A real-time RT-PCR assay for quantifying the fitness of tobacco etch virus in competition experiments.

Relative fitness determination has become a standard tool in experimental virus evolution studies. In this type of studies, the tested strain is mixed with a reference strain, which differs in an easy-to-score and genetically stable marker, and allowed to compete for a limited common pool of resources during a given number of generations. In this report, a TaqMan real-time PCR methodology is proposed for quantifying the relative fitness of tobacco etch potyvirus strains (TEV) in in planta mixed infections with a reference TEV strain. Two different forward primers along with a common reverse one are used into separated reactions mixes from the same RNA preparation. The reference strain, named TEV-PC1, was genetically engineered to carry a neutral marker in a highly conserved region of the RNA polymerase NIb gene. This marker allows tracking the frequency of both competitors during competition experiments by real-time quantitative PCR using specific primers. Both the reproducibility and sensitivity of the method have been explored. Reproducibility was assessed by running multiple competition experiments for the same genotype. Sensitivity was assessed by comparing the results of competition experiments against TEV-PC1 of 24 single-nucleotide substitutions mutants.