Dear Dr. Williams,

Thank you very much for submitting your manuscript "Molecular detection and characterisation of the first Japanese encephalitis virus belonging to genotype IV in Australia" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Geraldine Marie Foster, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Elvina Viennet

Section Editor

PLOS Neglected Tropical Diseases

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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: (No Response)

Reviewer #2: The objective is clearly stated. No concerns.

Reviewer #3: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? Yes

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

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

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

-Were correct statistical analysis used to support conclusions? Yes

-Are there concerns about ethical or regulatory requirements being met? No

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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: (No Response)

Reviewer #2: The results are clear and supported.

Reviewer #3: -Does the analysis presented match the analysis plan? Yes

-Are the results clearly and completely presented? Yes

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

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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: (No Response)

Reviewer #2: The conclusion is good and the discussion explains the public health relevance.

Reviewer #3: -Are the conclusions supported by the data presented? Yes

-Are the limitations of analysis clearly described? NA

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

-Is public health relevance addressed? Yes

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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: (No Response)

Reviewer #2: I did not see any edits.

Reviewer #3: Lines 132-136 and 228-233: Prior to reviewing this manuscript, I was not familiar with the utility of using embryonated chicken eggs to isolate avian-associated flaviviruses. Please consider adding some references describing this procedure and its relative success compared to other methods of isolation (i.e., cell culture and intracranial inoculation of suckling mice).

Lines 346-349 and Table 1: It is interesting that the Shao et al., 2018 RT-PCR assay detected JEV RNA in the right thalmus tissue sample but in no other tissue sample types. Do you happen to have an explanation for this finding (e.g., sequence mismatch in the primer-binding site)?

Lines 418-420: Has regular surveillance for JEV in mosquitoes, birds, and/or pigs been conducted in Timor-Leste, West Papua, and Papua New Guinea? After reading this sentence, I am unclear if regular surveillance for JEV is conducted, and the virus has not been detected or if regular surveillance has not been conducted. Please clarify.

Figures 2 and 3, and Supplementary figure 1: Please include bootstrap support values for the genotype-defining nodes.

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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: (No Response)

Reviewer #2: This paper looks ready to be published and contains interesting data.

Reviewer #3: In the manuscript “Molecular detection and and characterisation of the first Japanese encephalitis virus

belonging to genotype IV in Australia” (PNTD-D-22-01068), Sikazwe et al. reports the detection and whole genome sequencing of Japanese encephalitis virus (JEV) RNA recovered from a fatal case of JE that occurred in a resident of the Tiwi Islands, in the Northern Territory of Australia in 2021. The authors then perform an evolutionary analysis to examine the link between this new genotype IV sequence (first detection of genotype IV of JEV in Australia), other genotype IV sequences from Vietnam and Indonesia detected from 1979─2019, and a genotype IV sequence recovered from a large JE outbreak in southeastern Australia in 2022. The close phylogenetic relationship between the Tiwi Island sequence and recent Indonesian sequences suggest that Indonesia might be the origin of genotype IV viruses currently circulating in Australia.

This manuscript is very well-written, and the study was well-executed and scientifically sound. The introduction includes all relevant information necessary to understand the study, the methods are comprehensive, the results are well-organized, and the discussion nicely discusses possible origins of genotype IV sequences currently circulating in Australia, and the need for JEV vector competence studies and ongoing surveillance for the virus. Listed below are a few very minor comments.

Specific comments

Lines 132-136 and 228-233: Prior to reviewing this manuscript, I was not familiar with the utility of using embryonated chicken eggs to isolate avian-associated flaviviruses. Please consider adding some references describing this procedure and its relative success compared to other methods of isolation (i.e., cell culture and intracranial inoculation of suckling mice).

Lines 346-349 and Table 1: It is interesting that the Shao et al., 2018 RT-PCR assay detected JEV RNA in the right thalmus tissue sample but in no other tissue sample types. Do you happen to have an explanation for this finding (e.g., sequence mismatch in the primer-binding site)?

Lines 418-420: Has regular surveillance for JEV in mosquitoes, birds, and/or pigs been conducted in Timor-Leste, West Papua, and Papua New Guinea? After reading this sentence, I am unclear if regular surveillance for JEV is conducted, and the virus has not been detected or if regular surveillance has not been conducted. Please clarify.

Figures 2 and 3, and Supplementary figure 1: Please include bootstrap support values for the genotype-defining nodes.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

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Reproducibility:

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References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Attachments

Attachment

Submitted filename: Characterisation of Australian Japanese encephalitis virus genotype IV.docx

Dear Dr. Williams,

We are pleased to inform you that your manuscript 'Molecular detection and characterisation of the first Japanese encephalitis virus belonging to genotype IV acquired in Australia' 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,

Geraldine Marie Foster, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Elvina Viennet

Section Editor

PLOS Neglected Tropical Diseases

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