Investigating antiviral pathways in Atlantic salmon cells through interferon receptor knockouts via CRISPR-Cas9

Abstract

In Atlantic salmon (Salmo salar), infectious salmon anemia virus (ISAV) and infectious pancreatic necrosis virus (IPNV) evade host immune response through complex antagonistic mechanisms. Type I interferons (IFNs) play a pivotal role in antiviral defense by signaling through heterodimeric receptors to activate the JAK-STAT pathway and drives the expression of interferon-stimulated genes (ISGs). In this study, CRISPR-Cas9 was used to knock out (KO) interferon receptor genes (crfb1a, crfb5a, il10rb, ifngr2a) and a combined group of candidate receptors (crfb1a, crfb5a, il10rb, ifngr2a, il10r2) to investigate their roles and their impact on downstream signaling cascades with RNA sequencing. Recombinant IFNa was used to induce an antiviral state before challenging cells with ISAV and IPNV. The knockouts significantly disrupt downstream antiviral signaling, with two knockouts, crfb1a and crfb5a, showing pronounced effects. During ISAV infection, the crfb1a KO group exhibited a marked reduction in the expression of critical signaling genes such as stat1b, stat2, stat6, and irf3 during ISAV infection, while irf7 was upregulated during IPNV infection. The crfb5a KO group exhibited reduced stat2 expression in ISAV infection and upregulated irf7 during IPNV infection. Despite these disruptions, ISGs such as Mx and isg15 maintained their expression levels across all knockout groups, suggesting potential alternative signaling pathways. Pathway analysis further revealed upregulation of cellular processes like actin regulation and phagosome activity, which may compensate for impaired immune signaling. These findings highlight the distinct roles of IFN receptor genes in mediating antiviral responses and underscore the complexity of IFN signaling in Atlantic salmon.