A dengue type 2 reporter virus assay amenable to high-throughput screening

Li, Li-Hsin; Kaptein, Suzanne; Schmid, Michael; Zmurko, Joanna; Leyssen, Pieter; Neyts, Johan; Dallmeier, Kai

doi:10.1016/j.antiviral.2020.104929

Cited by 14 publications

(34 citation statements)

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“…The availability of such assays with no cross-reactivity could be of great benefit for differential serology to accurately and rapidly identify a specific flavivirus infection which is especially challenging in regions where several flaviviruses co-circulate. Finally, similar reporter assays could be optimized towards high-throughput antiviral screening campaigns, either as single (Li et al, 2020) (Zhang et al, 2020) or, by choice of compatible fluoroprotein reporters, multiplexed antiviral screens possibly allowing to identify pan-flavivirus antivirals.…”

Section: Discussionmentioning

confidence: 99%

“…C gene codons 2-34* were repeated with an alternative codon usage to avoid recombination during virus replication. The plasmids were recovered from yeast, transformed into Epi300T (Epicenter) competent E. coli cells and colonies were selected as described earlier (Li et al, 2020). The entire genome of ZIKV/mCherry and JEV/eGFP was verified by Sanger sequencing.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Infectious viruses were rescued from plasmid constructs by transfection into BHK-21J (pShuttle-YF17D, pShuttle-YFV/mCherry, pShuttle-JEV/eGFP and pShuttle-DENV2/mCherry) and Vero E6 (pShuttle-ZIKV/mCherry) cells using TransIT-LT1 Transfection Reagent (Mirusbio) following the manufacturer's instructions (Li et al, 2020;Mishra et al, 2020). Upon onset of cytopathic effect (CPE), the recombinant viruses were subsequently passaged on their corresponding cell lines to generate virus stocks.…”

Section: Viruses and Virus Titrationsmentioning

confidence: 99%

“…All 30 serum samples were assessed for selectivity and cross-reactivity using similar fluorescence-based SNT FLUO assays using four distinct reporter viruses, YFV/mCherry, DENV2/mCherry, ZIKV/mCherry and JEV/GFP (Figure 4b). DENV2/mCherry (Li et al, 2020) has been described earlier; ZIKV/mCherry and JEV/GFP were generated accordingly for this study. All test sera specifically neutralized only their corresponding cognate reporter virus without detectable cross-neutralizing activity towards the other flavivirus reporters (Figure 4c and supplementary figure 2).…”

Section: Assay Selectivitymentioning

confidence: 99%

“…Plasmid construction of the yellow fever 17D vaccine strain (pShuttle-YFV/mCherry) and Dengue virus type 2 NGC strain (pShuttle-DENV2/mCherry) reporter viruses stably expressing the red fluorescent protein mCherry have been reported previously (Li et al, 2020;Mishra et al, 2020). In a similar manner, Zika (pShuttle-ZIKV/mCherry) and Japanese encephalitis (pShuttle-JEV/eGFP) reporter viruses were generated for the current study (schematic representation in Figure 1 and Figure 4).…”

Section: Plasmid Constructionmentioning

confidence: 99%

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A high-throughput neutralization assay for yellow fever serodiagnostics

Rasulova

Vercruysse

Paulissen

et al. 2021

Preprint

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Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks, for surveillance and to evaluate vaccine efficacy in population-wide studies. All this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming and limited in throughput, classical plaque reduction neutralization test (PRNT) is still considered gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika and dengue viruses amenable for differential diagnostics.IMPORTANCEFast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance and monitoring of vaccine efficacy. Although classical PRNT still remains gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as a low throughput and an overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing large number of samples in a highly standardized manner and has the potential to be implemented in clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika and dengue viruses opening new avenues for differential diagnostics.

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Section: Discussionmentioning

confidence: 99%

Section: Plasmid Constructionmentioning

confidence: 99%

Section: Viruses and Virus Titrationsmentioning

confidence: 99%

Section: Assay Selectivitymentioning

confidence: 99%

Section: Plasmid Constructionmentioning

confidence: 99%

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