Figure 6

Analysis of recombinant virus genetic stability after serial passaging. (A) Schematics of viral regeneration and subsequent passages (10) of the YF 17D/Esa/5.1 glic virus obtained after RNA transfection. Two independent series of serial passages (at MOI of 0.02); P1 and P2 were analyzed by RT-PCR and flow citometry at passages 5 and 10 and are represented in all panels as 5P1, 10P1, 5P2 and 10P2. In these experiments the YF17D/E200-T3 virus was used as negative control for EGFP expression. (B) Electrophoretic analysis of RT-PCR amplicons from viral RNA extracted of samples from the supernatant of cultures used to derive the citometry data (C) according the passage history (A). The length of the main RT-PCR bands are shown on the left side. (C) The rate of double gated cells (YF+, EGFP+) over the total YF+ gated cells (YF+, EGFP+ plus YF+, EGFP- gated cells) corresponds to the percentage of cells infected by YF 17D/Esa/5.1 glic virus stably expressing the EGFP protein. The respective columns indicate the values for each of the viral passages.