(A) Flow cytometry analysis of expression of viral proteins in cells infected at high (MOI five as calculated by TCID50 on MDCK-SIAT1 cells) or low (MOI 0.1) initial infectious dose. Cells were concurrently stained for HA and NS1 proteins at 10 hr after initiation of infection, and then analyzed by flow cytometry. The level of HA protein was quantified in cells that were identified as infected based on being positive for NS1 protein (left), and the level of NS1 protein was quantified in cells that were identified as infected based on being positive for HA protein (right) While a higher dose leads to more cells expressing high amounts of viral protein, it does not greatly increase the amount of viral protein in either the low-expressing or high-expressing cells. Therefore, higher viral dose does not lead to a large continuous increase in viral protein production among all cells – rather, it mostly changes the proportions of cells that fall in different parts of the highly heterogeneous distribution. (B) Cells were co-infected with a mix of wild-type virus and pseudovirus in which the HA gene was replaced by GFP flanked by the terminal regions of the HA gene segment at an MOI of 0.1 for each virus. At 10 hr post-infection, cells were stained for NS1 and HA expression and analyzed by flow cytometry for these proteins and GFP. Cells could be annotated as infected by virions of the same type (wild-type infection indicated by presence of HA, or pseudovirus infection indicated by the presence of GFP) or both types of virions (indicated by presence of HA and GFP). Coinfected cells, like cells infected at a higher infectious dose, occupy different positions in the distribution of viral protein production but do not not exhibit a continuous increase in viral protein production. Data are shown for three independent replicates.