ECIS easily distinguishes between wells of cells infected with different amounts of FHV-1. (A) CRFK cells were plated in ECIS wells, and impedance was monitored for 24 h at 16,000 Hz. Wells were then infected with FHV-1 at the MOIs indicated. The inset shows normalized impedance (Z′) values following FHV-1 infection. (B) Half-maximal Z′ (Z′50) values for impedance curves based on data from panel A. Different letters indicate significantly different Z′50 values, as determined by one-way ANOVA.
FHV-1 infection induces dose-dependent changes in resistance and capacitance (A, B). Resistance (R) and capacitance (C) measurements at 4,000 and 64,000 Hz, respectively, following infection of CRFK cells with FHV-1 at the MOIs indicated at 24 hpp. The insets show normalized resistance (R′) and capacitance (C′) values following FHV-1 infection. (C) Comparison of half-maximal R′ (R′50) and C′ (C′50) values based on data from panels A and B. Different letters indicate MOI R′50 or C′50 values that are significantly different, as determined by one-way ANOVA. No significant differences between R′50 and C′50 values were observed at any MOI, as determined by Student t test.
ECIS identifies the growth impairments of recombinant FHV-1. (A) CRFK cells were infected with FHV-1-gD-DsRed or WT FHV-1 at an MOI of 3 or 0.01 to measure single-step (graphs i and ii) or multistep (graphs iii and iv) growth curves, respectively, with conventional viral infectivity assays. qPCR was used to quantify intracellular genomic viral DNA copy numbers, and standard plaque assays were used to quantify extracellular virus titers. (B) Normalized impedance (Z′) values of CRFK cells infected with FHV-1-gD-DsRed or WT FHV-1 at a high MOI of 3 (i) or a low MOI of 0.01 (iii). Half-maximal Z′ (Z′50) values for impedance curves based on data from graphs i and iii were determined (graphs ii and iv, respectively). *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
ECIS accurately determines the EC50 of the antiviral cidofovir. (A) CRFK cells were infected with MOI of 0.01 of FHV-1 and treated with 2-fold dilutions of cidofovir (ranging from 976 to 1.9 μM) at 24 hpp. Nontreated FHV1-infected and mock-infected CRFK cells were included as controls. The insert shows normalized impedance values (Z′) following FHV-1 infection and cidofovir treatment. For clarity, only Z′ values for a selected set of cidofovir concentrations are shown. (B) Dose-response curve of the Z′50 value of each cidofovir concentration used to determine the EC50. (C) Comparison of the EC50 of cidofovir as determined by ECIS in the present study to previously published EC50 values determined by standard plaque reduction assays (25–27). No significant difference between ECIS-based and previously published EC50s was observed.
Additional data for validation of ECIS as a tool with which to monitor alphaherpesvirus infections. (A) Graphs of the ratios of impedance (Z), resistance (R), and capacitance (C) of CRFK-containing wells to those of cell-free wells at 24 hpp, calculated and plotted as a function of frequency. The vertical broken blue line in each graph represents the frequency with the maximal difference between cell-containing and cell-free wells. (B) Light microscopy pictures of ECIS wells infected with FHV-1 at the MOIs indicated at different time points (0 to 48 hpi). Pictures in colored boxes represent the time points at which a substantial CPE was observed at the different MOIs. The black bars in the images are ECIS electrodes. Scale bar, 25 µm. Download FIG S1, PDF file, 0.4 MB.
Creation, by CRISPR/Cas9 genome engineering, and characterization of FHV-1-gD-DsRed. (A) Schematic representation of the site of introduction of DsRed Express2 at the C-terminal end of US6, with the target site and sequence of the sgRNA indicated. (B) Schematic map of the pJET1.2-FHV-1-DsRed donor vector showing the FHV-1 homology regions to drive the insertion of DsRed via homology-directed repair. (C) Confirmation of DsRed Express2 insertion at the targeted location. Four primer sets were used to amplify different regions around the insertion site from WT and edited viruses. (D) CRFK cells were infected at 10 PFU/coverslip with FHV-1-gD-DsRed (red) or WT FHV-1 and stained with an anti-FHV-1-antibody (green), and nuclei were counterstained with DAPI (blue). (E) Quantification of the area of viral plaques showing the median value and quartiles. ****, P < 0.0001. Download FIG S2, PDF file, 0.3 MB.
Evaluation of cidofovir cytotoxicity. (A) Calculation of cidofovir CC50 at 5 days posttreatment by MTT cell viability assay. (B) Calculation of cidofovir CC50 at 3 days posttreatment by ECIS. Dotted lines represent the CC50 values. (C) Comparison of the CC50 values determined by the two methods. *, P < 0.05. Download FIG S3, PDF file, 0.2 MB.