Abstract
The European sea bass (Dicentrarchus labrax) is an important farmed fish species in the Mediterranean area, very sensitive to the infection by encephalopathy and retinopathy virus (VERv), or Betanodavirus, which causes massive mortalities. Effective vaccines to fight the pathology are not yet available and in this work we describe a promising intraperitoneal immunization route against VERv of sea bass juveniles. We performed intraperitoneal and immersion immunization trials with a VERv (isolate 283.2009 RGNNV) inactivated by formalin, β-propiolactone and heat treatment. Interestingly, the intraperitoneal immunization with formalin-inactivated VERv induced a significant antigen-specific IgM production, differently from other inactivation protocols. However, the same formalin-inactivated antigen resulted in very low IgM antibodies when administered by immersion. Following the intraperitoneal injection with formalin-inactivated virus, the quantitative expression of the antiviral MxA gene showed a modulation of transcripts in the gut after 48 h and on head kidney after 24 h, whereas ISG12 gene was significantly up-regulated after 48 h on both tissues. In immersion immunization with formalin-inactivated VERv, a modulation of MxA and ISG12 genes after 24 h post-treatment was detected in the gills. An effective uptake of VERv particles in the gills was confirmed by immunohistochemistry using anti-VERv antibodies. Lastly, in challenge experiments using live VERv after intraperitoneal immunization with formalin-inactivated VERv, we observed a significant increase (81.9%) in relative survival percentage with respect to non-immunized fish, whereas immersion immunization resulted in no protection. Our results suggest that intraperitoneal immunization with formalin-inactivated VERv could be a safe and effective strategy to fight Betanodavirus infection in European sea bass.