Propylene glycol inactivates respiratory viruses and prevents airborne transmission

  1. Christine T Styles
  2. Jie Zhou
  3. Katie E Flight
  4. Jonathan C Brown
  5. Charlotte Lewis
  6. Xinyu Wang
  7. Michael Vanden Oever
  8. Thomas P Peacock
  9. Ziyin Wang
  10. Rosie Millns
  11. John S O'Neill
  12. Alexander Borodavka
  13. Joe Grove
  14. Wendy S Barclay
  15. John S Tregoning
  16. Rachel S Edgar

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Abstract

Viruses are vulnerable as they transmit between hosts, and we aimed to exploit this critical window. We found that the ubiquitous, safe, inexpensive and biodegradable small molecule propylene glycol (PG) has robust virucidal activity. Propylene glycol rapidly inactivates a broad range of viruses including influenza A, SARS‐CoV‐2 and rotavirus and reduces disease burden in mice when administered intranasally at concentrations commonly found in nasal sprays. Most critically, vaporised PG efficiently abolishes influenza A virus and SARS‐CoV‐2 infectivity within airborne droplets, potently preventing infection at levels well below those tolerated by mammals. We present PG vapour as a first‐in‐class non‐toxic airborne virucide that can prevent transmission of existing and emergent viral pathogens, with clear and immediate implications for public health.

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  1. Version published to 10.15252/emmm.202317932
  2. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    __We thank the reviewers for their detailed comments, constructive feedback and insightful suggestions to improve our research and manuscript. Our detailed responses are in bold. __

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary This paper by Styles et al describes the effect of propylene glycol (PG) on a range of enveloped viruses, focusing mainly on IAV and SARS-Cov2. The direct virucidal effect is shown both in vitro and in vivo, as well as the effect of vaporized PG on airborne IAV and SARS-Cov-2. This is a well-performed and very clear study.

    Major comments

    The claims and conclusions are in general well supported by the data, yet I have some comments.

    • Interestingly, there were variations in the effect of PG on the different pseudoviruses. Surprising as I assumed that the effect of PG would be mainly on the lipid envelope, as you also mention in the discussion. It is suggested that PG therefore also has an effect on the conformation of the surface proteins. This can be easily tested, does the addition of PG result in a changed recognition by antibodies targeting these surface proteins? This information would be very valuable for your manuscript.

    __Many thanks for this excellent suggestion. To address this we are working in collaboration with Dr Joe Grove (Centre for Virus Research, University of Glasgow) using immobilised purified pseudovirus particles +/- PG treatment. Immunostaining against pseudovirus glycoproteins and their HIV gagpol capsid core enables comprehensive analysis of particle composition and integrity. Our preliminary experiments since submission have shown treatment with >60% PG increased gagpol signal, indicating compromised envelope integrity of equivalent magnitude to direct permeabilisation (positive control condition). At PG concentrations between 25 – 60% we’ve observed a significant reduction in glycoprotein signal preceding substantial envelope disruption. We are repeating these experiments in parallel with infectivity assays to assess how these changes in glycoprotein antibody recognition and envelope permeabilization relate to pseudovirus entry. We anticipate these studies will be completed within 8 weeks. __

    __In addition, we are optimising cryo-EM protocols for analysis of SARS-CoV-2 virus particles +/- PG treatment to directly visualise the impact of PG on virus structure, in collaboration with Dr Paul Simpson (EM Centre, Imperial College). __

    Figure S1 shows that although the clinical scores are better, the viral load is not reduced by the addition of PG to the inoculum. This is not mentioned in the main text. This seems very relevant information and should be presented in the main text. Can you please elaborate on this.

    We agree that the virological and immunological findings are of interest, and have added the following to the main text “____Analysis of the remaining mice showed PG inhalation reduced clinical score and bronchioalveolar lavage cell counts on day 5, despite equivalent nasal and airway viral loads at this late time post-infection.” Examining longitudinal weight loss and survival was the primary objective of this experimental design, which only allowed analysis of BAL cell counts and viral loads in the remaining mice at the experimental end point (day 5 post-infection). As 3 mice in the IAV only group reached the severity limit on day 3, we did not have the statistical power to detect significant differences in clinical scores, lung infiltrates or viral loads. IAV burden in the nose and lung usually reach peak levels at day 2/3 post-infection, and further experiments to look at the magnitude of viral replication and immunopathology over the course of infection in PG-treated mice will be important for future translational studies. As such, we have added the following to the Fig. S1 legend “____Further investigations are required to determine whether PG treatment reduces inflammatory cell infiltrates or peak IAV loads on days 2/3 post-infection.”

    Optional: It would be very interesting to see the effect of vaporized PG on viral transmission between mice/ferrets of a highly infectious virus (eg Sendai virus). This would give an idea if the PG also works on more biological respiratory droplets.

    We wholeheartedly agree. To perform these additional studies requires additional funding and project licence amendments so unfortunately must lie beyond the scope of this study.

    Minor comments

    The caption of Fig1D mentions that the mice are inoculated with 50ml total volume. I presume this should be µl.

    __Absolutely, thank you for spotting this error and it is now corrected. __

    Please put the axis of the viral titer graph now in Fig s1 in PFU/lung or nasal cavity? This would be more informative.

    __Many thanks for this suggestion, now changed to PFU/lung along with BAL cells/lung. __

    Typo in 'PG has broad-spectrum virucidal activity' paragraph: titre should be titer.

    __Titre is the British English spelling whereas titer is the American English spelling. We will comply with the journal specification on American versus British English in the final text. __

    Is allergy to PG an issue that could have an effect on further development of this antiviral strategy?

    A recent review (Pemberton & Kimber ____https://doi.org/10.1016/j.yrtph.2023.105341____) of the evidence on this topic concludes “..the weight of evidence points to PG possessing no, or only extremely modest, skin sensitising properties.” There is anecdotal evidence of PG potentially acting as a very weak allergen in humans with underlying/pre-disposing skin conditions. Further clinical development of PG as a virucide would need to balance its significant benefits against this when “..the risk for sensitisation to PG on uncompromised skin seems to be extremely low.”

    Reviewer #1 (Significance (Required)):

    This study is highly relevant. It proposes an easy and possibly efficacious method to prevent airborne transmission of a broad range of (enveloped) viruses.

    A strength of this study is the set-up with the virus transmission tunnel. This nicely shows the effect of the vaporized PG on airborne IAV and SARS-Cov-2. A limitation is that the researchers did not analyze non-enveloped viruses, which should be done in a follow-up study.

    __Many thanks for these comments. Since submission we have extended our investigations to non-enveloped rotaviruses in collaboration with Dr Alex Borodavka (Department of Biochemistry, University of Cambridge). Preliminary experiments have shown that rotavirus infectivity decreased from ~108 PFU/mL to undetectable levels (2 PFU/mL) following incubation with 50% PG at 37oC for 1h compared to 0% PG control. We are now testing different temperatures, PG concentrations and incubation durations and will include this new data in our revised submission. __

    There does not seem to be any recent research investigating this compound as an antiviral tool. Although the compound was tested for its antimicrobial properties in the 1940s, this has not been continued for unclear reasons (?).

    __We speculate the rapid development and clinical testing of conventional antibiotics during this period (culminating the Nobel prize for Medicine for Flemming, Florey and Chain in 1945) is responsible for the abrupt cessation of research into PG as an antimicrobial despite its obvious efficacy. We were unaware of these studies when we commenced our research into PG and this literature seems to have flown completely under the radar. __

    This is very valuable research in this world recovering from the recent SARS-Cov-2 pandemic. More mechanistic studies need to be performed to understand how this compound exerts its antiviral and antimicrobial effect.

    A more specialized audience will be interested in this study.

    My field of expertise: RSV, PIV, antivirals

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    SUMMARY: This study investigates the inactivation potential of propylene glycol against a number of important human respiratory viruses, including influenza A virus and SARS-CoV-2. Potent antiviral activity of propylene glycol was demonstrated for viruses in bulk solutions, in 2uL droplets on a variety of surfaces, and within aerosols. Additionally, propylene glycol was added to viruses prior to infection of live animals, and this treatment significantly improved animal survival outcomes and reduced their weight loss and clinical scores compared to animals infected with non-treated virus. Levels of propylene glycol utilised in the study were substantially less than those classified as well-tolerated by mammals. Overall, authors demonstrate that propylene glycol is a strong yet safe virucide that could be used to control the spread of viral infections.

    MAJOR COMMENTS:

    Claims and conclusions are very well-supported by the data presented. Both the data presentation and method details are sufficient to reproduce the experiments. Authors might like to add precise gram concentrations of PG in addition to the percentages listed in the manuscript, either g/L or g/kg as appropriate. This would also allow easier comparison of the tested PG does to those published as safe doses for mammals, which are often given as g/kg.

    Many thanks for this suggestion. These calculations and the concentration are now in a new “supplementary calculations” section in the supplementary material and the Figure 1 legend directs readers to them: “____See supplementary materials for % PG solution (v/v) to g/L conversion and mouse PG dose in g/kg.”

    Replicates are more than adequate, and statistical analysis is mostly robust. I have one concern of statistical analyses performed in Figure 2D. In the SARS1 coronavirus panel of this figure, statistical analysis indicates that 10% PG causes significantly more infection (in RLU) than 0% PG. The same is true for the SARS-CoV-2 Alpha panel. The differences in infection level between 0% and 10% PG by eye are minimal, and authors should consider whether this statistical test is the most robust/appropriate, and whether these particular statistical differences are biologically meaningful to report. If analyses remain as is, authors should postulate why this small increase in infection level was observed for some lentivirus pseudotypes at 10% PG compared to controls.

    __Many thanks to the reviewer for highlighting this. Whilst Dunnet's post-test we employed does indeed suggest a significant difference between 10% and 0% PG for some of the pseudoviruses, another post-test (Bonferroni's) does not. The reviewer correctly questions the biological significance of these very small differences in luciferase activity. In our experience, effect sizes of less than half a log unit are not biologically meaningful and certainly we make no claims that 10% PG is affecting pseudovirus entry in these experiments. To avoid any ambiguity therefore, we have removed the "*" from the relevant graphs and have restricted reporting of statistical significance to those conditions with p

    OPTIONAL - The study could be further enhanced by preliminary mechanistic investigation into the action of propylene glycol (PG) - for example, authors postulate that PG may act via disruption of the lipid membrane, and this could be supported by EM imaging of a few PG-treated viruses. Alternatively, non-enveloped viruses could be tested for sensitivity to determine the importance of the envelope. Authors mention this in the discussion as being outside the scope, though testing one or two non-enveloped viruses would elevate the work substantially, as it opens the possibility for PG to also protect against non-enveloped respiratory viruses such as rhinovirus, as well as enteric viruses.

    __As explained in detail in our response to reviewer 1 and our revision plan, we are addressing these excellent suggestions in three ways for the revised manuscript: __

    • __Collaboration with Dr Joe Grove’s group to address PG's action on viral surface glycoproteins, virus composition and envelope integrity. __
    • EM imaging of PG-treated SARS-CoV-2 with Dr Paul Simpson.
    • Collaboration with Dr Alex Borodavka’s group to investigate PG-mediated virucidal activity against non-enveloped rotaviruses.

    OPTIONAL - All influenza work in this study used the lab-adapted isolate A/PR8. This is a very useful and representative virus, though the study may benefit from comparison of the PG-mediated inactivation kinetic of A/PR8 with that of a more recent clinical isolate of influenza A. This could be a useful supplementary figure.

    __Many thanks for this suggestion. We agree that examining the inactivation kinetics of different flu strains in vitro would be informative and will endeavour to include this in our revised manuscript if these experiments can be performed within an appropriate time frame. We know PG is effective against ____the 2009 pandemic strain H1N1 A/California/7/2009 as this was used for our in vivo experiments as listed in the methods section. For clarity we have added this information into the main text. __

    MINOR COMMENTS:

    Could authors generate (or reference) a robust correlation curve between IAV plaque area (px^2) that is calculated by the ColonyArea plugin, in relation to actual PFU/mL as counted by eye? This would help the reader to understand the decrease in viral titre that PG mediates from experiments conducted in the aerosol tunnel, e.g. does a decrease from 10^5 px^2 down to 10^3 px^2 equate to a 2-log10 reduction in PFU, or it is less than this?

    This correlation could also be used to generate a detection limit for Plaque Area (px^2) that is equivalent to 1 or 10 PFU/well, which could then be added to graphs in Figure 3, S4, S5. This will help the reader understand if detection of 10^3 px^2 (for example) is quite high, or if it is already close to detection limits.

    __The reviewer makes an interesting suggestion. As can be seen from the requested representation of those data (reviewer figure 1, replotted from Fig 3D), for lower nebulised input virus amounts where infection did not transmit beyond the first plate there is a very respectable linear correlation between plaque number and plaque area (R2=0.72, Spearman’s r=0.98, p2 – 103 px2 reflects the 1 – 10 plaque detection limit. However, when performing initial controls we found this relationship soon departs from linearity as input virus amount increases; essentially plaques begin to overlap with each other on the plates closest to the nebuliser. We are concerned it might be inappropriate to present PFU/well extrapolated from plaque areas collected from lower input virus experiments, since this could potentially mislead or confuse the reader about the fidelity and nature of the transmission tunnel experimental system. Moreover, for this particular type of assay plaque area affords a much greater dynamic range than PFU/well, which is essential for demonstrating the range of PG’s virucidal activity with higher input virus, range of vapor concentrations and over different transmission distances. Taking these considerations into account, and unavoidable idiosyncrasies of testing virus transmission under more “natural” conditions (e.g. direct virus deposition on cell monolayers prohibiting serial dilution for exact PFU determination), we have decided to continue reporting plaque area only for quantification of transmission tunnel data - the parameter that was actually measured. This accurately reflects the extensive cell clearance under control conditions, where the monolayer is completely disrupted, compared to the protective effect of PG vapor, where the monolayer is largely intact with limited plaques. We hope the reviewer understands our reasoning. __

    To communicate this, we have added the following sentence to the methods section:

    __“Plaque area shows a strong linear correlation with plaque number at lower viral inputs (R2=0.72, Spearman’s r=0.98, p

    Can authors specify if the PEG was mixed with virus immediately prior to inoculation of animals, or was it mixed ahead of time (e.g. 30 mins or 1 hour ahead of administration)? In this case, low-level inactivation of viruses in bulk solution may have occurred. Figure 1C shows minimal inactivation should have occurred with 20% PG over this time-course at room-temperature, but this detail would be helpful to specify.

    __Many thanks for bringing this oversight to our attention. IAV was mixed with PG immediately prior to inoculation of animals (

    Figure legend of Fig 1. D) currently reads "... inoculated with 50mL..." , should this be 50uL?

    __It should, thank you for spotting this error and it is now corrected. __

    Reviewer #2 (Significance (Required)):

    This study offers a robust proof of concept that PG can be applied to viral disinfection of a large range of enveloped viruses in a variety of settings (aerosol, fomites, in vivo, etc). This work now requires follow-on clinical studies to establish optimal and safe PG levels for viral inactivation in real-world settings, and comprehensive testing of safety risks from long term PG-exposure before this virucide can become clinically applied. This impact of this primary research could be considered broad and translational.

    The audiences anticipated to be interested in these results are varied, and include those in virology fields, medical personnel, aerosol scientists, disinfection experts, and public health/ policy makers.

    Field of expertise of reviewer: virology, animal infections, immunology, aerosol-borne influenza virus.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This study investigates the inactivation potential of propylene glycol against a number of important human respiratory viruses, including influenza A virus and SARS-CoV-2. Potent antiviral activity of propylene glycol was demonstrated for viruses in bulk solutions, in 2uL droplets on a variety of surfaces, and within aerosols. Additionally, propylene glycol was added to viruses prior to infection of live animals, and this treatment significantly improved animal survival outcomes and reduced their weight loss and clinical scores compared to animals infected with non-treated virus. Levels of propylene glycol utilised in the study were substantially less than those classified as well-tolerated by mammals. Overall, authors demonstrate that propylene glycol is a strong yet safe virucide that could be used to control the spread of viral infections.

    Major comments

    Claims and conclusions are very well-supported by the data presented. Both the data presentation and method details are sufficient to reproduce the experiments. Authors might like to add precise gram concentrations of PG in addition to the percentages listed in the manuscript, either g/L or g/kg as appropriate. This would also allow easier comparison of the tested PG does to those published as safe doses for mammals, which are often given as g/kg.

    Replicates are more than adequate, and statistical analysis is mostly robust. I have one concern of statistical analyses performed in Figure 2D. In the SARS1 coronavirus panel of this figure, statistical analysis indicates that 10% PG causes significantly more infection (in RLU) than 0% PG. The same is true for the SARS-CoV-2 Alpha panel. The differences in infection level between 0% and 10% PG by eye are minimal, and authors should consider whether this statistical test is the most robust/appropriate, and whether these particular statistical differences are biologically meaningful to report. If analyses remain as is, authors should postulate why this small increase in infection level was observed for some lentivirus pseudotypes at 10% PG compared to controls.

    OPTIONAL - The study could be further enhanced by preliminary mechanistic investigation into the action of propylene glycol (PG) - for example, authors postulate that PG may act via disruption of the lipid membrane, and this could be supported by EM imaging of a few PG-treated viruses. Alternatively, non-enveloped viruses could be tested for sensitivity to determine the importance of the envelope. Authors mention this in the discussion as being outside the scope, though testing one or two non-enveloped viruses would elevate the work substantially, as it opens the possibility for PG to also protect against non-enveloped respiratory viruses such as rhinovirus, as well as enteric viruses.

    OPTIONAL - All influenza work in this study used the lab-adapted isolate A/PR8. This is a very useful and representative virus, though the study may benefit from comparison of the PG-mediated inactivation kinetic of A/PR8 with that of a more recent clinical isolate of influenza A. This could be a useful supplementary figure.

    Minor comments

    Could authors generate (or reference) a robust correlation curve between IAV plaque area (px^2) that is calculated by the ColonyArea plugin, in relation to actual PFU/mL as counted by eye? This would help the reader to understand the decrease in viral titre that PG mediates from experiments conducted in the aerosol tunnel, e.g. does a decrease from 10^5 px^2 down to 10^3 px^2 equate to a 2-log10 reduction in PFU, or it is less than this?

    This correlation could also be used to generate a detection limit for Plaque Area (px^2) that is equivalent to 1 or 10 PFU/well, which could then be added to graphs in Figure 3, S4, S5. This will help the reader understand if detection of 10^3 px^2 (for example) is quite high, or if it is already close to detection limits.

    Can authors specify if the PEG was mixed with virus immediately prior to inoculation of animals, or was it mixed ahead of time (e.g. 30 mins or 1 hour ahead of administration)? In this case, low-level inactivation of viruses in bulk solution may have occurred. Figure 1C shows minimal inactivation should have occurred with 20% PG over this time-course at room-temperature, but this detail would be helpful to specify.

    Figure legend of Fig 1. D) currently reads "... inoculated with 50mL..." , should this be 50uL?

    Significance

    This study offers a robust proof of concept that PG can be applied to viral disinfection of a large range of enveloped viruses in a variety of settings (aerosol, fomites, in vivo, etc). This work now requires follow-on clinical studies to establish optimal and safe PG levels for viral inactivation in real-world settings, and comprehensive testing of safety risks from long term PG-exposure before this virucide can become clinically applied. This impact of this primary research could be considered broad and translational.

    The audiences anticipated to be interested in these results are varied, and include those in virology fields, medical personnel, aerosol scientists, disinfection experts, and public health/ policy makers.

    Field of expertise of reviewer: virology, animal infections, immunology, aerosol-borne influenza virus.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary

    This paper by Styles et al describes the effect of propylene glycol (PG) on a range of enveloped viruses, focusing mainly on IAV and SARS-Cov2. The direct virucidal effect is shown both in vitro and in vivo, as well as the effect of vaporized PG on airborne IAV and SARS-Cov-2. This is a well-performed and very clear study.

    Major comments

    The claims and conclusions are in general well supported by the data, yet I have some comments.

    • Interestingly, there were variations in the effect of PG on the different pseudoviruses. Surprising as I assumed that the effect of PG would be mainly on the lipid envelope, as you also mention in the discussion. It is suggested that PG therefore also has an effect on the conformation of the surface proteins. This can be easily tested, does the addition of PG result in a changed recognition by antibodies targeting these surface proteins? This information would be very valuable for your manuscript.
    • Figure S1 shows that although the clinical scores are better, the viral load is not reduced by the addition of PG to the inoculum. This is not mentioned in the main text. This seems very relevant information and should be presented in the main text. Can you please elaborate on this.
    • Optional: It would be very interesting to see the effect of vaporized PG on viral transmission between mice/ferrets of a highly infectious virus (eg Sendai virus). This would give an idea if the PG also works on more biological respiratory droplets.

    Minor comments

    • The caption of Fig1D mentions that the mice are inoculated with 50ml total volume. I presume this should be µl.
    • Please put the axis of the viral titer graph now in Fig s1 in PFU/lung or nasal cavity? This would be more informative.
    • Typo in 'PG has broad-spectrum virucidal activity' paragraph: titre should be titer.
    • Is allergy to PG an issue that could have an effect on further development of this antiviral strategy?

    Significance

    This study is highly relevant. It proposes an easy and possibly efficacious method to prevent airborne transmission of a broad range of (enveloped) viruses.

    A strength of this study is the set-up with the virus transmission tunnel. This nicely shows the effect of the vaporized PG on airborne IAV and SARS-Cov-2. A limitation is that the researchers did not analyze non-enveloped viruses, which should be done in a follow-up study.

    There does not seem to be any recent research investigating this compound as an antiviral tool. Although the compound was tested for its antimicrobial properties in the 1940s, this has not been continued for unclear reasons (?). This is very valuable research in this world recovering from the recent SARS-Cov-2 pandemic. More mechanistic studies need to be performed to understand how this compound exerts its antiviral and antimicrobial effect.

    A more specialized audience will be interested in this study.

    My field of expertise: RSV, PIV, antivirals

  5. Version published to 10.1101/2023.02.13.528349v1 on bioRxiv