Interferon regulatory factors (IRFs) have been confirmed as vital transcription factors for the immune system, which play a certain role in anti-infection defense, immune regulation, hematopoietic cell development, and maturation of the immune system. In this study, the cDNA of Interferon regulatory factor 5 (IRF5) was cloned from black-spotted frog, Pelophylax nigromaculatus , and termed PnIRF5. The results indicated, the full-length cDNA of PnIRF5 was 2090 bp, and a putative protein of 504 amino acids was encoded. The results of protein domain prediction suggested that PnIRF5 exhibited a DNA-binding domain (DBD), a middle region (MR), an IRF association domain (IAD), a virus activated domain (VAD), as well as two nuclear localization signals (NLSs). Phylogenetic analysis clustered PnIRF5 into the amphibian IRF5 subgroup in vertebrate IRF5 group. The results of qPCR show that PnIRF5 mRNA was expressed in all examined tissues, with higher levels were identified in the kidney, intestine, and lung. The gene expression of PnIRF5 was analyzed in kidney, spleen, and liver of black-spotted frog after challenged with Elizabethkingia miricola , and the maximum expression level of PnIRF5 in liver, spleen, and kidney were 1.80, 2.33 and 2.88 times that at 0 days post challenged, respectively. As indicated by the results, the PnIRF5 protein may take on critical significance in protecting black-spotted frog from E. miricola infection, and they may play a certain role in the immune response of black-spotted frog against bacteria.

The stimulator of interferon genes (STING) is a central and multifaceted mediator in innate immunity, and plays vital roles in defending against pathogen infection. In this study, we identified and functionally characterized the STING homolog from the Chinese giant salamander Andrias davidianus (AdSTING) for the first time. The open reading frame of AdSTING encodes a 362 amino acid protein with a predicted molecular mass of 41.6 kDa, which shares 31.1–46.7% of its sequence identity with STING homologs in other vertebrates. Structural analysis revealed that AdSTING possesses four predicted transmembrane domains (TMs) at the N-terminal, and a C-terminal domain (CTD) featuring a dimerization domain (DD), a c-di-GMP-binding domain (CBD), and a short C-terminal tail (CTT). Tissue distribution analysis showed that AdSTING mRNA was ubiquitously expressed in all examined tissues, with abundant expression in muscles, intestine, and thymus. During Andrias davidianus ranavirus (ADRV) infection, significant up-regulation of AdSTING expression was observed in the thymus, spleen, and kidney. Upon different stimuli in vitro, the expression of AdSTING was significantly induced by ADRV infection or polyinosin-polycytidylic acid (poly I:C) stimulation, but no obvious changes were observed during lipopolysaccharide (LPS) stimulation. Subcellular localization analysis revealed that AdSTING mainly localized in the cytoplasm in the Chinese giant salamander thymus cell line (GSTC) and co-localized with the endoplasmic reticulum (ER). Luciferase reporter assays confirmed the ability of AdSTING to activate the interferon-stimulated response element (ISRE) and interferon (IFN) promoter. Furthermore, overexpression of AdSTING effectively decreased ADRV infection, as evidenced by the reduction of virus titers and viral gene expression. Collectively, our findings underscore the pivotal role of AdSTING in the antiviral innate immunity of the Chinese giant salamander, offering insights into the functional evolution of STING in amphibians.