Characterization and Transcript Expression Analyses of Atlantic Cod Viperin

Viperin is a key antiviral effector in immune responses of vertebrates including the Atlantic cod (Gadus morhua). Using cloning, sequencing and gene expression analyses, we characterized the Atlantic cod viperin at the nucleotide and hypothetical amino acid levels, and its regulating factors were investigated. Atlantic cod viperin cDNA is 1,342 bp long, and its predicted protein contains 347 amino acids. Using in silico analyses, we showed that Atlantic cod viperin is composed of 5 exons, as in other vertebrate orthologs. In addition, the radical SAM domain and C-terminal sequences of the predicted Viperin protein are highly conserved among various species. As expected, Atlantic cod Viperin was most closely related to other teleost orthologs. Using computational modeling, we show that the Atlantic cod Viperin forms similar overall protein architecture compared to mammalian Viperins. qPCR revealed that viperin is a weakly expressed transcript during embryonic development of Atlantic cod. In adults, the highest constitutive expression of viperin transcript was found in blood compared with 18 other tissues. Using isolated macrophages and synthetic dsRNA (pIC) stimulation, we tested various immune inhibitors to determine the possible regulating pathways of Atlantic cod viperin. Atlantic cod viperin showed a comparable pIC induction to other well-known antiviral genes (e.g., interferon gamma and interferon-stimulated gene 15-1) in response to various immune inhibitors. The pIC induction of Atlantic cod viperin was significantly inhibited with 2-Aminopurine, Chloroquine, SB202190, and Ruxolitinib. Therefore, endosomal-TLR-mediated pIC recognition and signal transducers (i.e., PKR and p38 MAPK) downstream of the TLR-dependent pathway may activate the gene expression response of Atlantic cod viperin. Also, these results suggest that antiviral responses of Atlantic cod viperin may be transcriptionally regulated through the interferon-activated pathway

Characterization and transcript expression studies of interferon regulatory factors in Atlantic cod (Gadus morhua)

The interferon regulatory factor (IRF) family of genes encode a group of transcription factors which have important roles not only in regulating the expression of Type I interferons (IFNs) and other genes in the interferon pathway, but also in growth, development and regulation of oncogenesis. In this study, several IRF family members in Atlantic cod (Gadus morhua) were characterized at the cDNA and putative amino acid level, allowing for phylogenetic analysis of these genes in teleost fish, and the development of paralogue specific PCR primers which were used in semi-quantitative RT-PCR and Quantitative PCR (QPCR) analyses. Two Atlantic cod Irf10 splice variants were identified and named Irf10-v1 and Irf10-v2, and their presence was confirmed by sequencing of the Irf10 genomic region. RT-PCR showed that Irf7, Irf8 and both Irf10 transcripts were detected in 15 cod tissues, while Irf4a and Irf4b appeared to be absent in some tissues. RT-PCR in embryo and larval samples showed unique transcript expression profiles of IRFs during development and indicated potential stage specific roles that will be investigated in future studies. QPCR analysis of spleen expression expanded upon previous studies, confirming that all transcripts were responsive to stimulation by the viral mimic poly(I:C) and showing that all except Irf4a were responsive to killed Aeromonas salmonicida (ASAL). Temperature was observed to affect the responsiveness of all except Irf4a to poly(I:C) and/or ASAL, supporting earlier studies. The effect of increased temperature on immune responsiveness to pathogens is of particular interest to Atlantic cod aquaculture in Newfoundland, where fish experience seasonal fluctuations in temperature