Timsy Uppal, Ph.D. and Subhash C. Verma, Ph.D. University of Nevada, Reno, Reno NV 89557
Abstract: The global dissemination of COVID-19 infection caused by SARS-CoV-2, has posed an unprecedented healthcare COVID-19 control efforts in healthcare, home, and community settings, highlight the need for rapid, reliable, and effective SARS-CoV-2 inactivation Here, we determined the photocatalytic and virucidal activity of MACOMA™ Macoma™ PADS®2 photocatalytic film, activated by UV-A LED-12V-367nm (MA-FN 717836-1) lamp, against HCoV-OC43, a member of betacoronaviruses family, like SARS-CoV-2. Macoma™ PADS®2 coating coupled with UV-A LED radiation accelerated virus inactivation, compared to uncoated glass surface, when placed at a vertical distance of 2 feet (~14 inches) from virus samples for 10, 30, 60, and 120 minutes. Interestingly, 10 min of UV-A LED exposure effectively reduced 99% of live virus on Macoma™ PADS®2 coated surfaces, and further inactivated 99% of virus within 30 min. A complete virus inactivation was achieved within 60 min of UVA/Macoma™ PADS®2-exposure. These results confirmed that Macoma™ PADS®2 and UV-A LED-12V- 367nm (MA-FN 717836-1) based disinfection system is suitable for rapid, safe, and complete surface inactivation of human coronavirus.
Keywords: SARS-CoV-2; COVID-19; Macoma™ PADS®2, virus inactivation, UV-A LED-12V-367nm (MA-FN 717836-1), OC43
1. Introduction
COVID-19 infection caused by newly identified SARS-CoV-2, the most pathogenic human coronavirus, has led to an extraordinary threat to public health globally [1-3]. Clinically, SARS-CoV-2 infection is characterized by severe respiratory distress, fever (88%), dry cough (68%), shortness of breath (19%), and high rates of transmission [4]. Although vaccination and anti-virals are given top priority to combat COVID-19 spread, effective infection prevention and control (IPC) is a practical approach to reduce the transmission of this airborne A direct way to limit airborne virus spread is to inactivate them within a short time of their Among the widely used air sanitization techniques, Ultraviolet (UV) light exposure has attracted tremendous attention for several decades [5-7]. UV germicidal irradiation has been found to be suitable against a variety of microorganisms, and can cause critical viral DNA damage due to the absorbed UV photons/energy (4). For several years, different UV sources including low/medium pressure UV mercury lamps, UV light-emitting diodes (LEDs), and far UVC (200-240 nm) radiating excimer have been Among these, UV LEDs can generate different peak wavelengths ranging from 255-285 nm, ideal germicidal wavelengths, and provide reliable on-demand Additionally, UV light is often used in combination with photocatalytic materials, also called photocatalysts, including the widely used titanium dioxide (TiO2) Here, we evaluated the efficacy with which the Macoma™ PADS®2, (TiO2 coating) and UV-A LED-12V-367nm (MA-FN 717836-1) decontamination system inactivated human coronavirus, HCoV-OC43, when exposed in aerosol droplet Based on HCoV-OC43 results, Macoma™ PADS®2/UV exposure to virus kept at 1.2 feet distance, for even a short duration of 10 min, effectively inactivated the Since, all coronaviruses have comparable genomic size, we hypothesized that the findings from this study can be extrapolated to SARS-CoV-2 and other viruses.
2. Methods
Cells and Human The A549-hACE2 (HA-Flag) cells were obtained from BEI Resources and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals), 2 mM L-glutamine, 25 U/mL penicillin, and 25 µg/mL streptomycin and 1ug/mL The cells were grown at 370C and 5% CO2/95% air in a humidified cell culture incubator. HCoV-OC43 strain, is a human coronavirus and belongs to the family of betacoronaviruses, like SARS-CoV-2. HCoV-OC43 was obtained from BEI Resources (NIAID, NIH) and propagated in A549-ACE2 cells by infecting the cell monolayer with the virus for 2h at 340 Unattached virus was removed by washing with 1X PBS, followed by addition of fresh After 4 days, supernatant containing virus was harvested, cell debris were removed by centrifugation and virus was aliquotted, and stored at -800C until further Viral copies in the harvested supernatant were quantified by reverse transcriptase qPCR (RT-qPCR) using a standard curve as well as infectivity assay and the detection of virally infected cells through immunofluorescence All the assays were performed under BSL-2+ containment.
Macoma™ PADS® 2 and UV-A LED-12V-367NM Disinfection system. Macoma™ PADS®2 photocatalytic films deposited on 24mm x24mm glass coverslips, are water borne suspensions that consist of ~80% TiO2+20% mineral binder (inert, water-insoluble mineral carbonates, oxycarbonates, and hydrates). Thickness of the photocatalytic film is ~10-20 microns, and adhesion to the substrate is over UV source used in this study is a UV-A LED-12V-367nm (MA-FN 717836-1) lamp with two 16W UV-A LED-12V-367NM chips that emit ~500-350µW/cm2 energy on the test For the duration of this study, the UV-A LED-12V-367nm (MA-FN 717836-1) lamp was kept inside BSL-2+ containment and controlled using a manual switch on the instrument for each disinfection experiment. The sample was positioned 1.2 feet (14 inches) below the light, with UV light directed at the center of the sample for 10, 30, 60 and 120 The intensity of the light was measured with a UVA light meter.
Experiment Protocol. In order to test the efficacy of UV-A LED-12V-367nm (MA-FN 717836-1) activated Macoma™ PADS®2 photocatalytic film towards the inactivation of HCoV-OC43, 100 µL of the virus stock (equivalent to 2 x 106 calculated viral particles) were applied as 10 x 10 µl liquid droplets on top of both uncoated and Macoma™ PADS®2 coated coverslips placed inside each well of a 6-well culture plate, kept inside the BSL-2+ containment (Fig. 1). The applied virus in the culture medium with fetal bovine serum, resembled the protein composition of nasal secretions or respiratory The virus-containing 6-well plate was placed vertically below the UV-A LED-12V-367nm (MA-FN 717836-1)
lamp at the center and exposed to UVA radiation from 2 feet for specified times (10, 30, 60, and 120 min). Original untreated virus and virus-laden 6-well culture plate without UV treatment served as controls for calculating viral inactivation After each disinfection experiment, the mock-treated and UVA-treated viruses were recovered by adding 300 µl of culture medium on the surfaces and allowing it to resuspend for 15 min at 340C. The recovered viruses were either collected in a 1.5 mL eppendorf tube for further extraction of total viral RNA with Trizol reagent for viral genome quantitation using qRT-PCR or applied onto a permissive human lung carcinoma, A549-hACE2 cell monolayer for the detection of residual infectious virus through integrated cell culture PCR and localization of infected cells through immunofluorescence assay. Each assay was done in duplicates and each experiment was conducted three independent times.
RNA extraction and qPCR. For the detection of viral genomic RNA through RT-qPCR, control virus, UV- treated and untreated virus supernatants from both coated and uncoated coverslips (for intact viral genomic RNA) or from infected A549-hACE2 (HA-FLAG) cells (for infectious viral genomic RNA) were subjected for total RNA extraction using Trizol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s recommendation. An aliquot of extracted total RNA (1 µg) was used for cDNA synthesis using high capacity RNA to cDNA kit (Invitrogen, Carlsbad, CA). A fraction of synthesized cDNA (2 µL) was used for the relative quantification of viral genomic copies using TaqMan Fast Advanced master mix, and TaqMan OC43-specfic gene expression assay (FAM) in a RT-qPCR assay (ThermoFisher Scientific, Waltham, MA). Three ten-fold serial dilutions of HCoV-OC43 genomic RNA (BEI Resources, Cat.# NR-52727 with 2.0×108 genome equivalents/ml) were used for generating a standard curve to quantify the HCoV-OC43 viral copies in the virus preparations.
Virus Infectivity through Immunofluorescence Assay. Control or UV-treated viruses were added onto the human lung carcinoma, A549-hACE2 (HA-FLAG) cells for 2h (340C, 5%CO2). Following infection, the cells were incubated for 48h at 340C in a humidified chamber supplemented with 5%CO2. Infected cells were detected by using the immunofluorescence assay (IFA) of the nucleocapsid protein of HCoV-OC43 (Millipore Sigma). Infected cells were fixed using 3.2% formaldehye and permeabilized with 0.1% Triton X-100 for 10 min, washed (2x) and blocked (0.4% FSG-0.05% Triton X-100) for 45 mins at room temperature. Cell monolayers were washed (2x) and incubated with monoclonal mouse anti- HCoV-OC43 nucleocapsid antibody (1:1000; Millipore Sigma) overnight at 40C, followed by incubation with chicken anti-mouse Alexa Fluor 594 (1:1000; Molecular Probe, Carlsbad, CA), secondary antibody for 1h at RT in the dark. Finally, the nuclei were stained with DAPI (blue).
1:7000; ThermoFisher, Walthman, MA). Coverslips were mounted on the glass slides using prolong diamong antifade (ThermoFisher) and the slides were examined using Carl Zeiss microscope.
Statistical Data presented are an average of three independent experiments and the error bars represent the standard deviation across independent Statistical analyses were performed using Prism 0 software (Graphpad Inc.) and the p-values were calculated using 2-way ANOVA and the p-value cut offs for statistical significance were *, p<0.1; and **, p<0.01.
3. Results and Discussion
Quantification of infectious HCoV-OC43 We used an integrated cell culture PCR assay and analysis of infected cells to quantify the infectious The infected cells were detected by quantifying viral genomic RNA and by localizing HCoV-OC43 nuclocapsid protein through immunofluorescence Varying amounts of HCoV-OC43 virus, calculated using a standard curve (Fig. 2A) from our stock were added onto a permissive cell line, A549-hACE2 (infectivity assay) followed by the detection of viral genome copies after a 48h incubation. We found an excellent correlation between the amount of viral genome copies detected and the amount of infectious viruses added onto the cells at different dilutions (R2=0.9999) (Fig. 2B). We also confirmed the infectivity of our virus stock by the detection of viruses in the infected cells infected with different quantities of viral particles on A549-hACE2 and localizing HCoV-OC43’s nucleocapsid using immunofluorescence assay (localization of infected cells). Lack of any signals in control (uninfected cells) and specific localization of nucleocapsid signals confirmed the specificity of our assay (Fig. 2C). Further, cells infected with lower amounts of HCoV-OC43 virus showed proportionaly reduced number cells with nucleocapsid staining, as expected (Fig. 146 2B).
Virus inactivation assay to test the efficacy of Macoma™ PADS®2 film activated by UV-A LED-12V-367NM device. To test the inactivation efficiency of UV-A LED-12V-367NM irradiation coupled with Macoma™ PADS®2 film on HCoVs, HCoV-OC43 was used as a surrogate of the highly contagious, SARS-CoV-2 due to its high genomic sequence similarity. In this study, we tested the virus decontamination by depositing HCoV-OC43 viruses in a droplet form (10 droplets x 10µL each) into 6-well plate with uncoated and Macoma™ PADS®2 -coated coverslips, and exposing with UVA light emiting UV-A LED-12V-367NM unit placed 14 inches away, inside a BSL-2+containment for varying times, 10, 30, 60, and 120 min. Same amount of virus without UV treatment was used as a control. Untreated and UV-treated viruses were collected for the evaluation of virus inactivation efficacies by direct genomic RNA quantitation (highly sensitive RT-qPCR) and infectivity assay using permissive A549-ACE2 HCoV-OC43 viral copies in the UV-treated and untreated samples were calculated based on the standard curve generated using serial dilutions of known HCoV-OC43 genomic RNA (Fig. 3). The intact residual virus copies in UV-treated and untreated samples were calculated and compared to the original virus control, set to 100%. As expected, the copies of viral genomic RNA from UV-treated, Macoma™ PADS®2 coated coverslips declined 99-99.99% (2-4 log10 decrease) within 60 mins, and 100% within 120 min (Fig. 3A). We further performed infectivity assay to see whether there is any residual live virus. For this, UV-treated and untreated HCoV-OC43 viruses from uncoated and Macoma™ PADS®2-coated coverslips were added onto A549-ACE2 cells for 2 h at 340C to allow attachment and entry of the residual live virus. Quantitation of intracellular HCoV-OC43 genome copies through OC43 gene specific RT-qPCR provide highly accurate quantitation of any infectious virus present in the samples added onto the target cells as described in 3B. Calculated residual live virus copies based on the intracellular viral genomic copies, showed that UVA light activated Macoma™ PADS®2 film led to 2-fold (99%) and 4-fold (99.9%) reduction in the virus within 10 and 30 min and completely inactivated the infectious virus within 60 min, when placed 2-feet away (Fig. 3B). Additionally, we confirmed that Macoma™ PADS®2/UVA exposure completely inactivated the virus by adding the UVA/Macoma™ PADS®2 treated samples and localizing viral protein (indicator of infectious virus) through immunofluorescence staining for HCoV-OC43 nucleocapsid As shown in Fig. 4, there weren’t any red stained cells (stain to detect viral infected cells) in wells with 30 min UVA/Macoma™ PADS®2-exposed HCoV-OC43 virus (Fig. 4C). Same amount of virus processed similarly without UVA exposure showed red stained cells, as expected, confirming the lack of any infectious virus in UVA/Macoma™ PADS®2-treated An increase in the exposure time to 60 and 120 min completely abrogated infectious virus copies (Fig. 4D, and E). Overall these results confirm that the tested Macoma™ PADS®2/UV-A LED-12V-367NM system is effective in completely inactivating HCoV-OC43 and should perform at same efficiency for the inactivating other human coronaviruses.
4. Conclusion
Our results showed that HCoV-OC43 was effectively inactivated by Macoma™ PADS®2 and UV-A LED-12V-367NM based disinfection system within 30 Importantly, the device completely reduced the virus to background level within 1h, even when placed 2-feet distance away. Our findings confirm that Macoma™ PADS®2 and UV-A LED-12V-367NM based system offers a rapid, reliable, eco-friendly and effective disinfection tool to maximize virus inactivation and minimize viral spread.
Acknowledgments: Human Coronavirus, OC43 (HCoV-OC43) was obtained through BEI Resources, NIAID, NIH: Human Coronavirus, OC43, NR-52725.
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