Abstract
Variants of concern (VOCs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) exhibit high infectivity due to mutations, particularly in the spike protein, that facilitate enhanced binding of virus to human angiotensin-converting enzyme 2 (hACE2). The D614G mutation, situated in S1-domain, promotes the open conformation of spike protein, augmenting its interaction with hACE2. Activated water neutralizes pathogens by damaging biological molecules; however, its effect on mutated SARS-CoV-2 or VOCs requires further exploration. Here, the efficacy of nitric oxide (NO(x))-plasma activated water (PAW) in inhibiting infections by SARS-CoV-2 pseudovirus expressing D614G-mutated spike protein is investigated, which serves as a model for mutated SARS-CoV-2. Results demonstrated high prevalence of D614G mutation in SARS-CoV-2 and its VOCs. NO(x)-PAW is non-toxic to cells at high concentration, inhibiting infection by 71%. Moreover, NO(x)-PAW induced structural changes in S1-domain of spike protein, reducing its binding affinity and lowering clathrin-mediated endocytosis-related gene expression. Additionally, in silico analysis revealed NO(x) species in NO(x)-PAW played key role in impairing S1-domain function of the mutated SARS-CoV-2 pseudovirus by interacting directly with it. Collectively, these findings reveal the potent inactivation ability of PAW against mutated SARS-CoV-2 and suggest its potential application in combating emerging variants of SARS-CoV-2 and other viral threats.