a.......a.................at.... ........t...............gaag.a...................c.........c.acc...........ga....c......a..t..aa.... ................aa...a...ggg.......................c.........ac.............................a.a.....

3885 3763 3693 3655

S N C I

cattgcatccagtatgtgaccagtacactggacaatgcatgtgcaaacctaatgtgattggcctgcaatgtgatacatgtctgcctggatactatgggtt ......g....a........gt................g.......g.........g........................................... ...c.....a.t.c......a..gt...g........tg...........c............c.a........ct....g....c.......------...........c.g..ca..g.........................g...........g...t...........ca....a..........t.-------

3985 3863 3786 3748

S N C I

ataccacgatggcatttgcaagccatgtccatgtcatccgatgggaacacaaatgtgcaattcacagaccggacaatgtgactgtcacattggggtgtat .............................ag.........gc................g.c...................t........c.......... -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

4085 3963 3786 3748

S N C I

ggtgaacactgtgataaatgtaggcctggatatttcaatatacagacaggctgtgaagcctgcatgtgtcatcccaatcaatcaacatcacagcagtgta ......gca......c......t......................g.............a.........................t.............g ----------------------------------......c...tg...........c..........c...........c............g...... ----------------------------------.........................a................c........tg....t........

4185 4063 3852 3814

S N C I

atgaagacgggcagtgtgtctgtaaagaaggcttcacaggactaatgtgcacagtcgcaataccgctgcccactgaggccgaacctgaggaaccggagga .cc..............a................................................................g................. ..c...c..................g...........g............g.............a..a..g......c.t..g..g......------.. ......c...a.......c................................................a.....c........g........c...c....

4285 4163 3946 3914

S N C I

agaggaggacgagtacgactgtccagagtacgaggat------atcaccagcgctccgccgacaactgtgcctcctcccaggcgggaatgcaaaaagaag .....................c..c........a...gatacc.ca........a.....c..g..ca.......g....a.a.a............... g.......................c........a..ggagacc.ca........g.....c..g..c..t..g..a....a.a................. ..............................t......------...........g.....t.....cac...g..a....a.a.................

4379 4263 4046 4008

S N C I

accaccaccaccactgtggcacctacaacaaccaccaccactgtggcacccaccgagcctgagcctgaggaaccggaggaagaagagga---cgagtacg ...........---......................................................a..g..a..............---t....... ...........---cacca.ca..gt.g..c..........a..a...a.a..a.................g...........g.....gga........ ...........---------ca.cgt.g..---.....t..a...........------c........a.....a........g.....---t.......

4476 4357 4143 4087

S N C I

actgtccagagtacgagga------tatcaccagcgctccgccgacaactacacctcctcccaagagagaatgcaaaaagaagaccacca---------...................------............a.....c.....c.tg.....g...............................ccactgtggc .......c........a..ggagacc.ca........g.....c..g..cgtt..g..a........g......................---------.............t.....------............g.....t.....c..g..g..a.......................ac...--.----------

4560 4451 4233 4169

Fig. 3 (continued).

112

J.B. Jeong et al. / Aquaculture 255 (2006) 105–116

S N C I

-----------ccactgtggcacctacaacaaccaccaccactgtgccacctacaacaaccaccaccac---tgtggcacccaccgagcctgagcctgag aaccaccacca...................................g......................cac............................ -----------....cacca---..gt.g..c..........a..ag..---........ ........---...a........a............... ----------g....caat.--gt.g.....c..-.....t.ctcaa..----tg...g.----------------.....ag.t.gaa.a...a.c...

4646 4551 4313 4236

S N C I

gaaccggaggaagaagagga---tgagtacgactgtccggagtacgaggatatcaccagcgcgccgcccacaaccgtgccttctcccaagagggaatgca ..............g.....---c...........c..c.......................a............a.....c.g........a....... ..g...........g.....ggac..............c........a..-------------------------------------------------.c....c.ac.ca.t.tac----ct.t.gt.g.a.c..c-----t..aaga-------------------------------------------------

4743 4648 4363 4278

S N C I

aaaagaagacgaccaccactgtggcacctacaacaaccaccactgtggcacccaccgagcctgagcctgaggaaccggaggaagaagaggatgagtacga ..........c..............ga.c..c..c..................................................g.....c........ ----.g....c..------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

4843 4748 4372 4278

S N C I

ctgtccggagtacgaggatatcaccagcgctccgccgacaaccacgcctccgcccaagacggcatgcaaaaagaaacccaagccgccaatggttgtaacg ..................c..a........g..............a..............................................g..c.... ---------------------a........g.....c..g...gtt..g..a.......g..a..................a.ta..------..c...a ----------------------------..a..a..ac.tg.tg....ct.a.---------------------.g...g..------------------

4943 4848 4445 4311

S N C I

cccgtacatgacccacagactccaccacctggaaagcctgagcccgtggcaccccctcaagcaccagctgtaaagcccatgccagagactgaggcaccgc ..............................c.......................-----------.....g..g.......................... ...a....-..--.g.c.c.a......t..c..........a..t...c.......---------.....g...........------.......g.... ---------------------g....g.aacac.tc.....aaga.cac.....t---------------------------------.....a......

5043 4936 4527 4357

S N C I

aacgcgacgtagctattgtggcagtggtacacacccctgaaagagcaccgcctcaaaagccctcacagacccaggccccgcaacacgatgcgcctattgt ...........c...............................g........................................................ ...a.......c........t.c............a..............g.cgcggca...c.t..a--------.at...g.-t.gaaa...c..gcc ...a.......c.....................................---------a..a--c.t.--------.----tg.-------...c---tc

5143 5036 4618 4424

S N C I

ggcagtggtacacacccctgaaagagcaccgcct------ccctcacagactgaggccccaaagcataaggtacctgttaccacagtggtacacacccct ......................c.g.........------.......................a...............g................t... a.ag.caccg..aca.gtg...............gctgct.................a..gc.a..............cg.t.................. aca.cc.ca.....................a..g------------.ctg...c....t.---a..c.gacc.....c----------------------

5237 5130 4718 4487

S N C I

gaaagagcaccgcctgctgcgccctcacgcataccacctgccatccaaaatgaagtgcctgttgtgaaacaaagtactggtgaggaagatgaagatgatg ............................................................................................c....... .........a.....a.------------------------..g.................................c..a........a.....c.... -----------------------------------------...............a.....c..........................------.....

5337 5230 4794 4540

S N C I

tgcaccttggtgacattgggggtatgaagaaaggaggtctgttgatgggaattgtcattgggtcttgcatggttgccgctgcatttattatgtttgctat .....................................c...............................................c.............. c......c.............................c......................................a....................... .......c.............................c..............................................................

S N C I

aatagggcatctgtttttccgttttacccgctgtggtcaatatgatgttactac---tgaaccatgagtgctactgtactgcctgccataatctatgcat ......................................................tac....................................a...... ........................a.....................ac......---c............t.....---------....g...a....-........................a.............................cac................a.c--------a.t..gg..a..at--

5534 5430 4980 4730

S N C I

tgatcatcttttgtcctgtccctagtggtgtgtatgattttgaacatgattcatcttttgtcctagtgttttgaataaacatataactttacatctttgg a---.g............c.a..----..................--------------------------------------------------------------..........c.a..----------g.a.........--------------------------------------------------------------ga.gt.a..ac.a..---.aac--c....g.gct..tcacta.gc.ac.aaacta.gagtgc.act..gt.t..c.t---------------

5634 5467 5006 4919

S N C I

ctggtttataaagatggataggtatgtgctcaatcttttgtcctgtccctagtggtgtgtatgattttgaatatgattcatcttttgtcctgtctccggt ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------..t.--cc.gcca....g.gtatg

5734 5467 5006 4824

S N C I

gttttgaataaacatataactttacatcttt-ggctggtttattataaagatggataggtatgtgctcagaccaaacgacccacatctgtgggccatgta --------.........gc.a..........-.c.................................................................. --------............a.g...ct...-.....................................................t.............. tg...a..............a.g....t...t.........................a.................g.........t..............

S N C I

caagaaggcagtagcgtctttttggactgttgaggaggtggatctcagtaccgatgtacgccattggacaga ...a.........................................t.....t.................... t........c...........c.....g..c..............t.....t.................... .........c..t..............c.................t.....t....................

V2F 5437 5330 4894 4640

T2

RNRS gene 5833 5558 5097 4924

P 3R 5905 5630 5169 4996

K2 region to here

Fig. 3 (continued).

identity in the nucleotide sequence of the K2 region with that of RSIV Ehime-1 (GenBank accession number: BD143114) by base substitutions at seven points without deletions (or insertions) (data not shown) and consistent with the conclusion based on the analysis of the Pst I restriction fragment, which was that these two strains are the same (Jeong et al., 2003).

In comparing the nucleotide sequences of the K2 region, Sachun shared 92% identity with Namhae but only 76% and 72% identity, respectively, with CH-1 and ISKNV (Fig. 4). Two strains, CH-1 and ISKNV, isolated from fish in China, displayed a higher degree of sequence identity to one another, 79%, than to any other strains. No significant differences in the levels of

J.B. Jeong et al. / Aquaculture 255 (2006) 105–116

113

Sachun Namhae

92 % Namhae

CH-1

76 %

79 % CH-1

ISKNV

72 %

79 %

73 %

K2 region Sachun Namhae

CH-1

ISKNV

91 % (91 %)

Sachun Namhae Namhae

83 % 88 % (82 %) (86 %) 81 % 85 % (83 %) (86 %)

CH-1 CH-1 90 % (88 %)

ISKNV

ORF-1

93 % (92 %)

Namhae

75 % (71 %)

76 % (72 %)

71 % (68 %)

77 % (67 %)

CH-1 77 % (69 %)

ORF-2

Fig. 4. Comparative analysis of the K2 region of RSIV Sachun, Namhae, CH-1, and ISKNV. The homology of nucleotide (amino acid) sequences is expressed as percent identity.

homology were found when we used the nucleotide or amino acid sequences of the ORF regions, ORF-1 and ORF-2, within the K2 region in different iridovirus isolates, instead of the nucleotide sequences of the entire K2 region (Fig. 4). Phylogenetic trees based on the nucleotide sequences of three different regions of different RSIV isolates are shown in Fig. 5. Members of the Korean iridovirus group, Sachun and Namhae, were more closely related to one another than are the iridoviruses found in fish in China, CH-1 and ISKNV. 3.2. Differentiation and quantification of RSIV isolates in fish Two sets of primers, V1F/V1R and V2F/P3R, which can produce amplicons of less than 1 kb long by PCR, with the precise lengths of the amplicons differing for

the different variants of iridoviruses, were designed using the sequences of the conserved portions of the 3' flanking region of the ORF-1, ORF-1 itself, ORF-2, and the RNRS genes of the three different types of iridoviruses, respectively (Table 1, Fig. 2). As shown by electrophoresis after PCR amplification, we found that the resulting amplicons, 885, 780, and 723 bp with the V1F/V1R primer set (Fig. 6, A) and 509, 341, and 316 bp with the V2F/P3R primer set (Fig. 6, B) matched exactly the expected sizes from the three different viral DNA templates, Sachun, Namhae, and CH-1, respectively. In nine iridoviruses sampled from 1998 to 2003 for this study from different species of fish with suspicion of iridovirus infection in Korea, except for one Namhae type of 1999, all eight isolates appeared to be the Sachun type in two discriminative PCRs and nucleotide sequence of the amplicons (data not shown). Sachun Namhae ISKNV CH-1

0.06

0.05

0.04

0.03

0.02

0.01

0.00

Fig. 5. Phylogenetic tree of K2 region of four iridoviruses. Phylogenetic relationship was inferred using MEGA2 program. Branch lengths are proportional to the nucleotide differences.

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J.B. Jeong et al. / Aquaculture 255 (2006) 105–116

Fig. 6. Differentiation of RSIVs by PCR with same primers based on the variation of DNA nucleotide sequences in the K2 region. (A) product of PCR amplification with primers V1F and V1R; (B) product of PCR amplification with primers V2F and P3R; Lane 1 and 4, DNA of RSIV Sachun; Lane 2 and 5, DNA of RSIV Namhae; Lane 3 and 6, DNA of RSIV CH-1; M, 100 bp DNA ladder.

We propose that the Sachun isolate be considered to be a representative type of iridovirus in the aquatic farms of Korea. 4. Discussion Although many studies have been done to differentiate the iridovirus isolates using the nucleotide sequences of structural genomic regions (Oshima et al., 1996; Wabby and Kalmakoff, 1998; Kim et al., 2002; Marsh et al., 2002; Murali et al., 2002; Sudthongkong et al., 2002; Grizzle et al., 2003), such as the MCP gene, there have been few reports comparing both the structural genes and the intergenic regions in the Iridoviridae family. In this report, we present data on the differences between the iridovirus isolates Sachun, Namhae, CH-1, and ISKNV from Asian countries. For this work, we employed a genetic comparison of four different iridoviruses based on the sequences of nucleotides 4996-5905 of the K2 region, located between the DPOL and RNRS genes, which contained two ORFs of unknown function as well as long intergenic regions showing various lengths of repeating sequences (Fig. 3). At the present time, it is possible to use the published nucleotide sequences of the several members of the family Iridoviridae, LCDV, FV3, RSIV Ehime-1, and ISKNV, for the comparison of the complete genomic regions. However, LCDV (the type species of the Lymphocystivirus genus) and even FV3 (the type species of the Ranavirus genus) have been classified in a different branch in the phylogenetic trees of iridoviruses (Sudthongkong et al., 2002), and

therefore were not used in the comparisons made in this report. In comparing the nucleotide sequences of K2 region, different iridovirus isolates showed many insertions (or deletions) of up to 163 bases as well as base substitutions. Interestingly, the inserted (or deleted) nucleotide sequences were located entirely or partly in the repeating sequences present in the ORFs or intergenic regions, except for one insertion of 141 bp at position 3979 in Sachun (Fig. 3). We could not find any common features among the deleted nucleotide sequences, but the locations of the deletions were biased towards the 3′ ends in both ORF-1 and ORF-2 of the K2 region. These results agreed with the reported genomic structure of the iridoviruses: a single linear doublestranded DNA molecule that is terminally redundant (Delius et al., 1984; Darai et al., 1985). The numbers of inserted or deleted nucleotides in the ORFs were multiples of 3 and therefore did not induce frame shifting of the ORF-1 or ORF-2 regions. However, the structural or functional changes in used by inserted (or deleted) peptides in the resulting protein have not been studied. Although various repeating sequences have been identified in the genomes of numerous DNA and RNA viruses like poxviruses (Pickup et al., 1982), herpesviruses (Wadsworth et al., 1975), retroviruses (Reddy et al., 1980) and iridoviruses (Schnitzler et al., 1987; Fischer et al., 1988), little is known about the function of repeating sequences. It is only assumed that the repeating sequences might be associated with important regulatory functions during viral replication. Certainly, further studies are needed to evaluate the consequences of changed genomic information as a result of the insertion of repeating sequences or base substitutions. Such studies will require functional analyses of ORF-1, ORF-2, and intergenic regions in the evolution of iridoviruses. In a comparison of the nucleotide sequences, four iridovirus isolates of this study would be members of the genus tropical iridovirus in the Iridoviridae family (Sudthongkong et al., 2002). However, the Sachun isolate appears to be more closely related to the Namhae isolate, and the CH-1 or ISKNV isolates appear to be less closely related variants in this group of iridoviruses (Fig. 5). Geographical proximity of the isolated areas may influence the identity of iridovirus isolates. It appears that, for the study of iridoviral evolution in aquatic animals, the nucleotide sequence of the K2 region is a more suitable target than MCP or other structural genes used in other studies (Wabby and Kalmakoff, 1998; Marsh et al., 2002; Murali et al.,

J.B. Jeong et al. / Aquaculture 255 (2006) 105–116

2002; Sudthongkong et al., 2002; Grizzle et al., 2003) since it contains both conserved and diversified regions, and both ORFs and intergenic regions, which allow one to distinguish closely related iridovirus isolates. PCR with primers derived from the regions containing different lengths of insertions (or deletions) in the K2 region allowed us to distinguish the variants of iridoviruses isolated in Korea, possibly including ISKNV, by the different sizes of the resulting PCR products (Fig. 6). Until now, many iridoviruses isolated from diseased fish have been classified and distinguished by comparison of the nucleotide sequences of the MCP gene. However, the sequences of the MCP gene in iridoviruses has a very high degree of homology over its 1362 bp without size variation (Murali et al., 2002; Sudthongkong et al., 2002; Grizzle et al., 2003) and therefore it is rarely possible to distinguish iridovirus isolates based on the MCP gene. Other structural genes reported, like the DPOL, RNRS gene, and ATPase gene, also did not show enough variation (Oshima et al., 1996; Sudthongkong et al., 2002), with respect to both their sizes and nucleotide sequences, to design primers for discriminative PCR. Moreover even if the variable regions in the structural genes might allow one to design the primer sets for discriminative PCR of iridoviruses, there would be some problems, like limited numbers of variable regions to allow the design of discriminative primers or stringency controls for specificity, and applicability restricted to certain isolates. To our knowledge, except RFLP for the discrimination of Ranavirus (Grizzle et al., 2002) no studies have been done on the differentiation of the various iridoviruses using PCR with primers that produce different lengths of amplicons as in the present study. Additionally if we use two discriminative PCRs along with primers designed from separated variable regions within the ORF-1 region, which is assumed to be a structural gene (V1F/V1R), and in an intergenic region (V2F/P3R), more powerful and extensive analyses will be possible for the discrimination of various iridoviruses, including strains that we did not test in this study and even future variants. In PCR with serially diluted viral DNA from infected tissues as a viral template, the amount of DNA in the CH-1 infected spleen was much lower than that of Namhae or Sachun (data not shown). However, without other analyses, like controlling for the stages in viral infection or numbers of TCID50, fish species/ages or comparisons of the virulence of different viruses, it would be difficult to obtain a meaning information from the variations in the amount of viral DNA in infected tissues. A further survey designed to detect asymptom-

115

atic infections with iridovirus also might be needed, as CH-1 was isolated from sea perch that were considered to be healthy and which aquatic farmers had tried to import in the spring but which had been returned to China as a consequence of the finding of RSIV in the tissues. Moreover, we could not observe the typical symptoms of RSIVD or mass mortality in the sea perch that we sampled and had kept for four weeks in the laboratory for analysis. Some experiments are going on in our laboratory to clarify the situation related to asymptomatic infections with iridovirus in the cultured fish in Korea. Finally, it should be pointed out that there is a continuous evolution in various genomic regions of iridovirus and it may be possible to see the appearance of the iridovirus variants as different pathogenic types. Acknowledgements This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD)( R05-2003-000-10769-0). References Ahne, W., Schlotfeldt, H.J., Ogawa, M., 1989. Fish viruses: isolation of an icosahedral cytoplasmic deoxyribovirus from sheatfish (Silurus glanis). J. Vet. Med. 36, 333–336. Armstrong, R.D., Ferguson, H.W., 1989. Systemic viral disease of the chromide cichlid Etroplus maculatus. Dis. Aquat. Org. 7, 155–157. Chua, F.H.C., Ng, M.L., Ng, K.L., Loo, J.J., Wee, J.Y., 1994. Investigation of outbreaks of a novel disease, ‘sleepy grouper disease’, affecting the brown-spotted grouper, Epinephelus tauvina Forskal. J. Fish Dis. 17, 417–427. Chua, H.Y., Hsu, C.C., Peng, T.Y., 1998. Isolation and characterization of a pathogenic iridovirus from cultured grouper (Epinephelus sp.) in Taiwan. Fish Pathol. 33, 201–206. Danayadol, Y., Direkbusarakom, S., Boonyaratpalin, S., Miyazaki, T., Miyata, M., 1996. An outbreak of iridovirus-like infection in brown-spotted grouper (Epinephelus malabalicus) cultured in Thailand. The AAHRI Newsletter, vol. 5, p. 6. Darai, G., Anders, K., Koch, H.G., Delius, H., Gelderblom, H., Samalecos, C., Flugel, R.M., 1983. Analysis of the genome of fish lymphocystis disease virus isolated directly from epidermal tumors of pleuronectes. Virology 126, 466–479. Darai, G., Delius, H., Clarke, J., Apfel, H., Schnitzler, P., Flugel, R.M., 1985. Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus. Virology 146, 292–301. Delius, H., Darai, G., Flugel, R.M., 1984. DNA analysis of insect iridescent virus 6: evidence for circular permutation and terminal redundancy. J. Virol. 49, 609–614. Fischer, M., Schnitzler, P., Scholz, J., Rosen-Wolff, A., Delius, H., Darai, G., 1988. DNA nucleotide sequence analysis of the Pvu II DNA fragment L of the genome of insect iridescent virus type 6 reveals a complex cluster of multiple tandem, overlapping and interdigitated repetitive DNA elements. Virology 167, 497–506.

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