# Photodynamic Inactivation of Human Coronaviruses
## 4 Discussion
The precise covalent modifications of S following exposure to photoactivated PhytoQuin remain unknown, but ongoing experiments will investigate whether PDI increases protein carbonylation, a hallmark of protein oxidative damage, using 2,4-dinitrophenylhydrazine-based assays. Multiple mechanisms exist to mitigate ROS-induced changes in membrane integrity [48], which could alleviate any negative impacts of brief PhytoQuin treatments on living cells. Conversely, viral particles cannot repair oxidative damage. The most striking impact of PhytoQuin treatment on HCoV particles was the loss of vRNA after light treatment (Figure 4B), which is likely a direct result of ROS damage to the viral lipid envelope, which would expose the vRNP complexes to damaging RNases. While oxidation is known to directly damage nucleic acids [16], our current experiments did not measure whether the ROS produced during PhytoQuin-mediated PDI result in direct damage to nucleic acids. However, disruptive effects on cellular and liposomal membranes due to light-activated PSs have been previously documented [49; 50; 51; 52]. In our previous work, we did not observe any effect of PhytoQuin on herpesvirusus DNA when analyzing viral DNA copy number by qPCR following sequential PhytoQuin and deoxyribonuclease treatment [20]. This may be due to the presence of a more robust icosahedral capsid surrounding the DNA genome of herpesvirusures compared to the more flexible, N-associated vRNPs of HCoVs. However, PhytoQuin likely also damages herpesvirusus envelopes as earlier work demonstrated the ability of anthraquinones to disrupt HSV-1 virions [53]. Evidence of physical damage to viral particles due to PhytoQuin treatment was further supported by the appearance of high molecular weight forms of SARS-CoV-2 S on purified, pseudotyped lentiviruses following light exposure (Figure 5). Taken together, our data provide strong evidence for a light-dependent mechanism of action that involves damage to the virion exterior, to lipids and proteins alike, resulting in the inhibition of viral infection. While seemingly complex, PhytoQuin is primarily composed of emodin and resveratrol and their naturally occurring derivatives (Figure 6A). Polydatin is the precursor of resveratrol, and anthraglycoside B or physcon are glucopyranoside or methyl ether derivatives of emodin, respectively. Emodin and resveratrol are known antiviral polyphenic compounds which inhibit the replication of a number of respiratory viruses, includinginfluenza A virus [54; 55], rhinvirus [56], and respiratory syncytial virus [57; 58; 59], as well as several HCoVs including MERS-CoV, HCoV-229E and SARS-CoV-2 [60; 61; 62; 63; 64]. Despite this, there is no clear consensus on antiviral mechanism of action of resveratrol or emodin. Resveratrol and aloe-emodin, an isomer of emodin, were recently shown to be antimicrobial PSs [65; 66; 67]. These studies used blue LED photoactivation with comparable fluence to the white light LED source used herein; however, the concentrations for antimicrobial activity of photoactivated emodin (\(\geq\)27 \(\mu\)g/mL) or resveratrol (2 mg/mL) were significantly higher than those in 4 \(\mu\)g/mL of PhytoQuin. Our current study provides strong evidence that emodin is a photoactivatable antiviral that can be added to a growing list of antiviral PSs [68]. The near-complete inactivation of HCoV-OC43 by emodin (Figure 6B) suggests that, while a predominant factor, other constituents of the extract such as resveratrol may have minor contributions to PhytoQuin PDI. Interestingly, our previous work demonstrated similar antiviral effects against herpesvirusviruses and adenoviruses by PhytoQuin [20], but at doses four to ten-fold lower than those used herein for HCoVs. The HPLC trace of the original lot of PhytoQuin demonstrated a significantly higher concentration of emodin compared to the lot employed for our current CoV experiments. It is not yet known if emodin is also the primary bioactive component of PhytoQuin for neutralizing herpesviruses, adenoviruses, rhabdomy, or bacteria.