善家 孝介

Inducible nitric oxidase (iNOS) encoded by Nos2 is a representative IFNγ-inducible effector molecule that plays an important role in both innate and adaptive immunity. In the present study, we demonstrated that full-length NF-κB p105 (p105), which is a precursor of NF-κB p50 (p50), is required for full activation of IFNγ-induced iNOS expression in the RAW264.7 mouse macrophage cell line. In comparison to wild-type (WT) RAW264.7 cells, p105 KO RAW264.7 (p105 KO) cells completely lost IFNγ-induced iNOS expression. Despite the limited effect of exogenous expression of p50 in p105 KO cells on IFNγ-induced Nos2 promoter activity, p105 expression fully restored IFNγ-induced Nos2 promoter activity to a level comparable to that of WT cells, suggesting an important role for full-length p105 in IFNγ-induced iNOS expression. While the expression and phosphorylation of JAK1 and STAT1 were rather enhanced in p105 KO cells, the phosphorylation of c-Jun downstream of MAPK signaling was decreased. IFNγ-induced phosphorylation of ERK, a kinase for IFNγ-induced c-Jun phosphorylation, was not significantly reduced in p105 KO cells, although the nuclear activity of ERK was significantly decreased due to its reduced translocation to the nucleus. Expression of iNOS, nuclear translocation of ERK, and phosphorylation of c-Jun were restored by stable supplementation of p105 in p105 KO cells. These results suggest that p105 is required for the nuclear translocation of ERK and the subsequent phosphorylation of c-Jun, which are necessary for full activation of IFNγ-induced iNOS expression. Reduced nuclear translocation of ERK in p105 KO cells was also observed in the activation of ERK following serum starvation, further suggesting that the involvement of p105 in ERK nuclear translocation is not limited to IFNγ-stimulated cells but is a more general function of p105.

Abstract LPS interacts with TLR4, which play important roles in host-against-pathogen immune responses, by binding to MD-2 and inducing an inflammatory response. In this study, to our knowledge, we found a novel function of lipoteichoic acid (LTA), a TLR2 ligand, that involves suppression of TLR4-mediated signaling independently of TLR2 under serum-free conditions. LTA inhibited NF-κB activation induced by LPS or a synthetic lipid A in a noncompetitive manner in human embryonic kidney 293 cells expressing CD14, TLR4, and MD-2. This inhibition was abrogated by addition of serum or albumin. LTAs from different bacterial sources also inhibited NF-κB activation, although LTA from Enterococcus hirae had essentially no TLR2-mediated NF-κB activation. The TLR2 ligands tripalmitoyl-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4) and macrophage-activating lipopeptide-2 (MALP-2) did not affect the TLR4-mediated NF-κB activation. In bone marrow–derived macrophages from TLR2−/− mice, LTA inhibited LPS-induced IκB-α phosphorylation and production of TNF, CXCL1/KC, RANTES, and IFN-β without affecting cell surface expression of TLR4. LTA did not suppress IL-1β–induced NF-κB activation mediated through signaling pathways shared with TLRs. LTAs including E. hirae LTA, but not LPS, induced association of TLR4/MD-2 complexes, which was suppressed by serum. LTA also increased association of MD-2, but not TLR4 molecules. These results demonstrate that, under serum-free conditions, LTA induces association of MD-2 molecules to promote formation of an inactive TLR4/MD-2 complex dimer that in turn prevents TLR4-mediated signaling. The presence of LTA that poorly induces TLR2-mediated activation but inhibits TLR4 signaling provides insight into the role of Gram-positive bacteria in suppressing inflammation induced by Gram-negative bacteria in organs such as the intestines where serum is absent.

Heat shock proteins (HSPs) are molecular chaperones that have recently been shown to function as host factors (HFs) for virus multiplication in fish as well as in mammals, plants, and insects. HSPs are classified into families, and each family has multiple isoforms. However, no comprehensive studies have been performed to clarify the biological importance of these multiple isoforms for fish virus multiplication. Betanodaviruses are the causative agents of viral nervous necrosis in cultured marine fish and cause very high mortality. Although the viral genome and encoded proteins have been characterized extensively, information on HFs for these viruses is limited. In this study, therefore, we focused on the HSP70 and HSP90 families to examine the importance of their isoforms for betanodavirus multiplication. We found that HSP inhibitors (17-AAG, radicicol, and quercetin) suppressed viral RNA replication and production of progeny virus in infected medaka (Oryzias latipes) cells. Thermal stress or virus infection resulted in increased expression of some isoform genes and facilitated virus multiplication. Furthermore, overexpression and knockdown of some isoform genes revealed that the isoforms HSP70-1, HSP70-2, HSP70-5, HSP90-α1, HSP90-α2, and HSP90-β play positive roles in virus multiplication in medaka. Collectively, these results suggest that multiple isoforms of fish HPSs serve as HFs for betanodavirus multiplication.

Cryptocaryoniasis, caused by Cryptocaryon irritans, is a major threat to marine cage culture in tropical and subtropical waters; however, controlling the disease remains challenging. In this study, we constructed DNA vaccines encoding a cysteine protease of C. irritans (pcDNA3.1-cp2-full-myc and pcDNA3.1-cp2-partial-myc) and examined the protective efficacy of the vaccines. The results of the challenge experiment showed that the number of parasites recovered from fish immunized with the DNA vaccines was lower than that recovered from control fish (phosphate-buffered saline-injected and mock vector-injected groups); this difference was statistically significant when pcDNA3.1-cp2-full-myc was used for vaccination (p < 0.05). In addition, the cysteine protease was found to be relatively conserved among different isolates of the parasite. Thus, the protease may be a potential antigen candidate for the development of a vaccine against multiple strains of C. irritans.

In spite of the growing attention given to medaka (Oryzias latipes) as an excellent vertebrate model, an effective gene knockdown system has not yet been established using cultured cells of this fish species. In this study, a gene knockdown system using short interfering RNA (siRNA) in medaka cell lines was established through the optimization of transfection conditions. By extensive screening of several medaka cell lines and transfection reagents, OLHNI-2 cells and X-tremeGENE siRNA Transfection Reagent were selected as the best combination to achieve high transfection efficiency of siRNA without cytotoxic effect. Knockdown conditions were then refined using the endogenous heat shock protein 90 (Hsp90) genes as the siRNA targets. Among the parameters tested, cell density, serum concentration in the culture medium, and duration of transfection improved knockdown efficiency, where the target mRNA in cells transfected with each of the siRNAs was reduced from 12.0% to 26.7% of the control level. Our results indicate that the established knockdown system using siRNA is a promising tool for functional analysis of medaka genes in vitro.

In this study, we aimed to analyze the functions of highly expressed proteases at the theront (invasive stage) and trophont (parasitic stage) phases of Cryptocaryon irritans during the infection process using RNAi. We also analyzed the potential of these proteases as candidate antigens for vaccination against the parasite by infecting tiger pufferfish (Takifugu rubripes) with the recombinant proteases. Compared with the control group, RNAi against four proteases (including peptidases) genes reduced the number of parasites that infected and left the fish after complete development. The four recombinant proteases were then purified and evaluated as candidate vaccination antigens. The number of parasites recovered from fish immunized with the C. irritans recombinant proteins was lower than that recovered from control fish; this difference was statistically significant when a recombinant cysteine protease was used. These results suggest that the C. irritans proteases overexpressed at the theront and trophont stages are important for infection (including invasion, food uptake, digestion, and development) and can be targeted for the development of vaccines against cryptocaryoniasis.

<p>Cryptocaryon irritans has clear daily rhythms in protomont detachment from fish and theront excystment from tomonts. ​While the rhythms seem to be regulated by photoperiods, it has not been confirmed. ​We investigated whether the daily rhythms of the parasite would be controlled by giving different photoperiods of 12 h light and 12 h dark (06:00–18:00 L and 18:00–06:00 D; 15:00–03:00 L and 03:00–15:00 D) to infected fish and tomonts. ​Protomont detachment and theronts excystment mostly occurred in the last 3 h of the dark period and from 6 h to 3 h before the end of dark period, respectively, indicating that the rhythms are controlled by photoperiods.</p>

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.

We found that AKT1, a primary effector molecule of PI3K-AKT signaling, distinctively suppressed Toll-like receptor (TLR)-mediated MyD88-dependent and Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF)-dependent signaling by inhibiting NF-κB activation and IRF3 activity independently of its kinase activity. In AKT1 knockout RAW264.7 cells, lipopolysaccharide (LPS)-induced transcription and protein production of cytokines including IL-1β and TNF-α (regulated by the MyD88-dependent pathway), as well as IFN-β and RANTES (C-C motif chemokine ligand 5: CCL-5 regulated by the TRIF-dependent pathways) was enhanced compared to wild type cells. In response to LPS stimulation, AKT1 knockout cells also exhibited enhanced NF-κB and IFN-β promoter activities, which were reduced to a level comparable to that in wild type cells by complementation with either AKT1 or its kinase-dead mutant (AKT1-KD). Expression of AKT1 or AKT1-KD similarly suppressed NF-κB and IFN-β promoter activities induced by LPS and other TLR ligands in wild type cells. Analysis of NF-κB activation caused by transient expression of proteins involved in the MyD88-dependent pathway in TLR signaling revealed that AKT1 suppressed signaling that occurs between activation of IKKβ and that of NF-κB. In contrast, AKT1 appeared to suppress the IFN-β promoter through inhibition of IRF3 activity itself. These results demonstrate a novel, non-kinase function of AKT1 that inhibits TLR signaling, and suggest the multifunctional nature of AKT1.

We incubated tomonts of Cryptocaryon irritans in a hypoxic seawater environment (1.4–1.7 mg/L O2) (low dissolved oxygen DO) and examined their development using a acetocarmine whole-mount staining method we developed for nuclear staining. They showed little development and stayed in the dormant phase in the hypoxic environment. When transferred into the hypoxic environment after incubation in an oxic environment (air-saturated, 8.7–8.9 mg/L O2) for 1–4 days, their development stopped in 1 day. However, when dormant tomonts generated in the hypoxic environment were transferred to the oxic environment, they resumed development and released theronts. These results indicate that tomonts can become dormant when exposed to a hypoxic environment, but can resume development when exposed to an oxic environment at any developmental stage. When exposed to the oxic environment, tomonts recovered from 1-month dormancy and released as many theronts as control tomonts constantly incubated in the oxic environment. The infectivity of theronts from the recovered tomonts was similar to the control tomonts. Thermoclines prevent oxygen-rich surface seawater from reaching the bottom of water column and create a hypoxic sea floor environment in summer these thermoclines are broken down in autumn or after typhoons. The long-term viability of dormant tomonts in hypoxic environments may be a key factor in the autumn outbreaks of cryptocaryoniasis in floating net cages in temperate waters.

The ciliate protozoan Cryptocaryon irritans parasitizes marine fish and causes lethal white spot disease. Sporadic infections as well as large-scale outbreaks have been reported globally and the parasite's broad host range poses particular threat to the aquaculture and ornamental fish markets. In order to better understand C irritans' population structure, we sequenced and compared mitochondrial cox-1, SSU rRNA, and ITS-1 sequences from 8 new isolates of C irritans collected in China, Japan, and Taiwan. We detected two SSU rRNA haplotypes, which differ at three positions, separating the isolates into two main groups (I and II). Cox-1 sequences also support the division into two groups, and the cox-1 divergence between these two groups is unexpectedly high (9.28% for 1582 nucleotide positions). The divergence is much greater than that detected in Ichthyophthirius multifiliis, the ciliate protozoan causing freshwater white spot disease in fish, where intraspecies divergence on cox-1 sequence is only 1.95%. ITS-1 sequences derived from these eight isolates and from all other C irritans isolates (deposited in the GenBank) not only support the two groups, but further suggest the presence of a third group with even greater sequence divergence. Finally, a small Ka/Ks ratio estimated from cox-1 sequences suggests that this gene in C. irritans remains under strong purifying selection. Taken together, the C irritans species may consists of many subspecies and/or syngens. Further work is needed to determine if there is reproductive isolation between the groups we have defined. (C) 2017 Elsevier Inc. All rights reserved.

We investigated the development of the macronucleus of Cryptocaryon irritans, and the ingestion and digestion of host cells by the parasite. All developmental stages of the parasite except the theront (trophont, protomont and tomont) were examined with histological staining and/or whole mount staining. The sections were also subjected to in situ hybridization targeting the 18S rRNA gene of the host fish. The macronucleus developed negligibly, maintaining its four-segmented shape in trophonts, elongated and formed a massive nucleus in tomonts before cell division, and subsequently underwent repeated divisions to generate theronts. Denatured host cells, examined in situ hybridization, were accumulated in trophonts, and disappeared in tomonts by the beginning of cell division. Denatured host cells were also observed in the host tissue surrounding trophonts. They had condensed nuclei. Protomonts filled with denatured host cells presented a 180-bp DNA ladder in gel electrophoresis, suggesting apoptosis of the host cells. These results indicate that DNA synthesis occurs exclusively in the early stage of the tomont and that host cells are fed and accumulated, probably as apoptotic cells, in the trophont and are digested in the early tomont stage.

Cryptocaryon irritans is a parasitic ciliate that causes major economic losses in marine fish aquaculture globally. Despite the wide array of treatment methods, control of cryptocaryoniasis is still very challenging, especially in food fish culture. Thus, additional control methods against this parasite might be required to further reduce the occurrence of this disease. In this study, the swimming ability, excystment sequence, and distribution of theronts, the infective stage of C. irritans, were investigated in our effort to develop a physical control strategy. A video analysis for assessing the swimming ability of theronts showed diminishing mobility over time. The excystment of theronts primarily occurred during the dark period. Examination of the vertical distribution of theronts showed that they were mostly distributed at the 5 cm sampling point measured from the substrate, indicating that they have low upward swimming ability. From these results, we conclude that theronts possess limited mobility. Theronts also displayed an excystment pattern that might be influenced by photoperiod, since most theronts were released during the dark period of the day. Control strategies can be developed from these properties, such as increasing the water flow in a culture tank during the release period. Further, combined treatment methods against multiple stages of C. irritans can help minimize the occurrence of cryptocaryoniasis in culture facilities. (C) 2014 Elsevier B.V. All rights reserved.

The myxosporean parasite Kudoa septempunctata has been isolated from cultured olive flounder (Paralichthys olivaceus) and was recently identified as a cause of food poisoning in humans. Since the sporoplasm plays an important role in causing diarrhea by invading intestinal cells, the specific factors affecting the release of sporoplasm from spores should be determined. Thus, we investigated the effect of digestive and serum enzymes, fetal bovine serum (FBS), temperature, and the role of glucose in cell culture media on the release of sporoplasm. Sporoplasm release was observed in the groups treated with FBS and media containing glucose. In addition, 1,10-phenanthroline inhibited the release of sporoplasm in the FBS medium. These results indicate that K. septempunctata uses glucose for releasing its sporoplasm and that zinc or metalloprotease is related to the release mechanism. The present study provides important information for the development of agents to prevent sporoplasm release and the consequent food poisoning caused by K. septempunctata.

Proteases play important roles in parasite development and host parasite interactions. The protease of Kudoa spp. has been recognized as a key factor of severe proteolysis of fish muscle post-mortem however, there is little information available regarding the protease of Kudoa (K.) septempunctata, which was recently identified as a cause of food poisoning in humans. The present study was conducted to isolate and characterize proteases to elucidate the type of protease contained in the parasite and determine the optimal pH for protease activity. We confirmed the cysteine protease and metalloprotease produced by K. septempunctata. While the cysteine protease showed optimal activity at pH 5 that decreased rapidly with increasing pH, the optimal activity of metalloprotease was pH 7, and it remained stable from pH 6 to pH 8. These results indicate that the pH of cysteine protease is not proper for fish muscle postmortem, and that metalloprotease can act in human intestines. Overall, the present study provides important information that improves our understanding of the role of protease physiology and the subsequent food poisoning caused by K. septempunctata.

It remains unclear whether allergens are the same among the sibling species of Anisakis simplex sensu lato. This study was carried out to compare the amino acid sequences of three major (Ani s 1, 2 and 12) and one minor (Ani s 9) Anisakis allergens between A. simplex s.s. and Anisakis pegreffii. We found 2 (out of 163), 1 (out of 869), and 29 (out of 266) amino acid variable sites for Ani s 1, 2, and 12, respectively. However, as both intra- and inter-species variations were present at the same amino acid positions, no amino acid variations clearly distinguished the two sibling species. IgE-binding epitopes (Ani s 1) and a binding motif of human leukocyte antigen (Ani s 2 and 9) demonstrated by previous studies were conserved. The similarities of the amino acid sequences of the allergens indicate possible similar allergy-associated health risks in humans infected with or accidentally ingesting either Anisakis species. (C) 2013 Elsevier B.V. All rights reserved.

Background: Betanodaviruses, members of the family Nodaviridae, have bipartite, positive-sense RNA genomes and are the causal agents of viral nervous necrosis in many marine fish species. Recently, the viruses were shown to infect a few freshwater fish species including a model fish medaka (Oryzias latipes). Although virological study using cultured medaka cells would provide a lot of insight into virus-fish interactions in molecular aspects, no such cells have yet been tested for virus susceptibility. Results: We tested ten medaka cell lines for susceptibilities to redspotted grouper nervous necrosis virus (RGNNV). Although the viral coat protein was detected in all the cell lines inoculated, the levels of cytopathic effect development and viral propagation were quite different among the cell lines. Those levels were especially high in OLHNI-1 and OLHNI-2 cells, but were extremely low in OLME-104 cells. Some cell lines entered into antiviral state after RGNNV infections probably because of inducing an antiviral system. This is the first report to examine the susceptibilities of cultured medaka cells against a virus. Conclusion: OLHNI-1 and OLHNI-2 cells are candidates of new standard cells for betanodavirus study because of their high susceptibilities to the virus and their several advantages as model fish cells.

We have cloned and characterized rock bream double-stranded RNA-dependent protein kinase (PKR), which is a key component of type I IFN inducible innate immune system. Full-length rock bream PKR cDNA consists of 2115 bp ORF encoding 704 amino acids, 124 bp 5&apos; UTR, and 529 bp 3&apos; UTR. Conserved domain analysis revealed that rock bream PKR contained two tandem dsRBM and kinase domain consisted of 11 kinase sub-motifs, which are characteristics found in other PKRs. Rock bream PKR was constitutively expressed in the spleen of rock bream and, upon injection of poly I:C, up-regulated not later than 12 h post injection and returned to baseline level at 48 h post injection. Although the eIF2 alpha kinase activity of rock bream PKR was not examined in the present study, dsRNA inducible nature of rock bream PKR implies the possible important role of this gene in innate immune system of rock bream as suggested in zebrafish and flounder. (C) 2009 Elsevier B.V. All rights reserved.

In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream beta-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream beta-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream beta-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species.

Rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha: (rbTNF-alpha) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-alpha on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-alpha was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-alpha gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-alpha, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-alpha was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-alpha might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis. (C) 2009 Elsevier Ltd. All rights reserved.

Complementary DNAs (cDNAs) corresponding to three isoforms of rock bream (Oplegnathus fasciatus) Mx (RbMx1, RbMx2 and RbMx3) were cloned using RACE reactions. Analysis of deduced amino acid sequences revealed that the tripartite GTP-binding domain, the dynamine family signature and the leucine zipper repeat were present in all three rock bream Mx isoforms. Cloning of genomic DNA sequence and Southern blot analysis showed that three rock bream Mx isoforms were encoded by different genomic loci, and they were not alternative splicing variants, although some alternative splicing variants were found in RbMx1 and RbMx2. When comparing amino acid sequence identity, RbMx1 shares about 60-70% identities with other fish Mx proteins, whereas both RbMx2 and RbMx3 share slightly high identity of 70-90%. As a result of expression analysis using RT-PCR, RbMx1 was constitutively expressed in the spleen and kidney of rock bream yearling, but RbMx2 and RbMx3 were rarely detected in both organs. When injected with synthetic double-stranded RNA polyinosinic:polycytidylic acid (poly I:C), expression of all rock bream Mx isoforms was up-regulated in spleen and head kidney. RbMx1 was continuously up-regulated throughout experimental period of 72 h but RbMx2 and RbMx3 were down-regulated to almost non-detectable level at 48 h post-injection. (C) 2008 Elsevier Ltd. All rights reserved.

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 lambda 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 lambda P-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.

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.

In fish species, although many studies on the use of RNA interference (RNAi) for gene function analysis have been reported, almost of them have utilized in vitro transcribed or synthesized small interfering RNA (siRNA), and there are a few studies which examined vector based RNAi in fish species. in this study, we have identified U6 promoter of fugu (Takifugu rubripes), and utilized it for expression of short hairpin RNA (shRNA) in fish cell lines. Using Northern blot analysis, we confirmed successful transcription of shRNA by fugu U6 promoter in bluegill fry (BF-2) cells. The knock down assay targeting an exogenous EGFP reporter gene demonstrated that fugu U6 promoter expressed shRNA more efficiently than mouse U6 promoter in BF-2, grunt fin (CF), and Chinook salmon embryo (CHSE) cell lines. This study suggests that fugu U6 promoter driven shRNA expression vector can be novel tool for RNAi induction in fish cell lines. (C) 2008 Elsevier Inc. All rights reserved.

To determine whether Penaeus chinensis can be protected against white spot syndrome virus (WSSV) infection by intramuscular injection with long double-stranded RNAs (dsRNAs) as in other shrimp species and whether the protection degree by WSSV-specific dsRNAs is correlated with the roles of viral genes, P. chinensis juveniles were intramuscularly injected with long dsRNAs corresponding to VP28, VP28 1, protein kinase genes of WSSV, and an unrelated long dsRNA corresponding to a green fluorescence protein (GFP) gene. All shrimp injected with long dsRNAs including GFP dsRNA showed higher survival rates against WSSV infection than shrimp injected with PBS alone. Furthermore, shrimp injected with dsRNAs corresponding to VP28 and protein kinase showed higher survival rates than those injected with dsRNAs corresponding to VP281 and GFP. These results indicate that the introduction of long dsRNAs corresponding to viral proteins, which are essential for WSSV infection, is quite effective in blocking WSSV infection in P. chinensis., and suggest that dsRNA-mediated protection is a common feature across shrimp species. (c) 2006 Elsevier Ltd. All rights reserved.

Effects of water temperature on infection of the microsporidian Kabatana takedai were investigated in salmonids under field and experimental conditions. "Cysts" of K. takedai appeared in the heart and the skeletal muscle of wild juvenile masu salmon Oncorhynchus masou from the Chitose River, Hokkaido, during summer in 2003 and 2004, when the river water temperature exceeded 15 degrees C. Following the exposure of naive juvenile sockeye salmon Oncorhynchus nerka to river water (18-19 degrees C) for 3 days, the fish were transferred to separate tanks where the temperatures were set to 11, 13, 15 and 17 degrees C. Prevalence of infection reached more than 70% in the 13, 15 and 17 degrees C groups, but in the 1 VC group, it remained less than 30%. However, shifting a part of the 11 degrees C group to 18 degrees C increased the prevalence to 71% by 23 days after the elevation of the temperature. When juveniles were exposed to 11 degrees C river water and subsequently kept at 9 degrees C for 42 days, no development of K. takedai occurred even after the temperature was elevated to 15 degrees C. These results indicate that temperature manipulation is partially effective as a preventive method of takedai infection.