Selection and sequence analysis of resistant viruses.

The YFV 17D strain rescued from an infectious cDNA clone was used to infect Huh7.5 cells. Drug-resistant viruses were selected by multiple rounds of passaging of the viruses in cultures treated with increasing concentrations of BDAA. Briefly, Huh7.5 cells grown in a 12-well plate were infected either with wild-type YFV 17D at an MOI of 0.1 or in medium from a previous passage that contained virus and the compound. After incubation at 37°C for 1 h, the inocula were removed and replaced with fresh medium containing BDAA. For each passage, the cell culture was incubated for 3 days, during which time the cytopathic effect (CPE) was monitored as one of the readouts for the development of resistance. The viruses were passaged 6 times in the presence of 3 μM BDAA, followed by passage 6 times in the presence of 6 μM BDAA and 6 times in the presence of 8 μM BDAA. As a control, wild-type YFV 17D was passaged in the presence of 0.5% DMSO in parallel. The viruses in the supernatants of the 12th passage in the presence of 6 μM BDAA and 18th passage in the presence of 8 μM BDAA were evaluated for compound sensitivity in a yield reduction assay. To map drug resistance mutations, virus in the 18th passage was used to infect Huh7.5 cells. Total cellular RNAs were extracted on day 3 postinfection. YFV cDNA was synthesized and amplified by RT-PCR. Purified PCR products were used for direct DNA sequencing (Genewiz). Mutations were identified by sequence alignment with parental YFV 17D cDNA.