Fig 5. DI250 and DI250:A94G replication and packaging dynamics.

Clonal DI250 or DI250:A94G populations were co-passaged with WT PR8 for five serial passages each in duplicate lineages, individually. (A) The left panel displays DelVG copy numbers, the middle panel shows NP vRNA copy numbers for each condition, and the right panel presents the DelVG-to-NP ratio. By passage 3 (P3), DelVG levels were comparable across samples. From this point onward, intracellular (In, 6 hpi) and extracellular (Ex, 24 hpi) dynamics were tracked through passage 5 (P5). Replicate 1 is shown in solid color and replicate 2 in the same color with a patterned fill. (B) Packaging index was calculated across P3–P5 as the ratio of virion-associated DelVG at passage (P_{n+1}) to intracellular DelVG at passage (P_n). (C) DI250 and DI250:A94G plasmids were co-transfected at a 1:1 ratio in 293 cells together with the seven remaining PR8 plasmids and a PB2 mRNA–expressing plasmid. After 48 hours, the supernatant was treated with RNase, followed by RNA extraction, DNase treatment, and RT-PCR, before amplicon sequencing. The figure shows the relative proportions of the two DelVGs incorporated into virions, following the same color scheme as in other panels. Statistical significance was determined using an unpaired t-test. ns = not significant.