Red blood cells as antiviral responders in RGNNV-infected European sea bass: a transcriptomic study

Red-spotted grouper nervous necrosis virus (RGNNV) is the most widespread betanodavirus genotype, and European sea bass (Dicentrarchus labrax) is among the most susceptible species, with mortality approaching 100 % in larvae and juveniles. Teleost red blood cells (RBCs) are nucleated and immunocompetent, contributing to pathogen recognition, interferon signalling and antigen presentation, yet their role in the response to RGNNV has not been examined. Here we assessed whether European sea bass RBCs support RGNNV replication and how they respond transcriptionally to infection. Purified RBCs exposed to RGNNV ex vivo showed a significant increase in viral RdRp transcript levels between 1 and 3 days post-exposure, accompanied by marked cell aggregation that, to our knowledge, has not been described previously for betanodaviruses. In experimentally infected juveniles, viral RNA was detectable in circulating RBCs at 1 and 3 days post-infection, although levels in the brain exceeded those in RBCs by several orders of magnitude. RNA-sequencing identified 250 differentially expressed genes and enrichment of pathways related to antiviral innate immunity, pattern-recognition receptor signalling, type I interferon- and ISG15- dependent mechanisms, class I MHC-mediated antigen processing, and cellular stress and protein homeostasis. Among the upregulated genes, 19 canonical interferon-stimulated antiviral effectors were identified. These results suggest that European sea bass RBCs are susceptible to RGNNV and transcriptionally responsive to infection, supporting their role as immunocompetent cells contributing to the antiviral response.