In vitro comparison of viral replication and cytopathology induced by SARS-CoV-2 variants
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A myriad of coronaviruses cause diseases from a common cold to severe lung infections and pneumonia. SARS-CoV-2 was discovered to be the etiologic agent of the Coronavirus pandemic and many laboratory techniques were examined for virus culture and basic and applied research. Understanding the replication kinetics and characterizing the effect the virus has on different cell lines is crucial for developing in vitro studies. With the emergence of multiple variants of SARS-CoV-2, a comparison between their infectivity and replication in common cell lines will help give us a clear understanding of their characteristic differences in pathogenicity. In this study we compared the cytopathic effect and replication of Wild-Type (USA/WA1), Omicron (B.1.1.529), and Delta (B.1.617.2) variants on five different cell lines; VeroE6, VeroE6 cells expressing high endogenous ACE2, VeroE6 cells expressing human ACE2 and TMPRSS2, Calu3 cells highly expressing human ACE2 and A549 cells. This data will aid researchers with experimental planning and viral pathogenicity analysis and provide a baseline for testing any future variants.
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The reviewer and I are happy that the comments have been addressed in the revised manuscript and that it is now ready for publication.
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Comments to Author
n/a
Please rate the manuscript for methodological rigour
Satisfactory
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
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Both reviewers have recommended minor revisions. Please address these comments and make the necessary changes to your manuscript. A more specific title which describes on the outcomes of the research will be needed.
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Comments to Author
In this study the authors compared the cytopathic effect and replication of Wild type, Omicron, and Delta variants on 5 different cell lines: VeroE6, VeroE6 cells expressing high endogenous ACE2, VeroE6 cells expressing human ACE2 and TMPRSS2, Calu3 cells highly expressing human ACE2 and A549 cells. The study is useful for future experiment design and pathogenicity analysis. They conclude that for CPE or plaque-based assays, such as neutralization assays or antiviral testing assays, VeroE6/hACE22/TMPRSS2 would be a better choice over VeroE6 since the former cell line shows a higher sensitivity to SARS-CoV-2 infection. However, this reviewer has several concerns which need to be addressed: 1. In lines 23 and 43, please specify which strain as wild type or WT when it first appears. And keep consistent …
Comments to Author
In this study the authors compared the cytopathic effect and replication of Wild type, Omicron, and Delta variants on 5 different cell lines: VeroE6, VeroE6 cells expressing high endogenous ACE2, VeroE6 cells expressing human ACE2 and TMPRSS2, Calu3 cells highly expressing human ACE2 and A549 cells. The study is useful for future experiment design and pathogenicity analysis. They conclude that for CPE or plaque-based assays, such as neutralization assays or antiviral testing assays, VeroE6/hACE22/TMPRSS2 would be a better choice over VeroE6 since the former cell line shows a higher sensitivity to SARS-CoV-2 infection. However, this reviewer has several concerns which need to be addressed: 1. In lines 23 and 43, please specify which strain as wild type or WT when it first appears. And keep consistent when using WT or Wild Type in the manuscript. 2. In line 172, please correct "invitro", and also please check all the minor errors in the manuscript. 3. In lines 140, 183 and 186, please just use CPE only since the abbreviation has been clarified when it first appears. 4. In line 216, please change "cell entry" to "cell surface entry". 5. In Figure 2, the background for uninfected VeroE6/ACE2 cells is really "dirty", and it seems quite a lot of dead cells or other contaminants there, even has more dead cells or cell debris than PFU100 Delta variant, which makes it hard to compare how CPE was changed after virus infection. 6. Please indicate the numbers of technical repeats and the times of experiment repeats for each figure?
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
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Comments to Author
This is a generally well written, conducted and presented. This reviewer appreciated the use of real virus isolates and quantification of viability and viral RNA load. Their data on outcome of infection in different cells is not novel but may be helpful. However, a few points need addressed to maximise the paper. 1) Analysis of their results in light of viral sequencing is necessary. Is there anything unusual about your viral strains? It looks like your wildtype is unusual and likely may have the polybasic/furin cleavage sequence deleted like in many other isolates. 2) It is not clear how the virus is titred. Methods suggest tcid50 but through, pfu is used. Can the authors clarify? Also need to make clearer that your inoculum is titred on only cell type (?) so moi not necessarily what is suggested …
Comments to Author
This is a generally well written, conducted and presented. This reviewer appreciated the use of real virus isolates and quantification of viability and viral RNA load. Their data on outcome of infection in different cells is not novel but may be helpful. However, a few points need addressed to maximise the paper. 1) Analysis of their results in light of viral sequencing is necessary. Is there anything unusual about your viral strains? It looks like your wildtype is unusual and likely may have the polybasic/furin cleavage sequence deleted like in many other isolates. 2) It is not clear how the virus is titred. Methods suggest tcid50 but through, pfu is used. Can the authors clarify? Also need to make clearer that your inoculum is titred on only cell type (?) so moi not necessarily what is suggested by pfu axis on figure 3) The images of the calu3 and a549s would be helpful to see if they looked healthy like your viability scores suggest. 4) Reproducibility is not clear. How many times was each experiment repeated, and how (technical, biological, experimental?). 5) A limitations section in the discussion would help, covering: justification of time points, focus on viral load not infectivity, and no sequence analysis. A more specific title is needed
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
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
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