To further test this hypothesis, we analysed segment 2 RNA accumulation in cells infected with the ACC (as a representative of an AUG1 up-mutant), G28A and T32G viruses in comparison with WT and two mutant viruses (T30C and T72C) with no obvious packaging defects. All three RNA species (m-, c-and vRNA) were readily detectable in samples from cells infected with the WT, T30C and T72C viruses ( Figure 7A, lanes 2, 5 and 7) . However, the three viruses with apparent defects in segment 2 vRNA packaging produced much reduced quantities of vRNA and (with the exception of G28A), m-and cRNA also (lanes 3, 4 and 6). This defect was particularly apparent for segment 2, as more consistent levels of segment 7 vRNA were seen for all the viruses ( Figure 7A ). When replicate experiments were quantified, the three viruses with potential packaging defects produced <10% of the normal amount of segment 2 vRNA ( Figure 7B ).