Antiviral action of a functionalized plastic surface against human coronaviruses

Abstract

Viruses may persist on solid surfaces for long periods, which may contribute to indirect transmission. Thus, it is imperative to develop functionalized surfaces that will lower the infectious viral load in everyday life. Here, we have tested a plastic surface functionalized with tall oil rosin against the seasonal human coronavirus OC43 as well as severe acute respiratory syndrome coronavirus 2. All tested non-functionalized plastic surfaces showed virus persistence up to 48 h. In contrast, the functionalized plastic showed good antiviral action already within 15 min of contact and excellent efficacy after 30 min over 90% humidity. Excellent antiviral effects were also observed at lower humidities of 20% and 40%. Despite the hydrophilic nature of the functionalized plastic, viruses did not adhere strongly to it. According to helium ion microscopy, viruses appeared flatter on the rosin-functionalized surface, but after flushing away from the rosin-functionalized surface, they showed no apparent structural changes when imaged by transmission electron microscopy of cryogenic or negatively stained specimens or by atomic force microscopy. Flushed viruses were able to bind to their host cell surface and enter endosomes, suggesting that the fusion with the endosomal membrane was halted. The eluted rosin from the functionalized surface demonstrated its ability to inactivate viruses, indicating that the antiviral efficacy relied on the active leaching of the antiviral substances, which acted on the viruses coming into contact. The rosin-functionalized plastic thus serves as a promising candidate as an antiviral surface for enveloped viruses.IMPORTANCEDuring seasonal and viral outbreaks, the implementation of antiviral plastics can serve as a proactive strategy to limit the spread of viruses from contaminated surfaces, complementing existing hygiene practices. In this study, we show the efficacy of a rosin-functionalized plastic surface that kills the viral infectivity of human coronaviruses within 15 min of contact time, irrespective of the humidity levels. In contrast, non-functionalized plastic surfaces retain viral infectivity for an extended period of up to 48 h. The transient attachment on the surface or the leached active components do not cause major structural changes in the virus or prevent receptor binding; instead, they effectively block viral infection at the endosomal stage.

Keywords: antiviral surface; human coronavirus; plastic; tall oil rosin; virus persistence.

Fig 1

Persistence of HCoV-OC43 on (A) different plastic surfaces measured up to 72 h. Virus titer on various plastic surfaces—1.6 × 10⁴ PFU. (B) Standard LDPE and rosin-functionalized plastic measured up to 24 h of contact time with the surface. Virus titer on standard LDPE and rosin-functionalized plastic surface—1.6 × 10⁵ PFU. In both (A) and (B), a CPE assay was conducted to assess cell viability post-infection, and the treated samples and virus control were normalized against the cell control. The experiments were replicated three times, with each condition having three technical replicates. (C) Infectivity of HCoV-OC43 and SARS-CoV-2 on standard LDPE and rosin-functionalized plastic, measured up to 24 and 4 h of contact time, respectively, using RT-qPCR (see Materials and Methods). The qPCR experiment was conducted twice, each with three technical replicates. For all panels (A through C), the results are presented as average values + SEM. Statistical analysis employed one-way ANOVA, followed by the Bonferroni test (*P < 0.05, ***P < 0.001, and ****P < 0.0001). ns, not significant; VC, virus control; CC, cell control; and cyt, cytotoxicity.

Fig 2

The impact of lower humidity (A) 20% and (B) 40% on the antiviral activity of the rosin-functionalized plastic surface against HCoV-OC43. The experiments were conducted three times, with each condition including three technical replicates. The CPE results are presented as average values + standard error of the mean. Statistical analysis utilized one-way ANOVA, followed by the Bonferroni test (****P < 0.0001). ns, not significant; VC, virus control; and CC, cell control.

Fig 3

Detection of HCoV-OC43 viral RNA using qPCR from samples flushed from standard LDPE and rosin-functionalized plastic, measured at various contact time points. The experiments were conducted twice, including three technical replicates. VC, virus control.

Fig 4

Studying the impact of rosin-functionalized plastic and standard LDPE on the structure of HCoV-OC43 using (A) TEM and (B) AFM in liquid. The scale bar corresponds to 100 nm and 1 µm in the TEM and AFM images, respectively. In panel B, the blue circle highlights a doughnut-shaped virus. (C) The histogram derived from the AFM images illustrates the average size distribution of the height of individual viruses after being flushed from their respective surfaces. The average mean heights for the control virus, viruses flushed from standard LDPE, and rosin-functionalized plastic were 60.1 ± 1.6, 54.7 ± 0.8, and 59.9 ± 1.3 nm, respectively.

Fig 5

(A) HIM images of HCoV-OC43 on (i) standard LDPE and (ii) rosin-functionalized plastic. The viruses underwent a 1-h treatment on these surfaces before imaging with the HIM. Scale bar: 200 nm. (B) Camera-captured images of a water droplet on the surfaces of standard LDPE (left) and rosin-functionalized plastic (right) to measure the droplet contact angle. (i) Side view and (ii) top view.

Fig 6

Investigation of HCoV-OC43 viral genome release through (A) CryoEM and (B) qPCR. (A) Representative CryoEM micrographs of virus control and from viruses flushed from LPDE and rosin-functionalized plastic. The arrows indicate a prefusion conformation spike, while “N” indicates the nucleocapsid containing the genome. Over 700 images (each with two to three viruses) were examined and analyzed to study the viruses flushed from the rosin-functionalized plastic surface. (B) Viruses flushed from both the surfaces and control viruses were all sedimented by airfuge, and the amount of pelleted RNA was measured using qPCR.

Fig 7

The active component inside the functionalized surface leaches out into the media and can be detected using UV-vis spectroscopy and can inhibit the HCoV-OC43 virus infection. (A) The spectrum of the O/N rosin-functionalized plastic flushed sample (blue line) was normalized with the 1 h rosin-functionalized plastic flushed sample (black line). The reference spectrum of 0.0001% wt/vol conifer pitch in EtOH sample is shown in red. (B) Evaluation of the antiviral activity of 1 h flushed media against HCoV-OC43. The experiment was done once with three technical repeats. Results are plotted as average values + SEM. Statistical analysis employed one-way ANOVA, followed by the Bonferroni test (****P < 0.0001). VC, virus control and CC, cell control.

Fig 8

HCoV-OC43 virus binding onto its host cell receptors studied using (A) binding assay. The virus binding to host cell receptors was examined by facilitating synchronized virus binding onto the host cells, followed by recovery of the bound virus using RNA extraction, RT-PCR, and qPCR techniques. The experiment was performed three times with three technical replicates. VC, virus control. (B) Confocal microscopy. The viruses flushed from the surfaces were allowed to bind to MRC-5 cells for 1 h at RT and then internalize for another hour at 34°C. The virus-infected cells were fixed, permeabilized, and labeled with affinity-purified monoclonal rabbit anti-OC43-S protein, followed by a secondary goat anti-rabbit antibody (Alexa 555). The nucleus was stained blue with DAPI, and the cytoskeleton tubulin (gray) was labeled with a J2 primary antibody and goat anti-mouse secondary antibody (Alexa 488). Scale bar: 30 µm.

Fig 9

(A) Confocal microscopy images illustrating the entrapment of the virus within vesicles 15 h after infection, following flushing from the rosin-functionalized surface. Viruses from both surfaces and the control virus were allowed to infect cells for 15 h, followed by fixing, permeabilizing, and immuno-labeling with antibodies. The S protein of the virus is labeled in red, the cell nucleus in blue, and the cell cytoskeleton tubulin in gray. (B) Percentage of virus-infected cells calculated from confocal images using Cell Profiler. (C) Detection of dsRNA in HCoV-OC43-infected MRC-5 cells 15 h p.i. The nuclei are shown in blue and the dsRNA in green. dsRNA is present in the virus control and the viruses flushed from the standard LDPE surface; however, the viruses flushed from the rosin-functionalized plastic do not show any dsRNA. Scale bar: 30 µm.