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Cyclic GMP-AMP synthase (cGAS) is known as a DNA sensor for the initiation of innate immune responses in human and other mammals. However, the knowledge about fish cGAS is limited. In this study, we identified two paralogs of cGAS genes from grass carp (Ctenopharyngodon idellus), namely, CicGASa and CicGASb. Grass carp cGASa and cGASb share some conservative domains with mammalian cGASs; however, cGASb contains a unique transmembrane domain. Grass carp cGASa and cGASb responded to GCRV and poly (dA:dT) infection, but they played opposite roles in the regulation of type I IFN response, i.e. cGASa served as an activator for ISGs and NF-κB in a dose-dependent manner, while cGASb acted as an inhibitor. We found that cGASa and cGASb interacted with STING. Similarly, cGASa is an activator for IRF7, but cGASb inhibited IRF7 expression. Both cGASa and STING can protect cells from GCRV infection. Grass carp cGASb inhibited cGASa-induced type I IFN response by the competitive interaction with STING, suggesting that cGASb may be a negative regulator of cGASa-STING-IRF7 axis.

In order to breed new birds with strong disease resistance, it is necessary to first understand the mechanism of avian antiviral response. Interferon regulatory factor 7 (IRF7) is not only a member of type I interferons (IFNs) regulatory factor (IRFs) family, but also a major regulator of the IFN response in mammals. However, whether IRF7 is involved in the host innate immune response remains unclear in poultry, due to the absence of IRF3. Here, we first observed by HE stains that with the increase of the time of ALV-J challenge, the thymus was obviously loose and swollen, the arrangement of liver cell was disordered, and the bursa of fabricius formed vacuolated. Real-time PCR detection showed that the expression level of IRF7 gene and related immune genes in ALV-J group was significantly higher than that in control group (P

International audience; Tilapia lake virus (TiLV; genus: Tilapinevirus, family: Amnoonviridae) is a recently characterised enveloped virus with a linear, negative-sense single-stranded RNA genome, which causes high mortality in tilapia species. In the present study, we demonstrated that zebrafish (Danio rerio) larvae are susceptible to TiLV infection upon systemic injection. TiLV replicated in zebrafish larvae and caused their high mortality (of about 70%). Histopathological examination revealed that TiLV infection caused pathological abnormalities in zebrafish larvae that were well visible within the brain. Moreover, gene expression analysis revealed that TiLV infection induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I (ddx58), tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), and pro-inflammatory cytokine (il-1β). We also demonstrated the protective role of the recombinant zebrafish IFNϕ1 on the survival of zebrafish larvae during TiLV infection. Our results show the importance of type I IFN response during TiLV infection in zebrafish larvae and demonstrate that zebrafish is a good model organism to study interactions between TiLV - a newly emerging in aquaculture virus, and fish host.

Mre11A is considered as a cytosolic DNA receptor in mammals. However, it is rarely known about Mre11A in other vertebrates. Recently, a mammalian ortholog of Mre11A has been identified in grass carp (Ctenopharyngodon idellus) in our lab. Phylogenetic-tree analysis provided evidence for a close genetic relationship between C.idellus Mre11A and Carassius auratus Mre11A. The tissue expression profile of CiMre11A was detected, with a relatively higher level of expression in kidney, intestines, liver and spleen than that in other tissues after grass carp reovirus (GCRV) infection. Similarly, CiMre11A was also up-regulated in CIK cells after treatment with GCRV. Q-PCR and dual-luciferase assays indicated that the transcription levels of IFN1 and ISG15 were inhibited by CiMre11A knockdown, but were gradually augmented after CIK cells were transfected with increasing amounts of CiMre11A. Subcellular localization assays showed that a part of CiMre11A was translocated from the nucleus to the cytoplasm. Co-immunoprecipitation and co-localization assays demonstrated that CiMre11A interacts with CiSTING in response to GCRV infection. In CIK cells, the expressions of both IFN1 and ISG15 were acutely up-regulated by CiMre11A overexpression, as well as by co-overexpression of CiMre11A and CiSTING. CiMre11A and CiSTING induced the phosphorylation and cytoplasmic-to-nuclear translocation of IRF7 in CIK cells. The multiplication of GCRV in CIK cells was inhibited by the overexpression of CiMre11A and CiSTING.

The protein inhibitor of activated STAT (PIAS) proteins are important signal transduction modulator family and regulate the innate immune signaling pathway induced by certain transcription factors, including NF-κB, IRF3, and JAK/STAT. The PIAS protein mechanism that regulates innate immune response in mammals has been well described in the literature; however, whether the PIAS gene exists in ducks as well as the role of PIAS in duck IFN-β expression is still unclear. Here, we cloned duck PIAS (duPIAS), finding PIAS2 could repress IFN-β production. DuPIAS2 contains SAP-PINIT-RLD-S/T characteristic domains, and its overexpression could inhibit virus-induced IFN-β promoter activation. Moreover, duPIAS2 interacts with duck interferon regulatory factor 7 (IRF7) and inhibits IFN-β promoter activation induced by duck IRF7. Additionally, its inhibitory function does not rely on its SUMO E3 ligase activity but rather its C-terminal portion. The above results demonstrate that duPIAS2 is a repressor of IFN-β production induced by duck IRF7.

Interferon regulatory factors (IRFs) is an important family for IFN expression regulating while viral infection. IRF1, IRF3, and IRF7 are the primary regulators that trigger type I IFN response in mammals. However, IRF3, which has been identified as the most critical regulator in mammals, is absent in chickens, and it is unknown whether IRF1 is involved in type I IFN signaling pathways in IRF3-deficient chicken cells. Here, we identified chicken IRF1 (chIRF1) as a critical IFN-β mediator in response to viral infection. Overexpression of chIRF1 activated IFN-β intensively and suppressed AIV and NDV viral replication. Moreover, the mRNA levels of IFN-β and ISGs increased during chIRF1 overexpression. In addition, deletion mutant analysis revealed that the first four domains of chIRF1 are indispensable for IFN-β induction. Together, our studies demonstrate that chIRF1 is an important regulator of IFN-β and is involved in chicken antiviral innate immunity.

Interferon (IFN) system plays a vital role in the first line of defense against viruses. In this study, we first identified multiple transcripts of 15 IFN system genes, including PRRs ( TLR2 , TLR3 , RIG-I , and LGP2 ), PRR-mediated IFN signal pathway ( MyD88 , MITA , and MAVS ), IFN regulatory factors ( IRF1 , IRF3 , IRF7 , and IRF9 ), IFNs ( IFNφ1 and IFNφ3 ), and ISGs ( Mx and viperin ), and one transcript of TLR9 in de novo transcriptome assembly data of gibel carp head-kidney. Multiple nucleotide alignments and phylogenetic analysis of common region showed that the transcripts of every of the 15 IFN system genes were classified into two homologs with distinctly divergent sequences, indicating that hexaploid gibel carp may be an allopolyploid. During Carassius auratus herpesvirus ( Ca HV) infection, gibel carp resistant clone H significantly suppressed Ca HV replication with markedly less viral loads than those in highly susceptible clone A + and moderately resistant clone F. Then, qPCR analyses were performed to reveal their differential and dynamic expression changes during Ca HV infection in head kidney, spleen and liver among three gibel carp gynogenetic clones. Through qPCR and hierarchical clustering analysis, 8 genes, such as RIG-Is , LGP2s , IRF1-B , IRF3s , IRF7s , IRF9-B , Mxs , and viperins , were identified as candidate resistant-related genes. They remarkably increased their expression in immune tissues of three clones after Ca HV infection. Significantly, the up-regulation folds of these genes in clone A + , F and H were related to their resistance ability to Ca HV, progressively increasing from susceptible clone to resistant clone at 1 dpi. The positive correlation to the resistance ability suggested that resistant clone H immediately triggered stronger IFN response. IFNφ3 showed a different dynamic change and was sharply induced in moderately resistant clone F at 3 dpi. The other 5 IFN system genes ( TLR2 , TLR3 , TLR9 , MyD88 , and MITA ) maintained a low expression level after Ca HV challenge. Interestingly, the A or B copies/homologs of almost these IFN system genes exhibited differential transcript abundance in immune tissue after Ca HV challenge, suggesting A or B homologs might occur dominant or biased expression of homeologs during gibel carp evolution. These data provide candidate resistant-related genes for disease-resistance breeding of gibel carp.

A study was conducted to determine the transcriptome response of Atlantic cod (Gadus morhua) macrophages to the viral mimic, polyriboinosinic polyribocytidylic acid (pIC), using a 20K Atlantic cod microarray platform and qPCR. We identified 285 significantly up-regulated and 161 significantly down-regulated probes in cod macrophages 24 h after pIC stimulation. A subset of 26 microarray-identified transcripts was subjected to qPCR validation using samples treated with pIC or phosphate-buffered saline (control) over time (3, 6, 12, 24, 48 h), and 77% of them showed a significant response to pIC. The microarray and qPCR analyses in this study showed that pIC induced the expression of cod macrophage transcripts involved in RLR- and TLR-dependent pathogen recognition (e.g. tlr3, tlr7, mda5 and lgp2), as well as signal transducers (e.g. stat1 and nfkbia) and transcription activators (e.g. irf7 and irf10) in the MyD88-independent and dependent signalling pathways. Several immune effectors (e.g. isg15s, viperin, herc4, mip2 and ccl13) were significantly up-regulated in pIC-stimulated cod macrophages. The expression of some transcripts (e.g. irf7, irf10, viperin) was significantly up-regulated by pIC as early as 12 h. All pIC-induced transcripts had peak expression at either 24 h (e.g. tlr7, irf7, mip2) or 48 h (e.g. tlr3, lgp2, stat1). This study suggests possible roles of both vertebrate-conserved (e.g. tlr3 as an up-regulated gene) and fish-specific (tlr22g as a down-regulated gene) receptors in dsRNA recognition, and the importance of conserved and potentially fish-specific interferon stimulated genes in cod macrophages.

The development of macrophages is a highly regulated process requiring coordination amongst transcription factors. The presence/absence, relative levels, antagonism, or synergy of all transcription factors involved is critical to directing lineage cell fate and differentiation. While relative levels of many key myeloid transcription factors have been determined in mammalian macrophage differentiation, a similar set of studies have yet to be conducted in a teleost system. In this study, we report on the mRNA levels of transcription factors (cebpa, cjun, cmyb, egr1, gata1, gata2, gata3, lmo2, mafb, pax5, pu.1 and runx1) in sorted goldfish progenitor cells, monocytes, and macrophages from primary kidney macrophage cultures. The mRNA levels of runx1 and pu.1 were significantly higher, gata3 and pax5 mRNA levels were lower, in monocytes compared to progenitors, and the mRNA levels of cjun, egr1, gata2, gata3, mafb and pax5 were significantly decreased in macrophages compared to progenitor cells. The relative mRNA levels of the interferon regulatory factor family of transcription factors, irf1, irf2, irf5, irf7, irf8 and irf9 in sorted progenitors, monocytes and macrophages were also measured. In contrast to other irf family transcription factors examined, irf8 mRNA levels were increased in monocytes compared to progenitors by greater than three-fold, suggesting that irf8 is important for monopoiesis. Lastly, we show the differential regulation of myeloid transcription factor mRNA levels in sorted progenitor cells from 1, 2, or 3-day old cultures in response to the recombinant goldfish growth factors, rgCSF-1 and rgKITLA.

The interferon regulatory factor (IRF) family plays critical roles in a host's virus infection responses. In this study, two IRF family members, zfIRF5 and zfIRF7, are identified in zebrafish. The zfIRF5 protein encodes 297 amino acids without the carboxyl IRF3 domain. We suggest that zfIRF5 is a new kind of splicing variant, following the nine other kinds of IRF5 splicing variants found in mammals. The zfIRF7 protein is identified as a member of the IRF7 family, compared to the human IRF7 protein, the amino acid sequence of zfIRF7 only with 29% identity and devoid a virus activated domain (VAD). There zfIRF5/7 proteins are expressed in all 11 selective zebrafish tissues within 6-120h of embryonic development. Laser confocal microscopy shows that the full length the proteins are separately located in the cytoplasm. Mutation experiments show that the nuclear localization signals (NLS) of zfIRF7 and zfIRF-5 are at the N-terminal and C-terminals, respectively. In the assays, zfIRF7 expression increases during infectious spleen and kidney necrosis virus (ISKNV) infection and by poly(I:C) and LPS injections, both of which activate the transcriptional activity of L8G5-luc plasmids. The over-expression of zfIRF5/7 activates the interferon-stimulated response elements (ISRE) signal pathway. In addition, zfIRF7 can activate IFN-β, zfIRF5/7. Co-immunoprecipitation assays and laser co-confocal microscopy show that the two proteins could interact, and zfIRF7 may stimulate zfIRF5 to move into the nucleus. The co-expression of zfIRF5/IRF7 suppresses the transcriptional activities of IFN-β in HEK293T cells.