The early whole-blood transcriptional signature of dengue virus and features associated with progression to dengue shock syndrome in Vietnamese children and young adults

Abstract

Dengue is a pantropic public health problem. In children, dengue shock syndrome (DSS) is the most common life-threatening complication. The ability to predict which patients may develop DSS may improve triage and treatment. To this end, we conducted a nested case-control comparison of the early host transcriptional features in 24 DSS patients and 56 sex-, age-, and virus serotype-matched uncomplicated (UC) dengue patients. In the first instance, we defined the "early dengue" profile. The transcriptional signature in acute rather than convalescent samples (≤72 h post-illness onset) was defined by an overabundance of interferon-inducible transcripts (31% of the 551 overabundant transcripts) and canonical gene ontology terms that included the following: response to virus, immune response, innate immune response, and inflammatory response. Pathway and network analyses identified STAT1, STAT2, STAT3, IRF7, IRF9, IRF1, CEBPB, and SP1 as key transcriptional factors mediating the early response. Strikingly, the only difference in the transcriptional signatures of early DSS and UC dengue cases was the greater abundance of several neutrophil-associated transcripts in patients who progressed to DSS, a finding supported by higher plasma concentrations of several canonical proteins associated with neutrophil degranulation (bactericidal/permeability-increasing protein [BPI], elastase 2 [ELA2], and defensin 1 alpha [DEF1A]). Elevated levels of neutrophil-associated transcripts were independent of the neutrophil count and also of the genotype of the infecting virus, as genome-length sequences of dengue virus serotype 1 (DENV-1) (n = 15) and DENV-2 (n = 3) sampled from DSS patients were phylogenetically indistinguishable from those sampled from uncomplicated dengue patients (32 DENV-1 and 9 DENV-2 sequences). Collectively, these data suggest a hitherto unrecognized association between neutrophil activation, pathogenesis, and the development of DSS and point to future strategies for guiding prognosis.

FIG. 1.

Phylogenetic tree of DENV-1 consensus genome sequences from patients in this study. The tree (neighbor-joining method) contains consensus genome sequences deduced from plasma samples obtained from 32 DENV-1-infected patients with uncomplicated dengue (gray-highlighted tip labels) and 15 genomes sampled from DENV-1-infected patients with DSS (black-highlighted tip labels). The tree is midpoint rooted and contains sequences from other DENV-1 viruses for reference only (black-highlighted tip labels). Bootstrap values are shown on major branches.

FIG. 2.

Phylogenetic tree of DENV-2 consensus genome sequences from patients in this study. The tree (neighbor-joining method) contains genome sequences deduced from plasma samples obtained from 9 DENV-2-infected patients with uncomplicated dengue (gray-highlighted tip labels) and 3 genomes sampled from DENV-2-infected patients with DSS (black-highlighted tip labels). The tree is midpoint rooted and contains sequences from other DENV-2 viruses for reference only (black-highlighted tip labels). Bootstrap values are shown on major branches.

FIG. 3.

AcuteDengue_DE network. Network of known protein-protein and protein-DNA interactions encoded by genes differentially expressed in 80 acute dengue patients in comparison to 34 convalescent samples. Nodes encoded by upregulated genes are shown in red and by downregulated genes are shown in green. This network was generated using InnateDB (www.innateDB.com) and was visualized using the Cerebral 2.0 plug-in for Cytoscape 2.6.2, which was developed as part of the InnateDB project. This network has 289 nodes and 429 edges. The top 5 hubs (i.e., genes/proteins that are highly connected to other DE genes) in this network were identified as the transcription factors STAT1 and STAT2 (2× upregulated), the tyrosine kinase SRC (2× upregulated), PTPN6 (SHP1) (2.5× downregulated), and C1orf103 (2× upregulated).

FIG. 4.

Ingenuity Pathway Analysis (IPA) of differentially expressed genes between 24 DSS and 56 uncomplicated dengue patients. Twenty-one differentially expressed transcripts were analyzed using IPA. The following two significant networks were identified: cancer, cell cycle, and cell-mediated immune response (network 1, score of 31) (A) and antigen presentation, cell-mediated immune response, and humoral immune response (network 2, score of 21) (B). The lines between genes represent known interactions, with solid lines representing direct interactions and dashed lines representing indirect interactions. Differentially expressed genes are highlighted in red, and genes identified by IPA are not highlighted. The high scores associated with these networks indicate they were highly unlikely to be formed by chance.

FIG. 5.

Concentrations of secreted neutrophil-associated proteins in plasma samples. Concentrations of BPI (A), DEF1A (B), ELA2 (C), MPO (D), and albumin (E) in acute and convalescent dengue cases and, for reference, in patients with other febrile illnesses and in healthy-donor plasma samples. The box-and-whisker plots represent median and interquartile ranges. OFI, other febrile illnesses.