Serial Passage Competition Assay in HeLa Cells for Mixtures of Wild-Type and 3D-G64S Viruses Containing the 2C-F28S Temperature-Sensitive Attenuating Mutation

<div><p>One-to-one mixtures of 3D-G64S to WT virus containing the indicated 2C-F28S mutations were used to infect HeLa cells at an MOI of 0.1 PFU/cell at 32 °C. At 12 h post-infection, cell-associated RNA and virus were harvested, and the virus was titered to determine input for the next passage. This cycle was repeated three to five times, and representative gels of digestion products, with maps indicating the mutations at the 2C locus in the mixtures of viruses that were wild-type and 3D-G64S at the 3D locus, are shown: (A) wild-type, (B) 2C-F28S(1m), (C) 2C-F28S(3m). X denote the numbers of nucleotide substitutions at the 2C-F28S site, while O denotes the presence of the 3D-G64S mutation.</p><p>DOI: 10.1371/journal.ppat.0010011.g005</p>