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Generation and Application of a Luciferase Reporter Virus Based on Yellow Fever Virus 17D.

ABSTRACT: Yellow fever virus (YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in humans. Despite the availability of an effective vaccine, little is known about the replication mechanism of YFV, and there are still no available specific anti-YFV medicines. Herein, by introducing the Renilla luciferase gene (Rluc) into an infectious clone of YFV vaccine strain 17D, we generated a recombinant virus 17D-Rluc.2A via reverse genetics approaches. The 17D-Rluc.2A had similar plaque morphology and comparable in vitro growth characteristics with its parental strain. Importantly, the reporter luciferase was efficiently expressed in 17D-Rluc.2A-infected mammalian and mosquito cells, and there was a good linear correlation between intracellular luciferase expression and extracellular infectious virion reproduction. Furthermore, by a combination of the 17D-Rluc.2A reporter virus and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) technology, the conserved 5'-SLA element was shown to be essential for YFV replication, highlighting the capability of 17D-Rluc.2A in the investigation of YFV replication. At last, we demonstrated that two compounds with distinct anti-viral mechanisms can effectively inhibit the viral propagation in 17D-Rluc.2A-infected cells, demonstrating its potential application in the evaluation of anti-viral medicines. Taken together, the 17D-Rluc.2A serves as a useful tool for the study of YFV replication and anti-YFV medicine development.

SUBMITTER: Li D

PROVIDER: S-EPMC8692546 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Generation and Application of a Luciferase Reporter Virus Based on Yellow Fever Virus 17D.

Li Dan D Ye Jing-Long JL Liu Zhong-Yu ZY

Virologica Sinica 20210803 6

Yellow fever virus (YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in humans. Despite the availability of an effective vaccine, little is known about the replication mechanism of YFV, and there are still no available specific anti-YFV medicines. Herein, by introducing the Renilla luciferase gene (Rluc) into an infectious clone of YFV vaccine strain 17D, we generated a recombinant virus 17D-Rluc.2A via reverse genetics approaches. The 17D-Rluc.2A had similar plaq ...[more]

PMID: 34342842

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Project description:There is an urgent need for better diagnostic and analytical methods for vaccine research and infection control in virology. This has been highlighted by recently emerging viral epidemics and pandemics (Zika, SARS-CoV-2), and recurring viral outbreaks like the yellow fever outbreaks in Angola and the Democratic Republic of Congo (2016) and in Brazil (2016-2018). Current assays to determine neutralising activity against viral infections in sera are costly in time and equipment and suffer from high variability. Therefore, both basic infection research and diagnostic population screenings would benefit from improved methods to determine virus-neutralising activity in patient samples. Here we describe a robust, objective, and scalable Fluorescence Reduction Neutralisation Test (FluoRNT) for yellow fever virus, relying on flow cytometric detection of cells infected with a fluorescent Venus reporter containing variant of the yellow fever vaccine strain 17D (YF-17D-Venus). It accurately measures neutralising antibody titres in human serum samples within as little as 24 h. Samples from 32 vaccinees immunised with YF-17D were tested for neutralising activity by both a conventional focus reduction neutralisation test (FRNT) and FluoRNT. Both types of tests proved to be equally reliable for the detection of neutralising activity, however, FluoRNT is significantly more precise and reproducible with a greater dynamic range than conventional FRNT. The FluoRNT assay protocol is substantially faster, easier to control, and cheaper in per-assay costs. FluoRNT additionally reduces handling time minimising exposure of personnel to patient samples. FluoRNT thus brings a range of desirable features that can accelerate and standardise the measurement of neutralising anti-yellow fever virus antibodies. It could be used in applications ranging from vaccine testing to large cohort studies in systems virology and vaccinology. We also anticipate the potential to translate the methodology and analysis of FluoRNT to other flaviviruses such as West Nile, Dengue and Zika or to RNA viruses more generally.

Dataset Information

Generation and Application of a Luciferase Reporter Virus Based on Yellow Fever Virus 17D.

Publications

Generation and Application of a Luciferase Reporter Virus Based on Yellow Fever Virus 17D.

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