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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

 ,  ,  ,  ,  , 
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

In This Article

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 to severe neurological complications6. Concerningly, CHIKV has re-emerged as a major public health concern. Since the beginning of 2025 alone, over 220,000 cases have been reported across 14 countries, resulting in at least 80 deaths7. Although the mortality rate is low, a significant proportion of infected individuals develop chronic joint pain, which can lead to long-term disability and reduced mobility8. To develop targeted antiviral strategies, a more comprehensive understanding of alphavirus replication and host-pathogen interactions is essential. Meanwhile, some alphaviruses, like Semliki Forest virus (SFV), show low pathogenicity in humans, despite sharing high genetic and structural similarity with more virulent alphaviruses. This makes SFV an ideal model for studying alphaviruses without posing a high risk to human health9,10,11.

Traditional assays such as plaque assays, 50% tissue culture infectious dose (TCID50) assay, and quantitative polymerase chain reaction (qPCR) analysis are valuable for quantifying viral titers and gene expression but are limited to endpoint measurements and require cell lysis or fixation. As a result, they cannot directly capture the dynamics of infection within the same cells over time12,13. To address these limitations, real-time fluorescence imaging provides a powerful, non-destructive approach for monitoring infection kinetics across multiple time points. Using fluorescently tagged viruses, such as mCherry-labeled SFV, enables direct visualization and quantification of reporter virus signal and spread in living cells14,15. For the experiments described here, commonly used cell lines in arbovirus research were employed. U4.4 cells, derived from Aedes albopictus larvae, are mosquito cells that support efficient replication of many arboviruses and facilitate studies on vector-virus interactions16,17. In parallel, BHK-21 cells, a fibroblast-like mammalian cell line originating from baby hamster kidney tissue, provide a permissive vertebrate host system frequently used for viral propagation and molecular virology assays18.

This protocol describes the monitoring of viral reporter expression and the characterization of antiviral compounds in detail, using furin inhibitors as an example. Furin is a proprotein convertase found in many eukaryotes and plays a key role in activating precursors of growth factors, hormones, matrix metalloproteinases, plasma proteins, and receptors19,20. Many alphaviruses exploit furin to cleave and activate their surface proteins, a crucial step for successful replication20. Furin inhibitors are synthetically designed compounds that block furin activity, thereby reducing viral replication21,22,23. Given the limited availability of standardized protocols for automated fluorescence microscopy-based analysis of alphavirus infections using a cell-imaging multimode reader, a detailed workflow for characterizing antiviral compounds in aedine cells is provided. The protocol outlines steps for cell culture, virus production, infection, antiviral compound (furin inhibitor) treatment, fluorescence imaging, and image analysis. However, the methods described here can be readily adapted to other viruses and alternative fluorescent labels.

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Protocol

1. Biosafety

NOTE: The recombinant mCherry-tagged Semliki Forest virus (SFV-mCherry) used in this protocol is genetically modified24,25,26. Incorporation of a fluorescent reporter gene classifies it as a genetically modified organism, which makes it subject to additional containment and regulatory requirements. Work involving other alphaviruses, particularly more pathogenic viruses such as CHIKV, may require additional containment and permits.

  1. Clean the biosafety cabinet with an appropriate disinfectant and apply UV for at least 30 min before and after viral assays.
  2. Discard all contaminated material according to local biosafety regulations. Collect solid and liquid waste separately in autoclavable containers and autoclave before disposal.

2. Preparation of antiviral compounds

NOTE: Furin inhibitors MI-113027 (trans-4-(aminomethyl) cyclohexanecarboxyl-arginine-valine-arginine-4-amidinobenzylamide), MI-113127 (trans-4-(guanidinomethyl) cyclohexanecarboxyl-arginine-valine-arginine-4-amidinobenzylamide), and MI-114822,23 (4-(guanidinomethyl) phenylacetyl-arginine-tert.-leucine-arginine-4-amidinobenzylamide) used in this study, are examples of antiviral compounds selected for testing. Furin inhibitor MI-1148 was used as a negative control in the cytotoxicity assay and as a positive control in the antiviral assay.

  1. Dissolve antiviral compounds in an appropriate solvent. When possible, use sterile water to avoid cytotoxic effects. Dimethyl sulfoxide or methanol can be used to a maximum of 2% (v/v). Using stock solutions of 10 mM in sterile water is recommended.
  2. Store stock solutions at -20 °C, depending on stability and solubility, unless specified otherwise by the manufacturer.

3. Cell culture

  1. Culture U4.4 cells in Leibovitz's L-15 Medium supplemented with 10% fetal bovine serum (FBS), 1% MEM non-essential amino acids, 1% tryptose phosphate broth, 1% glutamine, and 1% penicillin/streptomycin (Pen/Strep) (seeding density: approx. 1 × 106 cells/T25 flask, culture volume: 5 mL). Maintain cells at 28 °C. Passage U4.4 cells at a 1:6 ratio once per week, but adjust seeding densities according to experimental needs.
    NOTE: Additional cell lines, such as C6/36 cells, are also compatible with this protocol. Alternative media and supplements may be used as appropriate. The inclusion of antibiotics is optional and may be omitted.
  2. Culture Baby hamster kidney (BHK-21) cells in Dulbecco's Modified Eagle's Medium supplemented with 10% FBS, 1% glutamine  or Static equilibrium, ΣFx=0, graph, force balance analysis, mechanical stability diagram.‑alanyl‑Static equilibrium, ΣFx=0, graph, force balance analysis, mechanical stability diagram.‑glutamine, and 1% Pen/Strep (seeding density: approx. 1 × 106 cells/T75 flask, culture volume: 10 mL). Maintain cells at 37 °C in a humidified incubator containing 5% CO2. Passage cells at a 1:12 ratio every 4-5 days, with flexibility to modify seeding densities as required.
  3. Ensure cells remain healthy and actively proliferating.

4. Virus production

  1. Preparation of SFV-mCherry (SFV6-2SG-mCherry) stocks in BHK-21 cells.
    1. Culture BHK-21 cells (seeding density: approx. 1 × 105 cells/well, culture volume: 500 µL) in a clear cell culture 24-well plate under the conditions described above.
    2. At approximately 90% confluency (target confluency: approx. 2 × 105 cells), transfect BHK-21 cells with the SFV6-2SG-mCherry plasmid using a transfection reagent in infection medium (DMEM supplemented with 1% Pen/Strep and 0.2% bovine serum albumin). Therefore, dilute 1 µL of transfection reagent in 25 µL of reduced serum medium.
    3. In a separate tube, dilute 0.5 µg plasmid DNA and 1 µL of a proprietary additive in 25 µL of reduced serum medium. Add the diluted DNA solution to the solution containing the diluted transfection reagent and incubate for 15 min at room temperature. Subsequently, add 50 µL of the resulting complex to the cells.
      NOTE: Other transfection reagents may be used, provided they are compatible with the specific cell line, and the instructions may vary depending on the reagent used.
    4. At 48 h post-transfection, collect the virus-containing supernatant using a pipette, aliquot 250 µL per DNA/RNA free tube, and store at -80 °C.
  2. Titration by TCID50 assay.
    1. Culture BHK-21 cells (seeding density: approx. 2 × 104 cells/well, culture volume: 100 µL) in a black imaging grade 96-well plate under the conditions described above.
    2. Thaw a virus aliquot, prepare tenfold serial dilutions of the virus sample in infection media (15 µL virus sample + 135 µL infection media), discard old media, and add 100 µL of the dilutions to cells.
    3. Incubate cells for 1 h at 37 °C in a humidified incubator containing 5% CO₂ and replace inoculum with 100 µL fresh infection medium afterward.
    4. At 48 h post-infection (hpi), assess viral replication by fluorescence measurement using a multimode imaging well-plate reader (detailed description in section 7) and determine the cytopathic effect caused by the virus infection. Calculate the TCID50 value based on the method of Reed-Muench28 and convert to estimated viral titers (PFU/mL) using the standard Poisson-based approximation that 1 TCID50 corresponds to 0.7 PFU.

5. Cytotoxicity determination

  1. Culture U4.4 cells (seeding density: approx. 3 × 104 cells/well, culture volume: 100 µL) in a clear cell culture 96-well plate under the conditions described above.
    NOTE: Outer wells (rows A and H; columns 1 and 12) were seeded with cells but excluded from experimental analysis to avoid edge effects, including increased evaporation.
  2. At 90% confluency (target confluency: approx. 6 × 104 cells), treat cells with 100, 50, 25, and 12.5 µM of furin inhibitors MI-1130 and MI-1131 in a total volume of 100 µL. Add MI-1148 (same concentrations as MI-1130 and MI-1131), untreated cells, and cells treated with water as negative controls and ionomycin (100 µM in the assay) as a positive control for cytotoxicity.
  3. At 48 h post-treatment (hpt), assess cell viability using a luminescent assay based on ATP quantification. Add an equal volume of the luminescent assay reagent to each well (1:1 relative to the culture medium), shake the plate for 2 min to ensure cell lysis, and incubate for 10 min in the dark before measuring luminescence.
    NOTE: Other cell viability assays based on different detection principles may also be used.
  4. Record luminescence in a black or white 96-well plate using the multimode reader.

6. Viral infection and treatment with an antiviral compound

  1. Culture U4.4 cells (seeding density: approx. 3 × 104 cells/well, culture volume: 100 µL) in a black imaging-grade 96-well plate under the conditions described above.
    NOTE: Outer wells (rows A and H; columns 1 and 12) were seeded with cells but excluded from experimental analysis to avoid edge effects, including increased evaporation.
  2. Infect cells (target confluency: approx. 6 × 104 cells) with SFV-mCherry at a multiplicity of infection (MOI) of 0.01 in non-supplemented L-15 medium using an inoculation volume of 100 µL, and incubate for 1 h at 28 °C.
    NOTE: Only low-passage reporter virus stocks should be used, particularly when working with very low MOIs and multi-day infections.
  3. Dilute the furin inhibitors MI-1130, MI-1131, and MI-1148 to final concentratios of 100, 50, 25, and 12.5 µM in a separate 96-well V-bottom plate using supplemented L-15 medium. Prepare 110 µL per well to allow for a 10% overage, and transfer 100 µL (final volume) onto the cells. For controls, use infected cells treated with the solvent of the antiviral compounds and infected cells containing the virus only as a negative control. Uninfected, untreated cells also need to be added to measure the background fluorescence.
  4. After the 1 h infection period, remove the inoculum and transfer 100 µL of the diluted furin inhibitors from the V-bottom plates to the black imaging-grade plates.
  5. Incubate cells at 28 °C.

7. Live-cell fluorescence imaging, quantitative image analysis, and data presentation

  1. Measurement of viral reporter expression via fluorescence microscopy (see Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3, Supplementary Figure 4, Supplementary Figure 5, Supplementary Figure 6, and Supplementary Figure 7 for program setup).
    1. At 24 and 48 hpi, stain cell nuclei by adding 8 µL of a cell-permeant nuclear counterstain solution per well. Incubate for 30 min at 28 °C.
      NOTE: Infection can be monitored at multiple timepoints. However, measurements at 48 h post-infection provide the strongest signal (see Supplementary Figure 8) and allow antiviral effects to be assessed most reliably. Readouts at 24 h or intermediate timepoints are feasible, but extending incubation beyond 48 h increases the risk of cell overgrowth. Select an appropriate cell confluency at the start of the experiment to ensure that cells do not become overgrown by the chosen time point for the readout. This will avoid the obstruction of an accurate cell count.
    2. After incubation (and in addition to the clear cover lid), add a clear view seal foil covering the top of the plate for safety.
      NOTE: When using an imaging device, it is essential to select the correct plate type. Illumination, integration time, and gain settings will vary depending on the reporter gene. It is highly recommended to perform preliminary measurements to determine the optimal parameters. Initial illumination, integration, and gain settings used to establish the protocol under the given conditions are provided in the Supplementary Figure 2, where the program setup is described in detail.
    3. Acquire whole-well images using image stitching (four fields per well at 4x magnification) using the multimode reader.
    4. Measure red fluorescence intensity using a Texas Red filter cube (Excitation: 586/15 nm; Emission: 647/57 nm).
    5. Determine the total cell count using the DAPI channel with the following settings (Excitation: 377/50 nm; Emission: 447/60 nm).
  2. Quantitative image analysis (see Supplementary Table 1 for raw data).
    NOTE: For the fluorescence measurements, a microplate imaging reader was used. Detailed parameters used for this assay can be found in Supplementary Figure 2.
    1. Process the images using appropriate imaging software.
    2. Using the data reduction panel, select Image Stitching to DAPI and Texas Red channels to create a single image from the wells.
    3. Add Image Statistics and select Total intensity on the Texas Red Stitched channel. Apply Image Preprocessing to remove background noise from the DAPI Stitched channel.
    4. Run Cellular Analysis on the Preprocessed DAPI Stitched channel.
    5. Calculate the mean red fluorescence intensity per cell by subtracting the mean fluorescence of the uninfected, untreated control wells from each treated well, then dividing the result by the total cell count in the corresponding well.
  3. Troubleshooting
    1. Monitor focus stability during the measurement, particularly for long imaging measurements or when using temperature-sensitive plates.
    2. Enable autofocus and increase autofocus frequency if focus drift is observed. Reduce acquisition speed or allow plates to equilibrate to the instrument's temperature before imaging if drift persists.
    3. Use imaging-grade plates to ensure consistent signal quality during acquisition. Prevent bleed-through and overexposure by avoiding excessive illumination and long integration times.
    4. Lower excitation intensity, shorten integration time, or reduce gain until signal saturation is avoided. Verify that fluorophore channels are well separated and adjust filter settings as needed.
      NOTE: Bleed-through between DAPI and mCherry is generally low but depends on the reporter construct used (e.g., GFP).
    5. Avoid over-confluency of cells to maintain proper infection kinetics and assay sensitivity. If over-confluency occurs, reduce incubation time before the measurement.
    6. Monitor dye-related effects on cell viability and signal quality, particularly at high concentrations. Decrease dye concentration or shorten incubation times if dye-related toxicity or oversaturated background is observed.
    7. Prepare and store dyes according to manufacturer recommendations to maintain signal stability.

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Results

In this work, a detailed protocol for monitoring alphavirus spread and the evaluation of antiviral compounds (e.g., furin inhibitors) using fluorescence microscopy in a multi-well plate format is presented. To demonstrate the visualization of the inhibitory effects on viral reporter expression, three previously synthesized furin inhibitors were selected for testing in the aedine cell line U4.4: MI-1148, MI-1130, and MI-1131. An overview of the experimental workflow is illustrated in Figure 1...

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Discussion

The capacity to observe alphavirus replication in real time offers a sequential and cumulative perspective on conventional endpoint tests. This multi-time live cell imaging protocol is a useful model for studying alphaviruses, as it provides a dynamic platform for observing viral spread.

The success of this protocol relies on several parameters. First, it is important to assess the cytotoxicity of the compounds before an antiviral assay to rule out false-positive results due to cell death. Onc...

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Disclosures

The authors have no conflict of interest.

Acknowledgements

We thank Prof. Dr. Andres Merits, University of Tartu, Estonia, and Prof. Dr. Andreas Pichlmair, Technical University of Munich, Germany, for providing pCMV-SFV6-2SG-mCherry. We thank Prof. Dr. Stefanie Becker, University of Veterinary Medicine, Hannover, Germany, for the C6/36 cells. We thank Prof. Dr. Torsten Steinmetzer, University of Marburg, Marburg, Germany, for providing the furin inhibitors. This work was supported by the Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz Program of the Hessian Ministry of Higher Education, Research, and the Arts through the LOEWE Centre for Translational Biodiversity Genomics (LOEWE-TBG) with funding code: LOEWE/1/10/519/03/03.001(0014)/52, and by the BMFTR (Project ASCRIBE-Grant Number 01KI2024).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Albumin Bovine Fraction V, pH 7.0SERVA Electrophoresis11930.03
BenchStable DMEMThermo Fisher ScientificA4192101
BHK-21 cellsCLS Cell Lines Service GmbH, Eppelheim, Germany603126
CELL CULTURE FLASK, 50 ML, 25 cm2, PS, Red filter screw cap, Clear, CELLSTAR, TC, sterileGreiner Bio-One690175
Cell culture microplate, 24 wells, PS, F-bottom, (Chimney well),Clear, CELLSTAR, TC, Lid with condensation rings, sterileGreiner Bio-One662160
Cell culture microplate, 96 wells, PS, F-bottom, (Chimney well), µCLEAR, Black, CELLSTAR, TC, Lid with condensation rings, sterileGreiner Bio-One655090
Cell culture microplate, 96 wells, PS, F-bottom, (Chimney well),Clear, CELLSTAR, TC, Lid with condensation rings, sterileGreiner Bio-One655180
Cell culture microplate, 96 wells, PS, V-bottom, ClearGreiner Bio-One651101
Cell scraperVWR734-2602
CellTiter-Glo Luminescent Cell Viability AssayPromegaG7573
Cytation 5 Cell Imaging Multimode ReaderBiotek
DAPI filter cube 1225100Biotekex: 377/50 nm; em 447/60 nm
Dulbecco's Modified Eagle's Medium (DMEM GlutaMAX)Thermo Fisher ScientificA4192102
Fetal Bovine SerumFisher Scientific11550356
Furin inhibitor MI-1130This study
Furin inhibitor MI-1131This study
Furin inhibitor MI-1148Hardes et al. 2015.  https://doi.org/10.1002/cmdc.201500103 
Gen5 Prime SoftwareBiotek
Ionomycin Calcium Salt, 99%Thermo Fisher ScientificJ60628
Leibovitz's L-15 Medium, GlutaMAX Supplement Thermo Fisher Scientific31415029
Lipofectamine 3000 Transfections reagentThermo Fisher ScientificL3000001
MEM non-essential amino acids (100x)Thermo Fisher Scientific11140050
Microplate, PS, 96 well, F-bottom, (Chimney well), Black, Fluotrac, Med.bidingGreiner Bio-One655076
NucBlue Live ReadyProbes Reagent (Hoechst 33342)Thermo Fisher ScientificR37605
Opti-MEMThermo Fisher Scientific31985062
Penicillin-Streptomycin (10, 000 U/mL)Thermo Fisher Scientific15140122
SFV6–2SG-mCherryProf. Dr. Andres Merits (University of Tartu, Estonia) and Prof. Dr. Andreas Pichlmair (Technical University of Munich, Germany)
Texas Red filter cube 1225102 Biotekex: 586/15 nm; em: 647/57 nm
Tryptose Phosphate Broth Thermo Fisher Scientific18050039
U4.4 cellsFriedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Greifswald, Germany
Viewseal Sealer, ClearGreiner Bio-One676070

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