Fig. 1

Establishment of a tractable virus producer cell line for KSHV r219. The 5r219 cell line was generated following infection of iSLK cells with KSHV.r219 virus derived from Vero cells. Clonal isolates that displayed the highest level of lytic induction were analyzed further. The cells were first treated with doxycycline (1 µg/ml) and fluorescence visualized by light microscopy. Red fluorescence was only observed after lytic induction (A). The supernatants from these cultures were harvested at 72 h, concentrated by centrifugation and the virus was used to infect monolayers of Vero or HEK-293T cells. Numerous GFP positive cells were observed 48 h post-infection indicative of KSHV virus infection and replication (B). In order to more precisely quantitate virus yields and the optimal time of virus production, 5r219 cells were induced with doxycycline (DOX) or doxycycline plus 1 mM sodium butyrate (DOX/NB) and virus supernatants harvested, 2- and 3-days post-induction, and used in qPCR assays to determine KSHV genome copies (C). This was also visualized using GFP fluorescence following infection of Vero cell monolayers with extracellular concentrated virus (harvested after 72 h) from induced 5r219 cells (D)