Exploring inactivation of SARS-CoV-2, MERS-CoV, Ebola, Lassa, and Nipah viruses on N95 and KN95 respirator material using photoactivated methylene blue to enable reuse

Abstract

Background: The COVID-19 pandemic resulted in a worldwide shortage of N95 respirators, prompting the development of decontamination methods to enable limited reuse. Countries lacking reliable supply chains would also benefit from the ability to safely reuse PPE. Methylene blue (MB) is a light-activated dye with demonstrated antimicrobial activity used to sterilize blood plasma. Decontamination of respirators using photoactivated MB requires no specialized equipment, making it attractive for use in the field during outbreaks.

Methods: We examined decontamination of N95 and KN95 respirators using photoactivated MB and 3 variants of SARS-CoV-2, the virus that causes COVID-19; and 4 World Health Organization priority pathogens: Ebola virus, Middle East respiratory syndrome coronavirus, Nipah virus, and Lassa virus. Virus inactivation by pretreating respirator material was also tested.

Results: Photoactivated MB inactivated all tested viruses on respirator material, albeit with varying efficiency. Virus applied to respirator material pre-treated with MB was also inactivated, thus MB pretreatment may potentially protect respirator wearers from virus exposure in real-time.

Conclusions: These results demonstrate that photoactivated MB represents a cost-effective, rapid, and widely deployable method to decontaminate N95 respirators for reuse during supply shortages.

Keywords: COVID-19; Decontamination; Hemorrhagic fever virus; Methylene blue; N95 respirator; Photochemical inactivation.

Fig 1

Methylene blue plus light inactivates SARS-CoV-2, EBOV, LASV, and NiV in tissue culture plates. To confirm that photoactivated methylene blue (MB) inactivates the tested viruses, serially diluted MB was combined with aliquots of the indicated viruses and exposed to bright light (50,000 lux) for 30 min. Control plates were stored in the dark. Titers of remaining infectious virus were determined by TCID₅₀. Values represent means and standard deviations of triplicate samples. Dotted line represents the limit of detection. icSARS-CoV-2, SARS-CoV-2 derived from an infectious clone. ND, not detected. (Color version of figure is available online.)

Fig 2

MB plus light inactivates SARS-CoV-2, EBOV, LASV, and NiV on N95 and KN95 respirator material. To investigate if photoactivated MB can decontaminate mask material, N95 or KN95 respirator coupons were inoculated with 10 µL virus, incubated for 20–60 min, treated with 20 µL of MB at the indicated concentrations, and exposed to ambient or bright (50,000 lux) light for 30 min. Control coupons were stored in the dark. Virus was eluted and quantified by TCID₅₀. icSARS-CoV-2, SARS-CoV-2 derived from an infectious clone.

Fig 3

SARS-CoV-2, MERS-CoV, EBOV, LASV, and NiV are inactivated on respirators pre-treated with MB and exposed to light. To investigate if MB pre-treatment inactivates the tested viruses on respirators, pre-treated N95 and KN95 respirator material was inoculated with coronaviruses (A) or hemorrhagic fever viruses (B). N95 and KN95 respirator material was pre-treated with 10 or 100 µM MB, dried, inoculated with 10 µL virus, and exposed for 30 min to ambient light or bright light (50,000 lux). Control coupons were stored in the dark. Virus was eluted and quantified by plaque assay or TCID₅₀. Dotted line represents the limit of detection. ND, not detected.

Fig 4

MB pretreatment of respirators is robust. To examine if MB pre-treatment of respirators loses potency after prolonged light exposure, material was treated with 10 µM MB, dried, and exposed to ambient light for up 252 h (mimicking 7–21 days of 12 h dark/light cycles). After pre-exposure, respirator material was inoculated with the indicated viruses and further exposed to ambient light for 30 min. Remaining virus was eluted and quantified by plaque assay. Dotted line represents the limit of detection. ND, not detected.