• Journal of Microbiology and Biotechnology
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• 제15권4호
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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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• 제24권11호
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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.

• 한국수산과학회지
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• 제47권3호
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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.

• 한국어병학회지
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• 제27권2호
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• pp.85-89
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• 2014

Water temperature is a key environmental factor controlling the epizootics of viral diseases in fish. High water temperature is associated with the rapid spread of rock bream iridovirus (RBIV) disease and with high mortality of RBIV infected fish. Although protection of fish against iridoviral disease by active immunization has been reported, little information is available concerning whether fish survived from an epizootic of iridoviral disease can naturally acquire resistance against the viral disease. In the present study, we have demonstrated that juvenile rock bream, which survived from a natural epizootic of RBIV, acquired resistance against recurrence or reinfection of RBIV, and this resistance was established during the subsequent low water temperature period. Furthermore, the possible involvement of the adaptive humoral immune response in the resistance of the juvenile rock bream was suggested by in vivo neutralization experiment.

• 한국식품위생안전성학회지
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• 제27권3호
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• pp.285-289
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• 2012

In this study, the veridical efficacy of an aquatic disinfectant tablet composed to calcium hypochlorite against red sea bream iridovirus (RBIV). A veridical efficacy was determined with the viability of RBIV contacted with the disinfectant in viral stock cultured in fat head minnow cell line. An aquatic disinfectant tablet and RBIV were reacted on the distilled water (DW), hard water (HW) or organic matter suspension (OM) condition. On DW and HW condition, RBIV was inactivated with 25,000 fold dilutions of an aquatic disinfectant tablet. With the investigation of the antiviral effect of the disinfectant on OM condition, RBIV was inactivated on 22,000 fold dilutions of an aquatic disinfectant tablet. As an aquatic disinfectant tablet possesses veridical efficacy against RBIV, the disinfectant solution can be used to limit the spread of cultured marine fish viral disease.

• Fisheries and Aquatic Sciences
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• 제9권4호
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• pp.146-152
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• 2006

Systemic infections of maricultured fishes by Megalocytivirus species have occurred over a broad area in South Korea, causing extensive economic loss. We developed digoxigenin-labeled nucleic acid probes against the 230-bp ATPase and 311-bp major capsid protein (MCP) of rock bream Oplegnathus fasciatus iridovirus (RBIV) using polymerase chain reaction, and an in situ hybridization (ISH) method to detect Megalocytivirus in formalin-fixed tissues of mariculture species (rock bream, sea bass, and olive flounder). ISH-positive cells were abundant in the hematopoietic and connective tissues of various organs, while brain tissue showed little or no signal. The ISH procedure can become an important diagnostic tool in complement with histopathological methods, and advances epidemiological studies on the origin and distribution of Megalocytivirus in mariculture.

• 한국어병학회지
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• 제23권3호
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• pp.273-280
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• 2010

To determine whether rock bream Oplegnathus fasciatus can be protected from rock bream iridovirus (RBIV) infection by intramuscular injection of long double-stranded RNAs (dsRNAs), we compared protective effect of virus-specific dsRNAs corresponding to major capsid protein (MCP), ORF 084, ORF 086 genes, and virus non-specific green fluorescent protein (GFP) gene. Furthermore, to determine whether the non-specific type I interferon (IFN) response was associated with protective effect, we estimated the activation of type I IFN response in fish using expression level of IFN inducible Mx gene as a marker. As a result, mortality of fish injected with dsRNAs and challenged with RBIV was delayed for a few days when comparing with PBS injected control group. However, virus-specific dsRNA injected groups exhibited no significant differences in survival period when compared to the GFP dsRNA injected group. Semi-quantitative analysis indicated that the degree of antiviral response via type I IFN response is supposedly equal among dsRNA injected fish. These results suggest that type I IFN response rather than sequence-specific RNA interference might involve in the lengthened survival period of fish injected with virus-specific dsRNAs.

• 한국어병학회지
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• 제32권1호
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• pp.1-8
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• 2019

Rock bream iridovirus (RBIV) is a member of the Megalocytivirus genus that causes severe mortality to rock bream (Oplegnathus fasciatus) with characteristic clinical signs of spleen enlargement. In this study, we assessed spleen size and RBIV copy number patterns in RBIV-infected rock bream to determine lethal and safe levels of virus copy number/spleen index that may define disease progress. We found that rock bream infected with RBIV ($1.1{\times}10^7virus\;copy\;number/100{\mu}l$) and held at 29, 26, 23 or $20^{\circ}C$ exhibited significantly higher levels of spleen size compared to $17^{\circ}C$. In dead condition (100% mortality at $20{\sim}29^{\circ}C$), the spleen index ($spleen\;weight/fish\;weight{\times}100$) and virus copy number were 3.00~5.38 and $10^6{\sim}10^8/{\mu}l$, respectively. Conversely, in survived condition (0% mortality at $17^{\circ}C$), spleen index and virus copy number was as low as not-infected control ($0.34{\sim}1.22/10^0{\sim}10^1/{\mu}l$, respectively). These findings suggest that spleen index can be an indicator of disease severity of RBIV disease.

• 한국어병학회지
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• 제34권1호
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• pp.9-15
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• 2021

Rock bream (Oplegnathus fasciatus) is a highly valued aquaculture species in Korea. However, the aquaculture industry suffers huge economic losses due to rock bream iridovirus (RBIV) infection in summer. The objective of this study was to determine genetic diversity and relationships of DNAs isolated from two groups of rock bream after RBIV infection using five microsatellite (MS) markers. The first group of fish died early and the second group of fish died later after RBIV infection. In this experiment, 90 fish (5.1±1.0 cm and 4.1±1.3 g) were injected with 50 μl of RBIV (10⁴ TCID⁵⁰/ml) and maintained at 26℃ for 15 days. Genomic DNAs were extracted from fins of 20 fish that died earlier or later after RBIV infection. These DNAs were subjected to genotyping using five MS markers (CA-03, CA3-05, CA3-06, CA-10, and CA3-36). Of these markers, CA3-05 (early death group), CA3-06 (late death group), and CA3-36 (both early and late death groups) showed different alleles distribution rates. In-depth studies are needed to provide valuable information for selecting RBIV-resistant fish. In conclusion, microsatellite marker distribution pattern differences between early- and late- death groups of rock bream after RBIV infection showing different RBIV susceptibilities were determined using MS markers and genotyping. Results of this study suggest that MS markers could be used to facilitate the selection of RBIV resistant rock bream.

• 한국어병학회지
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• 제36권2호
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• pp.191-211
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• 2023

Rock bream iridovirus (RBIV) causes high mortality and economic losses in rock bream (Oplegnathus fasciatus) aquaculture industry in Korea. Although, the immune responses of rock bream under RBIV infection have been studied, there is not much information at the different stages of infection (initial, middle and recovery). Gene expression profiling of rock bream under different RBIV infection stages was investigated using a microarray approaches. In total, 5699 and 6557 genes were significantly up- or down-regulated over 2-fold, respectively, upon RBIV infection. These genes were grouped into categories such as innate immune responses, adaptive immune responses, complements, lectin, antibacterial molecule, stress responses, DNA/RNA binding, energy metabolism, transport and cell cycle. Interestingly, hemoglobins (α and β) appears to be important during pathogenesis; it is highly up-regulated at the initial stage and is gradually decreased when the pathogen most likely multiplying and fish begin to die at the middle or later stage. Expression levels were re-elevated at the recovery stage of infection. Among up-regulated genes, interferon-related genes were found to be responsive in most stages of RBIV infection. Moreover, X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) expression was high, whereas expression of apoptosis-relate genes were low. In addition, stress responses were highly induced in the virus infection. The cDNA microarray data were validated using quantative real-time PCR. Our results provide novel inslights into the broad immune responses triggered by RBIV at different infection stages.