Photodynamic Inactivation of Bovine Coronavirus with the Photosensitizer Toluidine Blue O

Abstract

Coronaviruses (CoVs) belong to the group of enveloped positive-sense single-strand RNA viruses and are causative agents of respiratory, gastro-intestinal, and central nervous systems diseases in many host species, i.e., birds, mammals, and humans. Beta-CoVs revealed a great potential to cross the barrier between species by causing three epidemics/pandemics among humans in the 21st century. Considering the urgent need for powerful antiviral agents for decontamination, prevention, and treatment of BCoV infections, we turned our attention to the possibility of photodynamic inactivation with photosensitizers in combination with light irradiation. In the present study, we evaluated, for the first time, the antiviral activity of toluidine blue O (TBO) against Beta-coronavirus 1 (BCoV) in comparison to methylene blue (MB). First, we determined the in vitro cytotoxicity of MB and TBO on the Madin-Darby bovine kidney (MDBK) cell line with ISO10993-5/Annex C. Thereafter, BCoV was propagated in MDBK cells, and the virus titer was measured with digital droplet PCR, TCID₅₀ assay and plaque assay. The antiviral activity of non-toxic concentrations of TBO was estimated using the direct inactivation approach. All effects were calculated in MAPLE 15^® mathematical software by developing programs for non-linear modeling and response surface analysis. The median inhibitory concentration (IC₅₀) of TBO after 72 h of incubation in MDBK cells was 0.85 µM. The antiviral activity of TBO after the direct inactivation of BCoV (MOI = 1) was significantly stronger than that of MB. The median effective concentration (EC₅₀) of TBO was 0.005 µM. The cytopathic effect decreased in a concentration-dependent manner, from 0.0025 to 0.01 µM, and disappeared fully at concentrations between 0.02 and 0.3 µM of TBO. The number of virus particles also decreased, depending on the concentration applied, as proven by ddPCR analysis. In conclusion, TBO exhibits significant potential for direct inactivation of BCoV in vitro, with a very high selectivity index, and should be subjected to further investigation, aiming at its application in veterinary and/or human medical practice.

Keywords: antiviral activity; bovine coronavirus; light irradiation; photosensitizers; toluidine blue O.

Figure 1

Specific growth rate of MDBK cells at different initial seeding concentrations. Legend: X = absorbance of the cells in culture.

Figure 2

In vitro cytotoxicity of methylene blue and toluidine blue on MDBK cells after 24, 48 and 72 h exposure. Legend: Fa—antiviral activity (effect); MB—methylene blue; TBO—toluidine blue O; (a) Model and experimental data of Fa vs. concentration of MB after 24 h of exposure; (b) Model and experimental data of Fa vs. concentration of TBO after 24 h of exposure; (c) Model and experimental data of Fa vs. concentration of MB after 48 h of exposure; (d) Model and experimental data of Fa vs. concentration of TBO after 48 h of exposure; (e) Model and experimental data of Fa vs. concentration of MB after 72 h of exposure; (f) Model and experimental data of Fa vs. concentration of TBO after 72 h of exposure.

Figure 2

In vitro cytotoxicity of methylene blue and toluidine blue on MDBK cells after 24, 48 and 72 h exposure. Legend: Fa—antiviral activity (effect); MB—methylene blue; TBO—toluidine blue O; (a) Model and experimental data of Fa vs. concentration of MB after 24 h of exposure; (b) Model and experimental data of Fa vs. concentration of TBO after 24 h of exposure; (c) Model and experimental data of Fa vs. concentration of MB after 48 h of exposure; (d) Model and experimental data of Fa vs. concentration of TBO after 48 h of exposure; (e) Model and experimental data of Fa vs. concentration of MB after 72 h of exposure; (f) Model and experimental data of Fa vs. concentration of TBO after 72 h of exposure.

Figure 3

Histogram and total events from a ddPCR enumeration of the viral particles in the bovine coronavirus stock used for evaluation of PS antiviral activity. Legend: C01, C02 = dilution 10⁻³; D01, D02 = dilution 10⁻⁴; E01, E02 = dilution 10⁻⁵; F01, F02 = dilution 10⁻⁶.

Figure 4

Micrographs from the plaque assay for determination of the virus titer, 200× magnification.

Figure 5

Microscopic observation of the cytopathic effect of BCoV on MDBK cells after direct inactivation with MB + irradiation—100× magnification.

Figure 6

Microscopic observation of the cytopathic effect of BCoV on MDBK cells after direct inactivation with TBO + irradiation—100× magnification.

Figure 7

Evaluation of the viability of MDBK cells after exposure to MB or TBO in concentrations below the MNC: (a) comparison between the effects of MB with and without irradiation; (b) comparison between the effects of TBO with and without irradiation. Legend: MB—methylene blue; TBO—toluidine blue O; BCoV—bovine coronavirus; Co—untreated control.

Figure 8

Antiviral activity of MB and TBO in MDBK cells after direct inactivation of BCoV with irradiation—non-linear curves for calculation of the median effective concentrations. Legend: MB—methylene blue; TBO—toluidine blue O; EC₅₀—effective concentration 50%; m—hillslope; (a) Model and experimental data of antiviral activity (Fa) vs. applied concentration of MB; (b) Response surface analysis (RSA) of the antiviral activity of MB as a function of “Dose” (=concentration) and EC₅₀, m = const.; (c) Response surface analysis (RSA) of the antiviral activity of MB as a function of “Dose” (=concentration) and m, EC₅₀ = const.; (d) Model and experimental data of antiviral activity (Fa) vs. applied concentration of TBO; (e) Response surface analysis (RSA) of the antiviral activity of TBO as a function of “Dose” (=concentration) and EC₅₀, m = const.; (f) Response surface analysis (RSA) of the antiviral activity of TBO as a function of “Dose” (=concentration) and m, EC₅₀ = const.

Figure 9

Histograms from a ddPCR assay for enumeration of the viral particles in three TBO-treated samples and the virus control—dilution of the cDNA. Legend: (a) BCoV (virus control)—E02, E03 = dilution 10⁻⁴; F02, F03 = dilution 10⁻⁵; G02, G03 = dilution 10⁻⁶; H02, H03 = non template control (PCR water); (b) TBO 0.005 µM—E04, E05 = dilution 10⁻³; F04, F05 = dilution 10⁻⁴; G04, G05 = dilution 10⁻⁵; H04, H05 = negative control from the RNA isolation; (c) TBO 0.02 µM –E06, E07 = dilution 10⁻²; F06, F07 = dilution 10⁻³; G06, G07 = dilution 10⁻⁴; H06, H07 = control with untreated cells without BCoV; (d) TBO 0.15 µM—C08, C09 = no dilution; D08, D09 = dilution 10⁻¹; E08, E09 = dilution 10⁻²; (e) TBO 0.3 µM—C04, C05 no dilution; D04, D05 = dilution 10⁻¹; E04, E05 = dilution 10⁻².