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Two viral hemorrhagic septicemia virus (VHSV) isolates, VHSV-KR-CJA and VHSV-KR-YGH, were isolated from viral hemorrhagic septicemia disease outbreaks in flounder farms in South Korea. The VHSV-KR-CJA isolate was isolated from a flounder farm with high mortality (80%), while the VHSV-KR-YGH isolate was isolated from a flounder farm with low mortality (15%), suggesting that these isolates differ in virulence. The virulence of these isolates was evaluated in juvenile flounder via intraperitoneal injection. Consistent with their virulence in the field, mortality data revealed that the VHSV-KR-CJA isolate was highly pathogenic (cumulative mortality of 80%), while the VHSV-KR-YGH isolate was less pathogenic in flounder (cumulative mortality of 20%). To characterize the genotypes of these viruses, the full open reading frames (ORFs) encoding nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, nonstructural viral protein NV, and polymerase L of these viruses were sequenced and analyzed. Sequence analysis revealed that both isolates are genetically very similar (identical amino acid sequences for P, M, NV, and L and >99.7 and 99.8% amino acid sequence identity for N and G, respectively). Phylogenetic analysis indicated that both of these viruses belong to the Genotype IVa group, suggesting that they originated from a common ancestral virus. The low pathogenicity VHSV strain may potentially evolve to become a more pathogenic strain through only a few nucleotide substitutions. Further functional analyses of mutations in VHSV genes are necessary to identify factors that determine VHSV pathogenicity in flounder.

The cDNA encoding of a complement factor D/adipsin and kallikrein-like serine protease, designated PoDAK, was isolated from the olive flounder Paralichthys olivaceus. PoDAK cDNA encodes a polypeptide with 277 amino acids containing conserved catalytic triad residues of serine proteases. The amino acid sequence of PoDAK showed high similarity to the kallikrein-like protein of medaka, mammalian adipsin/complement factor D and tissue kallikrein homolog, KT-14 of trout, complement factor D of zebrafish, and shared 31.6-36.8% homology with complement factor D/adipsin known from other species, including mammals. Phylogenetic analysis revealed that PoDAK clustered with the kallikrein-like protein of medaka and mammalian adipsin/complement factor D and tissue kallikrein homolog KT-14 of trout. The expression of PoDAK mRNA was high in the gills and heart, moderate in muscle, liver, intestine, stomach, kidney, and spleen of healthy flounder, and increased in the kidney, liver, and spleen of flounder challenged by the viral hemorrhagic septicemia virus (VHSV) or Streptococcus iniae. In situ hybridization confirmed that PoDAK mRNA is localized in the kidney and heart of individuals infected with VHSV. Further investigations are needed to clarify the function of PoDAK in vivo and in vitro., (2009 Elsevier Ltd.)

Eukaryotic translation initiation factor 5A (eIF5A) is the only protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)-lysine). We isolated a 1385-bp eIF5A cDNA containing an open reading frame (ORF) of 468 bp, which encodes a protein of 155 amino acids with a conserved hypusine modification site, from the olive flounder Paralichthys olivaceus. Pairwise alignments revealed that flounder eIF5A had a high sequence identity with those of other known species including mammals. Real-time RT-PCR analysis showed the expression of eIF5A mRNA was constitutively detected in various tissues of healthy flounder. In HINAE cells or flounder kidney infected with the viral hemorrhagic septicemia virus (VHSV), the expression of eIF5A mRNA was slightly increased before cells showed cytopathic effects and then decreased when cells showed cytopathic effects. Treatment of N-guanyl-1,7-diaminoheptane (GC-7), a potent inhibitor of eIF5A hypusination, inhibited the expression of VHSV G protein in a dose-dependent manner suggesting a potential role for eIF5A and its hypusination in viral protein expression.

In 2003, 13 isolates of iridovirus were obtained from cultured flounders Paralichthys olivaceus during epizootics in Korea. The full open reading frames (ORFs) encoding the major capsid protein (MCP) (1362 bp) from the 13 flounder iridoviruses (FLIVs) were sequenced and the deduced amino acid sequences were phylogenetically analyzed. Phylogenetic analysis of the MCP revealed that all 13 FLIVs were the same species as rock bream iridovirus (RBIV), red sea bream iridovirus (RSIV), and infectious spleen and kidney necrosis virus (ISKNV), and were grouped into an unknown genus which was different from the 2 genera known to infect fish, Ranavirus and Lymphocystivirus. This is the first report on the isolation and phylogenetic analysis of the iridovirus of unknown genus from flounders during epizootics.