PNTD-D-25-01602

Shared viral burdens: Evidence of active Usutu virus circulation and multi-arbovirus exposure in migrant and resident birds at wintering locations in Nigeria

PLOS Neglected Tropical Diseases

Dear Dr. Atama,

Thank you for submitting your manuscript to PLOS Neglected Tropical Diseases. We sincerely apologize for the delay in the review process. We experienced difficulties in securing reviewers for your manuscript. We have now received one external review, and I have also carefully evaluated the manuscript.

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Kind regards,

Camille Lebarbenchon

Academic Editor

PLOS Neglected Tropical Diseases

Abdallah Samy

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

Additional Editor Comments:

The manuscript is well written, clear, and comprehensive. The study addresses an important topic, and the dataset collected represents a valuable contribution to the understanding of arbovirus circulation in wild bird populations in Africa.

I have only a minor comments related to the interpretation of some findings. In particular, I would suggest slightly tempering the interpretation of the detection of USUV RNA in feathers. While the Discussion is generally careful, some statements (including in the Abstract) may somewhat overextend the implications of RNA detection alone. Given that no infectious virus was isolated, it would be helpful to more clearly distinguish between evidence of viral RNA presence and evidence of active infection or transmission potential.

Similarly, the suggestion that migratory birds may contribute to long-distance dispersal of the virus, although plausible and supported by previous literature, could be framed more cautiously in light of the present data. Regarding the use of feathers as a sampling matrix, this is an interesting and potentially important observation. However, the conclusion that feathers may represent a suitable alternative to swabs appears to rely on assumptions about sample storage conditions that are not directly demonstrated.

Addressing these points would further strengthen the manuscript and ensure that the conclusions remain fully supported by the data.

Best regards,

Camille Lebarbenchon

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At this stage, the following Authors/Authors require contributions: Nnomzie Charles Atama, Joy Akpanta Ishong, Cora M Holicki, Yahkat Barshep, Felicity D Chandler, Anne van der Linden, Marion P G Koopmans, and Reina S Sikkema. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' comments:

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Methods

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Reviewer #1: No issues to report with the methodology

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Results

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Reviewer #1: Results are clearly described and appropriately analysed.

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Conclusions

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Reviewer #1: No concerns with the conclusions

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Editorial and Data Presentation Modifications?

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Reviewer #1: Minor revision

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Summary and General Comments

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Reviewer #1: Thank you for inviting me to review 'Shared viral burdens: Evidence of active Usutu virus circulation and multi-arbovirus exposure in migrant and resident birds at wintering locations in Nigeria' by Atama et al. The manuscript was very interesting, study well designed and very well written. Overall the manuscript warrants publication and used appropriate statistical analyses throughout.

I really only have a couple of very minor edits to suggest:

L41: Should 'was' be 'were'

L105-107: you dont specify that these were sampled in Nigeria, its implied, but might not be obvious

L108: When was SINV detected, if someone reads this paper in 20 years the detection wouldn't be recent, so best to be specific

L112: 253 migratory bird 'species'. Just to clarify that it isn't just a home for 253 birds

L175: You say swabs were shortly stored under 4oC. Can you be more specific, what was the window where they were out of cold storage, how many hours?

L193: We typically use ct 35 as a cut off, due to inability to sequence and corroborate the PCR. Can the authors confirm that the 6 positive USUV feathers were positive on both the screening and confirmatory PCR assays, and can they provide cts for both assays in the results section. This will provide important evidence for one of their main findings. Moreover, if the authors could provide the number of bp each assay should recover that would be beneficial.

L334: The low number of positives and very late cts dont immediately suggest active circulation. These could have been historic infections picked up from other localities prior to sampling in Nigeria. Moreover, given the broad geographic range of these arboviruses, the serological results could also be due to historic exposure in different areas. I agree transmission is likely to occur in Nigeria, but could the authors contextualise these findings appropriately throughout the ms, with some caveats.

L336: USUV RNA detected, by rt-PCR only, no sequencing or virus isolation to corroborate. Please ensure the result is accurately reported

L353:optimal cold storage. But could also be stage of infection? i.e. the bird may have been infected some time ago and almost cleared the infection?

L404: Here and more generally, you dont mention the utility of mosquito surveillance to help corroborate your results, obviously you didnt do it here, but it would be useful for future work?

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Reviewer #1: No

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Dear Atama,

We are pleased to inform you that your manuscript 'Shared viral burdens: Molecular evidence of Usutu virus infection and multi-arbovirus exposure in migrant and resident birds at wintering locations in Nigeria' 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.

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

Best regards,

Camille Lebarbenchon

Academic Editor

PLOS Neglected Tropical Diseases

Abdallah Samy

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

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