Fig 8. Competitive replication of HK and its 6 single-point HA mutants in HTBE cultures.

HTBE cultures were inoculated with the mixtures of HK and its 6 HA mutants containing 5 PFU (L, 12 replicate cultures), 20 PFU (M, 12 replicates) and 320 PFU (H, 6 replicates) of each virus. After 1-h incubation, the inoculum was removed, the cultures were incubated under ALI conditions, and the apical material was harvested at 72 h post-inoculation. Small filled circles show proportions of each HA genotype determined by next generation sequencing in the inoculated mixture (In) and in each replicate harvest in the L, M and H groups. Empty squares and error bars represent the mean values and confidence intervals of proportions. Asterisks depict statistical significance of the differences between proportion of the corresponding genotype in the harvest and in the inoculum. The proportions of the parent HK virus (shown on gray background) were inferred by subtracting proportions of the six HA variants from a theoretical value of 1. Because of the high intrinsic errors of this approach the data for HK were not analysed for statistical significance.