[EXSCINDED]Dear Dr. Lambrechts:

Thank you very much for submitting your manuscript "A Peridomestic Aedes malayensis Population Increases Risk of Yellow Fever Virus Transmission in Singapore" (PNTD-D-19-01129) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

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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: This study seeks to clarify the degree to which occurrence of Aedes malayensis in Singapore exacerbates the risk of yellow fever virus introduction to that country. The authors conducted a rigorous experimental study of vector competence with an Aedes aegypti control, and a field study to ascertain human attraction. It is the latter that is tripping me up a little, in that no unbaited traps were used to determine the number of Ae. malayensis that might have entered the net in the course of daily movement. Can the authors specify what the "other" mosquitoes captured via this method were? Were any of them species well known to not be attracted to humans?

Reviewer #2: Methods were appropriate and carefully described.

Reviewer #3: For the most part with the vector competence experiments. However, the host-seeking behavior study does not fulfil this criteria. Refer to attached document for full information.

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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: Statistical analyses are rigorous but I would ask that the authors delete text referring to "marginally insignificant" results and remove symbols to denote "P

-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: I am somewhat concerned about drawing strong conclusions about human attraction from the field data without an unbaited control; the authors could allay this concern if there are studies in the literature showing a clear difference.

Also, the authors should discuss the capture of a large number of Ae. albopictus in this urban park. This species has also been shown to be competent for YFV and to my mind poses the larger risk here.

Reviewer #2: Conclusions are supported by data, namely that Aedes malayensis is a component vector of yellow fever virus (YFV). YFV infection and dissemination rates in Ae. malayensis and Ae. aegypti were similar.

Reviewer #3: Refer to attached document

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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: Throughout, including title: Change to "Occurrence of a peridomestic Aedes malayensis population..." It is not the global existence of Ae. malayensis that exacerbates risk in Singapore, it is the occurrence of this species IN Singapore that exacerbates risk there.

Line 30: Higher than what? Clarify

Line 38: Change "a" to "one" and indicate 3 trap days- this was a very limited study and that should be made more clear.

Line 50: move "was" before "Ae. malayensis"

Line 51: move "experimentally" after "to"- infinitives may be split when necessary for clarity!

Summary: reconsider what has actually been demonstrated about human attraction based on comments above

Line 66: delete "largely"

Line 122: Change thereafter to hereafter

Line 177: In future, please provide volunteers with a motorized aspirator to enhance safety

Throughout: the term "insignificant" is not standard; please change to "not significant"

Line 271-272: move "were" to after "only"

Table 1: Please reformat to improve legibility (font is variable and some of it tiny); remove annotation of P

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 study highlights a very important risk factor for the spread of yellow fever into Asia. The experimental analysis of vector competence is very well done. The field study is both small scale and lacks a control-these limitations must be addressed.

Reviewer #2: Arbovirologists, epidemiologists and public health experts have long pondered, “why yellow fever has never appeared in Asia?” Certainly there were multiple opportunities in the past when Chinese explorers sailed to Africa and during the European colonial period when British, French, Dutch and ships of other nations (full of Ae. aegypti) made frequent journeys from Africa and tropical America to southeast Asia, India and the Pacific Islands. But, to our knowledge, yellow fever virus (YFV) never appeared or caused epidemics there, as happened periodically in Europe and the Americas.

Experimental studies have shown that Asian Ae. aegypti are similar to African and New World Ae. aegypti in their susceptibility and vector competence for YFV. Also humans and non-human primates of Asia origin are also susceptible. The most plausible explanation seems to be that most people living in Asia in the past and now have heterologous flavivirus antibodies due to natural or vaccine-induced infection with other flaviviruses like dengue and Japanese encephalitis viruses. Animal studies indicate that hamsters infected previously with JE and other flaviviruses have lower levels of viremia and modest liver pathology, when subsequently infected with YFV.

In this excellent study, the investigators have demonstrated that Ae. malayensis, an anthpophilic mosquito in Singapore, is a competent vector of YFV. Ae. malayensis occurs in some urban habitats that are not currently targeted in Ae. aegypti control programs, and they may contribute to “cryptic” arbovirus (DENV, CHIKV, ZIKV) transmission. But these findings do not increase the risk of YFV transmission in Singapore.

Ae. malayensis was first described in Singapore in 1962 and has subsequently been reported in Malaysia, Thailand, Cambodia, Vietnam and Taiwan. It probably occurs throughout southeastern Asia. So it is not a new introduction and has probably been in the region for thousands of years. Given that history and the multiple opportunities for introduction of YFV into the region in the past, is Singapore now at greater risk of a yellow fever outbreak that it was 200 or 400 years ago? If Ae. aegypti populations are maintained at a low level in Singapore, it may actually be at a lower risk, regardless of the presence of Ae. malayensis.

In the opinion of this reviewer, a more appropriate title for this article would be something like this:

“Aedes malayensis, a peridomestic mosquito in Singapore, is a competent vector of yellow fever virus.” In the discussion, the authors could speculate that this finding might facilitate the introduction of YFV into Singapore or other countries in the region where Ae. malayensis occurs.

Reviewer #3: Refer to attached document

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Attachments

Attachment

Submitted filename: YFV Ae. malayensis Singapore final.docx

Dear Dr. Lambrechts,

We are pleased to inform you that your manuscript, "A peridomestic Aedes malayensis population in Singapore can transmit yellow fever virus", has been editorially accepted for publication at PLOS Neglected Tropical Diseases.

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Best regards,

Elvina Viennet

Guest Editor

PLOS Neglected Tropical Diseases

David Harley

Deputy Editor

PLOS Neglected Tropical Diseases

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