In the presence of oseltamivir, the relative fitness of either single mutant dominates over the wild-type. A simulated competition experiment between WT-I223 and MUT-I223V shows a larger total

In the presence of oseltamivir, the relative physical fitness of either single mutant dominates above the wild-variety. A simulated competition experiment among WT-I223 and MGCD-265 hydrochloride MUT-I223V demonstrates a greater overall (1.three 107 to 1.nine 109 RNA copies/mL, p < 0.001) and infectious (4.5 104 to 1.3 107 PFU/mL, p < 0.001) viral titer, and fraction of infected cells (0.03 to 0.84, p < 0.001) for the MUT-I223V strain in the presence of 15nM oseltamivir. This replicative advantage will further increase at higher concentrations and supports the observations of the emergence of mutations at residue I223 in patients undergoing oseltamivir therapy [20, 21]. The WT strain used in this study was identical to strain A/Quec/144147/09 H1N1pdm09 analyzed in our previous work using the same protocols but different lots of cells, reagents, and media [18]. This provided us with a unique opportunity to evaluate the reproducibility of our observed experimental viral kinetics and of the parameters extracted by our analysis across experiments. A comparison of the parameters extracted by our method for the WT strain in the present experiments to those from our previous work revealed that almost all viral replication parameters had changed significantly, with the exception of the eclipse phase. We showed that the changes in the extracted parameters are genuine (correctly extracted by our analysis) and can be directly related to visible differences in the experimentally-observed virus kinetics exhibited by the strain in the two separate experiments. These changes are attributable to interexperimental variability. When comparing data across separate experiments performed days or even months apart, one expects significant variability or "batch effects" due to differences in the stock of cells, reagents or media used, personnel conducting the experiments, the overall experimental conditions such as atmospheric pressure, relative humidity, or temperature, an effect that is wellknown and recognized [26]. It is possible to control for these variations by, for example, having the same person run all arms of the study at the same time, performing the same experiments, with the same lot of cells, reagents, and media. In this work, the two sets of triplicates for the WT and MUT-I223V were all performed exactly in this manner (two sets of plates processed in the same manner, at the same time, by the same individual) so as to virtually eliminate this variability. However, to improve the likelihood that findings will hold true, the key results of an experiment should be reproducible, i.e. hold true despite reasonable inter-experimental variations (the investigators repeating the experiments, the lots of cells and media used), rather than being merely replicable, i.e. hold true only in the near absence of experimental variations [2730]. This will be the case if the sources of inter-experimental variability (e.g., temperature, precise content of bovine serum) affect all viral strains equivalently within one experiment. Then, the properties of one strain relative to another (e.g., strain A has a greater virus production rate than strain B) will be preserved between separate experiments, even if their actual, absolute parameter values change significantly. Experimentally, this would mean that, for example, if the peak viral titer of the WT is significantly above that of the MUT in one experiment, then this should remain true in any other experiment as well, irrespective of personnel or stock of experimental consumables. Thus, if inter-experimental variations affect all 11904527strains equivalently, then the fitness of one strain relative to another (e.g., strain A produces more virus than strain B) will be robust in the face of inter-experimental variability, and the results reported herein, in our previous work [18], and in the works of countless others using a similar infection system will hold and be robust.