# Photodynamic Inactivation of Human Coronaviruses
## 1 Introduction  
Coronaviruses (CoVs) are enveloped viruses with large positive-sense, single-stranded RNA genomes. Human CoVs occupy two genera, the alphacoronaviruses (HCoV-NL63 and HCoV-229E) and betacoronaviruses (HCoV-OC43, HCoV-HKU1, Middle East respiratory syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome-related coronavirus (SARS-CoV and SARS-CoV-2). CoV Spike (S) proteins enable attachment to diverse cell surface receptors and entry via receptor-mediated endocytosis [1]. Following uncoating in the cytoplasm, the CoV (+)ssRNA genome is translated into long polyproteins that are processed by viral proteases to yield non-structural proteins, including the viral RNA-dependent RNA polymerase (RdRp) and associated RNA modifying enzymes [2], as well as an RNA exonuclease enzyme that provides essential proofreading activity to ensure high-fidelity replication of the viral genome [3]. These viral enzymes assemble to form the replicase-transcriptase complex that orchestrates viral transcription and genome replication in discrete cytoplasmic compartments [4]. The RdRp uses the template (+)ssRNA genometo create full-length (\(-\))ssRNA intermediates that can be copied into full-length genomic (+)ssRNA, as well as subgenomic (\(-\))ssRNAs that can be copied into mRNAs that encode structural proteins: S, membrane, envelope, and nucleocapsid (N), as well as lineage-specific accessory proteins that aid immune evasion [5]. Newly synthesized viral genomes are coated with N proteins and assemble with the remaining structural proteins by budding into the ER-Golgi Intermediate Compartment (ERGIC). According to the longstanding model, progeny CoV virions escape the host cell by traversing the secretory pathway, but recent studies of SARS-CoV-2 and the related betacoronavirus mouse hepatitis virus have provided evidence for an alternative lysosomal egress pathway [6]. The emergence of SARS-CoV and MERS-CoV from animal reservoirs, followed by the current SARS-CoV-2 pandemic, has spurred efforts to develop safe and effective antivirals for coronavirus. Moreover, there is a growing recognition that pandemic preparedness requires the identification and development of broadly acting antivirals that can target outbreak-prone families of viruses [7]. Broadly acting antivirals must target highly conserved viral enzymes or structural components of these families of viruses and drug candidates must be tested against representative strains across the phylogenetic tree. One of the earliest examples of a broadly acting antiviral mechanism was photodynamic inactivation (PDI), a term first used 90 years ago by [PERSON] to describe the antiviral effects of methylene blue dye against bacteriophage [8]. [PERSON] borrowed the term \"photodynamic\" after observing that methylene blue only displayed antiviral activity in the presence of oxygen and light. [PERSON] and [PERSON] confirmed [PERSON]'s findings and extended them to include PDI of herpes simplex virus and vaccinia virus [9; 10]. Since these seminal discoveries, many additional photosensitizing molecules have been identified for use in PDI of microbes, including many natural products [11]. PDI mechanisms are now more fully understood, whereby exposure of a photosensitizer (PS) to visible light and oxygen results in the generation of singlet oxygen and other reactive oxygen species (ROS) [12; 13; 14]. The photosensitization process, coupling PS activation and subsequent oxidative reactions, occurs via two routes: type I and type II reactions. In type I reactions, the excited PS participates in electron transfer reactions involving oxygen, which result in the formation of superoxide and hydroxyl radicals. Conversely, type II reactions result in the formation of singlet oxygen from energy transfer between the excited PS and molecular oxygen.