Transcriptional Regulation and Signaling of Type IV IFN with Identification of the ISG Repertoire in an Amphibian Model, Xenopus laevis.

The type IV IFN (IFN-υ) is reported in vertebrates from fish to primary mammals with IFN-υR1 and IL-10R2 as receptor subunits. In this study, the proximal promoter of IFN-υ was identified in the amphibian model, Xenopus laevis, with functional IFN-sensitive responsive element and NF-κB sites, which can be transcriptionally activated by transcription factors, such as IFN regulatory factor (IRF)1, IRF3, IRF7, and p65. It was further found that IFN-υ signals through the classical IFN-stimulated gene (ISG) factor 3 (ISGF3) to induce the expression of ISGs. It seems likely that the promoter elements of the IFN-υ gene in amphibians is similar to type III IFN genes, and that the mechanism involved in IFN-υ induction is very much similar to type I and III IFNs. Using recombinant IFN-υ protein and the X. laevis A6 cell line, >400 ISGs were identified in the transcriptome, including ISGs homologous to humans. However, as many as 268 genes were unrelated to human or zebrafish ISGs, and some of these ISGs were expanded families such as the amphibian novel TRIM protein (AMNTR) family. AMNTR50, a member in the family, was found to be induced by type I, III, and IV IFNs through IFN-sensitive responsive element sites of the proximal promoter, and this molecule has a negative role in regulating the expression of type I, III, and IV IFNs. It is considered that the current study contributes to the understanding of transcription, signaling, and functional aspects of type IV IFN at least in amphibians.

Thioclava litoralis sp. nov., a novel species of alphaproteobacterium, isolated from surface seawater.

A Gram-negative, aerobic, rod-shaped, non-motile bacterium, designated as FTW29T, was isolated from surface seawater sampled in Futian district, Shenzhen, China. Growth of strain FTW29T was observed at 15-42 ℃ (optimum, 28-30 ℃), pH 4.0-9.0 (optimum, pH 5.5-7.5) and in the presence of 0.5-10% NaCl (optimum, 3.0% NaCl). Strain FTW29T showed 95.0-96.8% 16 S rRNA gene sequence similarity to various type strains of the genera Thioclava, Sinirhodobacter, Rhodobacter, Haematobacter and Frigidibacter of the family Paracoccaceae, and its most closely related strains were Thioclava pacifica DSM 10,166T (96.8%) and Thioclava marina 11.10-0-13T (96.7%). The phylogenomic tree constructed on the bac120 gene set showed that strain FTW29T formed a clade with the genus Thioclava, with a bootstrap value of 100%. The evolutionary distance values between FTW29T and type strains of the genus Thioclava were 0.17-0.19, which are below the recommended standard (0.21-0.23) for defining a novel genus in the family Paracoccaceae. In strain FTW29T, the major fatty acids identified were summed feature 8 (C18:1ω7c) and C16:0, and the predominant respiratory quinones were ubiquinone-10 and ubiquinone-9. The composition of polar lipids in strain FTW29T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid, two unidentified glycolipids and an unidentified lipid. The genome of strain FTW29T comprised one circle chromosome and six plasmids, with a G + C content of 61.4%. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain FTW29T and seven type strains of the genus Thioclava were 76.6-78.4%, 53.2-56.4% and 19.3-20.4%, respectively. Altogether, the phenotypic, phylogenetic and chemotaxonomic evidence illustrated in this study suggested that strain FTW29T represents a novel species of the genus Thioclava, with the proposed name Thioclava litoralis sp. nov. The type strain is FTW29T (= KCTC 82,841T = MCCC 1K08523T).

Microbulbifer bruguierae sp. nov., isolated from sediment of mangrove plant Bruguiera sexangula, and comparative genomic analyses of the genus Microbulbifer.

A Gram-negative, non-motile, strictly aerobic, rod-shaped bacterium, designated as H12T, was isolated from the sediments of mangrove plant Bruguiera sexangula taken from Dapeng district, Shenzhen, PR China. The pairwise 16S rRNA gene sequence analysis showed that strain H12T shared high identity levels with species of the genus Microbulbifer, with the highest similarity level of 98.5 % to M. pacificus SPO729T, followed by 98.1 % to M. donghaiensis CN85T. Phylogenetic analysis using core-genome sequences showed that strain H12T formed a cluster with type species of M. pacificus SPO729T and M. harenosus HB161719T. The complete genome of strain H12T was 4 481 396 bp in size and its DNA G+C content was 56.7 mol%. The average nucleotide identity and digital DNA-DNA hybridization values among strain H12T and type species of genus Microbulbifer were below the cut-off levels of 95-96 and 70 %, respectively. The predominant cellular fatty acids of strain H12T were iso-C15 : 0 (22.5 %) and C18 : 1 ω7c (13.9 %). Ubiquinone-8 was detected as the major respiratory quinone. The polar lipids of strain H12T comprised one phosphatidylglycerol, one phosphatidylethanolamine, one unidentified aminoglycophospholipid, one unidentified glycophospholipid, three unidentified glycolipids, two unidentified aminolipids, and one unidentified lipid. Based on polyphasic evidence, strain H12T represents a novel species of the genus Microbulbifer, for which the name Microbulbifer bruguierae sp. nov. is proposed. The type strain is H12T (=KCTC 92859T=MCCC 1K08451T). Comparative genomic analyses of strain H12T with strains of the genus Microbulbifer reveal its potential in degradation of pectin.

Identification and functional characterization of interleukin-22 (IL-22) in orange-spotted grouper (Epinephelus coioides).

In mammals, IL-22 is considered as a critical cytokine regulating of immunity and homeostasis at barrier surfaces. Although IL-22 have been functional characterization in different species of fish, the studies about distinct responses of IL-22 in different organs/tissues/cell types is rather limited. Here, we identified and cloned IL-22 gene (named as Ec-IL-22) from grouper (Epinephelus coioides). Ec-IL-22 gene was detected in all orangs/tissues examined, and was induced in intestine, gill, spleen, head kidney, and primary head kidney/intestine leukocytes following the stimulation of LPS and poly (I:C), as well as Vibrio harveyi and Singapore grouper iridovirus infection (SGIV). In addition, the stimulation of DSS could induce the expression of Ec-IL-22 in intestine and primary leukocytes from intestine. Importantly, the treatment of recombinant Ec-IL-22 induced the mRNA level of proinflammatory cytokines in primary intestine/head kidney leukocytes. The present results improve the understanding of expression patterns and functional characteristics of fish IL-22 in different organs/tissues/cell types.

Evolutionary and functional conservation of IRF7 in the Tibetan frog Nanorana parkeri.

BACKGROUND: The production of interferons (IFNs) is essential for the control of viral infections, and interferon regulatory factor 7 (IRF7) is considered as a vital regulator for the transcription of type I IFNs. Amphibians appear to possess a highly expanded type I IFN repertoire, consisting of intron-containing genes as observed in fish, and intronless genes as in other higher vertebrates. However, the knowledge on transcriptional regulatory mechanism of these two types of type I IFN genes is rather scarce in amphibians. METHODS AND RESULTS: A IRF7 gene named as Np-IRF7 was identified in Tibetan frog (Nanorana parkeri), and bioinformatic analysis revealed that the predicted protein of Np-IRF7 contains several important structural features known in IRF7. Expression analysis showed that Np-IRF7 gene was widely expressed and rapidly induced by poly(I:C) in different organs/tissues. Interestingly, luciferase reporter assay revealed that intronless IFN promoters were more effectively activated than intron-containing IFN promoter in Np-IRF7-transfected cells. Moreover, the overexpression of Np-IRF7 could induce the expression of ISGs and suppress the replication of FV3 in A6 cells. CONCLUSION: Np-IRF7 is indeed the ortholog of known IRF7, and IRF7 is structurally conserved in different lineages of vertebrates. Np-IRF7 played distinct roles in the activation of intron-containing and intronless type I IFN promoters, thus inducing the expression of interferon-stimulated antiviral effectors and providing a protection against ranavirus infection. The present research thus contributes to a better understanding of regulatory function of IRF7 in the IFN-mediated antiviral response of anuran amphibians.

Identification and characterization of two long-type peptidoglycan recognition proteins, PGRP-L1 and PGRP-L2, in the orange-spotted grouper, Epinephelus coioides.

Peptidoglycan recognition proteins (PGRPs) play an important role in innate immunity by recognizing components of pathogenic bacteria (such as peptidoglycan, PGN) and are evolutionarily conserved pattern recognition receptors (PRRs) in both invertebrates and vertebrates. In the present study, two long-type PGRPs (designed as Eco-PGRP-L1 and Eco-PGRP-L2) were identified in orange-spotted grouper (Epinephelus coioides), which is a major economic species cultured in Asia. The predicted protein sequences of both Eco-PGRP-L1 and Eco-PGRP-L2 contain a typical PGRP domain. Eco-PGRP-L1 and Eco-PGRP-L2 exhibited organ/tissue-specific expression patterns. An abundant expression of Eco-PGRP-L1 was observed in pyloric caecum, stomach and gill, whereas a highest expression level of Eco-PGRP-L2 was found in head kidney, spleen, skin and heart. In addition, Eco-PGRP-L1 is distributed in the cytoplasm and nucleus, while Eco-PGRP-L2 is mainly localized in cytoplasm. Both Eco-PGRP-L1 and Eco-PGRP-L2 were induced following the stimulation of PGN and have PGN binding activity. In addition, functional analysis revealed that Eco-PGRP-L1 and Eco-PGRP-L2 possess antibacterial activity against Edwardsiella tarda. These results may contribute to understand the innate immune system of orange-spotted grouper.

Dextran Sulfate Sodium Salt (DSS) induced enteritis in Orange-spotted grouper, Epinephelus coioides.

The enteritis is a common disease in fish farming, but the pathogenesis is still not fully understood. The aim of the present study was to investigate the inducement of Dextran Sulfate Sodium Salt (DSS) intestinal inflammation on Orange-spotted grouper (Epinephelus coioides). The fish were challenged with 200 µl 3% DSS via oral irrigation and feeding, an appropriate dose based on the disease activity index of inflammation. The results indicated that the inflammatory responses induced by DSS were closely associated with the expression of pro-inflammatory cytokines including interleukin 1ß (IL-1ß), IL-8, IL16, IL-10 and tumor necrosis factor α (TNF-α), as well as NF-κB and myeloperoxidase (MPO) activity. At day5 after DSS treatment, the highest levels of all parameters were observed. Also, the severe intestinal lesions (intestinal villus fusion and shedding), strong inflammatory cell infiltration and microvillus effacement were seen through histological examination and SEM (scanning electronic microscopy) analysis. During the subsequent 18 days of the experimental period, the injured intestinal villi were gradually recovery. These data is beneficial to further investigate the pathogenesis of enteritis in farmed fish, which is helpful for the control of enteritis in aquaculture.

Retroposition of the Long Transcript from Multiexon IFN-ß Homologs in Ancestry Vertebrate Gave Rise to the Proximal Transcription Elements of Intronless IFN-ß Promoter in Humans.

IFN-ß is a unique member of type I IFN in humans and contains four positive regulatory domains (PRDs), I-II-III-IV, in its promoter, which are docking sites for transcription factors IFN regulatory factor (IRF) 3/7, NF-κB, IRF3/7, and activating transcription factor 2/Jun proto-oncogene, respectively. In chicken IFN-ß and zebrafish IFNφ1 promoters, a conserved PRD or PRD-like sequences have been reported. In this study, a type I IFN gene, named as xl-IFN1 in the amphibian model Xenopus laevis, was found to contain similar PRD-like sites, IV-III/I-II, in its promoter, and these PRD-like sites were proved to be functionally responsive to activating transcription factor 2/Jun proto-oncogene, IRF3/IRF7, and p65, respectively. The xl-IFN1, as IFNφ1 in zebrafish, was transcribed into a long and a short transcript, with the long transcript containing all of the transcriptional elements, including PRD-like sites and TATA box in its proximal promoter. A retroposition model was then proposed to explain the transcriptional conservation of IFNφ1, xl-IFN1, and IFN-ß in chicken and humans.

Description and genomic characterization of Gallaecimonas kandeliae sp. nov., isolated from the sediments of mangrove plant Kandelia obovate.

The genus Gallaecimonas, proposed by Rodríguez-Blanco et al. (Int J Syst Evol Microbiol 60:504-509, 2010), is mainly isolated from marine environments. So far, only three species have been identified and characterized in this genus. In this study, a new Gallaecimonas strain named Q10T was isolated from the sediments of mangrove plant Kandelia obovate taken from Dapeng district, Shenzhen, China. Strain Q10T was a Gram-stain-negative, non-motile, strictly aerobic, rod-shaped bacterium, and grew with 0-8.0% (w/v) NaCl, at 10-45 °C and at pH 5.5-8.5. Phylogenetic analysis indicated that strain Q10T and the three Gallaecimonas species formed a clade in the tree, with 16S rRNA gene sequence similarities ranging from 96.0 to 97.0%. The major respiratory quinone is Q8. The polar lipids comprised aminolipid, aminophospholipid, diphosphatidylglycerol, glycolipid, phosphatidylethanolamine, phosphatidylglycerol, glycophospholipid and phospholipid. The predominant fatty acids are C16:0, C17:1ω8c, summed feature 3 (C16:1ω7c/C16:1ω6c), and iso-C16:0. The complete genome of strain Q10T is 3,836,841 bp with a G+C content of 62.6 mol%. The orthologous proteins analysis revealed 55 unique proteins in strain Q10T related to important biological processes, especially three frataxins related to iron-sulfur cluster assembly, which may play a pivotal role in environmental adaptability of this species. Based on polyphasic taxonomic data, strain Q10T is considered to represent a novel species within the genus Gallaecimonas, for which the name Gallaecimonas kandelia sp. nov. is proposed. The type strain is Q10T (=KCTC 92860T=MCCC 1K08421T). These results contribute to a better understanding of general features and taxonomy of the genus Gallaecimonas.

Comparative genomic analysis provides insights into taxonomy and temperature adaption of Aeromonas salmonicida.

Aeromonas salmonicida has long been known as psychrophiles since it is mainly isolated from cold water fish, and recent reports have revealed the existence of mesophilic strains isolated from warm sources. However, the genetic differences between mesophilic and psychrophilic strains remain unclear due to few complete genomes of mesophilic strain are available. In this study, six A. salmonicida (2 mesophilic and 4 psychrophilic) were genome-sequenced, and comparative analyses of 25 A. salmonicida complete genomes were conducted. The ANI values and phylogenetic analysis revealed that 25 strains formed three independent clades, which were referred as typical psychrophilic, atypical psychrophilic and mesophilic groups. Comparative genomic analysis showed that two chromosomal gene clusters, related to lateral flagella and outer membrane proteins (A-layer and T2SS proteins), and insertion sequences (ISAs4, ISAs7 and ISAs29) were unique to the psychrophilic groups, while the complete MSH type IV pili were unique to the mesophilic group, all of which may be considered as lifestyle-related factors. The results of this study not only provide new insights into the classification, lifestyle adaption and pathogenic mechanism of different strains of A. salmonicida, but also contributes to the prevention and control of disease caused by psychrophilic and mesophilic A. salmonicida.

Annonaceous Acetogenin Mimic AA005 Inhibits the Growth of TNBC MDA-MB-468 Cells by Altering Cell Energy Metabolism.

Triple-negative breast cancer (TNBC) is a serious health issue for women worldwide and there is still no suitable treatment option. AA005, a structurally simplified mimic of natural Annonaceous acetogenins, presents outstanding properties with impressive cytotoxicity and cell-type selective actions. The present study was aimed at evaluating the potential of AA005 as a therapeutic agent for TNBC. AA005 potently inhibited the growth of TNBC cells at 50 nM level. Inspired by the finding of the phosphatase and tensin homologue (PTEN) tumor suppressor, the effect of AA005 on aerobic glycolysis was investigated in TNBC MDA-MB-468 cells. A short-term AA005 exposure markedly suppressed mitochondrial function in MDA-MB-468 cells, thus activating the aerobic glycolysis to lessen the risk of decreased ATP generation in mitochondria. Prolonging the incubation time of AA005 clearly weakened the aerobic glycolysis in the cells. This was in part attributed to the PI3K-AKT pathway inactivation and subsequent declined glucose uptake. As a consequence, the energy supply was completely cut from the two major energy-producing pathways. Further experiments showed that AA005 resulted in irreversible damage on cell activity including cell cycle and growth, inducing mitochondrial oxidative stress and ultimately leading to cell death. In addition, the in vivo therapeutic efficacy of AA005 was proved on 4T1 xenograft tumor mice model. Our data demonstrate that AA005 exhibited a great potential for future clinical applications in TNBC therapy.

Functional characterization of a grouper nklysin with antibacterial and antiviral activity.

Natural killer lysin (Nklysin) is a small molecule antimicrobial peptide produced by natural killer cells and T lymphocytes and widely expressed in vertebrates. Homologues of Nklysin have been found in several fish, but only several of biological activity was identified. In this study, we characterized a Nklysin from grouper (Epinephelus coioides), and explored its expression pattern and biological function in bacterial infection. We also investigated the role of Nklysin in viral replication and maturation. The nklysin gene of grouper encodes a 169 amino acid, sharing 92.90% identity to H. septemfasciatus NKlysin protein, containing a saposin B domain and six well-conserved cysteine residues that necessary for antimicrobial activity by forming three intrachain disulfide bonds. Analysis of qRT-PCR revealed that nklysin gene widely expressed in all tested tissues with the higher expressions in spleen. After bacterial challenge, the nklysin gene expression significantly varied in different tissues. In addition, a large-scale of the recombinant Nklysin protein was secreted in Pichia pastoris strain GS115. The MIC assay showed that the Nklysin protein directly inhibited growth of several pathogens, including Proteus mirabilis, Bacillus subtilis, Salmonella typhi, Escherichia coli, Shigella sonnei and Streptococcus agalactiae. Further analysis showed the Nklysin protein over-expression might prevent viral genes transcriptions and replication in FHM cells. Our findings suggested that the Nklysin of grouper might be a potential agent for antibacterial and antiviral infection in the future.

Molecular cloning, expression analysis and functional characterization of NEDD4 from Nile tilapia (Oreochromis niloticus).

Neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) was a member of HECT E3 ubiquitin ligases, which participated in various biological processes. In this study, a NEDD4 was identified and analyzed in Nile tilapia, Oreochromis niloticus (OnNEDD4) and its open reading frame was 2781 bp, encoding 926 amino acids. Three conserved structure features were found in OnNEDD4, including C2 domain, WW domains and HECT domain. OnNEDD4 was constitutively expressed in all examined tissues and the highest expression level was observed in thymus. After Streptococcus agalactiae stimulation, OnNEDD4 was significantly induced in several tissues, including thymus, intestine, blood and gill. Moreover, yeast two-hybrid assay shown OnNEDD4 could interact with extracellular region of OnCD40, but this interaction didn't affect the phagocytosis of monocytes/macrophages (MO/MΦ) to S. agalactiae and A. hydrophila. Taken together, the present study suggested that OnNEDD4 participate in CD40-mediated immune response excluding phagocytosis.

In Primitive Zebrafish, MHC Class II Expression Is Regulated by IFN-γ, IRF1, and Two Forms of CIITA.

Mammalian CIITA isoforms are tightly regulated by independent promoters. These promotors are induced by IFN-γ through JAK-STAT signaling pathway. The induction of CIITA controls the expression of MHC class II (MHCII) and Ag presentation to the adaptive immune system. In the current study, to our knowledge, we first identified two independent promoters, p1 and p2, in the zebrafish (Danio rerio) that control the expression of the two variants of CIITA, CIITA variant 1 (CIITAv1), and CIITA variant 2 (CIITAv2), respectively. Moreover, although IRF1 in an IFN-γ signaling pathway induced CIITAv2, which has two ISRE motifs in its promoter, CIITAv1 expression was not induced by this signal. Further, the transcription of MHCII DAB was controlled by IRF1 via two distinct mechanisms: 1) the transcription of MHCII DAB was controlled by IRF1 indirectly through the two ISREs in p2; and 2) directly via the ISRE in MHCII DAB promoter. We also found that IRF1 associated with CIITAv1 and CIITAv2 via protein-protein interactions to synergistically drive the transcription of MHCII DAB. The IFN-γ-IRF1-CIITA-MHCII signaling cascade was functional in early life stages of CIITA-/- and IRF1-/- zebrafish. Our findings imply that the immune system develops early in fishes and that the IFN-γ signaling cascade-induced CIITA and MHCII DAB is conserved in teleost fishes and mammals.

Tilapia dsRNA-activated protein kinase R (PKR): An interferon-induced antiviral effector with translation inhibition activity.

The dsRNA-activated protein kinase R (PKR) is one of key antiviral effectors induced by interferons (IFNs), and its functions are largely unknown in tilapia, an important commercial fish species suffering from several viral infectious diseases. In the present study, a PKR gene named On-PKR was identified and cloned from Nile tilapia, Oreochromis niloticus. On-PKR gene was constitutively expressed in all tissues examined, with the highest expression level observed in head kidney and liver, and was rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). Importantly, the expression of On-PKR is induced by group I and group II IFNs with distinct induction kinetics in vivo: group I IFN elicits a relative delayed but sustained induction of On-PKR, whereas group II IFN triggers a rapid and transient expression of On-PKR. Moreover, the overexpression of On-PKR has been proven to inhibit the protein translation and virus replication in fish cells. The present study thus contributes to a better understanding of the functions of antiviral effectors in tilapia, and may provide clues for the prevention and therapy of viral diseases in fish.

[Polarized activation affects iron metabolism in macrophages].

The aim of this study was to investigate the effects of polarization program on the ability of macrophages to regulate iron metabolism. M1 and M2 macrophages were propagated in vitro from porcine alveolar macrophages 3D4/2 and polarized by cytokines. The 3D4/2 macrophages were treated with 20 ng/mL interferon gamma (IFN-γ) and 10 ng/mL interleukin-4 (IL-4) combined with 10 ng/mL macrophage colony-stimulating factor (M-CSF) to induce polarization to M1 and M2, respectively. After incubation for 24 h, the expression levels of inflammatory factors and iron-metabolism genes were determined using real-time qPCR, Western bot and immunofluorescence. The M1/M2 macrophages culture media supernatant was collected and used to treat porcine intestinal epithelial cells IPEC-J2. The proliferation ability of IPEC-J2 was detected using CCK-8 assay kit. Following exogenous addition of ammonium ferric citrate (FAC) to M1/M2 macrophages, the phagocytic function of macrophages was detected using fluorescein isothiocyanate-dextran (FITC-dextran) and flow cytometry. The results showed that, compared with control, M1 macrophages had higher mRNA levels of iron storage proteins (ferritin heavy and light polypeptide, i.e. FtH and FtL), hepcidin and lipocalin-2, as well as iron content. Moreover, iron enhanced the ability of M1 macrophages to phagocytize FITC-dextran. There was no significant change in these mRNA expression levels in M2 macrophages, but the mRNA expression levels of ferroportin and transferrin receptor were up-regulated. In addition, the conditioned media supernatant from M2 macrophages promoted cell proliferation of IPEC-J2. These findings indicate that M1 macrophages tend to lock iron in the cell and reduce extracellular iron content, thereby inhibiting the proliferation of extracellular bacteria. While M2 macrophages tend to excrete iron, which contributes to the proliferation of surrounding cells and thus promotes tissue repair.

Unique duplication of IFNh genes in Nile tilapia (Oreochromis niloticus) reveals lineage-specific evolution of IFNh in perciform fishes.

Fish appear to harbour a complex type I IFN repertoire containing subgroups a, b, c, d, e, f, and h, and IFNh is only reported in perciform fishes. However, no multiple copies of IFNh gene has been identified in fish to date. In this study, two IFNh genes named On-IFNh1 and On-IFNh2 were cloned from Nile tilapia, Oreochromis niloticus. The predicted proteins of On-IFNh1 and On-IFNh2 contain several structural features known in type I IFNs, and estimation of divergence time revealed that these two genes may have arisen from a much recent local duplication event. On-IFNh genes were constitutively expressed in all tissues examined, with the highest expression level observed in gill, and were rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). In addition, both recombinant On-IFNh1 and On-IFNh2 trigger a relative delayed but sustained induction of interferon-stimulated genes (ISGs), whereas recombinant On-IFNc elicits a rapid and transient expression of ISGs in vivo. The present study thus contributes to a better understanding of the functional properties of tilapia interferons, and also provides a new insight into the evolution of IFNh in fish.

Molecular and functional characterization of tilapia DDX41 in IFN regulation.

DEAD-box helicase 41 (DDX41) is a key cytosolic DNA sensor playing critical roles in the regulation of type I IFN responses, and their functions have been well-characterized in mammals. However, little information is available regarding the function of fish DDX41. In this study, a DDX41 gene, named On-DDX41, was identified in Nile tilapia, Oreochromis niloticus. The predicted protein of On-DDX41 contains several structural features known in DDX41, including conserved DEADc and HELICc domains, and a conserved sequence "Asp-Glu-Ala-Asp (D-E-A-D)". On-DDX41 gene was constitutively expressed in all tissues examined, with the highest expression level observed in liver and muscle, and was inducible after poly(I:C) stimulation. Moreover, the overexpression of On-DDX41 can elicit a strong activation of both zebrafish IFN1 and IFN3 promoter in fish cells treated with poly(dA:dT). The present study thus contributes to a better understanding of the functional properties of DDX41 in fish.

Identification and characterization of tilapia CRFB1, CRFB2 and CRFB5 reveals preferential receptor usage of three IFN subtypes in perciform fishes.

Type I interferons are a subset of cytokines playing central roles in host antiviral defense, and their effects depend on the interaction with the heterodimeric receptor complex. Surprisingly, two pairs of the receptor subunits, CRFB1 and CRFB5, and CRFB2 and CRFB5, have been identified in fish, but the studies about preferential receptor usage of different fish IFN subtypes are rather limited. In this study, the three receptor chains of type I IFNs named as On-CRFB1, On-CRFB2 and On-CRFB5 were identified in Nile tilapia, Oreochromis niloticus. These three genes were constitutively expressed in all tissues examined, with the highest expression level observed in muscle and liver, and were rapidly induced in liver following the stimulation of poly(I:C). Interestingly, it is possible that all three subtypes of tilapia IFNs are able to signal through two pairs of the receptor subunits, On-CRFB1 and On-CRFB5, and On-CRFB2 and On-CRFB5. More importantly, tilapia group I IFNs (On-IFNd and On-IFNh) preferentially signal through a receptor complex composed of On-CRFB1 and On-CRFB5, and group II IFNs (On-IFNc) preferentially signal through a receptor complex comprised of On-CRFB2 and On-CRFB5. The present study thus provides new insights into the receptor usage of group I and group II IFNs in fish.

Functional characterization of a group II interferon, IFNc in the perciform fish, Nile tilapia (Oreochromis niloticus).

Interferons are a family of class II α-helical cytokines playing vital roles in antiviral immune response, and little information is available to date regarding the interferon system of tilapia. In this study, a type I IFN gene, named On-IFNc, was identified in Nile tilapia, Oreochromis niloticus. The predicted protein of On-IFNc contains several structural features known in type I IFNs, and On-IFNc was clustered together with the known IFNc in fish into a separated clade in the phylogenetic tree. On-IFNc gene was constitutively expressed in all tissues examined, with the highest expression level observed in liver, and was rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). In addition, recombinant On-IFNc has been proven to markedly induce the expression of the antiviral effectors, Mx and viperin, the signalling components, STAT1, STAT2, and IRF9, and the transcription factors, IRF3 and IRF7, as well as the tyrosine phosphorylation of STAT1 and STAT2 in fish cells. Furthermore, recombinant On-IFNc has been proven to possess antiviral activity against ISKNV. The present study thus contributes to a better understanding of the functional properties of the type I IFN system in tilapia.