# Photodynamic Inactivation of Human Coronaviruses
## 3 Results
### PhytoQuin Exhibits Light-Dependent Antiviral Activity against HCoV-229E and HCoV-OC43  
Having established the baseline phototoxic threshold of PhytoQuin in Huh7.5 and BHK-21 cells, we next measured the antiviral activity of PhytoQuin against HCoV-229E and HCoV-OC43. HCoV-229E inocula were mixed with the indicated concentrations of DMSO or PhytoQuin and exposed to light at 49 J/cm\({}^{2}\), or kept in the dark, before being serially diluted for infecting Huh7.5 cells. The remaining viral infectivity after each treatment was determined using median tissue culture infectious dose (TCID\({}_{50}\)) assays. By exposing the HCoV-229E viral inoculum to DMSO and light, we observed that HCoV-229E is mildly photosensitive to 49 J/cm\({}^{2}\) LED exposure, displaying \(<\)3-fold decrease in viral titers compared to DMSO/Dark treated virus (Figure 2A). PhytoQuin incubated with HCoV-229E in the dark had little effect (\(<\)3-fold decrease relative to DMSO/Dark) across the full range of concentrations tested (Figure 2A). By contrast, a clear, dose-dependent reduction in viral titers was observed between 0.5 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 4 \(\mathrm{\SIUnitSymbolMicro g}\)/mL doses of photoactivated PhytoQuin; with all samples at the 4 \(\mathrm{\SIUnitSymbolMicro g}\)/mL dose reducing viral titers to the limit of detection (LOD, 1500 TCID\({}_{50}\)/mL) of the assay (Figure 2A). These data demonstrate that HCoV-229E is susceptible to PDI, with \(\sim\)1000-fold reductions in viral titers after a brief light treatment. Figure 1: Cell viability following DMSO and PhytoQuin treatment of Huh7.5 and BHK-21 cells. Huh7.5 cells (**A**) or BHK-21 cells (**B**) in low-serum infection media were exposed to increasing concentrations of DMSO (solvent, left panels) or PhytoQuin (right panels) with (49 J/cm\({}^{2}\), blue points; 98 J/cm\({}^{2}\), light blue points) or without (black points) LED light treatment. Cell viability was assayed at 48 h post-treatment using alamarBlue cell viability reagent. The vertical dotted lines indicate the highest concentrations of DMSO and PhytoQuin used in subsequent experiments. Data are plotted as averages \(\pm\) SEM from three independent experiments. The 50% cytotoxic concentration (CC\({}_{50}\)) values were calculated using Prism v9.2.0. Abbreviations: DMSO, dimethyl sulfoxide; LED, light-emitting diode; SEM, standard error of the mean. To determine whether the antiviral effects of PhytoQuin PDI were specific for the alphacoronavirus HCoV-229E or generalizable across diverse HCoVs, we next tested its activity on the distantly related betacoronavirus, HCoV-OC43. The viral inocula were exposed to sub-cytotoxic doses of 0.5-4 \(\mu\)g/mL PhytoQuin, either with or without 49 J/cm\({}^{2}\) LED light exposure, and then used to infect BHK-21 cells for TCID\({}_{50}\) assays. Compared to HCoV-229E, similar amounts of HCoV-OC43 were inactivated by PhytoQuin in a dose-dependent and light-dependent manner (Figure 2B). PhytoQuin (4 \(\mu\)g/mL) and light treatment led to an approximately 4 log\({}_{10}\) reduction in viral titer relative to DMSO-treated HCoV-OC43. Together, these data show that various HCoVs are highly susceptible to PhytoQuin PDI. Figure 2: PhytoQuin potently inactivates human coronaviruses 229E and OC43. (**A**) HCoV-229E was combined with 0.5% DMSO or PhytoQuin (0.5, 1, 2, or 4 \(\mu\)g/mL) prior to exposure to LED light in black tubes (“Dark”, black dots/lines) or clear tubes (“Light”, blue dots/lines) at 49 J/cm\({}^{2}\). The treated inocula were serially diluted and used to infect Huh7.5 cells and ittered using TCID\({}_{50}\) assays. (**B**) HCoV-OC43 was combined with 0.5% DMSO or PhytoQuin (0.5, 1, 2, or 4 \(\mu\)g/mL) prior to exposure to light as indicated for panel A. The treated inocula were serially diluted and used to infect BHK-21 cells and ittered using TCID\({}_{50}\) assays.