Search

CD2 is expressed on the surfaces of virtually all T cells and natural killer (NK) cells. In mammals, the CD2 molecule is 50 kDa. The cytoplasmic tail of CD2 interacts with CD2-associated protein (CD2AP), which plays an important role in mediating the trigger signal in outer magnetic pole cells. In this study, we identified CD2AP from rock bream and investigated its gene expression. The ORF of CD2AP (1950 bp) encodes 650 amino acids (aa). CD2AP has a Src homology 3 (SH3) domain. Quantitative real-time PCR analysis revealed that CD2AP shows higher expression in the gills and skin. Under experimental challenge, CD2AP gene expression was increased as relative to the control after 7 days. This result will improve our understanding of blood vessels in teleost fish, and will provide a basis for the study of CD2-related genes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the present study, the pathogenesis of lymphocystis disease virus (LCDV) and the immune gene expression patterns associated with this viral infection were determined in the flatfish Senegalese sole. The results indicate that LCDV spreads rapidly from the peritoneal cavity through the bloodstream to reach target organs such as kidney, gut, liver, and skin/fin. The viral load was highest in kidney and reduced progressively thorough the experiment in spite of the viral major capsid protein gene was transcribed. The LCDV injection activated a similar set of differentially expressed transcripts in kidney and intestine although with some differences in the intensity and time-course response. This set included antiviral-related transcripts (including the mx and interferon-related factors irf1, irf2, irf3, irf7, irf8, irf9, irf10), cytokines (il1b, il6, il8, il12 and tnfa) and their receptors (il1r, il8r, il10r, il15ra, il17r), chemokines (CXC-type, CC-type and IL-8), prostaglandins (cox-2), g-type lysozymes, hepcidin, complement fractions (c2, c4-1 and c4-2) and the antigen differentiation factors cd4, cd8a, and cd8b. The expression profile observed indicated that the host triggered a systemic defensive response including inflammation able to cope with the viral challenge., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tissue factor pathway inhibitor 2 (TFPI-2) is an analog of TFPI-1 and a potent endogenous inhibitor of tissue factor (TF)-mediated blood coagulation. Recent reports have proven that the C-terminal of TFPI-2 peptides in humans and several other vertebrates possesses antibacterial activity against Gram-positive and Gram-negative bacteria. In our previous study, we reported that the TFPI-2 peptide, TC38 in tongue sole (Cynoglossus semilaevis) was active against Micrococcus luteus. In this study, we further examine the antimicrobial spectrum, mechanism of action, and function of TC38 in tongue sole. Our results indicate that TC38 is active against the Gram-negative bacteria Vibrio ichthyoenteri, Vibrio litoralis, Vibrio parahaemolyticus, and Vibrio vulnificus, as well as the fish Megalocytivirus, infectious spleen and kidney necrosis virus (ISKNV). The mechanism of action of TC38 against V. vulnificus was explored. The results showed that TC38 killed V. vulnificus cells without lysis of the cell membrane. FITC-labeled TC38 was able to penetrate the cell membrane and bind to DNA and RNA, then disrupt cellular function, eventually leading to cell death. Administration of TC38 to tongue sole significantly improved its defense against V. vulnificus infection. Overall, these results indicate that TC38 is a novel peptide with a broad antimicrobial spectrum. Furthermore, the unique action of TC38 against V. vulnificus adds new insights to the mechanism of action of vertebrate TFPI peptides. Moreover, TC38 is an interesting antimicrobial agent that could be useful in the fight against pathogenic invasion in aquaculture., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

BNIP3 is a kind of BH3-only protein that induces both cell death and autophagy. Here, a BNIP3 gene (EcBNIP3) was identified from orange spotted grouper, Epinephelus coioides. EcBNIP3 possessed 236 amino acids residues, contained a conservative BNIP3 domain and a transmembrane region. Besides, EcBNIP3 expressed at a relative high level in heart and spleen. EcBNIP3 transcript was up-regulated after SGIV infection in vitro. Subcellular localization analysis revealed that EcBNIP3 was predominantly localized in the cytoplasm and co-localized with mitochondria. In addition, overexpression EcBNIP3 accelerated SGIV infection induced cell death but inhibited viral genes transcription. Taken together, these results provided new evidence that fish BNIP3 might involved in response to virus infection., (Copyright © 2017. Published by Elsevier Ltd.)

North American amphibians have recently been impacted by two major emerging pathogens, the fungus Batrachochytrium dendrobatidis (Bd) and iridoviruses in the genus Ranavirus (Rv). Environmental factors and host genetics may play important roles in disease dynamics, but few studies incorporate both of these components into their analyses. Here, we investigated the role of environmental and genetic factors in driving Bd and Rv infection prevalence and severity in a biodiversity hot spot, the southeastern United States. We used quantitative PCR to characterize Bd and Rv dynamics in natural populations of three amphibian species: Notophthalmus perstriatus, Hyla squirella and Pseudacris ornata. We combined pathogen data, genetic diversity metrics generated from neutral markers, and environmental variables into general linear models to evaluate how these factors impact infectious disease dynamics. Occurrence, prevalence and intensity of Bd and Rv varied across species and populations, but only one species, Pseudacris ornata, harbored high Bd intensities in the majority of sampled populations. Genetic diversity and climate variables both predicted Bd prevalence, whereas climatic variables alone predicted infection intensity. We conclude that Bd is more abundant in the southeastern United States than previously thought and that genetic and environmental factors are both important for predicting amphibian pathogen dynamics. Incorporating both genetic and environmental information into conservation plans for amphibians is necessary for the development of more effective management strategies to mitigate the impact of emerging infectious diseases.

As suggested by the Office International des Epizooties (OIE), fishes belonging to the genus Oplegnathus are more sensitive to megalocytivirus infection than other fish species including red sea bream (Pagrus major). To assess the roles of the innate immune response to these different susceptibilities, we cloned the genes encoding inflammatory factors including IL-8 and COX-2, and the antiviral factor like Mx from red sea bream for the first time and performed phylogenetic and structural analysis. Analysed expression levels of IL-1β, IL-8 and COX-2 and the antiviral factor like Mx genes performed with in vivo challenge experiment showed no difference in inflammatory gene expression or respiratory burst activity between red sea bream and rock bream (Oplegnathus fasciatus). However, the Mx gene expression levels in red sea bream were markedly higher than those in rock bream, suggesting the importance of type I interferon (IFN)-induced proteins, particularly Mx, during megalocytivirus infection, rather than inflammation-related genes. The in vitro challenge experiments using embryonic primary cultures derived from both fish species showed no difference in cytopathic effects (CPE), viral replication profiles, and inflammatory and Mx gene expression pattern between the two fish species., (© 2016 John Wiley & Sons Ltd.)

Water pollutants associated with agriculture may contribute to the increased prevalence of infectious diseases caused by ranaviruses. We have established the amphibian Xenopus laevis and the ranavirus Frog Virus 3 (FV3) as a reliable experimental platform for evaluating the effects of common waterborne pollutants, such as the insecticide carbaryl. Following 3 weeks of exposure to 10 ppb carbaryl, X. laevis tadpoles exhibited a marked increase in mortality and accelerated development. Exposure at lower concentrations (0.1 and 1.0 ppb) was not toxic, but it impaired tadpole innate antiviral immune responses, as evidenced by significantly decreased TNF-α, IL-1β, IFN-I, and IFN-III gene expression. The defect in IFN-I and IL-1β gene expression levels persisted after metamorphosis in froglets, whereas only IFN-I gene expression in response to FV3 was attenuated when carbaryl exposure was performed at the adult stage. These findings suggest that the agriculture-associated carbaryl exposure at low but ecologically-relevant concentrations has the potential to induce long term alterations in host-pathogen interactions and antiviral immunity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tripartite motif-containing 32 (TRIM32) has been demonstrated to pay vital roles in cancer, genetic disorders and antiviral immunity. However, the molecular functions of fish TRIM32 still remained largely unknown. Here, a novel TRIM32 gene from orange spotted grouper (EcTRIM32) was cloned and characterized. EcTRIM32 encoded a 685-aa protein which showed 93%, and 60% identity to large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Amino acid alignment showed that EcTRIM32 contained a conserved RING-finger domain, a BBOX domain and NHL domain. In healthy grouper, the transcript of EcTRIM32 was predominantly detected in brain, liver, intestine, spleen and skin. After injection with Singapore grouper iridovirus (SGIV) and polyI:C, the relative expression of EcTRIM32 in grouper spleen was differently regulated, suggested that EcTRIM32 was involved in antiviral immune response. In transfected grouper spleen (GS) cells, EcTRIM32 displayed bright fluorescence aggregates or spots in the cytoplasm. Notably, the deletion RING domain altered its precise localization and distributed throughout the cytoplasm in GS cells. In EcTRIM32 overexpressing cells, the replication of SGIV or red-spotted grouper nervous necrosis virus (RGNNV) was significantly inhibited compared to the vector control cells. Moreover, the overexpression of EcTRIM32 positively regulated the interferon immune response, evidenced by the significant increase of the expression level of interferon related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon-stimulated gene 15 (ISG15), interferon-induced 35-kDa protein (IFP35), MXI, TIR-domain-containing adaptor-inducing interferon-β (TRIF) and melanoma differentiation-associated protein 5 (MDA5). Further studies showed that overexpression of EcTRIM32 significantly enhanced the MDA5-mediated interferon immune response, but decreased stimulator of interferon genes (STING)-mediated interferon immune response. Meanwhile, the expression levels of pro-inflammation cytokines, including TNFα, IL-6 and IL-8 were up-regulated by the ectopic expression of EcTRIM32. We speculated that the regulation of IRF7, and pro-inflammation cytokines by EcTRIM32 overexpression might contribute critical roles in SGIV infection. In addition, the deletion of RING domain not only significantly weakened the antiviral roles of EcTRIM32, but also obviously affected the regulatory effects of EcTRIM32 on interferon immune and inflammation response. Together, our results firstly demonstrated that fish TRIM32 acted as an antiviral factor against both DNA and RNA virus infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cell cultures of higher organisms, especially cultures of human cells, are increasingly used in medical, pharmaceutical and scientific research. The main problem of cell cultures — non-lethal hidden contamination by mycoplasmas, viruses and outsider cell lines. As an available and reliable method for monitoring the purity of the cell cultures, we offer to use PCR kits designed and officially used in clinical diagnostics. We have tested 50 human cell lines using commercial diagnostic systems for detection of papilloma viruses, herpes viruses, adenoviruses, Mycoplasma hominis and total bacterial mass. Contamination in tested cell lines was not found. In the case of cell lines that contain integrated parts of viral genomes, the presence of the respective DNA sequences was confirmed. The proposed diagnostic systems can be effectively used to control the purity of cell lines, for qualitative detection of possible contamination, as well as for quantitative evaluations with calculation of viral load like it is practiced in clinical diagnostics.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adaptor molecule in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. In our previous study, the molecular characteristics of EtTRAF6 (TRAF6 from Epinephelus tauvina), the tissue distributions, expression patterns after challenging with bacterial and viral pathogens were investigated. Here we identified EtTRAF6 as an important regulator of virus-triggered signaling pathway. Overexpression of EtTRAF6-ORF and truncated forms of EtTRAF6, including EtTRAF6-C (delete the MATH domain), EtTRAF6-N (delete the RING domain) and EtTRAF6-MATH, inhibited IFN-β activity strongly in grouper spleen (GS) cells. Overexpression of EtTRAF6 repressed virus-induced production of type I IFNs. When EtTRAF6 cotransfected with EcIRF3 or EcIRF7, EtTRAF6 inhibited IRF-induced activation of IFN-β. Over-expressed EtTRAF6 inhibited the transcription of SGIV genes significantly in GS cells. Although TRAF6 has a role in apoptosis regulation, it is not known if EtTRAF6 has any role in apoptosis regulation. Strikingly, when over-expressed in fathead minnow (FHM) cells, EtTRAF6 protected them from cell death induced by SGIV. Therefore, these results suggest that TRAF6 may play a critical role in their response to SGIV infection, through regulation of a cell death pathway that is common to fish and humans., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

If the oxygen tension level is 21% in ambient air, it is only between 14% and 1% in vivo. Consequently, viral pathogens are exposed and must adapt to these fluctuating oxygen levels to colonize the host and cause diseases. The problem is that for many years, the virological studies have been performed at 21% oxygen levels and consequently this is a real handicap to have a correct view of the mechanistic aspects of human viral infections. In this brief review, we describe for some selected examples the interactions of human viruses with this relative hypoxia observed in vivo., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Tissue factor pathway inhibitor 1 (TFPI-1) is a serine protease inhibitor that inhibits tissue factor (TF)-mediated coagulation. The C-terminal region of TFPI-1 could be cleaved off and proved to be antimicrobial against a broad-spectrum of microorganism. In a previous study, a C-terminal peptide, TC24 (with 24 amino acids), derived from tongue sole (Cynoglossus semilaevis) TFPI-1, was synthesized and found antibacterial against Micrococcus luteus. In the present study, the antibacterial spectrum and the action mode of TC24 was further examined, and its in vivo function was analyzed. Our results showed that TC24 also possesses bactericidal activity against Staphylococcus aureus and Vibrio vulnificus. During its interaction with the target bacterial cells, TC24 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA and total RNA. In vivo study showed that administration of tongue sole with TC24 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. These results indicated that TC24 is a novel antimicrobial peptide against bacterial and viral pathogens, and that the observed effect of TC24 on bacterial RNA adds new insights to the action mechanism of fish antimicrobial peptides. Moreover, TC24 may play an important role in fighting pathogenic infection in aquaculture., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this review we discuss the role of mimiviruses as potential human pathogens focusing on clinical and evolutionary evidence. We also propose a novel antiviral immunomodulatory pathway controlled by interferon-β (IFN-β) and mediated by immune-responsive gene 1 (IRG1) and itaconic acid, its product. Acanthamoeba polyphaga Mimivirus (APMV) was isolated from amoebae in a hospital while investigating a pneumonia outbreak. Mimivirus ubiquity and role as protist pathogens are well understood, and its putative status as a human pathogen has been gaining strength as more evidence is being found. The study of APMV and human cells interaction revealed that the virus is able to evade the IFN system by inhibiting the regulation of interferon-stimulated genes, suggesting that the virus and humans have had host-pathogen interactions. It also has shown that the virus is capable of growing on IFN-α2, but not on IFN-β-treated cells, hinting at an exclusive IFN-β antiviral pathway. Our hypothesis based on preliminary data and published articles is that IFN-β preferentially upregulates IRG1 in human macrophagic cells, which in turn produces itaconic acid. This metabolite links metabolism to antiviral activity by inactivating the virus, in a novel immunomodulatory pathway relevant for APMV infections and probably to other infectious diseases as well.

Lymphocystis or lymphocystis disease virus (LCDV) is distributed worldwide and affects many fresh and marine water fish species. LCDV is commonly found in aquaria fish species but also in farmed fish species, among them the gilthead seabream (Sparus aurata L.). The immune status of gilthead seabream (S. aurata) specimens under a natural outbreak of LCDV was studied. The replication of the virus was demonstrated in infected fish, but not in control fish. The results showed decreased total serum IgM levels and increased innate cellular immune response (peroxidase and respiratory burst activities) of head kidney leucocytes in LCDV-infected fish, compared to the values obtained in uninfected specimens. In addition, transcription of antiviral genes (ifn and irf3) was down-regulated in the skin of LCDV-positive fish as well as genes involved in cellular immunity (csf1r, mhc2a, tcra and ighm) that were down-regulated in skin and head kidney of infected fish. By contrast, the transcription of nccrp1 was up-regulated in head kidney after LCDV infection. These present results show that head kidney leucocytes are activated to encounter the virus at the sites of replication., (© 2016 John Wiley & Sons Ltd.)

Tripartite motif 16 (TRIM16), has been demonstrated to act as a tumor suppressor through affecting cell proliferation and migration or tumorigenicity in carcigenesis. However, the roles of TRIM16 in immune response were unknown up to now. Here, we cloned a TRIM16-like gene (TRIM16L) from orange spotted grouper (EcTRIM16L) and investigated its roles in response to virus infection. EcTRIM16L encoded a 478 amino acid peptide which showed 72% and 29% identity to large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Sequence alignments indicated that EcTRIM16L shared the different gene structures with human TRIM16, evidenced by the presence of RING domain, but absence of the B-box domain. In transfected grouper cells, the green fluorescence mainly distributed in cytoplasm, and the deletion of SPRY domain affected the accurate localization of EcTRIM16L. In response to different stimuli, including infection with Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis (RGNNV), and transfection with b-DNA or poly I:C, the transcript of EcTRIM16L were differently regulated in grouper spleen cells. After incubation with SGIV, the ectopic expression of EcTRIM16L significantly enhanced the viral replication, demonstrated by the increase of cytopathic effect (CPE) severity and viral gene transcriptions. Simultaneously, we also found that overexpression of EcTIRM16L in vitro significantly weakened the expression of interferon related molecules, including interferon regulatory factor 3 (IRF3), IRF7, and melanoma differentiation-associated protein 5 (MDA5). Moreover, the ectopic expression of EcTRIM16L significantly decreased both MDA5-and mediator of IRF3 activation (MITA)-induced interferon immune responses. Further studies showed that the RING domain played more important roles in the molecular action of EcTIRM16L during grouper virus infection. Our data, for the first time, demonstrated that fish TRIM16L exerted negative regulation on the interferon immune response against DNA virus infection., (Copyright © 2016. Published by Elsevier Ltd.)

In mammalian cells, early defenses against infection by pathogens are mounted through a complex network of signaling pathways shepherded by immune-modulatory pattern-recognition receptors. As obligate parasites, the survival of viruses is dependent on the evolutionary acquisition of mechanisms that tactfully dismantle and subvert the cellular intrinsic and innate immune responses. Here, we review the diverse mechanisms by which viruses that accommodate DNA genomes are able to circumvent activation of cellular immunity. We start by discussing viral manipulation of host defense protein levels by either transcriptional regulation or protein degradation. We next review viral strategies used to repurpose or inhibit these cellular immune factors by molecular hijacking or by regulating their post-translational modification status. Additionally, we explore the infection-induced temporal modulation of apoptosis to facilitate viral replication and spread. Lastly, the co-evolution of viruses with their hosts is highlighted by the acquisition of elegant mechanisms for suppressing host defenses via viral mimicry of host factors. In closing, we present a perspective on how characterizing these viral evasion tactics both broadens the understanding of virus-host interactions and reveals essential functions of the immune system at the molecular level. This knowledge is critical in understanding the sources of viral pathogenesis, as well as for the design of antiviral therapeutics and autoimmunity treatments.

A novel grouper immune gene, EcVig was identified in orange-spotted grouper (Epinephelus coioides). We recently determined that EcVig expression can be induced by infection with nervous necrosis virus (NNV, an RNA virus), whereas NNV replication may be suppressed when EcVig was overexpressed. Although EcVig appeared to be involved in grouper antiviral activity, its immune effects have not been well characterized. In the present study, two PAMPs (pathogen-associated molecular patterns; lipopolysaccharides [LPS] and synthetic double-stranded RNA polyriboinosinic-polyribocytidylic acid [poly(I:C)]), as well as fish DNA virus (red sea bream iridovirus, RSIV; grouper iridovirus, GIV), were used to study EcVig responses in orange-spotted grouper. In addition, groupers were given recombinant type I interferon to determine whether EcVig expression was induced. Poly(I:C) rapidly induced substantial expression of EcVig, whereas LPS stimulation did not appear to have any effect in grouper intestine. Expression levels of total EcVig and other IFN-stimulated genes (ISGs) were all significantly increased after RSIV and GIV infection. Furthermore, stimulation of recombinant type I IFN also increased EcVig expression. We conclude that EcVig may be a novel IFN-stimulated gene that demonstrates an antiviral immune response., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tissue factor pathway inhibitors (TFPIs) are Kunitz-type serine protease inhibitors that reversibly regulate the blood coagulation induced by tissue factor. TFPI family contain two members, TFPI-1 and TFPI-2. Recent studies have shown TFPI-1 and TFPI-2 also play important roles in innate immunity, however, the potential function of teleost TFPI are very limited. In this study, we characterized two TFPI (CsTFPI-1 and CsTFPI-2) molecules from half-smooth tongue sole (Cynoglossus semilaevis), examined their tissue distributions and expression patterns under pathogens stimulation as well as investigated the antibacterial activity of the C-terminal peptides. Quantitative real time RT-PCR analysis showed that constitutive CsTFPI-1 expression occurred, in increasing order, in head kidney, intestine, brain, spleen, liver, skin, gills, heart, and muscle; CsTFPI-2 was expressed, in increasing order, in the gills, intestine, skin, head kidney, liver, brain, spleen, muscle, and heart. Under Vibrio anguillarum, Streptococcus agalactiae and fish megalocytivirus stimulation, both CsTFPI-1 and CsTFPI-2 expression increased significantly in a manner that depended on the pathogen, tissue type, and infection stage, which suggested CsTFPI-1 and CsTFPI-2 play important roles in anti-bacterial and anti-viral infection. Finally, C-terminal peptides of CsTFPI-1 and CsTFPI-2, were synthesized and proved to have antibacterial effect against Micrococcus luteus that were independent of host serum. Take together, these results indicate that CsTFPI-1 and CsTFPI-2 play important roles in antimicrobial immunity of this fish., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mitogen-activated protein kinase 7 (MKK7) is one of the major stress-activated protein kinase (SAPK)-activating kinases in response to environmental or physiological stimuli. Here a MKK7 named as Ec-MKK7 was identified from orange-spotted grouper, Epinephelus coioides. The full-length cDNA of Ec-MKK7 was 1853 bp, with an open reading frame (ORF) of 1272 bp encoding a putative protein of 423 amino acids. A characteristic S-K-A-K-T motif was contained in the domain of dual-specificity protein kinase, mitogen-activated protein kinase kinase 7 (PKc_MKK7). Intracellular localization showed that Ec-MKK7 was localized in both the cytoplasm and the nucleus of grouper spleen (GS) and/or grouper brain (EAGB) cells. Moreover, Ec-MKK7 was universally expressed in all examined tissues and showed expression modulation to challenges of lipopolysacchride (LPS), Singapore grouper iridovirus (SGIV) and polyriboinosinic polyribocytidylic acid (poly I:C) in vivo. A gene targeting strategy over-expressing Ec-MKK7 was performed to examine the activities of MKK7 to viral infection in vitro. Our data showed that Ec-MKK7 was involved in the evasion and replication of SGIV but played an antiviral role to the infection of nervous necrosis virus (NNV). All results demonstrated that Ec-MKK7 could play important roles in grouper innate immunity and show distinct functions on virus infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pentraxin 1 (PTX1) is a member of the pentraxin protein family, which plays important roles in the innate immunity of vertebrates. In fish, the biological function of PTX1 is essentially unknown. In this study, we examined the expression and function of a PTX homologue (CsPTX1) from the tongue sole, Cynoglossus semilaevis. CsPTX1 contains 223 amino acids and shares 49.3%-38.8% overall sequence identity with other known fish pentraxins. CsPTX1 is expressed in multiple tissues and is upregulated by bacterial and viral infection. CsPTX1 contains a pentraxin domain, which is known to bind extracellular antigens, and recombinant CsPTX1 (rCsPTX1) bound a wide range of Gram-positive and Gram-negative bacteria. rCsPTX1 also agglutinated all the bacteria tested in a Ca(2+)-dependent manner and the agglutinating capacity of rCsPTX1 was abolished in the absence of calcium. As well as its ability to agglutinate bacterial cells, rCsPTX1 displayed apparent bacteriostatic activity against Pseudomonas fluorescens in vitro by influencing the permeability of the microbial envelope. When introduced in vivo, rCsPTX1 enhanced the host's resistance to bacterial infection. These results indicate that CsPTX1 is a classic pattern recognition molecule that defends C. semilaevis against bacterial infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Laboratory of genetics and physiology 2 (LGP2), a member of RIG-I like receptor (RLR) family, plays crucial roles in modulating cellular antiviral response during viral infection. However, the detailed roles of LGP2 in different virus infection were controversial up to now. Here, we cloned a LGP2 gene from orange-spotted grouper (EcLGP2) and investigated its roles in response to grouper virus infection. EcLGP2 encoded a 678-aa protein which shared 83% identity to sea perch (Lateolabrax japonicas). Amino acid alignment showed that EcLGP2 contained three conserved domains, including a DEAD/DEAH box helicase domain, a helicase superfamily C-terminal domain and a C-terminal domain of RIG-I. In healthy grouper, the transcript of EcLGP2 could be predominantly detected in kidney, gill, fin, spleen and skin. Subcellular localization analysis showed that EcLGP2 distributed throughout the cytoplasm in grouper cells. Notably, the intracellular distribution of EcLGP2 was altered at the late stage of Singapore grouper iridovirus (SGIV) infection, but remained unchanged during red-spotted grouper nervous necrosis virus (RGNNV) infection. Moreover, overexpression of EcLGP2 in vitro significantly enhanced the viral replication of SGIV and RGNNV, evidenced by the acceleration of CPE occurrence and the up-regulation of the viral gene transcription or protein synthesis. Further studies indicated that overexpression of EcLGP2 decreased the expression level of interferon related molecules or effectors, including IRF3, IRF7, ISG15, IFP35, MXI, MXII, and MDA5, suggesting that the negative feedback of interferon immune response by EcLGP2 might contribute to the enhancement of RGNNV infection. Moreover, the expression levels of pro-inflammation cytokines, including IL-8 and TNFα were significantly decreased, but that of IL-6 was increased by the ectopic expression of EcLGP2. Thus, our results will contribute greatly to understanding the roles of fish LGP2 in innate immune response during iridovirus and nodavirus infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The tripartite motif (TRIM)-containing proteins have attracted particular attention to their multiple functions in different biological processes. TRIM13, a member of the TRIM family, is a RING domain-containing E3 ubiquitin ligase which plays critical roles in diverse cellular processes including cell death, cancer and antiviral immunity. In this study, a TRIM13 homolog from orange spotted grouper, Epinephelus coioides (EcTRIM13) was cloned and characterized. The full-length of EcTRIM13 cDNA encoded a polypeptide of 399 amino acids which shared 81% identity with TRIM13 homolog from large yellow croaker (Larimichthys crocea). Amino acid alignment analysis showed that EcTRIM13 contained conserved RING finger and B-box domain. Expression patterns analysis indicated that EcTRIM13 was abundant in liver, spleen, kidney, intestine and gill. Moreover, the transcript of EcTRIM13 in grouper spleen was differently regulated after injection with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C). Under fluorescence microscopy, we observed the tubular structure in wild type EcTRIM13 transfected cells, but the RING domain mutant resulted in the fluorescence distribution was changed and the bright punctate fluorescence was evenly situated throughout the cytoplasm, suggesting that the RING domain was essential for its accurate localization. Overexpression of EcTRIM13 in vitro obviously increased the replication of red spotted grouper nervous necrosis virus (RGNNV), and the enhancing effect of EcTRIM13 on virus replication was affected by the RING domain. Furthermore, the ectopic expression of EcTRIM13 not only negatively regulated the interferon promoter activity induced by interferon regulator factor (IRF) 3, IRF7, and melanoma differentiation-associated protein 5 (MDA5), but also decreased the expression of several interferon related factors. In addition, the overexpression of EcTRIM13 also differently regulated the transcription of pro-inflammatory factors. Together, our results firstly demonstrated that fish TRIM13 exerted negative regulation of antiviral response against nodavirus infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

While recent findings suggest that swine TTVs (TTSuVs) can act as primary or co-infecting pathogens, very little is known about viral immunity. To determine whether TTSuVs downregulate key host immune responses to facilitate their own survival, a swine macrophage cell line, 3D4/31, was used to over-express recombinant TTSuV1 viral particles or the ORF3 protein. Immune gene expression profiles were assessed by a quantitative PCR panel consisting of 22 immune genes, in cell samples collected at 6, 12, 24 and 48h post-transfection. Despite the upregulation of IFN-β and TLR9, interferon stimulated innate genes and pro-inflammatory genes were not upregulated in virally infected cells. The adaptive immune genes, IL-4 and IL-13, were significantly downregulated at 6h post-transfection. The ORF3 protein did not appear do not have a major immuno-suppressive effect, nor did it stimulate anti-viral immunity. Data from this study warrants further investigation into the mechanisms of TTV related immuno-pathogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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 277 amino 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 (2451 bp) 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) (12 h p.i.), Streptococcus iniae (12 h p.i.), rock bream iridovirus (RBIV) (6-12 h p.i.), and poly I:C (6 h 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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tripartite motif-containing 25 (TRIM25) has been demonstrated to exert crucial roles in the regulation of innate immune signaling. However, the roles of fish TRIM25 in antiviral immune response still remained uncertain. Here, a novel fish TRIM25 gene from orange spotted grouper (EcTRIM25) was cloned and its roles in grouper virus infection were elucidated. EcTRIM25 encoded a 734-aa protein which shared 68% identity to large yellow croaker (Larimichthys crocea). Amino acid alignment showed that EcTRIM25 contained three conserved domains, including a RING-finger domain, a B box/coiled-coil domain and a SPRY domain. In healthy grouper, the transcript of EcTRIM25 was predominantly detected in skin, spleen and intestine. After stimulation with Singapore grouper iridovirus (SGIV) or poly I:C, the relative expression of EcTRIM25 in grouper spleen was significantly increased at the early stage of injection. Subcellular localization analysis showed that EcTRIM25 distributed throughout the cytoplasm in grouper cells. Notably, the deletion RING domain affected its accurate localization and displayed microtubule like structures or bright aggregates in GS cells. After incubation with SGIV or red spotted grouper nervous necrosis virus (RGNNV), overexpression of full length of EcTRIM25 in vitro significantly decreased the viral gene transcription of SGIV and RGNNV. Consistently, the deletion of RING domain obviously affected the inhibitory effect of EcTRIM25. Furthermore, overexpression of EcTRIM25 significantly increased the expression level of interferon related signaling molecules, including interferon regulatory factor (IRF) 3, interferon-induced 35-kDa protein (IFP35), MXI, IRF7 and myeloid differentiation factor 88 (MyD88), suggesting that the positive regulation of interferon immune response by EcTRIM25 might affected RGNNV replication directly. Meanwhile, the expression levels of pro-inflammation cytokines were differently regulated by the ectopic expression of EcTRIM25. We proposed that the regulation of IRF7, MyD88 and pro-inflammation cytokines might contribute more important roles in SGIV infection. In addition, the RING domain of EcTRIM25 also played critical roles in the regulation of interferon immune and inflammation response. Together, our results will provide new evidences that the RING domain was essential for the antiviral action of fish TRIM25 against iridovirus and nodavirus infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The tripartite motif (TRIM)-containing proteins usually exert important regulatory roles during multiple biological processes. TRIM8 has been demonstrated to be a RING domain-containing E3 ubiquitin ligase which plays critical roles in inflammation and cancer. In this study, a TRIM8 homolog from grouper, Epinephelus coioides (EcTRIM8) was cloned, and its effects on fish virus replication were investigated. The full-length EcTRIM8 cDNA encoded a polypeptide of 568 amino acids with 92% identity to TRIM8 homolog from large yellow croaker (Larimichthys crocea). Sequence alignment analysis indicated that EcTRIM8 contained conserved RING finger, B-box and coiled-coil domain. Expression patterns analysis showed that EcTRIM8 was predominant in kidney, gill, fin, liver, spleen and brain. After challenging with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C), the EcTRIM8 transcript was significantly increased at the early stage of injection. Under fluorescence microscopy, we observed different distribution patterns of EcTRIM8 in grouper spleen (GS) cells, including punctate fluorescence evenly situated throughout the cytoplasm and bright aggregates. The ectopic expression of EcTRIM8 in vitro significantly inhibited the replication of SGIV and red spotted grouper nervous necrosis virus (RGNNV), evidenced by the obvious reduction in the severity of cytopathic effect (CPE) and the significant decrease in viral gene transcription and protein synthesis. Moreover, the transcription of the proinflammatory factors and interferon related immune factors were differently regulated by EcTRIM8 during SGIV or RGNNV infection. In addition, overexpression of EcTRIM8 significantly increased the transcription of interferon regulator factor 3 (IRF3) and IRF7, and enhanced IRF3 or IRF7 induced interferon-stimulated response element (ISRE) promoter activity. Together, our results firstly demonstrated that fish TRIM8 could exert antiviral function through the regulation of the expression of proinflammatory cytokines and interferon related transcription factors in response to fish viruses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Melanoma differentiation-associated gene 5 (MDA5) is a critical member of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family which can recognize viral RNA and enhances antiviral response in host cells. In this study, a MDA5 homolog from orange spotted grouper (Epinephelus coioides) (EcMDA5) was cloned, and its roles on grouper virus infection were characterized. The full-length EcMDA5 cDNA encoded a polypeptide of 982 amino acids with 74% identity with MDA5 homolog from rock bream (Oplegnathus fasciatus). Amino acid alignment analysis indicated that EcMDA5 contained three functional domains: two caspase activation and recruitment domain (CARDs), a DEAD box helicase-like (DExDc) domain, a helicase superfamily C-terminal domain (HELICc), and a C-terminal regulatory domain (RD). Upon challenge with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C), the transcript of EcMDA5 was significantly up-regulated especially at the early stage post-injection. Under fluorescence microscopy, we observed that EcMDA5 mostly localized in the cytoplasm of grouper spleen (GS) cells. Interestingly, during virus infection, the distribution pattern of EcMDA5 was significantly altered in SGIV infected cells, but not in red spotted grouper nervous necrosis virus (RGNNV) infected cells, suggested that EcMDA5 might interact with viral proteins during SGIV infection. The ectopic expression of EcMDA5 in vitro obviously delayed virus infection induced cytopathic effect (CPE) progression and significantly inhibited viral gene transcription of RGNNV and SGIV. Moreover, overexpression of EcMDA5 not only significantly increased interferon (IFN) and IFN-stimulated response element (ISRE) promoter activities in a dose dependent manner, but also enhanced the expression of IRF3, IRF7 and TRAF6. In addition, the transcription level of the proinflammatory factors, including TNF-α, IL-6 and IL-8 were differently altered by EcMDA5 overexpression during SGIV or RGNNV infection, suggesting that the regulation on proinflammatory cytokines by EcMDA5 were also important for RGNNV infection. Together, our results demonstrated for the first time that the inhibitory effect of fish MDA5 on iridovirus replication might be mainly through the regulation of proinflammatory cytokines., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

As a member of animal lectin family, galectin has the functions of pathogen recognition, anti-bacteria and anti-virus. In the present study, a galectin-1 homolog (EcGel-1) from grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcGel-1 is 504 bp, including a 408 bp open reading frame (ORF) which encodes 135 amino acids with a molecular mass of 15.19 kDa. Quantitative real-time PCR analysis indicated that EcGel-1 was constitutively expressed in all analyzed tissues of healthy grouper. The expression of EcGel-1 in the spleen of grouper was differentially up-regulated challenged with Singapore grouper iridovirus (SGIV), poly (I:C), and LPS. EcGel-1 was abundantly distributed in the cytoplasm in GS cells. Recombinant EcGel-1(rEcGel-1) protein can make chicken erythrocyte aggregation, and combine with gram negative bacteria and gram positive bacteria in the presence of 2-Mercaptoethanol (β-ME). Taken together, the results showed that EcGel-1 may be an important molecule involved in pathogen recognition and pathogen elimination in the innate immunity of grouper., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the four years following the first detection of ranavirus (genus Ranavirus, family Iridoviridae) infection in Dutch wildlife in 2010, amphibian mortality events were investigated nationwide to detect, characterize and map ranaviruses in amphibians over time, and to establish the affected host species and the clinico-pathological presentation of the disease in these hosts. The ultimate goal was to obtain more insight into ranavirus disease emergence and ecological risk. In total 155 dead amphibians from 52 sites were submitted between 2011 and 2014, and examined using histopathology, immunohistochemistry, virus isolation and molecular genetic characterization. Ranavirus-associated amphibian mortality events occurred at 18 sites (35%), initially only in proximity of the 2010 index site. Specimens belonging to approximately half of the native amphibian species were infected, including the threatened Pelobates fuscus (spadefoot toad). Clustered massive outbreaks involving dead adult specimens and ranavirus genomic identity indicated that one common midwife toad virus (CMTV)-like ranavirus strain is emerging in provinces in the north of the Netherlands. Modelling based on the spatiotemporal pattern of spread showed a high probability that this emerging virus will continue to be detected at new sites (the discrete reproductive power of this outbreak is 0.35). Phylogenetically distinct CMTV-like ranaviruses were found in the south of the Netherlands more recently. In addition to showing that CMTV-like ranaviruses threaten wild amphibian populations not only in Spain but also in the Netherlands, the current spread and risk of establishment reiterate that understanding the underlying causes of CMTV-like ranavirus emergence requires international attention.

Torque Teno Viruses (TTVs) are ubiquitous viruses which are highly prevalent in several mammalian species. Human TTV's are epidemiologically associated with several human disease conditions such as respiratory illnesses, auto-immune disorders and hepatitis. Recently it was found that swine TTV's (TTSuVs) can act as primary pathogens. The common occurrence of TTVs as environmental contaminants and the increasing interest in the use of swine organs for xenotransplantation lend importance to the question of whether TTV's can cross-infect across species. In this study, we examined human and swine sera by swine or human TTV-specific PCRs, to determine whether swine TTVs (TTSuV) DNA can be detected in humans and vice versa. Surprisingly, both human and TTSuV DNA were present in a majority of the samples tested. Transfection of human PBMC's with TTSuV1 genomic DNA resulted in productive viral infection which was sustained for the three serial passages tested. Lymphoproliferative responses in infected human PBMCs were diminished when compared to the controls. Furthermore, mild to moderate antibody responses against the TTSuV1 ORF2 protein was detected in 16 of the 40 human sera by ELISA. Therefore, these study findings provide initial and fundamental evidence for possible cross-species transmission of TTVs.

The world production of shrimp such as the Malaysian giant freshwater prawn, Macrobrachium rosenbergii is seriously affected by the white spot syndrome virus (WSSV). There is an urgent need to understand the host pathogen interaction between M. rosenbergii and WSSV which will be able to provide a solution in controlling the spread of this infectious disease and lastly save the aquaculture industry. Now, using Next Generation Sequencing (NGS), we will be able to capture the response of the M. rosenbergii to the pathogen and have a better understanding of the host defence mechanism. Two cDNA libraries, one of WSSV-challenged M. rosenbergii and a normal control one, were sequenced using the Illumina HiSeq™ 2000 platform. After de novo assembly and clustering of the unigenes from both libraries, 63,584 standard unigenes were generated with a mean size of 698bp and an N50 of 1137bp. We successfully annotated 35.31% of all unigenes by using BLASTX program (E-value <10-5) against NCBI non-redundant (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genome pathway (KEGG) and Orthologous Groups of proteins (COG) databases. Gene Ontology (GO) assessment was conducted using BLAST2GO software. Differentially expressed genes (DEGs) by using the FPKM method showed 8443 host genes were significantly up-regulated whereas 5973 genes were significantly down-regulated. The differentially expressed immune related genes were grouped into 15 animal immune functions. The present study showed that WSSV infection has a significant impact on the transcriptome profile of M. rosenbergii's hepatopancreas, and further enhanced the knowledge of this host-virus interaction. Furthermore, the high number of transcripts generated in this study will provide a platform for future genomic research on freshwater prawns., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In recent years, the tumor suppressor protein p53, which is crucial for cellular defense against tumor development, has also been implicated in host antiviral defense. In the present study, a 1555 bp full-length cDNA of p53 from mandarin fish (Siniperca chuatsi) (Sc-p53) was cloned and characterized. Quantitative real-time PCR assays revealed that Sc-p53 was expressed in all tissues examined, and it was most abundant in the gill and kidney. Recombinant Sc-p53 fused with a His·Tag was expressed in Escherichia coli BL21 (DE3) cells and a rabbit polyclonal antibody was raised against recombinant Sc-p53. In addition, the regulation of Sc-p53 gene expression after experimental viral infection was determined and characterized. The mRNA and protein expression of Sc-p53 were significantly up-regulated in the Chinese perch brain (CPB) cell line and mandarin fish after infection with infectious kidney and spleen necrosis virus (ISKNV). The results showed a biphasic expression pattern of Sc-p53 protein in CPB. However, a different expression pattern of Sc-p53 in response to S. chuatsi rhabdovirus (SCRV) infection was found. The mRNA expression of Sc-p53 was significantly up-regulated in CPB at 6 h and spleen of mandarin fish at 24 h post-infection. The protein expression of Sc-p53 was significantly up-regulated in CPB at 1 h, remained elevated at 4 h, and then decreased to control level at 8 h post-infection by SCRV. All of these data suggested that Sc-p53 plays a critical role in immune defense and antiviral responses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Early induction of proinflammatory cytokines is known to regulate the later immune responses to inhibit the progress of infectious diseases. In this study, proinflammatory cytokine gene expression has been studied in immune tissues to understand the early immune response induced by megalocytivirus in rock bream (Oplegnathus faciatus). For this, we have cloned interleukin (IL)-1β and IL-8 gene and performed the phylogenetic and structural analysis. Also the constitutive gene expressions of IL-1β and IL-8 were assessed in 12 organs and found to be the highest expression in tail fin and liver, respectively. The expressions of proinflammatory cytokine genes including IL-1β, IL-8, TNFα and Cox-2, and antiviral genes like Mx and IFN1 were analysed by stimulation with PAMPs and RBIV infection. In vitro study showed the highly up-regulated proinflammatory gene expressions in head kidney and the moderate up-regulation in spleen by LPS. Same concentration of polyI:C moderately upregulated IL-1β gene expression in head kidney but down-regulated IL-8 and TNFα gene expression in head kidney and spleen at 8 h. Mx and IFN1 gene expressions were highly upregulated by polyI:C in head kidney and spleen cells in vitro. By RBIV infection, proinflammatory gene expressions were initially up-regulated and later down-regulated in head kidney. In spleen, although mostly not significant, proinflammatory cytokine gene expressions were down-regulated by RBIV infection except up-regulation of Cox-2 gene expression by low concentration of RBIV at 24 h. Mx and IFN1 gene expressions were down-regulated by high dose of RBIV infection in vitro. In vivo study revealed that IL-8, TNFα, and IFN1 gene expressions were down-regulated in brain, head kidney, spleen, and gill while up-regulated in heart and liver, indicating differential proinflammatory and antiviral responses in the organs. It is supposed that down-regulation of proinflammatory gene expression in the immune organs may result in the failure of antiviral immune responses, causing high mortalities by megalocytivirus infection in rock bream., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The tripartite motif (TRIM)-containing proteins exert important immune regulatory roles through regulating different signaling pathways in response to different stimuli. TRIM39, a member of the TRIM family, is a RING domain-containing E3 ubiquitin ligase which could regulate cell cycle progression and apoptosis. However, the antiviral activity of TRIM39 is not explored. Here, a TRIM39 homolog from grouper, Epinephelus coioides (EcTRIM39) was cloned, and its effects on cell cycle progression and fish virus replication were investigated. The full-length EcTRIM39 cDNA was composed of 2535 bp and encoded a polypeptide of 543 amino acids with 70% identity with TRIM39 homologs from bicolor damselfish. Amino acid alignment analysis indicated that EcTRIM39 contained a RING finger, B-box and SPRY domain. Expression profile analysis revealed that EcTRIM39 was abundant in intestine, spleen and skin. Upon different stimuli in vivo, the EcTRIM39 transcript was obviously up-regulated after challenging with Singapore grouper iridovirus (SGIV), and polyinosinic-polycytidylic acid (poly I:C). Using fluorescence microscopy, we found that EcTRIM39 localized in the cytoplasm and formed aggregates in grouper spleen (GS) cells. The ectopic expression of EcTRIM39 in vitro affected the cell cycle progression via mediating G1/S transition. Moreover, the RING domain was essential for its accurate localization and effect on cell cycle. In addition, overexpression of EcTRIM39 significantly inhibited viral gene transcription of SGIV and red-spotted grouper nervous necrosis virus (RGNNV) in vitro, and the mutant of RING exerted the opposite effect. Together, our results demonstrated that fish TRIM39 not only regulated the cell cycle progression, but also acted as an important regulator of fish innate immune response against viruses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Small ubiquitin-like modifier (SUMO) is a group of proteins binding to lysine residues of target proteins and thereby modifying their stability, activity and subcellular localization. In the present study, two SUMO homolog genes (EcSUMO1 and EcSUMO2) from grouper (Epinephelus coioides) were cloned and characterized. The full-length sequence of EcSUMO1 was 749 bp in length and contained a predicted open reading frame of 306 bp encoding 101 amino acids with a molecular mass of 11.34 kDa. The full-length sequence of EcSUMO2 was 822 bp in length and contained a predicted open reading frame of 291 bp encoding 96 amino acids with a molecular mass of 10.88 kDa EcSUMO1 shares 44.55% identity with EcSUMO2. EcSUMO1 shares 99%, 90%, and 88% identity with those from Oreochromis niloticus, Danio rerio, and Homo sapiens, respectively. EcSUMO2 shares 98%, 93%, and 96% identity with those from Anoplopoma fimbria, D.rerio, and H. sapiens, respectively. Quantitative real-time PCR analysis indicated that EcSUMO1 and EcSUMO2 were constitutively expressed in all of the analyzed tissues in healthy grouper, but the expression of EcSUMO2 was higher than that of EcSUMO1. EcSUMO1 and EcSUMO2 were identified as a remarkably (P < 0.01) up-regulated responding to poly(I:C) and Singapore grouper iridovirus (SGIV) stimulation in head kidney of groupers. EcSUMO1 and EcSUMO2 were distributed in both cytoplasm and nucleus in GS cells. Over-expressed EcSUMO1 and EcSUMO2 enhanced SGIV and Red-spotted grouper nervous necrosis virus (RGNNV) replication during viral infection in vitro. Our study was an important attempt to understand the SUMO pathway in fish, which may provide insights into the regulatory mechanism of viral infection in E.coioides under farmed conditions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mitogen-activated protein kinase kinase 7 (MKK7) is a key signal transduction regulator in c-Jun N-terminal kinase (JNK) signaling pathway, which is involved in a wide range of physiological and pathological processes. In this study, we described the molecular cloning of a new member of MKK7 group from Litopenaeus vannamei named as LvMKK7. The full-length cDNA of LvMKK7 was 3093 bp in length, with an open reading frame (ORF) of 1440bp encoding a putative protein of 479 amino acids. LvMKK7 contained a conserved kinase domain of 261 amino acids in which there was a characteristic S-K-A-K-T motif as a potential target site of phosphorylation by MKKK. Moreover, subcellular localization showed LvMKK7 was located in both the cytoplasm and the nucleus of Drosophila S2 cells. Real-time PCR indicated that LvMKK7 was universally expressed in all tested tissues and its expression in hepatopancreas was responsive to the challenge of LPS, Poly (I:C), Vibrio parahaemolyticus, Staphhylococcus aureus and white spot syndrome virus (WSSV). In addition, co-immunoprecipitation assay demonstrated that LvJNK was phosphorylated and activated by LvMKK7, which suggested LvMKK7 was the upper regulator of LvJNK. Furthermore, RNAi-mediated knockdown of LvMKK7 enhanced the sensitivity of shrimps to V. parahaemolyticus infection. Overall, our results suggested that LvMKK7 may play important roles in the shrimp innate immunity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Chemokines are a large, diverse group of small cytokines that can be classified into several families, including the CC chemokine family, which plays a pivotal role in host defense by inducing leukocyte chemotaxis under physiological and inflammatory conditions. Here we studied 9 CC chemokines from half-smooth tongue sole (Cynoglossus semilaevis). Phylogenetic analysis divided these chemokines into four groups. The tissue specific expression patterns of the 9 chemokines under normal physiological conditions varied much, with most chemokines highly expressed in immune organs, while some other chemokines showing high expression levels in non-immune organs. In addition, the 9 chemokines exhibited similar or distinctly different expression profiles in response to the challenge of virus and intracellular and extracellular bacterial pathogens. These results indicate that in tongue sole, CC chemokines may be involved in different immune responses as homeostatic or inflammatory chemokines., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Toll-interacting protein (Tollip) is one of the important regulatory proteins of Toll-like receptor (TLR) signaling pathways. In previous studies, a Tollip sequence of grouper (Epinephelus coioides) was identified and the signal transduction functions of Tollip were studied. However, the response of Tollip to virus infection has not been characterized from grouper. In the present paper, the Tollip homolog (EtTollip) from grouper (Epinephelus tauvina) was cloned and its immune response to Singapore grouper iridovirus (SGIV) was investigated. EtTollip shares significant similarities to other mammalian Tollips, which contain a centrally localized protein kinase C conserved region 2 (C2) domain and a C-terminal CUE domain. After challenging with SGIV, the expression levels of EtTollip were altered in the spleen and head kidney of grouper. EtTollip mainly aggregated in the cytoplasm in a condensed state and was also distributed on the membranes of GS cells. EtTollip significantly inhibited the activities of NF-κB and IFN-β luciferase reporter when transfected into grouper spleen (GS) cells. SGIV can increase the activities of NF-κB and IFN-β luciferase reporter, especially to IFN-β. When transfected EtTollip with EcMyd88, the activity of NF-κB was increased, while transfected EtTollip with EcIRF3, the activity of IFN-β was significantly increased. Over-expressed EtTollip inhibited the transcription of SGIV genes significantly in GS cells, and silencing of EtTollip with siRNA led to increase of SGIV genes loads. Taken together, the results provide new insights in to the importance of Tollip as evolutionarily conserved molecule for grouper innate immunity against virus infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The emergence of an infectious viral disease caused by the Chinese giant salamander iridovirus (GSIV) has led to substantial economic losses. However, no more molecular information is available for the understanding of the mechanisms associated with virus-host interaction. In this study, de novo sequencing was used to obtain abundant high-quality ESTs and investigate differentially-expressed genes in the spleen of Chinese giant salamanders that were either infected or mock infected with GSIV. Comparative expression analysis indicated that 293 genes were down-regulated and 220 genes were up-regulated. Further enrichment analysis showed that the most enriched pathway is "complement and coagulation cascades", and significantly enriched diseases include "inherited thrombophilia", "immune system diseases", "primary immunodeficiency", "complement regulatory protein defects", and "disorders of nucleotide excision repair". Additionally, 30 678 simple sequence repeats (SSRs) from all spleen samples, 26 355 single nucleotide polymorphisms (SNPs) from the spleens of uninfected animals and 36 070 SNPs from the spleens of infected animals were detected. The large amount of variation was specific for the Chinese giant salamanders that were infected with GSIV. The results reported herein provided significant and new EST information that could contribute greatly in investigations into the molecular functions of immune genes in the Chinese giant salamander.

The interleukin (IL)-17 cytokine family participates in the regulation of many cellular functions. In the present study, we analyzed the genomic structure, expression, and promoter activity of four IL-17 members from the teleost fish tongue sole (Cynoglossus semilaevis), i.e. CsIL-17C CsIL-17D, CsIL-17F, and IL-17F like (IL-17Fl). We found that CsIL-17C, CsIL-17D, CsIL-17F, and CsIL-17Fl share 21.2%-28.6% overall sequence identities among themselves and 31.5%-71.2% overall sequence identities with their counterparts in other teleost. All four CsIL-17 members possess an IL-17 domain and four conserved cysteine residues. Phylogenetic analysis classified the four CsIL-17 members into three clusters. Under normal physiological conditions, the four CsIL-17 expressed in multiple tissues, especially non-immune tissues. Bacterial infection upregulated the expression of all four CsIL-17, while viral infection upregulated the expression of CsIL-17D and CsIL-17Fl but downregulated the expression of CsIL-17C and CsIL-17F. The 1.2 kb 5'-flanking regions of the four CsIL-17 exhibited apparent promoter activity and contain a number of putative transcription factor-binding sites. Furthermore, the promoter activities of CsIL-17C, CsIL-17D, and CsIL-17F, but not CsIL-17Fl, were modulated to significant extents by lipopolysaccharide, PolyI:C, and PMA. This study provides the first evidence that in teleost, different IL-17 members differ in expression pattern and promoter activity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Chemokines are a large family of chemotactic cytokines. Based on the arrangement of the first two cysteine residues, chemokines are divided into four groups, one of which is the CC chemokine group. In this study, we characterized a CC chemokine, CsCCL21, from half-smooth tongue sole (Cynoglossus semilaevis), and analyzed its activity. CsCCL21 contains two conserved N-terminal cysteine residues in a NCCL motif and is phylogenetically related to the CCL19/21/25 subgroup of CC chemokines. CsCCL21 was constitutively expressed in nine tissues and significantly upregulated by bacterial and viral infection. The recombinant CsCCL21 (rCsCCL21) induced migration of peripheral blood leukocytes. When the two conserved cysteine residues in the NCCL motif were mutated, the chemotactic activity of rCsCCL21 was abolished. rCsCCL21 enhanced the resistance of tongue sole against bacterial infection, but the mutant protein with NCCL mutation lacked this antibacterial effect. Taken together, these results suggest that CsCCL21 is a functional CC chemokine with the ability to recruit leukocytes and is involved in antibacterial immunity in a manner that requires the conserved NCCL motif., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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&#95;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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mammalian serum amyloid P component (SAP) recognizes a wide range of exogenous pathogenic substances and activates a complementary pathway leading to pathogen clearance. To determine the potential roles of SAP in the fish immune system, SAP (RbSAP2) gene was cloned from ESTs analysis of rock bream (Oplegnathus fasciatus), which consisted of a signal peptide and pentraxin domain. Phylogenetic analysis revealed that the RbSAP2 gene was classified with other known fish SAPs. RbSAP2 was highly expressed in the liver of healthy rock bream. Overall, pathogen exposure led to an induction of RbSAP2 in the liver and spleen, although this effect was not observed in the spleen following infection with Edwardsiella tarda. A high concentration of recombinant RbSAP2 (rRbSAP2) showed lower growth Streptococcus iniae than control in the absence of Ca(2+), whereas E. tarda growth was decreased by high concentration of rRbSAP in the presence of the Ca(2+). These results suggest that RbSAP plays an important role in the immune response against invading pathogens., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Stimulator of interferon genes (STING, also known as MITA, ERIS, MPYS or TMEM173) has been identified as a central component in the innate immune response to cytosolic DNA and RNA derived from different pathogens. However, the detailed role of STING during fish iridovirus infection still remained largely unknown. Here, the STING homolog from grouper Epinephelus coioides (EcSTING) was cloned and its effects on IFN response and antiviral activity were investigated. The full-length EcSTING cDNA was composed of 1590 bp and encoded a polypeptide of 409 amino acids with 80% identity to STING homolog from large yellow croaker. Amino acid alignment analysis indicated that EcSTING contained 4 predicated transmembrane motifs (TMs) in the N terminal, and a C-terminal domain (CTD) which consisted of a dimerization domain (DD), c-di-GMP-binding domain (CBD) and a C-terminal tail (CTT). Expression profile analysis revealed that EcSTING was abundant in gill, spleen, brain, skin, and liver. Upon different stimuli in vivo, the EcSTING transcript was dramatically up-regulated after challenging with Singapore grouper iridovirus (SGIV), lipopolysaccharide (LPS) and polyinosin-polycytidylic acid (poly I:C). Reporter gene assay showed that EcSTING activated ISRE, zebrafish type I IFN and type III IFN promoter in vitro. Mutant analysis showed that IFN promoter activity was mostly mediated by the phosphorylation sites at serine residue S379 and S387. Moreover, EcSTING induced type I and III IFN promoter activity could be impaired by overexpression of EcIRF3-DN or EcIRF7-DN, suggesting that EcSTING mediated IFN response in IRF3/IRF7 dependent manner. In addition, the cytopathic effect (CPE) progression of SGIV infection and viral protein synthesis was significantly inhibited by overexpression of EcSTING, and the inhibitory effect was abolished in serine residue S379 and S387 mutant transfected cells. Together, our results demonstrated that EcSTING might be an important regulator of grouper innate immune response against iridovirus infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Viruses are notorious for rapidly exchanging genetic information between close relatives and with the host cells they infect. This exchange has profound effects on the nature and rapidity of virus and host evolution. Recombination between dsDNA viruses is common, as is genetic exchange between dsDNA viruses or retroviruses and host genomes. Recombination between RNA virus genomes is also well known. In contrast, genetic exchange across viral kingdoms, for instance between nonretroviral RNA viruses or ssDNA viruses and host genomes or between RNA and DNA viruses, was previously thought to be practically nonexistent. However, there is now growing evidence for both RNA and ssDNA viruses recombining with host dsDNA genomes and, more surprisingly, RNA virus genes recombining with ssDNA virus genomes. Mechanisms are still unclear, but this deep recombination greatly expands the breadth of virus evolution and confounds virus taxonomy.

Peptidoglycan recognition proteins (PGRPs) are members of the innate immune system that interact with bacteria by binding to bacterial peptidoglycan. In this study, we examined the expression and function of a short type of PGRP, CsPGRP-SC2, from tongue sole (Cynoglossus semilaevis). CsPGRP-SC2 contains 164 amino acid residues and shares 54.5%-65.3% overall sequence identities with other teleost PGRPs. CsPGRP-SC2 possesses an amidase domain with a conserved zinc binding site. CsPGRP-SC2 expression occurred in multiple tissues and was upregulated by bacterial and viral infection. Purified recombinant CsPGRP-SC2 (rCsPGRP-SC2) was able to bind and agglutinate Gram-positive bacteria in a Zn(2+)-dependent manner. rCsPGRP-SC2 enhanced the uptake of the bound bacteria by host phagocytes and reduced bacterial dissemination and colonization in host tissues. These results indicate that CsPGRP-SC2 is an innate immune factor that participates in host defense against bacterial infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In mammals as well as in teleost, toll-like receptor 7 (TLR7) is known to be involved in antiviral immunity by recognizing viral RNA. However, the antibacterial potential of fish TLR7 is unclear. In this study, we analyzed the TLR7 of tongue sole (Cynoglossus semilaevis), CsTLR7, and examined its potential involvement in antibacterial immunity. CsTLR7 is composed of 1052 amino acid residues and shares 64.0%-75.9% overall sequence identities with known teleost TLR7. CsTLR7 possesses a toll/interleukin-1 receptor domain and six leucine-rich repeats. Constitutive expression of CsTLR7 occurred in relatively high levels in kidney, spleen and liver. Bacterial infection upregulated CsTLR7 expression, whereas viral infection downregulated CsTLR7 expression. Knockdown of CsTLR7 significantly enhanced bacterial dissemination in the tissues of tongue sole. Treatment of tongue sole with the imidazoquinoline compound R848 (TLR7 activator) and the endosomal acidification inhibitor chloroquine (TLR7 inhibitor) caused enhanced and reduced resistance against bacterial infection respectively. These results indicate that CsTLR7 plays an essential role in the antibacterial immunity of tongue sole., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Data regarding the influence of the APOBEC3B deletion on infectious diseases remain limited and shown discrepancies., Objectives: To characterize the APOBEC3B deletion polymorphism status and its association with prevalence of co-infection with blood-borne pathogens in Indonesian HIV-infected individuals., Materials and Methods: A total of 597 HIV-positive blood samples were tested for the hepatitis B virus (HBV), hepatitis C virus (HCV), Torque Teno virus (TTV), GB virus-C (GBV-C), and Toxoplasma gondii. Nucleic acid was extracted from plasma samples and used for the molecular detection of HIV RNA, HBV DNA, HCV RNA, TTV DNA, and GBV-C RNA, whereas HBsAg, anti-HCV, IgM and IgG anti-T. gondii were detected through serological testing. The APOBEC3B deletion polymorphism was genotyped by polymerase chain reaction (PCR)., Results: The deletion genotype was associated with HCV viremia (p<0.001) as well as elevated IgG anti-T. gondii (adjusted OR [aOR]=3.4). The deletion genotype was also associated with decreased levels of HBsAg (aOR=0.03), and anti-HCV (aOR=0.1). D/D was frequently found in HIV-infected individuals with CD4+T cells<14% (aOR=5.8). The intact genotype was associated with a reduced likelihood of a CD4+T cell count<200 cells/μL (aOR=0.2) but a higher prevalence of TTV co-infection (aOR=8.6)., Conclusions: The APOBEC3B deletion polymorphism was found to be associated with HBV, HCV, TTV, and T. gondii co-infection in Indonesian HIV-infected individuals., (Copyright © 2015 Elsevier B.V. All rights reserved.)