Identification of potential biomarkers in dengue via integrated bioinformatic analysis.

Dengue fever virus (DENV) is a global health threat that is becoming increasingly critical. However, the pathogenesis of dengue has not yet been fully elucidated. In this study, we employed bioinformatics analysis to identify potential biomarkers related to dengue fever and clarify their underlying mechanisms. The results showed that there were 668, 1901, and 8283 differentially expressed genes between the dengue-infected samples and normal samples in the GSE28405, GSE38246, and GSE51808 datasets, respectively. Through overlapping, a total of 69 differentially expressed genes (DEGs) were identified, of which 51 were upregulated and 18 were downregulated. We identified twelve hub genes, including MX1, IFI44L, IFI44, IFI27, ISG15, STAT1, IFI35, OAS3, OAS2, OAS1, IFI6, and USP18. Except for IFI44 and STAT1, the others were statistically significant after validation. We predicted the related microRNAs (miRNAs) of these 12 target genes through the database miRTarBase, and finally obtained one important miRNA: has-mir-146a-5p. In addition, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were carried out, and a protein-protein interaction (PPI) network was constructed to gain insight into the actions of DEGs. In conclusion, our study displayed the effectiveness of bioinformatics analysis methods in screening potential pathogenic genes in dengue fever and their underlying mechanisms. Further, we successfully predicted IFI44L and IFI6, as potential biomarkers with DENV infection, providing promising targets for the treatment of dengue fever to a certain extent.

Introduction

In the tropical and subtropical parts of the world, dengue fever virus (DENV) infection has become an increasingly common health concern. Due to the large geographic extent, increase in the number of cases, and severity of the disease, the DENV infection has evolved from a sporadic disease to a major public health problem with significant social and economic impacts[1-4]. Dengue is a mosquitoes-transmitted viral disease caused by a single-stranded RNA virus, which has four serotypes (DENV 1–4)[5]. DENV infection can cause various illnesses, such as breakbone fever, haemorrhagic fever, and shock syndrome[6]. Dengue divides into three phases: the febrile phase with acute onset of fever, the critical phase with metabolic acidosis and severe haemorrhage, and the recovery phase with resolved symptoms[3]. At present, some clinical trials have been conducted to reduce the effects and symptoms of dengue[7-9]. There are a few dengue vaccines but no specific antiviral treatment[3,5]. A DENV vaccine cannot elicit protection in naive individuals but only those with prior exposure, in addition, that is not equally protective against all four serotypes[10].

As one of the most viral diseases transmitted by arthropods that causes human morbidity and mortality, numerous studies have been performed to explore the pathogenesis of disease; The mainstream view is that immunity leads to cytokine storm, which leads to vascular leak and thus contributes to severe dengue disease in secondary infections[11]. However, many patients with DENV infection do not develop plasma leakage[12]. Plasma leak typically occurs in the critical phase[13], which is at the end of the acute phase[14]. Therefore, the febrile phase with or without the critical phase is the acute phase, which may lead to severe dengue[14]. A hypothesis based on molecular mimicry posits that some DENV-induced antibodies can cross-react with host proteins. A study verifies that the level of pre-existing anti-DENV antibodies is directly associated with the severity of secondary dengue disease in humans[11]. In sum, it still remains unclear, more research is needed to understand the potential pathogenesis in dengue.

In view of heterogeneity, biomarkers for reliably predict the development of severe dengue among symptomatic individuals are desperately needed in current research. The currently utilized warning signs to predict severe dengue are based on clinical parameters that appear late in the disease course and are neither sensitive nor specific. It promotes not only continued morbidity and mortality, but also ineffective patient triage and resource allocation[15]. Provided that we have had highly discriminating biomarkers, then developed a single, robust clinical algorithm, it will be broadly applicable across all age groups and in different locations[16], which is meaningful to predict severe dengue and differentiate dengue-infected diseases with similar clinical phenotypes.

Microarray data analysis can identify DEGs in dengue fever patients with differing disease severity[2]. In addition, an increasing amount of evidence indicates the potential role of microRNAs (miRNAs) in regulating DENV[17,18]. MiRNAs are small non-coding RNA molecules that can regulate gene expression by inhibiting messenger RNA (mRNA) translation or inducing mRNA degradation[17]. Recently, Pong et al. reported that, with a DENV-1 infection, 23 highly differentially expressed miRNAs jointly modulate the adaptive immune response involving TGF-β, MAPK, PI3K-Akt, Rap1, Wnt, and Ras signalling pathways[19].

In this study, we performed a biological information analysis using microarray data and identified the DEGs for the infected and normal samples. Subsequently, the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein–protein interaction (PPI) network, and miRNA-target gene interaction network were analysed to understand the molecular mechanisms underlying dengue fever. In conclusion, our study aimed to explore the molecular biomarkers of dengue based on bioinformatic analysis and provide candidate biomarkers for early diagnosis and therapeutic targets.

Materials and methods

Microarray data

The Gene Expression Omnibus is a public and functional genomics database that contains high throughput gene expression data, chips, and microarrays. In this study, the GSE28405[20], GSE38246[21], and GSE51808[22] microarray data were downloaded for analysis. Considering that the transcriptional profiles between the fever phase and convalescence phase in dengue patients are are quite different, we only included the data of samples collected in fever patients and the control group from these three data sets. Additionally, GSE28405, GSE38246, and GSE51808 are consisting of 26, 8, and 9 control samples and 31,105 and 28 infected samples in fever, respectively.

Data processing

We used the R 4.0.1 statistical software (https://www.r-project.org/) and a Bioconductor (http://bioconductor.org/biocLite.R) to process raw data and screen differentially expressed genes. The data of GSE28405 and GSE38246 were batch calibrated and standardized by using the limma package. Limma package contains particularly powerful tools for reading, standardizing and exploring such data, and its core component is to fit gene linear model to gene expression data to evaluate the ability of differential expression[23]. The data of GSE51808 was batch calibrated and standardized by using the affy package. The differentially expressed genes were then filtered using a limma package. The screening threshold was p-value < 0.05 and fold-change ≥ 1.5. The ggplot2 package was used to visualise the DEGs into a volcano map, while the pheatmap package was used to cluster the significant DEGs.

Function and pathway enrichment analysis of DEGs

The Gene Ontology (GO, http://www.geneontology.org) is a community-based bioinformatics resource. It provides information about genes and gene product functions and uses ontology to enhance biological knowledge[24]. The KEGG (https://www.kegg.jp/) is a database for the qualitative interpretation of genomic sequences and other biological data, including systematic, genomic, and chemical information as well as an additional human-specific category of health information[25]. The related biological functions and signal pathways were analysed using GO/KEGG enrichment and analysed again with the cluster Profiler software package, with p < 0.05 considered to be statistically significant.

PPI network construction and identification and validation of hub genes

Protein–protein interaction (PPI) network analysis plays a major role in predicting the function of interacting proteins. It is a feasible tool that can be used to understand cell function and disease mechanism[26,27]. The STRING database (http://string-db.org) focuses on providing a key assessment of protein–protein interactions by integrating a large number of known and predicted protein–protein association data[28,29]. A PPI network visualised by the Cytoscape software was constructed by using the STRING database. Furthermore, the cytohubba plug-in of Cytoscape software was used to analyze the interaction of proteins and screen out hub genes with a higher score in this analysis, which means that they have higher connectivity in PPI networks. The statistical significance of these genes was verified by GSE84331[30] microarray data analysed using GEO2R. GEO2R is an interactive network tool that allows users to compare two or more sets of samples in a GEO sequence to identify differentially expressed genes[31]. P < 0.05 was considered statistically significant.

MiRNA-target gene network

MicroRNA (miRNA) is a type of small endogenous non-coding RNA with 18–25 nucleotides. It is the main central regulatory factor at the post-transcriptional levels. It is involved in many biological processes such as cell cycle, cell differentiation, and apoptosis, among others[32,33]. The miRTarBase database contains manually managed and experimentally validated miRNA–gene interactions as well as detailed metadata, experimental methods, and conditions[32]. Accordingly, we constructed the miRNA–gene targeting relationship for overlapping differential genes and hub genes based on the miRTarBase database.

Results

Identification of DEGs

The datasets included are shown in Table 1. After analysing the GSE28405 dataset, we screened 668 DEGs, including 364 upregulated genes and 304 downregulated genes (Fig 1A); GSE38246 and GSE51808 were used to screen 1901 DEGs (924 upregulated and 977 downregulated) and 8283 DEGs (4165 upregulated and 4118 downregulated) respectively (Fig 1C and 1E). After screening the differential genes based on the volcano map, cluster analysis was carried out, as shown in Fig 1B, 1D and 1F. Finally, through a Venn analysis, 69 common DEGs were identified from three datasets, including 51 upregulated genes and 18 downregulated genes, which were subsequently used for further study (Fig 2 and Table 2).

Table 1

Details of the data sources from Gene Expression Omnibus(GEO) for this study.

Reference	GEO Series (GSE)	Sample	Sample size	Normal vs Infection	GEO Platform (GPL)
Tolfvenstam et al(2011)	GSE28405	Whole blood	57	26 vs 31	GPL2700 Sentrix HumanRef-8 Expression BeadChip
Popper et al(2012)	GSE38246	Peripheral blood mononuclear cell (PBMC)	113	8 vs 105	GPL15615 SMD Print_1430 hr1
Kwissa et al(2014)	GSE51808	Whole blood	37	9 vs 28	GPL13158 [HT_HG-U133_Plus_PM] Affymetrix HT HG-U133+ PM Array Plate
Chandele et al(2016)	GSE84331	Peripheral blood mononuclear cell (PBMC)	12	5 vs 7	GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array

Fig 1

Volcano map and heat map of differentially expressed genes (DEGs) in GSE28405(AB), GSE38246(CD), and GSE51808(EF).

(ACE) Red dots indicated up-regulated genes and blue dots indicated down-regulated genes. Black dots indicated the rest of the genes with no significant expression change. The threshold was set as followed: P<0.05 and |log2FC|≥2. FC: fold change. (BDF) Gene expression data is converted into a data matrix. Each column represents the genetic data of a sample, and each row represents a gene. The color of each cell represents the expression level, and there are references to expression levels in different colors in the upper right corner of the figure.

Fig 2

The intersection results of GSE28405, GSE38246, and GSE51808.

Table 2

Up-regulated genes and down-regulated genes of overlapping DEGs.

Overlapping DEGs		Gene terms
All	69	GOSR2, STAT1, MRPL17, THAP8, NR1H3, CBR1, MRPS18C, TOR3A, NAPA, BAK1, HIST1H4H, SIL1, BST2, TRIP6, C1QC, HIST1H2BD, DNASE2, C2, MAGED2, ISG20, SIGLEC1, IFI27L1, IFI35, TNNT1, SCO2, EPHB2, ATF5, CFB, OAS1, MT1F, OAS2, CTSD, IFI44, IFI6, HESX1, CD38, FDXR, MX1, KCTD14, C1QB, OAS3, LAG3, IFI44L, LGALS3BP, ISG15, CXCL10, LY6E, SPATS2L, TCN2, USP18, IFI27, CAMK1D, CMTM2, VENTX, FRY, ZFP36L2, SORL1, THBD, KLRB1, ITPKB, CIITA, CD22, MS4A1, LYST, CXCR5, PTGS2, STMN3, IVNS1ABP, TMEM71
Up-regulated	51	GOSR2, STAT1, MRPL17, THAP8, NR1H3, CBR1, MRPS18C, TOR3A, NAPA, BAK1, HIST1H4H, SIL1, BST2, TRIP6, C1QC, HIST1H2BD, DNASE2, C2, MAGED2, ISG20, SIGLEC1, IFI27L1, IFI35, TNNT1, SCO2, EPHB2, ATF5, CFB, OAS1, MT1F, OAS2, CTSD, IFI44, IFI6, HESX1, CD38, FDXR, MX1, KCTD14, C1QB, OAS3, LAG3, IFI44L, LGALS3BP, ISG15, CXCL10, LY6E, SPATS2L, TCN2, USP18, IFI27
Down-regulated	18	CAMK1D, CMTM2, VENTX, FRY, ZFP36L2, SORL1, THBD, KLRB1, ITPKB, CIITA, CD22, MS4A1, LYST, CXCR5, PTGS2, STMN3, IVNS1ABP, TMEM71

Abbreviation: DEGs, differentially expressed genes.

GO and KEGG pathway analysis

The detailed results of the GO enrichment analysis and KEGG pathway analysis of GSE28405, GSE38246, and GSE51808 are shown in Figs 3, 4 and 5. The type I interferon signaling pathway, DNA replication, and chromosome segregation were relatively enriched from these data sets in biological processes. In terms of cell components, the results showed that cytosolic ribosome, organellar large ribosomal subunit, mitochondrial ribosome, and mitochondrial large ribosomal subunit were significantly enriched. Concerning molecular function, ATPase activity, DNA-dependent ATPase activity, single-stranded DNA helicase activity, and catalytic activity, acting on DNA play an important role in the enrichment results relatively. For KEGG pathway enrichment analysis, the relatively enriched pathways were the coronavirus disease, cell cycle, DNA replication, and protein processing.

Fig 3

The GO enrichment analysis and KEGG pathways analysis of GSE28405.

Abbreviation: GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes.

Fig 4

The GO enrichment analysis and KEGG pathways analysis of GSE38246.

Abbreviation: GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes.

Fig 5

The GO enrichment analysis and KEGG pathways analysis of GSE51808.

Abbreviation: GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes.

Protein–protein interaction work of overlapped DEGs and identification and validation of key genes

To identify the potential interactions between overlapping DEGs, a PPI network was constructed on the STRING, consisting of 69 nodes (genes) and 174 edges (Fig 6A). The foremost module in the PPI network was identified by MCODE, and 12 genes were identified as hub genes (Fig 6B). After verifying with GSE84331, 10 genes (MX1, IFI44L, IFI44, IFI27, ISG15, STAT1, IFI35, OAS3, OAS2, OAS1, IFI6, USP18) were statistically significant (Fig 7). Among them, IFN inducible protein 44-like (IFI44L), and IFNα inducible protein 6 (IFI6) were found to have a higher score in the PPI network and a lower p-value in the analysis.

Fig 6

The PPI network of overlapping DEGs (A) and the the important module of PPI network (B).

Abbreviation: PPI, protein–protein interaction; DEGs, differentially expressed genes.

Fig 7

Verification of hub genes.

P-value < 0.01 is considered to be statistically significant. (***P<0.001).

The networks of miRNA-gene targeting relationship of 69 overlapping DEGs and 12 hub genes based on miRTarBase database are respectively presented in Fig 8A and 8B. According to the miRNA interactions and number and importance of target genes, has-mir-146a-5p was attained.

Fig 8

The miRNA-target gene network of overlapping DEGs (A) and hub genes (B) based on miRTarBase v8.0 database.

Abbreviation: miRNA, microRNA, DEGs, differentially expressed genes.

Discussion

DENV infection can further lead to recessive infection, dengue fever, and severe dengue fever[34,35]. It is mainly transmitted to humans through female Aedes mosquitoes. Aedes mosquitoes are widespread in the tropical and subtropical regions of the world, putting nearly two-thirds of the world’s population at risk[36,37]. Therefore, screening the potential biomarkers or exploring the related mechanisms through bioinformatics may contribute to the efficient diagnosis and treatment of dengue fever.

GO analysis can annotate genes and gene products involving cellular components, biological processes, and molecular functions[38]. In biological processes, DEGs are most enriched in type I interferon signaling pathway, DNA replication, and chromosome segregation. Dengue virus infection activates the innate immune system of the body to increase the secretion of interferon. Type I interferon signal transduction can fight against a variety of viruses that invade the human body. DENV can antagonize its signal transduction and promote its genome replication in host cells[39]. The imbalance of the cell cycle and mitotic cycle after DENV infection will affect DNA replication and cell proliferation. In terms of cell components, DEGs are significantly enriched in the cytosolic ribosome, organellar large ribosomal subunit, mitochondrial ribosome, and mitochondrial large ribosomal subunit, which is related to the enrichment of DNA replication and chromosome segregation in biological processes. Dengue virus genome replication in the cytoplasm of the host cell and take advantage of the host cell organelles to protein synthesis and assembly[40]. The protein is synthesized in the cytosolic ribosome, binding to a receptor protein such as the TOM complex on the outer membrane of the mitochondria, and is introduced into the mitochondria in an unfolded conformation, where it is eventually folded and assembled into the intrinsic structure[41]. In terms of molecular function, ATPase activity, DNA-dependent ATPase activity, single-stranded DNA helicase activity, and catalytic activity, acting on DNA play an important role in the enrichment results. ATP activation increases, generating cyclic adenosine phosphate (cAMP) under the action of adenylate cyclase. cAMP may trigger the fusion of secretory vesicles, again may by increasing vesicles and plasma membrane fusion between the diameter of the hole and opening time to adjust have fusion of secretory vesicles[42]. In this case, the diameter of the fusion hole and open time increasing, may increasing the dengue virus E protein involved in virus and nuclear fusion peptide in the somatic cell membrane fusion process, and promoting DENV through holes to promote infection in genetic material into cells.

Coronavirus disease 2019 (COVID-19) was found to be associated with dengue fever. It is hypothesized that dengue fever and COVID-19 share the same pathophysiological pathway, resulting in plasma leakage, thrombocytopenia, and coagulopathy are the hallmarks they both have[43]. Failing to diagnose dengue fever because of a false-positive test result for confirmed COVID-19, so we speculate that antibody cross-reactivity may exist in serology tests[44,45]. Cell cycle, DNA replication, and protein processing were significantly enriched in KEGG pathway enrichment analysis, all of which are closely related to cell division. Knockdown of cyclin-dependent kinase 8/19-cyclinC (Cdk8/19-cyclin C) reduced genome replication of the dengue virus and mitochondrial function in infected and uninfected cells as well as weakened glucose metabolism and autophagy to inhibit viral replication and metabolism[46]. Cyclin G-associated kinases (GAK) phosphorylated adaptor protein complexes (APs), thereby regulating membrane transport and promoting the dengue virus infection[47]. GAK inhibitors and their derivatives showed antiviral activity against the dengue virus[48]. XuM et al. found that the infection of Zika virus (ZIKV), a Flaviviridae similar to the dengue virus, was closely related to cell cycle regulation; Hammack et al. found that ZIKV suspends host DNA replication during the S phase and induces DNA damage response and enhanced virus replication, which may occur with the dengue virus as well[49]. Therefore, cell division is a significant pathway for virus replication and infection, including cell cycle, DNA replication, and protein processing.

The PPI could help us understand protein–protein interactions; the rich interaction in gene expression of the dengue virus underscores the potential role of regulating host gene expression during infection[50]. Screening the most important module and its verification showed higher degrees of IFI44L and IFI6 in the PPI network and lower p-values in the analysis, which potentially indicates their significant association with dengue fever. Remarkably, DENV-infected germ cells upregulated IFI44L by 130-fold confirmed in qRT-PCR, but not in ZIKV-infected germ cells[51]. It is uncovered that IFI44L supports different viruses replication, and negatively regulating type I IFN response induced[52]. IFI44L contributes to DENV infection due to low levels of type I IFN response showed in patients with severe dengue disease[53,54]. The canonical pathway of Type I IFN is activated by the IFN-stimulated gene like IFI6, which is up-regulated DENV infection[55]. Overall, IFI6 was demonstrated a high level of protection against DENV infection, by inhibited DENV2-induced autophagy and apoptosis[55-57]. They influence the occurrence and development of dengue virus infection through a complex and undefined network of interactions. MiRNA is a small non-coding RNA molecule, and there is an increasing evidence that the imbalance of miRNA results in many diseases. The MiRTarBase v8.0 database was used to predict miRNAs based on top the 12 hub genes, and miR-146a-5p was the core miRNA. miR-146a-5p, a type I IFN-mediated regulator targeting NF-kb, had a high correlation with the platelets and white blood cells count, especially in neutrophils and lymphocytes in initially diagnosed dengue fever[58,59]. Furthermore, activitied serum aspartate transaminase (AST) and alanine aminotransferase (ALT) also indicate miR-146a-5p affect liver complication in infected dengue[58]. Exosome miR-146a can act on different immune cells, making recipient cells more susceptible to many viral infections[60]. Surprisingly, some studies assessed that miR-146a-5p is associated with induced autophagy, which is a process in cell degradation and recycling for DENV replication[61-63]. Thus, miR-146a-5p has the potential to serve as a circulating biomarker for dengue pathogenesis.

This study has some limitations. Firstly, a small sample size would increase the error of research results to a certain extent. If the analysis can be based on large sample size, it is possible to more fully study the relationship between each central gene and pathway to improve the accuracy. Meanwhile, there are different samples in different data sets we included, such as whole blood and peripheral blood, which would cause some errors in our results. Finally, due to ethical issues and lack of funding, we did not conduct in vitro experiments to further verify our results but chose to use the results of another data set for validation analysis. This will also affect our conclusion to a certain extent.

Dengue is now the most important mosquito-borne disease after malaria and can cause serious complications such as bleeding or severe shock syndrome[64]. Therefore, it is urgent to study the pathogenesis of dengue fever. In conclusion, we have studied the microarray data of normal samples and DENV-infected samples through bioinformatics analysis to identify the differentially expressed genes after dengue infection. Another microarray data set was then used to verify genes of important modules, which resulted in 12 statistically significant hub genes. Their associated miRNAs were then predicted based on the miRTarBase database. Finally, we predicted that IFI44L, IFI6, and mir-146a-5p can be used as potential biomarkers of dengue infection, Our study may have potential implications for future prediction of disease progression in symptomatic dengue patients, and has important significance for the pathogenesis and targeted therapy of dengue.

9 Mar 2021

Dear Mr. Guo,

Thank you very much for submitting your manuscript "Identification of potential biomarkers in dengue via integrated bioinformatic analysis" 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. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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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: ALL CRITERIA ARE MET FOR METHODS

Reviewer #2: Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

- The objective of the study is to identify dengue infection biomarkers. In order to do so the authors analyze public data with a standard methodology.

Is the study design appropriate to address the stated objectives?

- Yes, it is. By analyzing available transcriptomes, filtering DEGs, proceeding with pathway enrichment and then looking for miRNAs, the authors are able to find potential biomarkers for dengue infection. However, without at least an in vitro experiment in KO or of some sort targeted pathways in cell line DENV-infected x control in order to validate what was found, the findings in this publication are susceptible to criticism.

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

- In fact, no. All three of the datasets chosen for this study have data from different timepoints of infection (post symptoms onset). During infections and diseases progression, patients’ transcriptional profile is in constant change and dengue infection is no exception. In fact, dengue infection is known to exhaust T and B lymphocytes (present in PBMCs, the samples from these studies) during initial stages of the infection but this changes in the convalescent stage. Since the transcriptional profile of each stage is so different, which of the samples were used and compared? There are no mentions to this point in the manuscript.

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

- Yes, the datasets combined have enough samples to ensure adequate power if all the samples are used. However, I do not believe a transcriptional profile from a convalescent patient would be equal from a patient in the initial stages of the infection. Since the study is trying to find biomarkers, by analyzing all of this data together the statistical tests should be removing potential DEGs from the initial stages just because they are not differentially expressed two weeks later. That being the case, should the authors take this into consideration, I cannot guarantee there are enough samples in these three datasets.

Were correct statistical analysis used to support conclusions?

- Yes, both limma and GEO2R are bioinformatics’ tools that are already published and are highly accepted by scientists.

Are there concerns about ethical or regulatory requirements being met?

- No, there are not.

Reviewer #3: Yes the objectives are clearly stated, still few things are lacking in the manuscript.

Yes the sample size is good enough to address the hypothesis.

1) As all the three gene expression data come from same disease condition but other factors are highly varying. The authors should explain in detail the significance and preselection condition for these datasets to consider in the study.

2) The author should state which method were used for data normalization.

3) What is the rational for selection of fold change value?

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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: all criteria are met

Reviewer #2: Does the analysis presented match the analysis plan?

- Yes, it does.

Are the results clearly and completely presented?

- Yes, they are.

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

- Yes, they are.

Reviewer #3: 1) Yes, The analysis is presented matches the analysis plan

2) In results section

Figure 5 is missing

Figure 5c is missing

Figure 6 an 7 description is missing in the text.

3) In discussion part references are missing.

The author either missed or overlooked some parts in the results and discussion, it should be carefully checked and edited.

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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: The conclusions are ok except for the microRNA part , which is not done correctly to predict microRNAs as biomarkers for deng.

Reviewer #2: Are the conclusions supported by the data presented?

- Yes

Are the limitations of analysis clearly described?

- No

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

- Yes, both in the introduction and in the conclusion

Is public health relevance addressed?

- Yes, identifying potential biomarkers for disease treatment (and in this case, infection control) are of extreme importance. Even more when talking about dengue, its relevance goes without saying given the number of infected people yearly.

Reviewer #3: 1) Yes the conclusion is well supported by the data presented, limitation of the study is clearly stated.

2) The author should add few points relating to future use of outcome in the conclusion section.

3) The author should corelate the study with public health importance in the conclusion section

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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: it is ok

Reviewer #2: I strongly suggest an English revisor in case the paper is accepted due to some English mistakes. Text without numerated lines so it is hard to reference, but some examples in the introduction are:

"There is a few dengue vaccine but no specific antiviral treatment" -> are … vaccines

"A DENV vaccine can not elicits protection" -> cannot elicit

"mainstream view is that immunity leads to cytokine storm cytokine storm" -> repetition

"many patients with DENV infection without developing plasma leakage" -> do not develop

"which is meaningful to predict sever dengue" -> severe

Also, there is an inconsistency (probably a typo in the abstract?):

"Through overlapping, a total of 66 differentially expressed genes (DEGs) were identified, of which 53 were upregulated and 24 were downregulated." (results 3.1 show 13 downregulated, 53+24 do not add up to 66)

Reviewer #3: Major revision is needed.

(As above stated points are significant to the study design)

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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: The study is ok; but with no new findings for pathogenesis of deng discovered except cell cycle which is already known, and the microRNA sectis not done rightlyion

Reviewer #2: As stated before, a differentiation in samples from different time (days) since symptoms onset is needed. Since transcriptional profiles are distinct, in order to find better biomarkers it would be required to at least not group convalescent blood samples with initial infection samples. Also, an in vitro experiment in cell lineage (jukarT, B-LCL, ...) in which the identified miRNA were targeted (either via KO by CRISPR/Cas) or maybe the mRNAs were silenced by siRNA would also be interesting. But i also understand that since no funding is linked to this research, the in vitro experiment may be out of question.

Reviewer #3: The study is a good and scientifically sound. Biomarkers are an important biological components in diagnosis and identification of disease and its severity. The DENV infection in tropical and sub-tropical region are big challenges to the health department. Identification of potential biomarkers will help us in diagnosis and identification of drug targets.

The current approach is insilico based analysis hence a clinical validation is needed.

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Reviewer #1: Yes: Mahmoud ElHefnawi

Reviewer #2: No

Reviewer #3: No

Figure Files:

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25 Apr 2021

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

8 Jun 2021

Dear Mr. Guo,

Thank you very much for submitting your manuscript "Identification of potential biomarkers in dengue via integrated bioinformatic analysis" 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.

When you are ready to resubmit, please upload the following:

[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.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[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,

Ahmed Mostafa

Associate Editor

PLOS Neglected Tropical Diseases

A. Desiree LaBeaud

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

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

Yes. The objective of the study is to identify dengue infection biomarkers. In order to do so the authors analyze public data with a standard methodology.

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

Yes, it is. By analyzing available transcriptomes, filtering DEGs, proceeding with pathway enrichment, protein-protein interaction and then looking for miRNAs, the authors are able to find potential biomarkers for dengue infection.

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

Yes, the authors specify all of it in the manuscript.

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

Yes, there is enough data from these datasets to account for any statistical analysis.

-Were correct statistical analysis used to support conclusions?

Yes, the packages and tools the group used are standard and widely accepted in the bioinformatics' field.

-Are there concerns about ethical or regulatory requirements being met?

No, as they are using public data there is nothing to worry about.

Reviewer #3: As per the authors reply over the reviewer comments, they are acceptable

--------------------

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 #2: -Does the analysis presented match the analysis plan?

Yes, it does.

-Are the results clearly and completely presented?

Yes, they are.

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

Yes, yes. The .tiff quality is excellent and the figures are disposed in an organized and clear way.

Reviewer #3: Most the author reply is acceptable, except figure 1 explanation

The threshold was set as followed: P<0.05 and |log2FC|≥2. FC: fold change

Review comment by author:

However, considering that it can ensure significant

differences and screen out more differential genes for further analysis and comparability

before data sets, this study took p < 0.05 and FC ≥ 1.5 when analyzing each dataset.

Because when fold change took 2, the number of DEGs obtained from individual data

sets are relatively small.

This both statements contradicts please clarify

Figure 4 and 5 quality is poor

--------------------

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 #2: -Are the conclusions supported by the data presented?

Yes, they proposed to find biomarkers for dengue infection and so they did.

-Are the limitations of analysis clearly described?

Yes, the authors perfectly describe it in the manuscript. Even though there are no in vitro nor in vivo experiments, and this may limit their findings, this is discussed and accounted for. Nonetheless, these findings are a great contribution for future studies and may be helpful for researches in dengue therapy.

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

Yes, they state it clearly to the readers.

-Is public health relevance addressed?

- Yes, identifying potential biomarkers for disease treatment (and in this case, infection control) are of extreme importance. Even more when talking about dengue, its relevance goes without saying given the number of infected people yearly.

Reviewer #3: The authors reply is acceptable

--------------------

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 #2: (No Response)

Reviewer #3: Minor revision

--------------------

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 #2: The authors did a great work with the revision. This sort of bioinformatics analysis is of utmost importance for identifying therapeutic targets and controlling diseases. There is one point I would like to address, though. In your first submission we discussed about different infection's stages and how it may affect the patients' transcriptional profile. I don't know about limma, but when analyzing RNA-seq with DESeq2 there is a test called Likelihood Ratio Test (LRT) that may be used (instead of the default wald test) to identify genes whose expression change overtime. There may be a similar or corresponding test that can be used on limma and may prove to be useful for your future analysis.

Reviewer #3: The authors reply is acceptable

--------------------

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Reviewer #2: Yes: Igor Salerno Filgueiras

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:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

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.

18 Jun 2021

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

7 Jul 2021

Dear Mr. Guo,

We are pleased to inform you that your manuscript 'Identification of potential biomarkers in dengue via integrated bioinformatic analysis' 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,

Ahmed Mostafa

Associate Editor

PLOS Neglected Tropical Diseases

A. Desiree LaBeaud

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

The quality of the figures must be improved "higher resolutions" in the published version of the manuscript

20 Jul 2021

Dear Mr. Guo,

We are delighted to inform you that your manuscript, "Identification of potential biomarkers in dengue via integrated bioinformatic analysis," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

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

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases