Metabolomics Approach for Investigation of Effects of Dengue Virus Infection Using the EA.hy926 Cell Line

Dengue, due to its global burden, is the most important arthropod-borne flavivirus disease, and early detection lowers fatality rates to below 1%. Since the metabolic resources crucial for viral replication are provided by host cells, detection of changes in the metabolic profile associated with disease pathogenesis could help with the identification of markers of prognostic and diagnostic importance. We applied 1 H nuclear magnetic resonance exploratory metabolomics to study longitudinal changes in plasma metabolites in a cohort in Recife, Brazil. To gain statistical power, we used innovative paired multivariate analyses to discriminate individuals with primary and secondary infection presenting as dengue fever (DF; mild) and dengue hemorrhagic fever (DHF; severe) and subjects with a nonspecific nondengue (ND) illness (ND subjects). Our results showed that a decrease in plasma low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) discriminated dengue virus (DENV)-infected subjects from ND subjects, and also, subjects with severe infection even presented a decrease in lipoprotein concentrations compared to the concentrations in subjects with mild infection. These results add to the ongoing discussion that the manipulation of lipid metabolism is crucial for DENV replication and infection. In addition, a decrease in plasma glutamine content was characteristic of DENV infection and disease severity, and an increase in plasma acetate levels discriminated subjects with DF and DHF from ND subjects. Several other metabolites shown to be altered in DENV infection and the implications of these alterations are discussed. We hypothesize that these changes in the plasma metabolome are suggestive of liver dysfunction, could provide insights into the underlying molecular mechanisms of dengue virus pathogenesis, and could help to discriminate individuals at risk of the development of severe infection and predict disease outcome. IMPORTANCEDengue, due to its global burden, is the most important mosquito-borne viral disease. There is no specific treatment for dengue disease, and early detection lowers fatality rates to below 1%. In this study, we observed the effects of dengue virus infection on the profile of small molecules in the blood of patients with mild and severe infection. Variations in the profiles of these small molecules reflected the replication of dengue virus in different tissues and the extent of tissue damage during infection. The results of this study showed that the molecules that changed the most were VLDL, LDL, and amino acids. We propose that these changes reflect liver dysfunction and also that they can be used to discriminate subjects with mild dengue from those with severe dengue. D engue, due to its global burden, is the most important arthropod-borne disease nowadays. Dengue is caused by dengue virus (DENV), a positive-strand RNA virus with four distinct serotypes: DENV serotype 1 (DENV1) to DENV4. Estimates of the DENV infection burden at global levels indicate approximately 40...

Section: Discussioncontrasting

confidence: 83%

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confidence: 99%

¹ H Nuclear Magnetic Resonance Metabolomics of Plasma Unveils Liver Dysfunction in Dengue Patients

El‐Bacha

Struchiner

Cordeiro

et al. 2016

“…Previously, a multiplatform approach was developed to measure the levels of a limited number of extracellular metabolites following DENV infection of the human endothelial hybrid cell line EA.hy926 (21). However, a comprehensive examination of host cellular metabolism during DENV infection of primary human cells has yet to be conducted.…”

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confidence: 99%

Dengue Virus Induces and Requires Glycolysis for Optimal Replication

Fontaine

Sanchez

Camarda

et al. 2015

259

272

Viruses rely on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. Dengue virus (DENV), a member of the Flaviviridae family, is one of the most important arthropod-borne human pathogens worldwide. We analyzed global intracellular metabolic changes associated with DENV infection of primary human cells. Our metabolic profiling data suggested that central carbon metabolism, particularly glycolysis, is strikingly altered during a time course of DENV infection. Glucose consumption is increased during DENV infection and depriving DENV-infected cells of exogenous glucose had a pronounced impact on viral replication. Furthermore, the expression of both glucose transporter 1 and hexokinase 2, the first enzyme of glycolysis, is upregulated in DENV-infected cells. Pharmacologically inhibiting the glycolytic pathway dramatically reduced DENV RNA synthesis and infectious virion production, revealing a requirement for glycolysis during DENV infection. Thus, these experiments suggest that DENV induces the glycolytic pathway to support efficient viral replication. This study raises the possibility that metabolic inhibitors, such as those that target glycolysis, could be used to treat DENV infection in the future. IMPORTANCEApproximately 400 million people are infected with dengue virus (DENV) annually, and more than one-third of the global population is at risk of infection. As there are currently no effective vaccines or specific antiviral therapies for DENV, we investigated the impact DENV has on the host cellular metabolome to identify metabolic pathways that are critical for the virus life cycle. We report an essential role for glycolysis during DENV infection. DENV activates the glycolytic pathway, and inhibition of glycolysis significantly blocks infectious DENV production. This study provides further evidence that viral metabolomic analyses can lead to the discovery of novel therapeutic targets to block the replication of medically important human pathogens. Viruses are obligate intracellular parasites that depend on the metabolic machinery of the host cell to supply the energy and macromolecules necessary for successful replication. In recent years, research has focused on investigating how virus infection alters host metabolism with the hope that these studies will provide insight into the metabolic requirements of viral replication. Viral modulation of the host cell metabolic profile has been examined for several viruses, including human cytomegalovirus (HCMV), herpes simplex virus 1 (HSV-1), hepatitis C virus, influenza A virus, human immunodeficiency virus type 1 (HIV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), vaccinia virus (VACV), and Epstein-Barr virus (1-11). These reports revealed that virus infection triggers dramatic changes in cellular metabolism, particularly in central carbon utilization pathways.Glucose and glutamine represent the two main carbon sources used to support the energetic and biosynthetic needs of mammalian cells. In...

“…HCMV was shown to induce multiple metabolic alterations, including upregulation of the tricarboxylic acid (TCA) cycle, fatty acid synthesis, pyrimidine nucleotide biosynthesis, and glycolysis. The metabolic effects of virus infection on human cells have now been investigated for several other viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), herpes simplex virus 1 (HSV-1), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and dengue virus (1,(3)(4)(5)(6)(7). These studies have identified numerous metabolic perturbations induced by virus infection.…”

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confidence: 99%

Vaccinia Virus Requires Glutamine but Not Glucose for Efficient Replication

Fontaine

Camarda

Lagunoff

2014

151

167

Viruses require host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Vaccinia virus (VACV) is a member of the Poxviridae family, and its use as a vaccine enabled the eradication of variola virus, the etiologic agent of smallpox. A global metabolic screen of VACV-infected primary human foreskin fibroblasts suggested that glutamine metabolism is altered during infection. Glutamine and glucose represent the two main carbon sources for mammalian cells. Depriving VACV-infected cells of exogenous glutamine led to a substantial decrease in infectious virus production, whereas starving infected cells of exogenous glucose had no significant impact on replication. Viral yield in glutamine-deprived cells or in cells treated with an inhibitor of glutaminolysis, the pathway of glutamine catabolism, could be rescued by the addition of multiple tricarboxylic acid (TCA) cycle intermediates. Thus, VACV infection induces a metabolic alteration to fully rely on glutamine to anaplerotically maintain the TCA cycle. VACV protein synthesis, but not viral transcription, was decreased in glutamine-deprived cells, which corresponded with a dramatic reduction in all VACV morphogenetic intermediates. This study reveals the unique carbon utilization program implemented during poxvirus infection and provides a potential metabolic pathway to target viral replication. IMPORTANCEViruses are dependent on the metabolic machinery of the host cell to supply the energy and molecular building blocks needed for critical processes including genome replication, viral protein synthesis, and membrane production. This study investigates how vaccinia virus (VACV) infection alters global cellular metabolism, providing the first metabolomic analysis for a member of the poxvirus family. Unlike most viruses examined to date, VACV does not activate glycolysis, and exogenous glucose is not required for maximal virus production. Instead, VACV requires exogenous glutamine for efficient replication, and inhibition of glutamine metabolism effectively blocks VACV protein synthesis. This study defines a major metabolic perturbation essential for the replication of a poxvirus and may lead to the discovery of novel antiviral therapies based on metabolic inhibitors.A ll viruses rely on host cellular metabolism for the energy and macromolecule synthesis required for their replication. Recently, significant focus has been placed on examining how virus infection alters the host cellular metabolic profile in the hopes of identifying metabolic pathways that are critical to virus life cycles. Viral metabolomic analyses were first conducted to determine changes in host cell metabolism caused by human cytomegalovirus (HCMV) lytic infection (1, 2). HCMV was shown to induce multiple metabolic alterations, including upregulation of the tricarboxylic acid (TCA) cycle, fatty acid synthesis, pyrimidine nucleotide biosynthesis, and glycolysis. The metabolic effects of virus infection on human cells have now been inves...