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

Lower Low-Density Lipoprotein Cholesterol Levels Are Associated with Severe Dengue Outcome

<div><p>Dengue virus (DENV) is a flavivirus of worldwide importance, with approximately 4 billion people across 128 countries at risk of infection, and up to 390 million infections and 96 million clinically apparent cases estimated annually. Previous <i>in vitro</i> studies have shown that lipids and lipoproteins play a role in modifying virus infectivity. However, the relationship between development of severe dengue and total cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), respectively, is unclear. We analyzed data from 789 laboratory-confirmed dengue cases and 447 other febrile illnesses (OFI) in a prospective pediatric hospital-based study in Managua, Nicaragua between August 2005 and January 2013, using three different classifications of dengue severity: World Health Organization (WHO) 1997, WHO 2009, and standardized intervention categories. Total serum cholesterol and LDL-C levels decreased over the course of illness and were generally lower with increasing dengue severity, regardless of classification scheme. Greater decreases in LDL-C than HDL-C were observed among dengue-positive patients compared to patients with OFI and among severe dengue compared to mild dengue cases. Furthermore, daily cholesterol levels declined with daily albumin blood levels. To examine the effect of cholesterol at presentation on subsequent risk of development of severe dengue, relative risks and 95% confidence intervals were calculated using multivariable modified Poisson models. We found that lower total serum cholesterol and LDL-C levels at presentation were associated with subsequent risk of developing dengue hemorrhagic fever/dengue shock syndrome using the WHO 1997 dengue severity classification, and thus that the reduction in LDL-C is likely driving the decreases observed in total serum cholesterol levels among dengue-positive patients. Our results suggest that cholesterol blood levels are important correlates of dengue pathophysiology and should be explored as part of a prognostic biomarker panel for severe dengue.</p></div

Characteristics of study participants by DENV infection status and disease severity classification criteria.

<p>Abbreviations: DENV, dengue virus; WHO, World Health Organization; OFI, other febrile illness; DF, dengue fever; DHF/DSS, dengue hemorrhagic fever/dengue shock syndrome; DWS, dengue with or without warning signs; SD, severe dengue; IC, standardized intervention categories.</p><p>Characteristics of study participants by DENV infection status and disease severity classification criteria.</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

Effect of cholesterol level at presentation on development of severe dengue outcome using the WHO 1997 disease severity classification.^*

<p>*Severe dengue outcome was modeled by cholesterol at presentation, age, gender, malnutrition and immune response using modified Poisson models with robust standard errors. For the WHO 1997 disease severity classification, severe dengue outcome was defined as DHF/DSS and mild dengue outcome, the reference group, was defined as DF.</p><p>Effect of cholesterol level at presentation on development of severe dengue outcome using the WHO 1997 disease severity classification.^{<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003904#t002fn001" target="_blank">*</a>}</p

Incidence of DENV infections, Managua, Nicaragua, 2004–2010.

<p>The number of DENV infections from 2004–2005 to 2007–2008 has been previously reported <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002462#pntd.0002462-Balmaseda3" target="_blank">[16]</a>. The number of inapparent DENV infections in this paper vary slightly from our previous report due to the implementation of new quality control procedures which resulted in several revisions to the inapparent infection data.</p

Eligibility flow chart.

<p>Of the 1,440 patients who presented to the hospital with suspected dengue, 1,236 met the eligibility criteria and were included in the analysis. Abbreviations: BMI, body mass index.</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

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