Myoung-Ae Park

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

Summary.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 manganese superoxide dismutase with potent antioxidant activity identified from Oplegnathus fasciatus: Genomic structure and transcriptional characterization

In this study, we describe the identification and characterization of manganese superoxide dismutase, an important antioxidant enzyme acting as the chief reactive oxygen species (ROS) scavenger, from rock bream Oplegnathus fasciatus (Of-mMnSOD) at genomic- and transcriptional-levels as well as the biological activity of recombinant protein. The Of-mMnSOD protein portrayed distinct MnSOD family features including signature motifs, metal association sites and the typical active site topology. It was also predicted to be localized in mitochondrial matrix. The Of-mMnSOD had a quinquepartite genome organization encompassing five exons interrupted by four introns. Comparison of its sequence and gene structure with that of other lineages emphasized its strong conservation among different vertebrates. The Of-mMnSOD was ubiquitously transcribed in different rock bream tissues with higher levels in blood cells and metabolically active tissues. Transcription of Of-mMnSOD was kinetically modulated in response to investigational challenges using mitogens (lipopolysaccharide and poly I:C) and live-pathogens (Edwardsiella tarda and rock bream irido virus) in blood cells and liver tissue. The purified recombinant Of-mMnSOD possessed potential antioxidant capacity and actively survived over a range of pH (7.5-11) and temperature (15-40 °C) conditions. Collectively, findings of this study suggest that Of-mMnSOD combats against oxidative stress and cellular damages induced by mitogen/pathogen-mediated inflammation, by detoxifying harmful ROS (O(2)(●-)) in rock bream.

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.

The toxic effects of ammonia exposure on antioxidant and immune responses in Rockfish, Sebastes schlegelii during thermal stress.

Rockfish, Sebastes schlegelii (mean weight 14.53 ± 1.14 cm, and mean weight 38.36 ± 3.45 g) were exposed for 4 weeks (2 weeks and 4 weeks) with the different levels of ammonia in the concentrations of 0, 0.1, 0.5, 1.0mg/L at 19 and 24°C. The ammonia exposure induced significant alterations in antioxidant responses. The activities of SOD, CAT, and GST were considerably increased by the ammonia exposure depending on water temperature, whereas the GSH level was notably decreased after 2 and 4 weeks. In the stress indicators, the cortisol and HSP 70 were significantly elevated by the exposure to ammonia depending on water temperature. In innate immune responses, the phagocytosis and lysozyme activity were notably decreased by ammonia exposure depending on water temperature after 2 and 4 weeks. The results suggest that ammonia exposure depending on water temperature can induce the considerable alterations in antioxidant responses, stress, and immune inhibition.

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

Summary.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 re-combinant VP2.

Three complement component 1q genes from rock bream, Oplegnathus fasciatus: genome characterization and potential role in immune response against bacterial and viral infections.

Complement component 1q (C1q) is a subcomponent of the C1 complex and the key protein that recognizes and binds to a broad range of immune and non-immune ligands to initiate the classical complement pathway. In the present study, we identified and characterized three novel C1q family members from rock bream, Oplegnathus fasciatus. The full-length cDNAs of C1q A-like (RbC1qAL), C1q B-like (RbC1qBL), and C1q C-like (RbC1qCL) consist of 780, 720 and 726 bp of nucleotide sequence encoding polypeptides of 260, 240 and 242 amino acids, respectively. All three RbC1qs possess a leading signal peptide and collagen-like region(s) (CLRs) in the N-terminus, and a C1q domain at the C-terminus. The C1q characteristic Gly-X-Y repeats are present in all three RbC1qs, while the CLR-associated sequence that enhances phagocytic activity is present in RbC1qAL ((49)GEKGEP(54)) and RbC1qCL ((70)GEKGEP(75)). Moreover, the coding region was distributed across six exons in RbCqAL and RbC1qCL, but only five exons in RbC1qBL. Phylogenetic analysis revealed that the three RbC1qs tightly cluster with the fish clade. All three RbC1qs are most highly expressed in the spleen and liver, as indicated by qPCR tissue profiling. In addition, all three are transcriptionally responsive to immune challenge, with liver expression being significantly up-regulated in the early phase of infection with intact, live bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus) and in the late phase of exposure to purified endotoxin (lipopolysaccharide). These data collectively suggest that the RbC1qs may play defense roles as an innate immune response to protect the rock bream from bacterial and viral infections.

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).

Identification of regulators of the early stage of viral hemorrhagic septicemia virus infection during curcumin treatment

The effect of curcumin pretreatment (15-240 μM) in fathead minnow cells infected with viral hemorrhagic septicemia virus (VHSV) was evaluated. Cell viability, apoptosis and viral copy number were analyzed using Cell Counting Kit-8 assay, Annexin V staining, and reverse transcription-PCR, respectively. Pretreatment with 120 μM curcumin showed an increase in viability (>90% of mock) of VHSV-infected cells and reduction in the copy number (0.2-log reduction in VHSV N gene expression), reactive oxygen species and apoptosis in the cells without cytotoxic effects. To understand the mechanisms underlaying the antiviral effects of curcumin pretreatment, a comparative proteomic analysis was performed in four samples (M, mock; C, curcumin-treated; V, VHSV-infected; and CV, curcumin-treated VHSV-infected) in triplicate. In total, 185 proteins were detected. The analysis showed that three proteins, including heat shock cognate 71 (HSC71), actin, alpha cardiac muscle (ACTC1) and elongation factor 1 (EEF1) were differentially expressed between V and CV samples. Network analysis performed by Ingenuity Pathways Analysis (IPA) showed that HSC71 was the primary protein interacting with fibronectin (FN) 1, actins (ACTB, ACTG, F-actin) and gelsolin (GSN) in both V and CV samples and thus is a strong target candidate for the protection from VHSV infection at the viral entry stage. Our proteomics data suggest that curcumin pretreatment inhibits entry of VHSV in cells by downregulating FN1 or upregulating F-actin. For both proteins, HSC71 acts as a binding protein that modulates their functions. Furthermore, consistent with the effect of a heat shock protein inhibitor (KNK437), curcumin downregulated HSC71 expression with increasing viability of VHSV-infected cells and inhibited VHSV replication, suggesting that the downregulation of HSC71 could be responsible for the antiviral activity of curcumin. In conclusion, this study indicates that the suppression of viral entry by rearrangement of the F-actin/G-actin ratio via downregulating HSC71 is a plausible mechanism by which curcumin pretreatment controls the early stages of VHSV infection.

Expression analysis and biological activity of moronecidin from rock bream, Oplegnathus fasciatus.

The piscidin-family, one of antimicrobial peptides (AMPs) mainly distributed in fish, is crucial effectors of fish innate immune response. Piscidin-family typically has broad-spectrum antimicrobial activity and the ability to modulate the immune response. In this study, we identified moronecidin (Rbmoro) included in piscidin-family from rock bream and investigated its gene expression using quantitative real-time PCR and biological activity (including antimicrobial and cytotoxic activity). The coding region of Rbmoro was 204 bp encoding 67 amino acid residues. Tertiary structure prediction of Rbmoro showed an amphipathic α-helical structure. Rbmoro gene was widely expressed in different tissues of healthy fish. Additionally, Rbmoro gene expression was induced in all tested tissues after infection with Edwardsiella tarda, Streptococcus iniae and red seabream iridovirus. We synthesized mature peptide of Rbmoro based on amino acid sequence of its AMP 12 domain, and the synthetic peptide appeared broad-spectrum antimicrobial activity to various bacteria. However, the synthetic peptide has weak haemolytic activity against fish erythrocytes. These results suggest that Rbmoro might play an important role in innate immune response of rock bream.

Piscidin: Antimicrobial peptide of rock bream, Oplegnathus fasciatus

The piscidin family consists of antimicrobial peptides (AMPs) that are mainly found in fish and are crucial effectors of fish innate immune responses. The piscidin family typically has broad-spectrum antimicrobial activity and can modulate immune responses. In this study, we cloned rock bream piscidin (Rbpisc) and investigated its gene expression and biological activity (including antimicrobial and cytotoxic activities). The coding region of Rbpisc consisted of 213 base pairs (bp) encoding 70 amino acid residues. The tertiary structure predicted for Rbpisc includes an amphipathic helix-loop-helix structure. The Rbpisc gene was highly expressed in the gills of healthy fish. The gene expression of Rbpisc increased in the gills after pathogen infection, while the expression was down-regulated in other tissues. A synthetic peptide based on the AMP 12 domain amino acid sequence of Rbpisc appeared to have broad-spectrum antimicrobial activity against various bacteria. However, the synthetic peptide exhibited weak haemolytic activity against fish erythrocytes. These results suggest that Rbpisc might play an important role in the innate immune responses of rock bream.