Ji Woong Jin

Analysis of proinflammatory gene expression by RBIV infection in rock bream, Oplegnathus faciatus.

Early induction of proinflammatory cytokines is known to regulate the later immune responses to inhibit the progress of infectious diseases. In this study, proinflammatory cytokine gene expression has been studied in immune tissues to understand the early immune response induced by megalocytivirus in rock bream (Oplegnathus faciatus). For this, we have cloned interleukin (IL)-1β and IL-8 gene and performed the phylogenetic and structural analysis. Also the constitutive gene expressions of IL-1β and IL-8 were assessed in 12 organs and found to be the highest expression in tail fin and liver, respectively. The expressions of proinflammatory cytokine genes including IL-1β, IL-8, TNFα and Cox-2, and antiviral genes like Mx and IFN1 were analysed by stimulation with PAMPs and RBIV infection. In vitro study showed the highly up-regulated proinflammatory gene expressions in head kidney and the moderate up-regulation in spleen by LPS. Same concentration of polyI:C moderately upregulated IL-1β gene expression in head kidney but down-regulated IL-8 and TNFα gene expression in head kidney and spleen at 8 h. Mx and IFN1 gene expressions were highly upregulated by polyI:C in head kidney and spleen cells in vitro. By RBIV infection, proinflammatory gene expressions were initially up-regulated and later down-regulated in head kidney. In spleen, although mostly not significant, proinflammatory cytokine gene expressions were down-regulated by RBIV infection except up-regulation of Cox-2 gene expression by low concentration of RBIV at 24 h. Mx and IFN1 gene expressions were down-regulated by high dose of RBIV infection in vitro. In vivo study revealed that IL-8, TNFα, and IFN1 gene expressions were down-regulated in brain, head kidney, spleen, and gill while up-regulated in heart and liver, indicating differential proinflammatory and antiviral responses in the organs. It is supposed that down-regulation of proinflammatory gene expression in the immune organs may result in the failure of antiviral immune responses, causing high mortalities by megalocytivirus infection in rock bream.

Increased Resistance to Quinolones in Streptococcus parauberis and Development of a Rapid Assay for Detecting Mutations in Topoisomerase Genes

To investigate the acquisition of quinolone resistance, we examined mutations in the quinolone resistance-determining region (QRDR) of type II topoisomerase genes in ciprofloxacin (CIP)-resistant clinical isolates and in vitro mutants of Streptococcus parauberis. The CIP-resistant clinical isolates had one base change responsible for a Ser-79→Thr in the QRDR of parC. However, the CIP-resistant in vitro mutants had an altered QRDR of parC (Ser-79→Ile) that differed from that of the isolates. None of the CIP-resistant S. parauberis clinical isolates or in vitro mutants exhibited amino acid changes in gyrA or gyrB. However, even though involvement in the increased resistance was not clear, an Arg-449→Ser mutation outside of the QRDR of parE was detected in CIP-resistant mutant 2P1. These results suggest that the topoisomerase IV gene, parC (and possibly parE, as well), is the primary ciprofloxacin target in S. parauberis. Additionally we established a high-resolution melting (HRM) assay capable of detecting the dominant mutation in four type II topoisomerase genes conferring ciprofloxacin resistance. These rapid and reliable assays may provide a convenient method of surveillance for genetic mutations conferring antibiotic resistance.

Detection of Fish Pathogenic Viruses in Seawater Using Negatively Charged Membranes

After an outbreak of viral disease in an aquafarm, release of virus (es) from infected fish into environmental seawater has been suspected. In the present study, we utilized a negatively charged membrane (HA type) as an efficient method for concentration and detection of fish pathogenic viruses, specifically, megalocytivirus and viral hemorrhagic septicemia virus (VHSV) present in field-collected seawater samples or inoculated into seawater artificially. Positively charged viruses adsorbed onto the negatively charged membrane and were eluted with 1 mM NaOH (pH 10.5) following rinsing with 0.5 mM (pH 3.0). Megalocytivirus and VHSV particles isolated using anegatively charged HA membrane from seawater inoculated with each virus at a concentration of 10 viral particles/mL were of sufficient quantity to show positive results in atwo-step PCR (or RT two-step PCR); however, despite it being negatively charged, a cellulose acetate (CA) membraneshowed negative results. In quantitative PCR, the detection limits of the HA membrane for megalocytivirus and VHSV in seawater were 1.20E+00 viral particles/mL and 1.22E+01 viralparticles/mL, respectively. The calculated mean recovery yields from 1 L seawater spiked with known concentrations of megalocytivirus and VHSV particles were 28.11% and 23.00%, respectively. The concentrate of a 1-L sample of culturing seawater from the aquatank of flounder suffering from VHSV showed clear positive results in PCR when isolated with an HA, but not a CA, membrane. Thus, viral isolation using an HA membrane is a practical and reliable method for detection of fish pathogenic viruses in seawater.

Characterization of the Repetitive Sequences Present in the ORF25 Genomic Region of Megalocytiviruses from Ornamental Fishes

The presence of ISKNV-like viruses in various freshwater ornamental fish species imported from Asia was confirmed by polymerase chain reaction(PCR) amplification of the ATPase(adenosine triphosphatase) gene. Interestingly, molecular analyses of the Open Reading Frame 25(ORF25) region of these isolates based on the ISKNV(Infectious spleen and kidney necrosis virus) genome revealed the presence of various repetitive sequences. ORF25 repeat sequence length had no effect on cumulative mortality of rock bream Oplegnathus fasciatus challenged with tissue homogenates of infected pearl gourami, Trichogaster leeri; silver gourami, Trichogaster microlepis; blue gourami, or Trichogaster trichopterus. All isolates induce cumulative mortalities after 12 days of infection, confirming that ORF25 polymorphism did not affect the pathogenicity of ornamental fish megalocytiviruses that cross infect rock bream, a seawater fish. Also, no statistically significant differences in spleen index or viral copy number in infected tissues was detected between isolates with varying ORF25 repeat sequence lengths. However, further studies are necessary to fully characterize the functional characteristics of these polymorphisms in megalocytivirus disease in ornamental fishes.Key Words : Polymorphism, Repetitive sequences, Freshwater ornamental fish, Megalocytivirus.