• Title, Summary, Keyword: Megalocytivirus

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Characterization of Asymptomatic Megalocytivirus Infection in farmed Rock Fish (Sebastes schlegeli) in Korea (양식 조피볼락 (Sebastes schlegeli)에서 megalocytivirus의 무증상적 감염과 특성 분석)

  • KWON, Woo-Ju;KIM, Young-Chul;YOON, Min-Ji;JEONG, Hyun-Do
    • Journal of Fisheries and Marine Sciences Education
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    • v.27 no.4
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    • pp.1184-1193
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    • 2015
  • Monitoring for megalocytivirus infection was conducted for ten months from March to December in 2013 in 15 aquatic farms culturing, red sea bream, rock bream, rock fish and black sea bream around Tongyoung coastal area in Korea, to assess spatial and temporal variability of detection prevalence, and to explore possible links with seawater temperature. In nested-PCR targeted major capsid protein (MCP) gene, asymptomatic megalocytivirus infection was detected in the externally healthy farmed fish with a significant prevalence in range from 0 to 58.3% for ten months. Higher prevalence of megalocytivirus (46.7% - 57.1%) was observed in high water temperature season from September to November than that in other months with lower prevalence of 0.0% to 20.0%. Even though an acute infection of megalocytivirus was occurred in rock bream (positive in the first PCR) with high mortality in one of fifteen farms, there was no expansion or transmission of the disease to the rock fish and red sea bream culturing in net cage just proximal to the rock bream cage in which disease outbreaked. Nucleotide sequence analysis of the cloned MCP gene isolated asymptomatically infected rock fish revealed that the megalocytivirus in this study was clustered together with the rock bream iridovirus (RBIV) under the subgroup II of the genus megalocytivirus (Iridoviridae), which is known to be the major megalocytivirus strain in Korea. The typical histopathological signs were not found in the spleen of rock fish asymptomatically infected by megalocytivirus. Experimental infection of rock bream with the spleen homogenate of the rock fish infected asymptomatically did not induce any mortality unlike the homogenate of infected rock bream with hih mortlity. However, these results may suggest that the asymptomatic infection of megalocytivirus in other fish species can be a potential risk threatening aquaculture industries as a transmission factor of megalocytivirus to susceptible fish species, especially rock bream.

Detection and Genetic Differentiation of Megalocytiviruses in Shellfish, via High-Resolution Melting (HRM) Analysis (HRM 분석법을 이용한 패류 내 Megalocytiviruses의 검출과 유전적 분석)

  • Kim, Kwang Il;Jin, Ji Woong;Kim, Young Chul;Jeong, Hyun Do
    • 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.

In situ Hybridization of a Megalocytivirus Using Nucleic Acid Probes against ATPase and the Major Capsid Protein of Rock Bream Iridovirus

  • Lee, Nam-Sil;Do, Jeong-Wan;Jung, Sung-Ju;Park, Mi-Seon;Kim, Jin-Woo;Kim, Yi-Cheong
    • Fisheries and aquatic sciences
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    • v.9 no.4
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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.

Quantitative analysis of the clinical signs in marine fish induced by Megalocytivirus infection (Megalocytivirus 감염 해산 어류에서 나타나는 임상증상의 정량적 변화 분석)

  • Jin, Ji-Woong;Cho, Hye-Jin;Kim, Kwang-Il;Jeong, Joon-Bum;Park, Gyeong-Hyun;Jeong, Hyun-Do
    • Journal of fish pathology
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    • v.24 no.2
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    • pp.53-64
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    • 2011
  • In quantitative studies of clinical signs, rock bream of adults and juveniles infected with Megalocytivirus IVS-1 isolated from rock bream (Oplegnathus fasciatus) in Korea showed average $4.49{\pm}1.13$ and $4.85{\pm}1.06$ of spleen index respectively. In challenge experiments, Megalocytivirus IVS-1 induced 100% cumulative mortality in both adult and juvenile rock bream. However we found 60% cumulative mortality in juvenile red sea bream (Pagrus major) even after 30 days of injection, which contradicted with the results of other laboratories. Interestingly, IVS-1 infected red sea bream of the same juvenile size with rock bream showed lower spleen index compared to that of rock bream. In real-time PCR, there was continuous increasing of the numbers of viral copies ($2.03{\times}10^7$ copies/mg) in the spleen of juvenile rock bream infected, which were different from those in adult rock bream showing plateau level after reaching to the peak level. Moreover, enlarged cell numbers in the infected spleen were also increased continuously in the juvenile but not in adult of rock bream, even decreased after reaching to peak level. Consequently, significant differences in clinical signs: cumulative mortality. spleen index and viral copy number were found between rock bream and red sea bream, but not between adult and juvenile rock bream. Certainly quantitative expression of clinical sign as in this study may be a way to compare the progression of megalocitiviral disease more accurately in different species or physiological conditions.

Cloning and Expression of the Cyclooxygenase-2 gene in the Rock bream, Oplegnathusfasciatus (돌돔, Oplegnathus fasciatus의 Cyclooxygenase-2 유전자의 cloning 및 발현분석)

  • Jin, Ji Woong;Kim, Do Hyung;Kim, Young Chul;Jeong, Hyun Do
    • Journal of fish pathology
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    • v.26 no.1
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    • pp.19-30
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    • 2013
  • Megalocytivirus is a major fish pathogen in marine aquaculture of Asian countries including Korea. Despite of many species affected by this pathogen, little is known interaction between megalocytivirus and the fish immune system. One of the cyclooxygenase isoforms, named COX-2, is playing an important role in immune regulation, and distinct from COX-1 isoform of constitutive activity. COX-2 enzyme is induced by various inflammatory signals, including injection of lipopolysaccharide or infection by pathogenic agents. We cloned COX-2 gene in rock bream using degenerated primers designed from reported sequences of other fish species in PCR followed with 5'- and 3'-end RACE-PCR. The full length of cDNA of rbCOX2 (rock bream COX-2) gene are 2655 bp and that translates into 609 amino acids. The rbCOX-2 genomic organization are found to span 10 exons separated by 9 introns. We also studied if the experimental infection of rock bream with megalocytivirus could affect the expression of COX-2 gene. When injected with LPS, expression of the COX-2 gene was reached peak level at 1 day post injection and showed 13.10 fold increased level compared with that of control. While, when injected with megalocytivirus, we were not able to find significantly increased COX-2 gene expression different from that of control. Cloned and analyzed COX-2 gene in rock bream will help to understand defence mechanisms in fish after viral infection and will also support the development of the measures for treatment and prevention of viral infection.

Genetic relatedness of Megalocytivirus from diseased fishes in Korea (국내 어류에서 분리된 Megalocytivirus의 유전형 분류 및 상관관계 분석)

  • Lee, Eun Sun;Cho, Miyoung;Min, Eun Young;Jung, Sung Hee;Kim, Kwang Il
    • 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.

Detection of Fish Pathogenic Viruses in Seawater Using Negatively Charged Membranes (Negatively Charged Membrane을 이용한 해수 중 어류질병바이러스의 검출)

  • Jee, Bo Young;Kim, Kwang Il;Lee, Soon Jeong;Kim, Ki Hong;Jin, Ji Woong;Jeong, Hyun Do
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.46 no.1
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    • pp.46-52
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    • 2013
  • After an outbreak of viral disease in an aquafarm, release of virus (es) from infected fish into environmental seawater has been suspected. In the present study, we utilized a negatively charged membrane (HA type) as an efficient method for concentration and detection of fish pathogenic viruses, specifically, megalocytivirus and viral hemorrhagic septicemia virus (VHSV) present in field-collected seawater samples or inoculated into seawater artificially. Positively charged viruses adsorbed onto the negatively charged membrane and were eluted with 1 mM NaOH (pH 10.5) following rinsing with 0.5 mM $H_2SO_4$ (pH 3.0). Megalocytivirus and VHSV particles isolated using anegatively charged HA membrane from seawater inoculated with each virus at a concentration of 10 viral particles/mL were of sufficient quantity to show positive results in atwo-step PCR (or RT two-step PCR); however, despite it being negatively charged, a cellulose acetate (CA) membraneshowed negative results. In quantitative PCR, the detection limits of the HA membrane for megalocytivirus and VHSV in seawater were 1.20E+00 viral particles/mL and 1.22E+01 viralparticles/mL, respectively. The calculated mean recovery yields from 1 L seawater spiked with known concentrations of megalocytivirus and VHSV particles were 28.11% and 23.00%, respectively. The concentrate of a 1-L sample of culturing seawater from the aquatank of flounder suffering from VHSV showed clear positive results in PCR when isolated with an HA, but not a CA, membrane. Thus, viral isolation using an HA membrane is a practical and reliable method for detection of fish pathogenic viruses in seawater.

Detection of Megalocytivirus in shellfish using PCR with various DNA extraction methods (다양한 PCR용 DNA 추출법에 의한 패류 내 Megalocytivirus의 검출)

  • Kim, Jin-Woo;Cho, Mi-Young;Jin, Ji-Woong;Kim, Ki-Hong;Jeong, Hyun-Do;Kim, Kwang-Il
    • Journal of fish pathology
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    • v.24 no.2
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    • pp.65-73
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    • 2011
  • In analysis of DNA viruses from the contaminated shellfish using PCR, preparation method of template DNA is an important factor to get enough copy number of viruses. In this study, we evaluated the efficiency of PCR template of Megalocytivirus (sT50mg-D) DNA obtained from 50 mg digestive gland homogenate of oyster using commercial method, and compared with that obtained from 5 g of the same tissues (T5g-D) after PEG precipitation procedures of virus. Both templates DNA suspended in the same volume of distilled water showed positive results by primary PCR with 35 cycles, and the presence of Megalocytivirus was confirmed in oysters collected from cultured farms in Korea. Moreover, PCR with sT50mg-D allowed us to discriminate the contaminated oyster individually, that can not be done in PCR with T5g-D prepared from the mixture of three different individual oyster to get 5 g digestive gland homogenate. In quantitative analysis with real time PCR, Megalocytivirus concentrations in 50 ${\mu}l$ templates prepared using 0.5~50 mg of one positive sample were appeared in the range 6.14E+00~1.2E+02/${\mu}l$. We were not able to get positive result using template DNA contained less than 6.14E+00 copies. Consequently, 2-step PCR performed with DNA extracts from oyster homogenate of small amount (sT50mg-D) i) was enough to detect the contaminated Megalocytivirus in shellfish, ii) allowed us to do the analysis for individual shellfish rather than mixture of several shellfish and iii) showed the presence of Megalocytivirus in oyster from Korea.

The First Report of a Megalocytivirus Infection in Farmed Starry Flounder, Platichthys stellatus, in Korea

  • Won, Kyoung-Mi;Cho, Mi Young;Park, Myoung Ae;Jee, Bo Young;Myeong, Jeong-In;Kim, Jin Woo
    • Fisheries and aquatic sciences
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    • v.16 no.2
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    • pp.93-99
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    • 2013
  • In 2009, a systemic megalocytivirus infection associated with high mortality was detected for the first time in cultured starry flounder Platichthys stellatus in Korea. Diseased starry flounder had pale bodies and gill coloring and enlarged spleens. Histopathological examinations revealed basophilic enlarged cells in various organs of diseased starry flounder. Polymerase chain reaction (PCR) was performed on tissue samples using three published primer sets developed for the red sea bream iridovirus. PCR products were detected for all primer sets, except 1-F/1-R, which are registered by the World Organization for Animal Health (OIE). The part of the gene corresponding to the full open reading frame encoding the viral major capsid protein (MCP) was amplified by PCR. PCR products of approximately 1,581 bp were cloned, and the nucleotide sequences were analyzed phylogenetically. The MCP gene of the starry flounder iridovirus, designated SFIV0909, was identical to that of the turbot reddish body iridovirus (AB166788).