Viperin as a putative antiviral effector in rainbow trout (Oncorhynchus mykiss) cells identified by CRISPR-Cas9 functional analysis following exposure to inactivated viral hemorrhagic septicemia virus

Abstract

Abstract Viperin is recognized for its potent broad-spectrum antiviral activity; however, its role in the antiviral immune response of rainbow trout ( Oncorhynchus mykiss ) remains poorly characterized. In this study, the contribution of viperin to the innate immune response against viral haemorrhagic septicaemia virus (VHSV) was investigated using CRISPR–Cas9 gene editing of the RTS11 cell line, a monocyte/macrophage-like cell line derived from rainbow trout spleen. Cells were edited via electroporation using ribonucleoprotein (RNP) complexes targeting viperin, achieving up to 50% editing efficiency. Edited cells and corresponding controls were subsequently stimulated with heat-inactivated VHSV, and RNA sequencing was performed to assess transcriptomic changes associated with partial viperin silencing. Transcriptomic analysis revealed disruption of canonical antiviral signalling pathways, particularly those associated with the MAVS–TBK1 axis, even in the absence of viral replication. While a robust inflammatory response mediated by TRAFs and NF-κB was maintained, genes involved in apoptosis, autophagy, and phagocytosis were predominantly downregulated, suggesting a shift toward cellular survival and adaptive responses. Alternative interferon-stimulated genes were also induced, including TRIM45, indicating activation of compensatory antiviral signalling pathways. Additionally, significant upregulation of S-adenosylmethionine synthetase (MAT) and cytidine deaminase (CDA) was observed, suggesting a compensatory metabolic response that may regulate nucleotide pools available to viral machinery while supporting viperin transcription and epigenetic antiviral regulation. Collectively, these findings demonstrate that viperin plays a central role in coordinating antiviral responses in rainbow trout macrophages and is essential for proper activation of classical IFN–ISG pathways. Even partial silencing of viperin, in the presence of a non-replicative viral stimulus, shifts macrophage responses toward compensatory inflammatory and metabolic mechanisms, indicating that loss of viperin may compromise direct antiviral defence.