Quantitative Fluorescence Imaging of Alphavirus Infection for Antiviral Screenings

Alejandra Centurión; Bodunrin Omokungbe; Cross Chambers; Sabrina Stiehler; Andreas Vilcinskas; Kornelia Hardes

doi:10.3791/69384

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Method Article

Quantitative Fluorescence Imaging of Alphavirus Infection for Antiviral Screenings

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DOI:

10.3791/69384

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August 14th, 2026

Alejandra Centurión^*¹^,² , Bodunrin Omokungbe^*¹^,² , Cross Chambers² , Sabrina Stiehler³ , Andreas Vilcinskas¹^,²^,³ , Kornelia Hardes¹^,²^,⁴

¹LOEWE Centre for Translational Biodiversity Genomics (LOEWE-TBG), ²Department of Pest and Vector Insect Control, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch of Bioresources, ³Institute for Insect Biotechnology, Justus-Liebig University, ⁴BMFTR Junior Research Group in Infection Research "ASCRIBE", Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch of Bioresources

^* These authors contributed equally

Summary

Here, a robust and adaptable protocol for the continuous visualization of alphavirus infections is presented. This protocol provides an approach for investigating alphavirus replication and assessing the efficacy of antiviral compounds at the cellular level based on fluorescence imaging.

Abstract

Fluorescence microscopy offers a highly sensitive and versatile approach for investigating alphavirus infection at the cellular level. By combining fluorescently labeled viruses with quantitative image analysis, this method enables detailed spatial and temporal characterization of infection dynamics, including the detection of subtle differences in replication kinetics and cell-to-cell spread. A central aim of this protocol is its application in antiviral screening assays. Image-based quantification of fluorescence intensity provides a robust and reproducible means to assess the efficacy of antiviral compounds, allowing early and sensitive detection of inhibitory effects in infected cells. This facilitates the identification of promising antiviral hits and supports the evaluation of dose-dependent responses. The approach is also well-suited for comparative studies of different alphavirus strains or mutants, as variations in replication behavior and dissemination patterns become readily apparent. Its flexibility, compatibility with multiple cell lines, and straightforward integration into automated imaging platforms makes the method scalable and suitable for high-throughput screening campaigns. Overall, this protocol advances the discovery and evaluation of antiviral strategies. Given that several alphaviruses cause significant human and veterinary diseases, lack approved antiviral therapies, and continue to expand geographically with emerging outbreaks, the identification of novel antivirals remains an urgent priority. Therefore, this fluorescence-based workflow represents a valuable and timely contribution to modern alphavirus research.

Introduction

Alphaviruses are enveloped, positive-sense, single-stranded ribonucleic acid (RNA) viruses belonging to the family Togaviridae¹^,²^,³. They are primarily transmitted by mosquitoes and are responsible for a range of diseases in humans and animals. The alphavirus genus includes several medically significant viruses, such as Chikungunya virus (CHIKV), Sindbis virus, Eastern equine encephalitis virus, and Venezuelan equine encephalitis virus⁴^,⁵. These pathogens cause symptoms ranging from febrile illness and rash t....