Objective:To evaluate reproductivity of nucleotide sequencing for quantitative determination of viral fitness. Methods:F gene fragments from respiratory syncytial virus (RSV) A2 strain

palivizumab escape mutants

F212 and MP4

were amplified by RT-PCR

gel purified

quantified by spectrophotometry and adjusted to same concentration. A2

F212 and MP4 RT-PCR products were mixed at different ratios and determined for nucleotide sequence. Electropherograms at sites with ambiguity were visually measured by using ABI EDIT software. Proportion of RT-PCR products from prototype virus or escape mutants represented viral fitness level. Molecular cloning was utilized to validate input ratios of RT-PCR products. Results:Dual peaks were seen at 816 and 828 positions in the F gene for A2/F212 and A2/MP4mixtures

respectively. Proportion of prototype virus and escape mutants were gained by measuring the heights of dual peaks. Measured ratios were similar to premixed ratios which were confirmed by molecular cloning. Conclusion:Nucleotide sequencing is reliable

easy assay for evaluation of viral fitness and practical for screening escape mutants and viral fitness in viral infections

such as HIV infection.