Analysis of the SARS-CoV-2 inactivation mechanism using violet-blue light (405 nm)

Abstract

The study evaluated the effects of violet-blue light (VBL) on cell viability and replication, carbonylation of three structural proteins (S, E, and N) and one non-structural protein (NSP13), and direct damage to the RNA of SARS-CoV-2. The virus was exposed to increasing doses of VBL along with influenza A and B viruses to compare their susceptibility. At the highest dose (21.6 J/cm²), SARS-CoV-2 was significantly more susceptible to VBL than the influenza viruses, with a reduction in viral titer of 2.33 log₁₀. Viral RNA did not show significant changes after exposure to VBL, as demonstrated by next-generation sequencing and real-time PCR quantification, suggesting that the inactivation process does not involve direct nucleic acid damage. To exclude the role of the culture suspension in the inactivation process, virus viability experiments were performed using different dilutions of Dulbecco's modified Eagle's medium (DMEM) in phosphate-buffered saline (PBS). The results indicated that the suspension medium played a secondary role in virus inactivation, as viability did not increase with increasing DMEM dilution. Subsequent tests with three different antioxidants (NAC, AsA, and SOD) at different concentrations prevented viral inactivation, from 99.99% to 85.43% (with SOD 0.003 mM). Carbonylation of S and E proteins was more pronounced when viruses were suspended in DMEM rather than PBS, although the tests demonstrated that the intrinsic properties of the viral membrane were a crucial element to consider in relation to its susceptibility to VBL.IMPORTANCELight-based disinfection methods are often used in combination with other cleaning methods due to their non-invasive nature, versatility, and environmental benefits. VBL is an effective approach as it induces the production of reactive oxygen species that reduce microbial viability. In this study, lipid peroxidation was identified as an important factor affecting the structural integrity and function of the viral envelope, reducing its ability to interact with host cells and consequently its ability to be infectious. The lipid envelope of SARS-CoV-2, composed mainly of glycerophospholipids and lacking cholesterol and sphingolipids, appears to be the critical factor in its susceptibility, distinguishing it from influenza viruses, which have a lipid profile richer in components that protect against oxidative stress.

Keywords: SARS-CoV-2 envelope; lipid peroxidation; protein carbonylation; reactive oxygen species; violet-blue light.

Fig 1

Effects of VBL exposure on the viral replication and protein expression of SARS-CoV-2. (A) VERO E6 cells stained with DAPI. (B) Expression of dsRNA. (C) Expression of viral S protein. (D) Expression of viral N protein. Cells were counted at 40× magnification in five random fields/slide for two replicates. Number of cells was expressed as total number; positive cells were expressed as percentage (%). Comparisons were performed by two-way ANOVA (*P < 0.05; **P < 0.01; ****P < 0.0001). CC, cell control; CV, virus control; VBL, VBL-exposed.

Fig 2

Intracellular localization of SARS-CoV-2 dsRNA and proteins. VERO E6 cells were infected with SARS-CoV-2 (CV) and SARS-CoV-2 after 90′ VBL exposure. Cells were stained at different time points (4, 8, and 12 h post-infection, P.I.) with NP-, S-, and dsRNA-antibody and examined by confocal microscopy.

Fig 3

Co-localization of SARS-CoV-2 S and N proteins. VERO E6 cells were infected with SARS-CoV-2 (CV) and SARS-CoV-2 after 90′ VBL exposure. Cells were stained at different time points (4, 8, and 12 h post-infection, P.I.) with NP- and S-antibody and examined by confocal microscopy. Merged signals (upper panels of each condition) and co-localized points (lower panels of each condition) are shown. Co-localized points appear white.

Fig 4

Viral load reduction of SARS-CoV-2 in different dilutions of culture medium exposed to VBL for 90′. The viral SN was diluted in PBS at different concentrations to reduce the presence of DMEM culture medium during VBL exposure. The results obtained showed a TCID₅₀/mL logarithmic reduction of the exposed samples with respect to CVs of 4.719 log₁₀ (for the SN dilution factor of 1:3), 4.543 log₁₀ (for the SN dilution factor of 1:20), and 2.620 log₁₀ (for the SN dilution factor of 1:1,000); percentage reductions achieved were 99.998%, 99.997%, and 99.76%, respectively. The dilution of the medium certainly affected virus survival, suggesting that its presence plays a secondary but relevant role in VBL-mediated viral inactivation. Results were expressed as the logarithm of TCID₅₀/mL and relative 95% confidence interval (CI) obtained using the improved Kärber.

Fig 5

Evaluation of antioxidant protection against VBL-induced inactivation of SARS-CoV-2 in different dilutions of culture medium. The viability of SARS-CoV-2, as measured by log₁₀ TCID₅₀/mL, is depicted in this figure after a 90-min exposure to VBL. The virus was incubated in DMEM diluted 1:3, 1:20, or 1:1,000 in PBS during irradiation, and various antioxidants were added at two concentrations. The goal was to ascertain whether these antioxidants could shield the virus from the oxidative stress caused by VBL. In the panels, the results are presented for (A) NAC at 5 mM (blue patterned bars) and 0.5 mM (white patterned bars), (B) AsA at 5 and 0.5 mM, and (C) SOD at 0.03 and 0.003 mM. The whiskers of the bars represent the 95% confidence interval. The titers of the non-exposed control virus (CV) are represented by red diamonds. The CV was incubated under identical conditions (medium dilution, antioxidant type, concentration, and time) without VBL exposure, and they served as a baseline for maximum viability. The titers of the CV range from 8 to 10 log₁₀ TCID₅₀/mL, according to the specific condition.

Fig 6

Protein carbonylation assay results. Detection of carbonylated proteins derivatized with DNPH and analyzed by Western blot in the presence (T) or absence (NT) of VBL exposure. (A) Protein N exposed to VBL showed no visible signs of carbonylation compared to unexposed controls and different incubation conditions with different dilutions of the culture medium. In general, slight protein oxidation seems to be visible in all samples, probably due to the experimental conditions under which the tests were carried out. (B) Protein E showed signs of carbonylation in samples exposed to VBL and incubated with DMEM medium dilutions of 1:3 and 1:20. In this case, the presence of higher medium concentrations seems to have affected the oxidative state of the protein. Again, a slight presence of protein oxidation is visible in all samples, probably due to the experimental conditions of the assay. (C) Protein S showed significant carbonylation in the 1:3 dilution of medium exposed to VBL, although a slight level of carbonylation is also present in the sample incubated with the 1:20 dilution. Again, it appears that the concentration of medium increases the degree of protein carbonylation in the presence of VBL. (D) Protein NSP13 showed signs of extensive carbonylation regardless of the variables examined. Again, the results could be due to a combination of factors related to the intrinsic properties of the protein and the experimental conditions under which the tests were carried out. Nevertheless, no differences were found between the samples exposed and those not exposed to VBL.

Fig 7

3D representation of the light source and VBL light distribution. (A) Light irradiance simulation on the plane realised with Ansys Speos software. The radio-photometric simulation was carried out considering the power of each light source (the nine LEDs at 405 nm), the distance of the light sources from the plane, and empirical measurements taken at various points on the plane using the Avantes ULS2048CL EVO spectrophotometer. As expected, the highest irradiance (section in green) was measured on the perpendicular plane section below the source (4.3 mW/cm²), while an average irradiance decrease of 83% (0.7 mW/cm²) was recorded on the lateral plane sections (section in blue). (B) Irradiance on the multi-well plate. The marked area on the plane (in green) represents the point, where the multi-well plate is positioned during the virus and protein exposure experiments. Before starting the experiments, the temperature at the point of maximum light exposure was measured to exclude it as a possible risk factor. The measured temperature was not different from room temperature (23°C, 50% relative humidity). (C) Detail of the light distribution on the plate.