Rock Bream Iridovirus Research Articles

Rock bream iridovirus (RBIV) replication in rock bream (Oplegnathus fasciatus) exposed for different time periods to susceptible water temperatures

Rock bream iridovirus (RBIV) is a member of the Megalocytivirus genus that causes severe mortality to rock bream. Water temperature is known to affect the immune system and susceptibility of fish to RBIV infection. In this study, we evaluated the time dependent virus replication pattern and time required to completely eliminate virus from the rock bream body against RBIV infection at different water temperature conditions. The rock bream was exposed to the virus and held at 7 (group A1), 4 (group A2) and 2 days (group A3) at 23 °C before the water temperature was reduced to 17 °C. A total of 28% mortality was observed 24–35 days post infection (dpi) in only the 7 day exposure group at 23 °C. In all 23 °C exposure groups, virus replication peaked at 20 to 22 dpi (106–107/μl). In recovery stages (30–100 dpi), the virus copy number was gradually reduced, from 106 to 101 with faster decreases in the shorter exposure period group at 23 °C. When the water temperature was increased in surviving fish from 17 to 26 °C at 70 dpi, they did not show any mortality or signs of disease and had low virus copy numbers (below 102/μl). Thus, fish need at least 50 days from peaked RBIV levels (approximately 20–25 dpi) to inhibit the virus. This indicates that maintaining the fish at low water temperature (17 °C) for 70 days is sufficient to eradicate RBIV from fish body. Thus, RBIV could be eliminated slowly from the fish body and the virus may be completely eliminated under the threshold of causing mortality.

Gene expression regulation of the TLR9 and MyD88-dependent pathways in rock bream against rock bream iridovirus (RBIV) infection

Rock bream iridovirus (RBIV), which is a member of the Megalocytivirus genus, causes severe mass mortalities in rock bream in Korea. To date, the innate immune defense mechanisms of rock bream against RBIV is unclear. In this study, we assessed the expression levels of genes related to TLR9 and MyD88-dependent pathways in RBIV-infected rock bream in high, low or no mortality conditions. In the high mortality group (100% mortality at 15 days post infection (dpi)), high levels of TLR9 and MyD88 expressions (6.4- and 2.4-fold, respectively) were observed at 8 d and then reduced (0.6- and 0.1-fold, respectively) with heavy viral loads at 10 dpi (2.21 × 107/μl). Moreover, TRAF6, IRF5, IL1β, IL8, IL12 and TNFα expression levels showed no statistical significance until 10 dpi. Conversely, in the low mortality group (28% expected mortality at 35 dpi), TLR9, MyD88 and TRAF6 expression levels were significantly higher than those in the control group at several sampling points until 30 dpi. Higher levels of IRF5, IL1β, IL8, IL12 and TNFα expression were also observed, however, these were not significantly different from those of the control group. In the no mortality group (0% mortality at 40 dpi), significantly higher levels of MyD88 (2 d, 4 d and 40 dpi), TRAF6 (2 dpi), IL1β (4 dpi) and IL8 (2 d and 4 dpi) expression were observed. In summary, RBIV-infected rock bream induces innate immune response, which could be a major contributing factor to effective fish control over viral transcription. MyD88, TRAF6, IL1β and IL8-related immune responses were activated in fish survivor condition (low or no mortality group). This is a critical factor for RBIV disease recovery; however, these immune responses did not efficiently respond in fish dead condition (high mortality group).

CpG ODN 1668 induce innate and adaptive immune responses in rock bream (Oplegnathus fasciatus) against rock bream iridovirus (RBIV) infection

Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream in Korea. CpG ODN 1668 showed promise as immunoprotective agents against RBIV infection in rock bream. In this study, we assessed innate/adaptive-related gene expression patterns in RBIV-infected rock bream with and without CpG ODN 1668 administration to determine important immune defense related factors that may affect fish survival. In the CpG ODN 1668+virus-injected group, virus copies were more than 7.4- to 790591-fold lower than in the virus-injected group at 4 d (8.79 × 104 and 6.58 × 105/μl, respectively), 7 d (5.30 × 102 and 2.29 × 107/μl, respectively) and 10 dpi (7.79 × 101 and 6.16 × 107/μl, respectively). Furthermore, in the CpG ODN 1668+virus-injected group, significantly higher levels of MyD88 (6 h, 1 d, 4 d and 7 dpi), IL1β (1 d, 2 d and 7 dpi) and perforin/granzyme (1 dpi) expression were observed, whereas these genes were not significantly expressed in the virus-injected group at that time points. Mx, ISG15 and PKR were significantly highly expressed at 4 d and 7 dpi and reduced when low viral loads at 10 dpi in the CpG ODN 1668+virus-injected group. Conversely, in the virus-injected group, Mx, ISG15 and PKR expression were significantly higher than the control group until 10 dpi. However, MHC class I, CD8, Fas, Fas ligand and caspases (3, 8 and 9) expression levels showed no statistically significant differences between virus- and CpG ODN 1668+virus-injected group. In summary, CpG ODN 1668 administration in fish induces innate immune response or cell death pathway, which could be a major contributing factor to effective fish control over viral transcription on 4 d to 10 dpi. Expression of MyD88, IL1β, perforin and granzyme-related immune gene response is critical factor for inhibition of RBIV replication.

Molecular insights of two STAT1 variants from rock bream (Oplegnathus fasciatus) and their transcriptional regulation in response to pathogenic stress, interleukin-10, and tissue injury

Signal transducers and activators of transcription 1 (STAT1) is critically involved in mediating cytokine-driven signaling, and triggers the transcription of target genes to activate cellular functions. Although the structural and functional aspects of STAT members have been well described in mammals, only limited information is available for the STAT genes in teleost fishes. In the present study, two variants of STAT1 genes (RbSTAT1 and RbSTAT1L) were identified from rock bream and characterized at the cDNA and genomic sequence levels. RbSTAT1 and RbSTAT1L were found to share a common domain architecture with mammalian STAT1. Phylogenetic analysis revealed that RbSTAT1 shows a common evolutionary trajectory with other STAT1 counterparts, whereas RbSTAT1L showed a separate path, implying that it could be a novel member of the STAT family. The genomic organizations of RbSTAT1 and RbSTAT1L illustrated a similar exon-intron pattern with 23 exons in the coding sequence. Transcription factor-binding sites, which are mostly involved in the regulation of immune responses, were predicted at the putative promoter regions of the RbSTAT1 and RbSTAT1L genes. SYBR Green qPCR analysis revealed the ubiquitous expression of RbSTAT1 and RbSTAT1L transcripts in different fish tissues with the highest level observed in peripheral blood cells. Significantly modulated transcripts were noted upon viral (rock bream iridovirus [RBIV]), bacterial (Edwardsiella tarda and Streptococcus iniae), and pathogen-associated molecular pattern (lipopolysaccharide and poly I:C) stimulations. The WST-1 cell viability assay affirmed the potential antiviral capacity of RbSTAT1 and RbSTAT1L against RBIV. A possible role of RbSTAT1 and RbSTAT1L in the wound healing process was revealed according to their modulated expression in injured fish. In addition, the transcriptional regulation of RbSTAT1 and RbSTAT1L was analyzed by qPCR following stimulation with rock bream interleukin-10. Taken together, these findings suggest that the STAT1-mediated Janus kinase/STAT pathway might at least in part be involved in the regulatory mechanisms underlying the immune defensive roles against microbial pathogens and the wound healing process.

Protective immunity against rock bream iridovirus (RBIV) infection and TLR3-mediated type I interferon signaling pathway in rock bream (Oplegnathus fasciatus) following poly (I:C) administration

In this study, we evaluated the potential of poly (I:C) to induce antiviral status for protecting rock bream from RBIV infection. Rock bream injected with poly (I:C) at 2 days before infection (1.1 × 104) at 20 °C had significantly higher protection with RPS 13.4% and 33.4% at 100 and 200 μg/fish, respectively, through 100 days post infection (dpi). The addition of boost immunization with poly (I:C) at before/post infection at 20 °C clearly enhanced the level of protection showing 33.4% and 60.0% at 100 and 200 μg/fish, respectively. To investigate the development of a protective immune response, rock bream were re-infected with RBIV (1.1 × 107) at 200 dpi. While 100% of the previously unexposed fish died, 100% of the previously infected fish survived. Poly (I:C) induced TLR3 and Mx responses were observed at several sampling time points in the spleen, kidney and blood. Moreover, significantly high expression levels of IRF3 (2.9- and 3.1-fold at 1 d and 2 days post administration (dpa), respectively), ISG15 and PKR expression (5.4- and 10.2-fold at 2 dpa, respectively) were observed in the blood, but the expression levels were low in the spleen and kidney after poly (I:C) administration. Our results showed the induction of antiviral immune responses and indicate the possibility of developing long term preventive measures against RBIV using poly (I:C).

Protective immunity against Megalocytivirus infection in rock bream (Oplegnathus fasciatus) following CpG ODN administration

Rock bream iridovirus (RBIV) disease in rock bream (Oplegnathus fasciatus) remains an unsolved problem in Korea aquaculture farms. CpG ODNs are known as immunostimulant, can improve the innate immune system of fish providing resistance to diseases. In this study, we evaluated the potential of CpG ODNs to induce anti-viral status protecting rock bream from different RBIV infection conditions. We found that, when administered into rock bream, CpG ODN 1668 induces better antiviral immune responses compared to other 5 CpG ODNs (2216, 1826, 2133, 2395 and 1720). All CpG ODN 1668 administered fish (1/5µg) at 2days before infection (1.1×107) held at 26°C died even though mortality was delayed from 8days (1µg) and 4days (5µg). Similarly, CpG ODN 1668 administered (5µg) at 2days before infection (1.2×106) held at 23/20°C had 100% mortality; the mortality was delayed from 9days (23°C) and 11days (20°C). Moreover, when CpG ODN 1668 administered (1/5/10µg) at 2/4/7days before infection or virus concentration was decreased to 1.1×104 and held at 20°C had mortality rates of 20/60/30% (2days), 30/40/60% (4days) and 60/60/20% (7days), respectively, for the respective administration dose, through 100 dpi. To investigate the development of a protective immune response, survivors were re-infected with RBIV (1.1×107) at 100 and 400 dpi, respectively. While 100% of the previously unexposed fish died, 100% of the previously infected fish survived. The high survival rate of fish following re-challenge with RBIV indicates that protective immunity was established in the surviving rock bream. Our results showed the possibility of developing preventive measures against RBIV using CpG ODN 1668 by reducing RBIV replication speed (i.e. water temperature of 20°C and infection dose of 1.1×104).

Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus

Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (P<0.05) altered transcription of RbSTAT2 was detected after immune challenge experiments with viral (rock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the cytoplasm and translocated near to the nucleus upon poly I:C administration. Altogether, these findings suggest that RbSTAT2 is involved in various biologically crucial mechanisms, and provides immune protection to the rock bream.

A homolog of teleostean signal transducer and activator of transcription 3 (STAT3) from rock bream, Oplegnathus fasciatus: Structural insights, transcriptional modulation, and subcellular localization

Signal transducer and activator of transcription 3 (STAT3) is one of the crucial transcription factors in the Janus kinase (JAK)/STAT signaling pathway, and it was previously considered as acute phase response factor. A number of interleukins (ILs) such as IL-5, IL-6, IL-9, IL-10, IL-12, and IL-22 are known to be involved in activation of STAT3. In addition, various growth factors and pathogenic or oxidative stresses mediate the activation of a wide range of functions via STAT3. In this study, a STAT3 homolog was identified and functionally characterized from rock bream (RbSTAT3), Oplegnathus fasciatus. In silico characterization revealed that the RbSTAT3 amino acid sequence shares highly conserved common domain architectural features including N-terminal domain, coiled coil domain, DNA binding domain, linker domain, and Src homology 2 (SH2) domains. In addition, a fairly conserved transcriptional activation domain (TAD) was located at the C-terminus. Comparison of RbSTAT3 with other counterparts revealed higher identities (>90%) with fish orthologs. The genomic sequence of RbSTAT3 was obtained from a bacterial artificial chromosome (BAC) library, and was identified as a multi-exonic gene (24 exons), as found in other vertebrates. Genomic structural comparison and phylogenetic studies have showed that the evolutionary routes of teleostean and non-teleostean vertebrates were distinct. Quantitative real time PCR (qPCR) analysis revealed that the spatial distribution of RbSTAT3 mRNA expression was ubiquitous and highly detectable in blood, heart, and liver tissues. Transcriptional modulation of RbSTAT3 was examined in blood and liver tissues after challenges with bacteria (Edwardsiella tarda and Streptococcus iniae), rock bream irido virus (RBIV), and immune stimulants (LPS and poly (I:C)). Significant changes in RbSTAT3 transcription were also observed in response to tissue injury. In addition, the transcriptional up-regulation of RbSTAT3 was detected in rock bream heart cells upon recombinant rock bream IL-10 (rRbIL-10) treatment. Subcellular localization and nuclear translocation of rock bream STAT3 following poly (I:C) treatment were also demonstrated. Taken together, the results of the current study provide important evidence for potential roles of rock bream STAT3 in the immune system and wound healing processes.

Expression Analysis of Lily Type Lectin Isotypes in the Rock Bream, Oplegnathus fasciatus: in the Tissue, Developmental Stage and Viral Infection.

ABSTRACTLectins belong to the pattern-recognition receptors (PRRs) class and play important roles in the recognition and elimination of pathogens via the innate immune system. Recently, it was reported that lily-type lectin-1 is involved when a pathogen attacks in the early immune response of fish. However, this study is limited to information that the lectin is involved in the innate immune response against viral infection. In the present study, the lily-type lectin-2 and -3 of Oplegnathus fasciatus (OfLTL-2 and 3) have been presented to be included B-lectin domain and two D-mannose binding sites in the amino acid sequence that an important feature for the fundamental structure. To investigate the functional properties of OfLTLs, the tissue distribution in the healthy rock bream and temporal expression during early developmental stage analysis are performed using quantitative real-time PCR. OfLTL-2 and 3 are predominantly expressed in the liver and skin, but rarely expressed in other organ. Also, the transcripts of OfLTLs are not expressed during the early developmental stage but its transcripts are increased after immune-related organs which are fully formed. In the challenge experiment with RBIV (rock bream iridovirus), the expression of OfLTLs was increased much more strongly in the late response than the early, unlike previously known. These results suggest that OfLTLs are specifically expressed in the immune-related tissues when those organs are fully formed and it can be inferred that the more intensively involved in the second half to the virus infection.

Production and characterization of monoclonal antibodies against recombinant ORF 049L of rock bream iridovirus

Abstract Iridovirus has been detected in more than 20 fish species, caused severe systemic diseases. Rock bream iridovirus (RBIV) is a known causative agent of epizootics in cultured Oplegnathus fasciatus in China. In this study, monoclonal antibodies (mAbs) against the recombinant RBIV protein ORF 049L were employed to further elucidate the pathogenesis of the virus. Briefly, mAbs against recombinant ORF 049L were prepared. Western blot analysis was performed to assess the ability of the mAbs to bind to the recombinant protein, and the sensitivity of mAbs to RBIV-infected rock bream was analyzed by dot blot analysis, an indirect immunofluorescence assay, and one-step PCR analysis. Eight mAbs against recombinant ORF 049L were developed, of which three (3F8, 2H11, and 2B7) were sensitive to RBIV-infected rock bream. These mAbs provide useful tools to analyze the location and replication in vivo of structural and functional proteins, and the pathogenesis of RBIV in aquatic organisms.

Complement factor D homolog involved in the alternative complement pathway of rock bream (Oplegnathus fasciatus): Molecular and functional characterization and immune responsive mRNA expression analysis.

The complement system serves conventional role in the innate defense against common invading pathogens. Complement factor D (CfD) is vital to alternative complement pathway activation in cleaving complement factor B. This catalytic reaction forms the alternative C3 convertase that is crucial for complement-mediated pathogenesis. In this study, rock bream (Oplegnathus fasciatus) CfD (OfCfD) was characterized and OfCfD mRNA expression was investigated. OfCfD encodes 277amino acids (aa) for a 30-kDa polypeptide. A domain analysis of the deduced OfCfD aa sequence showed a single serine protease trypsin superfamily domain, a serine active region, three active sites, and three substrate-binding sites. Pairwise sequence comparisons indicated that OfCfD has the highest identity (84.5%) with Oreochromis niloticus CfD. The phylogenetic tree revealed a common ancestral origin of CfD members, with fish CfD distinct from other vertebrate orthologs. The structural arrangement of the OfCfD gene (2451bp) contained five exons interrupted by four introns. A spatial transcriptional analysis indicated that OfCfD transcripts constitutively expressed in all of the examined rock bream tissues, and that they were highest in the spleen and liver. In addition, OfCfD transcripts were immunologically upregulated by lipopolysaccharide (LPS) (12h p.i.), Streptococcus iniae (12h p.i.), rock bream iridovirus (RBIV) (6-12h p.i.), and poly I:C (6h p.i.) in spleen tissue. OfCfD is a trypsin protease and its recombinant protein showed strong protease activity similar to that of trypsin, indicating its catalytic function in the alternative pathway. Together, our findings suggest that OfCfD might be involved in immune responses in rock bream.

Analysis of immune gene expression in rock bream spleen after rock bream iridovirus infection by transcriptome sequencing

The results of amputation free survival (AFS) of a cohort of 887 caucasian patients is shown.The data is based on further analyses of data presented in Jalkanen et al. (2016) [1]. The 36-month amputation free survival of patients divided in new crural vessel disease classification (Crural Index), aortoiliac TASC II classification, femoropopliteal TASC II classification and most severe segment is presented. Also, in depth demographic data is presented for each Crural Index group Jalkanen et al., 2016 [1].

Thioredoxin domain-containing protein 12 from Oplegnathus fasciatus: Molecular characterization, expression against immune stimuli, and biological activities related to oxidative stress.

Thioredoxin domain-containing protein 12 (TXNDC12) is a small, disulfide-containing protein that belongs to the thioredoxin (TXN) superfamily. In the present study, we identified and characterized a TXNDC12-like gene, designated OfTXNDC12, from rock bream, Oplegnathus fasciatus. OfTXNDC12 consists of seven exons interrupted by six introns. Comparative genomic structural analysis revealed that the TXNDC12 of vertebrates is a structurally conserved gene. The coding sequence of OfTXNDC12 comprises 522 bp, which encodes 173amino acid residues with the conserved thioredoxin active site motif, CGAC, and a probable C-terminal ER retrieval motif, GDEL. Transcriptional analysis of OfTXNDC12 showed the highest concentrations of the mRNA transcript in the liver, implying that it has a significant role in the liver under normal physiological conditions. In comparison, injection of lipopolysaccharide, Edwardsiella tarda, Streptococcus iniae, polyinosinic:polycytidylic acid (poly[I:C]) and rock bream iridovirus mostly triggered greater upregulation of OfTXNDC12 transcript levels in liver than in gill tissue, supporting its potential functional importance in the liver. Insulin disulfide reduction assay showed that the recombinant fusion protein (rOfTXNDC12) possesses significant thioredoxin activity. Treatment of LNCaP cells with the recombinant protein along with H2O2 revealed that rOfTXNDC12 increased the viability of cells and further supported its putative antioxidant capacity. Taken together, the results from our study suggest that OfTXNDC12 encodes for a potent antioxidant involved in redox regulation that shows significant responses to immune stimuli.

Low pathogenicity of flounder iridovirus (FLIV) and the absence of cross-protection between FLIV and rock bream iridovirus.

The genus Megalocytivirus is known to infect a wide range of cultured marine fish. In this study, we examined the pathogenicity of FLIV (Megalocytivirus from olive flounder, genotype III) and RBIV (Megalocytivirus from rock bream, genotype I) to their homologous and heterologous host species. Olive flounder (7.5±1.3cm) injected with FLIV [major capsid protein (MCP) gene copies, 6.8×103 -6.5×106 /fish] at 24°C did not die until 90days post-infection (dpi). The average virus replication in the spleen peaked (1.27× 106 /fish) at 20dpi. Rock bream (6.5±1.5cm) injected with FLIV (8.8×105 and 6.5×106 /fish of MCP copies) showed no mortality until 50dpi. The rock bream that survived after FLIV infection were rechallenged with RBIV at 50dpi had 100% mortality, showing that there is no cross-protection between FLIV and RBIV. Temperature shifting (26°C and 20°C at 12h intervals) did not cause FLIV-specific mortality into olive flounder, but higher virus copies were observed in the fish exposed to higher stocking density. This study demonstrates that FLIV and RBIV have different antigenic and pathogenic characteristics and that FLIV has low pathogenicity to olive flounder.

Cloning and expressional analysis of STAT1 and 3 genes in rock bream, Oplegnathus fasciatus

Rock bream (Oplegnathus fasciatus) aquaculture in Korea has been threatened by Rock bream iridovirus (RBIV), a megalocytivirus widely infected in various fish species. Major symptoms of RBIV infection is acute inflammation and enlargement of spleen. There is no available vaccine for RBIV though closely related red sea bream iridovirus has been successfully prevented by a commercial vaccine. In our previous study, proinflammatory cytokines and IFN1 gene expression was initially upregulated but later down-regulated by RBIV infection. This study was conducted to clone and analyse the expression of STAT (signal transducers and activators of transcription) 1 and 3. STATs are transcription factors involved in the regulation of immune genes including IFNs and the viral SOCS of megalocytivirus is known to play a key role to inhibit immune response by blocking STAT-JAK pathway. In phylogenetic analysis, rock bream STAT1 and 3 genes were highly homologous with those of mandarin fish, Siniperca chuatsi and yellow croaker, Larimichthys crocea. The expressional study of STAT1 and 3 genes was performed in head kidney upon RBIV infection and immune stimulants like LPS or polyI:C in vitro and in vivo. Head kidney cells were aseptically isolated from rock bream and treated with LPS, polyI:C, or different doses of RBIV and analyzed kinetics of gene expressions of rock bream. STAT1 gene expression was significantly upregulated by polyI:C at 4h and sustained at the high expression level until 24 h while upregulated by RBIV at 4 h but down-regulated at 24h. Meanwhile, STAT3 gene expression was not significantly affected by polyI:C though STAT3 gene expression was down-regulated at 24h by RBIV. In vivo study showed the down-regulation of STAT3 gene expression at day 4. Although viral SOCS is known to inhibit the STAT-JAK pathway at the protein level, the downregulation of STAT 1 and 3 gene expressions might be involved in the down-regulation of IFNI and Mx gene expression upon RBIV infection, resulting mass mortality of rock bream.

Analysis of immune gene expression in rock bream spleen after rock bream iridovirus infection by transcriptome sequencing

Rock bream (Oplegnathus fasciatus) aquaculture in Korea has been threatened by Rock bream iridovirus (RBIV), a megalocytivirus widely infected in various fish species. Major symptoms of RBIV infection is acute inflammation and enlargement of spleen. There is no available vaccine for RBIV though closely related red sea bream irido virus has been successfully prevented by a commercial vaccine. Without any effective vaccine for RBIV, it has been causing a huge economic loss in rock bream aquaculture industry in Korea. In this study, differential immune gene expression in spleen of rock bream was assessed by sequencing of whole transcriptome to understand rock bream immune system under RBIV infection at day 4. As a result, 402,848 contigs were obtained after de novo assembly and the fold change was counted for each contigs in infected versus noninfected fish. 96,015 contigs showed differential expression as fold change was higher or lower than 2. Among the differentially expressed contigs, immune related genes like interferon-induced protein 44 showed the fold change of 1582.5 while Fc receptor-like protein 5 showed the fold change of 83.8. IL-1, lamda chain of immunoglobulin (Ig) and Ig alpha chain were down-regulated, while interleukin-10, perforin-1 and inhibitor of nuclear factor kappa-b kinase  were upregulated at day 4 post RBIV infection. Interestingly, proteins in complement system (C1 to C9 (no C4), factor B) were upregulated but complement factor H (a regulator of complement system) was down-regulated, revealing the activation of alternative pathway. Differentially expressed genes were confirmed by Q-PCR and the expression was analysed in vitro and in vivo.

Antimicrobial response of galectin-1 from rock bream Oplegnathus fasciatus: Molecular, transcriptional, and biological characterization.

In this study, we describe the identification and characterization of a proto type galectin, galectin-1, from rock bream Oplegnathus fasciatus (OfGal-1). Galectins are evolutionarily conserved carbohydrate binding lectins that show a wide range of functions related to development and immune physiology. They have been identified as pattern recognition receptors of innate immune system that recognize a broad range of microbes. OfGal-1 cDNA comprised of 993 bp with an open reading frame of 408 bp that encodes 135 amino acids. A single carbohydrate recognition domain was present in the OfGal-1 amino acid sequence. The sequence comparison by multiple and pairwise alignments and the phylogenetic tree emphasized the strong evolutionary conservation of Gal-1. The typical β-sandwich structure was identified from the predicted tertiary structure. The constitutive expression of mRNA transcripts was detected in a wide range of tissues examined, with the highest expression in the heart. Immune challenges with live bacteria (Edwardsiella tarda and Streptococcus iniae), rock bream irido virus, and mitogens (lipopolysaccharide and poly I:C) modulated the expression of OfGal-1 mRNAs in the gills, head kidney, and liver. The recombinant OfGal-1 (rOfGal-1) strongly agglutinatinated the human erythrocytes, and this hemagglutination was inhibited by lactose and D-galactose. A wide range of bacteria (S. iniae, S. parauberis, Escherichia coli, Edwardsiella tarda, Vibrio anguillarum, Vibrio harveyi, and Vibrio tapetis) and a ciliate (Miamiensis avidus) were also effectively recognized by rOfGal-1. Significant antiviral activity against rock bream irido virus was also demonstrated by rOfGal-1. Collectively, results from the present study indicate that OfGal-1 can recognize a wide range of microbes and is a vital pattern recognition receptor in the innate immune system of rock bream.

Molecular, genomic, and expressional delineation of a piscidin from rock bream (Oplegnathus fasciatus) with evidence for the potent antimicrobial activities of Of-Pis1 peptide.

The piscidin family comprises a group of antimicrobial peptides (AMPs) that are vital components of teleost innate immunity. Piscidins protect the host from pathogens, through multifaceted roles as immunomodulators and anti-infective peptides. The present study reports the identification, and characterization of a putative piscidin homolog, Of-Pis1, from rock bream (Oplegnathus fasciatus). A combined genomic and transcriptomic approach revealed that the Of-Pis1 gene comprises 1396 nucleotides (nt), four exons, and three introns. The cDNA with the 213nt open reading frame encoded a 70-amino acid preprotein consisting of a signal peptide, a mature peptide, and a prodomain. Predicted mature Of-Pis1 was assumed to be a membrane-active AMP, based on the prediction of an amphipathic α-helical conformation with a net charge of+4. In addition, Of-Pis1 demonstrated significant similarities with other piscidin family members in terms of gene structure, sequence homology, and evolutionary relationship. Examination by quantitative real-time PCR (qPCR) of basal transcription of Of-Pis1 in the tissues of naïve rock bream, revealed predominant transcript levels in the gills, followed by the spleen, intestine, skin, and head kidney. In gill tissues, the temporally induced mRNA expression of Of-Pis1, upon invivo injection trials with lipopolysaccharide (LPS); polyinosinic:polycytidylic acid (poly I:C); and pathogens, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus (RBIV), was weak. In contrast, invivo flagellin administration led to a robust upregulation of Of-Pis1 in different tissues. Antimicrobial potency was determined by employing recombinant (rOf-Pis1), and synthetic (pOf-Pis1) peptides, in invitro assays. Recombinant overexpression inhibited the growth of bacteria expressing the rOf-Pis1 protein in a growth delay assay. The broad antimicrobial spectrum of pOf-Pis1 was evidenced by its potent activity against an array of microbes, including bacteria, fungi, and parasitic species. In addition, pOf-Pis1 showed no significant hemolytic toxicity against human erythrocytes. Collectively, the data presented in the current study improve our understanding of the piscidin AMP family, and the contribution of Of-Pis1 to the rock bream immunity.

Gene structure, molecular characterization and transcriptional expression of two p38 isoforms (MAPK11 and MAPK14) from rock bream (Oplegnathus fasciatus).

The p38 kinases are one of the four subgroups of mitogen-activated protein kinase (MAPK) superfamily which are involved in the innate immunity. The p38 subfamily that includes four members namely p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13), regulates the activation of several transcription factors. In this study, a p38β (OfMAPK11) homolog and a p38α (OfMAPK14) homolog of Oplegnathus fasciatus were identified at genomic level. Results clearly showed that both MAPK11 and MAPK14 are well-conserved at both genomic structural- and amino acid (aa)-levels. Genomic sequences of OfMAPK11 (∼ 15.6 kb) and OfMAPK14 (∼ 13.4 kb) had 12 exons. A comparison of exon-intron structural arrangement of these genes from different vertebrate lineages indicated that all the exon lengths are highly conserved, except their terminal exons. Full-length cDNAs of OfMAPK11 (3957 bp) and OfMAPK14 (2504 bp) encoded corresponding proteins of 361 aa and 360 aa, respectively. Both OfMAPK proteins harbored a Ser/Thr protein kinases catalytic domain (S_TKc domain) which includes an activation loop with a dual phosphorylation site (TGY motif) and several specific-binding sites for ATP and substrates. Molecular modeling of the activation loop and substrate binding sites of rock bream MAPKs revealed the conservation of crucial residues and their orientation in 3D space. Transcripts of OfMAPKs were ubiquitously detected in eleven tissues examined, however at different levels. The modulation of OfMAPKs' transcription upon pathogen-associated molecular patterns (PAMPs: flagellin, lipopolysaccharide and poly I:C) and pathogens (Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus) was investigated. Among the seven examined tissues, the flagellin-challenge upregulated the mRNA level of both OfMAPKs in the head kidney. Meanwhile, modulation of OfMAPK mRNA expression in the liver upon other immune-challenges varied in a time-dependent manner. Collectively, these results suggest that OfMAPKs are true members of p38 subfamily, which might be induced by different immune stimuli.

Characterization of rock bream (Oplegnathus fasciatus) complement components C1r and C1s in terms of molecular aspects, genomic modulation, and immune responsive transcriptional profiles following bacterial and viral pathogen exposure.

The complement components C1r and C1s play a crucial role in innate immunity via activation of the classical complement cascade system. As initiators of the pathogen-induced signaling cascade, C1r and C1s modulate innate immunity. In order to understand the immune responses of teleost C1r and C1s, Oplegnathus fasciatus C1r and C1s genes (OfC1r and OfC1s) were identified and characterized. The genomic sequence of OfC1r was enclosed with thirteen exons that represented a putative peptide with 704 amino acids (aa), whereas eleven exons of OfC1s represented a 691 aa polypeptide. In addition, genomic analysis revealed that both OfC1r and OfC1s were located on a single chromosome. These putative polypeptides were composed of two CUB domains, an EGF domain, two CCP domains, and a catalytically active serine protease domain. Phylogenetic analysis of C1r and C1s showed that OfC1r and OfC1s were evolutionary close to the orthologs of Pundamilia nyererei (identity = 73.4%) and Oryzias latipes (identity = 58.0%), respectively. Based on the results of quantitative real-time qPCR analysis, OfC1r and OfC1s transcripts were detected in all the eleven different tissues, with higher levels of OfC1r in blood and OfC1s in liver. The putative roles of OfC1r and OfC1s in response to pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus, RBIV) were investigated in liver and head kidney tissues. The transcription of OfC1r and OfC1s was found to be significantly upregulated in response to pathogenic bacterial and viral infections. Overall findings of the present study demonstrate the potential immune responses of OfC1r and OfC1s against invading microbial pathogens and the activation of classical signaling cascade in rock bream.