PONE-D-25-16759Molecular Epidemiology of hepatitis E virus in wastewater in Yaoundé, Cameroon: Toward optimal prevention and control of emerging zoonotic pathogensPLOS ONE

Dear Dr. META-DJOMSI,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 28 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Janin Nouhin, Ph.D.

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include a complete copy of PLOS’ questionnaire on inclusivity in global research in your revised manuscript. Our policy for research in this area aims to improve transparency in the reporting of research performed outside of researchers’ own country or community. The policy applies to researchers who have travelled to a different country to conduct research, research with Indigenous populations or their lands, and research on cultural artefacts. The questionnaire can also be requested at the journal’s discretion for any other submissions, even if these conditions are not met.  Please find more information on the policy and a link to download a blank copy of the questionnaire here: https://journals.plos.org/plosone/s/best-practices-in-research-reporting. Please upload a completed version of your questionnaire as Supporting Information when you resubmit your manuscript.”

3. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

4. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://d.docksci.com/first-evidence-of-the-hepatitis-e-virus-in-environmental-waters-in-colombia_59f0f730d64ab2a3b992cac0.html

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

5. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files.

6. We note that your Data Availability Statement is currently as follows: [All relevant data are within the manuscript and its Supporting Information files.]

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

7. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.   

8. Please amend your manuscript to include your abstract after the title page.

9. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

1. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license. 

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

2. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

10. Please include a copy of Table 1 which you refer to in your text on page 5.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General comments: This study describes the detection of HEV-3 in untreated wastewater collected monthly in 2023 in Yaoundé, Cameroon. The detection rate was 26.4% (19/72) of all sites, with significant differences in distinct locations and seasons. The study is well-designed, and the topic is important; however, some results are not appropriately presented. I have the following comments that the authors may consider.

Major comments:

� The current title seems too big. Consider removing “Toward optimal prevention and control of emerging zoonotic pathogens”.

� A brief introduction of genomic structure of HEV is suggested, since ORF1 gene has been mentioned in the main text. Additionally, the authors should also mention the classification of HEV-3 subtypes.

� Statistical analysis has not been described in Material and Methods.

� Figure 1: The texts are difficult to read. Please increase the resolution.

� Table 1: What is the meaning of “positif”?

� Line 126: This reviewer does not understand “In this study, 52.6% (10/19) of the samples showed a strong signal for HEV-RNA.” How did the authors determine a strong signal for HEV-RNA? Please specify this in more detail.

� Figure 2 is difficult to comprehend. Percentage (%) should be at least indicated in the Y axis.

� What is the subtype of these HEV-3 strains circulating in this region? Furthermore, the authors should conduct sequence analysis of HEV ORF1 fragments identified in this study. For example, the sequence identifies between them.

� Figure 3: In the phylogenetic tree, rat HEV-C1 strains are very divergent thus unnecessary to be shown; in contrast, representative HEV genotypes within Paslahepevirus balayani can be included in the phylogenetic analysis. In addition, please indicate the HEV-3 subtypes on the right of the tree for a better understanding (see PMID: 29514938).

� Discussion: As introduced in line 64, HEV-1 has been detected in Cameroon previously. Also, to the knowledge of this reviewer, both HEV-1 and HEV-2 are circulating in the neighbor country Nigeria (PMID: 30352598). The authors may discuss why no HEV-1/2 has been detected in the wastewater in this study.

� Discussion, line 202: The sentence is overstated. The findings in this study does not support the hypothesis of zoonotic transmission of HEV in Cameroon, unless identical HEV sequences could be identified in both humans and animals. Please revise it.

� Notably, references 19, 22, and 23 are identical! Please ensure that the references are properly cited.

Minor comments:

� Line 47: Humans are also hosts of HEV-3. Please refer to the relevant review (PMID: 32810801).

� Line 79: This sentence is figure legend which should be removed in the main text.

� Line 92: Define “ORF”.

� Line 93: The reference 2 is irrelevant.

� Line 106: Change “readability” to “accuracy”.

� Lines 110 and 115: “hepatitis E virus” has already been defined as “HEV” earlier.

� Lines 112-114: Please define the abbreviations.

� Lines 124-125: Remove this sentence which is a Table title.

� Line 137: Remove this sentence of figure legend.

� Line 140: It should be “Figure 3”.

� Lines 148-151: Remove the figure legend in the main text.

� Unify “HEV-3” and “HEV genotype 3” throughout the manuscript.

� Line 214: Delete “(2)”.

Reviewer #2: This study illustrates the interest of wastewater based epidemiology for HEV, an enteric virus that can be transmitted zoonotically. Despite the low number of water samples analysed this study indicates the predominance of HEV genotype 3 circulation in Yaounde, Cameroon, rather than HEV genotypes 1 or 2 that are strictly found in human. This topic is interesting but the manuscript needs corrections and can be improved.

Comments

1. Methods

The region targeted in ORF1 gene for PCR detection and sequencing must be indicated. What is the limit of detection of the assay ? Wastewater can contain PCR inhibitors ; how was the analytical process controlled ?

2. Results, line 116 – 123

There are discrepancies between the text and table 1 : eg BONAS detection rate at 10.5% (2/19) line 116 and 41.7% (5/12) in Table 1; no corresponding site for 15.8% (3/19) line 116; repetitions lines 121-123

3. Results line 126 :

Is the signal intensity sufficiently reliable ? Was the signal intensity reproducible in duplicate PCR experiments ? Fig 2a is not very useful.

4. The detection rate according to the different sites could be related to the population size. A potential relationship should be analysed.

5. Phylogenetic analysis

Reference HEV sequences (Smith et al J Gen Virol 2020) must be used for determining HEV-3 subtype. This is relevant because HEV subtype 3a was previously detected in Cameroon and a comparison with HEV 3 subtypes detected in animals could provide additional informations

6. Discussion :

HEV RNA was detected in Yaounde wastewaters. The source is probably from humans infected with HEV but an animal source is also possible (eg infected pigs)? This point should be documented and discussed.

7. Discussion, lines 165-167 : Beside foodborne transmission of HEV, waterborne transmission is a possibility. However, the detection of HEV in wastewater does not prove waterborne transmission because this reflects only excretion of the virus from infected individuals.

8. References

All the references must be checked. Duplicate and triplicate were detected (eg ref 16 and 30 ; ref 19, 22 and 23 with confusion between names and surnames)

9. Figures and Table :

- Fig 1 legend must give more detail

- Fig 3 is missing

- Table 1 : a column with the sampling area should be added.

10. Minor points in introduction

- Line 41 : add contaminated food

- Line 47 : five instead of four

- Line 53 : HEV4 mainly found in Asia

- Line 55-56 : persistent infectious lead to liver cirrhosis (and not the reverse)

- Line 63 : add reference 16

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Molecular surveillance of hepatitis e virus in wastewater in yaoundé, cameroon

PONE-D-25-16759R1

Dear Dr. META-DJOMSI,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Janin Nouhin, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: