Ficoll 1

Rainbow trout RBCs were obtained and purified as previously described (4 (link), 7 (link)). Briefly, RBCs extracted from the caudal vein were purified by 2 successive Ficoll density gradient centrifugations (7,206 g, Ficoll 1.007; Sigma-Aldrich, Madrid, Spain). Ficoll-purified RBCs were maintained in 25 cm² flasks (Nunc Roskilde, Denmark) with RPMI-1640 medium (Dutch modification) (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) gamma irradiated (Cultek, Madrid, Spain), 1 mM pyruvate (Gibco), 2 mM L-glutamine (Gibco), 50 μg/mL gentamicin (Gibco), 2 μg/mL fungizone (Gibco), 100 U/mL penicillin (Sigma-Aldrich), and 100 μg/mL streptomycin (Sigma-Aldrich) at 14°C for 24 h prior to experimentation.
The RTG-2 (rainbow trout gonad-2) cell line was purchased from the American Type Culture Collection (ATCC 50643) and maintained at 21°C in MEM medium (Sigma-Aldrich) containing 10% FBS, 1 mM pyruvate, 2 mM L-glutamine, 50 μg/mL gentamicin, and 2 μg/mL fungizone.
VHSV strain 07.71 (20 ) was purchased from the American Type Culture Collection (ATCC VR-1388) and cultured in fathead minnow epithelioma papulosum cyprini (EPC) (21 (link)) cells at 14°C as previously described (22 ).

Rainbow trout RBCs were obtained from the peripheral blood of fish sacrificed by overexposure to 0.3 g/L tricaine. RBCs were purified by 2 consecutive density gradient centrifugations (7206 g, Ficoll 1.007; Sigma-Aldrich). Purified RBCs were cultured in RPMI-1640 medium (Dutch modification) (Gibco, Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) gamma-irradiated (Cultek), 1 mM pyruvate (Gibco), 2 mM l-glutamine (Gibco), 50 μg/mL gentamicin (Gibco), 2 μg/mL fungizone (Gibco), 100 U/mL penicillin (Sigma-Aldrich), and 100 μg/mL streptomycin (Sigma-Aldrich) at a density of 10⁶ cells/mL and kept at 14 °C until use.

Fish (11.0 ± 0.8 cm, 29.3 ± 4.7 g) were randomly divided into two groups (20 fish per group): a virus-injected group and a PBS-injected group. The experimental group was injected i.p. with RBIV (100 μL/fish) containing 1.1 × 10⁷ MCP gene copies, and the control group was injected i.p. with PBS (100 μL/fish). Each group of fish were maintained at 23°C in the aquarium containing 250 L of UV-treated seawater. Blood (100 μL/fish) was collected from 8 fish at 7 dpi. Then, RBCs were purified by 2 consecutive density gradient centrifugations (7,206 g, Ficoll 1.007, Sigma-Aldrich). All rock bream experiments were carried out in strict accordance with the recommendations of the Institutional Animal Care and Use Committee of Chonnam National University (permit number: CNU IACUC-YS-2015-4).

RBIV-free rock bream individuals were obtained from a local farm. Thirty fish (11.2 ± 1.2 cm, 28.1 ± 3.2 g) were maintained at 23°C in an aquarium containing 250 L of UV-treated seawater. Fish were injected intraperitoneally (i.p.) with RBIV (100 μL/fish, 1.1 × 10⁷ MCP gene copies) or phosphate-buffered saline (PBS) (100 μL/fish) as a control. Blood (200 μL/fish) and organs (spleen, kidney, and liver) were collected from RBIV-infected rock bream individuals at 1, 2, 4, 7, and 10 days post infection (dpi) (4 fish per time point). RBCs were isolated from blood (100 μL/fish) and purified by 2 consecutive density gradient centrifugations (7,206 g, Ficoll 1.007, Sigma-Aldrich). For RBIV copy number analysis, genomic DNA was isolated from the RBCs, blood, spleen, kidney, and liver of each fish using High Pure PCR Template Preparation Kit (Roche) following standard protocol. A standard curve was generated to determine RBIV MCP gene copy number by RT-qPCR as described previously (11 (link)). Virus copy number was determined from 100 μL of total genomic DNA. Statistical analyses were performed using GraphPad Prism software version 5.0 (GraphPad Software, USA). One-way analysis of variance (ANOVA) was performed between conditions, with Tukey's multiple comparison test. P < 0.05 were considered to indicate statistical significance.

Rainbow trout RBCs were obtained from the peripheral blood of fish which died through overexposure to tricaine (tricaine methanesulfonate, Sigma-Aldrich, Madrid, Spain; 0.2 g/L), as previously described [8 (link)]. Briefly, peripheral blood was sampled from the caudal vein. Then, RBCs were purified by two consecutive density gradient centrifugations (7206 g, Ficoll 1.007; Sigma-Aldrich). Purity of RBCs of 99.9% was estimated by optical microscopy evaluation (Figure S1).