• Journal of fish pathology
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• v.34 no.2
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• pp.149-159
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• 2021

Genus Megalocytivirus cause red sea bream iridoviral disease (RSIVD) and scale drop disease (SDD). Based on the phylogeny of the major capsid protein (MCP) and adenosine triphosphatase (ATPase) genes, megalocytiviruses except for SDD virus (SDDV) could be three different genotypes, red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis (ISKNV), and turbot reddish body iridovirus (TRBIV). In this study, primary cells derived from the caudal fin of rock bream (Oplegnathus fasciatus) grew at 25℃ in Leibovitz's medium supplemented with 10% (v/v) fetal bovine serum and primocin (100 ㎍/mL). Rock bream fin (RBF) cells exhibited susceptibility to infections by different genotypes of megalocytiviruses (RSIV, ISKNV and TRBIV) with the appearance of cytopathic effects with an increase in the viral genome copy number. Furthermore, compared to grunt fin (GF) cells, even though 10 times lower number of RSIV genome copies were inoculated in RBF cells, viral genome copy number produced on RBF cells were 44 times higher than that of GF cells at 7 d post-inoculation. As the isolated RBF cells are sensitive to different genotypes of megalocytiviruses (RSIV, ISKNV and TRBIV), they can be used for future studies regarding in vitro viral infection and subsequent diagnosis.

• Journal of Marine Bioscience and Biotechnology
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• v.1 no.3
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• pp.163-169
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• 2006

In the survey of the samples infected by iridovirus, we found the natural outbreak of iridovirus disease in the ornamental fish, pearl gourami (Trichogaster leeri) in Korea. It was characterized by the appearance of enlarged cells and necrosis in the observation of the imprinted spleen cells under iridovirus. To determine the infection of iridovirus more accurately, PCR using 2 different primer sets for MCP and ATPase gene used for the diagnosis of iridovirus infection was done and found the produced fragments matched with the size of expectation. Partially determined nucleotide sequences of the MCP gene of the iridovirus isolated in this study showed very high homology (99.6%) with that of ISKNV as a reference strain. In challenge experiment with the iridovirus isolated from the moribund of pearl gourami, the pathogenicity of the isolated iridovirus was confirmed and suggested the potential of the risk associated the transfer of iridovirus from the ornamental fishes to the marine culturing fishes.

• Journal of fish pathology
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• v.23 no.1
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• pp.17-25
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• 2010

We have identified an iridovirus CH-1 from sea perch Lateolabrax sp. healthy externally and imported from China to Korea. In a comparison of the nucleotide sequences of the five different genomic regions, the CH-1 appears to be closely related to the ISKNV, IVS-1 and Ehime-1 strains detected in China, Korea and Japan respectively. In quantitative comparison of the viral DNA, level of CH-1 in tissue of imported fish was 10,000 times lower than that of IVS-1 strain presented in the infected rock bream Oplegnathus fasciatus of moribund stage. It allowed us to speculate the possibility of the asymtomatic iridovirus infection in the culturing sea perch. Such possibility of asymptomatic infection was supported by result of no appearance of dead fish with typical symptoms of iridoviral disease in keeping experiment of the imported sea pearch in laboratory for more than three weeks. Such asymptomatic infections with iridovirus were also found in spleen of the culturing and externally healthy sea perch of Korea by the presence of the iridoviral DNA in nested PCR.

• Journal of fish pathology
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• v.19 no.3
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• pp.197-206
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• 2006

The distribution of iridoviruses in five freshwater ornamental fishes, pearl gourami (Trichogaster leeri), dwarf gourami (Colisa lalia), silver gourami (Trichogaster microlepis), blue gourami (Trichogaster trichopterus sumatranus) and freshwater angelfish (Pterophyllum scalare), imported from Singapore was examined in 2004 and 2005. The presence of iridoviruses in 56 sample groups was determined using PCR technique and showed PCR positive in 11 sample groups. The proportion of fish infected by iridovirus was differed depending upon species; 31.8% (7/22) for pearl gourami, 18.2% (2/11) for dwarf gourami, 16.7% (1/6) for blue gourami, 9.1% (1/11) for silver gourami and 0% (0/6) for angelfish. In quantitative comparison of viral DNAs isolated from infected tissues, the DNA concentration of iridovirus in pearl gourami was higher than that in dwarf gourami. Although pearl gourami infected naturally by iridovirus showed 100% mortality in keeping experiment for 3 weeks, only 57% of those was positive in PCR. In the comparison of nucleotide sequences of the PstⅠ fragment known as the most variable genomic region, both iridoviruses isolated from pearl gourami and dwarf gourami showed identity more than 99% with infectious spleen and kidney necrosis virus (ISKNV) isolated from mandarinfish (Siniperca chuatsi).

• Korean Journal of Fisheries and Aquatic Sciences
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• v.44 no.4
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• pp.352-358
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• 2011

The presence of ISKNV-like viruses in various freshwater ornamental fish species imported from Asia was confirmed by polymerase chain reaction(PCR) amplification of the ATPase(adenosine triphosphatase) gene. Interestingly, molecular analyses of the Open Reading Frame 25(ORF25) region of these isolates based on the ISKNV(Infectious spleen and kidney necrosis virus) genome revealed the presence of various repetitive sequences. ORF25 repeat sequence length had no effect on cumulative mortality of rock bream Oplegnathus fasciatus challenged with tissue homogenates of infected pearl gourami, Trichogaster leeri; silver gourami, Trichogaster microlepis; blue gourami, or Trichogaster trichopterus. All isolates induce cumulative mortalities after 12 days of infection, confirming that ORF25 polymorphism did not affect the pathogenicity of ornamental fish megalocytiviruses that cross infect rock bream, a seawater fish. Also, no statistically significant differences in spleen index or viral copy number in infected tissues was detected between isolates with varying ORF25 repeat sequence lengths. However, further studies are necessary to fully characterize the functional characteristics of these polymorphisms in megalocytivirus disease in ornamental fishes.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.873-879
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• 2005

Red sea bream iridovirus (RSIV) obtained from infected rock bream was propagated by Bluegill fry-2 (BF-2) cell culture. The virus titer was determined as $10^{5.5}\;TCID_{50}/ml$ on confluent BF-2 cell monolayers. The integrin binding site of ORF 055L of infectious spleen and kidney necrosis virus (ISKNV) was selected for the construction of a primer to obtain the RSIV ORF 055L gene. The genes were amplified using RSIV gene lyzate by PCR. The homologies of the ORF 055L sequence of RSIV with ISKNV and rock bream iridovirus (RBIV) were approximately $96\%$ and $100\%$, respectively. DNA vaccine was constructed by cloning the ORF 055L of RSN into pcDNA 3.1 (+), containing a cytomegalovirus (CMV) promoter. For antibody production, pcDNA-055 DNA vaccine was injected to BALB/c mice. The production of antibodies against pcDNA-055 DNA vaccine was confirmed by the Western blot analysis. The antibodies produced by the pcDNA-055 DNA vaccine showed efficacy to neutralize the RSIV in the neutralization test in BF-2 cell culture.

• Fisheries and Aquatic Sciences
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• v.24 no.11
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• pp.351-359
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• 2021

The red sea bream iridovirus (RSIV) belonging to genus Megalocytivirus is responsible for red sea bream iridoviral disease (RSIVD) in marine and freshwater fishes. Although several diagnostic assays for RSIV have been developed, diagnostic sensitivity (DSe) and specificity (DSp) of real-time polymerase chain reaction (PCR) assays are not yet evaluated. In this study, we developed a TaqMan probe-based real-time PCR method and evaluated its DSe and DSp. To detect RSIV, the probe and primers were designed based on consensus sequences of the major capsid protein (MCP) genes from megalocytiviruses including RSIV, infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primers were shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A 95% limit of detection (LOD_95%) was determined to be 5.3 viral genome copies/µL of plasmid DNA containing the MCP gene from RSIV. The DSe and DSp of the developed real-time PCR assay for field samples (n = 112) were compared with those of conventional PCR assays and found to be 100% and 95.2%, respectively. The quantitative results for SYBR Green and TaqMan probe-based real-time PCR were not significantly different. The TaqMan probe-based real-time PCR assay for RSIV may be used as an appropriate diagnostic tool for qualitative and quantitative analysis.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.47 no.3
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• pp.241-246
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• 2014

Viruses in the genus Megalocytivirus have been subdivided into four subgroups. Among these subgroups 2 and 4, represented by the red sea bream iridovirus (RBIV) and the olive flounder iridovirus (FLIV), respectively, are non-exotic. subgroups 1 and 3, represented by the red sea bream iridovirus (RSIV) and the infectious spleen and kidney necrosis virus (ISKNV), respectively, have not been detected in Korea and are known as exotic. Shellfish are filter-feeders, and can thus filter and accumulate Megalocytivirus in their digestive glands, allowing us to track viral contamination in surrounding aquatic environment. In this study, we developed a high-resolution melting (HRM) analysis to differentiate among subgroups of Megalocytivirus accumulated in shellfish, and confirmed the convenience and efficiency of this method. More than two subgroups of Megalocytivirus were found in the digestive gland of a single shellfish. We classified all Megalocytivirus viruses from shellfish in Korea into subgroups 2 and 4, although proportions of subgroups were different among regions. Compared to nucleotide sequencing analysis, HRM analysis is a simple and rapid method for differentiating of Megalocytivirus subgroups.

• Journal of fish pathology
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• v.32 no.2
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• pp.49-57
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• 2019

In this study, we collected 39 megalocytiviruses isolated from diseased fish in Korea from 2012 to 2018. Major capsid protein (MCP) gene, a part of vascular endothelial growth factor (VEGF) gene and histidine triad motif-like protein (HIT) genes of Megalocytivirus were targeted for PCR amplification and analysis of those DNA nucleotide sequences. Korean strains revealed two genotypes (red sea bream iridovirus and turbot reddish body iridovirus types) based on the phylogeny of MCP gene. The red sea bream iridovirus type (RSIV-type) megalocytiviruses were divided into RSIV-subgroup 1 and 2. From the phylogenetic analysis of the VEGF genes, a genotypic variant of RSIV-type Megalocytivirus was identified. The HIT-like protein gene was detected in RSIVs, but not in TBRIV and ISKNV, suggesting that HIT-like protein gene may be specific in RSIV.