Cell culture and viruses
The TZM-bl cell line was obtained from the NIH AIDS Reagent Program (cat# ARP-8129) and maintained in high-glucose (4.5 g L−1) DMEM (Wisent cat# 319005-CL). MRC-5 was maintained in EMEM (ATCC cat# 30-2003) and Huh7 in low-glucose (1 g L−1) DMEM (Gibco cat# 11885-084). All media were supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Wisent cat# 098150), 100 U mL−1 penicillin and 100 µg mL−1 streptomycin (Fisher Scientific cat# 15140122), and all cell lines were kept at 37 °C in a humidified, 5% CO2 incubator.
HIV-1 IIIB stocks were generated through infection of the A3R5.7 T cell line (NIH ARP cat# 12386). In brief, cells were pelleted and resuspended in 1 mL of HIV-1 IIIB isolates for 4 h before fresh media was added. Virus-containing supernatants were harvested 10–12 days later, aliquoted, and frozen at − 80 °C. To quantify virus concentration and standardize input for infection assays, HIV-1 p24 capsid protein levels were measured using the AlphaLISA p24 detection kit following the manufacturer’s (PerkinElmer) instructions. Absorbance readings were performed on a Synergy NEO 2 multimode plate reader (BioTek) equipped with Gen 5 software (v. 3.08).
Wild-type (hCoV-229E) and EGFP-expressing (229E-EGFP) human coronavirus 229E were produced by infecting MRC-5 cells for 48 h (hCoV-229E) or Huh7 cells for 72 h (229E-EGFP) at 34 °C after which cell-free supernatants were aliquoted and stored at − 80 °C. Both virus stocks were titrated on Huh7 cells (2 × 104 cells/well; 96-well plate) by overlaying 50 µL of 1:10 serial dilutions of virus-containing supernatants in serum-free DMEM for a 1 h adsorption at 34 °C, as similarly described [14]. Following, inoculum was removed and replaced with 200 µL DMEM + 2% (v/v) FBS and infection was monitored for 5 days at 34 °C. TCID50 calculations based on observable cytopathic effect were done as previously described [15].
UV-LED specifications
The UV-LEDs were supplied as two sets, nine 275 nm LEDs in a 3 × 3 array, and twenty 380 nm LEDs in a 4 × 5 array. The LEDs were approximately 5 cm from the irradiated sample, with each array delivering between 0.4 and 0.6 mW/cm2 of UV light. The maximum irradiation time was 30 s, resulting in a total delivered dose for the combined arrays of 8 mJ/cm2 to 20 mJ/cm2 to the irradiated samples. The area illuminated was substantially larger than the irradiated sample, with the total lit area of the device approximately 10 cm by 20 cm, or a total of 200 cm2, resulting in a total areal dose of 1.6 J to 4 J.
Bacillus pumilus inactivation assay
Stainless steel discs inoculated with Bacillus pumilus spores were obtained through Mesa Labs (cat# DPSSC/3). Discs were exposed on both sides to the UV light for the specified times and cultured in tryptic soy both (Fisher Scientific cat# DF0370-17-3). Cultures were incubated for seven days at 33 °C under aerobic conditions. For turbidity measurements, samples were transferred into 96-well plates (FroggaBio cat# 92096) in duplicate and optical density (OD, 600 nm) measurements were determined using the Synergy Neo2 multimode plate reader. Final data presentation was done on Prism (GraphPad, v.9.1.2) for all experiments.
HIV-1 inactivation assay
For UV exposure, stock virus (82 ng mL−1 p24) was diluted and dispersed into 7 µL droplets, exposed to UV for 30 s, and then re-pooled for assaying by TZM-bl titration. TZM-bl cells were overlaid at 1.5 × 104 cells/well over the virus dilutions in 96-well plates. After 4 days of culture, cells were lysed and incubated in BriteLite Plus (Perkin Elmer cat# 6066766) for 10 min at room temperature, transferred to opaque white Opti Plates (Perkin Elmer) for luminescence read-out on the Synergy Neo2 multimode plate reader equipped with Gen5 (v.3.08) (BioTek).
hCoV-229E inactivation assays
For UV exposure of wild-type hCoV-229E, samples were prepared in serum-free DMEM (MOI 0.1, 0.01 and 0.0001), distributed in 7 µL droplets and exposed to UV for 30 s. Following exposure, virus droplets were picked up and diluted to 700 µL in serum-free DMEM. The entire inoculum was overlaid onto monolayers of 6 × 105 Huh7 cells plated in 6-well plates 24 h prior. Inoculated cells were incubated for 1 h at 34 °C to allow for adsorption before DMEM + 2% (v/v) FBS was added for a final volume of 2 mL/well. After 48 h of infection, monolayers were trypsinized, harvested and total RNA was extracted using the GENEzol TriRNA kit (Geneaid Biotech cat# 12183020). RNA purity and quantitation were done on the BioTek’s Synergy Neo2 multimode plate reader using the Take3 adapter. Real-time PCR was done using the TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher cat# 4444432). Optimized primer–probe TaqMan assays were obtained from Thermo Fisher for the detection of eukaryotic 18S rRNA (cat# 4453320, assay ID: Hs99999901_s1) and hCoV-229E (cat# 4331182, assay ID: Vi06439671_s1). PCR was performed on the Quant Studio 3 and Thermo Fisher’s cloud-based Relative Quantitation module was used for data analysis.
For experiments with EGFP-expressing hCoV-229E, stock virus was diluted 1:30 (akin to MOI 0.01) and irradiated as described above and used to infect monolayers of 2 × 104 Huh7 cells in 96-well plates. Inoculated cells were incubated for 1 h at 34 °C to allow for adsorption before DMEM + 2% (v/v) FBS was added for a final volume of 0.2 mL/well. After 72 h of infection, cells were collected, fixed (2% v/v PFA) and GFP fluorescence was analyzed on the BD LSR Fortessa, with analyses performed in FlowJo (v.10.7.2).