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.

Transcription of NOD1 and NOD2 and their interaction with CARD9 and RIPK2 in IFN signaling in a perciform fish, the Chinese perch, Siniperca chuatsi.

NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of nod1 and nod2 and their signal circle are less understood in teleost fish. In this study, with the cloning of card9 and ripk2 in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that nod1 and nod2 were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that nod1 and nod2 can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response.

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.

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.

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 characterization of nineteen cytokine receptor family B (CRFB) members, CRFB1, CRFB2, CRFB4-17, with three CRFB9 and two CRFB14 in a cyprinid fish, the blunt snout bream Megalobrama amblycephala.

The class II cytokine receptor family members are receptors of class 2 helical cytokines in mammals, and are named cytokine receptor family B (CRFB) in fish. In zebrafish, sixteen members, including CRFB1, CRFB2 and CRFB4-17 were reported. With the availability of genome sequence, a total of nineteen CRFBs was identified in the blunt snout bream (Megalobrama amblycephala), including CRFB1, CRFB2, CRFB4-17 with the presence of three CRFB9 isoforms, and two CRFB14 isoforms. These CRFB molecules contain well conserved features, such as fibronectin type III (FNIII) domain, transmembrane and intracellular domains as other class II cytokine receptors, and are phylogenetically grouped into thirteen clades with their homologues from other species of fish. The CRFB genes were constitutively expressed in organs/tissues examined in the fish. The finding of more CRFB members in the bream may provide clues to understand possible receptor-ligand interaction and their diversity from an evolutionary point of view.

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.

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.

FTR33, a member of fish-specific TRIM (finTRIM) subfamily, regulates negatively type I IFN antiviral immunity in zebrafish.

In mammals, the tripartite motif (TRIM) proteins have been identified as critical factors involved in various cellular processes, including antiviral immunity. In teleost fish, a subfamily of fish-specific TRIM (finTRIM, FTR) has emerged in genus- or species-specific duplication. In this study, a finTRIM gene, called ftr33, was identified in zebrafish (Danio rerio), and phylogenic analysis revealed that FTR33 is closely related with zebrafish FTR14. The FTR33 protein contains all conservative domains reported in other finTRIMs. The ftr33 has a constitutive expression in embryos and in tissues/organs of adult fish, and its expression can be induced following spring viremia of carp virus (SVCV) infection and interferon (IFN) stimulation. The overexpression of FTR33 significantly downregulated the expression of type I IFNs and IFN-stimulated genes (ISGs) both in vitro and in vivo, respectively, leading to the increased replication of SVCV. It was also found that FTR33 interacted with melanoma differentiation associated gene 5 (MDA5) or mitochondrial anti-viral signaling protein (MAVS) to weaken the promoter activity of type I IFN. It is thus concluded that the FTR33, as an ISG, in zebrafish can negatively regulate IFN-mediated antiviral response.

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.

Functional characterization of IL-18 receptor subunits, IL-18Rα and IL-18Rß, and its natural inhibitor, IL-18 binding protein (IL-18BP) in rainbow trout Oncorhynchus mykiss.

As an important proinflammation and immunomodulatory cytokine, IL-18 has been reported in several species of fish, but its receptor subunits, IL-18Rα and IL-18Rß, and its decoy receptor, IL-18BP, have not been functionally characterized in fish. In the present study, IL-18Rα, IL-18Rß and IL-18BP were cloned from rainbow trout Oncorhynchus mykiss, and they possess common conserved domains with their mammalian orthologues. In tested organs/tissues, IL-18Rα and IL-18Rß exhibit basal expression levels, and IL-18BP has a pattern of constitutive expression. When transfected with different combinations of chimeric receptors in HEK293T cells, recombinant IL-18 (rIL-18) can induce the activation of NF-κB only when pcDNA3.1-IL-18Rα/IL-1R1 and pcDNA3.1-IL-18Rß/IL-1RAP were both expressed. On the other hand, recombinant receptors, including rIL-18BP, rIL-18Rα-ECD-Fc and rIL-18Rß-ECD-Fc can down-regulate significantly the activity of NF-κB, suggesting the participation of IL-18Rα, IL-18Rß and IL-18BP in rainbow trout IL-18 signal transduction. Co-IP assays indicated that IL-18Rß may form a complex with MyD88, IRAK4, IRAK1, TRAF6 and TAB2 in HEK293T cells, indicating that IL-18Rß, in IL-18 signalling pathway, is associated with these signalling molecules. In conclusion, IL-18Rα, IL-18Rß and IL-18BP in rainbow trout are conserved in function and signalling pathway with their mammalian orthologues.

Identification of type II interferons and receptors in an osteoglossiform fish, the arapaima Arapaima gigas.

In mammals, type II interferon (IFN; i.e. IFN-γ) signalling transduces through its specific receptors IFN-γR1 and IFN-γR2. In an osteoglossiform fish, the arapaima Arapaima gigas, three type II IFNs, IFN-γ-like, IFN-γ and IFN-γrel, and their four possible receptor subunits IFN-γR1-1, IFN-γR1-2, IFN-γR2-1 and IFN-γR2-2 were identified in this study. The three type II IFN genes are composed of four exons and three introns, and they all contain IFN-γ signature motif and signal peptide, with the presence of potential nuclear localization signal (NLS) in IFN-γ-like and IFN-γ. The IFN-γR1-1, IFN-γR1-2, IFN-γR2-1 and IFN-γR2-2 are composed of seven exons and six introns, with predicted IFN-γR1-1 and IFN-γR1-2 proteins containing JAK1 and STAT1 binding sites, and IFN-γR2-1 and IFN-γR2-2 containing JAK2 binding sites. Gene synteny analysis showed that the type II IFN and their receptor loci are duplicated in arapaima. All these genes were expressed constitutively in all organs/tissues examined, and responded to the stimulation of polyI:C. The prokaryotic recombinant IFN-γ-like, IFN-γ and IFN-γrel proteins can significantly induce the upregulation of immune-related genes in trunk kidney leucocytes. The ligand-receptor relationship analyses revealed that recombinant IFN-γ-like, IFN-γ, and IFN-γrel transduce downstream signalling through IFN-γR1-1/IFN-γR2-1, IFN-γR1-2/IFN-γR2-2, and IFN-γR1-1, respectively, in xenogeneic cells with the overexpression of original or chimeric receptors. In addition, tyrosine (Y) 366 and Y377 in the intracellular region may be essential for the function of IFN-γR1-2 and IFN-γR1-1, respectively. The finding of type II IFN system in A. gigas thus provides different knowledge in understanding the diversity and evolution of type II IFN ligand-receptor relationships in vertebrates.

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.

Gene synteny, evolution and antiviral activity of type I IFNs in a reptile species, the Chinese soft-shelled turtle Pelodiscus sinensis.

Type I interferons (IFNs) are critical cytokines for the establishment of antiviral status in fish, amphibian, avian and mammal, but the knowledge on type I IFNs is rather limited in reptile. In this study, seven type I IFN genes, designed as IFN1 to IFN7, were identified from a reptile species, the Chinese soft-shelled turtle (Pelodiscus sinensis). These identified type I IFNs have relatively low protein identity, when compared with those in human and chicken; but they possess conserved cysteines, predicted multi-helix structure and N-terminal signal peptide. The Chinese soft-shelled turtle IFN1 to IFN5 have two exons and one intron, but IFN6 and IFN7 are the single-exon genes. Chinese soft-shelled turtle type I IFNs are located respectively on the two conserved reptile-bird loci, named as Locus a and Locus c, and are clustered into the four of the five reptile-bird groups (named as Groups I-V) based on phylogenetic evidence, due to the lack of IFNK in the turtle. Moreover, the Chinese soft-shelled turtle type I IFNs can be induced by soft-shelled turtle iridovirus (STIV) infection and show antiviral activity in soft-shelled turtle artery (STA) cells, except IFN6. In addition, due to the difference in genome organizations, such as the number of exons and introns of type I IFN genes from fish to mammal, the definition and evolution of 'intronless' type I IFN genes were discussed in lineages of vertebrates. Thus, the finding of type I IFNs on two different loci in P. sinensis sheds light on the evolution of type I IFN genes in vertebrates.

Identification and establishment of type IV interferon and the characterization of interferon-υ including its class II cytokine receptors IFN-υR1 and IL-10R2.

Interferons (IFNs) are critical soluble factors in the immune system and are composed of three types, (I, II and III) that utilize different receptor complexes IFN-αR1/IFN-αR2, IFN-γR1/IFN-γR2, and IFN-λR1/IL-10R2, respectively. Here we identify IFN-υ from the genomic sequences of vertebrates. The members of class II cytokine receptors, IFN-υR1 and IL-10R2, are identified as the receptor complex of IFN-υ, and are associated with IFN-υ stimulated gene expression and antiviral activity in zebrafish (Danio rerio) and African clawed frog (Xenopus laevis). IFN-υ and IFN-υR1 are separately located at unique and highly conserved loci, being distinct from all other three-type IFNs. IFN-υ and IFN-υR1 are phylogenetically clustered with class II cytokines and class II cytokine receptors, respectively. Therefore, the finding of this IFN ligand-receptor system may be considered as a type IV IFN, in addition to the currently recognized three types of IFNs in vertebrates.

Molecular characterization and transcriptional conservation of N-myc-interactor, Nmi, by type I and type II IFNs in mandarin fish Siniperca chuatsi.

N-myc-interactor (Nmi) belongs to interferon (IFN) stimulated genes (ISGs) and is involved in the regulation of physiological processes including viral infection, inflammatory response, apoptosis and tumorigenesis in mammals. However, the function of Nmi in teleost fish remains to be explored. In this study, an Nmi homologue was characterized from mandarin fish Siniperca chuatsi. The mandarin fish Nmi shares two conserved functional Nmi/IFP35 homology domains (NIDs) with mammalian Nmi protein in its C-terminal domain and a coiled coil region (CC) in its N-terminal domain, with its genomic DNA sequence consisting of nine exons and eight introns. Subcellular localization analysis shows that mandarin fish Nmi is a cytoplasmic protein and that its localization is dependent on the CC and NID1 regions. High and constitutive mRNA level of Nmi was observed in all examined tissues, with the highest level being observed in blood. In addition, the Nmi gene was significantly induced in various organs/tissues following the infection of infectious spleen and kidney necrosis virus (ISKNV), and its mRNA and protein level was also significantly induced in vitro after the treatment of IFNh, IFNc, as well as IFN-γ. The dual luciferase activity analysis indicated that the Nmi promoter was activated by the three type I IFNs through interferon-stimulated response element (ISRE) sites, and it can be also transcriptionally activated by IFN-γ via IRF1 which can activate the expression of Nmi through ISRE. Taken together, it is demonstrated in this study that the transcription of Nmi in mandarin fish can be regulated by type I and type II IFNs, thus confirming that Nmi in fish is also an ISG, and is involved in antiviral and IFN-induced innate immunity.

Presence of two RIG-I-like receptors, MDA5 and LGP2, and their dsRNA binding capacity in a perciform fish, the snakehead Channa argus.

Fish retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are critical RNA sensors in cytoplasm and are involved in antiviral innate immunity. However, some species of fish lack RIG-I gene, and the function of RLR members in RIG-I-absent fish is poorly understood. In the present study, MDA5, LGP2 and MAVS genes were identified in commercially important snakehead Channa argus. But, RIG-I gene was not found in this fish, and a systematic analysis of RLRs in available genome database of fish indicated the absence of RIG-I in the Acanthomorphata, Clupeiformes and Polypteriformes, suggesting that loss events of RIG-I gene may have occurred independently three times in the evolutionary history of fish. The MDA5, LGP2 and MAVS in snakehead have conserved protein domains and genomic location based on sequence, phylogenetic and syntenic analyses. These genes are constitutively expressed in healthy fish and can be induced by polyinosinic and polycytidylic acid (poly(I:C)) stimulation in vitro. It is further revealed that the snakehead MDA5 and LGP2 have binding capacity with dsRNA, such as poly(I:C), and MDA5 can interact with MAVS, implying the antiviral function of MDA5 in the RIG-I-absent fish.

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.