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The signal transducer and activator of transcription 1 (STAT1), a pivotal transcription factor in Janus kinase (JAK)-STAT signaling, regulates the expression of a wide range of immune-related genes, including interferon (IFN) regulatory factor 1 (IRF1). In this study, we found that IRF1 could induce STAT1 phosphorylation and in turn STAT1 activation. When IRF1 was transiently expressed in HEK293 cells, STAT1 phosphorylated at Y701, dimerized and bound to an oligonucleotide containing a gamma-activated sequence (GAS) derived from the IRF1 promoter. IRF1 expression also induced GAS-dependent promoter reporter activity, and phosphorylation of JAK1, a kinase upstream of STAT1. Although no direct interaction between IRF1 and STAT1 was observed, the transactivation domain of IRF1 was required for IRF1-mediated STAT1 activation, indicating the involvement of gene product(s) regulated by IRF1. Moreover, supernatants from cells expressing IRF1 induced phosphorylation of STAT1 and JAK1, and subsequent GAS binding by STAT1 that could not be blocked by treatment with antibodies against IFN-β or IFN-γ. IFN-γ-induced STAT1 phosphorylation persisted for up to 30 h following stimulation of HEK293, but declined in IRF1-deficient HEK293 cells. IRF1-promoter activity induced by IFN-γ was also reduced in IRF1-deficient HEK293 cells, which could be rescued by complementation with IRF1. Together these results indicate that IRF1 promotes DNA binding of STAT1, which can in turn participate in a positive feedback loop of JAK-STAT signaling.

All of the fully sequenced iridoviruses have an ORF resembling a putative RNase III gene. However, to the best of our knowledge, functional characterization of the iridovirus-encoded RNase III has not been done. In the present study, we have characterized the putative RNase III of rock bream iridovirus (RBIV), the major cause of mass mortality of cultured rock bream Oplegnathus fasciatus in Korea. RBIV RNase III has a single N-terminal endonuclease domain followed by a C-terminal double-stranded RNA (dsRNA) binding domain. The true presence of the predicted ORF encoding RNase III in RBIV was confirmed by temporal transcription analysis of the ORF in RBIV-infected grunt fin (GF) cells. Comparing the catalytic activity to that of previously reported RNase III proteins, including Escherichia coli RNase III, the present RBIV RNase III had different features in that: (1) the dsRNA substrate was cleaved by the RBIV RNase III at high concentrations of Mg(2+) (5-20 mM) at low salt concentration (50 mM), but the enzyme activity was completely inhibited at 200 mM NaCl (within physiological ranges) irrespective of Mg(2+) concentrations (0.5-20 mM); (2) the substrate dsRNA was cleaved at low concentrations of Mn(2+) (0.5-1 mM) at low salt concentration (50 mM) and was cleaved by increasing Mn(2+) (5-20 mM) at 200 mM salt. These features of RBIV RNase III are similar to E. coli RNase III devoid of the C-terminal dsRBD region. The exact role of the RNase III in RBIV replication is not known, and further studies are needed to elucidate whether the RNase III is involved in the suppression of host RNA interference, which attacks viral mRNAs, or in the processing of viral RNAs for effective replication.

We established a laboratory propagation method of Cryptocaryon irritans , a parasitic ciliate of marine fishes, with black molly Poecilia sp. as host fish, using small plastic aquaria. One cycle of the propagation usually takes one week. With this method, 1500–3000 protomonts are obtained from five challenged mollies every week, from which more than 100,000–200,000 theronts are obtained. Using this method, an isolate of C. irritans has been successfully maintained more than three years. This propagation method reduces labor for maintaining and propagating the parasite and will much contribute to researches on cryptocaryoniasis. The method is a laboratory propagation technique of Cryptocaryon irritans . Using small plastic aquaria and black molly as a host, the parasites can be stably propagated and maintained. An isolate of C. irritans has been successfully maintained more than three years.

A dual vector expressing the ghost-inducing PhiX174 lysis E gene and the bacterial DNA degrading staphylococcal nuclease A (SNA) gene was constructed to solve the problem of remnant antibiotic resistance genes and genomic DNA with intact pathogenic islands in the final product of Edwardsiella tarda ghosts (ETG). The SNA (devoid of secretion signal sequence and the nuclease B amino terminus sequence), fused with the 26 amino acid N-terminal sequence of the λ phage Cro gene, showed successful degradation of bacterial nucleic acids. Furthermore, the nuclease activity of SNA in E. tarda was enhanced by codon optimization of the SNA gene using site-directed mutagenesis. ETG were generated via coexpression of the SNA gene and lysis gene E under the control of each λP R promoter. The ghost bacteria generation system we describe is advantageous as it allows the use of a single plasmid, improves safety and vaccine purity by limiting residual genetic content from the ghost bacteria, and reduces production costs through cheap means of induction that use only temperature shifts. KEY WORDS: Edwardsiella tarda · Ghost bacteria · Staphylococcal nuclease A · Dual vector · Safety enhancement Resale or republication not permitted without written consent of the publisherDis Aquat Org 81: [249][250][251][252][253][254] 2008 Katinger et al. 1999, Panthel et al. 2002, Marchart et al. 2003.Recently, we have generated Edwardsiella tarda ghosts (ETG) by gene E mediated lysis (Kwon et al. 2005), and have demonstrated significantly higher protection against infection of E. tarda in tilapia and olive flounder immunized with ETG than in fish immunized with formalin-killed E. tarda (Kwon et al. 2006(Kwon et al. , 2007. In these studies, although the bacteria were inactivated by induction of E gene expression, large-sized genomic DNA or plasmid DNA was detected from the produced ETG, suggesting the presence of nonlysed or partially lysed inactivated cells within the ghost preparation. The presence of genomic DNA with intact pathogenic islands and/or antibiotic resistance genes in the ETG preparation would be problematic to use as a practical vaccine for cultured fish. In this study, we constructed a dual vector expressing both the ghost inducing PhiX174 E gene and the bacterial DNA degrading staphylococcal nuclease A (SNA) gene to minimize the presence of antibiotic resistance genes and genomic DNA with pathogenic islands in the ETG vaccine. We also examined the potential utility of the vector in producing safety-improved ETG. MATERIALS AND METHODSBacterial strains. Edwardsiella tarda FSW910410, isolated in 1991 from moribund olive flounder in a natural outbreak of edwardsiellosis on a commercial farm in Korea (Bang et al. 1992), was used. This strain is used in a commercial edwardsiellosis vaccine in Korea, and was kindly provided by the National Fisheries Research & Development Institute, Korea. For cloning of nuclease gene, Staphylococcus aureus KCCM 11335 was purchased from the Korean Culture Center of Microorganisms.Construc...