Nuclear factor 45 of half smooth tongue sole Cynoglossus semilaevis: gene structure, expression profile, and immunoregulatory property.

Nuclear factor 45 (NF45) is a component of the protein complex called nuclear factor of activated T-cells (NFAT), which in mammals regulates interleukin (IL)-2 expression. To date very little is known about fish NF45. In this study, we identified a NF45 (named CsNF45) from half smooth tongue sole Cynoglossus semilaevis and examined its gene organization, expression profile, and regulatory function. We found that CsNF45 is composed of 387 residues and shares 90.3%-97.9% overall sequence identities with the NF45 of human and teleosts. Genetic analysis showed that the genomic sequence of the ORF region of CsNF45 consists of 14 exons and 13 introns. Constitutive expression of CsNF45 occurred in multiple tissues including gill, muscle, brain, heart, liver, head kidney, spleen, and gut. Experimental infection with viral and bacterial pathogens upregulated the expression of CsNF45 in head kidney and spleen in a time-dependent manner. Transient transfection analysis showed that CsNF45 was localized in the nucleus and able to stimulate the activity of mouse IL-2 promoter. These results indicate that CsNF45 possesses immunoregulatory property and is possibly involved in host immune defense against bacterial and viral infection.

SmCCL19, a CC chemokine of turbot Scophthalmus maximus, induces leukocyte trafficking and promotes anti-viral and anti-bacterial defense.

Chemokines are classified into several different subfamilies, of which CC chemokines constitute the largest subfamily in teleost. The prominent structural characteristic of CC chemokines is the presence of an Asp-Cys-Cys-Leu (DCCL) motif. To date, cDNA sequences of several CC chemokines have been identified in turbot (Scophthalmus maximus), however, the activity and function of these putative chemokines remain unknown. In this study, we examined the biological effect of the turbot CC chemokine SmCCL19, which has been previously reported as KC70 and shown to be regulated in expression by bacterial infection. To facilitate functional analysis, recombinant SmCCL19 (rSmCCL19) and a mutant form of SmCCL19, SmCCL19M, that bears serine substitutions at the two cysteine residues of the DCCL motif were purified from Escherichia coli. Chemotactic analysis showed that rSmCCL19 induced migration of head kidney leukocytes in a dose-dependent manner, whereas rSmCCL19M caused no apparent cellular migration. To examine the in vivo effect of rSmCCL19, turbot were administered with rSmCCL19 or rSmCCL19M before being inoculated with viral and bacterial pathogens. Subsequent tissue infection analysis showed that the viral and bacterial loads in rSmCCL19-adminsitered fish were significantly reduced, whereas the pathogen loads in rSmCCL19M-adminsitered fish were largely comparable to those in the control fish. Consistent with these observations, significant inductions of immune relevant genes were observed in rSmCCL19-adminsitered fish but not in rSmCCL19M-adminsitered fish. Taken together, these results indicate that SmCCL19 recruits leukocytes and augments host immune defense in a manner that depends on the conserved DCCL motif.

Complete genome sequence and transcription profiles of the rock bream iridovirus RBIV-C1.

The family Iridoviridae consists of 5 genera of double-stranded DNA viruses, including the genus Megalocytivirus, which contains species that are important fish pathogens. In a previous study, we isolated the first rock bream iridovirus from China (RBIV-C1) and identified it as a member of the genus Megalocytivirus. In this report, we determined the complete genomic sequence of RBIV-C1 and examined its in vivo expression profiles. The genome of RBIV-C1 is 112333 bp in length, with a GC content of 55% and a coding density of 92%. RBIV-C1 contains 4584 simple sequence repeats, 89.8% of which are distributed among coding regions. A total of 119 potential open reading frames (ORFs) were identified in RBIV-C1, including the 26 core iridovirus genes; 41 ORFs encode proteins that are predicted to be associated with essential biological functions. RBIV-C1 exhibits the highest degree of sequence conservation and colinear arrangement of genes with orange-spotted grouper iridovirus (OSGIV) and rock bream iridovirus (RBIV). The pairwise nucleotide identities are 99.49% between RBIV-C1 and OSGIV and 98.69% between RBIV-C1 and RBIV. Compared to OSGIV, RBIV-C1 contains 11 insertions, 13 deletions, and 103 single nucleotide mutations. Whole-genome transcription analysis showed that following experimental infection of rock bream with RBIV-C1, all but 1 of the 119 ORFs were expressed at different time points and clustered into 3 hierarchical groups based on their expression patterns. These results provide new insights into the genetic nature and gene expression features of megalocytiviruses.

Construction and analysis of experimental DNA vaccines against megalocytivirus.

Iridoviruses are large double-stranded DNA viruses with icosahedral capsid. The Iridoviridae family contains five genera, one of which is Megalocytivirus. Megalocytivirus has emerged in recent years as an important pathogen to a wide range of marine and freshwater fish. In this study, we aimed at developing effective genetic vaccines against megalocytivirus affecting farmed fish in China. For this purpose, we constructed seven DNA vaccines based on seven genes of rock bream iridovirus isolate 1 from China (RBIV-C1), a megalocytivirus with a host range that includes Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus). The protective potentials of these vaccines were examined in a turbot model. The results showed that after vaccination via intramuscular injection, the vaccine plasmids were distributed in spleen, kidney, muscle, and liver, and transcription of the vaccine genes and production of the vaccine proteins were detected in these tissues. Following challenge with a lethal-dose of RBIV-C1, fish vaccinated with four of the seven DNA vaccines exhibited significantly higher levels of survival compared to control fish. Of these four protective DNA vaccines, pCN86, which is a plasmid that expresses an 86-residue viral protein, induced the highest protection. Immunological analysis showed that pCN86 was able to (i) stimulate the respiratory burst of head kidney macrophages at 14 d, 21 d, and 28 d post-vaccination, (ii) upregulate the expression of immune relevant genes involved in innate and adaptive immunity, and (iii) induce production of serum antibodies that, when incubated with RBIV-C1 before infection, significantly reduced viral loads in kidney and spleen following viral infection of turbot. Taken together, these results indicate that pCN86 is an effective DNA vaccine that may be used in the control of megalocytivirus-associated diseases in aquaculture.

Identification and analysis of the tissue factor pathway inhibitor 2 of Sciaenops ocellatus.

Tissue factor pathway inhibitor 2 (TFPI-2) is a structural homologue of TFPI, a potent inhibitor of tissue factor (TF)-mediated coagulation. Although TFPI-2 has been identified at sequence level in several fish species, no study on piscine TFPI-2 has been documented. In this report, we identified and analyzed a TFPI-2 homologue, SoTFPI2, from red drum Sciaenops ocellatus. The open reading frame of SoTFPI2 is 681 bp, which encodes a 226-residue protein that shares 59.2-82.3% overall sequence identities with known fish TFPI-2. SoTFPI2 possesses three tandem Kunitz domains and is negatively charged at the N-terminus and positively charged at the C-terminus. Expression of SoTFPI2 was detected, in increasing order, in spleen, muscle, gill, brain, liver, kidney, blood, and heart. Bacterial challenge and lipopolysaccharide treatment significantly upregulated SoTFPI2 expression in kidney in time-dependent manners. Recombinant SoTFPI2 purified from Escherichia coli inhibits the proteolytic activity of trypsin and exhibits bactericidal effect on a fish pathogen. Take together, these results indicate that SoTFPI2 is a biologically active serine protease inhibitor with antibacterial property and is likely to play a role in anti-bacterial infection.

Cloning and analysis of a ferritin subunit from turbot (Scophthalmus maximus).

Ferritin is an evolutionarily conserved protein that plays an important role in iron storage and detoxification. In this study, the gene encoding a ferritin H subunit homologue (SmFer1) was cloned from turbot (Scophthalmus maximus) and analyzed at the expression and functional levels. The open reading frame of SmFer1 is 534 bp and preceded by a 5'-untranslated region that contains a putative Iron Regulatory Element (IRE). The deduced amino acid sequence of SmFer1 shares extensive sequence identities with the H ferritins of a number of fish species and contains the ferroxidase center that is preserved in ferritin H subunits. Examination of tissue specific expression indicated that SmFer1 expression was most abundant in muscle, liver, and blood. Experimental infection with bacterial pathogens induced significant induction of SmFer1; however, the magnitudes of induction effected by Gram-negative pathogens were much higher than that induced by Gram-positive pathogen. Consistently, lipopolysaccharide (LPS) challenge drastically augmented SmFer1 expression. In addition to bacterial pathogens and LPS, poly(I:C) also induced a strong but transient induction of SmFer1 which differs in profile from those induced by bacterial pathogens. In vitro iron-chelating analysis showed that recombinant SmFer1 purified from Escherichia coli was able to bind ferrous iron in a concentration-dependent manner. To examine whether SmFer1, with its iron-chelating capacity, could have any effect on the infection of bacterial pathogens, purified recombinant SmFer1 was subjected to bacteriostatic analysis and proved to be able to inhibit the growth of the fish pathogen Listonella anguillarum which enhanced SmFer1 expression upon infection. Taken together, these results suggest that SmFer1 is likely to play a role in both iron storage and immune defense against microbial infections.

Effect of long-term cryopreservation on physiological characteristics, antioxidant activities and lipid peroxidation of red seabream (Pagrus major) sperm.

The aim of this study was to determine the effect of long-term cryopreservation on physiological characteristics, the antioxidant activities and lipid peroxidation of red seabream sperm which were respectively cryopreserved with 15% dimethylsulfoxide (Me(2)SO) for 1 month, 13 months, 26 months, 48 months and 73 months. The motility and fertility of post-thaw sperm decreased with the storage time going on. The highest motility (87.67±2.52%) was obtained in sperm cryopreserved for 1 month and the lowest (50.67±5.31%) was in sperm for 73 months. There were no significant differences (p<0.05) in fertilization rates of sperm cryopreserved for 1 month (71.33±8.84%), 13 months (69.22±1.02%) and 26 months (60.33±2.33%); however, the sperm fertility decreased significantly for 48 months (47.22±3.89%) and 73 months (39.56±0.69%) storage. In addition, superoxide dismutase (SOD) activities of sperm were at a stable level for less than 26 months storage, then, decreased significantly after 48 months storage. Catalase (CAT) activities of sperm cryopreserved for 13 months, 26 months, 48 months and 73 months were significantly lower than that for 1 month. There were no significant differences in the malondialdehyde (MDA) level of sperm for less than 13 months storage. After 26 months storage, the concentration of MDA increased significantly, and the highest concentration (3.22±0.05 nmol/mgprot) was obtained in 73 months storage sperm.

Ontogenetic development of digestive enzymes and effect of starvation in miiuy croaker Miichthys miiuy larvae.

The ontogenetic development of the digestive enzymes amylase, lipase, trypsin, and alkaline phosphatase and the effect of starvation in miiuy croaker Miichthys miiuy larvae were studied. The activities of these enzymes were detected prior to exogenous feeding, but their developmental patterns differed remarkably. Trypsin activity continuously increased from 2 days after hatching (dah), peaked on 20 dah, and decreased to 25 dah at weaning. Alkaline phosphatase activity oscillated at low levels within a small range after the first feeding on 3 dah. In contrast, amylase and lipase activities followed the general developmental pattern that has been characterized in fish larvae, with a succession of increases or decreases. Amylase, lipase, and trypsin activities generally started to increase or decrease at transitions from endogenous to exogenous feeding or diet changes, suggesting that these enzymatic activities can be modulated by feeding modes. The activities of all the enzymes remained stable from 25 dah onwards, coinciding with the formation of gastric glands and pyloric caecum. These results imply that specific activities of these enzymes underwent changes due to morphological and physiological modifications or diet shift during larval development but that they became stable after the development of the digestive organs and associated glands was fully completed and the organs/glands functioned. Trypsin and alkaline phosphatase were more sensitive to starvation than amylase and lipase because delayed feeding up to 2 days after mouth opening was able to adversely affect their activities. Enzyme activities did not significantly differ among feeding groups during endogenous feeding; however, all activities were remarkably reduced when delayed feeding was within 3 days after mouth opening. Initiation of larvae feeding should occur within 2 days after mouth opening so that good growth and survival can be obtained in the culture.

Nuclear factor 45 of tongue sole (Cynoglossus semilaevis): evidence for functional differentiation between two isoforms in immune defense against viral and bacterial pathogens.

Nuclear factor 45 (NF45) is known to play an important role in regulating interleukin-2 expression in mammals. The function of fish NF45 is largely unknown. In a previous study, we reported the identification of a NF45 (named CsNF45) from half smooth tongue sole (Cynoglossus semilaevis). In the present study, we identified an isoform of CsNF45 (named CsNF45i) from half smooth tongue sole and examined its biological properties in comparison with CsNF45. We found that CsNF45i is a truncated version of CsNF45 and lacks the N-terminal 38 residues of CsNF45. Genetic analysis showed that the CsNF45 gene consists of 14 exons and 13 introns, and that CsNF45 and CsNF45i are the products of alternative splicing. Constitutive expression of CsNF45 and CsNF45i occurred in multiple tissues but differed in patterns. Experimental infection with viral and bacterial pathogens upregulated the expression of both isoforms but to different degrees, with potent induction of CsNF45 being induced by bacterial pathogen, while dramatic induction of CsNF45i being induced by viral pathogen. Transient transfection analysis showed that both isoforms were localized in the nucleus and able to stimulate the activity of IL-2 promoter to comparable extents. To examine their in vivo effects, the two isoforms were overexpressed in tongue sole. Subsequent analysis showed that following viral and bacterial infection, the viral loads in CsNF45i-overexpressing fish were significantly lower than those in CsNF45-overexpressing fish, whereas the bacterial loads in CsNF45-overexpressing fish were significantly lower than those in CsNF45i-overexpressing fish. These results indicate that both CsNF45 and CsNF45i possess immunoregulatory properties, however, the two isoforms most likely participate in different aspects of host immune defense that target different pathogens.

Rock bream (Oplegnathus fasciatus) viperin is a virus-responsive protein that modulates innate immunity and promotes resistance against megalocytivirus infection.

Viperin in mammals is known to be an antiviral protein that inhibits the replication of diverse DNA and RNA viruses. In teleost, viperin homologues have been identified in a large number of species and, in some cases, are stimulated in transcription by viruses. However, the biological significance of fish viperin protein in antiviral immunity has not been investigated. In this study, we identified a viperin homologue from rock bream (Oplegnathus fasciatus) (named OfVip) and examined its expression pattern, subcellular localization, and immune effect. We found that OfVip expression occurred in eight tissues, and experimental challenge of rock bream with the viral fish pathogen megalocytivirus upregulated OfVip expression in kidney, liver, and spleen. OfVip was localized in the endoplasmic reticulum under normal physiological conditions, and viral infection induced subcellular redistribution of OfVip. Transient transfection of cultured fish cells with an OfVip-expressing plasmid caused enhanced cellular resistance against megalocytivirus challenge. Consistently, in vivo study showed that rock bream overexpressing OfVip exhibited significantly reduced viral loads in tissues following experimental infection with megalocytivirus. Furthermore, OfVip upregulated the expression of a wide range of immune genes, including those that are known to participate in antiviral immunity. Taken together, these results indicate for the first time that a teleost viperin is a virus-responsive protein that is modulated in subcellular localization by viral infection, and that viperin regulates the immune reactions of the host fish in a manner that augments resistance against viral infection.

Quantitative real time RT-PCR study of pathogen-induced gene expression in rock bream (Oplegnathus fasciatus): internal controls for data normalization.

Rock bream Oplegnathus fasciatus is an important economic fish species. In this study, we evaluated the appropriateness of six housekeeping genes as internal controls for quantitative real-time PCR (RT-qPCR) analysis of gene expression in rock bream before and after pathogen infection. The expression of the selected genes in eight tissues infected with Vibrio alginolyticus or megalocytivirus was determined by RT-qPCR, and the PCR data were analyzed with geNorm and NormFinder algorithms. The results showed that before pathogen infection, mediator of RNA polymerase II transcription subunit 8 and ß-actin were ranked as the most stable genes across the examined tissues. After bacterial or viral infection, the stabilities of the housekeeping genes varied to significant extents in tissue-dependent manners, and no single pair of genes was identified as suitable references for all tissues for either of the pathogen stimuli. In addition, for the majority of tissues, the most stable genes during bacterial infection differed from those during viral infection. Nevertheless, optimum reference genes were identified for each tissue under different conditions. Taken together, these results indicate that tissue type and the nature of the infectious agent used in the study can all influence the choice of normalization factors, and that the optimum reference genes identified in this study will provide a useful guidance for the selection of internal controls in future RT-PCR study of gene expression in rock bream.

The megalocytivirus-induced protein CsMig1 enhances Cynoglossus semilaevis resistance against viral infection.

Half-smooth tongue sole (Cynoglossus semilaevis) is an important economic fish species cultured in northern China. In this study, we identified and analyzed the expression and function of a megalocytivirus-induced gene, CsMig1, from tongue sole. The deduced amino acid sequence of CsMig1 is composed of 507 residues and contains no conserved domains. Blast analysis identified no close homologues of CsMig1. CsMig1 shares moderate sequence similarities in the N-terminal region with the Gig1 (i.e., grass carp hemorrhagic virus-induced gene) homologues of several teleost species. Quantitative real time RT-PCR analysis showed that constitutive CsMig1 expression occurred, in increasing order, in heart, spleen, muscle, kidney, liver, gill, and gut. Experimental infection with the viral pathogen megalocytivirus upregulated CsMig1 expression in kidney, spleen, and liver in time-dependent manners. Treatment of head kidney lymphocytes with the culture supernatant of megalocytivirus-stimulated cells significantly enhanced CsMig1 expression. When head kidney lymphocytes were transfected with the plasmid that constitutively expresses CsMig1, the cells exhibited significantly increased ability to resist megalocytivirus infection. Taken together, these results indicate that CsMig1 is a virus- and, possibly, interferon-induced novel immune factor that functions in the antiviral immunity of tongue sole.

Macrophage migration inhibitory factor of Sciaenops ocellatus regulates immune cell trafficking and is involved in pathogen-induced immune response.

Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine involved in immunoregulation and inflammation. In this study, we examined the expression and biological function of a MIF, SoMIF, from red drum Sciaenops ocellatus. SoMIF is composed of 115 residues and shares 85-99% overall sequence identities with the MIF of a number of teleost. SoMIF expression was detected in a wide range of tissues and upregulated by bacterial and viral infection in a time-dependent manner. In head kidney (HK) leukocytes, pathogen infection induced SoMIF expression, and the expressed SoMIF was secreted into the extracellular milieu. Recombinant SoMIF (rSoMIF) purified from Escherichia coli inhibited the migration of both HK monocytes and lymphocytes, and this inhibitory effect was abolished by the presence of anti-rSoMIF antibodies. When rSoMIF was administered into red drum, it stimulated the production of reactive oxygen species in HK monocytes both in the presence and absence of pathogen infection. In vivo infection study showed that compared to untreated fish, fish pre-treated with rSoMIF before bacterial infection exhibited significantly lower bacterial loads in blood, kidney, spleen, and liver. Taken together, these results indicate that SoMIF is a secreted protein that regulates immune cell trafficking and is involved in pathogen-induced immune response.

Megalocytivirus-induced proteins of turbot (Scophthalmus maximus): identification and antiviral potential.

Megalocytivirus is an important fish pathogen with a broad host range that includes turbot. In this study, proteomic analysis was conducted to examine turbot proteins modulated in expression by megalocytivirus infection. Thirty five proteins from spleen were identified to be differentially expressed at 2days post-viral infection (dpi) and 7dpi. Three upregulated proteins, i.e. heat shock protein 70 (Hsp70), Mx protein, and natural killer enhancing factor (NKEF), were further analyzed for potential antiviral effect. For this purpose, turbot were administered separately with the plasmids pHsp70, pMx, and pNKEF, which express Hsp70, Mx, and NKEF respectively, before megalocytivirus infection. Viral dissemination and propagation in spleen were subsequently determined. The results showed that the viral loads in fish administered with pNKEF were significantly reduced. To examine the potential of Hsp70, Mx, and NKEF as immunological adjuvant, turbot were immunized with a DNA vaccine in the presence of pHsp70, pMx, or pNKEF. Subsequent analysis showed that the presence of pNKEF and pHsp70, but not pMx, significantly reduced viral infection and enhanced fish survival. Taken together, these results indicate that NKEF exhibits antiviral property against megalocytivirus, and that both NKEF and Hsp70 may be used in DNA vaccine-based control of megalocytivirus infection. BIOLOGICAL SIGNIFICANCE: This study provides the first proteomic picture of turbot in response to megalocytivirus infection. We demonstrated that megalocytivirus infection modulates the expression of turbot proteins associated with various cellular functions, and that one of the upregulated proteins, NKEF, exhibits antiviral effect when overexpressed in vivo, while another upregulated protein, Hsp70, exhibits adjuvant effect when co-immunized with a DNA vaccine. These results add molecular insights into turbot immune response induced by megalocytivirus and provide candidate proteins with application potentials in the control of megalocytivirus-associated disease.

Selection of normalization factors for quantitative real time RT-PCR studies in Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus) under conditions of viral infection.

Disease outbreaks caused by iridoviruses are known to affect farmed flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus). To facilitate quantitative real time RT-PCR (qRT-PCR) analysis of gene expression in flounder and turbot during viral infection, we in this study examined the potentials of 9 housekeeping genes of flounder and turbot as internal references for qRT-PCR under conditions of experimental infection with megalocytivirus, a member of the Iridoviridae family. The mRNA levels of the 9 housekeeping genes in the brain, gill, heart, intestine, kidney, liver, muscle, and spleen of flounder and turbot were determined by qRT-PCR at 24h and 72h post-viral infection, and the expression stabilities of the genes were determined with geNorm and NormFinder algorithms. The results showed that (i) viral infection induced significant changes in the mRNA levels of the all the examined genes in a manner that was dependent on both tissue type and infection stage; (ii) for a given time point of infection, stability predictions made by the two algorisms were highly consistent for most tissues; (iii) the optimum reference genes differed at different infection time points at least in some tissues; (iv) at both examined time points, no common reference genes were identified across all tissue types. These results indicate that when studying gene expression in flounder and turbot in relation to viral infection, different internal references may have to be used not only for different tissues but also for different infection stages.

First characterization of a teleost Epstein-Barr virus-induced gene 3 (EBI3) reveals a regulatory effect of EBI3 on the innate immune response of peripheral blood leukocytes.

Epstein-Barr virus-induced gene 3 (EBI3) encodes a protein that in mammals is known to be a subunit of interleukin (IL)-27 and IL-35, both which regulate cytokine production and inflammatory response. To date, no studies on fish EBI3 have been documented. In this work, we report the identification of an EBI3 homologue, CsEBI3, from tongue sole (Cynoglossus semilaevis) and analysis of its expression and biological effect. CsEBI3 is composed of 245 amino acid residues and possesses a Fibronectin type 3 (FN3) domain that is preserved in lower and higher vertebrates. Expression of CsEBI3 was detected in a wide range of tissues, in particular those of immune relevant organs, and upregulated in a time-dependent manner by experimental challenge with bacterial and viral pathogens. Bacterial infection of peripheral blood leukocytes (PBL) enhanced CsEBI3 expression and caused extracellular secretion of CsEBI3. Purified recombinant CsEBI3 (rCsEBI3) stimulated the respiratory burst activity of PBL and upregulated the expression of IL-1ß, IL-8, Myd88, interferon-induced gene 15, CD28, and chemokines. In contrast, rCsEBI3M, a mutant CsEBI3 that lacks the FN3 domain failed to activate PBL and induced much weaker expression of the immune genes. Treatment of PBL with rCsEBI3, but not with the mutant rCsEBI3M, enhanced cellular resistance against bacterial invasion, whereas antibody blocking of CsEBI3 on PBL significantly reduced cellular resistance against bacterial infection. Taken together, these results indicate for the first time that a teleost EBI3 possesses immunoregulatory property in a manner that is dependent on the conserved FN3 domain, and that CsEBI3 is involved in the innate immune defense of PBL against microbial pathogens.

Overexpression of NF-κB inhibitor alpha in Cynoglossus semilaevis impairs pathogen-induced immune response.

IκBα is a member of the NF-κB inhibitor family that inhibits NF-κB activity by sequestering NF-κB in an inactive form in the cytosol. Unlike mammalian IκBα, which has been extensively studied, very little is known about the function of fish IκBα. In this study, we identified and analyzed an IκBα homologue, CsIκBα from half-smooth tongue sole (Cynoglossus semilaevis), a marine flatfish with important economic value. The deduced amino acid sequence of CsIκBα contains 308 residues and shares 58-82% overall sequence identities with the IκBα of a number of teleosts. In silico analysis identified in CsIκBα conserved domains that in mammals are known to be involved in phosphorylation, ubiquitination, and degradation of IκBα. Quantitative real time RT-PCR detected constitutive expression of CsIκBα in gut, spleen, liver, gill, heart, brain, muscle, and kidney. Experimental challenge with a bacterial pathogen-induced significant inductions of CsIκBα expression in head and trunk kidney, which, however, were transient and much lower in magnitude than that of interleukin-1ß. To examine the effect of unregulated overexpression of CsIκBα in a live fish model, tongue sole were administered via intramuscular injection with plasmid pCNCsIkBa, which constitutively expresses CsIκBα. PCR, RT-PCR, and immunohistochemistry analysis showed that pCNCsIkBa was able to translocate to internal tissues, where transcription and translation of the recombinant CsIκBα took place. Compared to control fish, fish administered with pCNCsIkBa were impaired in the ability to block bacterial dissemination and survival in kidney and exhibited significantly reduced expression of multiple immune genes. These results suggest the possible existence in tongue sole of a NF-κB-IκBα signaling pathway that is negatively regulated by CsIκBα and required for effective defense against bacterial infection.

Cynoglossus semilaevis ISG15: a secreted cytokine-like protein that stimulates antiviral immune response in a LRGG motif-dependent manner.

ISG15 is an ubiquitin-like protein that is induced rapidly by interferon stimulation. Like ubiquitin, ISG15 forms covalent conjugates with its target proteins in a process called ISGylation, which in mammals is known to play a role in antiviral immunity. In contrast to mammalian ISG15, the function of teleost ISG15 is unclear. In this study, we identified and analyzed the function of an ISG15 homologue, CsISG15, from tongue sole (Cynoglossus semilaevis). CsISG15 is composed of 162 residues and possesses two tandem ubiquitin-like domains and the highly conserved LRGG motif found in all known ISG15. Expression of CsISG15 occurred in a wide range of tissues and was upregulated in kidney and spleen by viral and bacterial infection. In vitro study with primary head kidney (HK) lymphocytes showed that megalocytivirus infection caused induction of CsISG15 expression and extracellular release of CsISG15 protein. Purified recombinant CsISG15 (rCsISG15) activated HK macrophages and enhanced the expression of immune genes in HK lymphocytes, both these effects, however, were significantly reduced when the conserved LRGG sequence was mutated to LAAG. Further study showed that the presence of rCsISG15 during megalocytivirus infection of HK lymphocytes reduced intracellular viral load, whereas antibody blocking of CsISG15 enhanced viral infection. Likewise, interference with CsISG15 expression by RNAi promoted viral infection. Taken together, these results indicate that CsISG15, a teleost ISG15, promotes antiviral immune response and that, unlike mammalian ISG15, CsISG15 exerts its immunoregulatory effect in the form of an unconjugated extracellular cytokine. In addition, these results also suggest a role for the LRGG motif other than that in protein conjugation.

Suppressor of cytokine signaling 3 inhibits head kidney macrophage activation and cytokine expression in Scophthalmus maximus.

Proteins of the suppressor of cytokine signaling (SOCS) family function as inducible feedback inhibitors of cytokine signaling via the JAK/STAT pathway. Although several SOCS isoforms have been identified in teleosts, their immunological functions remain largely unknown. In this study, we identified in turbot Scophthalmus maximus a SOCS homologue (named SmSOCS3) of the mammalian SOCS3 type. The deduced amino acid sequence of SmSOCS3 contains 205 residues and shares extensive overall identities (60-82%) with those of known fish SOCS3. In silico analyses revealed that, like typical SOCS3, SmSOCS3 possesses a kinase inhibitor region (KIR), a Src homology 2 (SH2) domain, and a SOCS box domain. Under physiological conditions SmSOCS3 expression was detected, in increasing order, in blood, brain, heart, kidney, liver, spleen, muscle, and gill. Experimental infection of turbot with a bacterial pathogen induced significant SmSOCS3 expression in kidney, spleen, liver, and gill in time-dependent manners. Examination of SmSOCS3 expression in head kidney (HK) macrophages showed that SmSOCS3 transcription was significantly upregulated in the presence of purified recombinant TNF-α. On the other hand, SmSOCS3 overexpression in HK macrophages inhibited the transcription of TNF-α as well as IL-1ß and CC-chemokine. In addition, SmSOCS3 overexpression significantly reduced macrophage respiratory burst activity, nitric oxide production, and bactericidal activity. Taken together, these results suggest that SmSOCS3 is a cytokine-inducible suppressor of pro-inflammatory cytokine signaling in HK macrophages and that regulated expression of SmSOCS3 is required for optimal innate immune response against bacterial infection.