• Korean Journal of Fisheries and Aquatic Sciences
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• v.52 no.6
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• pp.644-649
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• 2019

Killed vaccines, developed by inactivation with formalin, have been investigated for many fish viruses. In this study, the inactivation of viral hemorrhagic septicemia virus (VHSV) by formalin was investigated based on the infectivity titer. When viral cell culture supernatants were used, the infectivity titer decreased 1,000-fold at 1 d after treatment with 0.1% (v/v) formalin, but was below the detection limit at 7 and 14 d. Moreover, neither the N nor G gene were detectable by RT-PCR immediately after formalin treatment. In western blot analysis, N protein was not detected by rabbit antiserum against VHSV KR-9225 from 2 d after formalin treatment. On the other hand, when we used a virus that was purified and concentrated ~100 times, the infectivity titer was maintained at 10^6.05 TCID₅₀/mL, even at 14 d after formalin treatment, and no change in the viral structural proteins was observed. This study provides important data on the production and use of formalin-inactivated vaccines.

• 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.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.50 no.5
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• pp.621-623
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• 2017

Mariculture of rainbow trout Onchorhynchus mykiss has been initiated in or around olive flounder Paralichthys olivaceus farms, where viral hemorrhagic septicemia virus (VHSV) is often detected in some fish. In the present study, we investigated VHSV infection in seawater-reared rainbow trout because VHSV has never been detected in salmonids in Korea. A total of 104 adult fish were tested for the presence of VHSV by reverse transcription-polymerase chain reaction, followed by virus isolation with the fathead minnow caudal trunk cell line. Cytopathic effects were observed in two samples but the virus was identified as infectious hematopoietic necrosis virus. Thus, VHSV was not isolated from seawater-reared rainbow trout.

• 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.

• Journal of fish pathology
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• v.25 no.2
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• pp.117-121
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• 2012

The pathogenicity of viral hemorrhagic septicemia virus (VHSV) from olive flounder Paralichthys olivaceus was investigated with masu salmon Oncorhynchus masou fry. The cumulative mortality of fish challenged with FYeosu05 isolate at $10^{6.5}$ $TCID_{50}$/fish was 60%. No mortality was observed in fish challenged with the isolates at $10^{5.5}$ $TCID_{50}$/fish and in mock-challenged fish. The affected fish showed darkening of the body, expanded abdomen, pale gills and enlarged spleen. VHSV from $10^{6.3}$ to $10^{7.8}$ $TCID_{50}$/g-tissue was re-isolated from the dead fish. These results suggest that the VHSV from olive flounder is pathogenic to masu salmon fry, although with low virulence.

• Development and Reproduction
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• v.27 no.2
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• pp.67-75
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• 2023

Polymeric immunoglobulin receptor (pIgR) mediates the transfer of polymeric immunoglobulin to protect organisms and is one of the most important mucosal effectors. In this study, the developmental stage- and tissue-specific expression of pIgR were observed before virus inoculation in olive flounder. pIgR was gradually expressed until the formation of immune tissue, exhibiting high expression in the late juvenile period; thereafter, pIgR expression gradually decreased and exhibited high expression in the spleen and skin. Moreover, pIgR expression after viral hemorrhagic septicemia virus infection was high in the kidney and spleen tissues at high density and low at low density. The results of this study can provide a basis for future studies on breeding density, virus expression, and immune system studies in fish.

• Journal of fish pathology
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• v.31 no.1
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• pp.49-55
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• 2018

Tendency of viral hemorrhagic septicemia virus (VHSV) detection from 2001 to 2016 in Korea was studied based on 15 reported cases. Since the VHSV was first detected from cultured olive flounder (Paralichthys olivaceus) in Pohang in 2001, it has been continuously reported from olive flounder farms in various regions of the Korean coastal area. So far, the virus has been detected from 2 farmed fishes, 12 wild marine fishes and 2 marine bivalves. All the 67 isolates were belong to VHSV genotype IVa. The predisposing factor analysis from different olive flounder farms revealed that the VHSV were highly detected from the juveniles under 40 g in body weight, in the temperature range from 9.5 to $17^{\circ}C$ and during the period of March to June. Therefore, we recommend that farmers, need to exercise caution against VHSV infection in Spring.

• Journal of fish pathology
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• v.32 no.2
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• pp.59-67
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• 2019

We developed and subsequently characterized mouse antibodies (MAbs) against viral hemorrhagic septicemia virus (VHSV, genotype IVa), the causative agent of VHS. Five hybridoma clones secreting MAbs against VHSV were established. The MAbs recognized the glycoprotein (MAbs 2C10, 18H4, 23H6, and 30B7) and nucleocapsid protein (15E10) of VHSV by western blot analysis. All five MAbs reacted with VHSV-infected cells and tissue homogenates of VHSV-infected olive flounder (Paralichthys olivaceus) by western blot analysis. Whereas, no reactivity was observed in normal cells and tissue homogenates of normal olive flounder. Moreover, these MAbs reacted with VHSV, but did not react with other fish viruses (infectious hematopoietic necrosis virus, hirame rhabdovirus, spring viraemia of carp virus, infectious pancreatic necrosis virus, marine birnavirus, and nervous necrosis virus) by enzyme linked immunosorbent assay (ELISA). These results indicate that the MAbs are specific to VHSV and can be of value in VHSV detection.

• Development and Reproduction
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• v.23 no.3
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• pp.231-238
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• 2019

Interleukin-8 (IL-8) is an inflammatory cytokine that plays an important role in the inflammatory response through the activation of neutrophil cells. The expression of IL-8 was investigated in early developmental stages of the olive flounder and in tissues of 8-month-old individuals. The expression of IL-8 increased after the initiation of the immune system rather than at the early stage of development, and high expression was observed in the gills and spleen, the organs associated with immunity and metabolism. In addition, IL-8 expression after infection by viral hemorrhagic septicemia virus significantly increased in the fin, gill, muscles, and spleen. These results suggest that IL-8 is closely related to inflammation and immune regulation in the immune response of the olive flounder and may be used as a basis for studies on the immune systems of other fish.

• Fisheries and Aquatic Sciences
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• v.25 no.6
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• pp.320-326
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• 2022

Most of the emerging diseases that threaten humans are caused by RNA viruses which are extremely mutable during evolution. The fish RNA virus, viral hemorrhagic septicemia virus (VHSV) can infect a broad range of aquatic animal hosts, but the transmissibility of VHSV to mammals has not been thoroughly investigated. Therefore, our study aimed to investigate the potential adverse effects of VHSV in mammals. Briefly, the survival of VHSV was determined using only minimum essential media (MEM-2) and mammalian SNU-1411 and hepa-1c1c7s cells at 15℃ and 37℃. Mice (Mus musculus, 27.3 ± 1.9 g) were intravenously injected with VHSV (2.37E+05 TCID₅₀·mice^-1) in triplicate. Clinical signs and survival rates were examined at 14 days post-challenge, and infection was confirmed in the surviving mice. The 50% tissue culture infective dose (TCID₅₀) and polymerase chain reaction analysis were used to determine viral titers and the infection rate, respectively. The titer of VHSV suspended in MEM-2 at 15℃ was reduced by only one log after 8 days, whereas the virus maintained at 37℃ was inactivated 8 days post-inoculation (dpi). There were no recognizable cytopathic effects in either SNU-1411 or hepa-1c1c7s cells inoculated with VHSV at 15℃ and 37℃. VHSV in those cell lines at 37℃ was rapidly decreased and eventually inactivated at 12 dpi, whereas virus at 15℃ remained at low concentrations without replication. In vivo experiment showed that there were no signs of disease, mortality, or infection in VHSV-infected mice. The results of this study indicate that it is highly unlikely that VHSV can infect mammals including humans.