Relative freq MxA-, 54 hpi, replicate1
Frequency of Occurrence
Illumina Sequencing , Viral Competition
To confirm that the decreased MxA resistance of polymerase activity correlated with the effect of MxA on viral replication, we carried out competition experiments between wildtype and mutant viruses. Such competition experiments provide a sensitive and internally controlled way to measure the relative fitness of two viral variants. We used reverse genetics to generate influenza viruses carrying wildtype NP, the D51Dsyn mutation, or the D51N mutation. We mixed each mutant virus with wildtype virus at a 1:1 ratio of infectious particles, and infected MxA-expressing or non-expressing cells at a low MOI. At 10 and 54 hours post-infection, we isolated viral RNA and determined the frequency of each variant by Illumina deep sequencing. Experiments were performed in duplicate (replicates 1 and 2). Relative frequencies (mutant/WT) were determined for each of the conditions (MxA+, MxA-) at each time point for each replicate.