Quantification of viral load

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed on 5 μl RNA from each sample using CDC RT-PCR primers InfA Forward, InfA Reverse, and InfA probe, which bind to a portion of the influenza M gene (CDC protocol, 28 April 2009). Each reaction contained 5.4 μl nuclease-free water, 0.5 μl each primer/probe, 0.5 μl SuperScript III RT/Platinum Taq mix (Invitrogen111732, Carlsbad, CA) 12.5 μl PCR Master Mix, 0.1 μl ROX, 5 μl RNA. The PCR master mix was thawed and stored at 4°C, 24 hr before reaction set-up. A standard curve relating copy number to Ct value was generated based on 10-fold dilutions of a control plasmid run in duplicate.