Functional characterization of interferon regulatory factor 5 and its role in the innate antiviral immune response.

In mammals, type I interferons (IFNs) are primarily regulated by transcription factors of the IFN regulatory (IRF) family. Interferon regulatory factor 5 (IRF-5) plays pivotal roles in antiviral and inflammatory responses. In the present study, we found that zebrafish (Danio rerio) IRF5 is a key player in the regulation of the expression of type I IFN and its antiviral immune response. IRF5 was upregulated in zebrafish embryonic fibroblast cells (ZF4) when challenged with grass carp reovirus (GCRV). Moreover, the expression profiles of Mx, IFN, Viperin, and IRF7, but not IRF3, were upregulated by overexpression of IRF5 in Epithelioma papulosum cyprinid cells (EPCs). Luciferase assays revealed that the activation of the IFNϕ1 promoter was stimulated by overexpression of IRF5 and IRF5-△IAD (IRF5 lacking the IRF-associated domain), respectively. However, overexpression of IRF5 or IRF5-△IAD inhibited the activity of the IFNϕ3 promoter. IRF5-△DBD (lacking the DNA-binding domain) had no influence in the activation of the IFNϕ1 and IFNϕ3 promoters. Furthermore, the determination of the cytopathic effect (CPE) numbers and viral titers revealed that the viral concentration was reduced by ectopic expression of IRF5 in EPC cells. Ectopic expression of IRF5 in EPC cells could protect cells from GCRV and significantly inhibited GCRV virus replication. These data indicated that IRF5 could limit viral replication through an IFN-dependent pathway.

Transcriptome analysis of the critically endangered Dabry's sturgeon (Acipenser dabryanus) head kidney response to Aeromonas hydrophila.

Dabry's sturgeon (Acipenser dabryanus), as a living fossil, is considered a critically endangered aquatic animal in China. To date, the immune system of this species remains largely unknown, with limited available sequence information. In addition, increasing incidence of bacterial pathogenic diseases has been reported. Hence, the present study aimed to characterize comprehensively transcriptome profile of the head kidney from Dabry's sturgeon infected with Aeromonas hydrophila using Illumina platform. Over 42 million high-quality reads were obtained and de novo assembled into a final set of 195240 unique transcript fragments (unigenes), with an average length of 564 bp. Approximately 41702 unigenes were annotated in the NR NCBI database. Dabry's sturgeon unigenes had the highest number of hits with 14365 (34.45%) to Lepisosteus oculatus. The 195240 unigenes were assigned to three Gene Ontology (GO) categories: biological process, cellular component, and molecular function. Among them, 27770 unigenes were clustered into 26 Eukaryotic Orthologous Group (KOG) functional categories, and 36031 unigenes were mapped to 335 known Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. After A. hydrophila administration, 1728 differentially expressed unigenes were identified, including 980 upregulated and 748 downregulated unigenes. Further KEGG enrichment analysis of these unigenes identified 16 immune-related pathways, including the Toll-like receptor signaling pathway, chemokine signaling pathway, complement and coagulation pathway, RIG-I-like receptor signaling pathway, and NOD-like receptor signaling pathway. 20 DEGs were selected and their expression patterns are largely consistent with the transcriptome profile analysis, which clearly validated the reliability of the DEGs in transcriptome analysis. This work revealed novel gene expression patterns of Dabry's sturgeon host defense and contributes to a better understanding of the immune system and defense mechanisms of Dabry's sturgeon in response to bacterial infection. The results provide valuable references for studies in sturgeons that lack complete genomic sequences, and could also be helpful for the analyzing evolution among cartilaginous and teleost fish.

Bioinformatics and expression analysis of finTRIM genes in grass carp, Ctenopharyngodon idella.

The tripartite motifs (TRIMs) constitute a large family of proteins containing a Really Interesting New Gene (RING) domain, a B-box domain and coiled-coil region followed by different C-terminal domains. TRIM proteins play multiple roles in various cellular processes, including cell growth, differentiation, apoptosis and antiviral immunity. Fish novel large multigene TRIM genes (finTRIM/ftr) appear only in teleosts and play a vital role in antiviral responses. Phylogenetic analysis revealed the existence of different subsets of novel fish TRIM 14 genes (finTRIM14/ftr14), ftr51, ftr67, ftr72, ftr82, ftr83, and ftr99 in grass carp (Ctenopharyngodon idella), suggesting lineage-specific diversification events. Therefore, the number of finTRIM genes varies greatly among species. The ftr genes in grass carp, which are closely related to zebrafish and possess various evolutionary branches, have evolved faster than human TRIMs. The predicted protein domains were almost identical RING zinc finger domains, with the exception of ftr72, the B-box domain (excluding ftr67, ftr82, ftr83), and the B30.2 domain, which evolved under positive selection (with the exception of ftr67, and ftr72). The genes were predominantly expressed in the spleen, gill and head kidney. These findings indicate that the ftr genes in grass carp are involved diverse cellular processes, including innate immune responses.

Analysis of the expression patterns of the novel large multigene TRIM gene family (finTRIM) in zebrafish.

Tripartite motif (TRIM) proteins are receiving increased research interest because of their roles in a wide range of cellular biological processes in innate immunity. In zebrafish (Danio rerio), the functions of the finTRIM (ftr) family are unclear. In the present study, we investigated the expression pattern of ftr12, ftr51, ftr67, ftr82, ftr83, and ftr84 in zebrafish for the first time. The results showed that ftr12, ftr67, and ftr84 are maternally expressed in the oocyte and highly expressed at the early stage (0-4 hpf) of embryo (P < 0.05), suggesting their involvement in the embryonic innate defense system. The ftr82 gene was highly expressed at 8 hpf (P < 0.05), which implied that the embryos could synthesize their own immunity-related mRNAs. However, ftr51 and ftr83 were highest at 8 hpf (2.33 and 51.53 relative to ß-actin respectively) and might mediate embryonic development. The expression levels of ftr12, ftr51, and ftr67 were highest in the gill, intestines, and liver, respectively. Ftr82, ftr83, and ftr84 were predominantly expressed in the kidney, suggesting that these finTRIMs might play roles in both immunity and non-immunity-related tissue compartments. Zebrafish embryonic fibroblast (ZF4) cells were infected with Grass carp reovirus (GCRV) and Spring viremia of carp virus (SVCV). During GCRV infection, the expression of ftr12 was significantly upregulated from 12 h to 24 h; and ftr51 and ftr67 increased from 3 h to 12 h. The expressions of ftr82, ftr83, and ftr84 were only upregulated at 12 h, 12 h, and 24 h, respectively. All of these genes were significantly downregulated at 48 h (P < 0.05). Challenge with SVCV upregulated the expressions of ftr12 and ftr51 at 12 h and 48 h (P < 0.05), respectively, and ftr67 reached its highest expression level at 3 h. ftr82 showed only a slight upregulation at 6 h and 48 h, and ftr83 and ftr84 were consecutively increased, reaching their highest levels at 12 h (P < 0.05). Meanwhile, ftr67 and ftr83 were significantly downregulated at 48 h (P < 0.05). Our research demonstrated that ftr12, ftr51, ftr67, ftr82, ftr83, and ftr84 probably have important roles in innate immune responses and in non-immunity-related tissues.

Analysis of the expression patterns of the cytokine receptor family B (CRFB) and interferon gamma receptor (IFNGR) in Dabry's sturgeon (Acipenser dabryanus).

Teleost fish have more complex interferon receptor systems than mammals. In the present study, genes encoding four cytokine receptor family B (CRFBs) and two interferon gamma receptors (IFNGRs) in Dabry's sturgeon (Acipenser dabryanus) were identified by RNA-sequencing. Sequence analysis revealed that the Dabry's sturgeon CRFBs and IFNGRs contained several conserved characteristics features, including signal peptides and a transmembrane domain. Phylogenetic analysis suggested that they belong to the CRFB3, CRFB5, and IFNGR protein families, and were named CRFB3a, CRFB3b, CRFB5a, CRFB5b, IFNGR1, and IFNGR2. The expression patterns of the CRFB and IFNGR genes were investigated in Dabry's sturgeon. The expression levels of CRFB5a, CRFB5b, and IFNGR1 showed no significant changes, suggesting that those genes do not mediate embryonic development. By contrast, the high expression levels of CRFB3a, CRFB3b, and IFNGR2 in the fertilized egg, 16-cell phase, and initial blastula stage implied the existence of maternally expression in the oocyte and association with embryonic development. Tissue distribution analysis revealed that the CRFB and IFNGR proteins have potential functions in immune and non-immune tissue compartments. Comprehensive analysis in Dabry's sturgeon revealed that the expression fold changes of CRFB3a, CRFB3b, CRFB5a, and CRFB5b in Dabry's sturgeon stimulated with poly I:C were higher than those in fish administrated with lipopolysaccharide (LPS). Conversely, the fold changes IFNGRs mRNA levels stimulated with LPS were higher than those in fish administrated with poly I: C. CRFB5a and IFNGR2 genes showed the earliest responses to the poly I: C, and the CRFB5a and IFNGR1 genes showed the earliest responses to LPS. These results implied that CRFB5a has important role in the IFN immune response. Our findings indicated that the Dabry's sturgeon CRFB and IFNGR genes have important functions in antiviral and antibacterial immune responses. The differential responses of these genes to poly I: C and LPS implied differences in the defense mechanisms against viruses and bacteria.

First Study on Interferon Regulatory Factor in Sturgeon: Expression Pattern of Interferon Regulatory Factor in Dabry's Sturgeon Acipenser dabryanus.

Interferon regulatory factors (IRFs) are crucial regulators in initiating the host innate immune response against pathogen invasions. Dabry's sturgeon (Acipenser dabryanus) is particularly a valuable fish species found in the Yangtze River, China for which there is scarce immunological data. In the present study, we investigated the expression profile of sturgeon IRF genes. All adIRFs were composed of 8 exons and 7 introns, except adIRF1, which possessed 9 exons interrupted by 8 introns. Moreover, the predicted protein sequence has a DNA-binding domain (DBD) sharing high identity with spotted gar (Lepisosteus oculatus). Regarding the expression patterns, 5 adIRF genes were found to be constitutively expressed in all tissues examined, and were significantly higher in lymphoid organs (eg, blood, kidney, intestine, and spleen). Following Aeromonas hydrophila infection, the expression of adIRF1 and adIRF3 were upregulated in the spleen and caudal kidney, while both the adIRF5 and adIRF8 genes were downregulated in caudal kidney. In addition, adIRF4 was significantly upregulated at 3 h postinfection by A. hydrophila in the spleen and caudal kidney. These results suggest that adIRFs are related to the immune response to bacterial infection, which will help clarify the function of these IRFs and provide a fundamental basis for protecting the Dabry's sturgeon.