To examine genome packaging in the segment 2 mutant viruses directly, vRNA was extracted from equal plaque titres of virus. The segments were resolved and detected by Urea-PAGE and silver staining, and in all cases the expected pattern of seven vRNA segments were seen ( Figure 6C ; under these conditions, segments 1 and 2 comigrate). Obviously greater quantities of RNA were recovered from the ACC and TCC viruses (compare lanes 2, 6 and 8), a finding suggestive of an increased genome copy: PFU ratio and thus consistent with a raised virus particle: PFU ratio (30, 31) . However, the inability of this gel system to reliably separate the three largest genome segments hampered direct analysis of segment 2. In addition, the poor growth of the T32G virus made it difficult to extract sufficient vRNA to detect by this procedure (data not shown). We therefore used quantitative RT-PCR (qRT-PCR) to examine the copy number of segments in the mutant viruses. RNA was again extracted from equal PFU of virus and one step RT-qPCR was performed for segments 2, 3, 5 and 7. The amounts of each segment from the mutant viruses were normalized to that of the WT virus to derive a segment copy number:PFU ratio. The T30C AUG2 mutant and all AUG 3 and 4 mutants had similar levels of each of the segments tested to the WT virus, and also had equivalent amounts of each segment within each virus ( Figure 6D ). In contrast, most of the AUG1 up-mutants underincorporated segment 2. In addition, the ACC, CCC, TCC up-mutants as well as the AUG1 G28A and AUG2 T32G down-mutants showed several fold increases in the relative amounts of the other three segments. Since vRNA was extracted from equal numbers of infectious virus particles, these results are consistent with a specific packaging defect for segment 2 resulting in a higher number of defective virions and thus a higher segment copy number:PFU ratio of the other segments (30, 31, 42) . This is consistent with the hypothesis that the failure of the AUG1 mutants with an improved Kozak consensus to express elevated quantities of PB1 in infected cells results from lower delivery of the segment by infecting virions.