FIG. 2.
Genetic stability of rescued HEF mutants. The HEF mutants of C/JHB/1/66 were engineered to carry genetic markers. For each rescued virus, vRNA was extracted from virus particles after five passages on SK 93/2 cells at a MOI of 10−3 (lanes g to j). Amplification by RT-PCR was performed with primers specific for the HEF sequences flanking the mutated region (B). Amplification was performed in parallel on water (lane a) and with samples derived from the mock-transfected cell supernatant (lane f) as negative controls and on the counterpart vRNA expression plasmids (lanes b to e) as positive controls. After purification, PCR products were digested with EcoRV (C) or NgoMIV (D). A control reaction was carried out in the absence of reverse transcriptase (A) in order to ensure that the amplification product was derived from vRNA and not from plasmid carried over from transfected cells. Lanes b and g, wt virus; lanes c and h, 465 Ala mutant; lanes d and i, 284 Ile mutant; lanes e and j, 284 Ile and 465 Ala double mutant.