To determine genome copy numbers of released progeny influenza A particles (strains WSN, PR8/34, S-OIV Texas and Mexico), culture supernatants of infected MDCK cells (4610 5 cells/well in a 12-well plate format) were harvested and total RNA prepared using a QIAcube automated extractor and the QIAamp viral RNA mini kit reagent. Purified RNA was then subjected to quantitative real time (qRT) PCR analysis using an Applied Biosystems 7500 Fast real-time PCR system and the qRT-PCR TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems). Primers and probe are based on recent reports [79] and universally reactive with all influenza A strains including the recent S-OIV (H1N1) isolates. To generate a qRT-PCR standard, genome segment seven of influenza A/WSN was subcloned into pCR2.1-TOPO vector (Invitrogen) and copy numbers of the resulting standard calculated using Promega's BioMath Calculator tools (http://www.promega.com/ biomath/). For each TaqMan reaction, 10-fold serial dilutions of the linearized plasmid ranging from 10 7 to 10 1 were amplified in parallel.