Virus-inclusive single nucleus RNA-sequencing reveals two distinct endothelial response patterns in infectious salmon anaemia

Viral replication in endothelial cells is a hallmark of many viral diseases, underscoring the importance of understanding mechanisms that restrict viral replication and the associated consequences for vascular health. Pathogenic infectious salmon anaemia virus (ISAV, Isavirus salaris) targets endothelial cells of Atlantic salmon (Salmo salar L.), causing severe disease and economic losses in aquaculture. Using single nucleus RNA-sequencing at pre-clinical (12 days post infection, dpi) and clinical (16 dpi) stages of infection, we characterized the endothelial transcriptional response to ISAV at unprecedented resolution. ISAV RNA was predominantly detected in endothelial cells, which, along with mononuclear phagocytes, showed the highest number of differentially regulated genes at both time points. To retain statistical power, timepoint-specific analyses were performed on all endothelial subsets combined. Differentially expressed genes at 12 dpi were enriched for pathways related to NOD-like receptor signaling, antiviral responses, and regulation of apoptosis, but by 16 dpi shifted toward pathways associated with cellular senescence, apelin signaling, and insulin signaling. At both time points, we identified two distinct infection-related states: (1) a virus-permissive state, characterized by differential expression of genes involved in the regulation of small GTPase signalling and downstream processes including intracellular trafficking, cytoskeletal remodelling, and control of cell growth and death; and (2) a bystander phenotype, marked by activation of cytokine and chemokine signalling and degradation of viral products. This study is the first to capture cell type-specific responses to ISAV infection, and to characterize the in vivo endothelial response to active viral replication at single-cell resolution in any species.