• Journal of fish pathology
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• v.28 no.2
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• pp.113-116
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• 2015

A $0.45-{\mu}m$ membrane filter is generally used to remove bacterial contamination during virus extraction from fish samples. However, the number of fish viruses is drastically reduced after filtration with a $0.45{\mu}m$ filter. In this study, we investigated the effect of filters on virus infectivity titer and the change in virus titer and bacterial number following different centrifugation conditions to determine a suitable procedure for virus extraction from fish samples. $10^{4.05}$ and $10^{5.05}TCID_{50}/ml$ of infectious hematopoietic necrosis virus (IHNV) and $10^{4.05}$ and $10^{4.55}TCID_{50}/ml$ of Oncorhynchus masou virus (OMV) were not detectable after filtration with two types of $0.45-{\mu}m$ filters, except the IHNV titer was reduced by about 10 fold after filter use (company A). No significant difference was found in the virus titer following centrifugation at $880{\times}g$ (30 min) or $3,500{\times}g$ (30 min), whereas IHNV and OMV titers were reduced by about 10 and 10-1000 fold by centrifugation at $14,000{\times}g$ (30 min) and $14,000{\times}g$ (10 and 30 min), respectively. A total of 97.7-99.9% Escherichia coli were eliminated by centrifugation at $880 {\times}g$ (30 min) and $3,500{\times}g$ (30 min). These results show that fish viruses were affected by filtering, even though the effect differed by virus species and filter type. Therefore, centrifugation at $3,500{\times}g$ (30 min) and use of medium with antibiotics may be useful for virus extraction along with a reduction in bacteria.

• Korean Journal of Microbiology
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• v.42 no.4
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• pp.271-276
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• 2006

Previously we demonstrated that virus-inducible stress protein (VISP) is induced in fish cells by the infection of a fish rhabdovirus. In this paper, we investigated the subcellular localization of the VISP and determined the region of VISP responsible for the subcellular localization. The CHSE-214 cells were stained with monoclonal antibody raised against VISP and observed with confocal microscope to detect the endogenous VISP. The results showed that the VISP localizes to the perinuclear region as spots. A plasmid expressing VISP fused to enhanced green fluorescent protein (EGFP) was constructed. The transient expression of full-length VISP fused to EGFP in CHSE-214 cells confirmed the spot formation of the VISP at perinuclear region. To determine the region responsible for the perinuclear localization of the VISP, we constructed a series of deletion mutants and, by using these deletion mutants, we found that C-terminal region of the VISP (aa 612-710) is essential for the perinuclear distribution of VISP and that this region contained nuclear receptor binding motif (691-TLTSLLL-697). Our results suggest that VISP localizes to the perinuclear region and C-terminal regions are important for this localization. Further studies on the role of the perinuclear localization of VISP in IHNV growth mali reveal the novel mechanism of IHNV pathogenecity.

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

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2003.05a
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• pp.313-314
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• 2003

Antiviral DNA vaccine carrying a gene for a major antigenic viral protein have received considerable attention as a new approach in vaccine development. For fish viruses effects of DNA vaccine encoding viral G gene of infectious hematopoietic necrosis virus(IHNV) and viral hemorrhagic septicemia virus (VHSV)have been demonst.ated previously(Lapatra et al., 2001) Hirame rhabdovirus (HIRRV) causes hemorragic disease on flounder. (omitted)

• Fisheries and Aquatic Sciences
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• v.8 no.3
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• pp.185-187
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• 2005

We tested the in vivo ability of a DNA chip to detect virus-specific genes from virus-infected olive flounder Paralichthys olivaceus and rainbow trout Oncorhynchus mykiss. Target cDNA was obtained from total RNA of virus infected cell lines by reverse transcription (RT) and was labeled with fluorescent dye (Cy5-dUTP). The results show the successful detection of infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicaemia virus (VHSV) genes in the virus-infected fishes.

• Journal of Aquaculture
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• v.10 no.2
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• pp.171-178
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• 1997

Infectious pancreatic necrosis virus (IPNY) is an economically important fish pathogen since it causes the high-mortality disease in early stage of hatchery-reared fishes. In order to develop a rapid, sensitive and highly specific detection method for IPNV, reverse transcription-polymerase chain reaction (RT-PCR) was carried out using the oligonucleotide primers selected from the sequence of VP2, a major capsid polypertide of IPNV. As little as 40ng of purified IPNV dsRNA was detected by RT-PCR amplification, but no amplification products were obtained when nucleic acid genomes from other fish pathogens such as IHNV were used as RT-PCR templates. in situ RT-PCR methods are useful for the rapid and sensitive identification of IPNV.

• Journal of fish pathology
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• v.7 no.1
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• pp.7-11
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• 1994

The viral hemorrhagic septicemia of rainbow trout, Salmo gairdneri was studied. The hematocrit values of diseased fish were very low than those of normal fish. And, the GOT and GPT values of serum of diseased fish were a little high than those of normal fish. Cytopathic effect of viral agents(serum and organs of diseased fish) was observed with inverted phase contrast microscopy. After 24hrs infection, the cells were showed the cytopathic effect.

• Fisheries and Aquatic Sciences
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• v.8 no.3
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• pp.122-127
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• 2005

A DNA chip that rapidly and accurately detects the viral genes in rhabdovirus-infected fish was developed. The N, Ml, and G proteins of three rhabdovirus strains, infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), and flounder rhabdovirus (HIRRV), were selected for use as probes. The sequences of the corresponding genes were obtained, and probes were prepared by PCR using specific primer sets. The specificity of the probes was confirmed by cross PCR. The prepared probes were spotted on poly-L-lysine- or aminosilane-coated glass slides and hybridized with target DNA under several different conditions in order to determine the optimal hybridization temperature, glass-slide coating, and target cDNA concentration.

• Journal of fish pathology
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• v.33 no.2
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• pp.163-169
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• 2020

Snakehead rhabdovirus (SHRV) is different from other fish novirhabdoviruses such as viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and hirame rhabdovirus (HIRRV) in that it replicates at high temperatures. Therefore, the delivery of foreign proteins to fish living at high water temperature would be possible by using recombinant SHRVs. In the present study, to evaluate the possible use of SHRV as a vehicle for foreign proteins delivery, we generated a recombinant SHRV that contains an enhanced-GFP (eGFP) gene between nucleoprotein (N) and phosphoprotein (P) genes (rSHRV-A-eGFP), and another recombinant SHRV expressing two heterologous genes by inserting an eGFP gene between N and P genes, and mCherry gene between P and M genes (rSHRV-AeGFP-BmCherry). Epithelioma papulosum cyprini (EPC) cells infected with the recombinant SHRVs showed strong fluorescence(s), suggesting the possible availability of recombinant SHRVs for the development of combined vaccines by expressing multiple foreign antigens.

• Journal of fish pathology
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• v.11 no.2
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• pp.129-136
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• 1998

In March 1997, a new rhabdovirus was isolated from moribund cultured Japanese flounder Paralichthys olivaceus in sea water tank and cage culture systems in Kyung-Nam and Chun-Nam province, Korea. At temperature $15^{\circ}C$ the virus replicated and induced cytopathic effects (CPE), which progressed to eventual cytolysis, in susceptible cell lines, including RTG-2 and EPC. The CHES-214 cell line was refractory. Virus particles were bullet-shaped and measured $70nm{\times}100$ to 150 nm in size. The isolate was sensitive to pH 3, to diethyl ether, and to heat ($50^{\circ}C$ 5 min, $60^{\circ}C$ 1 min). Viral replication was not inhibited by $10^{-4}$ M 5-iododeoxyuridine. Virus infectivity was reduced by anti-HRV (8401-H) rabbit serum, but can not reduced by antisera against infectious hematopoietic necrosis virus (IHNV), chum salmon reovirus (CSV), retrovirus of salmonid (RVS) and infectious pancreatic necrosis virus (IPNV). HRV virus antigen was detected by fluorescent antibody test (FAT) in the cytoplasm of infected EPC cell. Purified isolates virions were composed of: polymerase (L), glycoprotein (G), nucleoprotein (N) and 2 matrix proteins (M1 and M2). Based upon their relative mobilities, the estimated molecular weights of the proteins were: L, 160 kDa; G, 55 kDa; N, 45 kDa; M1, 26 kDa; and M2, 22 kDa.