Gentamycin sulfate

A frozen stock of EEDV containing 1 × 10⁶ viral copies/mL was prepared through homogenization of skin tissue collected from naturally infected lake trout as described by Shavalier (2017). For this study, the infectious inoculum was prepared by combining 1.6 mL EEDV stock with 1.4 mL sample diluent (pH 7.525 ± 0.025) containing 458 mL Minimal Essential Medium (MEM; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), 7 mL 1 M tris buffer, 1 mL gentamycin sulfate (Sigma-Aldrich), 5 mL penicillin/streptomycin (Invitrogen), and 5 mL Amphotericin B (Thermo Fisher Scientific), resulting in a final working virus concentration of 4.5 × 10⁵ viral copies/mL.
Fish were anesthetized as described above and then injected intracoelomically (IC) with 100 µL/fish of either sample diluent (NC group) or EEDV infectious inoculum (EEDV group). After injection, fish were returned to their respective tanks and monitored daily. Throughout the study, moribund fish were euthanized using an overdose dose of MS-222 (0.25 mg/mL, buffered with sodium bicarbonate at dose of 0.5 mg/mL), after which a gross necropsy was performed. Percent cumulative mortality was calculated by dividing total mortalities through each study period by the starting number of fish in each group (infected and control).

Both RAW264.7 macrophages and THP-1 cells were seeded at 1 × 10⁵ cells per well of a 24-well plate 48 h prior to infection. THP-1 cells were differentiated for 24 h in the presence of 200 nM phorbol 12-myristate 13-acetate (PMA) to activate macrophages. After activation, the medium was removed, and the cells washed prior to infection to remove dead or non-adherent cells and left for a further 24 h. RAW264.7 macrophages were treated with 100 ng/ml lipopolysaccharide to induce an activated state 24 h after seeding. Infections for both cell types were carried out in RPMI media supplemented with 3% FCS and L-glutamate. Infections were carried out at an MOI of 10. After 1 h the bacteria that had not been internalized were killed by adding 50 µg/ml gentamycin sulfate (Sigma-Aldrich) and the infection allowed to proceed.

Albumax I and RPMI-1640 were purchased from Fisher Scientific^® (Leicestershire, UK). All solvents used were HPLC-grade or higher quality and purchased from Sigma-Aldrich (St. Louis, Missouri USA). Radiolabeled isoprenic precursors [1-(n)-³H] geranylgeranyl pyrophosphate triammonium salt {[1-(n)-³H]-GGPP; 14 Ci/mmol}, [1-(n)-³H] phytol (20 Ci/mmol), and {[1-(n)-³H] phytyl-PP ([³H]-phytyl-PP; 20 Ci/mmol) were obtained from Amersham-Pharmacia Biotech (Buckinghamshire, UK). Adenosyl-L-methionine, S-[methyl-³H] ([³H]-SAM, 82 Ci/mmol) and [1-(n)-³H] FPP triammonium salt ([³H]-FPP 23 Ci/mmol) were purchased from Perkin Elmer^® (Waltham, Massachusetts, EUA). PK, α-tocopherol, γ-tocopherol, phytol, phytyl-PP, GGPP, UQ-8, UQ-9 and MK-4 pure standards were purchased from Sigma-Aldrich. Saponin, hypoxanthine, gentamycin sulfate, D-sorbitol, glucose, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and another reagent not cited here were also purchased from Sigma-Aldrich.