Tryptose phosphate broth

The reference SVCV strain VR-1390, isolate stock of the INRA laboratory (34 (link), 35 ), was propagated in epithelioma papulosum cyprinid (EPC) cells grown in Glasgow’s modified Eagle’s medium (GMEM)–25 mM HEPES (Eurobio) supplemented with 10% fetal calf serum (FCS; Eurobio), 1% tryptose phosphate broth (Eurobio), 2 mM l-glutamine (PAA), 100 µg/mL penicillin (Biovalley), and 100 µg/mL streptomycin (Biovalley) in the absence of CO₂. The SVCV CAPM V 539 strain (36 (link)) was propagated in common carp brain (CCB) cells at 27°C or in EPC cells at 20°C. EPCs were grown in MEM medium (Gibco) supplemented with 10% FCS, 2 mM l-glutamine, 100 µg/mL penicillin, and 100 µg/mL streptomycin in the presence of CO₂. For CCBs, the same medium was supplemented with 3.5 g/L d-glucose and 1% non-essential amino acids (Gibco). Virus titers were determined by the method of Reed and Muench (37 (link)) and were given as plaque-forming units (pfu).

Zebrafish were raised in the fish facilities of Institut National de la Recherche Agronomique (Jouy-en-Josas, France). Epithelioma papulosum cyprini (EPC) cell line (ATCC^® CRL-2872™) was maintained in Glasgow’s modified Eagle’s medium-HEPES 25 mM medium (Eurobio) supplemented with 10% fetal bovine serum (FBS, Eurobio, produced and distributed in France under the veterinary authorization FR 91 692 200), 1% tryptose phosphate broth (Eurobio), 2mM l-glutamine (PAA) and antibiotics 100 µg/mL penicillin (Biovalley), 100 µg/mL streptomycin (Biovalley). Transfection experiments, viral production, and titration were performed in EPC cells. The novirhabdoviruses infectious hematopoietic necrosis virus 32-87 (IHNV) and viral hemorrhagic septicemia virus 07-71 (VHSV) and the vesiculovirus spring viremia of carp virus (SVCV) were produced at 14°C on EPC in GMEM media supplemented with 2% FBS, 5% tryptose, and 2mM l-glutamine. Cytopathic effect was evaluated 72 h postinfection after cell fixation with 10% formol prior to coloration in 2% crystal violet.