Isolation of RNA and real-time PCR

Approximately 250 mg of frozen lung or kidney tissue was homogenized in 1.0 mL TRIzol reagent using gentleMACS M tubes and a gentleMACS dissociator (Miltenyi Biotec) on the RNA setting. Serum samples were added directly to TRIzol reagent. Whole blood samples were added directly to TRIzol LS reagent. RNA was extracted using Qiagen miRNeasy Mini kit per the manufacturer’s instructions. The concentration of the extracted RNA was determined using a NanoDrop 8000 instrument and standardized to a final concentration of 10 ng/μl. Real-time PCR was conducted on a BioRad CFX thermal cycler using an Agilent Brilliant II QRT-PCR one-step master mix. ANDV primer/probe sequences are described in [21]. HTNV forward primer 5’- GCT GGC GTA TAG CCT TTG AC-3’, reverse primer 5’- AGT TAG CCT CCT TGG TGG TC-3’ and probe 5’- /56-FAM/ATG CCA CTT /ZEN/ GCC GCT GCC GT /3IABkFQ/ -3’. Cycling conditions were 30 min at 48°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Data acquisition occurs following the annealing step.