Project description:Infection with dengue viruses (DENVs) leads to a spectrum of disease outcomes. The pathophysiology of severe versus non-severe manifestations of DENV infection may be driven by host responses, which could be reflected in the transcriptional profiles of peripheral blood immune cells. We conducted genome-wide microarray analysis of whole blood RNA from 34 DENV-infected children in Nicaragua collected on days 3–6 of illness, with different disease manifestations. Gene expression analysis identified genes that are differentially regulated between clinical subgroups. The most striking transcriptional differences were observed between dengue patients with and without shock, especially in the expression of mitochondrial ribosomal proteins associated with protein biosynthesis. In the dengue hemorrhagic fever patients, one subset of differentially expressed genes encode neutrophil-derived anti-microbial peptides associated with innate immunity. We analyzed 44 HEEBO arrays on which were hybridized RNA amplified from whole blood collected into PAXgene tubes. 34 samples were collected from DENV-infected patients who presented between days 3-6 of illness. Six convalescent samples collected 14-19 days after onset of symptoms were from two dengue fever patients, one dengue hemorrhagic fever patient and three dengue shock syndrome patients. Additionally, samples from four normal healthy individuals were also analyzed.
Project description:We looked at the whole-blood transcriptional profiling on dengue patients sampled within 72h of fever presentation and compared the signatures with autologous samples drawn at defervescence and convalescence and to control patients with fever of other etiology. Our data show that the early response in patients mimics those previously only described in vitro and suggests that this innate immune responses may initiate the later adaptive immune responses. Total RNA obtained from whole-blood of dengue patients with fever presentation, defervescence and convalescence are compared. Also, dengue patients with fever presentation are compared with control patients which are with fever of other etiology. 119 samples are analysed, which include 31 dengue patients and 26 control patients.
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 Deciphering host responses contributing to dengue shock syndrome (DSS), the life-threatening form of acute viral dengue infections, is required to improve both the differential prognosis and the treatments provided to DSS patients, a challenge for clinicians. Methodology/Principal Findings Based on a prospective study, we analyzed the genome-wide expression profiles of whole blood cells from 48 matched Cambodian children: 19 progressed to DSS while 16 and 13 presented respectively classical dengue fever (DF) or dengue hemorrhagic fever grades I/II (DHF). Using multi-way analysis of variance (ANOVA) and adjustment of p-values to control the False Discovery Rate (FDR<10%), we identified a signature of 2959 genes differentiating DSS patients from both DF and DHF, and showed a strong association of this DSS-gene signature with the dengue disease phenotype. Using a combined approach to analyse the molecular patterns associated with the DSS-gene signature, we provide an integrative overview of the transcriptional responses altered in DSS children. In particular, we show that the transcriptome of DSS children blood cells is characterized by a decreased abundance of transcripts related to T and NK lymphocyte responses and by an increased abundance of anti-inflammatory and repair/remodeling transcripts. We also show that unexpected pro-inflammatory gene patterns at the interface between innate immunity, inflammation and host lipid metabolism, known to play pathogenic roles in acute and chronic inflammatory diseases associated with systemic vascular dysfunction, are transcriptionnally active in the blood cells of DSS children. Conclusions/Significance We provide a global while non exhaustive overview of the molecular mechanisms altered in of DSS children and suggest how they may interact to lead to final vascular homeostasis breakdown. We suggest that some mechanisms identified should be considered putative therapeutic targets or biomarkers of progression to DSS. Whole blood genome-wide expression profiles of Cambodian children (3-15 year old) infected with dengue virus, having different clinical outcomes were compared. The studied cohort included 16 acute dengue fever samples, 13 acute dengue hemorrhagic fever samples and 19 acute dengue shock syndrome samples, classified according to the 1997 WHO criteria and randomised for age, gender, viral serotype and day of blood sampling after onset of fever. Microarray data were normalised using the quantile method. Multi-way ANOVA was used to compared the three clinical groups, at several False Discovery Rate of 10. Unsupervised Hierarchical Clustering (TreeView) showed that DSS patients clustered together (17 out of 19), identifying a gene-signature of DSS. Bio-informatics-based analysis using the demonstration version 7.1 of Ingenuity Pathway Analysis software (IPA; Ingenuity Systems, www.ingenuity.com) associated with manual and litterature-based analysis was carried out to identify the most relevant functional processes associated with the identified DSS gene expression profile. This was done by combining most informative canonical pathways identified using IPA, genes having the strongest association with the disease phenotype based on ANOVA analysis, and similarities to molecular patterns altered in other systemic inflammatory processes associated with endothelial dysfunction.
Project description:Dengue patient whole blood samples were analyzed during onset of disease, at defervescence and at early convalescence. Availability of samples at very early time points of disease allows to study the onset of the innate immune response. The main objective of the study is to understand early events during acute dengue disease, and how these events can eventually lead to the obsereved pathology including vascular leakage, thrombocytopenia and lyphocytopenia. Patients with suspected dengue disease gave a blood sample within 72h after onset of fever (DK1). The sample was used to confirm dengue infection by RT-PCR and RNA was stored for microarray. The patients returned for a second sample between 4 and 7 days after onset of fever (DK2) and 15-25 days after onset of fever (DK3). These longitdinal samples were all analyzed by microarray for inter-sample comparison and accross sample comparison. In total eleven patients were analyzed at the three time points indicated.
Project description:We measured fever-day-specific genome-wide transcript abundance patterns in 105 peripheral blood mononuclear cell (PBMC) samples collected from 41 children hospitalized with dengue virus (DENV) infection in Nicaragua, and from 8 healthy controls. DF1 = primary DF; DF2 = secondary DF; DHF = Dengue Hemorrhagic Fever; DSS = Dengue Shock Syndrome. Samples were collected from pediatric patients with DENV infection at the Hospital Infantil Manuel de Jesus Rivera (HIMJR) in Managua, Nicaragua. Enrollment criteria consisted of hospitalized patients younger than 15 years of age whose parents or guardians completed the informed consent process, and for children 6 years and older who provided assent, and who presented with acute febrile illness of less than 7 days duration with one or more of the following symptoms or signs at the time of evaluation: headache, arthralgia, myalgia, retro-orbital pain, positive tourniquet test, petechiae, and signs of bleeding. Children were considered to have dengue if DENV was detected by RT-PCR or by isolation in cell culture, if an IgM ELISA indicated seroconversion between the acute and convalescent sample, and/or if there was a > 4-fold increase in DENV-specific antibodies between the acute and convalescent phase. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood within four hours of collection and stored in Trizol at -80 oC disease_state_design
Project description:We investigated the abundances and transcriptomic changes of immune cells at several time points over the course of dengue virus infection. The PBMC samples were obtained from one dengue fever (DF) and one dengue hemorrhagic fever (DHF) patients (8 samples in total). The samples were harvested at two and one days before defervescence (febrile phase), at defervescence (critical phase), and two-week convalescence. Single-cell RNA-seq libraries were prepared using the 10x genomic protocol and were sequenced using the Illumina Hiseq platform. One healthy control sample analysed by the same protocol was included.
Project description:Dengue viruses cause two severe diseases that alter vascular fluid barrier functions, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). While the mechanisms that lead to vascular permeability are unknown, the endothelium plays a central role in regulating fluid and cellular efflux from capillaries. Thus, dysregulation of endothelial cells functions by dengue virus infection may contribute to pathogenesis and severe disease. We used microarrays to investigate the effect of dengue virus infection on gene expression within primary human endothelial cells at various times post infection and identified numerous upregulated antiviral and immune response genes.