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Interferon regulatory factors (IRFs) are transcription factors involved in immune responses, such as pathogen response regulation, immune cell growth, and differentiation. IRFs are necessary for the synthesis of type I interferons through a signaling cascade when pathogen recognition receptors identify viral DNA or RNA. We discovered that irf3 is expressed in the early embryonic stages and in all immune organs of adult zebrafish. We demonstrated the antiviral immune mechanism of Irf3 against viral hemorrhagic septicemia virus (VHSV) using CRISPR/Cas9-mediated knockout zebrafish (irf3 -KO). In this study, we used a truncated Irf3 protein, encoded by irf3 with a 10 bp deletion, for further investigation. Upon VHSV injection, irf3- KO zebrafish showed dose-dependent high and early mortality compared with zebrafish with the wild-type Irf3 protein (WT), confirming the antiviral activity of Irf3. Based on the results of expression analysis of downstream genes upon VHSV challenge, we inferred that Irf3 deficiency substantially affects the expression of ifnphi1 and ifnphi2. However, after 5 days post infection (dpi), ifnphi3 expression was not significantly altered in irf3 -KO compared to that in WT, and irf7 transcription showed a considerable increase in irf3 -KO after 5 dpi, indicating irf7 's control over ifnphi3 expression. The significantly reduced expression of isg15 , viperin , mxa , and mxb at 3 dpi also supported the effect of Irf3 deficiency on the antiviral activity in the early stage of infection. The higher mortality in irf3 -KO zebrafish than in WT might be due to an increased inflammation and tissue damage that occurs in irf3 -KO because of delayed immune response. Our results suggest that Irf3 plays a role in antiviral immunity of zebrafish by modulating critical immune signaling molecules and regulating antiviral immune genes. • The irf3 mutant zebrafish was generated using CRISPR/Cas9-mediated genome editing. • The irf3 knockout zebrafish showed increased susceptibility to VHSV infection. • Irf3 depletion significantly affected the transcription of ifnphi1 and ifnphi2 upon VHSV challenge. [ABSTRACT FROM AUTHOR]

All organisms have evolved sophisticated antioxidant networks and enzymes to counteract reactive radicals, among which thioredoxin (Trx) systems are especially noteworthy. Thioredoxin domain-containing protein 17 (TXNDC17) is a ubiquitously expressed enzyme with oxidoreductase activity belonging to the Trx protein family. This study successfully uncovered and analyzed the TXNDC17 gene in Scomber japonicus (SjTXNDC17). The gene consists of a 372-base-pair coding sequence that encodes a protein of 123 amino acids, with an estimated molecular weight of 14.1 kDa. Structural analysis revealed that SjTXNDC17 contains a TRX-related protein 14 domain with two redox-responsive cysteine residues in the 42WCPDC46 motif. Spatial expression analysis indicated that SjTXNDC17 had the highest constitutive expression in the brain. Stimulation with polyinosinic-polycytidylic acid (poly I:C), Vibrio harveyi , and Streptococcus iniae , significantly upregulated the mRNA levels of SjTXNDC17 in the head kidney. The antioxidant activity of the recombinant SjTXNDC17 protein was evidenced by 2,2-Diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging, insulin reduction, and cupric ion-reducing antioxidant capacity assays. SjTXNDC17 overexpression in fathead minnow (FHM) cells significantly reduced reactive oxygen species (ROS) levels and decreased apoptosis. The anti-apoptotic effect was driven by the upregulation of the Bcl2 gene and the downregulation of the Bax gene, as well as the suppression of JNK signaling pathway genes. Moreover, overexpression of SjTXNDC17 facilitated M2 polarization and suppressed nitric oxide production in macrophages. Collectively, these results demonstrate that SjTXNDC17 plays a crucial role in both the immune response and cellular redox balance in Scomber japonicus. • Thioredoxin domain-containing protein 17 (SjTXNDC17) was identified in chub mackerel. • Pathogen stimulation significantly increased SjTXNDC1 7 mRNA levels in head kidney. • The recombinant SjTXNDC17 protein showed antioxidant activity. • Overexpression of SjTXNDC17 reduced ROS levels and apoptosis in FHM cells. • SjTXNDC17 overexpression promoted M2 polarization and lowered NO production. [ABSTRACT FROM AUTHOR]

Membrane-associated Ring-CH 5 (MARCH5) is a mitochondrial E3 ubiquitin ligase playing a key role in the regulation of mitochondrial dynamics. In mammals, MARCH5 negatively regulates mitochondrial antiviral signaling (MAVS) protein aggregation during viral infection and hampers downstream type I interferon signaling to prevent excessive immune activation. However, its precise functional role in the teleost immune system remains unclear. This study investigated the molecular characteristics and immune response of the MARCH5 ortholog in Amphiprion clarkii (A. clarkii ; AcMARCH5). The predicted AcMARCH5 protein sequence consists of 287 amino acids with a molecular weight of 32.02 kDa and a theoretical isoelectric point of 9.11. It contains four C-terminal transmembrane (TM) domains and an N-terminal RING cysteine-histidine (CH) domain, which directly regulates ubiquitin transfer. Multiple sequence alignment revealed a high level of conservation between AcMARCH5 and its orthologs in other vertebrate species. Under normal physiological conditions, AcMARCH5 showed the highest mRNA expression in the muscle, brain, and kidney tissues of A. clarkii. Upon stimulation with polyinosinic:polycytidylic acid (Poly I:C), lipopolysaccharide (LPS), and Vibrio harveyi , AcMARCH5 expression was drastically modulated. Functional assays showed that overexpression of AcMARCH5 in fathead minnow (FHM) cells downregulated antiviral gene expression, accompanied by enhanced viral hemorrhagic septicemia virus (VHSV) replication. In murine macrophages, AcMARCH5 overexpression markedly reduced the production of pro-inflammatory cytokines in response to poly I:C treatment. Additionally, AcMARCH5 exhibited an anti-apoptotic effect in H 2 O 2 -treated FHM cells. Collectively, these results suggest that AcMARCH5 may play a role in maintaining cellular homeostasis under disease and stress conditions in A. clarkii. • The membrane-associated Ring-CH5 (MARCH5) from Amphiprion clarkii (AcMARCH5) was identified and characterized. • Temporal expression of AcMARCH5 in blood, spleen and head kidney is drastically modulated by various immune stimuli. • AcMARCH5 overexpression suppressed antiviral gene expression and promoted VHSV viral replication. • AcMARCH5 overexpression reduced pro-inflammatory cytokine production in Poly I:C stimulated macrophages. • AcMARCH5 showed an anti-apoptotic property during H 2 O 2 induced oxidative stress. [ABSTRACT FROM AUTHOR]