A Practical Framework for ASFV Disinfectant Evaluation: Differentiating Cytopathic Effects from Cytotoxicity via Integrated Analytical Methods

Figure 1. An overview of the experimental design.

Figure 2. Damage scores in Vero cells exposed to ASFV and disinfectant treatments. Bright-field images of Vero cells were captured following treatment with ASFV alone (A), disinfectant only (D), or ASFV plus disinfectant (AD) at various dilutions (175×, 275×, 375×) and observed daily from 1 to 6 days post inoculation (DPI). Images were acquired at a total magnification of 200× using an Olympus IX73 inverted microscope equipped with a 20×/0.40 objective. All images are shown at the same scale for comparative purposes; therefore, individual scale bars are not included. Scoring system for cell damage: 0: No visible damage; cells appear healthy; 1: Minimal damage; slight rounding or detachment of a few cells; 2: Mild damage; increased rounding and detachment of cells (~10–20%); 3: Moderate damage; significant rounding, detachment, and cell loss (~30–50%); 4: Severe damage; widespread cell rounding and detachment (~50–80%); 5: Complete damage; almost all cells are detached or destroyed (>80%).

Figure 3. Antigen detection results for ASFV with different disinfectant treatments. (A) Lateral flow assay (LFA) results for ASFV detection after treatment with various disinfectants. Band intensities were quantified using ImageJ, and values are shown next to each band. Positive results are highlighted in red, and negative results are indicated in black. (B) The quantification of band intensities from (A), focusing on the groups outlined in black. The strongest signal, “Supernatants-6” from the ASFV-only group (190.24), was set as 100%, and all other values were normalized accordingly. Asterisks indicate statistically significant differences between groups (*** p < 0.001), determined by a one-way ANOVA.

Figure 4. Detection of ASFV antigen (p54) and cellular integrity under different treatment conditions. Vero cells were treated with ASFV alone, ASFV combined with disinfectants at dilution rates of 175×, 275×, and 375×, or left untreated (N.C.). Cells were examined by bright-field microscopy, an indirect immunofluorescence assay (IFA; anti-p54, green), and DAPI staining (blue). The ASFV antigen was detected only in the ASFV-only group. Nuclear damage increased with higher disinfectant concentrations, while the 375× dilution group showed improved nuclear preservation. All images were acquired at the same magnification using an Olympus IX73 inverted microscope equipped with a 20×/0.40 Ph1 objective (total magnification: 200×). Images were cropped for layout consistency.

Figure 5. LDH release and MTT assay-based cytotoxicity analysis across experimental groups. (A) Daily LDH release (%) measured over six days (DPI 1–6) to assess cytotoxicity. (B) MTT assay absorbance values on DPI-6 to evaluate cell viability. (C) Cumulative LDH-based cytotoxicity (%) across the experimental period. (D) MTT assay-based cell viability (%) on DPI-6. Statistically significant differences were observed between the 375× + ASFV group and the D 375× group (p < 0.01), as determined by a one-way ANOVA followed by post hoc analysis. Asterisks (**) indicate p < 0.01.

Figure 6. A systematic workflow to differentiate CPEs and cytotoxicity.