Efficient disinfection of SARS-CoV-2-like coronavirus, pseudotyped SARS-CoV-2 and other coronaviruses using cold plasma induces spike protein damage

Abstract

Coronavirus disease 2019 (COVID-19) has become a worldwide public health emergency, and the high transmission of SARS-CoV-2 variants has raised serious concerns. Efficient disinfection methods are crucial for the prevention of viral transmission. Herein, pulse power-driven cold atmospheric plasma (CAP), a novel sterilization strategy, was found to potently inactivate SARS-CoV-2-like coronavirus GX_P2V, six strains of major epidemic SARS-CoV-2 variants and even swine coronavirus PEDV and SADS-CoV within 300 s (with inhibition rate more than 99%). We identified four dominant short-lived reactive species, ONOO^-, ¹O₂, O₂^- and·OH, generated in response to CAP and distinguished their roles in the inactivation of GX_P2V and SARS-CoV-2 spike protein receptor binding domain (RBD), which is responsible for recognition and binding to human angiotensin-converting enzyme 2 (hACE2). Our study provides detailed evidence of a novel surface disinfection strategy for SARS-CoV-2 and other coronaviruses.

Keywords: Cold pressure plasma; Coronavirus; Peroxynitrite anion; Pseudotyped variants; SARS-CoV-2; Short-lived reactive species.

Graphical abstract

Fig. 1

Schematic diagrams. (A) Schematic diagram of virus inactivation. (B) Schematic diagram of the plasma jet device for virus treatment.

Fig. 2

Effects of plasma on the elimination of SARS-CoV-2-like coronavirus GX_P2V. (A) Inhibition (%) of GX_P2V at various volumes by Ar-fed CAP. The calculation of inhibition was based on the titer of the corresponding volume of GX_P2V with Ar treatment. (B) Inhibition (%) of GX_P2V at various Ar-fed CAP exposure durations. (C) Plaques produced by GX_P2V after CAP treatment for 180 s (D) Plaques produced by GX_P2V after CAP treatment for 10 s (E) Plaques produced by GX_P2V without CAP treated.

Fig. 3

Effect of plasma on SARS-CoV-2 wild type and variants. Pseudotyped SARS-CoV-2 wild type and five strains of variants, B.1.351 (Beta), P.1 (Gamma), B.1.526 (Lota), B.1.1.7 (Alpha), B.1.617.2 (Delta) and C.37 (lambda), were subjected to the plasma jet.

Fig. 4

Disinfection effect of Ar-fed CAP on coronavirus SADS-CoV and PEDV. (A, C) Inhibition (%) of SADS-CoV and PEDV at various volumes by Ar-fed CAP. (B, D) Inhibition (%) of SADS-CoV and PEDV at various Ar-fed CAP exposure durations.

Fig. 5

GX_P2V images captured by TEM. (A) Transmission electron microscopy (TEM) images of GX_P2V after CAP exposure for 3 min (B) TEM images of GX_P2V not treated with CAP. The bars in A1, A2, B1 and B2 represent 200 nm.The bars in A3 and B3 represent 50 nm.

Fig. 6

CAP effectively inhibit GX_P2V adsorption and entry into cells and reduce the binding ability of SARS-CoV-2 RBD to hACE2, rather than degrading viral RNA. (A) The viral inactivation ability of CAP was analyzed by viral attachment assay. The virus with Ar-fed CAP treated at 60 s and 180 s was added to Vero E6 cells for attachment at 4 ℃ and for entrey at 37 ℃. The same volume of GX_P2V with only Ar treatment was used as a control. ***p < 0.001,****p < 0.0001. (B) Binding activities of the SARS-CoV-2 RBD treated with Ar-fed CAP for different durations were analyzed by ELISA. (C) SDS-PAGE analysis of RBDs with CAP-treated and untreated. (D) Viral copy number was detected by RT-qPCR. Specific primers for the S protein were employed for cDNA amplification in RT-qPCR analysis.

Fig. 7

Detection of active substances produced by plasma. (A) ¹O₂ content produced by plasma jet in different duration of exposure. (B) O₂^- content produced by plasma jet in different duration of exposure. (C) Trend of·OH content produced by plasma jet in different duration of exposure. (D) Trend of ONOO^- content produced by CAP plasma jet in different duration of exposure.

Fig. 8

Functional examination of short-lived reactive species by adding quenchers to the GX_P2V and SARS-CoV-2 RBDs. (A) The titers of GX_P2V premixed with quencher after 180 s of treatment with CAP were measured using TCID50 assay. * *p < 0.01. (B) Analysis of binding between RBD premixed with quencher and hACE2 after 180 s of CAP treatment by ELISA. *p < 0.05, **p < 0.01.

Fig. 9

Mechanism of RONS generated in Ar-fed CAP damaging S protein. Reactive species generated by plasma jet disslove into liquid and cross-react in liquid phase. Predominant RONS ONOO^- and O₂^- caused oxidation to tyrosine, tryptophan and histidine that located at RBD and NTD, which may impair the binding ability of RBD to cell receptor ACE2 and the function of NTD. The viral genome remained intact after 3 min-CAP treatment.