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. 2023 Dec 26;11(1):42-52.
doi: 10.1021/acsphotonics.3c00828. eCollection 2024 Jan 17.

Mechanisms of SARS-CoV-2 Inactivation Using UVC Laser Radiation

George Devitt  1   2   3 , Peter B Johnson  1   3 , Niall Hanrahan  1   3 , Simon I R Lane  1   3 , Magdalena C Vidale  2 , Bhavwanti Sheth  2 , Joel D Allen  2 , Maria V Humbert  4   5 , Cosma M Spalluto  4   6 , Rodolphe C Hervé  2 , Karl Staples  4   7   6 , Jonathan J West  3   8 , Robert Forster  9 , Nullin Divecha  2 , Christopher J McCormick  4 , Max Crispin  2 , Nils Hempler  9 , Graeme P A Malcolm  9 , Sumeet Mahajan  1   3   10
Affiliations

Mechanisms of SARS-CoV-2 Inactivation Using UVC Laser Radiation

George Devitt et al. ACS Photonics. .

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has had a tremendous impact on humanity. Prevention of transmission by disinfection of surfaces and aerosols through a chemical-free method is highly desirable. Ultraviolet C (UVC) light is uniquely positioned to achieve inactivation of pathogens. We report the inactivation of SARS-CoV-2 virus by UVC radiation and explore its mechanisms. A dose of 50 mJ/cm2 using a UVC laser at 266 nm achieved an inactivation efficiency of 99.89%, while infectious virions were undetectable at 75 mJ/cm2 indicating >99.99% inactivation. Infection by SARS-CoV-2 involves viral entry mediated by the spike glycoprotein (S), and viral reproduction, reliant on translation of its genome. We demonstrate that UVC radiation damages ribonucleic acid (RNA) and provide in-depth characterization of UVC-induced damage of the S protein. We find that UVC severely impacts SARS-CoV- 2 spike protein's ability to bind human angiotensin-converting enzyme 2 (hACE2) and this correlates with loss of native protein conformation and aromatic amino acid integrity. This report has important implications for the design and development of rapid and effective disinfection systems against the SARS-CoV-2 virus and other pathogens.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
SARS-CoV-2 virus surface inactivation by 266 nm UVC continuous wave laser. (A) Representative set of images from a plaque assay showing the dose (horizontal) and serial dilution (vertical). (B) Dose-dependence of SARS-CoV-2 virus to 266 nm UVC radiation, from two independent experiments performed in triplicate.
Figure 2
Figure 2
UVC dose dependence of ssRNA and rSARS-CoV-2 S protein integrity. (A) Schematic depicting the effect of UVC radiation on SARS-CoV-2 cellular entry and reproduction. (B) Dose- dependent RNA inactivation. Figure depicts RT-qPCR product off ∼2kb RNA template, and projected 30kb RNA template as a percentage of nonirradiated RNA control. n = 3, plotted data represents mean and SD from 6 cycle threshold readings. (C) UV absorption spectrum of rSARS-CoV-2 S. Arrows indicate UVC laser wavelengths used in this study. (D) Experimentally determined 227 and 266 nm UV dose requirements for 99.9% replication inhibition from the 2231 base MS2 amplicon, and theoretical model extrapolated using Poisson statistics for larger 13 588 and ∼30 kb SARS-CoV-2 genomes. (E) Dose-dependence of rSARS-CoV-2 S foldedness ratio to UVC radiation determined by UV–vis absorption spectroscopy. Foldedness ratio = A280/A275 nm + A280/A258 nm. (F) Dose-dependence of rSARS-CoV-2 S unfolding ratio to UVC radiation determined by UV–vis absorption spectroscopy. Unfolding ratio = A280/A230 nm. (G) Dose-dependence of SARS-CoV-2 S oxidation/aggregation ratio to UVC radiation determined by UV–vis absorption spectroscopy. Oxidation/aggregation ratio = A320/A280 nm. n = 2, the plotted data represent the average and SD from 10 absorbance readings for native rSARS-CoV-2 S and 3–4 absorbance readings per UVC condition.
Figure 3
Figure 3
Surface plasmon resonance (SPR) analysis of rSARS-CoV-2 trimeric spike glycoprotein shows the impacts of UVC treatment on hACE2 binding. (A) Sensogram depicting the binding of solubilized recombinant SARS-CoV-2 to serial dilutions of the hACE2 receptor with dilutions ranging from 200 nM to 3.125 nM. Data represent an average of three analytical repeats. RU = resonance units (proportional to the number of SARS-CoV-2 S protein molecules bound to the surface). (B) Comparison of the calculated Rmax (the maximum observed binding signal) values for each UVC treatment condition expressed as percentage of the nonirradiated protein. (C) Binding affinity, measured as equilibrium dissociation constant or KD values, for all of the SPR experiments performed. This parameter was calculated using a 1:1 binding model using Biacore Evaluation software. The response for the 2200 mJ dose for 227 and 266 nm was not sufficient to accurately determine KD. (D–I) SPR experiments were performed identically to panel A except an equal amount of S protein was treated with the dosage indicated.
Figure 4
Figure 4
UVC dose-dependent loss of rSARS-CoV-2 spike protein conformation. Readouts of rSARS-CoV-2 spike protein conformation induced by 227 nm (A–H) or 266 nm (I–P) radiation (A,I) Bis-ANS binding measured by fluorescence emission at 490 nm. n = 2, the plotted data represent the average and SD from 4 fluorescence readings. (B,J) Fluorescence emission ratio 330/425 nm representing tryptophan/NFK fluorescence. (C,K) Fluorescence emission spectra from 300 to 500 nm representing changes in intrinsic tryptophan fluorescence. n = 1, plotted data represents the average from 3 fluorescence readings. (D,L) Particle height analysis of atomic force microscopy (AFM) images. 3–4 images were used for each condition. n = 2, the plotted data represent the average and SD from 2 to 4 AFM images. (E–H, M–P) Representative tapping-mode AFM images of SARS-CoV-2 spike protein bound to a mica surface after irradiation with 0 mJ (E,M), 100 mJ (F,N), 600 mJ (G,O), 2200 mJ (H,P). Images are 5 × 5 μm2, scale bars represent 1 μm and z-scale is equal to 0–30 nm.
Figure 5
Figure 5
Mechanism of UVC-induced rSARS-CoV-2 S conformational damage. Raman spectra of rSARS-CoV-2 S protein following irradiation by 227 nm (A–C) or 266 nm (D–F) light. (A,D) Amide I second derivative spectra from 1645 to 1700 cm–1 (B,E) Disulfide region spectra from 462 to 558 cm–1. (C,F) Tryptophan indole ring spectra from 750 to 772 cm–1. (G–H) 1-Dimensional principle component analysis (PCA) scores plot of Raman spectra for 227 nm (G) and 266 nm (H) irradiated rSARS-CoV-2 S. Each solid diamond represents the PC score of a single spectrum. Hollow diamonds represent mean score. (I) PC loadings spectra representing the spectral variation responsible for the score across the given PC axis. (J) Schematic depicting conformational damage induced by 227 and 266 nm radiation determined by Raman spectroscopy. n = 2, plotted spectra represent the class means of 10–15 spectra per class.
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