Identification and molecular profiling of a novel homolog of cystatin C from rock bream (Oplegnathus fasciatus) evidencing its transcriptional sensitivity to pathogen infections.

Cystatins are reversible inhibitors of cysteine proteases which show an omnipresent distribution in the life on earth. Although, cystatins with mammalian origin were well characterized and their roles in physiology were reported in details, those from teleostean origin are still underrepresented in literature. However, role of cystatins in fish physiology and immune defense is highlighted in few recent reports. In this study, a cystatin C holmologue from rock bream (Oplegnathus fasciatus); termed RbCytC was identified and molecularly characterized. The complete coding sequence of RbCytC was 387 bp in length, which codes for a polypeptide with 129 amino acids, including a signal peptide of 19 amino acids. The consensus cystatin family signatures including a G residue, turning up towards the N-terminus region, QVVAG motif, locating at the middle of the sequence and the PW motif at the c terminal region was found to be well conserved in RbCytC. Phylogenetic analysis using different cystatin counterparts affirmed the close evolutionary relationship of RbCytC with its teleostan homologs which belong to family 2 cystatins. The predicted molecular model of RbCytC resembled most of the structural features of empirically elucidated tertiary structures for chicken egg white cystatin. According to the qPCR assays, RbCytC showed detectable expression in all fish tissues used in the experiment, with markedly pronounced expression level in liver. Moreover, its basal mRNA expression was up-regulated in liver and spleen tissues by experimental rock bream iridovirus infection, whereas down regulated in the same tissues, post live Edwardsiella tarda injection. Collectively, outcomes of our study validate the structural homology of RbCytC with known cystatin C similitudes, especially those of teleosts and suggest its potential roles in proteolytic processes of rock bream physiology as well as in host immune defense mechanisms.

Molecular cloning, inducible expression with SGIV and Vibrio alginolyticus challenge, and function analysis of Epinephelus coioides PDCD4.

Programmed cell death 4 (PDCD4) in mammals, a gene closely associated with apoptosis, is involved in many biological processes, such as cell aging, differentiation, regulation of cell cycle, and inflammatory response. In this study, grouper Epinephelus coioides PDCD4, EcPDCD4-1 and EcPDCD4-2, were obtained. The open reading frame (ORF) of EcPDCD4-1 is 1413 bp encoding 470 amino acids with a molecular mass of 52.39 kDa and a theoretical pI of 5.33. The ORF of EcPDCD4-2 is 1410 bp encoding 469 amino acids with a molecular mass of 52.29 kDa and a theoretical pI of 5.29. Both EcPDCD4-1 and EcPDCD4-2 proteins contain two conserved MA3 domains, and their mRNA were detected in all eight tissues of E. coioides by quantitative real-time PCR (qRT-PCR) with the highest expression in liver. The expressions of two EcPDCD4s were significantly up-regulated after Singapore grouper iridovirus (SGIV) or Vibrio alginolyticus infection. In addition, over-expression of EcPDCD4-1 or EcPDCD4-2 can inhibit the activity of the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), and regulate SGIV-induced apoptosis. The results demonstrated that EcPDCD4s might play important roles in E. coioides tissues during pathogen-caused inflammation.

Molecular characterization, expression and function analysis of Epinephelus coioides MKK4 response to SGIV and Vibrio alginolyticus infection.

Mitogen-activated protein kinase 4 (MKK4), a member of the MAP kinase family, play important roles in response to many environmental and cellular stresses in mammals. In this study, three MKK4 subtypes, EcMKK4-1, EcMKK4-2 and EcMKK4-3, were obtained from grouper Epinephelus coioides. The open reading frame (ORF) of EcMKK4s are obtained and the EcMKK4s proteins contain highly conserved domains: a S_TKc domain, a canonical diphosphorylation group and two conserved MKKK ATP binding motifs, Asp-Phe-Gly (DFG) and Ala-Pro-Glu (APE). EcMKK4s could be found both in the cytoplasmic and nuclear. The EcMKK4s mRNA were detected in all E. coioides tissues examined with the different expression levels, and the expression were up-regulated during SGIV (Singapore grouper iridescent virus) or Vibrio alginolyticus infection. EcMKK4 could significantly reduce the activation of AP-1 reporter gene. The results suggested that EcMKK4s might play important roles in pathogen-caused inflammation.

Functional differences in the products of two TRAF3 genes in antiviral responses in the Chinese giant salamander, Andrias davidianus.

Tumour necrosis factor receptor associated factor 3 (TRAF3) is a crucial transducing protein for linking upstream receptor signals and downstream antiviral signalling pathways. Previous studies mostly clarified the functions of TRAF3 in mammals, birds and fish, but little is known about the characterization and function of TRAF3 in amphibians. In this study, the molecular and functional identification of two TRAF3 genes, AdTRAF3A and AdTRAF3B, were investigated in the Chinese giant salamander Andrias davidianus. The complete open reading frames (ORFs) of AdTRAF3A and AdTRAF3B were 1698 bp and 1743 bp in length, encoding 565 and 580 amino acids, respectively. Both AdTRAF3A and AdTRAF3B deduced proteins contained a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Phylogenetic analysis showed that the AdTRAF3 protein clustered together with other known TRAF3 proteins. Gene expression analysis showed that AdTRAF3s were broadly distributed in all examined tissues with similar distribution patterns. AdTRAF3s in the blood or spleen positively responded to Giant salamander iridovirus (GSIV) and poly (I:C) induction but exhibited distinct response patterns. Silencing AdTRAF3A/B remarkably suppressed the expression of IFN signalling pathway-related genes when leukocytes were treated with DNA virus and the viral RNA analogue. Moreover, overexpression of AdTRAF3A may induce the activation of the IFN-ß promoter, and the zinc finger, coiled coil and MATH domains of AdTRAF3A were essential for IFN-ß promoter activation. However, the overexpression of AdTRAF3B significantly suppressed IFN-ß promoter activity, and its inhibitory effect was enhanced when the RING finger or MATH domain was deleted. Furthermore, AdTRAF3A rather than AdTRAF3B significantly induced NF-κB activation, implying that AdTRAF3A may function as an enhancer in both the IFN and NF-κB signalling pathways. Taken together, our results suggest that the two TRAF3 genes play different crucial regulatory roles in innate antiviral immunity in Chinese giant salamanders.

Fish RIP1 Mediates Innate Antiviral Immune Responses Induced by SGIV and RGNNV Infection.

Receptor interacting protein 1 (RIP1) is an essential sensor of cellular stress, which may respond to apoptosis or cell survival and participate in antiviral pathways. To investigate the roles of fish RIP1 in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, a RIP1 homolog from orange-spotted grouper (Epinephelus coioides) (EcRIP1) was cloned and characterized. EcRIP1 encoded a 679 amino acid protein that shares 83.28% identity with that of Perca flavescens and contained a homologous N-terminal kinase (S-TKc) domain, a RIP isotype interaction motif (RHIM), and a C-terminal domain (DD). EcRIP1 was predominantly detected in immune tissues, and its expression was induced by RGNNV or SGIV infection in vitro. Subcellular localization showed that EcRIP1 was distributed in the cytoplasm with point-like uniform and dot-like aggregation forms. Overexpression of EcRIP1 inhibited SGIV and RGNNV replication and positively regulated the expression levels of interferon (IFN) and IFN-stimulated genes and pro-inflammatory factors. EcRIP1 may interact with grouper tumor necrosis factor receptor type 1-associated DEATH domain protein (EcTRADD) to promote SGIV-induced apoptosis, and interact with grouper Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-ß (EcTRIF) and participate in Myeloid Differentiation Factor 88 (MyD88)-independent toll-like receptor (TLR) signaling. EcRIP1 may also interact with grouper tumor necrosis factor receptor-associated factors (TRAFs) as intracellular linker proteins and mediate the signaling of various downstream signaling pathways, including NF-κB and IFN. These results suggest that EcRIP1 may inhibit SGIV and RGNNV infection by regulating apoptosis and various signaling molecules. Our study offers new insights into the regulatory mechanism of RIP1-related signaling, and provides a novel perspective on fish diseases mediated by RIP1.

Cloning and characterization of tumor necrosis factor superfamily 15 in rock bream, Oplegnathus fasciatus; phylogenetic, in silico, and expressional analysis.

Tumor necrosis factor superfamily (TNFSF)15 is a member of TNFSF which shares a high homology with other TNFSFs, especially lymphotoxin (LT)-α in teleost. In this study, we have cloned a putative TNFSF15 gene in rock bream which was highly homologous with other fish TNFSF15 and performed bioinformatic analysis to confirm the membership. The RB-TNFSF15 cDNA consists of 3192 bp (193 bp of 5'-untranslated region (UTR), 732 bp of ORF, and 2267 bp of 3'-UTR) and encodes a polypeptide of 243 amino acids containing a predicted TNF superfamily signature with 43-61% identities with fish TNFSF15. The predicted 3D structure was similar to human TNFSF15 with ß barrel structure containing 10 ß strands and 1 α helix while human LT-α and ß contain 10 ß strands and 2 α helices. Consequently, the synteny and phylogenetic analysis of fish TNFSF15 genes and structural similarity of the predicted protein to mammalian TNFSF15 implicate that they can be identified as TNFSF15. In healthy rock bream, RB-TNFSF15 gene expression level was the highest in fin and the lowest in blood. In vitro, TNFSF15 gene expression was up-regulated by lipopolysaccharide, polyinosinic:polycytidylic acid (poly I:C) and rock bream iridovirus (RBIV) in head kidney, while up-regulated by poly I:C and RBIV at later time in spleen. In vivo, RB-TNFSF15 gene expression was up-regulated in head kidney, liver and blood after vaccination with a formalin inactivated RBIV. After challenging with RBIV, RB-TNFSF15 gene expression was up-regulated in unvaccinated group at day 3 post-infection in head kidney. In gill, it was significantly up-regulated in vaccinated group at day 1 post-challenge and all groups at day 7, indicating that RB-TNFSF may play a key role in mucosal immunity during viral infection. Since the regulation mechanism of TNFSF15 gene expression in fish has not yet been elucidated, the present study will help to understand the roles of TNFSF15 in fish immune system.

Transcriptomic profiles reveal that inactivated iridovirus and rhabdovirus bivalent vaccine elicits robust adaptive immune responses against lethal challenge in marbled sleepy goby.

Oxyeleotris marmoratus iridovirus (OMIV) and Oxyeleotris marmoratus rhabdovirus (OMRV) are the two major causative agents of disease leading to massive mortality and severe economic losses in marbled sleepy goby (Oxyeleotris marmoratus) industry. It's urgent to develop an effective vaccine against these fatal diseases. In this study, we developed bivalent inactivated vaccine against OMIV and OMRV and evaluated its protective effect in Oxyeleotris marmoratus. The intraperitoneally vaccinated fish were protected against challenge with OMIV and OMRV with both relative percent survival (RPS) of 100%. In addition, deep RNA sequencing was used to analyze the transcriptomic profiles of the spleen tissues at progressive time points post-vaccination with bivalent inactivated vaccine and challenge with OMIV and OMRV infection. Results showed that adaptive immune response was induced in Oxyeleotris marmoratus injected with bivalent inactivated vaccine. Furthermore, robust adaptive immune responses were also detected in vaccinated fish at 7 d and 2 d post-challenge with OMIV and OMRV. Taken together, these results indicated that bivalent inactivated vaccine activated adaptive immune responses in Oxyeleotris marmoratus, and provided protection against OMIV and OMRV lethal challenge.

Grouper TRADD Mediates Innate Antiviral Immune Responses and Apoptosis Induced by Singapore Grouper Iridovirus (SGIV) Infection.

Tumor necrosis factor (TNF) receptor type 1-associated DEATH domain protein (TRADD) is a TNFR1-associated signal transducer and an essential component of the TNFR1 complex that is involved in activating both apoptotic and nuclear factor (NF)-κB pathways as an adaptor. It also is required for TNFR-1-initiated neuronal apoptosis following in vitro infection with virus as an essential component of the antiviral response. To date, few studies have investigated the function of TRADD in lower vertebrates and its antiviral response to DNA virus infection. In the present study, a TRADD gene (named as EcTRADD) from the orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The full-length cDNA of EcTRADD consists of 1,370 base pairs (bp) and contains a 44 bp 5'-terminal untranslated region (UTR), a 450 bp 3'-UTR including a poly (A) tail, and an 876 bp open reading frame encoding a putative 291 amino acid protein. EcTRADD has two conserved domains of N-terminal domain (TRADD-N) and a death domain (DD). EcTRADD was detected in all examined tissues. EcTRADD was up-regulated in the spleen after infection with Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that EcTRADD and EcTRADD-DD exhibited a clear pattern of discrete and interconnecting cytoplasmic filaments resembling the death-effector filaments, while EcTRADD-N was observed in the cytoplasm. After infection with SGIV, EcTRADD, and EcTRADD-DD were transferred to the nucleus. Overexpression of EcTRADD and its domains inhibited replication of SGIV in vitro. Both EcTRADD and EcTRADD-DD induced the caspase-dependent apoptosis in control and infected cells, while EcTRADD-N inhibited the apoptosis. Additionally, EcTRADD and EcTRADD-DD significantly promoted activation of NF-κB and reporter gene p53, whereas EcTRADD-N had no significant effect on p53. The results may provide new insights into the role of fish TRADD in fish virus infection.

Fish Autophagy Protein 5 Exerts Negative Regulation on Antiviral Immune Response Against Iridovirus and Nodavirus.

Autophagy is an important biological activity that maintains homeostasis in eukaryotic cells. However, little is known about the functions of fish autophagy-related genes (Atgs). In this study, we cloned and characterized Atg5, a key gene in the autophagy gene superfamily, from orange-spotted grouper (Epinephelus coioides) (EcAtg5). EcAtg5 encoded a 275-amino acid protein that shared 94 and 81% identity to seabass (Lates calcarifer) and humans (Homo sapiens), respectively. The transcription level of EcAtg5 was significantly increased in cells infected with red-spotted grouper nervous necrosis virus (RGNNV). In cells infected with Singapore grouper iridovirus (SGIV), EcAtg5 expression declined during the early stage of infection and increased in the late stage. Fluorescence microscopy revealed that EcAtg5 mainly localized with a dot-like pattern in the cytoplasm of grouper cells. Overexpression of EcAtg5 significantly increased the replication of RGNNV and SGIV at different levels of detection, as indicated by increased severity of the cytopathic effect, transcription levels of viral genes, and levels of viral proteins. Knockdown of EcAtg5 decreased the replication of RGNNV and SGIV. Further studies showed that overexpression EcAtg5 activated autophagy, decreased expression levels of interferon related cytokines or effectors and pro-inflammatory factors, and inhibited the activation of nuclear factor κB, IFN-sensitive response element, and IFNs. In addition, ectopic expression of EcAtg5 affected cell cycle progression by hindering the G1/S transition. Taken together, our results demonstrated that fish Atg5 exerted a crucial role in virus replication by promoting autophagy, down-regulating antiviral IFN responses, and affecting the cell cycle.

Negative regulation of the interferon response by finTRIM82 in the orange spotted grouper.

Tripartite motif (TRIM) proteins have been demonstrated to exhibit critical functions in multiple cellular processes, including development, carcinogenesis, and programmed cell death, and are also widely recognized to be important antiviral restriction factors or modulators of immune and inflammatory signaling pathways. However, in teleosts, additional TRIM members have been identified and their functions remain largely unknown. Here, a novel finTRIM gene from orange spotted grouper (EcfinTRIM82) was cloned and characterized. Sequence analysis indicated that EcfinTRIM82 encoded a 575 amino acid peptide which shared 94% and 82% identity with Asian sea bass (Lates calcarifer), and zebrafish (Danio rerio) finTRIM82, respectively. EcfinTRIM82 contained three conserved domains, including a RING, B-Box, and SPRY domain. Using fluorescence microscopy, we found that green fluorescence aggregates were observed in the cytoplasm of EcfinTRIM82-EGFP transfected grouper spleen (GS) cells. As the infection proceeded, EcfinTRIM82 transcription was significantly upregulated in Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) infected GS cells. This suggests that EcfinTRIM82 might be involved in fish virus infection. The in vitro overexpression of EcfinTRIM82 in GS cells significantly enhanced the replication of SGIV and RGNNV, evidenced by increased expression of viral genes, including the SGIV major capsid protein (MCP), VP19, ICP-18, RGNNV coat protein (CP), and RNA-dependent RNA polymerase (RdRp). Furthermore, the ectopic expression of EcfinTRIM82 significantly decreased the expression of interferon (IFN)-related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon stimulated gene 15 (ISG15), ISG56, IFP35, and myxovirus resistance gene (MXI), suggesting that EcfinTRIM82 regulated viral replication via the negative regulation of the host IFN response. In addition, EcfinTRIM82 overexpression substantially decreased the level of proinflammatory cytokine transcription. Furthermore, the ectopic expression of EcfinTRIM82 significantly weakened the melanoma differentiation-associated protein 5 (MDA5), mediator of IRF3 activation (MITA) and mitochondrial antiviral-signaling (MAVS) protein-induced IFN response by detecting the transcription of interferon related cytokines and the promoter activity of IFN. Together, our results demonstrate that finTRIM82 negatively regulates the innate antiviral immune response against grouper virus infection.

Fish Cholesterol 25-Hydroxylase Inhibits Virus Replication via Regulating Interferon Immune Response or Affecting Virus Entry.

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-induced gene that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC), which exerts broad-spectrum antiviral function. To investigate the roles of fish CH25H in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, we cloned and characterized a CH25H homolog from orange-spotted grouper (Epinephelus coioides) (EcCH25H). EcCH25H encoded a 271-amino-acid polypeptide, with 86 and 59% homology with yellow croaker (Larimichthys crocea) and humans, respectively. EcCH25H contained a conserved fatty acid (FA) hydroxylase domain and an ERG3 domain. EcCH25H expression was induced by RGNNV or SGIV infection, lipopolysaccharide (LPS) or poly (I:C) treatment in vitro. Subcellular localization showed that EcCH25H and mutant EcCH25H-M were distributed in the cytoplasm and partly colocalized with the endoplasmic reticulum. SGIV and RGNNV replication was decreased by EcCH25H overexpression, which was reflected in the reduced severity of the cytopathic effect and a decrease in viral gene transcription, but replication of both viruses was increased by knockdown of EcCH25H. Besides, the antiviral activity was dependent on its enzymatic activity. Treatment with 25HC significantly inhibited replication of SGIV and RGNNV. EcCH25H overexpression positively regulated the IFN-related molecules and proinflammatory cytokines, and increased both IFN and ISRE promoter activities. Moreover, 25HC treatment significantly suppressed SGIV and RGNNV entry into host cells. The similar inhibitory effect on SGIV entry was observed in EcCH25H overexpression cells. Taken together, our findings demonstrated that EcCH25H inhibited SGIV and RGNNV infection by regulating IFN signaling molecules, and might also influence viral entry via an effect on cholesterol.

Functional characterization of Cystatin C in orange-spotted grouper, Epinephelus coioides.

Cystatin C is an endogenous inhibitor of cysteine proteases and widely exist in organisms. Several studies in mammals have showed that Cystatin C plays critical role in the immune defense against microorganisms. It is also well known that some fish Cystatin C have important immune regulation functions in inflammatory responses. However, the function of fish Cystatin C in virus infection as well as its underlying molecular mechanisms remain to be elucidated. In the present study, a Cystatin C gene termed Ec-CysC was identified from orange-spotted grouper, Epinephelus coioides. The full-length of Ec-CysC cDNA was 817 bp with a 387 bp open reading frame (ORF) that encoded a 129-amino acid (aa) protein, including 18-aa signal peptide and 111-aa mature polypeptide. The deduced amino acid of Ec-CysC shared three conserved domains containing Glycine at the N-terminus region, QVVAG motif in the middle and PW motif near the C-terminus region. Transcription analysis of the Ec-CysC gene showed its expression in all twelve examined tissues including liver, spleen, kidney, brain, intestine, heart, skin, muscle, fin, stomach, gill and head kidney. Its expression following stimulation with Singapore grouper iridovirus (SGIV) was further tested in spleen, the relative expression of Ec-CysC was significantly up-regulated at 12 h post-infection. The subcellular localization experiment revealed that Ec-CysC was mainly distributed in the cytoplasm in Grouper Spleen (GS) cells. In vitro, Overexpression of Ec-CysC in GS cells significantly reduced the expression of viral genes, namely, ORF162, ORF049 and ORF072. Meanwhile, we found that overexpression of Ec-CysC resulted in upward trend of expression of inflammatory cytokines TNF-a, IL-1ß and IL8 during SGIV infection. Further, SGIV-inducible apoptosis and Caspase-3 activity were also weakened by overexpression Ec-CysC in fathead minnow (FHM) cells. These results indicated that Ec-CysC might have a deeper involvement in fish immune defense, and played important roles in inflammation and apoptosis induced by SGIV.

p38b and JAK-STAT signaling protect against Invertebrate iridescent virus 6 infection in Drosophila.

The fruit fly Drosophila melanogaster is a powerful model system for the study of innate immunity in vector insects as well as mammals. For vector insects, it is particularly important to understand all aspects of their antiviral immune defenses, which could eventually be harnessed to control the transmission of human pathogenic viruses. The immune responses controlling RNA viruses in insects have been extensively studied, but the response to DNA virus infections is poorly characterized. Here, we report that infection of Drosophila with the DNA virus Invertebrate iridescent Virus 6 (IIV-6) triggers JAK-STAT signaling and the robust expression of the Turandots, a gene family encoding small secreted proteins. To drive JAK-STAT signaling, IIV-6 infection more immediately induced expression of the unpaireds, a family of IL-6-related cytokine genes, via a pathway that required one of the three Drosophila p38 homologs, p38b. In fact, both Stat92E and p38b were required for the survival of IIV-6 infected flies. In addition, in vitro induction of the unpaireds required an NADPH-oxidase, and in vivo studies demonstrated Nox was required for induction of TotA. These results argue that ROS production, triggered by IIV-6 infection, leads to p38b activation and unpaired expression, and subsequent JAK-STAT signaling, which ultimately protects the fly from IIV-6 infection.

DNA vaccine encoding myristoylated membrane protein (MMP) of rock bream iridovirus (RBIV) induces protective immunity in rock bream (Oplegnathus fasciatus).

Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream (Oplegnathus fasciatus) in Korea. In this study, we investigated the potential of viral membrane protein to induce antiviral status protecting rock bream against RBIV infection. We found that fish administered with ORF008L (myristoylated membrane protein, MMP) vaccine exhibited significantly higher levels of survival compared to ORF007L (major capsid protein, MCP). Moreover, ORF008L-based DNA vaccinated fish showed significant protection at 4 and 8 weeks post vaccination (wpv) than non-vaccinated fish after infected with RBIV (6.7 × 105) at 23 °C, with relative percent survival (RPS) of 73.36% and 46.72%, respectively. All of the survivors from the first RBIV infection were strongly protected (100% RPS) from re-infected with RBIV (1.1 × 107) at 100 dpi. In addition, the MMP (ORF008L)-based DNA vaccine significantly induced the gene expression of TLR3 (14.2-fold), MyD88 (11.6-fold), Mx (84.7-fold), ISG15 (8.7-fold), PKR (25.6-fold), MHC class I (13.3-fold), Fas (6.7-fold), Fas ligand (6.7-fold), caspase9 (17.0-fold) and caspase3 (15.3-fold) at 7 days post vaccination in the muscle (vaccine injection site). Our results showed the induction of immune responses and suggest the possibility of developing preventive measures against RBIV using myristoylated membrane protein-based DNA vaccine.

The first report of cathepsin A gene in a teleost (rock bream, Oplegnathus fasciatus): An investigation of immune functions upon infection with several pathogens.

We isolated and characterised a cDNA encoding the lysosomal protective protein (serine protease) cathepsin A (CTSA) from rock bream (Oplegnathus fasciatus). The full-length rock bream CTSA (RbCTSA) cDNA (1814 bp) contains an open reading frame of 1419 bp that encodes 472 amino acids. Alignment of multiple CTSA protein sequences revealed that the active site serine and histidine residues were well-conserved among the other CTSA sequences. RbCTSA is highly expressed in the peripheral blood leukocytes, kidney, spleen, liver, intestine, gill, heart, brain, stomach, and eye. RbCTSA expression was also examined in several tissues, including whole kidneys and spleens, under bacterial and viral challenge. In general, all of the examined tissues that were infected with Edwardsiella tarda, Streptococcus iniae, or red sea bream iridovirus (RSIV) exhibited significant upregulation of RbCTSA expression compared to the controls. Our results reveal that RbCTSA may be involved in the immune responses of rock bream.

Characterization of galectin-1 from Chinese giant salamanders Andrias davidianus and its involvements during immune response.

Galectins are considered as a multifunctional protein which play essential roles in cell adhesion and apoptosis, inflammation, tumor progression and immune response. In spite of extensive studies of galectin importance in immune system among different animals, few studies have been devoted to their functions in amphibian. In the present study, we characterized one proto type of galectin (named AdGal1) from Chinese giant salamander Andrias davidianus and studied its function in immune response. AdGal1 cDNA possesses an open reading frame of 598 bp, which encodes a putative galectin of 134 amino acids containing one carbohydrate recognition domains (CRDs). The constitutive expression of mRNA transcripts was detected in a wide range of tissues, with the highest expression in kidney. Immune challenges with Aeromonas hydrophila and Chinese giant salamander iridovirus (GSIV), the transcript level of AdGal1 in kidney was significantly upregulated. The mature protein of AdGal1 was successfully expressed and purified in Escherichia coli BL21 (DE3). The recombinant AdGal1 (rAdGal1) could show bind activity to different Gram negative and Gram positive bacteria. It could also strongly agglutinate different kinds of bacteria at different concentrations. Collectively, these data from the present study indicate that AdGal1 is a vital pattern recognition receptor to recognize different microbes in the innate immune system of Andrias davidianus.

[Prokaryotic expression and antiserum preparation for major antigenic epitope region of major capsid protein of Chinese giant salamander (Andrias davidianus) iridovirus].

Objective To express the fusion protein of major antigenic epitope region of major capsid protein (MCP) of Chinese giant salamander (Andrias davidianus) iridovirus (CGSIV) and prepare the rabbit antiserum. Methods Using the genomic DNA of CGSIV Lueyang strain (CGSIV-LY) as a template, the gene fragment of major antigenic epitope region of MCP was amplified by PCR and cloned into the prokaryotic vector pET-21a(+) to construct the prokaryotic expression recombinant plasmid pET-21a-MCP. The recombinant plasmid was transformed into Escherichia coli BL21(DE3). His-tagged fusion protein was induced by IPTG. After identified by SDS-PAGE and Western blot analysis, the recombinant protein was purified by nitrilotriacetic acid (Ni-NTA) agarose resin. New Zealand rabbits were immunized with the purified recombinant protein to generate antiserum. Specificity and titer of the antiserum were determined by Western blotting and indirect ELISA, and then the antiserum was used to detect the CGSIV in the infected EPC cells by indirect immunofluorescence assay. Results The recombinant protein with the relative molecular mass of 29 000 was expressed. The prepared rabbit antiserum had a good specificity and a high titer. Indirect immunofluorescence assay showed that the antiserum could recognize CGSIV in the infected EPC cells. Conclusion The fusion protein of major antigenic epitope region of MCP of CGSIV is successfully expressed and the rabbit antiserum with a high titer and a good specificity been prepared.

Differential autophagic effects triggered by five different vertebrate iridoviruses in a common, highly permissive mandarinfish fry (MFF-1) cell model.

Autophagy of five vertebrate iridoviruses, including one megalocytivirus (infectious spleen and kidney necrosis virus, ISKNV) and four ranaviruses (Chinese giant salamander iridovirus, CGSIV; Tiger frog virus, TFV; Grouper iridovirus, GIV; and Largemouth bass virus, LMBV) were investigated in a common, highly permissive mandarinfish fry (MFF-1) cell model. The results showed marked autophagosome formation in GIV- and LMBV-infected cells but not in ISKNV-, CGSIV- and TFV-infected MFF-1 cells. Strong evidence for the autophagosomes was provided by transmission electron microscopy, the detection of mandarinfish microtubule-associated protein 1 light chain 3B (mLC3)-based fluorescent dot formation and mLC3-I/mLC3-II conversion was provided by Western blotting. Pharmacological tests indicated that autophagy plays an antiviral role during GIV or LMBV infection. Collectively, our data are the first to show that antiviral autophagic effects can be triggered by GIV and LMBV but not by ISKNV, TFV and CGSIV in a common susceptible cell model. These results suggest that differential host-virus interaction strategies may be utilized against different vertebrate iridoviruses; they also indicate the potential effectiveness of an antiviral treatment that modulates autophagy to control iridoviral infections, such as GIV and LMBV.

Cloning of the Major Capsid Protein (MCP) of Grouper Iridovirus of Taiwan (TGIV) and Preliminary Evaluation of a Recombinant MCP Vaccine against TGIV.

Fish iridoviruses cause systemic diseases with high mortality in various species of wild and farm-raised fish, resulting in severe economic losses. In 1998, we isolated a new epizootic iridovirus in cultured grouper (Epinephelus sp.) in Taiwan, thus named as grouper iridovirus of Taiwan (TGIV). We report here the cloning of TGIV major capsid protein (MCP). Phylogenetic analysis of the iridoviral MCPs confirmed the classification of TGIV into the Megalocytivirus genus. Recombinant TGIV MCP and GIV MCP were then generated to produce polyclonal antibodies. Western blot analysis revealed that the two antisera were species-specific, indicating no common epitope shared by the MCPs of the two viruses. We further assayed the potency of a subunit vaccine containing recombinant TGIV MCP. The vaccine effectively protected grouper from TGIV infection. The result demonstrated that MCP is a suitable antigen for anti-TGIV vaccines.

Comprehensive identification and profiling of host miRNAs in response to Singapore grouper iridovirus (SGIV) infection in grouper (Epinephelus coioides).

microRNAs (miRNAs) are an evolutionarily conserved class of non-coding RNA molecules that participate in various biological processes. Employment of high-throughput screening strategies greatly prompts the investigation and profiling of miRNAs in diverse species. In recent years, grouper (Epinephelus spp.) aquaculture was severely affected by iridoviral diseases. However, knowledge regarding the host immune responses to viral infection, especially the miRNA-mediated immune regulatory roles, is rather limited. In this study, by employing Solexa deep sequencing approach, we identified 116 grouper miRNAs from grouper spleen-derived cells (GS). As expected, these miRNAs shared high sequence similarity with miRNAs identified in zebrafish (Danio rerio), pufferfish (Fugu rubripes), and other higher vertebrates. In the process of Singapore grouper iridovirus (SGIV) infection, 45 and 43 miRNAs with altered expression (>1.5-fold) were identified by miRNA microarray assays in grouper spleen tissues and GS cells, respectively. Furthermore, target prediction revealed 189 putative targets of these grouper miRNAs.