Ebastine

The drug screening assay was performed using the Sigma Library of Pharmacologically Active Compounds (LO4200, LOPAC¹²⁸⁰, Sigma‐Aldrich), comprising 1280 compounds dissolved in DMSO arrayed across 16 plates. V‐bottom 96‐well microtitre plates (Matrix) containing 2.5 μl of compound in each well at 2.5 mM (or 1.5 mM) were prepared for screening. Initial hit compounds and additional compounds of interest were cherry‐picked from the Sigma LOPAC master plates (5‐mM concentration) and used for retests and counter screening assays. Compounds selected for follow‐up experiments were purchased separately: ivermectin (Sigma I8898) and ebastine (Sigma E9531).

Six-week-old female C57BL/6 or Sash mice were infected i.p. with MuLV/Friend (1 × 10⁹ copies) for indicated times. In some mice, C48/80 (1 mg/kg) was administered along with infection, and Ebastine (5 mg/kg) or Loratadine (10 mg/kg) (both from Sigma-Aldrich) was administered 1 day before infection and treatment was continued each day during infection. At necropsy, spleen, blood or bone marrow (BM) were harvested, and total cellular RNAs were extracted with TRIzol reagent (Life Technologies) and then reverse transcribed to cDNA with ReverTra Ace qPCR RT Master Mix with genome DNA Remover Kit (TOYOBO). Viral replication was quantified by measuring the expression of viral gag gene with PCR quantification using the GoldStar TaqMan Mixture (CWBiao). PCR was performed using the Thunderbird SYBR qPCR Mix (TOYOBO) on the ABI 7900HT Real-time PCR system (Applied Biosystems), with an initial denaturation step for 10 min at 95°C, amplification with 40 cycles of denaturation (95°C, 15 s), annealing and extension (60°C, 1 min). The primers for gag, forward, 5′-CTC TTT CTC CGA GGA CCC AG- 3′, reverse, 5′-GTC ATT GGG CAG CTG AGT TG- 3′, and the probe: 5′-FAM- ACA GCT TTG ATC GAG TCC GTT CTC CT-TAMRA- 3′.