Oxygen-dependent laser inactivation of murine norovirus using visible light lasers

Authors: Kingsley, David; JOHNSON, ANTHONY - University Of Maryland; ROBINSON, KUIS - University Of Maryland; PEREZ, RAFAEL - Former ARS Employee; BASALDUA, ISAAC - University Of Maryland; BURKINS, PAUL - University Of Maryland; MARCANO, ARISTIDES - Delaware State University

Submitted to: Virology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/29/2018
Publication Date: 7/31/2018
Citation: Kingsley, D.H., Johnson, A., Robinson, K., Perez, R., Basaldua, I., Burkins, P., Marcano, A. 2018. Oxygen-dependent laser inactivation of murine norovirus using visible light lasers. Virology Journal. 15:117. https://doi.org/10.1186/s12985-018-1019-2.
DOI: https://doi.org/10.1186/s12985-018-1019-2

Interpretive Summary: High intensity blue visible spectrum light has been a subject of investigation for nonthermal inactivation of pathogens in the health care and food safety fields. Previous work demonstrated that murine norovirus, a human norovirus research surrogate, was inactivated by 425 nm femtosecond pulsed light. At that time, impulsive stimulated Raman scattering, essentially thought to generate capsid vibrations, was proposed as the mechanism of action. Here we show that inactivation is not wavelength-specific and not dependent on pulsed laser light since a continuous wave (non-pulsed) laser also inactivates the virus. Experiments with a chemical oxygen scavenger show that reducing dissolved oxygen concentration limits the inactivation by blue laser light, inferring that reactive oxygen species are in fact the cause of inactivation rather than vibrations.

Technical Abstract: Previous work indicated that a ultrashort pulse (USP) 425 nm laser is capable of inactivating murine norovirus (Virol. J. 11:20), perhaps via an impulsive stimulated Raman scattering (ISRS) mechanism, and does not substantially damage human plasma proteins (PLOS One 9:11). In this study, we evaluate whether inactivation is specific to the USP wavelength of 425nm, or if it occurs at other visible wavelengths, using a tunable mode-locked Ti-Sapphire laser that has been frequency doubled to generate femtosecond pulses at wavelengths of 400, 408, 425, 450, 465, and 510 nm. Greater than 3 log inactivation of MNV pfu occurs after irradiation with 150 mW of 408, 425 or 450 nm femtosecond-pulsed light for 3 hrs. Thus results suggest that the mechanism by which a laser inactivates the virus is not wavelength-specific. Furthermore, we also show that irradiation using a continuous wave (CW) laser of similar power at 408 nm also yields substantial MNV inactivation indicating that inactivation is does not require a USP. Use of photosensitizers, riboflavin, rose bengal and methylene blue that generate singlet oxygen substantially improves the efficiency of the inactivation. The results indicate a photochemical mechanism of the laser-induced inactivation where the action of relatively low power blue laser light generates singlet oxygen. Results implicate formation of short-lived reactive oxygen species such as singlet oxygen by visible laser light as the cause of virus inactivation rather than via an ISRS mechanism which induced resonant vibrations.