The vRNA content of virus particles was determined by silver staining as previously described (30, 31) . Quantitative RT-PCR (qRT-PCR) for segments 2, 3, 5 and 7 was also performed on RNA extracted from equal PFU using the QIASymphony system (Qiagen) as previously described (30, 31) . Reverse transcriptase primer extension analysis of RNA from infected or transfected cells was performed as described (32) , with the exception that SuperscriptIII (Invitrogen) was used and reverse transcription was performed at 50 C. Reaction conditions, primer and probe sequences are available upon request. Quantification was performed by densitometry of scanned X-ray films using Image J (Research Services Branch, NIH). Values were corrected with respect to a loading control (cellular 5S ribosomal RNA) and normalized to those of WT virus.