Abstract
Flavivirus outbreaks require fast and reliable diagnostics that can be easily adapted to newly emerging and re-emerging flaviviruses. Due to the serological cross-reactivity among flavivirus antibodies, neutralization tests (NT) are considered the gold standard for sero-diagnostics. Here, we first established wild-type single-round infectious virus replicon particles (VRPs) by packaging a yellow fever virus (YFV) replicon expressing Gaussia luciferase (Gluc) with YFV structural proteins in trans using a double subgenomic Sindbis virus (SINV) replicon. The latter expressed the YFV envelope proteins prME via the first SINV subgenomic promoter and the capsid protein via a second subgenomic SINV promoter. VRPs were produced upon co-electroporation of replicon and packaging RNA. Introduction of single restriction enzyme sites in the packaging construct flanking the prME sequence easily allowed to exchange the prME moiety resulting in chimeric VRPs that have the surface proteins of other flaviviruses including dengue virus 1–-4, Zika virus, West Nile virus, and tick-borne encephalitis virus. Besides comparing the YF-VRP based NT assay to a YF reporter virus NT assay, we analyzed the neutralization efficiencies of different human anti-flavivirus sera or a monoclonal antibody against all established VRPs. The assays were performed in a 96-well high-throughput format setting with Gluc as readout in comparison to classical plaque reduction NTs indicating that the VRP-based NT assays are suitable for high-throughput analyses of neutralizing flavivirus antibodies.
Highlights
To establish a yellow fever virus (YFV) reporter replicon allowing easy detection of replication, the majority of the YFV structural protein gene region was replaced by a cassette encoding a Gaussia luciferase (Gluc) reporter
Gluc in the supernatant was measured at several time points after electroporation yielding increasing luciferase levels over time, thereby confirming efficient replication of the replicon (Figure 1B)
We demonstrated that wild-type and chimeric flavivirus reporter replicon particles can be produced by packaging a YFV reporter replicon expressing Gluc with flavivirus structural proteins expressed by a double subgenomic Sindbis virus (SINV) expression vector
Summary
Flaviviruses, the majority of which are arthropod-borne, represent an ongoing threat to public health [1]. Some of the well-known flaviviruses include yellow fever virus (YFV), dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV), which cause widespread morbidity and mortality. Despite an available vaccine against yellow fever (YF) [2], several outbreaks have been described over the last years. Reoccurring YF cases involving sylvatic cycles have been described in Brazil since
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