A Fluorescent Reporter Virus Toolkit for Interrogating Enterovirus Biology and Host Interactions

Figures

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  Figure 1. Generation and characterization of CVB3 reporter viruses. (A) Schematic representation of the reporter virus constructs. eGFP or mCherry was inserted at three different sites across the CVB3 proteome: upstream of the P1 precursor (mCh:P1 and GFP:P1), between the P1 and P2 precursors (P1:GFP and P1:mCh), and between the P2 and P3 precursors (P2:GFP and P2:mCh). The additional amino acid sequences inserted to generate the 3C (black) or 2A (gray) cleavage sites are underlined. (B) Titration of passage 0 CVB3 variants in HeLa-H1 cells using a fluorescence-based method. Bars represent the mean of ≥2 technical replicates, shown as black dots. ND, not detected. (C) Comparison of titers obtained by the fluorescence-based or plaque-based assays for three CVB3 reporters (n = 3). Statistical significance was assessed using paired t-tests on log-transformed data. * p < 0.05, *** p < 0.001.

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  Figure 2. Analysis of CVB3 reporter virus fitness. (A) Single-cycle virus production. Viral titration in HeLa-H1 cells after infection at a multiplicity of infection (MOI) of 5. Bars represent the mean of three biological replicates, shown as black dots. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (p < 0.05) on log-transformed data. Groups sharing the same letter are not significantly different. (B) Representative images of plaque morphology for CVB3 GFP:P1, P1:GFP, and P1:mCh reporter viruses. (C) Schematic representation of the fluorescence-based competition assay. mCherry- and eGFP-expressing reporter viruses (colored in red or green, respectively) are mixed at a 1:1 ratio and used to infect cells. Images are taken at 8 hpi and 16 hpi to measure the number of cells infected with each virus following initial infection and viral spread, respectively. Relative fitness can then be calculated by dividing the number of fluorescent cells at 16 hpi by those at 8 hpi for each reporter virus. (D,E) Relative viral spread (16 hpi versus 8 hpi) comparing GFP-encoding viruses (GFP:P1 or P1:GFP) against an mCherry-encoding control virus (P1:mCh; D),…

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  Figure 3. Fluorescent reporter viruses for screening neutralizing antibodies. (A) Schematic representation of the sera neutralization assay workflow. A fixed amount of virus is incubated with serial dilutions of sera or monoclonal antibodies (Treatments: A, B, C, D) before infecting cells. No-sera (virus-only) and no-virus (cells-only) controls are included to determine maximum infectivity and background fluorescence levels, respectively. The number of fluorescent cells or average well fluorescent intensity is quantified following a single cycle of infection (e.g., 8 hpi). Background fluorescence (cells-only control) is subtracted from all virus-infected cells. Subsequently, the relative signal in each infected well is then standardized to that of untreated, virus-infected controls (virus-only control) followed by curve-fitting to determine the concentration resulting in the desired inhibition level (e.g., IC50, IC90, etc.). (B) Neutralization of WT and mutant reporter viruses by a monoclonal antibody (mAb). Data represent mean ± SD from two independent experiments.

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  Figure 4. Antiviral screening and mechanism of action studies using fluorescent viruses. (A) Dose–response curve of interferon (IFN) sensitivity for the WT CVB3 fluorescent reporter virus. (B) Sensitivity of GFP- and mCherry-expressing viruses to ribavirin treatment determined at 16 h post-infection (hpi). Cell viability was assessed in parallel using the resazurin reduction (Alamar blue) assay (red line). (C,D) Growth curve for P1:nLuc (C) and P2:mCh (D) in HeLa-H1 cells. Cells were transfected with in vitro-transcribed RNA and either mock-treated or treated with 0.5 mM GuHCl, and reporter signal was quantified at 2 h intervals. The experiment was performed in triplicate and mean values ± SD are shown. Statistical significance was assessed using t-tests on log-transformed data. ns, p > 0.05; *, p < 0.05; **, p < 0.001.

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  Figure 5. A quantitative, fluorescence-based recombination assay. (A) Schematic diagram of CVB3 acceptor and donor vRNAs, along with the recombinant vRNA product. The acceptor genome (blue) encodes eGFP but is rendered replication-incompetent by deletion of the catalytic triad in the viral polymerase (ΔGDD). The donor genome (orange) is a subgenomic replicon in which the capsid region has been replaced with mCherry. Following co-transfection, recombination can yield a replication-competent genome encoding the eGFP reporter. The color gradient (from blue to orange) between the 3′ end of the VP1-coding region and the ΔGDD indicates the crossover region within which recombination must occur to produce a functional genome. (B) Live-cell fluorescence and phase contrast images from the cell-based recombination assay. In vitro transcribed vRNAs were transfected into BHK-21 cells and reporter expression was examined at 10 hpt to quantify the number of eGFP- or mCherry-positive cells. (C) Supernatants collected at 10 hpt from (B) were used to infect HeLa-H1 cells and the fluorescence and phase contrast images at 8 and 20 hpi are shown. (D,E) Quantification of the number of eGFP- or mCherry-positive…