Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

Tuplin, A.; Evans, D. J.; Buckley, A.; Jones, I. M.; Gould, E. A.; Gritsun, T. S.

For growth curves, monolayers of PS cells in 96-well plates were infected with viruses at a multiplicity of infection (moi) of 1 PFU/cell, in quadruplicates. The inoculum (30 ml) was removed after 1 h, the monolayer washed thoroughly and replaced with 200 ml of media containing 2% serum. Media (10 ml) was collected at different time-points (8, 12, 16 and 24 h post-infection) and stored frozen at À70 C, before virus quantification by plaque assay. For cytopathic effect (cpe), PS cell monolayers were infected in 96-well plates at an moi of 1 PFU/cell, in quadruplicates, and stained with naphthalene black after 72 h.