Quantitative RT-PCR
RNA was isolated from tissue homogenates using Qiagen RNeasy kit according to the manufacturer’s instructions. RNA was treated with DNAse, then cDNA was synthesized using random primers and MultiScribe reverse transcriptase (Applied Biosystems). A single reverse-transcriptase master mix was used to reverse-transcribe all samples in order to minimize differences in reverse-transcription efficiency using the following conditions: 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. For all primers except WNV, qRT-PCR was performed using Power SYBR Green PCR mastermix, and data are reported as ΔCt (Cttarget − CtGAPDH). Viral RNA was isolated from serum using Qiagen Viral RNA kit. A standard curve of viral genome with known PFU and serum samples were quantified using TaqMan reagents and the primers and probe listed in Table 1.