Project description:CEM.NKR.DC-SIGN cells were infected in vitro with DENV1 (strain WestPac74) for 18 hours, after which either total viable cells or viable cells expressing surface DENV1 NS1 were isolated by flow cytometric sorting. Sorted cells were processed for scRNAseq analysis using either a standard Oligo(dT) primer, or an Oligo(dT) primer supplemented with a DENV-specific RT primer
Project description:Low abundance mRNAs are more difficult to examine using microarrays than high abundance mRNAs due to the effect of concentration on hybridization kinetics, signal-to-noise ratios, and interference between signals from mRNA isoforms. Individual sequences in LCRs are more highly represented than in the mRNA population from which they are derived, leading to favorable hybridization kinetics. LCR targets permit the measurement of abundance changes that are difficult to measure using oligo dT priming for target synthesis. An oligo dT-primed target and three LCRs detect twice as many differentially regulated genes as could be detected by the oligo dT-primed target alone, as serum-starved fibroblasts responded to the reintroduction of serum. Thus, this target preparation strategy considerably increases the sensitivity of spotted cDNA microarrays. Keywords: parallel sample
Project description:Controlled dengue human challenge studies present a unique opportunity to address many longstanding questions in the field of flavivirus biology. These fundamental questions include defining the early immunological signatures of infection, the host/environmental factors that impact disease severity, and the role of preexisting immunity on the development of symptomatic viral infection. However, while several controlled dengue human challenge studies have been performed and appear to clinically recapitulate may features of mild natural DENV infection, limited data are available on how the immunological and transcriptional response elicited by these attenuated challenge viruses compares to the profile associated with a natural, unattenuated DENV infection. To bridge this knowledge gap, we performed scRNAseq analysis on longitudinally collected PBMC samples obtained from 3 individuals (8 time points per subject) enrolled in the SUNY/WRAIR DENV-1 controlled human challenge study. In addition, 3 time points (two acute infection time points, one control time point) from two individuals experiencing a natural DENV-1 infection were analyzed and computationally integrated with the challenge model dataset. This temporally integrated dataset contains a total of 171,208 cells and 22 statistically distinct populations corresponding to all major anticipated leukocyte subsets. While all identified cell populations demonstrated significant and consistant perturbations in their transcriptional profile in response to either natural or experimental DENV infection, conventional monocytes respond most robustly to infection across all subjects and study groups from an unbiased transcriptional perspective. Using these data, core sets of genes that were consistently induced by either natural or experimental DENV were identified, and the overlap between the two arms of the study assessed.
Project description:The objective of this analysis was to determine the transcriptional signature associated with experimental DENV-1 infection in human volunteers. Nine flavivirus naive volunteers were challenged with an attenuated DENV-1 strain - 45AZ5 - and blood collected for RNA extraction and transcriptional analysis on days 0, 8, 10, 14, and 28 post challenge using PAXgene collection tubes. Total RNA was isolated from the collection tubes and subjected to RNAseq analysis to identify genes and gene sets that were differentially expressed across the infection time course.
Project description:The objective of this analysis was to determine the transcriptional signature associated with experimental primary DENV-3 infection in human volunteers. Nine flavivirus naive volunteers were challenged with an attenuated DENV-3 strain - CH53489 - and blood collected for RNA extraction and transcriptional analysis on or around study days 0, 6, 8, 10, 14, and 28 post challenge using PAXgene collection tubes. Total RNA was isolated from the collection tubes and subjected to RNAseq analysis to identify genes and gene sets that were differentially expressed across the infection time course.