Surfing the Waves of SARS-CoV-2: Analysis of Viral Genome Variants Using an NGS Survey in Verona, Italy

Round 1

Reviewer 1 Report

Tonon et al. collected the SARS-CoV-2-positive samples from pandemic (January 2020) to June 2023 in Italy. viral RNAs extracted and swab samples were analyzed by NGS and virus lineages were identified. BA.2, BQ.1, BA.5.1, BA.5.2, and, XBB.1 are the variants in circulation during 2020 to 2023. The data provides new insights to the SARS-CoV-2 genome evolution. The topic is interesting and important. Please see the suggestions below.

Major concern:

1.       Since the main findings in the study is about the virus/ variants in circulating in Italy, it would be beneficial for the readers to see the phylogenetic tree in the main text. Please move the phylogenetic tree from supplementary figure1 to the main text. Please use BA.2, BA5.1. and XBB.1 in the figure legend to keep consistency with the text.

2.       The authors mentioned the decreased vaccine effectiveness in the introduction section, line 48. Since viruses were collected and sequenced, it would be better to perform neutralization assay to test vaccine effectiveness against the variants.

Minor concern:

1.       About figure 4a. the authors showed the trend on variants prevalence (15%). On the Y-axle, numbers from 0 to 70 are shown in percentage? If so, please add “%” to avoid confusion. Also for Figure 4b.

Author Response

Thanks for your suggestions, in the attachment file you will find our point-by-point response.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Specific Comments

-Introduction (Lines 28-44, Page 2)

Generalization and Citation: The introduction broadly states the impact of genomic mutations and recombination events on coronavirus evolution without specific examples relating to SARS-CoV-2. Including more specific examples of mutations within the SARS-CoV-2 genome that have significantly altered transmissibility or immune evasion would strengthen the background. Additionally, ensure all statements are backed by recent and relevant citations.

-Materials and Methods (Lines 45-89, Pages 2-3):

Study's design: Please clarify the type of study design employed in this research (e.g., cohort, cross-sectional, case series, etc.). Understanding the study design is essential for accurately evaluating the methodology and its outcomes.

Sample Selection Bias (Lines 72-78, Page 3): The criteria for sample selection based on Ct values (<32) could introduce bias in the study, as samples with higher Ct values (indicating lower viral loads) might also contain relevant viral variants. It would be beneficial to discuss the potential impact of this selection bias on the study's findings and conclusions.

Ethical Approval: The statement regarding the lack of need for specific ethical approval because of Italian regulations is vague. Ethical considerations, especially in retrospective studies involving human samples, require clear justification and reference to specific guidelines or regulatory frameworks. Clarification and expansion on this point would enhance the manuscript's ethical transparency.

-Results (Lines 90-207, Pages 3-7)

Data Interpretation and Representation (Figure 1, Lines 105-111, Page 3): The interpretation of epidemiological waves and their representation in Figure 1 could be improved by providing statistical analysis or models that support the identification of these waves as significant increases in cases. Additionally, comparing the data from Verona with national data in a more analytical manner could provide insights into the significance of regional variations observed.

Variant Prevalence (Lines 158-173, Page 6): The discussion on the prevalence of different variants and their implications lacks depth. While the identified variants are listed, a more thorough analysis of the potential reasons behind the prevalence changes and their impact on public health measures in Italy would enrich the discussion.

-Discussion (Lines 204-331, Pages 7-9)

Comparison with Other Studies (Lines 209-237, Page 7): The discussion makes broad comparisons with national data and previous pandemic events without specific references. Including direct comparisons with other studies that have analyzed variant prevalence and its impacts on COVID-19 wave dynamics would strengthen the argument.

Limitations (Lines 312-319, Page 9): While the manuscript mentions limitations such as the end of mandatory medical surveillance and the bias in sequencing samples, further discussion on how these limitations could affect the results' interpretation and the generalizability of the study findings is necessary. Suggestions for future studies to overcome these limitations would be helpful.

Conclusion (Lines 321-331, Page 9)

Broad Statements: The conclusion provides a broad summary without directly linking back to the study's specific findings and their implications for public health policy or future research directions. Tailoring the conclusion to more explicitly reflect the study's contributions and potential impacts would enhance its effectiveness.

General comments:

-Consistent Terminology and Abbreviations: The manuscript shows an inconsistent application of terminology and abbreviations, especially when discussing SARS-CoV-2 variants. Variants are at times referred to by their Pango lineage designations (e.g., BA.1, BA.2) and at other times by their common names (e.g., Omicron), with no uniform explanation or usage. To enhance readability and ensure consistency, establish and adhere to a specific convention for naming variants throughout the document.

-Sentence Structure and Grammar: Some complex sentences in the manuscript could be simplified to improve clarity. For example:

Original: "The sequencing of SARS-CoV-2-positive samples allowed epidemiological surveillance to monitor the evolution of the viral genome even though the sampling is relatively low."

Revised: "Despite limited sampling, sequencing SARS-CoV-2-positive samples enabled the monitoring of viral genome evolution through epidemiological surveillance."

Author Response

Thanks for your suggestions, in the attachment file you will find our point-by-point response.

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

This is an updated version, and the authors have taken the suggestions into consideration and modified the manuscript. No concerns detected in this version. 

Reviewer 2 Report

All relevant amendments have been performed.