Temporal Dynamics of the Transcriptional Response to Dengue Virus Infection in Nicaraguan Children

<div><p>Dengue is the most prevalent mosquito-borne human illness worldwide. The ability to predict disease severity during the earliest days of the illness is a long-sought, but unachieved goal.</p> <p>We examined human genome-wide transcript abundance patterns in daily peripheral blood mononuclear cell (PBMC) samples from 41 children hospitalized with dengue virus (DENV) infection in Nicaragua, as well as 8 healthy control subjects. Nine patients had primary dengue fever (DF1), 11 had dengue fever with serologic evidence of prior DENV infection, i.e., secondary dengue fever (DF2), 12 had dengue hemorrhagic fever (DHF), and 9 had dengue shock syndrome (DSS). We identified 2,092 genes for which transcript abundance differed significantly between patients on days 3–6 of fever and healthy subjects (FDR<1%). Prior DENV infection explained the greatest amount of variation in gene expression among patients. The number of differentially expressed genes was greatest on fever day 3 in patients with DF1, while the number in patients with DF2 or DHF/DSS was greatest on day 5. Genes associated with the mitotic cell cycle and B cell differentiation were expressed at higher levels, and genes associated with signal transduction and cell adhesion were expressed at lower levels, in patients versus healthy controls. On fever day 3, a set of interferon-stimulated gene transcripts was less abundant in patients who subsequently developed DSS than in other patient groups (p<0.05, ranksum). Patients who later developed DSS also had higher levels of transcripts on day 3 associated with mitochondrial function (p<0.01, ranksum). These day 3 transcript abundance findings were not evident on subsequent fever days.</p> <p>In conclusion, we identified differences in the timing and magnitude of human gene transcript abundance changes in DENV patients that were associated with serologic evidence of prior infection and with disease severity. Some of these differential features may predict the outcome of DENV infection.</p> </div

Transcripts with different abundance in each patient group compared to healthy individuals, by fever day.

<p>Significant differences in transcript abundance were identified using SAM <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001966#pntd.0001966-Tusher1" target="_blank">[32]</a> and a false discovery rate of 1%. Resampling was used to control for differences in sample size <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001966#pntd.0001966-Beck1" target="_blank">[33]</a>. Red bars: transcripts more abundant in dengue patients than in healthy individuals; green bars: transcripts more abundant in healthy individuals.</p

Transcripts whose abundance differed in patients with primary DF and secondary DSS on day 3.

<p>A) Abundance levels for 291 transcripts whose abundance differed between subjects with DF1 and subjects with DSS on fever day 3 (FDR<20%) were normalized to the median expression in healthy controls, and organized using k-means clustering. Gray triangles mark gene clusters that were annotated using expression relative to healthy individuals (u, significantly higher; o, not significantly different; d; significantly lower than healthy controls). B–E) Median expression of each gene cluster depicted in (A). Each data point represents a single patient. § indicates significant differences compared to patients with DSS (p<0.05, ranksum).</p

Characteristics of study participants with dengue.

<p>Note: Data are median (range) unless otherwise indicated.</p

Gene expression in dengue patients on day 4 of fever.

<p>A) Hierarchical clustering of PBMC expression profiles from healthy individuals and dengue patients on fever day 4. Unsupervised clustering based on expression of 10,075 transcripts meeting quality control criteria (see Methods and Materials). Samples are colored by serologic status and WHO-defined clinical status. All patients with DHF or DSS had secondary DENV infection. Numbered horizontal bars refer to sample clusters described in the text. B) Values associated with principal component 1 from singular value decomposition of the data in (A). H = healthy control; DF1 = dengue fever, primary DENV infection; DF2 = dengue fever, secondary DENV infection; DHF = dengue hemorrhagic fever; DSS = dengue shock syndrome. §: differs from DF1 (p≤0.01, rank sum).</p

Differential gene expression between dengue patients and healthy controls, and among patient groups.

<p>A) Hierarchical clustering of 2,092 transcripts that differ in abundance in at least one patient group on fever days 3, 4, 5, or 6 when compared to healthy individuals. Median transcript abundance for each group on each of days 3 through day 7 (5 columns per group) is presented. Genes organized using hierarchical clustering, after normalizing to median value of healthy controls. Red indicates more abundant than controls; green indicates less abundant. Gray columns separate each group; the black column represents median value for healthy controls. Vertical lines and numbers 1–4 correspond to gene clusters discussed in the text. B) Median expression of each gene cluster, by day of illness and patient group.</p

Temporal patterns of transcript abundance for genes associated with disease severity on fever day 3.

<p>A) Transcripts less abundant in DF1 patients and more abundant in DSS patients, compared to healthy controls (DF1dDSSu, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001966#pntd-0001966-g005" target="_blank">Figure 5</a>). B) PBMC transcripts induced by type-I interferons <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001966#pntd.0001966-Waddell1" target="_blank">[37]</a>. Symbols indicate median expression of all patients in the indicated clinical group sampled on that fever day. Light blue diamonds, DF1; dark blue squares, DF2; orange triangles, DHF; red circles, DSS. Error bars are defined by the median absolute deviation. Transcript abundance is calculated relative to the median abundance in healthy controls (dotted line). § indicates significant difference compared to DSS patients (p<0.05, ranksum).</p

Transcriptome Patterns from Primary Cutaneous <em>Leishmania braziliensis</em> Infections Associate with Eventual Development of Mucosal Disease in Humans

<div><h3>Introduction</h3><p>Localized Cutaneous Leishmaniasis (LCL) and Mucosal Leishmaniasis (ML) are two extreme clinical forms of American Tegumentary Leishmaniasis that usually begin as solitary primary cutaneous lesions. Host and parasite factors that influence the progression of LCL to ML are not completely understood. In this manuscript, we compare the gene expression profiles of primary cutaneous lesions from patients who eventually developed ML to those that did not.</p> <h3>Methods</h3><p>Using RNA-seq, we analyzed both the human and <em>Leishmania</em> transcriptomes in primary cutaneous lesions.</p> <h3>Results</h3><p>Limited number of reads mapping to <em>Leishmania</em> transcripts were obtained. For human transcripts, compared to ML patients, lesions from LCL patients displayed a general multi-polarization of the adaptive immune response and showed up-regulation of genes involved in chemoattraction of innate immune cells and in antigen presentation. We also identified a potential transcriptional signature in the primary lesions that may predict long-term disease outcome.</p> <h3>Conclusions</h3><p>We were able to simultaneously sequence both human and <em>Leishmania</em> mRNA transcripts in primary cutaneous leishmaniasis lesions. Our results suggest an intrinsic difference in the immune capacity of LCL and ML patients. The findings correlate the complete cure of <em>L. braziliensis</em> infection with a controlled inflammatory response and a balanced activation of innate and adaptive immunity.</p> </div

Genes involved in the most relevant biological activities according to Ingenuity Pathway Assay.

<p>The bars indicate the significance levels of the up-regulated genes presence based on the normal event (yellow line). Black and gray bars represent LCL and ML samples, respectively. LCL: Localized Cutaneous Leishmaniasis. ML: Mucosal Leishmaniasis.</p

Global transcriptional expression patterns.

<p>Hierarchical clustering was performed using the Euclidean distance method (Gene Cluster 3.0) with complete linkage for both samples and genes assays. Intensity of gene expression increases from black to yellow. The heat map in the left panel includes the 18,577 genes detected by RNA-seq and shows that the samples were correctly clustered into the two clinical phenotypic groups, ML and LCL. The right panel represents the same assay using the 1208 genes selected by the dimensional reduction step performed by the <i>t-test</i>, confirming that the samples could still be clustered into LCL and ML groups. LCL: Localized Cutaneous Leishmaniasis. ML: Mucosal Leishmaniasis.</p