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. 2019 Feb 13;116(10):4611–4618. doi: 10.1073/pnas.1818494116

Fig. 5.

Fig. 5.

HA segments carrying H5 or H7 packaging signals were present less frequently than control Pan/99 HA segments in virus particles. Triplicate stocks of HA_H3PS, HA_H5PS, HA_H7PS, and control Pan/99 viruses were grown in embryonated eggs. Each of the resultant 12 stocks was processed individually by concentration through a sucrose cushion. RT ddPCR was used to enumerate segment copy number in the resultant virus preparations. (A and B) HA copy numbers were normalized to PB2 copy numbers and then normalized to the control Pan/99 values. n = 3 for each virus (three technical replicates with ddPCR performed in duplicate for each). Independent sets of PB2 and HA primers were used to generate data shown in A and B. Data are plotted as mean with SD and analyzed using one-way ANOVA with Dunnett’s multiple comparisons. (A) **P < 0.0001, *P = 0.0119, n.s., not significant; (B) **P = 0.0013, n.s., not significant. (C) All segment copy numbers were normalized to PB2 copy numbers and then normalized to the control Pan/99 values. n = 3 for each virus (three technical replicates with ddPCR performed in duplicate for each). HA data were acquired using the primer set in A and at a separate time from the remaining seven segments. Data are plotted as mean with SD and analyzed within segments using two-way ANOVA with Dunnett’s multiple comparisons; ***P < 0.0001, **P < 0.0005, *P < 0.01.