• Journal of fish pathology
• /
• v.30 no.2
• /
• pp.149-154
• /
• 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 Microbiology
• /
• v.34 no.3
• /
• pp.263-263
• /
• 1996

Infection of fish cells with IHNV resulted in gradual increase in cytosolic free $Ca^{2+}$ concentration $([Ca^{2+}]_i)$ in CHSE, gradual decrease in $[Ca^{2+}]_i$ in FHM, and no significant change in RTG cells. The degree of $[Ca^{2+}]_i$ increase or decrease was dependent on the amount of infectious virus, and these $[Ca^{2+}]_i$ variations were maximal at 16 hours after virus infection (p. i.) in both cell lines. When the fish cells were infected with inactivated IHNV, evident variation in $[Ca^{2+}]_i$ was not observed. Thus, infectivity of IHNV appears to correlate with changes in $[Ca^{2+}]_i$ in virus-infected cells. These IHNV-induced $[Ca^{2+}]_i$ changes were partially blocked by cycloheximide, but not affected by cordycepin. It seems to be that virus-induced $Ca^{2+}$ variations were more related with protein synthesis than RNA synthesis. Various $Ca^{2+}$ related drugs were used in search for the mechanisms of the $[Ca^{2+}]_i$, changes following IHNV infection of CHSE cells. Decreasing extracellular $Ca^{2+}$ concentration or blocking $Ca^{2+}$ influx from extracellular media inhibited the IHNV-induced increase in $[Ca^{2+}]_i$, in CHSE cells. Similar results were obtained with intracellular $Ca^{2+}$ blockers. Thus it is suggested that both the extracellular and the intracellular $Ca^{2+}$ sources are important in IHNV-induced $[Ca^{2+}]_i$ increase in CHSE cells.

• Journal of fish pathology
• /
• v.26 no.3
• /
• pp.283-287
• /
• 2013

The susceptibility of marine medaka, Oryzias dancena to fish pathogenic viruses (infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV), hirame rhabdovirus (HIRRV), infectious hematopoietic necrosis virus (IHNV), and lymphocystis disease virus (LCDV)) was investigated. The cumulative mortalities of fish immersed with IPNV (experimental condition: $15^{\circ}C$ sea water (SW)), VHSV ($15^{\circ}C$ SW), HIRRV ($15^{\circ}C$ fresh water (FW)) were 30%, 40% and 60%, respectively. In the fish immersed with IPNV ($15^{\circ}C$ FW, $18^{\circ}C$ FW and SW), VHSV ($15^{\circ}C$ FW, $18^{\circ}C$ FW and SW), HIRRV ($15^{\circ}C$ SW), IHNV ($15^{\circ}C$ FW and SW), LCDV ($15^{\circ}C$ FW and SW, $18^{\circ}C$ FW and SW), and mock-challenged group, mortality rate was less than 10%. IPNV, VHSV and HIRRV were re-isolated from the dead fish. These results suggest that marine medaka is susceptible to IPNV, VHSV and HIRRV, although their susceptibility depends on the environmental conditions.

• Development and Reproduction
• /
• v.24 no.4
• /
• pp.277-285
• /
• 2020

The disease-causing koi herpes virus (KHV), also known as cyprinid herpesvirus-3 (CyHV3), causes mass mortality of koi and carp. Koi (Cyprinus carpio) is a host for KHV, one of 12 virus species in the Alloherpesviridae family. We examined the effects of KHV disease koi (KK), and on koi×red common carp (KR) and red common carp×koi (RK) cross, using a virus challenge test. The infected fish had clinical signs that included gill necrosis and skin lesions. The RK and KR were highly more resistant (cumulative mortality: RK; 6% and KR; 8%) to KHV infection than KK fish (cumulative mortality: 28%). KHV DNA was confirmed in the tissues of all dead fish in groups by use of polymerase chain reaction (PCR), and the presence of the KHV protein in kidney was confirmed by immunohistochemistry. Histological analysis showed severe gill lesions and fusion of the lamellae in KK fish, but less severe damage in RK fish. In immunohistochemistry analysis, the KHV protein localized in the cytoplasm of infected kidney cells of KK, but the cross groups had lower levels of KHV antigen. Our data indicate that the cross groups had increased resistance to KHV disease.

• Journal of fish pathology
• /
• v.13 no.1
• /
• pp.61-66
• /
• 2000

A method was developed for concentrating fish pathogenic virus from sea water using membrane ultrafiltration system and centricon. The method consists of passing large volumes (Ca. 20 liter) of sea water through ultrafiltration (PAN) filter followed by cross-flow filtration method and centrifugation use the centricon (Plus-20). This procedure permitted the processing of 20 liter of sea water which resulted in a 20,000-fold reduction in the volume of water and greater than 90% recovery of the seeded MABV.

• Journal of fish pathology
• /
• v.22 no.2
• /
• pp.173-177
• /
• 2009

Lymphocystis disease virus (LCDV) was detected from olive flounder Paralichthys olivaceus, painted glass fish Chanda baculis, gourami Trichogaster leeri and rockfish Sebastes schlegeli, and proteins of the viruses were compared. The major capsid protein (MCP) gene-specific primer sets successfully amplified approximately 1300 bp nucleotides from the olive flounder and 600 bp nucleotides from painted glass fish, gourami and rockfish isolates, respectively. In western blotting analysis using anti-LCDV mouse polyclonal serum, major antigenic proteins had 21, 26, 45, 50, 80, 110 and 120 kDa in olive flounder, 26, 47 and 80 kDa in painted glass fish, 26, 46, 80 and 92 kDa in gourami, 26, 44, 49, 80 and 105 in rockfish, respectively. All the marine and freshwater isolates showed only common antigens of approximately 26 kDa and 80 kDa. These results suggest that antigenic protein profiles of LCDVs may vary depending upon fish species.

• Journal of fish pathology
• /
• v.9 no.2
• /
• pp.177-183
• /
• 1996

A new RNA virus isolated from abnormally swimming behavior has caused mortalities in salmonid fish (Oh et al., 1995 a), A reverse transcriptase (RTase) activity of the virus was determined by using poly r(A) : oligo d(T) as templete : primer. This RTase activity was associated with virus particles of buoyant density of 1.16 g/ml. The virus particles in sucrose fractions were enveloped and were about 85 nm diameter with central electron-dense core. The brain and kidney samples of artificially infected fish showed RTase activity. Virus particle associated proteins about 120, 80, 65, 61, 48, 42, 35, 30, 25, 19, and 15 kDa were observed when examined by polyacrylamide gel electrophoresis analysis. This study showed the presence of a new retrovirus in salmonid fish, which tentatively called RVS (Retrovirus of salmonid).

• Journal of fish pathology
• /
• v.32 no.1
• /
• pp.1-8
• /
• 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.

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

• Journal of fish pathology
• /
• v.25 no.3
• /
• pp.257-262
• /
• 2012

A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was evaluated to monitor infectious hematopoietic necrosis virus (IHNV) from artificially infected rainbow trout Oncorhynchus mykiss. The cumulative mortalities of fish challenged with IHNV at $10^{6.5}\;TCID_{50}$/fish, $10^{5.5}\;TCID_{50}$/fish and $10^{4.5}\;TCID_{50}$/fish were 40%, 0% and 0%, respectively. Dead fish and survivors at 16 and 28 d post-challenge in each group were employed for IHNV detection by RT-LAMP assay and virus isolation using BF-2 cells. IHNV from $10^{4.3}$ to $10^{6.8}\;TCID_{50}/ml$ was isolated from all the dead fish and also detected in all of the examined dead fish by RT-LAMP assay. In survivors at 16 d, 60% (3/5 fish, $10^{2.8}-10^{5.05}\;TCID_{50}/ml$), 20% (1/5 fish, $10^{1.05}\;TCID_{50}/ml$) and 60% (3/5 fish, $10^{1.05}-10^{4.8}\;TCID_{50}/ml$) were found to be IHNV-positive by virus isolation in fish challenged with IHNV at $10^{6.5}\;TCID_{50}$/fish, $10^{5.5}\;TCID_{50}$/fish and $10^{4.5}\;TCID_{50}$/fish, respectively, while 20% (1/5 fish), 0% (0/5 fish) and 20% (1/5 fish) were IHNV-positive by RT-LAMP assay. No IHNV was detected in the survivors at 28 d and control fish. These results indicate that the RT-LAMP assay is useful for detection of IHNV in diseased fish although it is not enough to monitor virus in IHNV-survivors.