Outbreak of hirame rhabdovirus infection in cultured spotted sea bass Lateolabrax maculatus on the western coast of Korea.

In this study, we determined the cause of a disease outbreak in spotted sea bass, Lateolabrax maculatus reared in culture cages on the western coast of Korea in 2013. The major signs in the diseased fish exhibited were haemorrhaging on the membranes of the abdomen, gastrointestinal organs and opercular gills, as well as an enlarged spleen. No external morphological signs of infection were visible, except for a darkening in colour. No parasites or pathological bacteria were isolated from the diseased fish; however, epithelioma papulosum cyprini (EPC) cells inoculated with tissue homogenates from the diseased fish showed cytopathic effects (CPEs). Virus particles in the EPC cells were bullet-shaped, 185-225 nm long and 70-80 nm wide, characteristic of Rhabdoviridae. Polymerase chain reaction analyses of homogenized tissues from the diseased fish and supernatants of cell cultures with CPEs indicated specific, 553-bp-long fragments corresponding to the matrix protein gene of the hirame rhabdovirus (HIRRV). Phylogenetically, the HIRRV phosphoprotein gene of spotted sea bass was more closely related to phosphoproteins from Chinese and Polish HIRRV strains than from other Korean strains. To our knowledge, this is the first report of HIRRV infection in cultured spotted sea bass.

Characterization of virus distribution in Rock Bream (Oplegnathus fasciatus; Temminck and Schlegel) infected with megalocytivirus.

The distribution of virus-infected cells in the organs of Rock Bream naturally infected with megalocytivirus is reported. Examination of sections of liver, spleen and kidney stained by haematoxylin and eosin (HE) and periodic acid Schiff (PAS) revealed the presence of swollen and degenerate cells having morphology consistent with leucocytes. Many of these cells were shown to contain viral DNA by in-situ hybridization (ISH). Cells containing viral DNA were also found in the connective tissue of other organs in which there was no prominent infiltrate of degenerate leucocytes. Viral DNA was also found in the cytoplasm of leucocytes in blood smears. Transmission electron microscopy (TEM) confirmed the presence of viral particles in the cells within tissue and free within blood. The tissue distribution of virus in this infection is suggested to reflect the infiltration of virus-infected leucocytes.

Sequence variation in the gene encoding the major capsid protein of Korean fish iridoviruses.

Ten iridoviruses were isolated from cultured fish from various regions in Korea; 7 from rock bream, 1 from red sea bream, 1 from sea bass, and 1 from rockfish. The full open-reading frame (ORF) encoding the major capsid protein (MCP) (1362 bp) from ten iridoviruses were sequenced and the nucleotide sequences were phylogenetically analyzed. Phylogenetic analysis revealed that the ten Korean isolates were classified into one cluster. However, their sequences were not identical and, based on the nucleotide sequence variation, they could be further divided into two subgroups. While nine Korean isolates were similar to the Japanese isolate red sea bream iridovirus (RSIV), one isolate was distinct from other iridovirus isolates. These results suggest that a diversity of iridoviruses exist in Korea and that a new variant strain has emerged.

A novel 90-kDa stress protein induced in fish cells by fish rhabdovirus infection.

A 90 kDa cellular protein in a fish cell, CHSE-214, showed increased expression by the infection of infectious hematopoietic necrosis virus (IHNV), heat shock, 2-mercaptoethanol, copper sulfate, and cadmium sulfate, and was detected in various kinds of cells such as human, rat, and mouse cells. The molecular mass of the 90 kDa protein was different from those of the hsp90 and grp94. In addition, all the anti-stress protein MAbs did not react with the 90 kDa protein. Finally, the subcellular distribution of the 90 kDa protein, determined by Western blots of subcellular fractions, was found to be mainly nuclear, both in normal and IHNV-infected CHSE-214 cells. The present results indicate that the 90 kDa protein is a kind of stress protein. However, based on its molecular mass, antigenic characteristics, and subcellular distribution, it is likely that this protein is a novel stress protein that has not been previously described in animal systems, especially in fish systems.

Monoclonal antibodies raised against infectious haematopoietic necrosis virus (IHNV) G protein and a cellular 90 kDa protein neutralize IHNV infection in vitro.

Immune sera were obtained from four rainbow trout that had survived natural infection by infectious haematopoietic necrosis virus (IHNV), and five monoclonal antibodies (MAbs) were prepared against a Korean isolate of IHNV, IHNV-PRT. These immune sera and MAbs were characterized in terms of IHNV-neutralizing properties and reactivity in Western blots with the viral proteins of IHNV-PRT. All five MAbs and four immune sera neutralized IHNV-PRT to various extents. Antibodies in these immune sera recognized two structural proteins of IHNV, G and M1, and one protein with a molecular mass of 90 kDa. Of the five MAbs, three (AB9, AF6 and AG6) recognized the IHNV G protein, and the other two (AB7 and BC2) recognized the 90 kDa protein. The 90 kDa protein was found to be a cellular protein constitutively expressed at low levels in fish cells and expression of this protein was augmented by infection with IHNV and heat shock. MAbs specific to four stress proteins, hsp60, hsp70, hsp90 and grp94, failed to bind to this 90 kDa protein. MAbs AB9 and AB7 reacted fairly broadly with six different IHNV strains. Together, these results indicate that (1) two IHNV proteins, G and M1, and a 90 kDa cellular protein are immunogenic, (2) G and the 90 kDa proteins contain neutralizing epitopes, and (3) the epitopes recognized by MAbs AB9 and AB7 are conserved among the six different IHNV strains.

Comparison of the immunogenicity of recombinant VP2 and VP3 of infectious pancreatic necrosis virus and marine birnavirus.

Recombinant proteins of truncated viral protein-2 (VP2) (aa 79-359) and VP3 of infectious pancreatic necrosis virus (IPNV) and marine birnavirus (MABV) were expressed in E. coli and their immunogenicities in fish were investigated. The recombinant proteins from IPNV were used to immunize rainbow trout and those from MABV to immunize flounder. The sera from the immunized fishes were assayed for antibody by ELISA and a neutralization test. Both the recombinant VP2 and VP3 produced antibodies in fish but the VP3 antibody titers were higher than that of the VP2 of IPNV and MABV. These results indicate that the recombinant VP3 is more immunogenic than the recombinant VP2.

Immune-stimulating properties of polysaccharides from Phellodendri cortex (Hwangbek).

Heteropolysaccarides were isolated from the Korean medicinal plant, Phellodendri cortex (Hwangbek), by hot water and alkali extractions. The extracted polysaccharides were fractionated into eight fractions and they are mainly composed of D-N-acetylglucosamine, D-galactose, D-mannose, and D-glucose. Among the polysaccharide fractions, Fr.-2 showed a potent B-lymphocyte-stimulating activity in a system using polyclonal antibody forming cells in C57BL/6XC3H mice at dosages of 2-10 mg. On the basis of their solubility in aqueous ethanol, four fractions of Fr.-2-1 to Fr.-2-4 were further obtained from the Fr.-2, and Fr.-2-3 was divided into Fr.-2-3-1, 2, 3, and 4 by DEAE cellulose chromatography. The main activity was found in Fr.-2-3-2, which contained 100% (w/w) of carbohydrates and further purified to Fr.-2-3-2-2 by gel filtration chromatography using TSK Gel HW50S. Fr.-2-3-2-2, having a molecular weight of about 230 kDa, showed the highest B-cell-stimulating activity and the half-maximal concentration for B-lymphocyte-stimulating activity was ca. 2.2 microg/ml.