Cb300

Blood samples were used to analyze whole-blood compound levels and were obtained either from tail vein during the in-life part into EDTA tubes (CB300, Sarstedt, Germany) or from trunk blood on the day of necropsy into EDTA Eppendorf tubes (Milian SA, CatNoTOM-14, Fisher Scientific, Wohlen, Switzerland), frozen on dry ice and stored at −80 °C until analysis.
The brain was removed immediately after decapitation, rinsed with saline and sectioned sagitally down the midline. The left half of the cerebellum was used to analyze compound level and was placed into a glass tube (Chromacol, 125 × 5-SV T051, Welwyn Garden City, United Kingdom), weighed and frozen in dry-ice. The left half of the forebrain (without olfactory bulb) was used for Aβ analysis, and was frozen on a metal plate on dry ice and placed into protein Lo-bind tube (003 0108.116, Eppendorf, Hamburg, Germany).
Ventral and dorsal skin were taken to analyze compound level, weighed and frozen on dry-ice.

Wars2^V117L/V117L mice were generated and genotyped as previously described (Potter et al., 2016 (link); Agnew et al., 2018 (link)). Tissues and plasma were collected in experiments previously described (Agnew et al., 2018 (link)). Briefly, 4-month-old male and female Wars2^V117L/V117Land Wars2^+/+ mice (n = 5–7) were humanely killed by terminal anaesthesia, and retro-orbital blood was collected into lithium-heparin microvette tubes (CB300, Sarstedt, Numbrecht, Germany). Death was confirmed by cervical dislocation and mice were then dissected and kidney, liver, muscle, heart, iWAT, gWAT, and BAT collected. Tissues were directly placed in cryotubes and snap frozen in liquid nitrogen and samples were stored at −70°C before subsequent analyses by qPCR and Western blot.

Groups of ferrets (n=3 each) were inoculated with 1×10⁵ pfu of 2019-nCoV/USA-WA1/2020 in 1 ml (0.5 ml per nare). At 12 hours after infection, three groups of ferrets were treated b.i.d. with vehicle (1% methylcellulose) or MK-4482/EIDD-2801 at a dose level of 5 mg/kg or 15 mg/kg, respectively. At 36 hours after infection, a fourth group of ferrets began receiving b.i.d. treatment with MK-4482/EIDD-2801 at a dose of 15 mg/kg. Compound was administered via oral gavage in 1% methylcellulose. After treatment onset, b.i.d. dosing was continued until four days after infection. Nasal lavages were performed on all ferrets every 12 hours. Blood samples were obtained every two days after infection and stored in K₂-EDTA tubes (Sarstedt CB 300). CBC analysis was performed on each blood sample in accordance with the manufacturer’s protocols. After CBC analysis, red blood cells were lysed with ACK buffer (150 mM NH₄CL, 10mM KHCO₃, 0.01 mM EDTA pH 7.4) and PBMCs were harvested and stored at −80°C in RNAlater until further qPCR analysis was performed. Four days after infection, all ferrets were euthanized and organs harvested to determine virus titers and the presence of viral RNA in different tissues.