2.2. Cell Cultures and Virus

Rainbow trout were sacrificed by overexposure to tricaine methanesulfonate (Sigma-Aldrich, Madrid, Spain) at 0.3 g/L. Peripheral blood was sampled from the caudal vein using insulin syringes (NIPRO, Bridgewater, NJ, USA). Approximately 100 µL of blood was diluted in RPMI-1640 medium (Dutch modification) (Gibco, Thermo Fischer Scientific Inc., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Cultek, Madrid, Spain), 1 mM pyruvate (Gibco), 2 mM l-glutamine (Gibco), 50 µg/mL gentamicin (Gibco), 2 µg/mL fungizone (Gibco), and 100 U/mL penicillin/streptomycin (Sigma-Aldrich). Then, RBCs were purified by two consecutive density gradient centrifugations with Histopaque 1077 (7206g, Ficoll 1.007; Sigma-Aldrich). Finally, RBCs were washed twice with RPMI 2% FBS. An RBC purity of 99.99% was estimated by optical microscopy evaluation. Then, purified RBCs were cultured in the above indicated medium at a density of 10⁷ cells/mL in cell culture flasks at 14 °C overnight.

For autophagy assays, RBCs were treated with niclosamide (Sigma-Aldrich) after three hours post-exposure (hpe) to VHSV and then incubated for the time and at the concentration indicated for each assay. Similarly, the proteasome inhibitor MG132 (Sigma-Aldrich) was added after three hpe to VHSV and then incubated for the time and at the concentration indicated for each assay.

Viral hemorrhagic septicemia virus (VHSV-07.71) [36] was purchased from the American Type Culture Collection (ATCC, VR-1388) and propagated in fathead minnow epithelioma papulosum cyprini EPC cells (ATCC, CRL-2872) at 14 °C, as previously reported [37].