Propagation and isolation of ranaviruses in cell culture

The optimal in vitro propagation procedure for a panel of ranavirus isolates and the best method for isolation of Epizootic haematopoietic necrosis virus (EHNV) from organ material in cell-culture were investigated. The panel of ranavirus isolates included: Frog virus 3 (FV3), Bohle iridovirus (BIV), Pike-perch iridovirus (PPIV), European catfish virus (ECV), European sheatfish virus (ESV), EHNV, Doctor fish virus (DFV), Guppy virus 6 (GF6), short-finned eel virus (SERV) and Rana esculenta virus Italy 282/102 (REV 282/102). Each isolate was titrated in five cell lines: bluegill fry (BF-2), epithelioma papulosum cyprini (EPC), chinook salmon embryo (CHSE-214) rainbow trout gonad (RTG-2) and fathead minnow (FHM), and incubated at 10, 15, 20, 24 and 28 °C for two weeks.BF-2, EPC and CHSE-214 cells performed well and titers obtained in the three cell lines were similar, whereas FHM and RTG-2 cells consistently produced lower titers than the other cell lines at all temperatures. The optimal temperature for propagating the isolates collectively to high titers in vivo was 24 °C.Additionally, three established methods for re-isolation of virus from EHNV-infected organ material were compared. Challenged fish were sampled twice weekly and 7 organs were processed separately according to the three methods. Samples incubated on BF-2 cells at 22 °C for 2 weeks + 1 week sub-cultivation (method 1) provided more positive results than the other 2 methods and when using the EPC cell line. Virus was most frequently isolated from the kidney, followed by brain, muscle, heart, liver, gills and lastly spleen.