Quantitative evaluation of viral fitness by nucleotide sequence determination
- VernacularTitle:核苷酸序列测定法定量评价病毒相对适合度
- Author:
Yao ZHAO
- Publication Type:Journal Article
- Keywords:
Respiratory syncytial virus;
Fitness;
Assay;
Nucleotide sequence determination
- From:
Journal of Chongqing Medical University
2007;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
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.
