Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98105-8070, United States.
J Virol Methods. 2013 Dec;194(1-2):7-13. doi: 10.1016/j.jviromet.2013.07.062. Epub 2013 Aug 8.
Cell culture growth competition assays of human immunodeficiency virus type 1 (HIV-1) are used to estimate viral fitness and quantify the impact of mutations conferring drug resistance and immunological escape. A comprehensive study of growth competition assays was conducted and identified experimental parameters that can impact measurements of relative fitness including multiplicity of infection, viral input ratio, number, timing and interval of time points used to evaluate selective outgrowth, and the algorithm for calculating fitness values. An optimized protocol is developed here that is a multi-point growth competition assay that resolves reproducibly small differences in viral fitness. The optimized protocol uses an MOI of 0.005, a consistent ratio of mutant: parental viruses (70:30), and a multipoint [1+s 4,7] algorithm that uses data points within the logarithmic phase of viral growth for assessing fitness differences.
人免疫缺陷病毒 1 型（HIV-1）的细胞培养生长竞争测定用于估计病毒适应性，并定量评估赋予耐药性和免疫逃逸的突变的影响。对生长竞争测定进行了全面研究，并确定了可能影响相对适应性测量的实验参数，包括感染复数、病毒输入比、用于评估选择性生长的时间点的数量、时间和时间间隔，以及计算适应性值的算法。这里开发了一种优化方案，即多点生长竞争测定法，可重复且明确地测定病毒适应性的微小差异。优化方案使用 0.005 的感染复数、突变体：亲本病毒的恒定比例（70:30），以及多点[1+s 4,7]算法，该算法使用病毒生长对数期内的数据点来评估适应性差异。
