Development and characterization of megalocytivirus persistently-infected cell cultures for high yield of virus.

Megalocytivirus infection is a major threat in rock bream aquaculture in Korea. To produce a highly concentrated megalocytivirus, primary cells, established cell line and persistently infected cell line were used in this study. Megalocytivirus was inoculated in primary fin cell cultures of red sea bream (Pagrus major), rock bream (Oplegnathus fasciatus), olive flounder (Paralichthys olivaceus) and black sea bream (Acanthopagrus schlegelii) and produced at similar concentrations of 108.99 - 9.88 viral particles/mL in all cultures while produced 107.31 viral particles/mL in grunt fin (GF) cell line. Since only red sea bream fin culture was amenable to subculturing for more than 100 times, it was established into Pagrus major fin (PMF) cell line. A persistently infected PMF cell line (PI-PMF) was obtained by continuous subculturing every 7 days as a batch culture system (PI-PMF-B) after infecting with megalocytivirus. Virus in supernatant of PI-PMF-B was maintained at high concentrations throughout over 50 consecutive subcultures in a relatively narrow range from 108.33 to 108.94 viral particles/mL with high level of CPE. For a more efficient and convenient production, a semi-batch culture system (PI-PMF-S) was developed in which culture media were exchanged at intervals of 3 days without subculturing for more than 50 media exchanges. Despite low virus productivity in a single cell (specific virus productivity, SVP), total cell number was increased in PI-PMF-S, allowing us to efficiently obtain a much higher concentration of virus (108.56 to 109.75 viral particles/mL) than in PMF-B. This is the first study to report detailed new methods for continuous and efficient production of high concentrations of megalocytivivrus with characterization of viral propagation in persistently infected cells.

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)-1ß and IL-8 gene and performed the phylogenetic and structural analysis. Also the constitutive gene expressions of IL-1ß 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-1ß, 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-1ß 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.

Characterization of a new beta-lactamase gene from isolates of Vibrio spp. in Korea.

PCR was performed to analyze the beta-lactamase genes carried by ampicillin-resistant Vibrio spp. strains isolated from marine environments in Korea between 2006 and 2009. All 36 strains tested showed negative results in PCR with the primers designed from the nucleotide sequences of various known beta-lactamase genes. This prompted us to screen new beta-lactamase genes. A novel beta-lactamase gene was cloned from Vibrio alginolyticus KV3 isolated from the aquaculture water of Geoje Island of Korea. The determined nucleotide sequence (VAK-3 beta-lactamase) revealed an open reading frame (ORF) of 852 bp, encoding a protein of 283 amino acids (aa), which displayed low homology to any other beta-lactamase genes reported in public databases. The deduced 283 aa sequence of VAK-3, consisting of a 19 aa signal peptide and a 264 aa mature protein, contained highly conserved peptide segments specific to class A beta-lactamases including the specific amino acid residues STFK (62-65), SDN (122-124), E (158), and RTG (226-228). Results from PCR performed with primers specific to the VAK-3 beta-lactamase gene identified 3 of the 36 isolated strains as V. alginolyticus, Vibrio cholerae, and Photobacterium damselae subsp. damselae, indicating the utilization of various beta-lactamase genes including unidentified ones in ampicillin-resistant Vibrio spp. strains from the marine environment. In a mating experiment, none of the isolates transfered the VAK-3 beta-lactamase gene to the Escherichia coli recipient. This lack of mobility, and the presence of a chromosomal acyl-CoA flanking sequence upstream of the VAK-3 beta- lactamase gene, led to the assumption that the location of this new beta-lactamase gene was in the chromosome, rather than the mobile plasmid. Antibiotic susceptibility of VAK-3 beta-lactamase was indicated by elevated levels of resistance to penicillins, but not to cephalosporins in the wild type and E. coli harboring recombinant plasmid pKV-3, compared with those of the host strain alone. Phylogenetic analysis showed that VAK-3 beta-lactamase is a new and separate member of class A beta-lactamases.