Figure 2. The viral stocks in our experiments are relatively pure of defective particles.

(A) Our viral stocks have a higher ratio of infectious particles to HA virion RNA compared to a high-defective stock propagated at high MOI. HA viral RNA was quantified by qPCR on virions. Error bars $\pm$ S.D., n = 6 (qPCR replicates). (B) Our viral stocks have a higher ratio of infectious particles to particles capable of expressing HA protein. A549 cells were infected at an MOI of 0.1, and the percentage of cells expressing HA protein at 9 hr post-infection was quantified by antibody staining and flow cytometry. (C) Our viral stocks are less immunostimulatory than virus propagated at high MOI when used at the same number of infectious units as calculated by TCID50. Note that this fact does not necessarily imply that they are more immunostimulatory per virion, as the high-MOI stocks also have more virions per infectious unit as shown in the first two panels. Measurements of IFNB1 transcript by qPCR normalized to the housekeeping gene L32 in A549 cells at 10 hr post infection at an MOI of 0.5. Error bars $\pm$ S.D., n = 3. Note that MOIs were calculated by TCID50 on MDCK-SIAT1 cells, whereas the experiments in this figure involved infection of A549 cells.

Figure 2—figure supplement 1. Full flow cytometry data for Figure 2B.

A549 cells were infected at an MOI of 0.1 as calculated by TCID50 on MDCK-SIAT1 cells. (A) Uninfected gating control. (B) Cells infected with the wild-type virus stock used in our experiments. (C) Cells infected with synonymously barcoded virus stock used in our experiments. (D) Cells infected with a stock of wild-type virus propagated at a high MOI, and therefore enriched in defective particles.