Project description:Background: Dengue virus (DENV) infection often leads to acute illness lasting 2-7 days with severity ranging from dengue fever (DF) to hemorrhagic fever (DHF) and fatal dengue shock syndrome (DSS). The dynamic changes of host responses on the gene transcription level that accompany DENV infection and differences between DF and DHF cases have been poorly understood, particularly for South American population. Methodology/Principal Findings: Supported by a longitudinal active surveillance program for dengue transmission in Maracay, Venezuela, we conducted a prospective study to investigate host responses in dengue patients. Blood specimens and clinical information were collected on a daily basis from febrile cases confirmed with DENV infection from their first day of enrollment to early defervescence together with one convalescent sample. A total of 49 and 13 study participants were defined as DF and DHF cases respectively based on their clinical and hematological information. Using convalescent specimens as baseline, day-to-day gene expression was evaluated ex vivo in peripheral blood mononuclear cells of the study participants. Two waves of gene expression were detected: the first wave peaked at day 1 from the onset of fever (day 0) then declined at days 3-4; the second wave emerged from day 4 and peaked around day 5-6. Genes associated with innate immune process, including type I interferon signaling, cytokine-mediated signaling, chemotaxis, and antiviral responses, dominated the first wave; whereas genes involved in cell cycle processes, including cell division, mitosis, DNA replication, chromosome, and spindle organization dominated the second wave. Measureable genomic markers predicted early acute, late acute and convalescent phases with 91% accuracy. Gene signatures expressed in early acute phase predicted disease severity (DF vs DHF) with 96% accuracy. Conclusions/Significance: Our study established a dynamic pattern of detailed host immune responses to DENV infection and revealed genomic signatures valuable for diagnostics purposes.

Project description:Background: We report the detailed development of biomarkers to predict the clinical outcome under dengue infection. Transcriptional signatures from purified peripheral blood mononuclear cells were derived from whole-genome gene-expression microarray data and validated by quantitative PCR and tested in independent samples. Methodology/Principal Findings: The study was performed on patients of a well-characterized dengue cohort from Recife, Brazil. The samples analyzed were collected prospectively from acute febrile dengue patients who evolved with different degrees of disease severity, classic dengue fever or dengue hemorrhagic fever (DHF) and compared with similar samples from other non-dengue febrile illnesses. The DHF samples were collected 2-3 days before the presentation of the plasma leakage symptoms. Differentially-expressed genes were selected by univariate statistical tests as well as multivariate classification techniques. The results showed that at early stages of dengue infection, the genes involved in effector mechanisms of innate immune response presented a weaker activation on patients who later developed hemorrhagic fever, whereas the genes involved in apoptosis were expressed in higher levels. Conclusions/Significance: Some of the gene expression signatures displayed estimated accuracy rates of more than 95%, indicating that expression profiling with these signatures may provide a useful means of DHF prognosis at early stages of infection The samples correspond to blood samples from 26 patients from a cohort in Brazil, divided into three classes, DHF, DF, ND, as follows: 18 were confirmed dengue 3, genotype III cases, among which 10 were diagnosed as dengue hemorrhagic fever (DHF) and 8 as classical dengue fever (DF), and 8 control samples (ND) from febrile patients confirmed to be not infected with dengue. None of the DHF patients presented vasculopathy signs and symptoms at the time the samples used in the functional genomic characterization were collected. At the time of collection the patients referred approximately 5 days of disease and the absence of fever was reported two to three days after enrollment. The samples from the DF, DHF and ND patients were matched to avoid significant differences regarding patient age, gender, dengue infection history and days of symptoms among the groups.

Project description:Background: Dengue virus (DENV) infection often leads to acute illness lasting 2-7 days with severity ranging from dengue fever (DF) to hemorrhagic fever (DHF) and fatal dengue shock syndrome (DSS). The dynamic changes of host responses on the gene transcription level that accompany DENV infection and differences between DF and DHF cases have been poorly understood, particularly for South American population. Methodology/Principal Findings: Supported by a longitudinal active surveillance program for dengue transmission in Maracay, Venezuela, we conducted a prospective study to investigate host responses in dengue patients. Blood specimens and clinical information were collected on a daily basis from febrile cases confirmed with DENV infection from their first day of enrollment to early defervescence together with one convalescent sample. A total of 49 and 13 study participants were defined as DF and DHF cases respectively based on their clinical and hematological information. Using convalescent specimens as baseline, day-to-day gene expression was evaluated ex vivo in peripheral blood mononuclear cells of the study participants. Two waves of gene expression were detected: the first wave peaked at day 1 from the onset of fever (day 0) then declined at days 3-4; the second wave emerged from day 4 and peaked around day 5-6. Genes associated with innate immune process, including type I interferon signaling, cytokine-mediated signaling, chemotaxis, and antiviral responses, dominated the first wave; whereas genes involved in cell cycle processes, including cell division, mitosis, DNA replication, chromosome, and spindle organization dominated the second wave. Measureable genomic markers predicted early acute, late acute and convalescent phases with 91% accuracy. Gene signatures expressed in early acute phase predicted disease severity (DF vs DHF) with 96% accuracy. Conclusions/Significance: Our study established a dynamic pattern of detailed host immune responses to DENV infection and revealed genomic signatures valuable for diagnostics purposes. Total 64 subjects representing 51 DF (dengue fever) and 13 DHF (dengue hemorrhagic fever) cases were used to study gene expression during the course of dengue acute illness. Sample information regarding Patient No., Infecting serotype, fever status (fever days or defervescent (df) days), and disease severity (DF vs DHF) were provided. The date for a sample for gene chip hybridization and image scanning was marked as Scan date. Samples from study subjects were collected once a patient was enrolled. Over 200 samples were collected. Two genechip platforms were used, the HG-focus and HG-U133plus2. A total of 168 samples were assayed on the HG-focus platform, 2 were excluded for further analysis as they failed on quality control (i.e. VFP-0003_G7_DF, VFP-0029_G1_DF samples). A total of 101 samples were assayed on the HG-U133plus2 platform, 4 were excluded for further analysis as they failed on quality control (i.e. VFP_0186_G4_DF, VFP-0213_G3_DF, VFP_0213_G5_DF, VFP_0220_G3_DHF). Data generated by HG-focus platform were used to explore significantly expressed genes in individual Gs, phases, and for prediction analysis for disease phases. Whereas data generated by HG-U133plus2 were first used to assess the reproducibility of results generated by the HG-focus platform. Secondarily, they were used to perform prediction analysis for disease severity. Samples were grouped into stages (G) based on the timepoint when they were collected: G0=day0 (on the day of fever onset); G1=day 1 (one day after fever onset); G2=day2 (two days after fever onset)M-bM-^@M-&until G5; G6=day6-10 (6-10 days after fever onset); G7= convalescent time point (around day 28 after the first sample). Samples were also grouped into early acute (G0-G3), late acute (G4-G6) and convalescent phases (G7). References for the protocols; PMID 12803996 and 20078211

Project description:Dengue virus (DENV) infection is associated with a range of clinical manifestations, from self-limiting dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Although DENV-specific CD4 T cells are capable of producing inflammatory cytokines and can acquire cytotoxic functions in previously DENV-exposed individuals, much less is known related to DENV-specific CD4 T cells during acute DENV infection and disease. In this study, we set out to characterize the immune signatures of DENV-specific CD4 T cells during acute infection and investigate whether DENV-specific CD4 T cell response differs between DF and DHF. Previous studies suggest that increased IL-10 level in the blood is associated with severe dengue disease. Here we show evidence of DENV-specific IL-10-producing CD4 T cells during acute DENV infection. More specifically, an IL-10+IFN-γ+ double positive (DP) CD4 T cell population was prominent in acute hospitalized DENV cases but disappeared at the convalescent stage. RNA-sequencing analysis revealed that these cells were associated with gene signatures that marginally overlapped with previously identified signatures of cytotoxic CD4 T cells and type 1 regulatory T (Tr1)-like cells. However, the majority of the genes differentially expressed by DP cells were not related to cytotoxic CD4 T cells or Tr1-like cells. These genes include IL-21, IL-22, CD86, CD109, and CCR1, which are involved in T cell activation, function, cytokine signaling, and migration. Notably, the magnitude of DP cell response was higher in DHF than DF, whereas the gene expression profile of DP cells was similar between DF and DHF. Finally, our data show that DENV-specific DP cells are largely homogeneous based on the expression of 31 selected markers. Overall, this study reveals unique phenotypes of virus-specific IL-10/IFN-γ co-producing CD4 cells and indicates that the quantity but not quality of these cells may be positively correlated with dengue disease severity.

Project description:Dengue virus (DENV) infects hundreds of millions of people annually, yet there is only a limited knowledge of the host immune response to dengue. Here, we used a systems biological approach to perform a detailed analysis of the innate immune response to DENV infection in the whole blood samples of acutely infected humans in Bangkok, Thailand. Transcriptomic analysis revealed that genes encoding pro-inflammatory mediators and type I IFN related proteins, were associated with high levels of virus during the first few days of infection. Individuals with low or negative viremia at the late stage of fever were enriched with genes associated with pathways involved in cell cycle, proliferation, cell metabolism and translational control. Meta-analysis showed significant enrichment in genes specific for innate cells (monocytes, macrophages and DCs) in the specimens with high VL and enrichment in genes specific for NK cells, CD4+ and CD8+ T cells as well as B cells in specimens with low VL. Furthermore, flow cytometric analysis revealed an expansion in the numbers of CD14+CD16+ monocytes and depletion of CD14dimCD16++ cells and BDCA-1+ myeloid DC in blood. Consistent with this, in a non-human primate model, infection with DENV boosted the numbers of CD14+CD16+ monocytes in the blood and in secondary lymphoid organs. In vitro, freshly isolated blood monocytes infected with DENV up regulated CD16 and mediated robust differentiation of resting B cells to CD27++CD38++ plasmablasts and IgG and IgM secretion. Taken together, these data provide a detailed picture of the innate response to dengue infection in humans, and highlight an unappreciated role for CD14+CD16+ monocytes in promoting the differentiation of plasmablasts and mediating antibody response to DENV. We analyzed whole blood samples from 28 dengue patients (DF n=18, DHF=10) hospitalized at the Siriraj Hospital in Bangkok, Thailand during the season of 2009 The specimens were acquired between days 2 and 9 after onset of symptoms (acute illness), and for 19 patients (DF n=13, DHF=6) also at the convalescence at 4 weeks or later after discharge. Additionally, blood was sampled from 9 healthy, non-infected donors to provide controls for transcriptomic and immunological analysis.

Project description:Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. Background Dengue is a mosquito-borne viral infection causing a major public health problem globally. Dengue virus (DENV) is the causative agent of dengue fever (DF) and dengue hemorrhagic fever (DHF) and includes four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). DENV-2 and DENV-3 have been associated with severe dengue disease, consequently, laboratory testing for DENV is needed to confirm the diagnosis of DENV infection, serotype and to differentiate dengue from other febrile tropical illnesses. In addition, surveillance of mosquitoes infected with DENV is needed to monitor the infection rates within vector mosquito populations harboring specific serotype to provide an early warning sign to predict epidemics. Results In this work we have applied microarray analysis to simultaneously determine the serotype of multiple RNA samples from human or mosquitoes. The proposed microarray method can be used for i) rapid and reliable dengue diagnosis; ii) serotyping and iii) surveillance of mosquitoes infected with dengue. These microarrays were useful to confirm the presence of DENV-2 in 94 serum samples, DENV-3 in three samples from Juchitan, Oaxaca and one case from Juchitan, Oaxaca contained DENV-2 and -3. Moreover by using these microarrays we also determined DENV in pools of gravid females mosquitoes collected in several sites of nineteen Mexican states in 2005. Mosquito pools from 31 cities in the states of Yucatan, Campeche, Tabasco, Chiapas, Veracruz, Oaxaca, Guerrero, Tamaulipas and Colima were infected with DENV-2, six cities in Yucatán, Tabasco, Morelos, Tamaulipas, Colima, and Nayarit with DENV-1, three from Tabasco, Veracruz and Oaxaca with DENV 3 and two with two serotypes simultaneously (Ciudad Mante with DENV-1 and DENV-2, and Tavela with DENV-2 and DENV-3). Conclusion Here we show the success of applying microarrays assay to provide a consistently robust qualitative detection of dengue serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) in serum samples from patients or in pools of gravid female mosquitoes collected in the field of nineteen Mexican states. Interestingly, we did not detect any mosquito or serum sample containing DENV-4.

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Innate Lymphoid Cells Activation and Transcriptomic Changes in Response to Human Dengue Infection

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