Quantitative Fluorescence Imaging of Alphavirus Infection for Antiviral Screenings

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 Togaviridae^1,2,3. 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 virus^4,5. 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 health^9,10,11.

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 time^12,13. 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 cells^14,15. 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 interactions^16,17. 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 receptors^19,20. 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 replication^21,22,23. 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.

Protocol

1. Biosafety

NOTE: The recombinant mCherry-tagged Semliki Forest virus (SFV-mCherry) used in this protocol is genetically modified^24,25,26. 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-1148^22,23 (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 ‑alanyl‑‑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.

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.

Prior to assessing antiviral efficacy, it is essential to evaluate cytotoxicity to ensure that any reduction in viral replication is not due to cell death by the compound. Therefore, U4.4 cells were treated at 90% confluency with the inhibitors at 100, 50, 25, or 12.5 µM. Untreated cells and cells treated with water (solvent control) served as negative controls, while ionomycin was used as a positive control. The cell viability was assessed at 48 h post-treatment using a luminescent assay based on ATP quantification. For the furin inhibitors tested, no significant reduction in cell viability was observed (Figure 2).

Given the lack of cytotoxicity, the compounds were subsequently evaluated for antiviral activity. For this reason, U4.4 cells were infected with SFV-mCherry for 1 h, followed by treatment with the same furin inhibitors at the same concentrations. Infected, untreated cells and infected, water-treated cells served as negative controls for antiviral activity, while uninfected, untreated cells were used to assess background fluorescence. At 48 h post-treatment, viral reporter expression was quantified by fluorescence microscopy. Prior to imaging, 8 µL of a cell-permeant nuclear counterstain solution was added to each well and incubated for 30 min to stain cell nuclei. The red fluorescence signal corresponding to the expression of the viral reporter and the total number of nuclei were quantified from processed fluorescence images. Among the tested inhibitors, MI-1148 demonstrated potent antiviral activity, significantly reducing viral reporter expression in a dose-dependent manner (Figure 3). In contrast, MI-1130 and MI-1131 showed no observable inhibition of viral reporter expression at any of the tested concentrations. The raw data and analysis of the antiviral assay are provided in Supplementary Table 1.

The representative results demonstrate that the protocol reliably identifies compounds with antiviral activity, as shown by the strong reduction in reporter signal observed with MI-1148. In contrast, the inhibitors MI-1130 and MI-1131 did not decrease reporter expression, indicating that their potent furin inhibition, reflected by picomolar inhibition constants, did not translate into antiviral activity under these conditions (Figure 4). These findings highlight the assay's ability to distinguish active compounds from inactive ones and to support the identification of potent and relevant lead structures.

Figure 1: Experimental workflow. A schematic overview of the experimental procedure used to evaluate the antiviral activity of furin inhibitors in the aedine cell line U4.4. The workflow includes compound preparation, cell seeding, infection with mCherry-tagged Semliki Forest virus, compound treatment, fluorescence microscopy for real-time visualization, and quantification of viral reporter expression. This illustration was created with BioRender. Please click here to view a larger version of this figure.

Figure 2: Cytotoxic effects of furin inhibitors in U4.4 cells. At approximately 90% confluency, U4.4 cells were treated with MI-1148, MI-1130, and MI-1131 at 100, 50, 25, and 12.5 µM. Untreated cells and cells treated with water served as negative controls, while ionomycin (100 µM) was used as a positive control. At 48 h post-treatment, the cell viability was assessed via ATP quantification. The data were normalized to the untreated control and expressed as percentage (%). The mean cell viability (n=3) is shown, and the error bars represent the coefficient of variation. The dotted line represents the cytotoxicity cut-off set at 80%. n.d. = not detectable. Data representation was performed with GraphPad Prism v9.5.1. Please click here to view a larger version of this figure.

Figure 3: Antiviral activity of furin inhibitors against the mCherry-tagged Semliki Forest virus (SFV) in the aedine cell line U4.4. At approximately 90% confluency, cells were infected with SFV-mCherry for 1 h, followed by treatment with MI-1148, MI-1130, and MI-1131 at 100, 50, 25, and 12.5 µM. At 48 h post-treatment, viral reporter expression was quantified via fluorescence microscopy. Prior to imaging, 8 µL of a cell-permeant nuclear counterstain solution was added to each well and incubated for 30 min to stain the cell nuclei. Fluorescence images were acquired using a Texas Red filter for viral signal and a DAPI filter for nuclei. Image analysis was performed to quantify total red fluorescence intensity (indicating viral replication) and the total number of cells per well. Data (n = 4) are presented as total red intensity per cell (total virus signal / total cell count). Error bars represent standard deviation. Statistical significance compared to SFV-infected control, using one-way ANOVA and Dunnett's multiple comparisons test: **** = P<0.0001; ns = P>0.05. Data representation and statistical analysis were performed with GraphPad Prism v9.5.1. Please click here to view a larger version of this figure.

Figure 4: Representative fluorescence images showing the antiviral activity of furin inhibitors against the mCherry-tagged Semliki Forest virus (SFV) in the aedine cell line U4.4. The cells at approximately 90% confluency were infected with SFV-mCherry for 1 h, followed by treatment with MI-1148, MI-1130, and MI-1131 at concentrations of 100, 50, 25, and 12.5 µM. Infected, untreated cells and infected, water-treated cells served as negative controls, while uninfected, untreated cells were included as background controls. The reporter signal was assessed 48 h post-treatment by fluorescence microscopy using a Texas Red filter to capture the mCherry signal. Scale bar = 2000 µm. Please click here to view a larger version of this figure.

Supplementary Table 1: Raw data of the total intensity of virus signal and the cell count. Please click here to download this File.

Supplementary Figure 1: Procedure steps used on the imaging reader for the representative results. First, a set on temperature (red) matching the culture conditions of U4.4 cells. Then, three independent image action steps (orange) for measurements of DAPI, Texas Red, and Brightfield. Please click here to download this File.

Supplementary Figure 2: Image settings in the procedure steps used on the imaging reader for the representative results. The first settings to modify are: magnification (4x) and image size (Full WFOV). The fluorophore to be measured must be selected, and the Illumination, Integration time, and Gain parameters must be modified accordingly. Finally, the image montage (2x2) and overlap (30 µm x 30 µm) must be selected. Please click here to download this File.

Supplementary Figure 3: Data reduction steps used on the imaging reader for the representative results. The first step is Image stitching (blue) for DAPI and Texas Red channels. Second, Image Statistics (purple) gives the measurement for total intensity. Then, the image preprocessing step (green) is required to remove background noise from the DAPI channel. Finally, the Cellular Analysis (yellow) is performed on the preprocessed stitched image of DAPI. Please click here to download this File.

Supplementary Figure 4: Image stitching in the data reduction steps used on the imaging reader for the representative results. The most relevant setting is the downsizing of the final image (80%), because if it is lower, it affects the resolution of the image and therefore, the final measurement of the parameters. Please click here to download this File.

Supplementary Figure 5: Image statistics in the data reduction steps used on the imaging reader for the representative results. It is important to select the correct channel (Stitched: Fluorophore), choose a minimum threshold (2500), and finally, the parameter to be measured (Total Intensity). For a more accurate measurement, an Image Plug can be made. Please click here to download this File.

Supplementary Figure 6: Image preprocessing in the data reduction steps used on the imaging reader for the representative results. The most relevant setting is the rolling ball parameter (30 µm), giving priority to fine results. Please click here to download this File.

Supplementary Figure 7: Cellular Analysis in the data reduction steps used on the imaging reader for the representative results. It is important to choose the channel that has the Image Preprocessed. Choose to divide the touching objects and fill holes with masks. It is critical to choose the correct range of object selection (5-50 µm). Finally, for refined results, an Image Plug can be made. Please click here to download this File.

Supplementary Figure 8: Antiviral activity of furin inhibitors against mCherry-tagged Semliki Forest virus (SFV) in the aedine cell line U4.4. At approximately 90% confluency, cells were infected with SFV-mCherry for 1 h, followed by treatment with MI-1148, MI-1130, and MI-1131 at 100, 50, 25, and 12.5 µM. At 24 h (red bars) and 48 h (green bars) post-treatment, viral reporter expression was quantified via fluorescence microscopy. Error bars represent standard deviation (n = 4). Please click here to download this File.

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. Once the evaluation for cytotoxic effects has been carried out, the antiviral activity of the compounds can be subsequently evaluated. For the test compounds, no cytotoxicity was observed. MI-1130 and MI-1131 showed no significant impact on viral reporter expression, whereas MI-1148, used as a positive control, effectively suppressed it. Additionally, the timing of infection in relation to cell seeding is crucial. Cells must reach adequate confluency to ensure a reproducible infection without overgrowth. It is also important to optimize the multiplicity of infection depending on the cell line to observe a gradual infection without compromising the cell viability. Finally, the frequency of image acquisition must be carefully determined to allow visualization of gradual viral reporter expression without large jumps. For most applications, acquiring images every eight hours, for example, provides an effective balance between temporal detail and sample integrity.

The main strength of this protocol is its adaptability. In this experiment, we used SFV-mCherry and U4.4 cells to demonstrate the monitoring of viral reporter expression and inhibition after treatment with furin inhibitors. However, the protocol can be optimized for the fluorescent analysis of alternative viruses with different reporter markers, such as GFP, as well as other host cell types, including primary cells and differentiated cell lines. Although the protocol focuses on compound treatment after infection, the workflow can also be readily adapted for time-of-addition experiments, including pre-treatment, co-treatment, immediate post-infection treatment, or delayed treatment, to further explore the mechanism of antiviral action. The protocol was initially used to compare the functionality of the RNAi machinery of C6/36 and U4.4 cells¹⁴. It has been further extended to examine how dsRNA directly targeting the virus inhibits viral reporter expression¹⁵.

There are potential challenges that can be encountered while using this protocol. Reporter viruses, although very useful for numerous applications in vivo²⁹ and in vitro³⁰, can also show lower virulence³¹ and genetic instability of the reporter genes³². Cell detachment caused by over-confluency can obstruct clear visualization and imaging, thus affecting the cell count. Prolonged exposure to live-cell dyes (e.g., Hoechst 33342 for nuclei) may alter cell behavior or viability¹⁵. Furthermore, experiments involving highly pathogenic alphaviruses, such as CHIKV, may require higher biosafety measures and corresponding protocol adaptations. Additionally, focus drift can be observed in some cases due to the variability of the plates as well as the use of different plates. For example, fluorescence signals in transparent cell culture plates can bleed from wells into their adjacent wells, and white plates can cause overexposure. In both cases, the accuracy of the measurement of the fluorescent signal will be affected.

This protocol offers several advantages over conventional virological assays. For example, plaque assays and TCID₅₀ assays provide endpoint data on virus titers¹². More so, qPCR offers sensitive detection of viral RNA, but it cannot distinguish between replication-competent viruses and non-infectious viral particles or residual nucleic acids. In contrast, this kind of fluorescence live imaging enables continuous, non-invasive assessment of viral progression, spread, and treatment effects within the same population of cells over time.

Beyond the examination of furin inhibitors as antiviral compounds, our protocol can be adapted for other applications. It is particularly well-suited for antiviral drug screening, enabling the quantification of dose-dependent responses to various compounds. It provides a platform for studying host-pathogen interactions, including the timing and localization of viral entry, replication, and spread. It can be used for the comparison between reporter viruses, different strains, or different alphaviruses. Additionally, it can be coupled with approaches such as gene knockdown using siRNA or dsRNA. Thus, this system can help identify and validate critical targets and antiviral compounds for viral replication. However, this fluorescence-based assay represents an initial screening approach rather than a standalone confirmatory method; further investigations, including assays with wild-type viruses, will be necessary to substantiate the screening results.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Albumin Bovine Fraction V, pH 7.0	SERVA Electrophoresis	11930.03
BenchStable DMEM	Thermo Fisher Scientific	A4192101
BHK-21 cells	CLS Cell Lines Service GmbH, Eppelheim, Germany	603126
CELL CULTURE FLASK, 50 ML, 25 cm2, PS, Red filter screw cap, Clear, CELLSTAR, TC, sterile	Greiner Bio-One	690175
Cell culture microplate, 24 wells, PS, F-bottom, (Chimney well),Clear, CELLSTAR, TC, Lid with condensation rings, sterile	Greiner Bio-One	662160
Cell culture microplate, 96 wells, PS, F-bottom, (Chimney well), µCLEAR, Black, CELLSTAR, TC, Lid with condensation rings, sterile	Greiner Bio-One	655090
Cell culture microplate, 96 wells, PS, F-bottom, (Chimney well),Clear, CELLSTAR, TC, Lid with condensation rings, sterile	Greiner Bio-One	655180
Cell culture microplate, 96 wells, PS, V-bottom, Clear	Greiner Bio-One	651101
Cell scraper	VWR	734-2602
CellTiter-Glo Luminescent Cell Viability Assay	Promega	G7573
Cytation 5 Cell Imaging Multimode Reader	Biotek
DAPI filter cube 1225100	Biotek		ex: 377/50 nm; em 447/60 nm
Dulbecco's Modified Eagle's Medium (DMEM GlutaMAX)	Thermo Fisher Scientific	A4192102
Fetal Bovine Serum	Fisher Scientific	11550356
Furin inhibitor MI-1130			This study
Furin inhibitor MI-1131			This study
Furin inhibitor MI-1148			Hardes et al. 2015.  https://doi.org/10.1002/cmdc.201500103
Gen5 Prime Software	Biotek
Ionomycin Calcium Salt, 99%	Thermo Fisher Scientific	J60628
Leibovitz's L-15 Medium, GlutaMAX Supplement	Thermo Fisher Scientific	31415029
Lipofectamine 3000 Transfections reagent	Thermo Fisher Scientific	L3000001
MEM non-essential amino acids (100x)	Thermo Fisher Scientific	11140050
Microplate, PS, 96 well, F-bottom, (Chimney well), Black, Fluotrac, Med.biding	Greiner Bio-One	655076
NucBlue Live ReadyProbes Reagent (Hoechst 33342)	Thermo Fisher Scientific	R37605
Opti-MEM	Thermo Fisher Scientific	31985062
Penicillin-Streptomycin (10, 000 U/mL)	Thermo Fisher Scientific	15140122
SFV6–2SG-mCherry			Prof. Dr. Andres Merits (University of Tartu, Estonia) and Prof. Dr. Andreas Pichlmair (Technical University of Munich, Germany)
Texas Red filter cube 1225102	Biotek		ex: 586/15 nm; em: 647/57 nm
Tryptose Phosphate Broth	Thermo Fisher Scientific	18050039
U4.4 cells			Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Greifswald, Germany
Viewseal Sealer, Clear	Greiner Bio-One	676070