Abstract
BackgroundYellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases.ResultsWe developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction--for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi).ConclusionsThe SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods.
Highlights
Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas
A set of published primers targeting the conserved 5’ non-translated region (NTR) and capsid gene junction of YFV was selected on the basis of their specificity, relative conservation across strains, primer compatibility and assay efficiency [9]
Optimal assay condition was established at an annealing temperature of 58°C using 500 nmol of each of the primers (YFS and YFAS) in a 10 μL of reaction scale
Summary
Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. Yellow fever (YF) is one of the most important arboviruses affecting humans in many tropical areas of subSaharan Africa and the Americas. The Yellow fever virus (YFV) is the prototype member of the genus Flavivirus in the family Flaviviridae. The genome of YFV is a linear, non segmented, positivesense strand of RNA of approximately 11 kb.
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