Photodynamic Inactivation of Bovine Coronavirus with the Photosensitizer Toluidine Blue O
Maya Margaritova Zaharieva,
Pelagia Foka,
Eirini Karamichali,
Alexander Dimitrov Kroumov,
Stanislav Philipov,
Yana Ilieva,
Tanya Chan Kim,
Petar Podlesniy,
Yordan Manasiev,
Vesselin Kussovski,
Urania Georgopoulou,
Hristo Miladinov Najdenski
Affiliations
Maya Margaritova Zaharieva
Department of Infectious Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. G. Bonchev Str., 1000 Sofia, Bulgaria
Pelagia Foka
Department of Microbiology, Laboratory of Molecular Virology, Hellenic Institute Pasteur, Vasilissis Sofias 127, 11521 Athens, Greece
Eirini Karamichali
Department of Microbiology, Laboratory of Molecular Virology, Hellenic Institute Pasteur, Vasilissis Sofias 127, 11521 Athens, Greece
Alexander Dimitrov Kroumov
Department of Infectious Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. G. Bonchev Str., 1000 Sofia, Bulgaria
Stanislav Philipov
Chair Human Anatomy, Histology, General and Clinical Pathology and Forensic Medicine, Faculty of Medicine, Hospital Lozenetz, Sofia University “St. Kliment Ohridski”, 2 Kozyak Str., 1407 Sofia, Bulgaria
Yana Ilieva
Department of Infectious Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. G. Bonchev Str., 1000 Sofia, Bulgaria
Tanya Chan Kim
Department of Infectious Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. G. Bonchev Str., 1000 Sofia, Bulgaria
Petar Podlesniy
Institute of Biomedical Research of Barcelona, CSIC, Rosselló, 161, 7ª Planta, 08036 Barcelona, Spain
Yordan Manasiev
Evgeni Budevski Institute of Electrochemistry and Energy Systems, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Vesselin Kussovski
Department of Infectious Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. G. Bonchev Str., 1000 Sofia, Bulgaria
Urania Georgopoulou
Department of Microbiology, Laboratory of Molecular Virology, Hellenic Institute Pasteur, Vasilissis Sofias 127, 11521 Athens, Greece
Hristo Miladinov Najdenski
Department of Infectious Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. G. Bonchev Str., 1000 Sofia, Bulgaria
Coronaviruses (CoVs) belong to the group of enveloped positive-sense single-strand RNA viruses and are causative agents of respiratory, gastro-intestinal, and central nervous systems diseases in many host species, i.e., birds, mammals, and humans. Beta-CoVs revealed a great potential to cross the barrier between species by causing three epidemics/pandemics among humans in the 21st century. Considering the urgent need for powerful antiviral agents for decontamination, prevention, and treatment of BCoV infections, we turned our attention to the possibility of photodynamic inactivation with photosensitizers in combination with light irradiation. In the present study, we evaluated, for the first time, the antiviral activity of toluidine blue O (TBO) against Beta-coronavirus 1 (BCoV) in comparison to methylene blue (MB). First, we determined the in vitro cytotoxicity of MB and TBO on the Madin–Darby bovine kidney (MDBK) cell line with ISO10993-5/Annex C. Thereafter, BCoV was propagated in MDBK cells, and the virus titer was measured with digital droplet PCR, TCID50 assay and plaque assay. The antiviral activity of non-toxic concentrations of TBO was estimated using the direct inactivation approach. All effects were calculated in MAPLE 15® mathematical software by developing programs for non-linear modeling and response surface analysis. The median inhibitory concentration (IC50) of TBO after 72 h of incubation in MDBK cells was 0.85 µM. The antiviral activity of TBO after the direct inactivation of BCoV (MOI = 1) was significantly stronger than that of MB. The median effective concentration (EC50) of TBO was 0.005 µM. The cytopathic effect decreased in a concentration-dependent manner, from 0.0025 to 0.01 µM, and disappeared fully at concentrations between 0.02 and 0.3 µM of TBO. The number of virus particles also decreased, depending on the concentration applied, as proven by ddPCR analysis. In conclusion, TBO exhibits significant potential for direct inactivation of BCoV in vitro, with a very high selectivity index, and should be subjected to further investigation, aiming at its application in veterinary and/or human medical practice.
