Review Reports - West Nile Virus in Italy: An Update of the Viral Strains Circulating in the Late 2022 Epidemic Season

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The aim of this study was to investigate the peculiar West Nile virus scenario characterized by the Italian West Nile disease epidemic since its onset through the use of qRT‒PCR, sequencing, and phylogenetic analysis of the detected strains. The manuscript presents significant insights into this topic. However, I believe that the manuscript could benefit from improved cohesion and additional information in the background section to emphasize the necessity of conducting this study.

Specific comments

Each paragraph in the introduction section should focus on a single topic, placing background information, the monitoring plan, and the purpose of the study in separate paragraphs to improve readability. The idea that strong and early viral circulation characterizes the 2022 Italian vector season has already been demonstrated in other articles in the background section. Therefore, additional details on the innovation and necessity of this study are needed.

I believe that the methodology section requires additional details. For instance, specific procedures for conducting qRT‒PCR runs should be included, and the versions of the Illumina sequencing platform employed should be specified. Moreover, there is a noticeable absence of a description regarding the incorporation of quality control measures for the sequencing data.

Other comments

Line 42-44：Please add a reference.

Line 48-50：Add some reference to this sentence.

Line 52：What is SoHO?

Line 56：588 human cases or 588 in human cases? Please maintain a consistent format when referring to quantities.

Line 92：Please indicate the full name of the USUV the first time it appears.

Line 105：What was the rationale for choosing Koutango virus as an outgroup?

Line 140：Cx. Pipiens should be italicized.

Line 187-188: provide specific information on earlier start of viral circulation.

Author Response

Thank you for your thorough review of our manuscript. We appreciate the time and effort you invested in providing your feedback.
Please find our responses in the following text:

Specific comments

We split in different paragraphs the introduction and we add additional detail about the necessity of this study (Line 68) as suggested.

We improved the paragraph with specific procedures

Other comments We made the revision suggested.You can find the new lines in the following draft

Line 42-44：Please add a reference.

Same line

Line 48-50：Add some reference to this sentence.

Line 54

Line 52：What is SoHO?

The term SoHO includes blood, tissues, cells and any other substances of human origin like human breast milk and microbiota. Only solid organs intended for transplantation will remain regulated separately under the Organs Directive (Directive 2010/45/EU).

Line 56：588 human cases or 588 in human cases? Please maintain a consistent format when referring to quantities.

Line 58-59

Line 92：Please indicate the full name of the USUV the first time it appears.

Line 96

Line 105：What was the rationale for choosing Koutango virus as an outgroup?

We choose an outgroup capable to root the tree with a lineage clearly distinct from WNV lineage 2 but close enough to allow good sequence alignment. We could indeed have chosen any other lineage of WNV (among those sufficiently distinct from lineage 2) but we preferred Koutango virus both because it turns out to be a viable outgroup also for the phylogeny study of WNV lineage 1 and because it is one of the few WNVs that remains confined (for now) only to Africa.

Line 140：Cx. Pipiens should be italicized.

Done

Line 187-188: provide specific information on earlier start of viral circulation.

Line 213-214

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents the sequencing results for WNV strains circulating in Tuscany and Sicily from samples collected in the late 2022 epidemic season. Two samples were positive for WNV lineage 1 and the other two samples were for WNV lineage 2. New strains were identified by sequencing, and they were published on NCBI. Then phylogenetic tree analyses of WNV lineages 1 and 2 were carried out, separately, including new strains and sequences from different geographic regions. Figures 1 and 2 have low resolution and it was not possible to confirm the results described by the authors. As mentioned by the authors, WNV strains from Tuscany and Sicily had not been sequenced previously, so the results of the manuscript are interesting.

I have some comments on the manuscript which are listed below.

- Line 51-53: “The main goal of the programme is to … limit its spread and the human infections through the SoHO transmission.”

Add a reference to this part and explain the meaning of “SoHO transmission”.

- Line 54: “An intense and early viral circulation has characterized the 2022 vector season.”

Revise this sentence, the meaning is not clear.

- Line 59-60: “(see Italian Epidemiological reports https://west-59 nile.izs.it/j6_wnd/home)”t

Reports available on the website are references and should be cited and added in the “References” section.

- Lines 62-63: This paragraph describes the purpose of the present work, but the main results/findings, such as new strain identification, are not highlighted.

- Line 68: Subitem 2.1 should be reorganized into “Sample collection”, “Real-time PCR assay” and “Next-generation sequencing (NGS)” subitems. A brief description of each protocol used in the work should be included.

- Line 122: “Illumina sequencing” is not commonly used, replace with “Next-generation sequencing (NGS)” at this line and other lines mentioning this term.

- Line 160: Subitem “3.2. Figures” is unnecessary. The figures appear when it is cited in the main text when the results are presented.

- “Discussion” section: Some websites are cited in the main text and should be considered as references as mentioned before.

Comments on the Quality of English Language

In general, the quality of the English language is adequate, but it is recommended for a moderate revision to improve the manuscript.

Author Response

Thank you for your thorough review of our manuscript. We appreciate the time and effort you invested in providing your feedback.
Please find our responses in the following text:

We tried to improve the resolution of our figure without success. We suggest to add it in supplementary materials.

I have some comments on the manuscript which are listed below.

We made the revision suggested.You can find the new lines in the following draft

- Line 51-53: “The main goal of the programme is to limit its spread and the human infections through the SoHO transmission.”

Line 52-54

Add a reference to this part and explain the meaning of “SoHO transmission”.

Done

- Line 54: “An intense and early viral circulation has characterized the 2022 vector season.”

Revise this sentence, the meaning is not clear.

Line 56

- Line 59-60: “(see Italian Epidemiological reports https://west-59 nile.izs.it/j6_wnd/home)”t

Reports available on the website are references and should be cited and added in the “References” section.

Done

- Lines 62-63: This paragraph describes the purpose of the present work, but the main results/findings, such as new strain identification, are not highlighted.

Line 64-69

Done

- Line 122: “Illumina sequencing” is not commonly used, replace with “Next-generation sequencing (NGS)” at this line and other lines mentioning this term.

Done

- Line 160: Subitem “3.2. Figures” is unnecessary. The figures appear when it is cited in the main text when the results are presented.

Done

- “Discussion” section: Some websites are cited in the main text and should be considered as references as mentioned before.

Done

In general, the quality of the English language is adequate, but it is recommended for a moderate revision to improve the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript, West Nile Virus in Italy: An Update of the Viral Strains Circulating in the Late 2022 Epidemic Season by Valleriani et al., reports that two strains of West Nile virus were detected in Sicily and Tuscany of Italy and suggests based on their gene sequences that the virus was introduced to the regions likely from other European countries.

This is a fairly well written article. All sections are enjoyable to read. The introduction provides adequate information to understand the study. The materials and methods section needs a description how the Illumina sequencing was performed, though. The results section describes the figures precisely. The discussion section is congruent to the results and concludes adequately. Over all, the manuscript is well written and I recommend publication with a minor revision. I have a few minor suggestions as follows:

(1) Line 17, revise the sentence to be “West Nile Virus infection represents one of the major public health concerns in Europe,”.

(2) Line 27, revise the sentence to be “West Nile virus (WNV, Flaviviridae family- Flavivirus genus) infection is a mosquito-borne zoonosis capable of causing disease and death in humans and animals.”

(3) Lines 28 – 29, revise the sentence to be “In the last decades, WNV infections have represented one of the major public health concerns in Europe, and Italy is one of the most affected countries since 2008.”

(4) Line 30, I suggest to remove the sentence as it is just a claim without any supporting data.

(5) Lines 54 thru 58, revise the sentences to be “The 2022 vector season was characterized by an early and intense viral circulation. Mosquitoes and birds were found to be infected with WNV as early as June, with the first human case being reported on June 19th. By the end of 2022, the Italian surveillance system had recorded 588 human cases of WNV infection, with 82 cases in equids, 157 confirmed cases in residential birds (carrion crow, magpie, and Eurasian jay), and 210 cases in wild birds.”

(6) Lines 58 thru 60, Replace the number of WNV positive mosquito pools with infection rate in percent because 246 WNV positive mosquito pools may be an alarmingly high number for the people in Italy, but it may not be the case for the people elsewhere. Also, I suggest to remove the word, genome, after WNV unless the method of detection was indeed the whole genome sequencing.

(7) Lines 93 and 94, describe how the Illumina Sequencing was performed instead of referring to the paper by Diagne et al, 2023. With the current description, readers may think that the Illumina sequencing was performed using the products of qRT-PCR. Also, the first paragraph in the results section should be moved to this place in the methods section.

(8) Lines 117 through 122, I suggest to move the paragraph to the methods section where Illumina sequencing is described.

(9) Please define what USUV and WND stand for. The abbreviations occur multiple times, but the explanation is nowhere to be found. Does USUV stand for Usutu virus? Does WND stand for West Nile Disease?

(10) Line 192, A space is needed between “1” and “was”.

Comments on the Quality of English Language

Some minor revision is needed as I indicated in my review comments.

Author Response

Thank you for your thorough review of our manuscript. We appreciate the time and effort you invested in providing your feedback.
Please find our responses in the following text:

We made the revision suggested.You can find the new lines in the following draft

Line 17, revise the sentence to be “West Nile Virus infection represents one of the major public health concerns in Europe,”.

Same line

Line 27, revise the sentence to be “West Nile virus (WNV, Flaviviridae family- Flavivirus genus) infection is a mosquito-borne zoonosis capable of causing disease and death in humans and animals.”

Same line

Lines 28 – 29, revise the sentence to be “In the last decades, WNV infections have represented one of the major public health concerns in Europe, and Italy is one of the most affected countries since 2008.”

Same line

Line 30, I suggest to remove the sentence as it is just a claim without any supporting data.

Done

Lines 54 thru 58, revise the sentences to be “The 2022 vector season was characterized by an early and intense viral circulation. Mosquitoes and birds were found to be infected with WNV as early as June, with the first human case being reported on June 19th. By the end of 2022, the Italian surveillance system had recorded 588 human cases of WNV infection, with 82 cases in equids, 157 confirmed cases in residential birds (carrion crow, magpie, and Eurasian jay), and 210 cases in wild birds.”

Line 56-60

In the context of monitoring and managing the total pools tested for vector-borne diseases (WNV and USUV), determining the exact number of pools (and thus calculating a reliable percentage) becomes a challenging task. This is primarily due to the fact that provinces send only the positive pools to the central reference center for further analysis. Consequently, the total number of pools conducted remains unknown as only the positive cases are reported. Additionally, it is worth noting that the entomological surveillance efforts are contingent upon the specific risk level of each territory. The intensity and scope of surveillance activities may vary based on the perceived risk, making it different for each region.

Also, I suggest to remove the word, genome, after WNV unless the method of detection was indeed the whole genome sequencing.

Done

Lines 93 and 94, describe how the Illumina Sequencing was performed instead of referring to the paper by Diagne et al, 2023. With the current description, readers may think that the Illumina sequencing was performed using the products of qRT-PCR. Also, the first paragraph in the results section should be moved to this place in the methods section.

Done from line 99

Lines 117 through 122, I suggest to move the paragraph to the methods section where Illumina sequencing is described.

Done

(10) Line 192, A space is needed between “1” and “was”.

Done

Some minor revision is needed as I indicated in my review comments.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author has made effective modifications to the manuscript, and I have no further concerns.

Author Response

Thank you for your revision.

Reviewer 2 Report

Comments and Suggestions for Authors

The author's response letter has addressed most of comments from the first round of revision.

I have comments to improve the manuscript.

- Please provide complete descriptions for website references according to “Instructions for Authors” (https://www.mdpi.com/journal/zoonoticdis/instructions). Example: line 54, reference [4]. Check all website references.

- “Materials and Methods” section has improved, and each sub-section description is adequate.

- “Results” section was not improved as suggested in the first round of revision and few changes were added in the revised version. The authors changed figures (comment is below) and the text was not updated. Please explain and highlight changes because I cannot evaluate this section in the revised version.

- Figures 1 and 2 were substituted for one figure in the revised version and look different from previous figures. The authors did not explain to the reviewers the reason for this change in the revised version of the manuscript.

- Figure caption “…2 WNV L1 sequences added 193 as outgroups (FJ483548 Italy 2008 and MW627239 Italy 2020)...” Please highlight these sequences in the Figure to help find out the localization.

The figure file was sent as a separate file of the text and it presented good resolution and is readable.

Comments on the Quality of English Language

I suggest minor editing of English language.

Author Response

Fixed website references [4;7;22]

I sincerely apologize for the omission in explaining the accidental deletion of Figure 1 in the revised version of our manuscript.The removal of Figure 1 occurred due to an error during the revision process, where it was mistakenly omitted.To rectify this situation and ensure the completeness of information, we reintegrated Figure 1 into the next version of the manuscript.Thank you for your understanding.

- Figure caption “…2 WNV L1 sequences added 193 as outgroups (FJ483548 Italy 2008 and MW627239 Italy 2020)...” Please highlight these sequences in the Figure to help find out the localization.

In the given figure, the exclusion of outgroup sequences is intentional to enhance visualization. The abundance of sequences, particularly including outgroups, complicates the readability of the tree. For completeness, this information has also been added to the caption of the image (Lines 209-210).

The figure file was sent as a separate file of the text and it presented good resolution and is readable