Photochemical Inactivation of Porcine Epidemic Diarrhea Virus Using Methylene Blue: A Study on Photo‐Oxidative Antiviral Chemistry

ABSTRACT Porcine epidemic diarrhea virus (PEDV), a highly contagious Alphacoronavirus , continues to cause severe economic losses in global swine production due to its high virulence and limited vaccine protection. In this study, a methylene blue (MB)‐based photodynamic therapy (PDT) was investigated as a chemical–photonic antiviral strategy against the pathogenic PED‐3 strain. MB, a red‐absorbing phenothiazinium dye with a high singlet oxygen ( 1 O 2 ) quantum yield, was photoactivated using a low laser dose (1.28 J/cm 2 ) to induce reactive oxygen species (ROS). Under these mild photochemical conditions, PEDV was completely inactivated in suspension, while infection assays in Vero cells demonstrated that 2–20 µM MB‐PDT reduced viral RNA by 3.69–6.02 log 10 and infectious titers by 1.77–4.69 log 10 TCID 50 at low viral load (multiplicity of infection [MOI] = 0.001). At a higher MOI (0.01), 20 µM MB‐PDT delayed cytopathic effects by 24 h, reducing RNA and viral titers by 3.07 and 2.46 log 10 , respectively. A significant elevation of intracellular ROS confirmed efficient photochemical activation of MB. These findings provide the first evidence that MB‐mediated PDT can chemically and photophysically inactivate PEDV under low‐energy irradiation, supporting its potential as a safe, cost‐effective, and environmentally compatible antiviral approach for veterinary applications.