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    Reply to the reviewers

    Dear Dr. Monaco,

    Thank you for reviewing our manuscript entitled ‘Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells’. We are pleased to see that the reviewers make suggestions that will strengthen the paper. With cases of COVID-19 rising at dramatic levels in some parts of the world, we are anxious to see our results published in a peer-review journal.

    Please find below a detailed response to the comments is shown in bold. We can perform the additional experiments and make changes to the manuscript within 3 weeks of a journal agreeing to consider our paper.

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

    **Summary:**

    Pickard et al. present in the manuscript entitled "Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells" a new screen of FDA approved drugs against SARS-CoV-2. The authors based their screen on Vero and HUH7 cell lines. The methods applied for screening including the SARS-Cov-2-ΔOrf7a-NLuc modified virus are properly designed and preformed. This is an interesting study that finds several potential drugs that might be effective as anti SARS-CoV-2 therapies. However, such experiments have been done throughout the last year and the novelty and importance of these findings are questionable.

    Regarding this point, there are several studies that have attempted to identify compounds that impact on SARS-Cov2 infection; however, these do not specifically focus on the replication of the virus (studies have used viability markers and staining of viral proteins but many of the compounds identified exert their effects on the virus uptake). Whilst SARS-CoV2-Nluc viruses have been developed these have been used for infection studies to measure the amount of virus taken up by cells and have not further explored how they impact on virus replication. Therefore, we feel that our study shows that a reporter virus can be used to reflect virus replication.


    **Major comments:**

    1. Most the experiments presented are only done twice, while in the screen itself it should not be a problem, for verifying the drugs identified at least three experiments are suggested (Figure 5 and Supplemental Figure 6) __At the time of submission there was an urgent need to make our data accessible to the scientific community. Therefore, we performed some experiments with n=2. We used n=2 to validate the screen and each time we got the same experimental outcome. We would perform further repeats for the figures mentioned for publication. __

    To strengthen the results of the screen, the wild type virus should also be tested for plaque reduction assay with these nine drugs.

    __We will perform these experiments and present these data in the manuscript. We have already performed immunostaining of WT-virus infected cells and could include this as an alternative. __


    Identification of antivirals is important for SARS-CoV-2 and other coronaviruses, regardless of the presence or effectiveness of vaccines. I think the abstract and introduction should be written to emphasize this point (instead of trying to underestimate the vaccine effectiveness). Similarly, the authors ignore the relative failures of known antivirals (known to inhibit SARS-CoV-2 replication in vitro like Remdesavir) in clinical trials and suggest starting clinical trials with their screen results. I think that this suggestion is premature and require several more studies (including animals studies) before initiating clinical trials.

    __We will re-write this section of the manuscript. We have identified all compounds that have been evaluated in the AGILE clinical trial, and these compounds failed to show a patient benefit and also failed to impact on virus replication in human cells. __


    **Minor comments.**

    1. The errors bars are not defined throughout all the figures. I am not sure that error bars are even meaningful if experiments only done twice, I recommend showing the two results for each point. We will add additional repeats or as the reviewer suggests we could add the two points.

    Figure 1E and the tables especially supp tables 3 and 4? don't have legends.

    __Apologies, this will be amended. __


    Most graphs will benefit from presenting the results in logarithmic scale (all Luc counts/ qPCRs).

    This can be changed if editors agree.

    P6 in the Generation of functional SARS-CoV-2 virus section - a reference is missing "It has been reported that this aids the recovery of replicative virus (Insert ref 3)"

    Apologies, this will be amended.

    Reviewer #1 (Significance (Required)):

    This is a well performed drug screen on two cell lines that identified new potential FDA approved drugs as anti-SARS-CoV-2 inhibitors. There are several studies that already been published or distributed as preprints that have done similar experiments in other cells lines including more relevant lung epithelial cells (for example PMC743673). This study does not verify the screen results by additional methods. However, in the current pandemic situation this study could be important and interesting to follow up.

    I am a virologist; my expertise is in viral host interactions within infected cell.

    __We were unable to identify the paper which is referred to in the reviewer’s comments. We would aim to highlight further in the text that using the reporter virus, we are able to screen and identify compounds that impact on virus replication unlike many of the other studies. __


    **Referee Cross-commenting**

    No problem with the other comments

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ **Summary:**

    In this manuscript, the authors report on the creation of a luciferase-encoding SARS-CoV-2 (deleted for orf7a) and the use of this virus to test infectability of multiple cell lines as well as perform a drug repurposing screen in two cell lines (Vero E6 and Huh7). Of the 35 drugs that blocked the virus replication they further identify 9 drugs that have a (mild) effect on replication when administered 24 hours post infection.

    An important note here is that many studies which have identified potential therapeutics for SARS-Cov2 have performed experiments whereby cells are pre-treated with compounds prior to infection. We have been able to performed the same experiments and many of the drugs were unable to prevent replication after infection. The 9 compounds we have identified retain the ability to inhibit replication when applied post-infection. This sets our study apart from other screens that have been conducted for SARS-Cov2.


    **Major comments:**

    1. Figure 2: What's the difference between "Luminescence counts above noise" in Fig 2B and "Luminescence counts per second" in Figure 2C,D ? It seems like there is no difference in luminescence between 1 PFU and 100 PFU (and if anything, the bassline for 1PFU is higher, >1.5M, compared to 100 PFU where is below 1M). One would expect more luminescence in the 100 PFU experiment, as seen in Fig 2B. Also in Fig 2B it does not mention how many replicates, or what does the **** stands for. __Thank you for the comment. The difference in “luminescence counts above noise” and “luminescence counts per second” is set out in Figure 2A. When adding more virus the baseline level should increase, as also demonstrated in Supplemental Figure 3. However, the degree of background luminescence varies between virus batches, presumably due to the degree of cell lysis in each sample. You will note in the Supplemental figure that the baseline levels for our P4 viral stock is lower than P1. We performed the experiments in Figure 2C using virus P1 virus stocks and for Figure 2D we used P4 virus. For clarity this information will be included in the figure legend and the data presented at luminescence over background. __

    The authors do not explain why deleting orf7a was needed to generate the NLuc virus. Was there a rational for this?

    __Orf7a has been successfully removed from SARS-CoV and SARS-CoV2 in order to incorporate traceable proteins such as fluorescent or bioluminescent proteins. We describe this at the start of the results section. __“Orf7a has previously been removed in SARS-CoV and SARS-CoV-2 and yielded infectious and replicative virus particles (Thi Nhu Thao* et al, 2020; Xie et al, 2020a; Xie et al*, 2020b)”.

    Figure 5C - IC50 should be properly determined from compounds where the lowest concentration tested was still inhibitory (such as LY2835219 and panobinostat).

    __These experiments can be conducted, within 2 weeks. However we do not feel that this would provide additional information to the reader. The aim of these figures is to demonstrate that there are dose dependent effects of these compounds on the replication of SARS-CoV2. __

    Supplementary tables must be provided in an excel or similar file format. The PDF version is both unreadable and does not allow other researchers to probe the dataset for their own interests.

    This would be amended during revision of our submission.

    **Minor comments:**

    1. Intro: "SARS-CoV-2 infection in patients with COVID-19 can result in pulmonary distress, inflammation, and broad tissue tropism". Broad tissue tropism is not a result of infection, please rephrase. Patients with COVID-19 are reported to have liver and kidney damage. This could be a direct result of SARS-CoV2 infection or indirectly via the cytokine storm. Our data shows that kidney and liver cells are highly susceptible to SARS-CoV2 infection and support replication, in culture. We thank the reviewer for their comment and we will rephrase this statement and cite relevant literature.

    Fig S1D - why are the MOI different for WT (moi 0.1) and NLuc mutant (moi 1) ?

    __This was used to demonstrate the lack of replication of the WT virus in lung epithelial cells, the same MOI used in Vero cells demonstrates that the levels of the nucleocapsid protein increases when compared to other cell types. We have also used an MOI of 10 for the NLuc virus to be able to detect the NLuc protein. This information would be added to the figure legend. __


    Fig S3 - using volume of virus in ul is problematic, as it doesn't allow for proper comparison between the passages. The author would express the virus amount in PFU or MOI.

    This will be amended


    Fig S5 - in panel A - what do the colors represent? What is 0-1?. The number of repetitions for each panel should be indicated.

    Apologies, relative expression should have been added alongside the scale. N=3 for this experiments this will be added to the figure legend.


    The "NLuc activity as a marker of virus replication" and "SARS-CoV-2 replication screen validation" are largely overlapping and should be edited.

    We would combine these sections.


    Methods: "Generation of functional SARS-CoV-2 virus" - the author confuse "virus" with "plasmid". They should also include the reference marked "(Insert ref 3)"

    Apologies, this will be amended


    Reviewer #2 (Significance (Required)):

    1. My main concern is that a very similar, if not identical, NLuc encoding virus has been reported in October 2020 (https://www.nature.com/articles/s41467-020-19055-7#Sec9). While the authors cite this paper, they only do so to say that "Orf7a has previously been removed in SARS-CoV and SARS-CoV-2 and yielded infectious and replicative virus particles", without mentioning this was done to generate the same NLuc carrying virus reported in their work. Thus the generation of this virus is not a "new tool" as the authors would seem to suggest. __Whilst this is not the first use of a NLuc SARS-CoV2 virus, this is the first time that the virus has been utilised to screen for compounds that effect replication. The study mentioned does not screen nor monitor the replication of the virus, the authors do monitor the capability of the virus to infect cells only during the first 24 hours. __

    While drug repurposing screens have been performed, the addition validation in Vero E6 and Huh7 cells is of some interest to those working on anti-viral therapies, given that the authors change their supplementary tables to a format that can be accessible by other researchers.

    This will be amended for the submission.


    My expertise: I study virus-host interactions (not coronaviruse). In the last year I have been involved in several drug repurposing efforts against SARS-CoV-2.

    **Referee Cross-commenting**

    No problem with the other comments.

    Read the original source
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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, the authors report on the creation of a luciferase-encoding SARS-CoV-2 (deleted for orf7a) and the use of this virus to test infectability of multiple cell lines as well as perform a drug repurposing screen in two cell lines (Vero E6 and Huh7). Of the 35 drugs that blocked the virus replication they further identify 9 drugs that have a (mild) effect on replication when administered 24 hours post infection.

    Major comments:

    1. Figure 2: What's the difference between "Luminescence counts above noise" in Fig 2B and "Luminescence counts per second" in Figure 2C,D ? It seems like there is no difference in luminescence between 1 PFU and 100 PFU (and if anything, the bassline for 1PFU is higher, >1.5M, compared to 100 PFU where is below 1M). One would expect more luminescence in the 100 PFU experiment, as seen in Fig 2B. Also in Fig 2B it does not mention how many replicates, or what does the **** stands for.
    2. The authors do not explain why deleting orf7a was needed to generate the NLuc virus. Was there a rational for this?
    3. Figure 5C - IC50 should be properly determined from compounds where the lowest concentration tested was still inhibitory (such as LY2835219 and panobinostat).
    4. Supplementary tables must be provided in an excel or similar file format. The PDF version is both unreadable and does not allow other researchers to probe the dataset for their own interests.

    Minor comments:

    1. Intro: "SARS-CoV-2 infection in patients with COVID-19 can result in pulmonary distress, inflammation, and broad tissue tropism". Broad tissue tropism is not a result of infection, please rephrase.
    2. Fig S1D - why are the MOI different for WT (moi 0.1) and NLuc mutant (moi 1) ?
    3. Fig S3 - using volume of virus in ul is problematic, as it doesn't allow for proper comparison between the passages. The author would express the virus amount in PFU or MOI.
    4. Fig S5 - in panel A - what do the colors represent? What is 0-1?. The number of repetitions for each panel should be indicated.
    5. The "NLuc activity as a marker of virus replication" and "SARS-CoV-2 replication screen validation" are largely overlapping and should be edited.
    6. Methods: "Generation of functional SARS-CoV-2 virus" - the author confuse "virus" with "plasmid". They should also include the reference marked "(Insert ref 3)"

    Significance

    1. My main concern is that a very similar, if not identical, NLuc encoding virus has been reported in October 2020 (https://www.nature.com/articles/s41467-020-19055-7#Sec9). While the authors cite this paper, they only do so to say that "Orf7a has previously been removed in SARS-CoV and SARS-CoV-2 and yielded infectious and replicative virus particles", without mentioning this was done to generate the same NLuc carrying virus reported in their work. Thus the generation of this virus is not a "new tool" as the authors would seem to suggest.
    2. While drug repurposing screens have been performed, the addition validation in Vero E6 and Huh7 cells is of some interest to those working on anti-viral therapies, given that the authors change their supplementary tables to a format that can be accessible by other researchers.

    My expertise: I study virus-host interactions (not coronaviruse). In the last year I have been involved in several drug repurposing efforts against SARS-CoV-2.

    Referee Cross-commenting

    No problem with the other comments.

    Read the original source
    Was this evaluation helpful?
  3. 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:

    Pickard et al. present in the manuscript entitled "Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells" a new screen of FDA approved drugs against SARS-CoV-2. The authors based their screen on Vero and HUH7 cell lines. The methods applied for screening including the SARS-Cov-2-ΔOrf7a-NLuc modified virus are properly designed and preformed. This is an interesting study that finds several potential drugs that might be effective as anti SARS-CoV-2 therapies. However, such experiments have been done throughout the last year and the novelty and importance of these findings are questionable.

    Major comments:

    1. Most the experiments presented are only done twice, while in the screen itself it should not be a problem, for verifying the drugs identified at least three experiments are suggested (Figure 5 and Supplemental Figure 6)
    2. To strengthen the results of the screen, the wild type virus should also be tested for plaque reduction assay with these nine drugs.
    3. Identification of antivirals is important for SARS-CoV-2 and other coronaviruses, regardless of the presence or effectiveness of vaccines. I think the abstract and introduction should be written to emphasize this point (instead of trying to underestimate the vaccine effectiveness). Similarly, the authors ignore the relative failures of known antivirals (known to inhibit SARS-CoV-2 replication in vitro like Remdesavir) in clinical trials and suggest starting clinical trials with their screen results. I think that this suggestion is premature and require several more studies (including animals studies) before initiating clinical trials.

    Minor comments.

    1. The errors bars are not defined throughout all the figures. I am not sure that error bars are even meaningful if experiments only done twice, I recommend showing the two results for each point.
    2. Figure 1E and the tables especially supp tables 3 and 4? don't have legends.
    3. Most graphs will benefit from presenting the results in logarithmic scale (all Luc counts/ qPCRs).
    4. P6 in the Generation of functional SARS-CoV-2 virus section - a reference is missing "It has been reported that this aids the recovery of replicative virus (Insert ref 3)"

    Significance

    This is a well performed drug screen on two cell lines that identified new potential FDA approved drugs as anti-SARS-CoV-2 inhibitors. There are several studies that already been published or distributed as preprints that have done similar experiments in other cells lines including more relevant lung epithelial cells (for example PMC743673). This study does not verify the screen results by additional methods. However, in the current pandemic situation this study could be important and interesting to follow up.

    I am a virologist; my expertise is in viral host interactions within infected cell.

    Referee Cross-commenting

    No problem with the other comments

    Read the original source
    Was this evaluation helpful?