Dear Dr. Inizan,

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Additional Editor Comments:

Dear Dr. Catherine Inizan,

Thank you for submitting your manuscript titled "Development of a competition assay to assess the in vitro fitness of dengue virus serotypes using an optimized serotype-specific qRT-PCR" to PLOS ONE.

After careful evaluation by four reviewers, we regret to inform you that your manuscript requires major revisions before it can be considered for publication. Three reviewers have identified significant issues, especially with the methodology and the clarity of the results, which need to be addressed in detail. One reviewer recommended rejection. However, with substantial revisions, we believe the manuscript has the potential to be reconsidered for publication.

Please revise your manuscript to address all the comments and concerns raised by the reviewers. Specifically, focus on the following areas:

Methodology: Provide further clarification and detail regarding the experimental design and analysis. Reviewers highlighted the need for more robust justification for some of the methods used.

Data Interpretation: The presentation and interpretation of your results need to be clearer, particularly in how the data support your conclusions.

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Best regards,

André Ricardo Ribas Freitas

Academic Editor

PLOS ONE

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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?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: No

Reviewer #4: Partly

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

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: I Don't Know

Reviewer #4: No

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: The manuscript by Griffon and colleagues entitled “Development of a competition assay to assess the in vitro fitness of dengue virus serotypes using an optimized serotype-specific qRT-PCR” is interesting research.

In identifying DENV serotype is difficult, when there is a multiple serotype infection. Due to difficulty in identifying; most researchers report single serotype infection rather than multiple serotype infection. This research is quite useful. It will be much better, if the author has validated their claims with the serum sample containing multiple DENV serotypes. Below are the comments that will help to strengthen the manuscript.

Critical comments:

1. In order to avoid discrepancies related to suitability of using serum samples due to viral RNA degradation; it will be best to address the time span in which the samples were collected, stored and reused. Also indicate how these samples were stored in the methods section.

2. The authors have mentioned on the genotypes of DENV-1, 2, 3 & 4 for viral isolates. How the genotypes were identified and by using which gene? Are these from serum samples from your study? Whether these DENV strains were used in Huh7 cultures? Whether these strains were sequenced completely? Include these details in the manuscript.

3. There is no evidence of phylogenetic tree for the mentioned genotypes of DENV-1, 2, 3 & 4. Please provide details in methods and results section.

4. How many serum samples were used in this experiment? How long the serum samples were stored before RNA extraction? How these samples were stored? Indicate in which experiments these samples has been used? Include this information at appropriate places in methods section.

5. How the primers/probes were designed? Which gene was used for designing the primers/probes? Whether the designed primers/probes were from gene/genome sequences available from data repository or from your own sequencing or from other researcher’s publication? Please, include the details appropriately.

6. Competition assay – As per the methods, 2 x 105 HuH7 cells were seeded and after 24 hours viral isolates were introduced. Whether these cells attain confluence in 24 hrs? The cell population might also affect the titer value of viral load, right? How this was avoided?

7. The result section has no way to identify whether experimental observation is from human serum or cell culture. Please indicate wherever appropriate.

8. The competition assay shown interesting results. Whether the author validated in human serum samples? It is requested the author should validate the competition assay observation in their collected human serum samples containing one or more DENV serotypes.

9. Whether competition assay can help in identifying different genotypes within serotypes? Explain in the discussion section.

10. The discussion is superficial. It will be better to do an in-depth discussion. Compare your observation with other researchers’ article and discuss.

11. The authors have provided two genome data from GenBank. What is the purpose of providing it? If you used in your experiments, provide where and how. Similarly, you have provided data from GISAID, please include in which experiment you have used it or what is the importance of these sequence? Include at appropriate places.

12. Since the serum samples from before and after COVID pandemic i.e., from 1995-2021 was included in your study, did the author perform any COVID testing in these samples? If so, is there any difference in DENV viral load or serotype difference in COVID positive serum samples? It is advised to include COVID analysis status (as acknowledgement or wherever possible) in this manuscript which might be helpful for the researchers to know whether COVID has impact on DENV infection.

Specific comments:

Line 104-106: how the genotypes were identified? Provide details.

Line 114: Provide the primer/probe list as supplementary data.

Line 240-242: The sentence ended with a citation. From the information, it is a statement from your observation. Avoid providing citation for your statements unless you have already published article replated to this. It is suggested that the author should reframe the sentence.

Line 114 & 203: How many sets of primers/probes were used? The authors have mentioned two different articles (Warrilow et al., 2002, Ito et al., 2004) as reference. Clarify and rewrite.

Table 2: Lacks information on gene, gene location, repository name and ID from which the sequence was obtained.

The preprinted supporting methods section does not provide any valid information, it is just the repetition of the manuscript. Avoid submitting this or provide valid information for supporting additional methods which are not provided in the main manuscript.

Reviewer #2: This is an interesting manuscript but the authors need to make clear for the reviewers and all readers.

1. The authors described that the authors developed a new multiplex RT-PCR system for quantification of DENV for competition assay. Did the authors make validation about the cross reactivity f the primers and probes with other serotypes of the virus as well as the other flaviviruses. Please describe clearly at the revised manuscript.

2. Regarding the ethical issues, did the authors take written informed consent from the patients or please briefly describe about the justification for not taking ethical clearance.

3.The authors described the the concentration of the virus by mixing supernatants and the authors described that the results showed expected results. The authors should make confirmation of the results with another method such as immunofluorescence based focus assay method and the authors already established this system.

4. Regarding the competition assay, the authors must clear describe how many and how did the author check the competition assay such DENV1 and DENV-2 or DENV1 and DENV-3 or DENV2 and DENV-4. This point did not clear to the reviewers.

5. The authors did not describe how many samples were used in this study .

6. Why did the author used HuH cells for competitive assess and please briefly describe the justification.

7. For the competition assay, did the authors check different genotypes within one DENV serotypes. How did you do for two different genotypes competition assay because your RT-PCR system did not support for this competition assays,

Reviewer #3: This manuscript tried to introduce a novel competition assay of dengue virus serotypes using an optimized serotype-specific qRT-PCR. My major concerns are as follows:

1. The authors should provide more detailed results to support the specificity of the assays. From Figure 3, I could infer that the manual threshold adjustment is not reliable. Because fluorescence intensity could be influenced by many factors not restricted to the matching degree between the primers and the template. However, the delta Cp value between matched and unmatched primer pairs is relatively reliable. Recommend the authors set up a reliable threshold of the delta Cp value to support the specificity.

2. Could the author explain why the efficiencies of the qRT-PCR are so high and looks unnormal?

3. The authors should enroll more samples to validate the performance of the developed assays. and provide the related statistical analysis.

Reviewer #4: Introduction

- Line 44 - 45: DENV serotypes share between 34 and 73% identity at the nucleotide sequence level [Blok J et al., 1985].

Please check (https://doi.org/10.1186/s12864-019-6311-z). Inter-serotype nucleotide identity is around ~60-70% while polypeptide sequence identity might be as low as ~30%.

-Line 47-48: However, the mechanisms underlying DENV serotypes co-circulation and replacements are misunderstood.

To be misunderstood could mean "to fail to understand somebody/something correctly." Please consider using another word or rephrase the sentence.

-Line 48-51: Viral replicative fitness may contribute to DENV serotype replacements. It is therefore of prime importance to set up experimental models to assess the relative replicative fitness of strainsfrom different serotypes.

DENV serotype replacements could not be primarily attributed to replicative fitness. Host immune response and herd immunity might play more important roles in serotype replacements [https://doi.org/10.1073/pnas.1120621109]. Please provide more references in the introduction or the discussion to convince that replicative fitness evidently contribute to serotype replacements in human (or mosquito) population. If there is no evidence, please at least provide a use case for assaying replicative fitness in vitro that could contribute to the field (e.g. vaccine or drug development).

-Line 52-53: The study of viral replicative fitness has become an expanding field of research [Wargo et al., 2012].

The referenced paper was from 2012 (>10 years ago). Please provided a newer reference that the study of viral replicative fitness is still relevant today, especially in dengue research.

Materials and Methods

-Please add a "statistical analyses" part/sub-section that at least includes:

1) Sample size calculation (How do you justify that the number of samples or repeats are enough to accurately evaluate your assay?)

2) Describe statistical tests used in this study. If possible, explain why the tests were selected. For example, chi-square tests were used for testing the difference in proportions. Why Chi-square tests was preferred to Fisher's test?

3) Specify important statisitcal parameters. For example, the statistical significant level was set at 0.05 (i.e. p < 0.05).

4) Specify statistical software used in this study including version and company.

Results

-Khi² test (Line 260 and 263) and Chi² tests (Line 279, 285 and 288) should be changed to chi-squared tests or chi-square.

-Please report raw p-values in three decimal places (e.g. 0.001 - 0.009). The p-values could be reported as p<0.001 if they are smaller than 0.001.

-Since the authors propose a part this work as a quantification assay, the precision of the quantification must be presented. The coefficient of variation in a percentage form (%CV) is usually used for precision and repeatability evaluation of quantitative PCR assays. Please calculate %CV for all quantitation assays presented in this study.

-Line 245 - 247: This assay allowed to determine the sensitivity of this serotype-specific qRT-PCR, which was higher than 5.08x101, 5.16x101, 7.14x101 and 1.36x101 genome copies/µL for DENV-1, -2, -3 and -4 respectively.

What are these reported numbers? The lower limit of detection (LLOD) or the lower limit of quantification (LLOQ)? Please describe how these numbers were derived in the materials and methods. One way to calculate LLOD is to perform PCR with serially diluted samples with known concentrations in replicates (preferably >= 9 replicates). Then calculate a probability of detection curve by logistic regression. Finally, calculate a concentration (for dilution) that have a probability of detection = 95%. For LLOQ, %CV must be derived first. Then acceptable %CV must be specified (e.g. %CV <= 35%). Finally, the lowest concentration with %CV < acceptable %CV must be calculated for LLOQ. (LLOQ is the lowest concentration that could be repeatedly measured with a acceptable range of variations or errors).

- Comparison of the replicative fitness of two DENV serotypes and Figure 4

While the authors proposed this work for a quantification assay, the main results were reported as proportions (%), not absolute quantification results. The use of these assays might be limited since there already are several serotype-specific or pan-serotype quantification assays that report the quantification results in absolute forms (i.e. copies/ml or log10-copies/ml). In addtion, digital RT-PCR could directly report an absolute count of dengue genomes without standard curves.

The quantification of dengue virus with qRT-PCR could have a variation up to +/- 1 log10-copies so any difference < 1 log10-copies might just be an error of the quantification. It would be interesting to see the variation (or %CV) of the quantification assays proposed here.

Figure 4C is the only panel in Figure 4 that show results as absolute counts. However, the scale of the y-axis is misleading as the viral load has a skewed distribution. The y-axis should be changed to a log10-scale.

Also were experiments for comparing the replicative fitness performed in replicates? If so, please specify the number of replicates in the materials and methods. If there were no replicates, how are the error bars in Figure 4D and 4E derived?

Discussion

- Line 302-306: Of importance, this optimized multiplex serotype-specific qRT-PCR may be used in diagnostic laboratories to identify the infecting DENV serotype and could easily be deployed on the field using field-compatible real-time thermocyclers, albeit possibly requiring fluorochrome adjustments.

1) There is no need for serotyping in clinical settings. The doctors do not need to know serotype. Dengue-infected patients confirmed by PCR will be treated the same regardless of serotype.

2) There is no need for dengue quantification in epidemiological study. Serotyping results might be useful for surveillance as the replacement of the current predominant serotype may indicate the new outbreak cycle.

3) The quantification results are generally and only used in research.

4) The point-of-care PCR that could identify the serotype of dengue virus plus detecting other arboviruses (e.g. Zika virus, Chikungunya virus) is now commercially available for clinical use (https://www.sdbiosensor.com/product/product_view?product_no=23001).

Conclusion

Line 345-347: Such in vitro assessment of viral fitness may allow to evaluate the contribution of DENV replicative fitness to DENV serotype replacements in dengue-endemic or endemo-epidemic settings.

- Again, serotype replacements generally caused by host immune response and herd immunity. Please provide evidence that replicative fitness causes serotype replacements in real-life.

Figures

- There are no descriptions or annotations of all figures.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes:  Jianguo Li

Reviewer #4: No

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Dear Dr. Inizan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’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 by Nov 13 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 plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

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Julian Ruiz-Saenz

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: The revised manuscript by Griffon and colleagues entitled “Development of a competition assay to assess the in vitro fitness of dengue virus serotypes using an optimized serotype-specific qRT-PCR” is much improved compared to the previous version. The authors’ efforts in revising the manuscript are commendable, and their responses to the queries are satisfactory.

Few things to consider…

Critical comments:

Why was the envelope (E) gene chosen for serotype-specific RT-PCR in DENV infection? Please provide a brief explanation in the Introduction section or a detailed explanation in the discussion section.

Specific comments:

Line 109-115: Please include the justification for using HuH7 cell line in this paragraph. This will help in understanding why HuH7 cells were used in the study.

Line 374-376: //… a DENV-1/DENV-2, a DENV-1/DENV-4, a DENV-2/DENV-3, a DENV-2/DENV-4 and a DENV-3/DENV-4//. Please replace ‘a’ with ‘1’.

Line 383-386: //Regarding the co-infections, while the routine technique detected two co-infections (DENV-2/DENV-3 and DENV-2/DENV-4) out the five co-infections historically determined at the time of sample collection, the current optimized qRT-PCR detected three co-infections (DENV-1/DENV-4, DENV-2/DENV-3, DENV-3/DENV-4)//. Since you describe the co-infections as ‘historically determined,’ does this mean they were previously reported by other researchers, or are they based on your current observations? Please clarify and include this information.

Line 394-395: // Only one sample typed as DENV-2 by the routine technique was identified as DENV-3 positive by our technique, yielding one false positive//. Do you think this discrepancy could be due to sequencing of a specific gene rather than the complete genome? Discuss…

Figure 4: Please provide color legend within the figure for D, E, F, & G.

Reviewer #2: Thank you very much for your great improvement of your manuscript and satisfactory revision. No more revision required.

Reviewer #3: I have no more questions, it seems that the authors do a good job and all my concerns have been addressed.

Reviewer #4: For a formatted version please see the attached file.

Overall:

The authors have addressed the technical concerns and revised the manuscript such that the methodological aspects now meet the standards of PLOS ONE. Nevertheless, I remain unconvinced of the broader applicability of the reported assay. Its utility appears to be restricted to laboratory-controlled co-infection experiments in cell culture, which represent highly artificial conditions. As such, the assay is unlikely to have meaningful relevance to serotype replacement or other processes of epidemiological importance in natural settings. To strengthen the manuscript, the authors may consider clarifying the potential practical applications of this assay, while avoiding references to serotype replacement caused by differences in viral replication levels. For example, could it be used to titrate each serotype in a live attenuated dengue vaccine so that the vaccine will induce balanced immune response against all four serotypes? (Again, antigenicity of each serotype is also important for balancing immune response.) The authors may focus more on co-infection study. For example, Quintero-Gil et al. (https://doi.org/10.3855/jidc.3978) evaluated replicative fitness of two DENV serotypes infecting a mosquito cell line and a live mosquito.

There are several studies reporting that each serotype of DENV intrinsically replicates at different rates. Tricou et al. (https://doi.org/10.1371/journal.pntd.0001309), Douyen at al. (https://doi.org/10.1093/infdis/jir014), and Voung et al. (https://doi.org/10.7554/eLife.92606.3) reported that DENV-1 shows higher early/febrile-phase viral loads than DENV-2 and DENV-3 in dengue patients. However, serotype replacement still occurs among all four serotypes. Therefore, the ability of viral replication may have limited relevance, or even be irrelevant, to the serotype replacement observed in epidemiological surveillance.

The authors try to convince that the difference in replicative fitness play a very important role in serotype replacement. However, the citations included actually demonstrated replication fitness changes that might contribute to “genotype”, “lineage” and “clade” replacement, not serotype replacement. Genotype, lineage and clade are more difficult to identify requiring virus genome sequencing or specialized PCR. The assay proposed by the authors cannot identify genotype of DENV or evaluate replicative fitness of genotypes within the same serotype in a co-infection. (The authors mention this in Line 544-547).

REF 6: Ko, H.-Y., et al., Emergence and increased epidemic potential of dengue variants with the NS5V357E mutation after consecutive years of transmission. iScience, 2024. 27(11): p. 110899. � The article reported an investigation of clade/strain replacement of DENV-2, not the serotype replacement.

REF 35: Inizan, C., et al., Viral evolution sustains a dengue outbreak of enhanced severity. Emerg Microbes Infect, 2021. 10(1): p. 536-544. � The article focuses on DENV-1 evolution (i.e., genotype or strain replacement), not serotype replacements.

REF 47: Gomgnimbou, M.K., et al., Utilization of novel molecular multiplex methods for the detection and, epidemiological surveillance of dengue virus serotypes and chikungunya virus in Burkina Faso, West Africa. Molecular Biology Reports, 2024. 51(1): p. 906. � The article does not address replicative fitness or provide any information regarding virus load quantification.

REF 48: O'Connor, O., et al., Potential role of vector-mediated natural selection in dengue virus genotype/lineage replacements in two epidemiologically contrasted settings. Emerg Microbes Infect, 2021. 10(1): p. 1346-1357. � The article investigated genotype/lineage replacements within a serotype, not serotype replacement.

REF 54: Vu, T.T., et al., Emergence of the Asian 1 genotype of dengue virus serotype 2 in Vietnam: in vivo fitness advantage and lineage replacement in South-East Asia. PLoS Negl Trop Dis, 2010. 4(7): p. e757 � The article investigated lineage replacement within DENV-2, not serotype replacement.

Abstract:

Line 27 – 29: The qRT-PCR was specific, and had a limit of detection below 7.52, 1.19, 3.48 and 1.36 genome copies/µL, an efficiency of 1.993, 1.975, 1.902, 1.898 and a linearity (R²) of 0.99975, 0.99975,0.9985, 0.99965 for DENV-1, -2, -3 and -4 respectively.

Please report R² values with the same/consistent decimal places.

Discussion:

Line 451- 453: This serotype-specific qRT-PCR offers rapid, sensitive, and specific detection of DENV, allowing serotype differentiation and viral load quantification, which are crucial for efficient epidemiological surveillance [39-41].

The authors state that both serotype differentiation and viral load quantification are essential for effective epidemiological surveillance. While serotype differentiation is indeed widely recognized and applied in epidemiological analyses, viral load quantification is not typically used for this purpose. Of the three references cited, all specifically demonstrate the application of serotype data in epidemiological studies, but none provide evidence for the use of viral load quantification in this context. Consequently, the statement in lines 451–453 is not entirely accurate. To resolve this, either “viral load quantification” in the sentence should be removed, or an additional citation must be provided that clearly demonstrates its role in epidemiological surveillance.

REF 39: Singh, K., et al., Identification of Dengue virus serotype and genotype: A comprehensive study from AIIMS Patna, Bihar. Indian Journal of Medical Microbiology, 2025. 53: p. 100789. � Serotyping was done, but viral load quantification was not done. This reference does not support that “viral load quantification” is crucial for epidemiological surveillance.

REF 40: Dieng, I., et al., Multifoci and multiserotypes circulation of dengue virus in Senegal between 2017 and 2018. BMC Infectious Diseases, 2021. 21(1): p. 867. � Serotyping was done, but viral load quantification was not done. This reference does not support that “viral load quantification” is crucial for epidemiological surveillance.

REF 41: Dupont-Rouzeyrol, M., et al., Epidemiological and molecular features of dengue virus type-1 in New Caledonia, South Pacific, 2001–2013. Virology Journal, 2014. 11(1): p.61. � Serotyping was done, but viral load quantification was not done. This reference does not support that “viral load quantification” is crucial for epidemiological surveillance.

Line 512 – 514: To improve dengue outbreak prevention and the surveillance of antiviral resistance [46], it is crucial to strengthen genomic surveillance but also to better assess viral replicative fitness [47].

REF 47: Gomgnimbou, M.K., et al., Utilization of novel molecular multiplex methods for the detection and, epidemiological surveillance of dengue virus serotypes and chikungunya virus in Burkina Faso, West Africa. Molecular Biology Reports, 2024. 51(1): p. 906. � The article does not address replicative fitness or provide any information regarding virus load quantification.

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Development of a competition assay to assess the in vitro fitness of dengue virus serotypes using an optimized serotype-specific qRT-PCR

PONE-D-24-55413R2

Dear Dr. Inizan,

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Reviewer #1: The authors have addressed all the queries raised.

Only a few English grammar and syntax errors remain in the manuscript; these should be rectified before publication.

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