• Fisheries and Aquatic Sciences
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• v.1 no.2
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• pp.187-191
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• 1998

Chemotherapy can not be applied for the control of fish viral diseases because viruses depend on host machinery for their replication. Although new control strategies including vaccination are under development, avoidance of virus introduction by rapid and correct diagnosis is the best way of fish viral disease control. Although observation of virus particles with an electron microscope is an easy method for virus detection, it take a few days for the sample preparation. In order to shorten the sample preparation time, microwave radiation was applied in the procedure. With this method, 15 seconds was enough for fixation of virus infected fish samples or cultured cells inoculated with infectious hematopoietic necrosis virus, which takes 2-4 hours with routine methods. Also four minutes was enough for polymerization of embedding resin which takes 24-48 hours with routine methods. Samples prepared with microwave were good enough for direct electron microscopic observation and immunogold labeling assay.

• Journal of fish pathology
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• v.21 no.3
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• pp.181-188
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• 2008

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to detect and identify four fish viruses, fish iridovirus, viral hemorrhagic septicaemia virus (VHSV), viral nervous necrosis virus (VNNV), hirame rhabdovirus (HRV). Four viruses were detected by PCR with each specific primers. Identification of iridovirus was achieved by digesting the PCR amplified fragment with a restriction enzyme ApaⅠ. It was possible to distinguish positive from false positive PCR amplicons of VHSV by RFLP of PstⅠ or HindⅢ restriction enzymes. VNNV was identified using RFLP of BamHⅠrestriction enzyme and HRV was identified by XbaⅠ restriction enzyme. This approach can be used for more rapid, simple and specific diagnosis of fish viral diseases.

• Journal of fish pathology
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• v.30 no.2
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• pp.149-154
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• 2017

Mouse monoclonal antibodies (MAbs) were produced by using viral hemorrhagic septicemia virus (VHSV, genotype IVa) as an immunogen, isolated from diseased olive flounder (Paralichthys olivaceus). Four hybridoma clones secreting MAbs against VHSV were established. The MAbs were recognized the nucleoprotein (MAb 4), phosphoprotein (MAb 1) and matrix protein (MAbs 2 and 3) of VHSV by western blot analysis. Among them, the MAbs 1 and 4 strongly reacted with the VHSV-infected FHM cells, but not normal FHM cells. In enzyme linked immunosorbent assay, the four MAbs reacted with the VHSV, but not different six fish viruses (infectious hematopoietic necrosis virus, hirame rhabdovirus, spring viraemia of carp virus, infectious pancreatic necrosis virus, marine birnavirus and nervous necrosis virus). These results indicate that the MAbs are useful for diagnosis of VHSV infection.

• Journal of fish pathology
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• v.36 no.2
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• pp.389-394
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• 2023

Infectious hematopoietic necrosis virus (IHNV) is s significant viral pathogen affecting cultured rainbow trout (Oncorhynchus mykiss) in Korea. In this study, five monoclonal antibodies (mAbs) (IHNV-1, 2, 3, 4, and 5) were produced using purified IHNV. Reactivities of these mAbs were analyzed by western blot (WB), enzyme-linked immunosorbent assay (ELISA), and indirect fluorescent antibody test (IFAT). These mAbs recognized glycoprotein (69 kDa, IHNV-1), nucleocapsid protein (39 kDa, IHNV-3, 4, and 5), or phosphoprotein (27 kDa, IHNV-2) of IHNV by WB analysis. ELISA results indicated that these five mAbs were specific to IHNV without showing any cross-reactivity against other fish viruses (hirame rhabdovirus, infectious pancreatic necrosis virus, and viral hemorrhagic septicemia virus). IFAT demonstrated specific fluorescence signals of IHNV-infected epithelioma papulosum cyprini (EPC) cells, whereas no reactivity of normal EPC cells was observed. These mAbs can be very useful for immuno-diagnosis of IHNV infection.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.50 no.5
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• pp.547-552
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• 2017

The viral hemorrhagic septicemia virus (VHSV) has an extensive host range, and infects farmed and wild fish inhabiting both freshwater and marine ecosystems. Enzyme-linked immunosorbent assay (ELISA) is highly useful in diagnosing viral hemorrhagic septicemia. However, ELISA shows high, non-specific background reaction with fish antibodies. In this study, we optimized the antigen and antibody concentrations used for detecting specific antibodies in VHSV-infected olive flounder to reduce non-specific binding, and improve the sensitivity of ELISA. The results suggested that OD (optical Density) values were valid when ELISA was performed with $0.1{\mu}g/well$ of virus, involving blocking with blocking buffer (Roth, Roti-Block), 1:300-1:600 dilution with flounder antisera, and 1:1000 dilution with anti-flounder IgM and HRP-conjugated goat anti-mouse IgG for detecting the VHSV antibody in flounder sera. Furthermore, 11 different VHSV strains isolated in Korea from 2012 to 2016 were used to infect the fish. The results showed no correlation between viral pathogenicity and antibody production. This research is a basic study on the application of antibody detection in the diagnosis of viral hemorrhagic septicemia in the olive flounder.

• Korean Journal of Environmental Biology
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• v.39 no.3
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• pp.259-265
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• 2021

A diagnostic test for viral hemorrhagic septicemia virus (VHSV), which infects more than 80 species of freshwater and marine fish at home and abroad, causing mass mortality, was conducted to provide quantitative data on the amount of virus expression in various tissues of flounder in chronological order. The tissues were collected in chronological order after the intraperitoneal injection of 3.0E+07 tissue culture infective dose₅₀ (TCID₅₀) per 0.1mL per fish of VHSV to randomly selected flounder. As a result of relative quantification through real-time PCR, the highest levels of virus expression were found in the spleen, kidney, gill, and liver on day 5. This study proved that the spleen was an appropriate site for the final diagnosis of VHSV in the early stages of infection and will provide important information for the diagnosis of legal infectious diseases in Korea.

• Journal of fish pathology
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• v.35 no.2
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• pp.241-246
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• 2022

White spot syndrome virus (WSSV) is a prevalent and virulent pathogen affecting cultured whiteleg shrimp (Litopenaeus vannamei) in Korea. In this study, seven monoclonal antibodies (mAbs) (10A12, 16C3, 17G4, 21G5, 22C4, 23B6 and 24G6) were produced by using purified WSSV. The reactivity of these mAbs was analysed by Western blot (WB), indirect immunofluorescence (IIF), and lateral flow immunochromatographic assay (LFIA). WB analysis demonstrated that three mAbs (17G4, 22C4, and 23B6) reacted specifically to VP28 with an approximate molecular weight of 24 kDa, mAb 16C3 reacted with approximately 17 kDa. IIF analysis demonstrated specific fluorescence signals on gill tissues of WSSV-infected shrimp, with five mAbs (10A12, 16C3, 22C4, 23B6, and 24G6), pleopods from WSSV-infected shrimp were used for LFIA, where, two mAbs (21G5 and 22C4) exhibited positive reaction. In conclusion, it can be inferred that the mAbs usage and specificity depends on the nature of assay used for diagnosis.

• Journal of fish pathology
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• v.35 no.1
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• pp.129-133
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• 2022

The full genome sequence of a Korean white spot syndrome virus (WSSV, isolate: WSSV-GoC18) is presented here. We obtained a total of 12,320,554 reads with 291,172 bases, 170 gene, and 170 coding DNA sequence, which were assembled in 1 contig. Phylogenetic analysis revealed that the WSSV-GoC18 was closely related to Chinese isolate (WSSV-PC) and distinctly different with previously reported a Korean isolate (WSSV K-LV1). The complete genome sequence of WSSV isolates will be of great help in molecular epidemiological studies, contributing to molecular diagnosis and disease prevention in shrimp aquaculture.

• Journal of fish pathology
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• v.9 no.2
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• pp.169-176
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• 1996

An indirect double antibody enzyme-linked immunosorbent assay (ELISA) was developed for rapid detection of a new virus isolated from abnormally swimming salmonid fish, RVS (Retrovirus of salmonid). Results using brain tissue homogenates, and infected cell cultures are described. The sensitivity of the methods is $10^{2.6}$ $TCID_{50}/100{\mu}l$ of the examined cell culture fluid. The specificity was confirmed by the ELISA inhibition test and virological examinations. Viral antigen could be detected in artificially infected fish tissue homogenates. The assay will allow the diagnosis of RVS-infected fish within a day.

• Journal of fish pathology
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• v.10 no.2
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• pp.87-95
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• 1997

Baculovirus associated with white spot syndrome (WSBV) is the causative agent of a disease with high mortalities and causes severe damage to shrimp cultures. In this study, we analyzed a recombinant clone (E3) obtained from a viral genomic library to characterize the causative agent in diseased shrimp Penaeus chinensis with white spot syndrome. According to the analysis of nucleotide sequence of E3, this clone did not showed considerable sequence homology with those of other known viruses, including baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), indicating that WSBV is a novel virus causing a serious disease in P. chinensis. Based on the sequence of E3 clone, a pair of PCR primers was designed. After 30 cycles of amplification, a specific product of the expected size was detected only if the total nucleic acids extracted from the diseased shrimp was used as a template DNA, suggesting that this method can be used to diagnose the virus infection in diseased shrimp.