Abstract
Since coronaviruses can remain infectious on different inanimate surfaces for several hours or even days, the possibility of indirect fomite transmission through infected objects and surfaces cannot be ruled out. We describe a method for the photodynamic disinfection of inanimate surfaces infected with severe acute respiratory syndrome coronavirus 2, Omicron variant strain. Application of only 5 µM photosensitizer octakis(cholinyl)zinc phthalocyanine followed immediately by 7 min irradiation with light emitting diode (LED) light 692 nm (12.5 mW cm−2) results in complete inactivation of the virus on polystyrene and glass surfaces, while 10 min irradiation lead to complete eradication of the virus also on Al-foil and medical mask fabric. A photodynamic technique is being considered to combat the spread of coronaviruses.
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1. Introduction
The causative agent of the current pandemic coronavirus disease COVID-19 is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus that belongs to the β-coronavirus genus with a single-stranded positive-sense ribonucleic acid (RNA) genome. RNA-containing viruses, which are responsible also for outbreaks of influenza and Ebola are characterized by a high mutation rate and extreme genetic diversity [1]. As a consequence, to date, several variants of SARS-CoV-2, including Omicron and its subvariants, have succeeded each other as the main pathogen circulating in the human population. The Omicron (B.1.1.529) variant is characterized by a high number of mutations and is at least three times more infectious than the original SARS-CoV-2 [2] and spreads several times faster than any previous variant [3]. Immune-escape mutations that increase the risk of re-infection and the reduced effectiveness of vaccines against new variants of SARS-CoV-2 are a serious challenge [4]. To successfully fight the pandemic, we need a multi-path approach, in which there should be a place for vaccination, antiviral drugs, and measures aimed at preventing and controlling the spread of the pathogen [5].
In patients with COVID-19, SARS-CoV-2 exits the epithelial cells of the respiratory tract into the extracellular fluid and is then released into the environment in aerosols (⩽100 µm) and droplets (>100 µm) when breathing (1–320 droplets per liter of exhaled air), speaking (4–600 per liter), coughing (24–23 600 droplets per liter, one cough generates about 3000 droplets) [6]. The virus is especially actively released into the external environment by 10%–20% of patients, the so-called super-emitters. The probability of the presence of SARS-CoV-2 in respiratory aerosol particles is estimated at about 3%, and in droplets—about 25%, with most of the droplets containing a single virion [7].
Aerosols, depending on the aerodynamic radius, settle within 2 m (50–100 µm) or more (10–50 µm) and can cause transmission of the virus by inhalation, while respiratory droplets quickly settle on nearby surfaces and thus can participate in indirect transmission [7]. Viable SARS-CoV-2 has been detected in air samples from a hospital room taken at a distance of 2–4.8 m from COVID-19 patients at concentrations of 6–74 TCID50 units l−1 of air [8]. Detection of SARS-CoV-2 on hospital surfaces showed that 26.66% of surfaces were positive for SARS-CoV-2 RNA, all of them in close proximity to the patient [9]. In the intensive care unit, 86% of samples from the stethoscope and bed rail were positive [10]. The role of fomite transmission in the spread of coronavirus infection through contaminated objects and surfaces is debated [11, 12]. However, such indirect transmission of the latest SARS-CoV-2 variant Omicron seems possible, as the percentage of super-emitters among those infected with Omicron has increased dramatically compared to the wild-type strain, and the critical dose of virus copies to become potentially infectious has decreased from 500 copies for wild-type virus or 300 copies for Delta to 100 copies for Omicron [13].
Droplets exist before drying from a few milliseconds at a size of 10 µm to a few tenths of a second at a size of 100 µm [6]. Temperature and humidity have a decisive influence on the further maintaining of the infectivity of the virus in the dried state. In general, at room temperature, human coronaviruses are reported to remain infective for 9 d, and animal coronaviruses for up to 28 d [14]. In the dried state at room temperature, SARS-CoV-2 persists on stainless steel with mean half-lives of 5.6 h and on plastics—6.8 h [15]. SARS-CoV-2 dried on glass retained viability for over 3–4 d at room temperature (22 °C–25 °C) and for 14 d at 4 °C but lost viability within 1 d at 37 °C [16].
Since fomites can contribute to the spread of coronavirus infections, especially nosocomial infections, cleaning/disinfection of surfaces is essential [17]. Decontamination of surfaces utilizes quaternary ammonium compounds [18] and other disinfectants, ozone exposure [19], and germicidal ultraviolet C (UVC, 100-280 nm) [20]. However, photodisinfection is not limited to antimicrobial effects of UVC 254 nm, and can be induced by almost all optical wavelengths [21], from far-UVC 222-nm [22] to high intensity violet light [23] and visible light activating production of reactive oxygen species (ROS) by photosensitizers (PS), namely photodynamic disinfection [24].
Antimicrobial potential of photosensitized ROS generation is realized in the photodynamic antimicrobial chemotherapy of localized dermatological and dental infections, decontamination of blood and blood products, TiO2 photocatalytic disinfection. The use of PSs for the prevention and treatment of infections has been expected to expand with novel PS, delivery strategies, and clinical carriers [25].
In our previous studies, octakis(cholinyl)zinc phthalocyanine (ZnPcChol8+) demonstrated electrostatic binding to bacterial cells [26, 27] and enveloped viruses [28, 29], and readily sensitized targeted pathogens in suspensions when activated with far red light [30–33]. Given the large overall positive charge of ZnPcChol8+, it can be expected that the molecules of this PS will effectively compete with the cations contained in the droplets for binding sites on SARS-CoV-2 virions and potentiate photodynamic inactivation. In this work, we contaminated the surfaces of various materials with the SARS-CoV-2 variant Omicron virus in a rich culture medium, dried the virus, and showed that the PS solution is effective in photodynamic disinfection of such surfaces.
2. Materials and methods
2.1. Photosensitize and LED source
PS octakis(cholinyl)zinc phthalocyanine (ZnPcChol8+), synthesized in the Research Institute of Organic Intermediates and Dyes (Russia) [34], was used for virus inactivation at concentration of 5 µM. The molecular structure of ZnPcChol8+ is given in [32]. The light source was built on six light-emitting diodes supplied with heat removal. Its emission spectrum, with a spectral maximum at 692 nm, was close to the absorption spectrum of ZnPcChol8+ [32]. The intensity of light reaching the probes was measured with PM160T Wireless Power Meter (THORLABS GmbH, Dachau, Germany) and made up 12.5 mW cm−2.
2.2. Coronavirus and cell culture
To study the effectiveness of photodynamic inactivation of the SARS-CoV-2 pandemic coronavirus on model inanimate surfaces the Omicron variant strain (laboratory number 8.97) was chosen. Virus was propagated in Vero E6 cells (monkey kidney cells) grown in Dulbecco's Modified Eagle Medium (DMEM/F12, Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS, Capricorn Scientific, Germany) and 50 µg ml−1 gentamicin sulfate (Biolot, St. Petersburg, Russia)at 37 °C under 5% CO2. Virus stock was harvested and filtrated through a 0.22 µm filter and stored at −80 °C until use. In all experiments, cells that were not infected with the virus were included as controls.
All SARS-CoV-2 experiments were performed according to the standard protocols at the BSL 3 Laboratory (Federal Research Center of Fundamental and Translational Medicine (CFTM), Novosibirsk, Russia).
2.3. Virus dried samples, photodynamic inactivation and titration
A virus-containing liquid (100 µl) was applied to a surface (Al-foil, cover glass, medical mask fabric, polystyrene) 1 × 1 cm2 in size and exposed in a laminar with the flow turned on for 1 h to dry the sample. A solution of PS ZnPcChol8+ at a concentration of 5 µM in double distilled water (100 µl) was applied to the dried sample and irradiated with LED system for 2, 5, 7 and 10 min. A sample of the surface with a dried virus without further processing and without irradiation was used as a control. Then, the samples were placed in a test tubes with 900 µl of Hanks solution (Biolot, St. Petersburg, Russia) and mixed on a vortex obtaining a virus-containing liquid in dilution 10−1, after which a series of tenfold dilutions (10−1–10−5) of the virus-containing liquid was prepared.
The obtained dilutions were added to the plate with a daily monolayer of VeroE6 cells. For this purpose, VeroE6 cell culture was seeded into a 96-well plate (Techno Plastic Products AG, TPP, Switzerland) at a density of 30 000 cells/well in DMEM (DMEM/F12, Capricorn Scientific, Ebsdorfergrund, Germany) culture medium with 10% FBS (Capricorn, South America), 100 μg ml−1 of gentamicin and were incubated in a CO2 incubator for 24 h at a temperature of 37 °C. In 24 h, after visual control of the state and density of the cell monolayer, Vero E6 cells were washed twice with Hanks solution (Biolot, St. Petersburg, Russia) and inoculated with tenfold dilutions of the virus using eight wells for each dilution. After 60 min incubation to absorb the virus, the supernatant was removed and DMEM (Capricorn) with 2% FBS (Capricorn, South America) and 100 μg ml−1 gentamicin was added. The plates were incubated in a CO2 incubator at 37 °C. Then, for 7 d, the state of the cell monolayer was assessed daily by light microscopy to identify the specific cytopathic effect of the SARS-CoV-2 pandemic coronavirus on Vero E6 cells, manifested as apoptotic changes in the monolayer of infected cells, changes in cell morphology, loss of intercellular contacts and subsequent death of infected cells with the formation of apoptotic bodies. The virus titer was calculated by the Kerber method [35] and expressed in tissue culture infectious dose log10TCID50/ml.
The experiments were carried out in triplicate.
3. Results
The following controls were used in each experiment:
- a sample of the surface with a dried virus without further processing, without irradiation;
- a sample of the surface with a dried virus, followed by application of 100 µl of Hanks solution and irradiation with LED;
- a sample of the surface with a dried virus, followed by application of 100 µl of ZnPcChol8+ solution and exposure without irradiation.
The titration results are presented in table 1. As can be seen from the table, the virus titers after drying it on various surfaces for an hour did not significantly differ from each other and from the initial virus titer, which was 3.38 ± 0.13 log10TCID50/ml. Irradiation of surfaces with the dried virus for 10 min without application of PS ZnPcChol8+, as well as the exposure of ZnPcChol8+ for 10 min in the dark did not change the infectivity of the virus.
Table 1. Titers of SARS-CoV-2 dried on different types of surfaces and exposed to photosensitizer (PS) and/or LED irradiation.
| Surface | Virus dried on the surface without further processing and irradiation | Virus dried on the surface with application 100 µl of Hanks solution and irradiation with LED | Virus dried on the surface with application 100 µl of PS and exposure without irradiation | Virus dried on the surface with application 100 µl of PS and irradiation with LED for 2–10 min | |||
|---|---|---|---|---|---|---|---|
| Time of irradiation, min | |||||||
| 2 | 5 | 7 | 10 | ||||
| Metal (Al-foil) | 3.29 ± 0.11 | 3.50 ± 0.15 | 3.38 ± 0.07 | 2.67 ± 0.08 | 1.25 ± 0.15 | 1.00 ± 0.15 | 0 |
| Fabric (medical mask) | 3.38 ± 0.07 | 3.38 ± 0.07 | 3.21 ± 0.04 | 2.13 ± 0.27 | 1.63 ± 0.13 | 1.33 ± 0.17 | 0 |
| Poly-styrene | 3.00 ± 0.07 | 3.17 ± 0.18 | 3.25 ± 0.13 | 2.17 ± 0.11 | 1.21 ± 0.08 | 0 | 0 |
| Glass | 3.16 ± 0.03 | 3.25 ± 0.13 | 3.38 ± 0.19 | 2.38 ± 0.15 | 1.67 ± 0.08 | 0 | 0 |
Virus titers are expressed in log10TCID50/ml, (M ± m).
Treatment of the virus dried on any of the used surfaces with PS ZnPcChol8+ followed by irradiation for 2 (1.50 J cm−2) and 5 min (3.75 J cm−2) lead to a decrease in the infectivity of the virus by 10 and 100 times, respectively.
Virus titers with PS ZnPcChol8+ on fabric (medical mask) or metal (Al-foil) upon 7 min (5.25 J cm−2) irradiation did not differ significantly from those upon irradiation for 5 min, while 7 min irradiation of virus samples dried on polystyrene and glass surfaces led to complete inactivation of virus infectivity.
Application of PS ZnPcChol8+ followed by irradiation for 10 (7.50 J cm−2) min lead to the complete loss of infectivity of the virus on all types of model surfaces. Figure 1 shows the corresponding photographs of Vero E6 cells monolayers.
Figure 1. The monolayers of Vero E6 cells 72 h after infection with SARS-CoV-2 untreated (a), treated with 5 µM Zn-PcChol8+ (b), irradiated with 7.50 J cm−2 LED light at 692 nm (c), treated with 5 µM Zn-PcChol8+ and irradiated with LED 7.50 J cm−2 (d), and uninfected Vero E6 monolayer (e). Photographs were made with magnification ×20.
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4. Discussion
In this study, coronavirus SARS-CoV-2 was exposed on different types of inanimate surfaces of 1 square centimeter, that is, on Al-foil, glass, fabric and polystyrene. Drying samples for 1 h in a laminar flow at room temperature did not significantly affect the infectivity of the virus. This is consistent with the much longer persistence of SARS-CoV-2 in the dried state on tested surfaces [14, 15].
PS ZnPcChol8+ in the form of a solution in a volume of 100 µl was applied to dried virus samples, transferring the viral particles into a wet and/or resuspended state. Irradiation of samples with PS for 2 and 5 min reduced the infectivity of the virus (a decrease in titer by one and two orders of magnitude, respectively), but did not lead to complete eradication. However, treatment with 5 µM ZnPcChol8+ followed by 7 min irradiation results in complete inactivation of the virus on polystyrene and glass surfaces, while 10 min irradiation lead to complete eradication of the virus on any of the surfaces used in this study. The virus dried on surfaces followed by the application of Hanks' solution without PS, as well as the virus exposed to PS for 10 min in the dark, retained almost the same infectivity as compared to the control sample. We recently showed [32] that 2 min (1.50 J cm−2) irradiation with the same LED source in the presence of 5 μM Zn-PcChol8+ lead to a complete loss of SARS-CoV-2 infectivity in suspension with initial virus titer 4.38 ± 0.38 log10TCID50/ml. A significantly lower degree of photodynamic inactivation with the same PS concentration and light dose of the virus after drying and rehydration in PS solution (titer decrease by 1 log) may be associated with adsorption of medium components on the surface of viral particles during drying. Evaporation of water leads to the change in virus microenvironment [36]. Increased salt concentration can lead to rigidification of lipid bilayer and its decreased elasticity [37], thus influencing efficacy of oxidative destruction of viral membrane. Some of these components can remain tightly bound to the surface of virus particles after being resuspended into the water medium, and prevent PS binding or act as singlet oxygen traps, thereby protecting the virus from critical oxidative damage. ROS can oxidize with high rate constants components of the DMEM culture medium, such as aromatic and sulfur-containing amino acids. Competitive reactions of ozone with media components were assigned as one of the potential mechanisms that reduce the effectiveness of SARS-CoV-2 inactivation [38]. Bovine coronavirus has been shown to be sensitive to the high intensity violet light 405 nm after centrifugation in the culture medium and resuspension in a tenfold volume of phosphate buffered saline (PBS) [23]. This may just indicate the binding of endogenous PS from the culture medium to viral particles. The virus in dried samples was less sensitive to 405 nm compared to that in suspensions [23]. As for the direct inactivation of SARS-CoV-2, at an ultraviolet C intensity of 0.849 mW cm−2, the decrease in the infectivity of the virus to below detectable levels also occurred with a more than two fold increase in the exposure time (dose) of the dried virus compared to the wet one [39]. Since the virus dried in the culture medium is a prototype of the virus settled with respiratory droplets and dried on the external surfaces, the data obtained must be taken into account when transferring the modes of virus photoinactivation in solutions to viruses in the environment. Reassuring is the fact that as we have shown in this study, increasing the time (dose) of irradiation leads to the complete virus photodynamic eradication on all four types of treated surfaces.
In terms of possible practical application, the procedure of photodynamic disinfection is quite simple, utilizes low micromolar PS concentrations, low-intensity far red light from a LED source, and the infected surface can be irradiated immediately after applying PS. This follows from the electrostatic nature of the binding of polycationic PS molecules to negatively charged regions on the surface of virions [29], which in model experiments proceeded in the millisecond time range. In suspensions, Zn-PcChol8+ caused photodynamic inactivation of not only SARS-CoV-2 [32], but also bovine coronavirus [33] and avian influenza virus [31] with close efficiency. Considering that enveloped viruses easily develop resistance to antiviral therapies, alternative methods of combating these pathogens are of undoubted practical interest in terms of photodynamic disinfection and controlling infectious diseases [24].
5. Conclusion
Coronavirus SARS-CoV-2, which remains infective when dried at room temperature on the surface of foil, cover glass, medical mask fabric or polystyrene, turned out to be sensitive to photodynamic inactivation with octakis(cholinyl)zinc phthalocyanine when wetted with a PS solution and irradiated with low-intensity far-red light from a LED source.
Acknowledgments
The reported study was funded by RFBR, Project Number 20-04-60084. Virological experiments were partly supported by Project RSF 19-74-10055-P. Work of G A Meerovich was partly supported by Ministry of Education and Science grant No. 075-15-2020-912.