INTRODUCTION
During the past few decades, the threat of infectious diseases has been on the rise. A recent example is the COVID-19 pandemic, which was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing high mortality (
1). Development of strategies to combat the transmission of these contagious pathogens is currently a global priority. During the COVID-19 pandemic, preventive strategies like the use of face masks, travel restrictions, surface disinfection, hand sanitization, and mass vaccination were used (
2). Vaccines reduced morbidity and mortality to a large extent but did not help eliminate the virus. Thus, to complement these strategies, we need novel antiviral surfaces that lower the virus load directly on contaminated surfaces, decreasing the probability of virus transmission.
Respiratory viruses, like coronaviruses, are transmitted mainly through large droplets and small aerosols from infected people when they breathe, cough, sneeze, and converse. Every cough or sneeze produces about 3,000 or 40,000 droplets, respectively, with an average viral load of 7 × 10
6 copies/mL, subject to the size of the droplet, severity of infection, and type of respiratory activity (
3). Smaller aerosols can stay in the air for a longer duration and actively transmit the virus, while larger droplets quickly settle on inanimate objects and contaminate the surface (
3). Contaminated fomite surfaces can be classified into two categories, i.e., porous and non-porous surfaces. Studies have suggested that viruses stay infectious on porous surfaces only from minutes to hours due to the capillary action of the pores and faster evaporation rate from the surface (
4). On the other hand, viruses may stay active on non-porous surfaces for days to several weeks (
5–7).
To reduce the viral load on non-porous surfaces like plastic, several disinfectants or surfactants, such as quaternary ammonium, sodium hypochlorite, and hydrogen peroxides, are used (
8). However, due to the health hazards associated with the regular use of strong disinfectants, laborious repetitive cleaning, crazing in plastics, and a direct, negative impact on biodiversity, we need safer and more sustainable options (
9,
10). Some strategies have been developed in the past to design self-cleaning plastics with anti-microbial properties (
11). However, these methods can be expensive and toxic to humans and the environment (
12). An alternate approach is to explore natural organic products as potential antivirals.
Coniferous tree-derived resin and rosin compounds have been explored for their therapeutic potential in the past (
13). Rosins come from tall oil, which is a side stream product obtained by a process called kraft pulping of coniferous tree barks (
14). Tall oil contains a mixture of 5%–50% fatty acids, 15%–55% resin acids, and 5%–35% unsaponifiable and neutral materials. Rosin components have demonstrated excellent anti-inflammatory, antitumor, antifungal, and antibacterial activities (
15–19). The antiviral nature of rosin and its components has also been recognized, for example, against influenza, respiratory syncytial virus, SARS-CoV-2, and herpes simplex virus (
20–22). Despite the widespread use of rosin compounds in various industrial applications, such as adhesives, rubbers, paints, coatings, soaps, detergents, paper cutting, varnishes, and emulsions, their potential as antiviral agents in applications has been relatively overlooked (
14).
In our study, we show that a plastic surface functionalized with tall-oil rosin effectively inactivates the seasonal human coronavirus OC43 (HCoV-OC43) and the more virulent SARS-CoV-2. Through molecular virology assays and different imaging modalities, we demonstrate that the rosin-functionalized plastic leads to efficient loss of virus infectivity by actively releasing rosin.
MATERIALS AND METHODS
Cells
Human lung fibroblast (MRC-5) cells and green monkey kidney epithelial (Vero E6) cells were both obtained from the American type of culture collection (ATCC, Manassa, VA, USA). Both cell lines were propagated in Eagle’s Minimum Essential Medium (MEM) (Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (Gibco, Paisley, UK), 1% L-GlutaMAX (Gibco, Paisley, UK), 1% antibiotics (penicillin/streptomycin) (Gibco, Paisley, UK) and stored in a humidified 5% CO2 incubator at 37℃.
Viruses
Severe acute respiratory syndrome coronavirus 2 used in our experiments was kindly provided by the University of Helsinki. The SARS-CoV-2 virus (SARS-CoV-2/Finland/1/2020) was an isolate from the first COVID-19 patient in Finland (GenBank:
MT020781.2) (
23). HCoV-OC43 (ATCC VR1558) was obtained from the American Type Culture Collection (Manassa, VA, USA). The virus was cultured in MRC-5 cells containing 2% MEM with a multiplicity of infection (MOI) of 2. The inoculum was replaced after 2 h of infection with fresh 2% MEM. The supernatant containing the cultured crude virus was collected 2 days post-infection (p.i.). The cell debris was pelleted using a swing-out rotor at 2,700 ×
g for 3 min at room temperature (RT) (Heraeus Megafuge 1.0 R, Germany). The supernatant was flash frozen and stored as a crude working stock at −80°C.
Purification of HCoV-OC43 virus
The purification protocol for the HCoV-OC43 virus was adapted and modified from Dent et al. (
24). A subconfluent layer of MRC-5 cells cultivated in a 175 cm
3 flask was infected with HCoV-OC43 (ATCC) at an MOI of 3 for 72 h, after which the supernatant was collected and the cell debris was pelleted by centrifugation at 10,000 ×
g at 4℃ for 20 min. The virus in the supernatant was precipitated using 10%, wt/vol polyethylene glycol 6000 and 2.2% of NaCl (
25). The solution was stirred for 30 min at 4℃, after which the precipitate was centrifuged at 10,000 ×
g at 4℃, for 30 min. The pellet was dissolved in 3 mL HEPES saline buffer [1 mM HEPES pH 6.7, 0.9% NaCl (wt/vol)] and stored on ice. The solution containing the virus appeared viscous in consistency. The virus was concentrated by pelleting through a stepwise sucrose gradient (10%–20%–30%) at 100,000 ×
g at 4 ℃ for 2 h. The gradient was prepared by adding 3 mL of 30% sucrose to the bottom, followed by the same amount of 20% and then 10% on the top. The pellet was dissolved in 100 µL of cold HEPES saline buffer and stored at −80 ℃. The infectivity of the virus batch was calculated using the end-point titration method (
26).
Plastic samples
We used two types of plastic surfaces for our study: low-density polyethylene (standard LDPE) and PREXELENT, generously provided by Premix Ltd. The plastic surfaces were dimensioned as 1 cm2. PREXELENT is a functional plastic that is prepared by incorporating 10 wt% of rosin extracted from coniferous trees into standard LDPE (patent WO2018229190A1). Throughout our article, we will refer to PREXELENT as rosin-functionalized plastic and in the figure sets as rosin. Standard LDPE was chosen as a control to compare the results of the rosin-functionalized plastic. “Conifer pitch,” which is the active ingredient inside the rosin-functionalized plastic, was also provided in powder form by Premix Ltd. Additionally, a variety of industrial grade plastic varieties like acrylonitrile butadiene styrene (ABS), polyamide (PA6), polycarbonate (PC), polypropylene (PP), and polymethyl methacrylate (PMMA) were obtained via Aikolon Ltd. Prior to the experiments, all samples were sterilized with 70% ethanol for 30 s.
Infectivity of HCoV-OC43 studied on different surfaces using a cytopathic effect inhibition assay and RT-qPCR
The persistence of HCoV-OC43 and the antiviral effect of the rosin-functionalized plastic against HCoV-OC43 were studied using the cytopathic effect (CPE) inhibition assay, modified from Schmidtke et al. (
27). The experiments were conducted following the international standard ISO 21702, which is a test method to determine the antiviral activity on plastic surfaces, with slight modifications. Ethanol-sterilized plastic samples were added to a 12-well plate with relative humidity (RH) of ~92%, maintained using 6 mL of ddH
2O. The RH maintained inside the plate assembly was measured with a commercial RuuviTag wireless sensor (Ruuvi Ltd, Finland). MRC-5 cells were seeded at a density of 15,000 cells/well in a 96-well flat-bottomed microtiter plate (Sarstedt, Numbrecht, Germany) and allowed to grow overnight (O/N) in an incubator at 37°C, with 5% CO
2. On the following day, a droplet of 5 µL of HCoV-OC43 virus (1.60 × 10
4–10
5 PFU) was loaded onto the different plastic surfaces and incubated for different time points (24, 48, and 72 h) at RT and ~92% RH inside the 12-well plates. We used several controls in the experiment (i) an equivalent amount of virus without any surface treatment was used as a positive control. (ii) MRC-5 cells without any virus infection were used as a negative control (or mock infection). (iii) Additionally, a sample control without the addition of the virus was used to test the cytotoxicity of the surfaces on MRC-5 cells. A glass coverslip was gently placed on the virus-loaded surfaces to ensure maximum contact of the virus with the surface. At the end of each incubation time point, the surface was rinsed with 995 µL of 2% MEM, followed by gentle rocking of the 12-well plate for 1 min to facilitate the detachment of the virus from the surface. The medium containing the flushed virus was added to the cultured MRC-5 cells (MOI: 0.01–0.1) in a 96-well plate. The cells were incubated for 5 days in a humidified 5% CO
2 incubator at 34°C until the cytopathic effect (rounding and detachment of cells) was seen. After the development of the cytopathic effect, the cells were washed twice with phosphate buffer saline (PBS), after which they were stained with a CPE stain (containing 0.03% crystal violet, 2% ethanol, and 3.5% formaldehyde in ddH
2O) for 12–15 min. Next, the cells were washed twice with ddH
2O to get rid of excess stain, and finally, the cells were lysed for 5 min to elute the stain out of healthy cells with the help of CPE lysis buffer (0.03 M sodium citrate and 1 N HCl in 47.5% ethanol). Thereafter, the viable cells were quantified by measuring the absorbance of the stain in the 96-well plate at 570 nm (Victor X4 2030 Multilabel Reader, PerkinElmer). The amount of infectious viral RNA in the sample was also quantified using RT-qPCR from the cell culture supernatant collected 3 days p.i. The protocol for RT-PCR and qPCR has been elaborately described in “Quantification of HCoV-OC43 viral RNA directly flushed from the surfaces using RT-qPCR” section.
The effect of humidity on the antiviral function of the rosin-functionalized plastic was studied similarly as above, with the exception that the incubation of the virus-loaded surface was done inside a custom-made chamber fitted in a laminar in the BSL2 facility, capable of accurate measurements of temperature and relative humidity (Kenttäviiva Ltd, Finland). In addition, the coverslip was not used on the surface in order to allow various humidities to act on the surface and on the viruses. During the experiment, the RH was controlled to either 20% or 40% and the incubation times were shorter (5, 15, 30, and 60 min) compared to those tested earlier.
Persistence of SARS-CoV-2 measured using RT-qPCR
The persistence studies of SARS-CoV-2 on the standard LDPE and rosin-functionalized plastic were carried out in a Biosafety level-3 unit. Vero E6 cells were grown in a 96-well flat-bottomed microtiter plate, with each well containing 50,000 cells. The cell cultures were maintained for 24 h at 37°C in an environment with 5% CO2. On the following day, a 5 µL droplet (5 × 103 PFU) of the SARS-CoV-2 was added on top of both the surfaces and incubated for different time points (5 min, 30 min, 1 h, 2 h, and 4 h) at RT and ~92% RH. At the end of each time point, the surface containing the virus was flushed with 2% MEM and gently mixed for 1 min to detach any virus from the surface. The flushed virus was then added to the Vero E6 cells at an MOI of 0.005. The cells were then placed in a humidified incubator with 5% CO2 at a temperature of 34°C for a 3-day incubation period. After 3 days post-infection, the supernatant was collected and the viral RNA was extracted.
The RNA from the supernatant was extracted using the Chemagic viral DNA/RNA kit (Perkin Elmer) and the Chemagic 360 instrument. For the RNA extraction, a master mix containing Poly (A) RNA, Proteinase K, and Lysis buffer 1 was mixed in the proportion mentioned inside the kit by the manufacturer. The samples were initially diluted 10 times in ddH2O. The diluted samples and the master mix were added into deep-well Chemagic plates. In a different low-well plate, 15 µL of magnetic beads was pipetted, and in a deep-well Chemagic plate, 80 µL of elution buffer was added. The RNA was extracted using the Chemagic 360 machine as per the manufacturer’s instructions. The extracted RNA was stored at −80°C. After the RNA isolation, a TaqMan-based real time RT-PCR assay was used to transcribe the viral RNA, amplify it, and detect the SARS-CoV-2 genes using the SARS-CoV-2 RT-qPCR reagent kit (Perkin Elmer, Turku, Finland).
In order to compare the relative amounts of viral RNA (number of infective viruses) present in the culture supernatant after 3 days of cultivation in cells, we used the Cq values obtained from the qPCR experiment. Cq values obtained from the qPCR reaction of the virus flushed from the standard LDPE and rosin-functionalized plastic surfaces were placed into the following self-derived equation to obtain the difference in the RNA amount, ΔRNA:
where ΔCq is the Cq value difference between the mean of the rosin-functionalized plastic surface to the mean of the standard LDPE surface. A log value of ΔCq was then calculated to describe the difference in virus amounts.
Statistical analysis
The results from the CPE assays were plotted as a column graph of cell viability, and the statistical analysis was performed using GraphPad Prism software 6 (GraphPad Software, San Diego, CA, USA). The CPE results were normalized against the mock infection and are presented as mean + standard error of the mean (SEM). The statistical significance was calculated by performing a one-way analysis of variance (ANOVA) of all test samples against the virus control followed by the Bonferroni tests (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
Quantification of HCoV-OC43 viral RNA directly flushed from the surfaces using RT-qPCR
A droplet of 5 µL of HCoV-OC43 (1.60 × 105 PFU) was loaded onto the standard LDPE and rosin-functionalized surface and incubated for different time points (1, 5, 15, and 30 min and 1, 2, and 4 h) at RT, ~92% RH inside 12-well plates. A similar amount of virus without any surface treatment was used as a positive control. At the end of each time point, the virus on the surface was rinsed with 995 µL of culture media (2% MEM) and gently agitated for 1 min to facilitate the detachment of the virus from the surface. RT-qPCR was then used to measure the amount of viral RNA in the flushed samples. Processing of the flushed samples to measure the viral RNA was done through the following steps: (i) the samples were diluted five times with RNase-free water (J71768, Thermo Fisher Scientific, Geel, Belgium) and heat treated for 5 min at 75°C to release the RNA from the viruses. (ii) Reverse transcription (RT’) was carried out to make cDNA from the viral RNA using 20U M-MLV reverse transcriptase enzyme (Promega, WI, USA), RT-buffer (Promega, WI, USA), RNase-free water (J71768, Thermo Fisher Scientific), 4U RNAsin ribonuclease inhibitor (Promega, WI, USA), 0.5 mM dNTPs (Promega, WI, USA), and 1.2 µM reverse primer (5′-AATGTAAAGATGRCCGCGTATT) (Merck, Darmstadt, Germany). The RT’ reaction was carried out for 1 h at 42°C, and then the RT’ enzyme was inactivated at 70°C for 10 min. (iii) Finally, the cDNA was amplified using qPCR. A master mix containing SYBR Green Supermix (BioRad), 600 nM forward (5′-TGTTAGGCCRATAATTGAGGAC) (Merck, Darmstadt, Germany) and reverse primers (5′-AATGTAAAGATGRCCGCGTATT) (Merck, Darmstadt, Germany) and the RNase-free water was prepared as per the manufacturer’s protocol. cDNA template (5 µL) from the RT’ reaction was added to the prepared master mix and run through the Touch Thermal Cycler (BioRad C100, BioRad, Helsinki, Finland). The amplification steps were as follows: 10 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 50°C, 5 s at 72°C, 1 min at 95°C, followed by cooling at 12°C for 10 min.
Transmission electron microscopy
Purified HCoV-OC43 (10 µL of 1.09 × 10
8 PFU) was added on the sterilized standard LDPE and rosin-functionalized plastic and incubated for 1 h at RT with ~92% RH. Post-incubation, 10 µL of PBS was added to the surface and the droplet was gently mixed. A virus control of the same amount without any surface treatment was also prepared. Formvar-coated copper grids were glow discharged (EMS/SC7620 mini-sputter coater), and negative staining was done as previously described (
28). Briefly, 5 µL of virus (control, standard LDPE, and rosin-functionalized plastic) was added onto the copper grids for 30 s and blotted with Whatman paper (Whatman 3 MM). The samples were negatively stained using 1% (wt/vol) phosphotungstic acid for 10 s and blotted. The samples were dried O/N before imaging in a JEOL JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a LaB
6 filament. The images were captured using a bottom-mounted Quemesa 4,008 × 2,664-pixel CCD camera.
Atomic force microscopy
A volume of 10 µL of purified HCoV-OC43 virus (1.09 × 107 PFU) was deposited on the standard LDPE and rosin-functionalized plastic surfaces and incubated for 1 h at RT with a RH of ~92%. Post-incubation, 10 µL of 2.5% glutaraldehyde (GA) was added to fix the virus and maintain the morphology. Post-fixation, the droplet containing the virus was transferred onto the silicon surface that was pretreated with air (oxygen/nitrogen) plasma (Electron Microscopy Sciences, Hatfield, PA, USA) and 0.01% poly-l-lysine for 15 min to enhance the attachment of the viruses to the surface. Virus without any surface treatment was used as a control and was added directly onto poly-l-lysine-coated silicon chips for atomic force microscopy (AFM) measurements. Bruker Dimension Icon atomic force microscope (Bruker Corporation, Billerica, MA, USA) and Scanasyst fluid tips (nominal radius 20 nm) were used during the liquid AFM measurements. Peak force quantitative nanomechanical mapping mode was used in all the measurements. Imaging was done in PBS (scan size of 5 µm, scan rate varying between 0.332 and 0.939 Hz, and peak force amplitude between 100 and 200 nm). The peak force setpoint was kept below 1 nN and the scan rate, peak force, and peak force amplitude were adjusted to minimize the forces between the tip and the virus to obtain the best image quality. Bruker analysis software 1.9 version was used to plot and analyze the data. In addition, the data were plotted and tested for statistical significance in Origin Pro 2022. Viruses that appeared in large clusters were excluded from the measurements.
Helium ion microscopy
A 5 µL droplet of HCoV-OC43 virus (5.45 × 106 PFU) was loaded onto the standard LDPE and rosin-functionalized plastic surfaces on ice and fixed in 2 mL of 2.5% GA + 0.1 M sodium cacodylate (NaCaC) solution for 30 min. After three washes with 0.1 M NaCaC solution, 3 min each, the surfaces were stained for 30 min with 1% OsO4 prepared in 0.1 M NaCaC. After two washes with 0.1 M NaCaC solution, the surfaces were subjected to increasing ethanol series (50%, 70%, 96%, and 99%) and dried using a critical point dryer (Leica Microsystems EM CPD300). The samples were stored in a vacuum chamber until they were taken for helium ion microscopy (HIM) (Zeiss Orion Nanofab) imaging, where the acceleration voltage of 30 kV and an aperture of 10 mm were used. Spot size 6 was used, giving an ion current of 0.2 pA. It was possible to image the plastic samples without a flood gun, but on the rosin-functionalized plastic, the charge compensation was used with line averaging. Imaging was done from an 85° tilted angle. The aspect ratio of the particles was calculated by measuring their maximum height and the width of the single particles from the images.
Hydrophobicity measurements
A water droplet of 40 µL was added on top of the standard LDPE and rosin-functionalized plastic. Images of the droplet were taken from the side and top angle using a smartphone camera (Xiaomi MI 8). The contact angle was measured from the base of the surface using Inkscape drawing software (Inkscape Project 2020).
Cryo-electron microscopy
Purified HCoV-OC43 virus (5 µL, 5.45 × 10
7 PFU) was added to the surfaces and incubated for 1 h at RT with ~92% RH. Post-incubation, 5 µL of HEPES saline was added to the surface and the droplet was gently mixed. A virus control without any surface treatment and with the same amount was also prepared. A total of 10 µL of the virus collected from the surface was exposed to ultraviolet (UV)-C irradiation at 100 mJ cm
−2 using a UVP crosslinker CL-1000 (Analytik Jena, Jena, Germany) at A
254 nm for inactivation before plunge freezing (
29). The virus samples were loaded on a glow-discharged Quantifoil electron microscopy grid with 2 nm carbon film R1.2/1.3, mesh 300 and incubated for 1 min before plunging at 22°C, 85% humidity, and 1.5 s blotting time with the Leica EM GP plunger. Cryo-electron microscopy (CryoEM) data were collected using a FEI Talos Arctica field emission transmission electron microscope (TEM), equipped with a Falcon III direct electron detector at the Instruct-FI Centre’s CryoEM Core Facility. Data were collected in counting mode at 150,000× nominal magnification at a 0.97 Å/pixel sampling rate using a total dose of 30 e
− Å
−2. Movies were motion corrected using Relion’s motion correction (
30–36) implemented in Scipion 3.0.12 (
37,
38) and the contrast transfer function was estimated using ctffind4 (
39–41). Images were further adjusted in Corel Draw 2023 to improve the contrast of viral features.
Sedimentation assay
Genome release from the virus capsid was studied using ultracentrifugation. For this experiment, 5 µL of the purified HCoV-OC43 virus (2 × 106 PFU) was added to the surface with a glass coverslip and incubated for 1 h at RT with ~92% RH. Post-incubation, the samples were flushed with 2% MEM and collected into polyallomer centrifuge tubes (Beckman, Palo Alto, CA, USA). The flushed content was ultracentrifuged (A-95 rotor, 20 psig, 30 min) using the Beckman Coulter Airfuge centrifuge (Brea, CA, USA). A virus of the same amount, without any pretreatment on the surface, was used as an experimental control. Post-centrifugation, the supernatant was collected in a separate tube, and the pellet was redissolved in 2% MEM. The RNA from both the pellet and supernatant was isolated using the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany, ref. 52906). The isolated RNA was converted into cDNA and amplified using RT-PCR and qPCR as described above. The cDNA was diluted 100 times before performing the qPCR.
Binding assay
MRC-5 cells were cultured in a 6-well plate with a flat glass bottom (Fisher Scientific) at a density of 350,000 cells/well and incubated O/N at 37°C. On the subsequent day, 10 µL of the purified HCoV-OC43 virus (1.89 × 107 PFU) was added to the surface with a glass coverslip and incubated for 1 h at RT with ~92% RH. Post-incubation, the samples were flushed with 2% MEM and the flushed virus was added to the MRC-5 cells on ice for 1 h while rocking. After three washes with 0.5% BSA/PBS, 5 min each, the 6-well plate was subjected to three cycles of freeze-thaw by alternating between −80°C and RT to detach the cells from the bottom of the plate. The viral RNA from the virus bound to cells was isolated using the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany, ref. 52906). The isolated RNA was converted into cDNA and amplified using RT-PCR and qPCR as described above. The cDNA was diluted 1,000 times before performing the qPCR.
Immunofluorescence and confocal microscopy
Confocal microscopy was applied to study the different stages of the virus infection cycle. Purified HCoV-OC43 virus (5 µL, 2 × 106 PFU) was added to the surfaces and incubated for 1 h at RT with ~92% RH. Viruses flushed from the surfaces were added onto cells (MOI: 100) into 96-well plates and kept at RT for 1 h, followed by 1 h at 34°C. Infection was allowed to proceed for 1 or 15 h at 34°C and then the cells were fixed with 4% paraformaldehyde in PBS. Cells were permeabilized with 0.2% Triton X-100 for 5 min, followed by antibody labeling with affinity-purified rabbit monoclonal antiserum against the S protein (1:16 diluted), mouse monoclonal antisera against human beta-tubulin (#sc-58886, Santa Cruz Biotechnology) or J2 (Scicons, Hungary) against the dsRNA. The secondary antibodies used were Alexa Fluor 555 goat polyclonal IgG against rabbit (catalog no. A-21429; Thermo Fisher Scientific) and Alexa Fluor 488 goat polyclonal IgG against mouse (catalog no. A-21121; Thermo Fisher Scientific). The second PBS wash was supplemented with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen/Molecular Probes, ref. D3571) (1:40,000) in PBS to stain the cellular nucleus. All antibody dilutions were made in 3% BSA in PBS. The cells were imaged using a Nikon A1R confocal microscope. Images were taken with a 40× objective and numerical aperture of 1.25. Three lasers were used: a 405 nm diode laser, a 488 nm multiline argon laser, and a 561 nm sapphire laser. The virus spike protein and dsRNA channels were adjusted with the cell control, such that the cell control had a very low signal. Several images were taken using the automation option in the software to accumulate at least 700 cells per case. The montages from the images were made with Fiji2 (ImageJ). Cell profiler 4.2.1 was used to quantify the amount of infection in the wells. The nuclei were selected as primary objects, and the infected cells were then identified as secondary objects if they were present around the previously identified primary object (nuclei). The primary objects were identified using the Otsu thresholding method. Once the primary and secondary objects were classified, the software automatically calculated the area and the intensity of the secondary objects, which were then exported as an Excel data sheet. At this point, manual thresholding was done to differentiate infected cells from non-infected cells having some background signal. The data were subsequently plotted as a column graph and represented as infection (%).
UV-vis spectroscopy
To measure the rosin content secreted to the flush medium, rosin-functionalized plastic was flushed with ddH2O for 1 h and O/N using gentle rocking. As a reference of the patented compound embedded inside the rosin-functionalized plastic, Premix Ltd. provided us with a powdered sample of elemental conifer pitch (secreted and cured oleoresin mixture) that was dissolved in 94%, vol/vol of ethanol and 10-fold serial dilutions were made to obtain a final concentration of 0.0001%, wt/vol of conifer pitch. The UV-vis spectra of the flushed material without any further dilution were measured with LAMBDA 850 UV-Vis spectrometer (Perkin Elmer Life Sciences) and Quartz cuvettes (Hellma) with optical pathlength of 1 cm. The measured raw spectra were background corrected with the measured spectrum of EtOH using Origin 2022 software.
DISCUSSION
Respiratory viruses can spread through direct and indirect transmission routes. While various strategies are in place to limit direct transmission, such as travel restrictions, usage of face masks, and routine sanitization practices, there are fewer options available to address the indirect route of transmission through fomite surfaces. Repeated surface disinfection and the use of biocidal agents are commonly employed measures. The indirect route of virus transmission depends on several factors, most notably, the ability of the virus to remain active on the surface of a material. It is thus important to prepare for future outbreaks by designing antiviral surfaces that would effectively reduce the spread in the community. Functionalizing surfaces with biologically derived materials from nature to reduce indirect transmission is still relatively uncommon. However, due to their natural origin, such materials hold great promise as sustainable alternatives for microbial inactivation.
When studying antiviral surfaces, it is essential to first have a detailed understanding of the persistence of the virus on different fomite surfaces. Among the different known fomite surfaces, plastic demands particular attention. Although plastic is susceptible to degradation by disinfectants and UV-C, different grades of plastic have widespread use in household, commercial, and industrial settings. In the past, several studies have demonstrated good persistence of different human coronavirus species on plastic surfaces. For instance, Warnes and colleagues reported infective HCoV-229E on PTFE and polyvinyl chloride plastic surface for 5 days at 21°C and RH of 40%–50% (
42). Independent studies by Duan et al. (
43) and Rabenau et al. (
44) reported the persistence of SARS-CoV (strain P9 and FFM1) on plastic at RT for up to 4 and 6 days, respectively (
43,
44). Rabenau and colleagues reported further that the HCoV-229E could persist on plastic only for 24 h at RT. These variations in coronavirus persistence can be attributed to the interplay of multiple factors such as environmental conditions (temperature, relative humidity, wind speed, etc.), type and surface topology of the plastic, virus type and concentration, and mode of deposition used in the experiments (
11). While some publications do not specify the type of plastic being used in the study and generalize it as just “plastic,” the type of polymers may affect the surface properties, such as hydrophobicity/hydrophilicity, which could also affect the persistence. While we lack standardized methods and conditions for testing, all the previous studies suggest that non-functionalized plastic retains the infectivity of coronaviruses well.
We focused here to explore the antiviral properties of a plastic surface functionalized with rosin (resin acid mixture) against human coronaviruses. HCoV-OC43 was chosen as a model for the study due to its structural and genetic similarity to the more virulent SARS-CoV-2 (
45), allowing for work in a BSL-2 laboratory, although we also verified the results with SARS-CoV-2 as well. All the non-functionalized surfaces sustained high virus infectivity for the first 48 h, which was expected in the light of the previous studies (
42–44). However, strikingly, the rosin-functionalized plastic exhibited remarkable antiviral efficacy against both the seasonal coronavirus and SARS-CoV-2 already within 15 min of contact time. Previously, several efforts have been made to add antiviral properties to surfaces, e.g., through functionalization with biocidal compounds (
46), antiviral polymers (
47,
48), metallic and nanoparticle coatings (
49), and surface texturing (
46,
50). In a study performed by Zhou and colleagues (
50), an antiviral plastic surface designed by nanopatterning and coating of silver nanoparticles showed antiviral effect against SARS-CoV-2 after 1 h of contact time. In another recent study by Butot and colleagues (
51), the antiviral activity of quaternary ammonium compounds (QACs) and copper-coated polyethylene terephthalate surface against HCoV-229E and SARS-CoV-2 was shown. Both surfaces were able to reduce viral titers, but the QAC-coated surface did not work after one round of washing. Here, we present a more sustainable solution using rosin-functionalized plastic. This surface showed an excellent 3–4 log reduction of SARS-CoV-2 and low toxicity against cells.
The survival time of the virus on a surface is influenced by various factors, among which RH plays a crucial role (
11). RH can vary significantly across different seasons and indoor environments, impacting virus persistence. Previous studies have reported diverse patterns of virus persistence under different conditions of RH. For instance, Biryukov and colleagues (
52) observed a linear decrease in SARS-CoV-2 persistence on an ABS plastic surface with increasing RH. In contrast, Morris and colleagues (
53) found that the SARS-CoV-2 persisted for a longer period at extreme humidities, indicating a U-shaped relation with RH. However, we observed little effect of RH on the antiviral efficacy of the rosin-functionalized surface. The virus was inactivated at an earlier time point at lower humidities of 20% and 40% compared to higher humidity levels of 92%. However, rosin-functionalized plastic showed excellent inactivation within the first 30 min of contact with the functionalized surface in all tested humidities.
To gain a better understanding of the antiviral mechanism of the rosin-functionalized plastic, our study employed advanced imaging techniques such as AFM, TEM, HIM, and cryoEM to investigate the status and morphology of the flushed viruses. Interestingly, our investigations did not reveal any apparent structural changes in the virus, which could explain the observed loss of infectivity. This suggested that the rosin-functionalized surface had minimal impact on the size, shape, and physical dimensions of individual virus particles. Additionally, we investigated the structure of the virus on rosin-functionalized plastic using HIM. Notably, we observed distinct differences between the virus on the functionalized and the non-functionalized surfaces. On the functionalized surface, the viruses appear flatter in shape. This observation could be explained by the contact angle measurements, which suggested that the functionalized plastic was more hydrophilic (contact angle: 55°) compared to the non-functionalized one (contact angle: 95°). The hydrophilic nature of the functionalized plastic caused the droplets containing the virus to spread out, resulting in the flattening of the viruses. Despite this flattening, flushed viruses from rosin-functionalized surface did not appear different from the viruses flushed from the non-functionalized plastic, suggesting that the hydrophilicity may play a minor role in killing the virus infectivity.
It is known that viruses tend to persist for longer periods on hydrophobic surfaces as opposed to hydrophilic surfaces (
4). This behavior has been explained by Paton and colleagues (
54), who proposed that on hydrophobic surfaces, droplets dry in a beaded shape, creating a higher volume-to-surface ratio. This drying process brings the virus particles, organic material, and salts in proximity, offering a protective environment against desiccation. Owen and colleagues (
55) suggest further that faster desiccation from hydrophilic surfaces can potentially alter the pH and salt concentration inside the droplet, thereby affecting the stability of the lipids in the viral envelope. However, the hydrophilicity of the rosin-functionalized surface does not solely explain the observed antiviral effect. Namely, the quantification of viral RNA using qPCR demonstrated that the virus added to the surface was easily flushed off into the surrounding media, indicating a weak and temporary interaction between the virus and the hydrophilic surface.
Our imaging results as well as the fact that even short periods of encounter with the functionalized surface caused a strong antiviral effect, both pointed out to the direction that active rosin leaching out from the surface could be the cause of action. Indeed, we could demonstrate through absorption spectroscopy that the active ingredient inside the functionalized plastic leached out into the flush medium. Importantly, the leached flush was independently capable of causing the antiviral effect. Thus, it is likely that the leached rosin components contribute to most of the antiviral effect.
Previously, in addition to SARS-CoV-2, other enveloped viruses such as influenza A virus and respiratory syncytial virus have been shown to be affected by products derived from tall oil. However, non-enveloped viruses like encephalomyocarditis virus (EMCV) were not affected (
20). Bell and colleagues, therefore, proposed that the lack of efficacy against non-enveloped viruses suggests the involvement of the lipids in the viral envelope by inhibiting the fusion between the viral envelope and host cell membranes. As bacteria also have membranes, it is no wonder that resin could have effects also on bacterial survival. Indeed, Sipponen and colleagues (
56) showed the effect of Norway spruce-derived resin salves on bacterial membranes. The interaction of the resin salve caused the thickening of the bacterial membrane in
Staphylococcus aureus. They additionally reported changes in the branching of the fatty acids and an altered membrane potential. Another study by Jokinen and Siponen (
57) also demonstrated the antibacterial activity of refined spruce resin in treating chronic wounds. Tall oil rosin is a mixture of eight closely related resin acids, of which abietic acid and dehydroabietic acid have been more heavily studied for their antimicrobial properties. In a study by Agudelo-Gomez and colleagues (
21), they not only showed antiherpetic activity (HHV-1 and HHV-2) of commercial abietic acid and dehydroabietic acid but also developed their analogs that displayed enhanced antiviral activity. Another study by Fonseca and colleagues (
58) demonstrated the antiviral activity of dehydroabietic acid and their analogs against varicella-zoster virus and cytomegalovirus. We have preliminary results showing excellent antiviral efficacy of the rosin-functionalized plastic against the African Swine Fever Virus (J. D. Hemmink, S. Shroff, N. Chege, M. Haapakoski, L. K. Dixon, and V. Marjomäki, unpublished results). As this virus is known to be very difficult to decontaminate, these results suggest that rosin-functionalized plastic may also serve as an antiviral surface for more stable viruses and a larger variety of enveloped viruses. Furthermore, rosin chemicals are biobased, easy to apply to conventional polymer matrices, available in bulk, and very affordable, especially in comparison to several proposed nanomaterial applications. So, the industrial applications of this approach have promise in locations where surface contact plays a role in the transfer of pathogens like public spaces, hospitals, shops, transport, etc.
Using confocal microscopy, we show that, despite the 1 h encounter on the rosin surface with HCoV-OC43 and the resulting total block in infectivity, the viruses were successful in binding to MRC-5 cells. This indicates that the leached rosin components were not altering the functionality of the spike proteins to promote receptor binding. This is in line with our cryoEM evaluation of the flushed viruses, which showed intact spikes that were mostly in the prefusion stage. As the binding to receptors was not inhibited, the virions were able to proceed and enter cytoplasmic vesicles. However, importantly, post entry, the viruses were stuck inside vesicles and did not proceed with replication and translation in the cytoplasm, which was demonstrated by confocal labeling of replication intermediates. It thus seems obvious that the block in infectivity was in the endosomal membrane fusion step that occurs between the viral envelope and the endosomal membrane. The two major sites on the virus surface involved in membrane fusion are the S2 subunit of the viral spike protein and the viral lipid-rich envelope (
59). The S2 subunit has two heptad repeats HR1 and HR2 that form a six-helix bundle, which are responsible for membrane fusion and genome release. In addition to a possible direct effect on spike proteins and their processing, the viral membrane is likely to be affected due to interactions between the envelope lipids and the bioactive rosin components. This study was not able to pinpoint the mechanism of action by the leaching rosin; however, it did confirm its effective response in inactivating viruses on treated surfaces. It will be remarkably interesting to study and identify in the future what exact steps in the fusion process are affected by the rosin components.
Through this study, we demonstrate that the plastic surface functionalized with rosin effectively compromises the infectivity of coronaviruses. Importantly, the rosin-functionalized surface leaches enough rosin components to kill the fusion capability of coronaviruses when they have been in contact with the surface even for a relatively brief time. Due to the conserved nature of membrane fusion of enveloped viruses from different virus families, this rosin-functionalized plastic might serve as a broadly acting antiviral solution for a larger variety of viruses. Altogether, we propose that the rosin-functionalized plastic is a promising candidate as an antiviral surface for enveloped viruses.