Isolation and Characterization of a Novel Gammaherpesvirus from a Microbat Cell Line

Shabman, Reed S.; Shrivastava, Susmita; Tsibane, Tshidi; Attie, Oliver; Jayaprakash, Anitha; Mire, Chad E.; Dilley, Kari E.; Puri, Vinita; Stockwell, Timothy B.; Geisbert, Thomas W.; Sachidanandam, Ravi; Basler, Christopher F.

Isolation and propagation of BGHV8 virions. Strict biosafety level 2 precautions were followed for all studies involving live virus. Supernatant obtained from a confluent 75-centimeter flask of MVI-it cells was harvested and clarified by centrifugation at 3,500 rpm for 20 min. Supernatant was transferred to uninfected Vero cells (ATCC CCL-81) seeded in 6-well plates. At either 18 or 42 h after supernatant transfer, supernatant was harvested and stored and cells were fixed in 2% paraformaldehyde (PFA). Phase-contrast images captured evidence of cytopathic effect, and Hoechst staining (Thermo Fisher) captured evidence of syncytium formation. To generate working stocks of BGHV8, MVI-it supernatant was passaged onto confluent flasks of Vero cells, and supernatant was harvested once complete CPE was apparent. Viral titers of BGHV8 were calculated by either plaque assay or 50% tissue culture infectious dose (TCID 50 ) assay on Vero cells. For plaque assay, serial dilutions of clarified supernatant were used to infect fresh Vero cells in 6-well plates for 2 h. Infection medium was removed, and cells were overlaid with Avicel-591. Briefly, a 2.4% Avicel solution was mixed 1:1 with 2Ï« DMEM and 4 ml of this mixture was added to each well of a 6-well plate. Three to 4 days postinfection, the Avicel overlay was removed, and cells were washed with phosphate-buffered saline (PBS), fixed with 4% PFA, and stained with crystal violet to visualize plaques. For TCID 50 studies, Vero cells were seeded in 96-well plates. Serial 10-fold dilutions of BGHV8 stocks were added in replicates of 7 adjacent columns. When complete CPE was visible at lower dilutions, cells were fixed with PFA and stained with crystal violet.