Factors affecting relative fitness measurements in pairwise competition assays of human immunodeficiency viruses.
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.
Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98105-8070, United States.
Erinn C
EC
Lanxon-Cookson
J Victor
JV
Swain
Siriphan
S
Manocheewa
Robert A
RA
Smith
Brandon
B
Maust
Moon
M
Kim
Dylan
D
Westfall
Morgane
M
Rolland
James I
JI
Mullins
D002478:Cells, Cultured; D024882:Drug Resistance, Viral; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D009154:Mutation; D014776:Virus Cultivation / Q000379:methods; D014779:Virus Replication
Growth competition; HIV-1; Replication capacity; Viral fitness