Seok-Ryel Kim

Genetic variation and geographic distribution of megalocytiviruses.

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae have caused mass mortalities in marine and freshwater fish in Asian countries. In this study, partial major capsid protein (MCP) gene of seven Japanese and six Korean megalocytiviruses was sequenced and compared with the known megalocytiviruses to evaluate genetic variation and geographic distribution of the viruses. Comparison of MCP gene nucleotide sequences revealed sequence identity of 92.8% or greater among these 48 isolates. A phylogenetic tree clearly revealed three clusters: genotype I including nine Japanese isolates, thirteen Korean isolates, one Chinese isolates, one Thailand isolate and one South China Sea isolate; genotype II including five freshwater fish isolates in Southeast Asian countries and Australia; and the remaining genotype III mainly consisted of flatfish isolate in Korea and China. This suggests that viruses belonging to the genotype I widely distribute among various fish species in many Asian countries. Conversely, the epidemic viruses belonged to genotype II and III are may be still locally spreading and constrained in their prevalence to the limited host fish species, i.e., genotype II viruses mainly distribute in Southeast Asian countries, whereas genotype III viruses distribute in flatfish species in Korea and China.

Detection of hepatitis a virus from oyster by nested PCR using efficient extraction and concentration method

The molecular methods using polymerase chain reaction have been proposed as useful tools for the identification of viral pathogens in food and water. However, the PCR-based methods are highly dependent on the methods of virus concentration and nucleic acid purification due to the low sensitivity of PCR in the presence of PCR inhibitors. We developed TPTT [tris elution buffer-PEG-TRIzol-poly(dT) magnetic bead] protocol in order to detect hepatitis A virus (HAV) inoculated in oyster digestive glands. The detection limit of HAV precipitated with zirconium hydroxide was 105 fold less sensitive in a nested PCR than that precipitated the HAV supernatant twice with PEG/NaCl (16% polyethylene glycol 6,000, 0.525 M NaCl) in a 1:2 (v/v) ratio, which provided an efficient detection of 0.0148 PFU/g from approximately 0.05 g of oyster homogenate. This method is efficient for potential use in the detection of HAV from shellfish and is more sensitive than most currently published tests.

Lymphocystis disease virus persists in the epidermal tissues of olive flounder, Paralichthys olivaceus (Temminch & Schlegel), at low temperatures.

Olive flounder artificially infected with lymphocystis disease virus (LCDV) were reared at 10, 20 and 30 degrees C for 60 days, to compare LCD-incidence. In the fish reared at 20 degrees C, lymphocystis cells appeared on the skin and fins at 35 days post-challenge, and the cumulative LCD-incidence was 80% at 60 days. High levels of LCDV, with a mean polymerase chain reaction (PCR) titre of 10(6) PCR-U mg(-1) tissue, were detected in the fins and skin of LCD-affected fish at 20 degrees C, but were not detected in the spleen, kidney, brain and intestinal tissues of these fish. No LCD clinical signs were observed in the fish reared at 10 degrees C and 30 degrees C; however, a low level of LCDV (10(3) PCR-U mg(-1) tissue) was detected in the fins and skin of these fish. By increasing the rearing temperature from 10 to 20 degrees C, lymphocystis clusters appeared on the skin and fins of the fish with no previous LCD clinical signs within 33 days after the temperature change. It was shown that permissive cells for LCDV infection exist in the epidermis of olive flounder. At low temperatures, small amounts of LCDV were able to persist over a period extended for a further 45 days in the fish epidermis, even though the fish showed no LCD clinical signs. The optimum growth temperature of LCDV is near 20 degrees C.

Distribution of marine birnavirus in cultured olive flounder Paralichthys olivaceus in Korea

Surveys of marine birnavirus (MABV) were undertaken in cultured olive flounder Paralichthys olivaceus from the south and west coastal areas and Jeju in Korea during the period January 1999 to April 2007. MABV was detected in all seasons from the fry, juveniles and adult fish from the areas examined. Evident cytopathic effects of the virus including rounding and cell lysis were observed in chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells, but not in fathead minnow (FHM) and epithelial papilloma of carp (EPC) cells. Nucleotide sequences of the VP2/NS junction region of the Korean isolates showed 97.8%∼100% similarity, and they belonged to the same genogroup. Cross neutralization tests with serotype-specific rabbit antisera against MABV strains exhibited a close antigenic relationships between strains, and were distinct from infectious pancreatic necrosis virus (IPNV) strains. Coinfection of MABV with bacteria (Streptococcus iniae, Vibrio spp.) and viruses (nervous necrosis virus, lymphocystis disease virus, viral hemorrhagic septicemia virus) was observed.

A mu class glutathione S-transferase from Manila clam Ruditapes philippinarum (RpGSTμ): cloning, mRNA expression, and conjugation assays.

Glutathione S-transferases (GSTs) are enzymes that catalyze xenobiotic metabolism in the phase II detoxification process. GSTs have a potential for use as indicators or biomarkers to assess the presence of organic and inorganic contaminants in aquatic environments. In this study, a full-length cDNA of a mu (μ) class GST (RpGSTμ) was identified from Manila clam (Ruditapes philippinarum) and biochemically characterized. The 1356 bp of the cDNA included an open reading frame of 651 bp encoding a polypeptide of 217 amino acid residues with a molecular mass of 25.04 kDa and an estimated pI of 6.34. Sequence analysis revealed that the RpGSTμ possessed several characteristic features of μ class GSTs, such as a thioredoxin-like N-terminal domain containing binding sites for glutathione (GSH), a C-terminal domain containing substrate binding sites, and a μ loop. The recombinant RpGSTμ (rRpGSTμ) protein exhibited GSH-conjugating catalytic activity towards several substrates, and significantly strong activity was detected against 4-nitrophenethyl bromide (5.77 ± 0.55) and 1-chloro-2,4-dinitrobenzene (CDNB, 3.19 ± 0.05). Kinetic analysis as a function of GSH and CDNB concentrations revealed relatively low Km values of 1.03 ± 0.46 mM and 0.56 ± 0.20 mM, respectively, thereby indicating a GSH-conjugation attributed with high rates. The optimum pH and temperature for the catalytic activity of the rRpGSTμ protein were 7.7 and 37°C, respectively. The effect of two inhibitors, Cibacron blue and hematin, on the activity of rRpGSTμ was evaluated and the IC50 values of 0.65 μM and 9 μM, respectively, were obtained. While RpGSTμ transcripts were highly expressed in gills and hemocytes, a significant elevation in mRNA levels was detected in these tissues after lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly I:C) and live bacterial (Vibrio tapetis) challenges. These findings collectively suggest that RpGSTμ functions as a potent detoxifier of xenobiotic toxicants present in the aquatic environment, and that its mRNA expression could be modulated by pathogenic stress signal(s).

Molecular aspects, genomic arrangement and immune responsive mRNA expression profiles of two CXC chemokine receptor homologs (CXCR1 and CXCR2) from rock bream, Oplegnathus fasciatus.

The CXCR1 and CXCR2 are the prototypical receptors and are the only known receptors for mammalian ELR+ (Glu-Leu-Arg) CXC chemokines, including CXCL8 (interleukin 8). These receptors transduce the ELR+ chemokine signals and operate the downstream signaling pathways in inflammation and innate immunity. In this study, we report the identification and characterization of CXCR1 and CXCR2 genes from rock bream fish (OfCXCR1 and OfCXCR2) at the molecular level. The cDNA and genomic DNA sequences of the OfCXCR1 and OfCXCR2 were identified from a transcriptome library and a custom-constructed BAC library, respectively. Both OfCXCR genes consisted of two exons, separated by an intron. The 5-flanking regions of OfCXCR genes possessed multiple putative transcription factor binding sites related to immune response. The coding sequences of OfCXCR1 and OfCXCR2 encoded putative peptides of 355 and 360 amino acids (aa), respectively. The deduced aa sequences of OfCXCR1 and OfCXCR2 comprised of a G-protein coupled receptors (GPCR) family 1 profile with a GPCR signature and a DRY motif. In addition, seven conserved transmembrane regions were predicted in both OfCXCRs. While our multiple alignment study revealed the functionally significant conserved elements of the OfCXCR1 and OfCXCR2, phylogeny analyses further confirmed their position in teleost sub clade, in which they manifested an evolutionary relatedness with other fish counterparts. Based on comparative analyses, teleost CXC chemokine receptors appear to be distinct from their non-fish orthologs in terms of evolution (both CXCR1 and CXCR2) and genomic organization (CXCR2). Quantitative real-time PCR (qPCR) detected the transcripts of OfCXCR1 and OfCXCR2 in eleven examined tissues, with higher levels in head kidney, kidney and spleen highlighting their crucial importance in immunity. In vitro stimulation of peripheral blood leukocytes (PBLs) with concanavalin A (Con A) resulted in modulation of OfCXCR2 transcription, but not that of OfCXCR1. In addition, the magnitude of the OfCXCR1 and OfCXCR2 transcripts in head kidney and spleen was differentially increased after the in vivo administration of immune stimulants, LPS and poly I:C and in the infection models injected with rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. These lines of evidence suggest that these receptors may play an important role(s) in immune responsive signaling during pathogenesis of rock bream.

Expression profile of cystatin B ortholog from Manila clam (Ruditapes philippinarum) in host pathology with respect to its structural and functional properties

Cystatins are a well-characterized group of cysteine protease inhibitors, which play crucial roles in physiology and immunity. In the present study, an invertebrate ortholog of cystatin B was identified in Manila clam (Ruditapes philippinarum) (RpCytB) and characterized at the molecular level, demonstrating its inhibitory activity against the well-known cysteine protease, papain. The complete coding sequence of RpCytB (297 bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11 kDa and a theoretical isoelectric point of 5.9. The derived peptide was found to harbor typical features of cystatin proteins, including the Q-X-V-X-G motif, which was identified as QLVAG in RpCytB. Phylogenetic analysis of RpCytB revealed close evolutionary relationships with its invertebrate counterparts, especially those from mollusks. Recombinant RpCytB (rRpCytB) was overexpressed as a fusion with maltose binding protein (MBP) in Escherichia coli BL21 (DE3) cells. Purified rRpCytB fusion protein exhibited a detectable inhibitory activity against papain, while the control MBP showed an almost constant negligible activity. While quantitative RT-PCR detected ubiquitous RpCytB expression in all tissues examined, the expressions in hemocytes and gills were relatively higher. Upon in vivo immune challenge with lipopolysaccharide (LPS), the expression of RpCytB in gills and hemocytes was down-regulated. Similar challenges with poly I:C and intact Vibrio tapetis bacteria revealed a complicated transcriptional regulation, wherein mRNA expression levels fluctuated over time of exposure. Moreover, a precise induction of RpCytB expression after bacterial infection was detected in gills by in situ hybridization. Collectively, our findings in this study indicate that RpCytB expression is sensitive to host pathological conditions and may contribute cysteine protease inhibitory activity to modulate the immune response.

A Novel Screening Method of Dextran Binding Antibody Using Phage Display Libraries

Sephadextrade bead agarose electrophoresis (SBAE) was used to enrich the higher dextran binding phages. Phage collection obtained after the 2nd round of SBAE was 3.5 fold higher color intensity than that of phage collections obtained after the 1st round. DNA sequencing of phage collection (SBAE-2R) confirmed that human origin antibody was present. The PCR products of lambda, k light chains and heavy chain from phage collection (SBAE-2R) were approximately 420 bp, 550 bp and 600 bp.