PONE-D-19-19136

Molecular epidemiological characteristics of dengue virus carried by 34 patients in Guangzhou in 2018

PLOS ONE

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[The research work was supported by the National Nature Science Foundation of China (81803813),

376 the Science & Technology Planning Project of Guangzhou (No.201804010029), the Special Foundation

377 supported by the Central Government for Higher Education Research Program (2013-2015): Project

378 Number: A1-AFD016141A02-14, A1-AFD018181A28) and the State Major Research Program for

379 Innovation Team of Guangdong Province (Project Number: E1-KFD015151K02).]

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Additional Editor Comments (if provided):

This a phylogenetic study of dengue virus in China.  The population size is quite small; therefore, robust inferences are very difficult.  The phylogenetic analyses are well described and appropriate for this type of study.

It would have been very helpful for the authors to evaluate dengue virus from other years as well.  Restricting the study to a single year – even an outbreak year – limits usefulness of the data collected.  For instance, did the virus evolve in 2018 in such a way as to be different from previous years?  If so, what are the virologic differences across the viral genome?

Are other dengue virus sequences available from southern China in the years before 2018?  These should be included in the phylogenetic analyses.

Serum samples from 170 individuals with dengue were evaluated, so why are genotypic data presented for only 34?  How are these 34 different / similar to the 55 individuals with positive blood samples?

How large are the E gene sequences that were analyzed?  This should be stated explicitly.

Lines 150-152 state that a set of global dengue references were used.  How many DENV-1 and DENV-2 references were included?

How was the propagation path evaluated?  This part of the analysis is not described in the Methods.

For the 5 sequence clusters, the authors should calculate the median genetic distance and compare that to sequences that are not in clusters.  It is important for the readers to have some quantified measure of similarity / dissimilarity amongst sequences within a cluster.

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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: No

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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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: No

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

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Reviewer #1: Yes

Reviewer #2: Yes

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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: Liao et al. studied 34 DENV strains, including 29 DENV-1 strains and 5 DENV-2 strains, isolated from blood samples from dengue fever patients at Guangzhou in 2018. They amplified the envelope genes of these isolates by RT-PCR and performed phylogenetic analysis. The found DENV isolates of 2018 in Guangzhou were mainly imported from Southeast Asian countries. Propagation paths based on phylogeographical analysis suggested potential local DENV transmission in Guangzhou.

The authors have carried out RT-PCR, sequencing and phylogenetic analysis of E genes of 34 DENV strains isolated from DF patients at Guangzhou in 2018. Overall, the observation that DENV isolates in Guangzhou were mainly imported from Southeast Asian countries did not bring new insights to the field. The evidence of possible local DENV transmission in Guangzhou over the years was weak. Moreover, there several places in the introduction, methods and results need to be clarified. Several references were inappropriate or irrelevant. See specific comments. There should be addressed to improve this manuscript.

Specific comments:

1) Line 28: “, including 29 DENV-1 strains and 5 DENV-2 strains, isolated from a blood sample from…”? Should be blood samples.

2) Lines 55-59: “DENV, as a member of the genus Flavivirus in the family Flaviviridae, is an enveloped, single stranded, positive-sense RNA virus [8]. The DENV genome is approximately 11,000 nucleotides in length and encodes three structural proteins, namely, the capsid (C), premembrane (prM), and envelope (E) proteins, and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) in a single open reading frame (ORF) [9,10].” References 8, 9 and 10 are not appropriate for the citation here.

3) Lines 60-63: “There are five DENV serotypes…classified into different subtypes [11]”? Five DENV serotypes are controversial due to lack of evidence in humans. Major literature states 4 DENV serotypes. There is no citation of 5 serotypes and Reference 11 is inappropriate here. Different subtypes?

4) Line 83: “Guangdong Province sees a continuous increase in the number of patients”?

5) Lines 69 to 77: They provided a review of DENV epidemiology in Guangdong up to 2014. What happened between 2015 and 2017 before their study in 2018?

6) Lines 99-100: “IgM and IgG enzyme-linked immunosorbent assay (ELISA) kits were used to confirm dengue infection”. Please describe which commercial kits were used or in-house assays.

7) Lines 100-101: “A total of 170 serum samples were available”. These are likely acute samples. Which days post onset of symptoms were the samples collected?

8) Lines 112-113: “The amplified PCR products…” What are the PCR conditions? Reference 24 was real-time RT-PCR and not relevant here.

9) Line 117: “DENV-2 standard Hawaii strains”?

10) Table 1 “Primer sequence, and size of RT-PCR product…” There is no information of the size of RT-PCR product. At least, the genome positions of primers should be presented. There is no reference of these.

11) Table 2: The genome positions of primers should be presented.

12) Lines 150-152: “…we set up a global dataset that involved all samples and DENV-1 and DENV-2 sequences available from the GenBank…”? How many sequences from the GenBank were selected in the analysis and what are selection criteria? Do they include representative sequences from different genotypes within each serotype?

13) Lines 201-202: “… the entropy-based index presented by Xia. The ….Iss<iss.c)..” by="" entropy-based="" index="" is="" presented="" what="" xia="">14) Lines 336-338: “Since Aedes albopictus is the main medium for DENV transmission…eggs and offspring through winter [34]”. Aedes albopictus is the main medium for DENV? Reference 34 is about program interface and is an irrelevant citation.

15) Lines 332-333: “Others were localized strains after vertical transmission”. Vertical transmission has been reported to be very insufficient. Is vertical transmission the only mechanism to explain localized strains? How about silent transmission or sporadic cases plus under-reporting?

Reviewer #2: Liao et al . determined 34 DENV (29 DENV-1 and 5 DENV-2) sequences of E region from isolated virus in Guangzhou, Guangdong Province, China during 2018. They concluded that DENV in Guangzhou was mainly imported from Southeast Asian countries from the phylogenetic analysis but there are several points to be clarified before they reach to the conclusion.

Major concerns.

1. DENV-1 is sub-divided into five genotypes: U, II II, IV and V. DENV-2 is classified as six genotypes: Asian I, Asian II, Asian/American, Cosmopolitan, American and sylvatic. These genotypes should be assigned in Figure 2 and specify the genotypes the presented cluster I-V belongs to.

2. In Table 3, do location columns really indicate the place of infection?

Is there any questioner asking travel history?

3. There are several reports from China. (1) Lin F et al. (https://doi.org/10.1007/s00705-019-04266-1) already reported the DENV-2 genotype Cosmopolitan in same Guangdong province in 2015. (2) Yu H et al. https://doi.org/10.1038/s41598-019-43560-5.) also reported DENV-2 genotype Cosmopolitan in Zhejiang Province, in 2017. (3) Cao et al. (https://doi.org/10.1371/journal.pone.0213353) reported DENV-1 genotype I and V in 2015. (4) Zou et al. (https://doi.org/10.1371/journal.pntd.0007202) also reported DENV-1 genotype I and V in 2014. Many Asian I viruses were deposited to NCBI from China. Before reached to the conclusion that the origin of the virus isolated in this study derived from Southeast Asian countries, the author should pay more attention to the pre-existing Chinese viruses in phylogenetic analysis. Can you deny the possibility from Yunnan or other part of China?

4. The sequences used in molecular clock tests were deviated to recent sequences.

5. Did the author calculate the Bayes factor for phylogeographic reconstruction in Figure 3? What was the cut-off value?

6. In line 173, why the ULRC was selected?

7. In line 174, why the Bayesian skyline coalescent model was selected?

Minor points:

1. The lines in the phylogenetic tree were faint in Figure 2.

2. In line 61, the reference is required for DENV-5.

3. The accession numbers of the reference sequences used in Figure 2-8 should be listed in the supplemental tables.

 </iss.c)..”>

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Reviewer #1: No

Reviewer #2: No

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Molecular epidemiological characteristics of dengue virus carried by 34 patients in Guangzhou in 2018

PONE-D-19-19136R1

Dear Dr. Li,

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

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Jason Blackard, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

None

Reviewers' comments:

None

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. 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 #2: Yes

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

Reviewer #2: Yes

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4. 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 #2: Yes

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5. 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 #2: Yes

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6. 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 #2: (No Response)

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Reviewer #2: No