Zebrafish and Fathead Minnow Cell Cultures for Virus Propagation

Zebrafish liver (ZFL) cells were obtained from a pool of 10 adult zebrafish livers, whose sex were not recorded (American Type Culture Collection, ATCC, www.atcc.com). ZFL cells were cultured at 28°C in 5% CO₂ in Ham’s F12 nutrient mixture medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). Epithelioma papulosum cyprini (EPC) cells were derived from fathead minnow (Pimephales promelas) without sex record [42 (link)], which is belongs to Cyprinidae and is also phylogenetically closely related to zebrafish. EPC cells were obtained from China Center for Type Culture Collection (CCTCC) and were maintained at 28°C in 5% CO₂ in medium 199 (M199) (Invitrogen) supplemented with 10% FBS. SVCV, a negative ssRNA virus, was propagated in EPC cells until a CPE was observed, and then cell culture fluid containing SVCV was harvested and centrifuged at 4 × 10³g for 20 min to remove the cell debris, and the supernatant was stored at -80°C until used. CaHV was provided by Prof. Q. Y. Zhang (Institute of Hydrobiology, Chinese Academy of Sciences) and propagated by intraperitoneal injection into healthy gibel carp. The isolation method of CaHV was used as previously described [43 (link)].