Antiviral activity of salt-coated materials against SARS-CoV-2

  1. Christopher M. Coleman
  2. Belinda Wang
  3. Yixin Wang
  4. Emmanuel Tapia-Brito
  5. Ziwei Chen
  6. James Riffat
  7. Saffa Riffat
  8. Rachael Tarlinton
  9. Amir Ghaemmaghami

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Abstract

The SARS-CoV-2 pandemic demonstrated the importance of human coronaviruses and the need to develop materials to prevent the spread of emergent respiratory viruses. Coating of surfaces with antiviral materials is a major interest in controlling spread of viruses, especially in high-risk or high-traffic areas. A number of different coatings for surfaces have been proposed, each with their own advantages and disadvantages. Here we show that simple salt coating on a range of surfaces, including a novel biomass aerogel can reduce the infectivity of SARS-CoV-2 placed onto the surface. This suggests that a simple to apply coating could be applied to a range of materials and have an antiviral effect against SARS-CoV-2, as well as other potential emerging viruses.

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  2. Version published to 10.1099/acmi.0.000492.v5 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000492.v4 on Access Microbiology
  5. Access Microbiology

    Comments to Author

    1. Methodological rigour, reproducibility and availability of underlying data Whilst I appreciate that the authors have not attempted to conduct these experiments in line with ISO standard methods there are some fundamental principles that need to be adhered to in order to be able to validate the results. As I understand it, the controls used in the experiments (against which the reduction in viral titre after exposure to the materials is measured) is virus suspension that has not been exposed to the material. As all porous material will absorb virus instantly, the ISO standard for textiles requires the control to be virus recovered from the textile immediately post-exposure as this gives a much more realistic comparison to measure the antiviral effect of the textile. This has not been done here and so it should be acknowledged that the base line viral titre used is likely to be higher than it should be. In addition the only non-coated material used is face mask which is not a valid control for the other materials. Several times in the manuscript reference is made to a one-log drop in viral activity being in line with ISO standards. A one-log drop is equivalent to 90% reduction in activity, this is a minimally accepted value and most products that claim to be anti-bacterial or virucidal are expected to achieve a 3 log drop e.g "kills 99.9% of all know germs". I have checked both of the ISO standards cited (ISO 21702 and ISO 18184) and I cannot see any reference to a one-log drop in these? Please can the source of this claim be clarified? Materials - it appears that different sizes of the test materials were used? 12cm x 10cm and 15cm2 - and different methods for coating the materials - soaking and spraying - how can these be compared? L.130 - Vero E6 growth media should be changed to MEM + 10% FCS (if that is what was used) L.131 isn't clear - why were the virus stocks used? Do the authors mean the washings from the material? "applied to vero cells in 96 well plates with TCID50 assay" doesn't make sense? Maybe for TCID50 assay? L.136 what size of material was used for the RT-PCR experiments? What volume was recovered? What volume was tested in the assay? L.142-143 - what is the "no material control"? just virus? Is there a material without salt coating control? As mentioned above this may in itself absorb virus resulting in a reduced titre for recovered washings? L. 168-170 - I am unsure of the importance of Schmallenberg virus replication in the same cell line as SARS-CoV-2? Is this just convenience to avoid having to propogate 2 cell lines? There is no direct comparison being made between the 2 viruses and indeed substantially different titres and exposure times have been used so why is this relevant? Table 1 - the only test material control used appears to be uncoated face mask? This cannot act as a control for the other materials and is therefore only relevant for numbers 1 and 2 L.221-223 - Agreed - the only "fair" way to conduct this test is to use uncoated material of each type as the control and compare virus recovered from that material with virus recovered from the coated material. Most materials will cause a reduction in viral titre over time. 2. Presentation of results Generally the results are clearly presented. The below suggestions would help: What Ct values were obtained for the RT-PCR? only the relative ratios compared to no-material control are presented. L.234-235 RT-PCR is not more sensitive that TCID50 - they measure different things. RT-PCR detects viral RNA whether from a live or inactivated virus particle. TCID50 is a measure of viral infectivity. This should be amended. L.235 - 252 - Again, in the absence of test material controls it is difficult to draw conclusions about the RT-PCR data. The results are highly variable and show no correlation with time of exposure. The most likely explanation is variability in the absorbance of each test material and in volume recovered after exposure. Was any external control (e.g. MS2 RNA) used to ensure the assay worked consistently? Could some comment on this be made? 3. How the style and organization of the paper communicates and represents key findings The methods are difficult to follow in parts and would be hard to replicate. I do not think it is justified to conclude that either the data (in conjunction with TCID50) show it is non-infectious virus (L.247-250) or that some of the materials….cause complete degradation….(L.251-252) this is just speculation - maybe include in the discussion rather than results. 4. Literature analysis or discussion L.28 Insert "usually": The seasonal hCoVs, such as hCoV-299E and hCoV-OC43, usually cause mild….. L.31 as far as I am aware the first SARS virus is just called "SARS" not "SARS CoV-1"? L.34-35 - It may be true that the reason for the geographical restriction of MERS is due to the zoonotic host - although the original source of MERS is still debated - The virus is now endemic in camels in the Middle East and transmission to humans occurs from close contact with the camels with poor human-human spread limiting its geographical spread (not highly restricting it though, it has been found in humans in 27 different countries). However, this is not true for SARS. SARS did spread human-human so the geographical habitat of civet cats is not a factor. It was contained by quarantining humans. Why it hasn't re-emerged isn't understood. But if the argument used here were true it would keep re-emerging wherever humans were in contact with civet cats. Suggest rewriting this paragraph to reflect the above and remove "highly" restricted L.35 "civet" not "civit" L.41 "coronaviruses emerging into the human population hCoVs, causing…." This line doesn't make sense - change to …"coronaviruses emerging into the human population as human coronaviruses (hCoVs), causing…." L.41-42 - update to the latest COVID figures from the WHO dashboard? L.45 - I disagree with the statement that there was "relatively little interest in hCoVs as human pathogens" - they are well recognised as causing significant morbidity and even mortality in some patient groups and the economic cost of the "common cold" is huge. It would be better to say something like: Human coronaviruses have been recognised as a significant cause of the "common cold" since the 1960's when they were first identified, but despite the emergence of SARS in 2002-3 had not been seen as having major pandemic potential L.61 - (even under relaxed regulatory requirements that promote rapid development) - this is an incorrect and potentially dangerous statement. The regulatory requirements were not relaxed. They were speeded up - at financial risk to the manufacturers who began vaccine production before results of phase III clinical trials had been obtained. This was the main reason for the accelerated production of the vaccines, there was no relaxation in terms of safety evaluation of the vaccines. This statement must be removed or significantly amended. 5. Any other relevant comments How practical would a salt coated surface or textile be? Wouldn't the salt be easily removed via normal day-day activities? Apart from a brief citation to reference 9 little discussion of the many existing anti-viral coatings for face masks (e.g. copper) that emerged early in the pandemic is made. There are numerous examples in the literature and on commercial websites.

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  6. Access Microbiology

    Comments to Author

    The previous reviewer comments have all been addressed and I am happy to recommend this manuscript for acceptance.

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  7. Version published to 10.1099/acmi.0.000492.v3 on Access Microbiology
  8. Access Microbiology

    Please deposit the data underlying the work in the Society’s data repository Figshare account here: https://microbiology.figshare.com/submit. Please also cite this data in the Data Summary of the main manuscript and list it as a unique reference in the References section. When you resubmit your article, the Editorial staff will post this data publicly on Figshare and add the DOI to the Data Summary section where you have cited it. This data will be viewable on the Figshare website with a link to the preprint and vice versa, allowing for greater discovery of your work, and the unique DOI of the data means it can be cited independently. Before we send your manuscript back to reviewers following your revision, please ensure all data underpinning your work (for example, data that went into figures 3 and 4), are shared via FigShare (or other similar platform) and detailed in the Data Summary section.

  9. Version published to 10.1099/acmi.0.000492.v2 on Access Microbiology
  10. Access Microbiology

    Comments to Author

    In this manuscript the authors suggest an approach to modifying face masks to provide an antiviral property. This is likely still of interest to many, given the rise in interest in respiratory viruses. However, there are some scientific queries and clarifications that need to be addressed. Overall: In parts the writing needs clarity/more scientific language. Method needs more detail. Discussion on applicability of methods used. Specific comments: L31: 'cannot spread between humans well in normal situations' - please provide detail, what is a normal situation? What is 'well' relative too? Does not spread efficiently? L49 - repetition of 'adopted'. L52 - efficacy of what? L53 - is it any virus? viruses spread via respiratory droplets? L61 - can you describe what you mean by 'salt coating of surfaces'? What kind or surface? L83/84 - how long where they soaked? in what volume? what temperature was the oven? L84 - watch out for tense - 'A small humidifier is...' should be 'was' instead of 'is'? L105 - how big were the pieces? What is 'main source material' - please clarify. L105 - need full detail on the materials. Were they analysed at all? Supplier? size? were they sterile? L112 - bring the ISO method to the top. Please be clear what elements of this you followed and what you didnt. E.g. choice of virus is different. L118 - 'entire surface' - what surface? how big? L133 - Please describe this method. Figure 2 - there was no method for SEM? Please add. L152 - this paragraph is confusing - please clarify. L159 - Please use more accurate language than 'hit' L162 - 'based on the ISO standards' what standards? L163 - what is a 'no material control', please clarify. L168 - what does 'tested over a longer time course post-surface contact' mean? L211 - onwards - can you comment on the applicability of a 1log drop for RT PCR as a measure? L239 - 'used as a coating'. Also reptation of word 'used'. General comment for discussion - is a salt coating practical for this use? Would the humidity/moisture from breathing through a mask have an impact on the salt coating? Can you comment on the applicability of your data to the real world? At the moment it looks like data from a multiwell plate experiment is being used to suggest antiviral action of a mask.

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  11. Access Microbiology

    This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. Please ensure all data is uploaded to a repository as per the mandatory Access Microbiology Open Data Policy. This includes data that was used to generate your figures.

  12. Access Microbiology

    Comments to Author

    General Comments: Abstract section needs to be expanded upon so that the reader gets a sense for the direction the article will take and what the authors found. For example, was there a single coating that was more effective in all test materials? Did the material type have any impact on antiviral activity given similar coatings? What else did the authors conclude from the study? How does this information add to the existing body of knowledge? Descriptions of fabric treatments in the Materials section needs to include more procedural detail so that the testing could be repeated by others, if desired. For example, what were the fabric swatch sizes, salt solution volumes, and drying oven temperature? Also, Appendix 1 needs to indicate if by "face mask" this means melt-blown fabric since nonwoven fabric is listed. It would be better to have SEM photomicrographs seen in Figure 2 show the common treatment, that being 30% KCl:NaCl (50:50 mix). It is difficult to compare the relative distribution of salt coating from one material to the other when four different treatments are shown. The authors need to provide a rationale for testing the outer, middle, and inner layers of the "face mask" in Appendix 1 using the Schmallenberg virus. This process was not mentioned in the Materials section. Also, it should be specified if non-woven and melt-blown fabric samples seen in Figure 1 are intact 3-layers or a single separated layer. The pore sizes shown in Figure 2 appear to be much too large to ensure adequate contact of aerosolized virus particles with incorporated salt material. The Centers for Disease Control and Prevention (CDC) states that the average particle size of SARS-CoV-2 is around 0.1 micrometer (µm). Also, the minimum size of a respiratory particle that can contain SARS-CoV-2 is calculated to be approximately 9.3 μm.* Judging by the 100 µm size bar, the pore sizes in these materials, with the possible exception of MBF-30% NaCL, would be much too large for an appreciable numbers of virus particles to come into contact with the salt-coated material. Physical contact would be required to for virus inactivation via desiccation. An additional practical concern would be the breathability (ease of breathing) using a mask coated with NWF-NACL&KCL 30%. This would be questionable given that the provided SEM photomicrograph is representative of several examined fields. This type of limitation should also be discussed since this same issue is mentioned in one of their provided references.** The authors should supply the commercial source of face mask materials. *Lee BU. Minimum Sizes of Respiratory Particles Carrying SARS-CoV-2 and the Possibility of Aerosol Generation. Int J Environ Res Public Health. 2020 Sep 23;17(19):6960. doi: 10.3390/ijerph17196960 ** Rubino I, Oh E, Han S, Kaleem S, Hornig A, Lee SH, Kang HJ, Lee DH, Chu KB, Kumaran S, Armstrong S, Lalani R, Choudhry S, Kim CI, Quan FS, Jeon B, Choi HJ. 2020. Salt coatings functionalize inert membranes into high-performing filters against infectious. Specific Comments: Lines 25-29 - Suggest mentioning that the geographic restriction was most likely related to a limited geographical distribution of zoonotic sources for SARS-CoV-1 (civet cats) and MERS-CoV (dromedary camels). Line 32 - Recommend changing "well in normal situations" to "easily under normal conditions". Line 35 - The zoonotic source of SARS-CoV-2 has not been definitively established. Recommend sticking to statements indicating the relative ease by which this virus spreads from human-to-human. Lines 39-40 - Suggest describing the typical affliction brought on by hCoVs, pre- SARS-CoV-2, that being a head cold that was not geographically restricted in its incidence. hCovs were deemed responsible for ~20% of common colds until the appearance of SARS-CoV-2. Lines 52-53 - The authors should provide examples of attempts to incorporate antiviral materials (e.g., metallic nanoparticles) into facemasks and what types of masks (e.g., cloth-single vs cloth double layer) were used in these attempts. Lines 84-85 - The authors should specify the amount of salt solution applied to the biomass aerogel material. The same goes for non-woven fabrics and melt-blown fabrics soaked in salt solutions. Additionally, how big were salt-treated fabric sizes used for testing? Line 105-107 - The authors should explain the reason 2 greatly different microplates were used for testing either SARS-CoV-2 or SBV. This implies a rather large difference in sample size used to test either virus. Line 115 - Viruses are nonliving, so the word "active" should be used in lieu of "live". Line 163 - Was a material control also included? A lowering of the virus titer should be compared to an untreated material control and not to a "no material" control. It cannot be reasonably assumed that the material by itself does not possess some antiviral activity. Fabric material may be chemically treated to improve durability (shelf life). Line 212 - The authors should state the reason why detecting SARS-CoV-2 RNA degradation is important. Line 225 - What is the importance of the finding that noninfectious "fragments of SARS-CoV-2 RNA can remain on the surfaces for longer periods of time"? Line 243 - The authors should provide an example of a commercially available Bioaerogel mask.

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    No: Not Applicable

  13. Version published to 10.1099/acmi.0.000492.v1 on Access Microbiology