Development of a Real-Time Reverse Transcription-PCR Assay for Global Differentiation of Yellow Fever Virus Vaccine-Related Adverse Events from Natural Infections

Abstract

Yellow fever (YF) is a reemerging public health threat, with frequent outbreaks prompting large vaccination campaigns in regions of endemicity in Africa and South America. Specific detection of vaccine-related adverse events is resource-intensive, time-consuming, and difficult to achieve during an outbreak. To address this, we have developed a highly transferable rapid yellow fever virus (YFV) vaccine-specific real-time reverse transcription-PCR (RT-PCR) assay that distinguishes vaccine from wild-type lineages. The assay utilizes a specific hydrolysis probe that includes locked nucleic acids to enhance specific discrimination of the YFV17D vaccine strain genome. Promisingly, sensitivity and specificity analyses reveal this assay to be highly specific to vaccine strain(s) when tested on clinical samples and YFV cell culture isolates of global origin. Taken together, our data suggest the utility of this assay for use in laboratories of varied capacity for the identification and differentiation of vaccine-related adverse events from wild-type infections of both African and South American origin.

Keywords: 17D vaccine; diagnostic; locked nucleic acids; real-time RT-PCR; yellow fever virus.

FIG 1

Standardization and specificity of yellow fever 17D vaccine virus-specific locked nucleic acid hydrolysis probes. Real-time RT-PCR locked nucleic acid probes were designed to be specific to a single nucleotide polymorphism identified in the yellow fever virus (YFV) vaccine genome. The specificity of primer set 8 was optimized by testing RNA from YFV strains 17D (vaccine) and Asibi (parental strain) at an annealing temperature (T_a) of 60°C or 64°C. RFU, relative fluorescence unit.

FIG 2

Limit of detection analysis of yellow fever 17D vaccine virus-specific real-time RT-PCR assay. Probit regression analysis of the yellow fever 17D-specific assay was performed at an annealing temperature of 64°C using serial dilutions of in vitro transcribed RNA copies. Solid circles represent positive test results. Dotted lines represent the 95% confidence interval. C_q, quantification cycle.