Figure 3. Recovery of infectivity of FMDV mutants expressing one VPg, upon serial passages in BHK–21 cells.

2×10⁶ BHK-21 cells were transfected with 100 ng (series A) (panels A, B, C) or 500 ng (series B) (panels D, E, F) of the FMDV transcripts indicated in the boxes. Cells and cell culture supernatants were collected at 72 h post-transfection (passage 0). Successive passages were carried out by infecting BHK–21 cells with the viruses from the cell culture supernatant of the previous passage, and samples were collected when cytopathology was complete (generally 20 to 30 hours pi). A, D, Viral infectivity as a function of passage number; plaque development was for 48 hours. B, E, Number of genomic RNA molecules in the cell culture supernatants. C, F, Specific infectivity calculated as the number of PFU per 10¹⁰ viral RNA genomes, from the data given in A, B and D, E. Measurements were carried out in triplicate and standard deviations are given. Procedures for titration of infectivity and determination of viral RNA levels are described in Materials and Methods.