a, Intersegment RNA interaction map for the Udorn virus, with the interaction between the PB1 and NA segments highlighted in dark blue. Structure prediction for the highlighted PB1–NA interaction is shown on the right. The circled nucleotides highlight the bases that differ between the Udorn and Wyo03 strains. b, Competitive reverse-engineering of influenza viruses. Six plasmids encoding H1N1 background segments are transfected together with an H3N2 NA segment-encoding plasmid and PB1 segment-encoding plasmids from both the H1N1 and H3N2 strains. The origin of the PB1 segment in the progeny viruses is determined using RT–qPCR. c, Cosegregation between H1N1 or H3N2 PB1 segments and H3N2 NA segments. P values as indicated; analysis of variance (ANOVA) with Sidak correction for multiple testing; n = 5 (Ud-NA), n = 3 (Mem71-NA and PC73-NA) and n = 8 (Wyo03-NA) biologically independent experiments. The centre of the bar plot represents the mean; the error bars indicate the s.e.m. d, Preferential Wyo03 PB1 and NA segment cosegregation is recovered by substituting the four nucleotides that differ in Wyo03-NA from those in the PB1-interacting region of Udorn-NA (highlighted in a) for a Udorn-like sequence. P values as indicated; ANOVA with Sidak correction for multiple testing; n = 7 (PR8 PB1 versus Wyo03 PB1 competitions) and n = 8 (PR8 PB1 versus Udorn PB1 competitions) biologically independent experiments. The centre of the bar plot represents the mean; the error bars indicate the s.e.m. e, Substitution of the four Udorn-like nucleotides regenerates a strong intersegment RNA interaction between the H3N2-origin PB1 and NA segments in reassortant viruses.