Viral inactivation of murine coronavirus via multiple gas plasma-derived reactive species

The recent pandemic has highlighted the urgent need to elucidate the pathophysiological mechanisms underlying viral effects in humans and is driving the search for innovative antiviral therapies. Several studies have investigated the ability of gas plasma, a partially ionized gas that simultaneously generates several reactive species, to be a new antiviral tool. However, several aspects of the mechanisms of antiviral action of gas plasma remained elusive. In this study, we, for the first time, used a gas plasma device approved for medical purposes and routinely applied in the clinics, especially for wound healing, to test its antiviral activity against a murine corona-virus in vitro (MHV-GFP), a research model analogous to human coronaviruses such as SARS-CoV-2. For this, we established a novel high-content imaging assay that gave quantitative and kinetic information about infection and reduced viral activity in murine fibroblasts (17Cl-1) host cells. Gas plasma treatment delayed viral infectivity and reduced overall infection and toxicity in 17Cl1 cells. Various antioxidants at different concentrations were screened to identify ROS relevant to antiviral effects. Catalase provided no virus protection, and DMSO, mannitol, histidine, Trolox, and ascorbic acid only modestly reduced gas plasma virucidal efficacy. By contrast, glutathione, tyrosine, and cysteine showed profound but not complete protection of MHV from gas plasma-derived reactive species, suggesting pivotal roles of superoxide radicals and peroxynitrite gas in plasma-driven viral inactivation. At extended gas plasma exposure times, fewer intact MHV RNA were detected, indicative of reactive species-driven RNA modifications or degradation as an additional mechanism of action. Virus particle size changes measured by electron microscopy were moderate. Collectively, we identified the potent antiviral activity of a clinically approved argon plasma jet along with potential mechanisms of action.