Figure 1. Design of new real-time RT-PCRs for differentiation between vaccine and wild-type YFV. A) YFV genomic representation (GenBank accession no. DQ100292) with real-time RT-PCR target sites, indicated by arrowheads, and identity plot of all complete YFV sequences available in GenBank as of May 24, 2017. Plots were done in SSE version 1.2 (11) using a sliding window of 200 and a step size of 40 nt. Target sites of the eventually selected assays are indicated by filled arrowheads; all other designed assays excluded after preliminary testing by open arrowheads. Of the real-time RT-PCR assays developed in this study, 1 assay targets only 1 genomic region, whereas the other assay targets 2 different genomic regions of vaccine and wild-type YFV strains. Both PCRs are duplex assays in which vaccine and wild-type YFV RNA are detected by lineage-specific probes. We called the assay targeting only 1 genomic region a single-target assay and the assay targeting 2 separate genomic regions a dual-target assay, even though the term dual-target commonly refers to detection of 2 different genes of a single pathogen, which is not the case in this study. B) Alignment of real-time RT-PCR oligonucleotide binding sites with YFV 17DD and American wild-type strains. The 100% consensus sequences were generated in Geneious (Biomatters Ltd., Auckland, New Zealand) and mapped to respective PCR primers and probes. Potential nucleotide mismatches are indicated by asterisks. D = A/G/T, M = A/C, R = A/G, W = A/T, Y = C/T. Black indicates a mismatch with all American wild-type strains, gray a mismatch with some American wild-type strains, based on the complete genetic information of American YFV strains and YFV vaccine strains available in GenBank as of March 24, 2017. C, capsid; E, envelope; Fwd, Forward; NS, nonstructural protein; prM, precursor membrane; Rev, reverse; RT-PCR, reverse transcription PCR; UTR, untranslated region; YFV, yellow fever virus.