Lineage-Specific Real-Time RT-PCR for Yellow Fever Virus Outbreak Surveillance, Brazil

Abstract

The current yellow fever outbreak in Brazil prompted widespread yellow fever virus (YFV) vaccination campaigns, imposing a responsibility to distinguish between vaccine- and wild-type YFV-associated disease. We developed novel multiplex real-time reverse transcription PCRs that differentiate between vaccine and American wild-type YFV. We validated these highly specific and sensitive assays in an outbreak setting.

Keywords: Americas; Brazil; real-time RT-PCR; reverse transcription PCR; surveillance; vaccine safety; vector-borne infections; viruses; yellow fever virus.

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 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.

Figure 2

Validation of new real-time RT-PCRs for differentiation between vaccine and wild-type YFV. A) Effects of target competition on YFV real-time RT-PCRs. Mean cycle threshold (C_t) values are plotted against IVT concentrations. Triplicates were tested for each datum point. B) Validation of the assays with clinical matrices. Spiked viruses were vaccine strain 17D and the American genotype 2 wild-type strain BOL88/1999. RNA purification was performed using the MagNA Pure 96 Viral NA Small Volume Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. C) Clinical validation. Clinical specimens (serum, liver, whole blood, and plasma) from 11 YFV-infected patients were tested. RNA was extracted using the MagMAX Pathogen RNA/DNA Kit (Thermo Fisher, São Paulo, Brazil) and serial dilutions of the RNA were tested using the new assays and a YFV reference assay (12). Viral loads were determined for clinical specimens using a commercially available quantitative real-time RT-PCR (Bio Gene Research Yellow Fever PCR kit; Bioclin, Minas Gerais, Brazil), following the manufacturer´s instructions. Standard curves and sample copies per millileter were calculated using an in-house IVT standard. IVT, in vitro transcript; RT-PCR, reverse transcription PCR; YFV, yellow fever virus.