A test apparatus was designed, optimized, fabricated and calibrated to enable accurate and controlled UV-C treatment of test samples. A collimated beam setup was fabricated based on a dual chamber construction. The top chamber contains the UV-C light source, the electronic driver, and a shutter system to control the exposure times of the samples while keeping the lamp output stable. Samples were treated in the bottom chamber using deep UV-C light generated with a classical Mercury type TUV PLL 35W light source, generating a peak wavelength at 254 nm. Multiple sensor-based safety measures were applied to protect the user against incidental exposure to the UV-C radiation. The irradiance level for three different lamps inside the treatment chamber was measured using a calibrated UV-C sensor system (Spectroradiometer GL Optic Spectis 5.0 Touch with detector GL Opti Probe 5.1.50), which provided irradiance patterns and levels from which optimal treatment locations could be deduced.
Virus inactivation procedures
All experiments were performed in the biosafety level 4 laboratory of the National Emerging Infectious Diseases Laboratories of Boston University. A volume of 100 µl SARS-CoV-2 (7.33 x 103 PFU/ml) (USA/WA1-2020)11 was plated onto the surface of 60 mm plastic tissue culture dishes (TPP) in 5 µl aliquots. The virus was allowed to dry for approximately 2 hours on a subset of the dishes while the rest were processed immediately in the prototype UV-C device. Briefly, a pair of dishes (one to be treated and one control wrapped tightly in aluminum foil) were placed in the center of the device at an irradiance level of 0.849 mW/cm2, and towards the side of the UV-C device, respectively. The dishes were UV-C-treated for either 0.8, 2, 3, 4, 5, 6, 9, 15, 30 or 120 seconds, with each treatment time tested in triplicate. Dishes containing dried virus were treated in the same manner. Following treatment, the wet and dried virus were resuspended in 1.9 ml or 2 ml, respectively, of high glucose Dulbecco's Modified Eagle Medium (DMEM)(Gibco) containing 0.04 mM phenol red, 1 x antibiotic-antimycotic (Gibco), 1 x non-essential amino acids (Gibco), 1 x GlutaMAX-I (Gibco), 1 mM sodium pyruvate (Gibco) and 2% fetal bovine serum (FBS)(Gibco). The resuspended virus was then serially diluted from 1 x 100 to 1 x 10-2.5 using half-logarithmic dilutions for the crystal violet plaque assay, or from 1 x 100 to 1 x 10-5 using 10-fold dilutions for the anti-SARS-CoV-2 antibody plaque assay. A back-titration of the virus was included for each experiment.
Confirmation of virus inactivation by plaque assay
a) Plaque identification using crystal violet
Vero E6 cells maintained in high glucose DMEM (Gibco) supplemented with 1 x GlutaMAX-I, 1 mM sodium pyruvate, 10% FBS (Gibco) and 1 x non-essential amino acids (Gibco) were seeded into 6-well CellBIND plates (Corning) at a density of 8.0 x 105 cells per well. The cells were incubated at 37°C and 5% CO2 overnight. The media was removed from each well and 200 µl of each dilution prepared from resuspended virus was added to the respective wells of a 6-well plate. One well containing only DMEM with 2% FBS was included as a control on each plate. A back-titer of the virus used to prepare the 60 mm dishes was performed in triplicate by inoculating each well of a 6-well plate with 1 x 10-2 to 1 x 10-6 dilutions of the virus, respectively. Plates were incubated at 37°C and 5% CO2 for 1 hour with intermittent rocking. Cells were then overlaid with 2 ml of a 1:1 solution of 2.5% Avicel RC-591 (DuPont Nutrition and Health) and 2 x Temin's Modified Eagle Medium (Gibco) without phenol red, supplemented with 10% FBS (Gibco), 2 x antibiotic-antimycotic (Gibco) and 2 x GlutaMAX-I (Gibco). The cells were incubated at 37°C and 5% CO2 for 2 days. Plates were fixed in 10% neutral buffered formalin (ThermoFisher Scientific), followed by staining with 0.2% Gentian Violet (Ricca Chemical) in 10% neutral buffered formalin. The number of plaques per virus dilution were determined by eye and used to calculate the titer of the virus using the following formula:
Virus titer in PFU/ml = Number of plaques / (virus dilution in well x volume plated in ml)
The statistical package Microcal Origin® was used to analyze the data. A detailed explanation of the statistical methods used is provided with the results.
Additional data supporting the findings of this study are available from the corresponding author upon request.