Posted in Dengue, Resource

Gene Expression Patterns of Dengue Virus-Infected Children from Nicaragua Reveal a Distinct Signature of Increased Metabolism

Pathways and genes that are differentially regulated in DF, DHF and DSS patients. Source from Loke et al., PLOS NTD, 2010.

Identifying signatures of host genome-wide transcriptional patterns can be a tool for biomarker discovery as well as for understanding molecular mechanisms and pathophysiological signatures of disease states.

In this study by Loke et al., transcriptional profiling analysis of pediatric patients from Nicaragua with a predominantly DENV-1 infection was performed, and the gene signatures between healthy, dengue fever (DF), dengue haemorrhaigic fever (DHF) and dengue shock syndrome (DSS) were compared. Enrolment criteria consisted of hospitalised patients younger than 15 years of age. Whole blood was collected during acute illness (days 3-6).

Unsupervised clustering reveal that DHF and DF patients cluster distinctly from the DSS patients. Interestingly, many of the genes that separate these two groups are involved in ‘protein biosynthesis’ and ‘protein metabolism and modification’. A large number of mitochondrial ribosomal proteins and ‘nucleic acid binding’ were also flagged (See Figure above).

Genes related to metabolism, oxidative phosphorylation, protein targeting, nucleic acid metabolism, purine and pyrimidine metabolism, electron transport, DNA metabolism and replication, and protein metabolism and modification were differentially regulated by DF, DHF and DSS patients, reflecting a shared signature of DENV-1 infection.

On the other hand, the biological processes differentially expressed by DSS patients were protein metabolism and modification, intracellular protein traffic, pre-mRNA processing, mRNA splicing, nuclear transport, protein-lipid modification and protein folding.

Of note, the changes in metabolism genes cannot be seen in vitro. Instead, interferon signatures were upregulated.

Data is deposited in Gene Expression Omnibus (GEO) under GSE25226.

Posted in Dengue, Resource

A 20-Gene Set Predictive of Progression to Severe Dengue

Methodology employed by Robinson et al., Cell Reports, 2019. The 20-gene set was used to distinguish between individuals with severe and mild dengue

The gene signatures predictive of severe dengue disease progression is poorly understood.

The study by Robinson et al., utilise 10 publicly available datasets and divided them into 7 “discovery” and 3 “validation” datasets. In the discovery datasets, a total of 59 differentially expressed genes (FDR < 10%, effect size > 1.3-fold) was detected between patients who progress to DHF and/or DSS (DHF/DSS) versus patients with an uncomplicated course (dengue fever).

An iterative greedy forward search to the 59 genes revealed a final set of and 20 differentially expressed genes (3 over-expressed, 17 under-expressed) in DHF/DSS (Gene list as shown in figure above). A dengue score for each sample was obtained by subtracting the geometric mean expression of the 17 under-expressed genes from the geometric mean expression of the 3 over-expressed genes.

The 20-gene dengue severity scores distinguished DHF/DSS from dengue fever upon presentation and prior to the onset of severe complications with a summary area under the curve (AUC) = 0.79 in the discovery datasets. The 20-gene dengue scores also accurately identified dengue patients who will develop DHF/DSS in all three validation datasets.

To further validate this signature, the authors tested a cohort of prospectively enrolled dengue patients in Colombia. The 20-gene dengue score, measured by qPCR, distinguished severe dengue from dengue with or without warning signs (AUC = 0.89) and even severe dengue from dengue with warning signs (AUC = 0.85).

Finally, the 20-gene set is significantly downregulated in natural killer (NK) and NK T (NKT) cells, indicating the role of NK and NKT cells in modulating severe disease outcome.

Dataset deposited under Gene Expression Omnibus (GEO): GSE124046

Posted in Dengue, Resource

Immunotranscriptomic profiling the acute and clearance phases of a human challenge dengue virus serotype 2 infection model

Differentially expressed genes at day 8 and 28 after rDEN2Δ30 infection. Source from Hanley JP et al., Nature Communications, 2021.

rDEN2Δ30 is a recombinant serotype 2 virus based on the American genotype 1974 Tonga DENV2 virus, which has been partially attenuated by deletion of 30 nucleotides in the 3′ untranslated region of the RNA genome (Δ30). rDEN2Δ30 infection is known to induce modest viremia in all flavivirus-naive subjects and a mild, transient non-pruritic rash in 80% of recipients.

rDEN2Δ30 infection could hence be a suitable model to evaluate molecular signatures responsible for asymptomatic or mild DENV-2 infection.

In this study by Hanley JP et al., RNA-seq was performed on whole blood collected from rDEN2Δ30-infected subjects at 0, 8, and 28 days post infection. rDEN2Δ30-induced reproducible but modest viremia and a mild rash as the only clinically significant finding in DENV-naive subjects.

Principal component analysis reveal minimal overlap between baseline (day 0) and peak viremia (day 8). The day 28 data (post viremia) partially overlapped with the baseline (day 0) and acute (day 8) timepoints. Pathways enriched in the type I and type II interferon and antiviral responses were upregulated at day 8, whereas pathways controlling translational initiation were downregulated. NF-κB, IL-17 signaling pathways, apoptosis, toll-like receptor signaling, response to viruses, ribosomes, and defense responses were also differentially regulated at day 28.

Myeloid cells including monocytes and activated dendritic cells were significantly increased during acute infection and returned to baseline. In contrast, regulatory T cells (Tregs) were significantly decreased during acute stage.

Gene ontology pathway analysis revealed that the viremia-tracking set of genes was enriched for both response to and regulation of type I and II interferon pathways, including JAK/STAT signaling. Genes encoding for proteins that directly inhibit viral genome replication and involved in protein ubiquitination and catabolism, especially ISG15 pathway, tracked with viremia. Day 28 revealed more varied pathways, including protein ubiquitination, cell migration, cytoskeletal reorganization, and angiogenesis.

Baseline transcript signatures can potentially predict whether the subjects would develop rash after rDEN2Δ30 infection. Higher baseline expression of myeloid nuclear differentiation antigen (MNDA), and cell surface associated cellular processes such as tetraspanin CD37, integral membrane 2B (ITM2B), and genes involved in autophagy (VMP1) was associated with protection from rash. These genes are mostly related to myeloid responses, membrane regulation, autophagy, K63 ubiquitination, and cell morphogenesis.

Transcriptomic signatures modulated by rDEN2Δ30 infection and severe dengue are distinct. Only one gene family, the guanine binding protein (GBP1/2) genes was differentially regulated in both severe dengue and during mild rDEN2Δ30 infection.

Data deposited im Gene Expression Omnibus under accession number GSE152255

Posted in Dengue, Resource

Increased adaptive immune responses and proper feedback regulation protect against clinical dengue

Genes related to antigen presentation were significantly increased in the asymptomatic compared to the symptomatic dengue individuals. Manuscript by E Simon-Lorière et al., Science Translational Medicine, 2017.

Dengue infections can be asymptomatic, symptomatic, or occasionally progress to severe dengue, a life-threatening condition characterised by a cytokine storm, vascular leakage, and shock. However, the molecular and immunological mechanisms underlying asymptomatic dengue virus (DENV) infection remains largely unknown.

In the publication, E Simon-Lorière et al recruited DENV infected children in Cambodia. Nine individuals remained strictly asymptomatic at the time of inclusion and during the 10-day follow-up period. PBMCs from 8 asymptomatic DENV-1 viremic individuals and 25 symptomatic dengue patients were used for further gene expression analysis.

Asymptomatic individuals have an increase in the percentage of CD4+ T cells and a decrease in CD8+ T cells compared to symptomatic dengue individiuals. However, CD14+ monocytes, Lin-CD11c+ dendritic cells, CD19+ B cells, and CD335+ natural killer cells are not significantly different between asymptomatic and symptomatic individuals.

Transcriptomic signatures were distinct between asymptomatic and symptomatic individuals. The top pathways that diverge the most between asymptomatic and clinical dengue individuals were related to immune processes. Notably, the transcriptomic differences cannot be explained by differences in viral load or immune status.

The innate immune responses were not significantly different between the asymptomatic and symptomatic individuals. Instead, the most significantly activated pathway in asymptomatic individuals was related to “nuclear factor of activated T-cells (NFAT) mediated regulation of immune response.” These genes include CIITA, CD74 and various human leukocyte antigen (HLA) genes, where their expression differences were also validated at the protein levels (See figure on top).

Protein kinase Cq (PKCq) signaling in T lymphocytes was also highly activated in asymptomatic viremic individuals. Genes upregulated included AKT3, SOS1, PAK1, and SLAMF6, as well as T-cell costimulatory pathways such as ICOS-ICOSL, and CD28 and CTLA4 signaling in cytotoxic T-cells.

In contrast, genes related to B-cell activation, differentiation and plasma cell development (BLIMP-1, IRF4) were downregulated in asymptomatic individuals. This finding is correlated with the reduction in antibody production in the asymptomatic individuals.

Data is saved in Gene Expression Omnibus under accession number GSE100299