Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism

Qi Liu; Amita Gupta; Ayse Okesli-Armlovich; Wenjie Qiao; Curt R. Fischer; Mark Smith; Jan E. Carette; Michael C. Bassik; Chaitan Khosla

The design of the pDENV-Luc infectious clone derived from dengue serotype 2 (DENV-2) strain 16681 was described in detail by Marceau et al (Marceau et al., 2016) . The plasmid was linearized with XbaI and in vitro transcribed into the genomic RNA of DENV-Luc virus using the T7 Megascript Kit (Ambion) in the presence of m7G(5')ppp(5')G RNA Cap Structure Analog (NEB). 5 μg DENV-Luc RNA was electroporated into 2X10 6 Vero cells. The transfected cells were resuspended with Dulbecco's modified Eagle's medium (DMEM; Invitrogen) with 10% FBS and 100 U/ml penicillin-streptomycin and transferred into a T-175 flask incubated at 37 °C with 5% CO2. Supernatants were collected and replenished with fresh medium every 24 h from day 17 to 24 post-transfection, pooled together, clarified by centrifugation and stored in aliquots at −80°C. The amount of infectious DENV-Luc virus in the stock was titrated using the TCID50 assay and calculated by the Spearman & Kärber algorithm as described previously (Hierholzer and Killington, 1996) .