PNTD-D-25-01092

Serological and viral prevalence of Oropouche virus (OROV): A systematic review and meta-analysis from 2000-2024 including human, animal, and vector surveillance studies

PLOS Neglected Tropical Diseases

Dear Dr. Whelan,

Thank you for submitting your manuscript to PLOS Neglected Tropical Diseases. After careful consideration, we feel that it has merit but does not fully meet PLOS Neglected Tropical Diseases'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 within 60 days Nov 16 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 plosntds@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pntd/ 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 editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Rebecca C. Christofferson

Academic Editor

PLOS Neglected Tropical Diseases

David Safronetz

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

Journal Requirements:

1) We ask that a manuscript source file is provided at Revision. Please upload your manuscript file as a .doc, .docx, .rtf or .tex. If you are providing a .tex file, please upload it under the item type u2018LaTeX Source Fileu2019 and leave your .pdf version as the item type u2018Manuscriptu2019.

2) Please upload all main figures as separate Figure files in .tif or .eps format. For more information about how to convert and format your figure files please see our guidelines: 

https://journals.plos.org/plosntds/s/figures

3) We have noticed that you have uploaded Supporting Information files, but you have not included a list of legends. Please add a full list of legends for your Supporting Information files after the references list.

4) Thank you for stating "Study data will be made available through the interactive and open-access ArboTracker dashboard and data platform, accessible at 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. 

5) Please ensure that the funders and grant numbers match between the Financial Disclosure field and the Funding Information tab in your submission form. Note that the funders must be provided in the same order in both places as well.

6) Please amend your Competing Interests statement in the online submission form. Please declare all competing interests beginning with the statement "I have read the journal's policy and the authors of this manuscript have the following competing interests:"

Note: If there are no competing interests to declare, please state "The authors have declared that no competing interests exist". 

6) As required by our policy on Data Availability, please ensure your manuscript or supplementary information includes the following:

- A numbered table of all studies identified in the literature search, including those that were excluded from the analyses.

- For every excluded study, the table should list the reason(s) for exclusion.

- If any of the included studies are unpublished, include a link (URL) to the primary source or detailed information about how the content can be accessed.

- A table of all data extracted from the primary research sources for the systematic review and/or meta-analysis. The table must include the following information for each study:

  - Name of data extractors and date of data extraction

  - Confirmation that the study was eligible to be included in the review.

  - All data extracted from each study for the reported systematic review and/or meta-analysis that would be needed to replicate your analyses.

  - If data or supporting information were obtained from another source (e.g. correspondence with the author of the original research article), please provide the source of data and dates on which the data/information were obtained by your research group.

  - If applicable for your analysis, a table showing the completed risk of bias and quality/certainty assessments for each study or outcome.  Please ensure this is provided for each domain or parameter assessed. For example, if you used the Cochrane risk-of-bias tool for randomized trials, provide answers to each of the signalling questions for each study. If you used GRADE to assess certainty of evidence, provide judgements about each of the quality of evidence factor. This should be provided for each outcome.

  - An explanation of how missing data were handled.

This information can be included in the main text, supplementary information, or relevant data repository.

Note: If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Reviewers' Comments:

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The methods section should be described in greater detail. For instance, what type of generalized linear mixed-effects models were used? What distribution and link function were applied? This information is particularly relevant and is currently not adequately reported.

The authors should make a greater effort to identify and understand the sources of heterogeneity among the studies so that appropriate conclusions can be drawn from the results. A meta-regression analysis may be appropriate in this regard.

Reviewer #2: This is not a hypothesis-driven study

I am not concerned about the ethical or regulatory requirements being met.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The analysis does match the analysis plan.

It is important to specify the within-country locations of the included studies. Generalizing results at the country level may overstate the findings. For example, reporting seroprevalence of 24.4% for Brazil or 14.5% for Colombia may be misleading. The studies reporting the highest seroprevalence in Brazil for instance (Azevedo et al., 2004; Vasconcelos et al., 2006) were conducted in the state of Pará, which accounts for only about 4% of the Brazilian population. Greater effort should be made to account for the within-country origin of the studies and, more importantly, the local characteristics of those areas.

Was any additional analysis performed to investigate the high heterogeneity observed in the results? Based on those values, can the authors reasonably justify grouping these studies together? Probably authors should explore alternative grouping of the results

Reviewer #2: Yes

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusions should be refined to reflect a more accurate presentation of the results. It does not seem reasonable to use highly localized studies to draw country-wide conclusions.

Only by considering further refinement of results I would confidently state that conclusions are important for the understanding of the topic

Reviewer #2: Yes

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Figures: Figure legends should be self-explanatory. For example, clarify what the numbers in brackets represent.

I suggest to authors improve literature support of the statements, specially in the introduction. For example,

First paragraph of introduction: “In addition, early data raise the concern that OROV’s vector range may be expanding both geographically and into other carrying vectors beyond the biting midge, for example Culex mosquitoes” This sentence needs a reference.

Second paragraph of introduction: “Additionally, evidence of infection in animal reservoirs like birds and mammals remains poorly defined, complicating efforts to assess zoonotic spillover risks”. Needs a reference.

Reviewer #2: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This is a relevant study. However, a major concern is the broad generalization of the results. Grouping studies solely by country without considering their local context can be misleading. For instance, stating that febrile individuals in Brazil have an Oropouche seroprevalence of 24.4% is problematic. Closer examination reveals that most of the studies reporting the highest values were conducted in municipalities located in highly sylvatic areas of Brazil (e.g., Amazonas and Pará). Such values should be attributed to those specific regions rather than the entire country, as the current approach may significantly distort the interpretation of the results.

Additionally, the reported heterogeneity is extremely high—above 95%. This issue does not appear to be adequately addressed, especially given the presence of very low estimates (e.g., prevalence between 0.2% and 3.5%). Further analyses are warranted to explain and understand the sources of variability. Possible approaches include meta-regression or more detailed intra-group analyses. It is also possible that important local variables, not considered in the current analysis, could explain part of this variability.

Reviewer #2: This manuscript presents a systematic review and meta-analysis of OROV serological and viral prevalence across human, animal, and vector populations from 2000-2024. The authors identify 71 articles and provide pooled prevalence estimates for humans, as well as a descriptive synthesis for animal and vector data. The review is well-motivated, the methods are largely appropriate, and the open-access data dashboard (ArboTracker) is a valuable resource. The manuscript would benefit from clarifications in data presentation, additional detail in the methods, and a more explicit discussion of certain limitations.

Major Comments

1. The manuscript states that 71 articles were included, but only 61 (human) and 96 (vector) studies were sampled after 2000 for meta-analysis. The text should explicitly state which data were meta-analyzed vs. only described, and why the 2000 cutoff was chosen. Please also clarify how multiple estimates from the same source were handled, e.g., were they pooled within-study or treated as separate entries?

2. The I2 values for the pooled estimates are extremely high (≥ 92-99%). While heterogeneity is acknowledged, the manuscript would benefit from more formal exploration, e.g., meta-regression or sensitivity analysis by test type, sampling frame, or outbreak vs. non-outbreak context.

3. As diagnostic assay performance is a major confounder (particularly cross-reactivity in ELISA/HAI), stratifying or adjusting by assay type could improve interpretability.

4. The limitations section mentions cross-reactivity and poor reporting of assay details, but the impact on pooled prevalence is not quantified. Could you provide a summary table indicating which studies used confirmatory neutralization tests vs. screening assays only? This would allow readers to assess confidence in different subsets of the data.

5. The descriptive synthesis of animal and vector prevalence is valuable but currently feels secondary. The text could be strengthened by summarizing the geographic overlap between human and vector detection. Commenting on the time gap in vector studies, e.g., no studies after 2018 in certain groups, and discussing vector competence evidence in more detail, referencing the limited number of studies showing OROV RNA in non-traditional vectors.

6. The automated decision-rule approach is innovative, but for transparency, the manuscript should provide a brief explanation of the six automated items vs. two manually reviewed items. An example of how different combinations map to low/moderate/high ratings. Clarify if human, vector, and animal studies were assessed using the exact same criteria.

7. Figure 2 and Figure 3: Consider adding the number of studies (n) and total sample size directly in the plot captions or alongside the pooled estimates.

8. Figure 5: Label n and sample size. For all forest plots, displaying raw proportions alongside CIs would make them easier to interpret.

9. Some forest plots (esp. Figure 2) are difficult to read due to density; splitting by country or using facet plots could help.

Minor Comments

1. Introduction “…OROV may additionally be transmitted sexually and…” I may reword this because it is too early to speculate.

2. Line 172: "i.e., with fever and related symptoms" replace “i.e.” with “that is” or rephrase for smoother readability.

3. Ensure consistent terminology: the manuscript alternates between “non-human animals” and “animal hosts”. Pick one and define early.

4. Some tables (e.g., Table 2) contain a lot of detail; consider moving full species lists to supplementary materials for readability.

5. The Discussion could explicitly mention that prevalence estimates in humans may be biased upward in febrile cohorts due to convenience sampling.

6. The conclusion mentions “increasing severity” of OROV epidemics; severity metrics are not presented in this paper, so this should be reworded unless referencing external data.

**********

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.]

Figure resubmission:

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Ms Whelan,

We are pleased to inform you that your manuscript 'Serological and viral prevalence of Oropouche virus (OROV): A systematic review and meta-analysis from 2000-2024 including human, animal, and vector surveillance studies' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Rebecca C. Christofferson

Academic Editor

PLOS Neglected Tropical Diseases

David Safronetz

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

***********************************************************

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; line-height: 16.0px; font: 14.0px Arial; color: #323333; -webkit-text-stroke: #323333}span.s1 {font-kerning: none