Interaction of Nmi and IFP35 Promotes Mutual Protein Stabilization and IRF3 and IRF7 Degradation to Suppress Type I IFN Production in Teleost Fish.

IFN-stimulated genes (ISGs) can act as effector molecules against viral infection and can also regulate pathogenic infection and host immune response. N-Myc and STAT interactor (Nmi) is reported as an ISG in mammals and in fish. In this study, the expression of Nmi was found to be induced significantly by the infection of Siniperca chuatsi rhabdovirus (SCRV), and the induced expression of type I IFNs after SCRV infection was reduced following Nmi overexpression. It is observed that Nmi can interact with IRF3 and IRF7 and promote the autophagy-mediated degradation of these two transcription factors. Furthermore, Nmi was found to be interactive with IFP35 through the CC region to inhibit IFP35 protein degradation, thereby enhancing the negative role in type I IFN expression after viral infection. In turn, IFP35 is also capable of protecting Nmi protein from degradation through its N-terminal domain. It is considered that Nmi and IFP35 in fish can also interact with each other in regulating negatively the expression of type I IFNs, but thus in enhancing the replication of SCRV.

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.

SIRT6 positively regulates antiviral response in a bony fish, the Chinese perch Siniperca chuatsi.

SIRT6, a key member of the sirtuin family, plays a pivotal role in regulating a number of vital biological processes, including energy metabolism, oxidative stress, and immune system modulation. Nevertheless, the function of SIRT6 in bony fish, particularly in the context of antiviral immune response, remains largely unexplored. In this study, a sirt6 was cloned and characterized in a commercial fish, the Chinese perch (Siniperca chuatsi). The SIRT6 possesses conserved SIR2 domain with catalytic core region when compared with other vertebrates. Tissue distribution analysis indicated that sirt6 was expressed in all detected tissues, and the sirt6 was significantly induced following infection of infectious haemorrhagic syndrome virus (IHSV). The overexpression of SIRT6 resulted in significant upregulation of interferon-stimulated genes (ISGs), such as viperin, mx, isg15, irf3 and ifp35, and inhibited viral replication. It was further found that SIRT6 was located in nucleus and could enhance the expression of ISGs induced by type I and II IFNs. These findings may provide new information in relation with the function of SIRT6 in vertebrates, and with viral prevention strategy development in aquaculture.

Characterization of type II IFNs and their receptors in a cyprinid fish, the blunt snout bream Megalobrama amblycephala.

Type II interferons (IFNs) are a key class of molecules regulating innate and adaptive immunity in vertebrates. In the present study, two members of the type II IFNs, IFN-γ and IFNγ-rel, were identified in the blunt snout bream (Megalobrama amblycephala). The open reading frame (ORF) of IFN-γ and IFNγ-rel was found to have 564 bp and 492 bp, encoding 187 and 163 amino acids, with the first 26 and 24 amino acids being the signal peptide, respectively. IFN-γ and IFNγ-rel genes showed a high degree of similarity to their zebrafish homologues, being 76.9 % and 58.9 %, respectively. In the phylogenetic tree, IFN-γ and IFNγ-rel were clustered with homologous genes in cyprinids. In blunt snout bream, IFN-γ and IFNγ-rel were constitutively expressed in trunk kidney, head kidney, spleen, liver, heart, muscle, gill, intestine and brain and were significantly up-regulated by poly (I:C) induction in head kidney, spleen, liver, gill and intestine. Using recombinant proteins of IFN-γ and IFNγ-rel, the surface plasmon resonance (SPR) results showed that IFN-γ was bound to CRFB6, CRFB13 and CRFB17, but mainly to CRFB6 and CRFB13, whereas IFN-γrel bound mainly to CRFB17 and had no affinity with CRFB6. These results contribute to a better understanding on type II IFNs and their receptor usage in teleost fish.

Molecular cloning and functional analyses of C-C motif chemokine ligand 3 (CCL3) in mandarin fish Siniperca chuatsi.

Chemokines are critical molecules involved in immune reaction and immune system homeostasis, and some chemokines play a role in antiviral immunity. It is not known if the C-C motif chemokine ligand 3 (CCL3), a member of the CC chemokine family, possesses antiviral properties in fish. In this study, a ccl3 was cloned from the mandarin fish (Siniperca chuatsi), and it has an open reading frame (ORF) of 276 base pairs, which are predicted to encode a 91-amino acid peptide. Mandarin fish CCL3 revealed conserved sequence features with four cysteine residues and closely relationships with the CCL3s from other vertebrates based on the sequence alignment and phylogenetic analysis. The transcripts of ccl3 were notably enriched in immune-related organs, such as spleen and gills in healthy mandarin fish, and the ccl3 was induced in the isolated mandarin fish brain (MFB) cells following infection with infectious spleen and kidney necrosis virus (ISKNV). Moreover, in MFB cells, overexpression of CCL3 induced immune factors, such as IL1ß, TNFα, MX, IRF1 and IFNh, and exhibited antiviral activity against ISKNV. This study sheds light on the immune role of CCL3 in immune response of mandarin fish, and its antiviral defense mechanism is of interest for further investigation.

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.

Molecular cloning and functional analysis of polymeric immunoglobulin receptor, pIgR, gene in mandarin fish Siniperca chuatsi.

Polymeric immunoglobulin receptor (pIgR) can bind and transport immunoglobulins (Igs), thus playing a role in mucosal immunity. In this study, pIgR gene was cloned in mandarin fish, Siniperca chuatsi, with the open reading frame (ORF) of 1011 bp, encoding 336 amino acids. The pIgR protein consists of a signal peptide, an extracellular domain, a transmembrane domain and an intracellular region, with the presence of two Ig-like domains (ILDs) in the extracellular domain, as reported in other species of fish. The pIgR gene was expressed in all organs/tissues of healthy mandarin fish, with higher level observed in liver and spleen. Following the immersion infection of Flavobacterium columnare, pIgR transcripts were detected in immune related, especially mucosal tissues, with significantly increased transcription during the first two days of infection. Through transfection of plasmids expressing pIgR, IgT and IgM, pIgR was found to be interacted with IgT and IgM as revealed by co-immunoprecipitation and immunofluorescence.

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.

Molecular and functional characterization of zinc finger aspartate-histidine-histidine-cysteine (DHHC)-type containing 1, ZDHHC1 in Chinese perch Siniperca chuatsi.

In the present study, the zinc finger aspartate-histidine-histidine-cysteine (DHHC)-type containing 1 (ZDHHC1) gene was identified in a commercial fish, the Chinese perch Siniperca chuatsi. The ZDHHC1 has five putative transmembrane motifs and conserved DHHC domain, showing high amino-acid identity with other teleost fish, and vertebrate ZDHHC1 loci are conserved from fish to human. In vivo expression analysis indicated that ZDHHC1 gene was constitutively transcribed in all the examined organs/tissues, and was induced following infectious spleen and kidney necrosis virus (ISKNV) infection. It is further observed that ZDHHC1 interacts with MITA and the overexpression of ZDHHC1 in cells resulted in the upregulated expression of ISGs, such as Mx, RSAD2, IRF3 and type I IFNs such as IFNh and IFNc, exhibiting its antiviral function in fish as reported in mammals.

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.

Four type I IFNs, IFNa1, IFNa2, IFNb, IFNc, and their receptor usage in an osteoglossomorph fish, the Asian arowana, Scleropages formosus.

In fish, type I IFNs are classified into three groups, i.e. Group I, Group II and Group III, which are further divided into seven subgroups according to the number of conservative cysteines, phylogenetic relationship, and probably their receptor complexes. In the present study, four type I IFNs and four cytokine receptor family B members (CRFBs) were identified in the Asian arowana, Scleropages formosus, an ancient species in the Osteoglossomorpha with commercial and conservation values. According to multiple sequence alignment and phylogenetic relationship, the four type I IFNs are named as IFNa1, IFNa2, IFNb and IFNc, with the former two belonging to Group I, and the latter two to Group II. The four receptors are named as CRFB1, CRFB2, CRFB5a and CRFB5b. The IFNs and their possible receptor genes are widely expressed in examined organs/tissues, and are induced following the stimulation of polyinosinic polycytidylic acid (polyI:C) in vivo. It was found that IFNa1, IFNa2, IFNb and IFNc use preferentially the receptor complexes, CRFB1 and CRFB5b, CRFB1 and CRFB5b, CRFB2 and CRFB5a, and CRFB2 and CRFB5b, respectively, indicating the evolutionary diversification in the interaction of type I IFNs and their receptors in this ancient fish species, S. formosus.

Expression and antibacterial analysis of galectin-8 and -9 genes in mandarin fish, Siniperca chuatsi.

Galectin-8 and galectin-9 belong to tandem repeat-type galectins, and in the present study, these two genes were cloned in mandarin fish Siniperca chuatsi. The open reading frame (ORF) of the mandarin fish galectin-8 and galectin-9 contains 942, and 1008 bp, encoding 313 and 335 amino acids, respectively. As a conserved feature, an N-terminal carbohydrate recognition domain (CRD), and a C-terminal CRD were observed in each of the two galectins in mandarin fish. In healthy fish, galectin-8 and -9 were constitutively expressed in all organs/tissues examined, and their expression can be induced following the stimulation of LPS and poly(I:C). It is obvious that galectin-8 had a higher increase at mRNA level following the stimulation of poly(I:C). It is further demonstrated that mandarin fish galectin-8 inhibited the growth of Flavobacterium columnare and Streptococcus agalactiae, and in addition to the two species of bacteria, galectin-9 inhibited also the growth of Edwardsiella piscicida, which provides the basis for further understanding their antibacterial role in immune response of mandarin fish.

Identification of type I IFNs and their receptors in a cyprinid fish, the topmouth culter Culter alburnus.

In fish, type I IFNs are classified into three groups, i.e. group one, group two and group three, and further separated into seven subgroups based on the number of conserved cysteines and phylogenetic relationships. In the present study, four type I IFNs, named as IFNϕ1, IFNϕ2, IFNϕ3, IFNϕ4, as reported in zebrafish, were identified in a cyprinid, the topmouth culter, Culter alburnus, a species introduced recently into China's aquaculture. These IFNs may be classified as IFNa, IFNc, IFNc and IFNd in a recent nomenclature, with IFNa and IFNd having two cysteines in group one, and IFNc four cysteines in group two. These IFNs, together with their possible receptors, IFNϕ1, IFNϕ2, IFNϕ3, IFNϕ4, and CRFB1, CRFB2 and CRFB5 have an open reading frame (ORF) of 540, 552, 567, 516 bp, and 1572, 1392, 1125 bp, respectively. These IFNs have high amino acid sequence identities, being 91.1-93.6% and 66.9-77.3%, with those in grass carp and zebrafish, respectively, and are expressed constitutively in organs/tissues examined in the fish. The expression of these IFNs can be further induced following poly (I:C) stimulation. However, the possible function of these IFNs and their signalling pathway are of interest for further research.

Unique Composition of Intronless and Intron-Containing Type I IFNs in the Tibetan Frog Nanorana parkeri Provides New Evidence To Support Independent Retroposition Hypothesis for Type I IFN Genes in Amphibians.

In vertebrates, intron-containing and intronless type I IFN genes have recently been reported in amphibian model species Xenopus tropicalis and X. laevis. However, whether intronless type I IFNs in amphibians are the ancestral genes of type I IFNs in amniotes or just represent the independent divergence in amphibians is unknown or even uninvestigated. In this study, both intron-containing and intronless type I IFN genes, as well as their receptor genes, were identified in the Tibetan frog Nanorana parkeri The evidence obtained from homology, synteny, phylogeny, and divergence time showed that intronless type I IFN genes in N. parkeri and in Xenopus might have arisen from two independent retroposition events occurred in these two lineages, and the retrotransposition causing the generation of intronless type I IFN genes in amniotes is another independent event beyond the two in amphibians. It can then be proposed that intronless type I IFNs in N. parkeri and Xenopus may not be the ancestral genes of intronless type I IFNs in amniotes but may just represent two independent bifurcations in the amphibian lineage. Furthermore, both intronless and intron-containing type I IFNs in N. parkeri showed strong ability in inducing the expression of IFN-stimulated genes and the strong antiviral activity against frog virus 3. The present study thus provides the evolutionary evidence to support the independent retroposition hypothesis for the occurrence of intronless type I IFN genes in amphibians and contributes to a functional understanding of type I IFNs in this group of vertebrates.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in fish: current knowledge and future perspectives.

Retinoic acid-inducible gene I (RIG-I) -like receptors (RLRs) are found conservatively present in teleost fish. All three members, RIG-I, MDA5 and LGP2, together with the downstream molecules such as MITA, TRAF3 and TBK1, have been identified in a range of fish species. However, it is unexpected that RIG-I has not been reported in fish of Acanthopterygii, and it would be important to clarify the presence and role of the RIG-I gene in a broad range of taxa in Teleostei. RLRs in fish can be induced in vivo and in vitro by viral pathogens as well as synthetic dsRNA, poly(I:C), leading to the production of type I interferons (IFNs) and the expression of IFN-stimulated genes (ISGs). Bacterial pathogens, such as Edwardsiella tarda, and their components, such as lipopolysaccharide are also found to induce the expression of RLRs, and whether such induction was mediated through the direct recognition by RLRs or through crosstalk with other pattern recognition receptors recognizing directly bacterial pathogen-associated molecular patterns awaits to be investigated. On the other hand, RLR-activated type I IFN production can be negatively regulated in fish by molecules, such as TBK-1-like protein and IRF10, which are found to negatively regulate RIG-I and MAVS-activated type I IFN production, and to block MITA or bind ISRE motifs, respectively. It is considered that the evolutionary occurrence of RLRs in fish, and their recognized ligands, especially those from their fish pathogens, as well as the mechanisms involved in the RLR signalling pathways, are of significant interest for further investigation.

Two type II IFN members, IFN-γ and IFN-γ related (rel), regulate differentially IRF1 and IRF11 in zebrafish.

Two members of type II IFNs have been identified in fish, i.e. an IFN-γ gene as in other vertebrates and a unique IFN-γ related (IFN-γ rel) gene being solely present in fish. However, the signalling pathways involved in the down-stream signalling of type II IFNs in fish remains poorly described. In this study, the type II IFNs mediated IRF1 was investigated in zebrafish, and the true homologous gene of mammalian IRF1 in fish was revealed despite the report of so-called IRF1a and IRF1b in zebrafish. As revealed in overexpression analysis, zebrafish IFN-γ had a higher induction ability than IFN-γ rel in relation with the expression of IRF1. IFN-γ stimulated the expression level of STAT1a and also STAT1b, but they had opposite trends with the increase of time; enhancement of STAT1a waned after 12 h post injection of plasmids; whereas STAT1b expression increased continuously. Zebrafish IRF1 gene promoter contained several putative transcription factor binding sites, including GAS and NF-κB motifs. Luciferase assay revealed that the GAS site was essential in the IFN-γ triggered IRF1 expression. In contrast, IRF11 contained neither GAS nor NF-κB elements, and did not respond to IFN-γ induction. It is considered that STAT1a and STAT1b are structurally and functionally similar to STAT1α and STAT1ß in mammal respectively, and that IRF11, although used to be nominated as IRF1a, is not the orthologue of mammalian IRF1, but IRF1b in zebrafish should be the orthologue.

NOD2 in zebrafish functions in antibacterial and also antiviral responses via NF-κB, and also MDA5, RIG-I and MAVS.

NOD2/RIPK2 signalling plays essential role in the modulation of innate and adaptive immunity in mammals. In this study, NOD2 was functionally characterized in zebrafish (Danio rerio), and its interaction with a receptor-interaction protein, RIPK2, and RLRs such as MDA5 and RIG-I, as well as the adaptor, MAVS was revealed in fish innate immunity. The expression of NOD2 and RIPK2 in ZF4 cells has been constitutive and can be induced by the infection of Edwardsiella tarda and SVCV. The NOD2 can sense MDP in PGN from Gram-negative and -positive bacteria. It is further revealed that the NOD2 and RIPK2 can activate NF-κB and IFN promoters, inducing significantly antiviral defense against SVCV infection. As observed in the reduced bacterial burden in RIPK2 overexpressed cells, RIPK2 also has a role in inhibiting the bacterial replication. The overexpression of NOD2 in zebrafish embryos resulted in the increase of immune gene expression, especially those encoding PRRs and cytokines involved in antiviral response such as MDA5, RIG-I, and type I IFNs, etc. Luciferase reporter assays and co-immunoprecipitation assays demonstrated that zebrafish NOD2 is associated with MDA5 and RIG-I in signalling pathway. In addition, it is further demonstrated that RIPK2 and MAVS in combination with NOD2 have an enhanced role in NOD2-mediated NF-κB and type I IFN activation. It is concluded that teleost fish NOD2 can not only sense MDP for activating innate immunity as reported in mammals, but can also interact with other PRRs to form a network in antiviral innate response.

Higher antiviral response of RIG-I through enhancing RIG-I/MAVS-mediated signaling by its long insertion variant in zebrafish.

As an intracellular pattern recognition receptor (PRR), the retinoic acid-inducible gene-I (RIG-I) is responsible for the recognition of cytosolic viral nucleic acids and the production of type I interferons (IFNs). In the present study, an insertion variant of RIG-I with 38 amino acids inserted in the N-terminal CARD2 domain, as well as the typical type, named as RIG-Ia and RIG-Ib respectively were identified in zebrafish. RIG-Ia and RIG-Ib were all up-regulated following the infection of a negative ssRNA virus, the Spring Viremia of Carp Virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, indicating the RLR may have a role in the recognition of both viruses and bacteria. The over-expression of RIG-Ib in cultured fish cells resulted in significant increase in type I IFN promoter activity, and in protection against SVCV infection, whereas the over-expression of RIG-Ia had no direct effect on IFN activation nor antiviral response. Furthermore, it was revealed that both RIG-Ia and RIG-Ib were associated with the downstream molecular mitochondrial antiviral signaling protein, MAVS, and interestingly RIG-Ia when co-transfected with RIG-Ib or MAVS, induced a significantly higher level of type I IFN promoter activity and the expression level of Mx and IRF7, implying that the RIG-Ia may function as an enhancer in the RIG-Ib/MAVS-mediated signaling pathway.

Melanoma differentiation-associated gene 5 in zebrafish provoking higher interferon-promoter activity through signalling enhancing of its shorter splicing variant.

Melanoma differentiation-associated gene 5 (MDA5) is one of the three members in the retinoic acid-inducible gene I-like receptor (RLR) family, which are cytoplasmic pathogen recognition receptors recognizing intracellular viruses. In the present study, MDA5 and its spliced shorter forms, named as MDA5a and MDA5b, were identified in zebrafish. MDA5a and MDA5b can be up-regulated in cell lines following the infection of a negative ssRNA virus, the spring viraemia of carp virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, implying that the RLR may also be able to sense elements released from bacteria. The over-expression of MDA5a and MDA5b in fish cells resulted in significant induction of type I interferon promoter activity and enabled the protection of transfected cells against SVCV infection. Furthermore, the shorter spliced form, MDA5b when co-transfected with MDA5a or mitochondrial antiviral signalling protein (MAVS), induced a significantly higher level of interferon promoter activity, indicating that MDA5b may function as an enhancer in the interaction between MDA5 and MAVS.

IFN-υ and its receptor subunits, IFN-υR1 and IL10RB in mallard Anas platyrhynchos.

Type IV interferon (IFN) has been shown to be a cytokine with antiviral activity in fish and amphibian. But, it has not been cloned and characterized functionally in avian species. In this study, type IV IFN, IFN-υ, and its 2 possible receptors, IFN-υR1 and IL10RB, were identified from an avian species, the mallard (Anas platyrhynchos). Mallard IFN-υ has a 531 bp open reading frame (ORF), encoding 176 amino acids (aa), and has highly conserved features as reported in different species, with an N-terminal signal peptide and a predicted multi-helix structure. The IFN-υR1 and IL10RB contain 528 and 343 aa, respectively, with IFN-υR1 protein containing JAK1 and STAT binding sites, and IL10RB containing TYK2 binding site. These 2 receptor subunits also possess 3 domains, the N-terminal extracellular domain, the transmembrane domain, and the C-terminal intracellular domain. Expression analysis indicated that IFN-υ, IFN-υR1 and IL10RB were widely expressed in examined organs/tissues, with the highest level observed in pancreas, blood, and kidney, respectively. The expression of IFN-υ, IFN-υR1 and IL10RB in liver, spleen or kidney was significantly upregulated after stimulation with polyI:C. Furthermore, recombinant IFN-υ protein induced the expression of ISGs, and the receptor of IFN-υ was verified as IFN-υR1 and IL10RB using a chimeric receptor approach in HEK293 cells. Taken together, these results indicate that IFN-υ is involved in the host innate immune response in mallard.