UV-C Lethal Doses for Sars-Cov-2

The rapid and continuous spread of SARS-CoV-2, responsible for COVID-19, has been challenging global health systems and many strategies have been proposed to face the COVID-19 pandemic crisis [1]. In this scenario, ultraviolet lamps emitting ultraviolet C (UV-C) germicidal radiation (peak emission at 254 nm) are in the spotlight to provide efficient and sustainable disinfection of air, liquids and surfaces (e.g., plastics, fabrics, metals, etc). However, UV light with wavelengths greater than 180 nm can cause health adverse effects as eye damage, skin cancer and ageing, and UV-C should be not used in inhabited environments. Herein, we established the inactivation kinetics and reported the UV-C lethal doses (LD) for SARS-CoV-2.

A twenty-four-well plate was seeded with 2×105/mL Vero cells (ATCC CCL-81) for a final volume of 500 μL/well. Cells were kept in the DMEM High Glucose (DMEN-HG) culture medium (Sigma-Aldrich, USA) supplemented with 10 % bovine fetal serum, 100 units/mL penicillin and 100 μg/mL streptomycin. Subsequently, the plate was incubated at 37 ◦C with 5% CO2 for 24 h, and then the culture medium was completely removed and replaced by 750 μL of DMEM-HG without supplementation [2,3]. An aliquot of the SARS-CoV-2 stock, previously characterized by Araujo et al. [4], was thawed and 100 μL were diluted in 900 μL of DMEM-HG without supplementation. Then, 200 μL of this dilution were placed in wells of a 24-well plate, which were exposed to the UV-C lamp (UV surface, Biolambda, Brazil) placed 30 cm above the plate to allow a uniform irradiance over the plate wells (2.2±0.2 mW/cm2). The light was delivered by 2, 30 and 120 s corresponding to doses of 4.4, 66 e 264 mJ/cm2, respectively. Controls were not submitted to irradiation.
After exposure to UV-C light, aliquots of 83.4 μL were placed into the plates containing the previously seeded Vero cells and incubated for 1 h at 37 ◦C with 5% CO2 for viral adsorption. Thereafter, 166.6 μL of DMEM-HG medium containing 12 % fetal bovine serum was added and the plate was incubated for 48 h at 37 ◦C with 5% CO2.

After that, 100 μL of medium from each well was removed and placed into a lysis buffer solution to proceed with the extraction of the viral RNA using the MagMAX™ CORE Nucleic Acid Purification Kit (Thermo Fisher). After extraction, the number of copies of SARS-CoV-2 per mL was obtained using the RT-qPCR technique. Results were normalized in relation to controls for the calculation of viral inhibition rates of each sample. For the viral inactivation kinetics, we used the methodology reported by Sabino et al. [5].

UV-C inactivation kinetics and lethal doses for SARS-CoV-2 are presented at Fig. 1 and Table 1, respectively. We verified that within less than a second, UV-C irradiation was able to inactivate more than 99 % of SARS-CoV-2 viral particles. In fact, LD90 and LD99.999 were achieved at 0.016 and 108.714 mJ/cm2 (0.01 and 49.42 s) respectively.

References

[1] N. Zhu, D. Zhang, W. Wang, X. Li, B. Yang, J. Song, et al., China novel coronavirus investigating and research team (2020). A novel coronavirus from patients with pneumonia in
China, N. Engl. J. Med. 382 (8) (2019) 727–733.
[2] Y. Shi, D.H. Yang, J. Xiong, J. Jia, B. Huang, Y.X. Jin, Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs, Cell Res. 15 (2005) 193–200.
[3] C. TaS¸tan, B. Yurtsever, G. Sir KarakuS¸, D. D˙Ilek KanÇa˘Gi, S. Dem˙Ir, S. Abanuz, et al., SARS-CoV-2 isolation and propagation from Turkish COVID-19 patients, Turk. J. Biol. 44
(2020) 192–202.
[4] D.B. Araujo, R.R.G. Machado, D.E. Amgarten, F.M. Malta, G.G. de Araujo, C.O. Monteiro, et al., SARS-CoV-2 Isolation From the First Reported Patients in Brazil and Establishment of
a Coordinated Task Network, Mem´orias do Instituto Oswaldo Cruz, 2020, https://doi.org/10.1590/0074-02760200342.

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