Genome-wide expression profiling deciphers host responses altered during dengue shock syndrome and reveals the role of innate immunity in severe dengue.

BACKGROUND: Deciphering host responses contributing to dengue shock syndrome (DSS), the life-threatening form of acute viral dengue infections, is required to improve both the differential prognosis and the treatments provided to DSS patients, a challenge for clinicians. METHODOLOGY/PRINCIPAL FINDINGS: Based on a prospective study, we analyzed the genome-wide expression profiles of whole blood cells from 48 matched Cambodian children: 19 progressed to DSS while 16 and 13 presented respectively classical dengue fever (DF) or dengue hemorrhagic fever grades I/II (DHF). Using multi-way analysis of variance (ANOVA) and adjustment of p-values to control the False Discovery Rate (FDR<10%), we identified a signature of 2959 genes differentiating DSS patients from both DF and DHF, and showed a strong association of this DSS-gene signature with the dengue disease phenotype. Using a combined approach to analyse the molecular patterns associated with the DSS-gene signature, we provide an integrative overview of the transcriptional responses altered in DSS children. In particular, we show that the transcriptome of DSS children blood cells is characterized by a decreased abundance of transcripts related to T and NK lymphocyte responses and by an increased abundance of anti-inflammatory and repair/remodeling transcripts. We also show that unexpected pro-inflammatory gene patterns at the interface between innate immunity, inflammation and host lipid metabolism, known to play pathogenic roles in acute and chronic inflammatory diseases associated with systemic vascular dysfunction, are transcriptionnally active in the blood cells of DSS children. CONCLUSIONS/SIGNIFICANCE: We provide a global while non exhaustive overview of the molecular mechanisms altered in of DSS children and suggest how they may interact to lead to final vascular homeostasis breakdown. We suggest that some mechanisms identified should be considered putative therapeutic targets or biomarkers of progression to DSS.

[Activated eosinophils: techniques to characterize them]. / Etude de l'activation du polynucléaire éosinophile. Apport des méthodologies classiques et récentes à la caractérisation d'une cellule complexe.

The deleterious role thought to be played by eosinophils in many situations is linked to their ability to secrete various inflammatory substances, mainly toxic proteins and lipid mediators, in body tissue. This ability is a particular feature of activated eosinophils, which have undergone numerous metabolic, functional, and phenotypic changes from their resting state. Characterizing the properties of these activated cells is an essential step in improving our understanding of their contributions to local inflammatory response, as both regulatory and effector cells. Improvements in existing methods as well as the development of new technical approaches have facilitated the ex vivo and in vitro study of activated eosinophils and their contribution to various disease states.

Dengue virus infection of human microvascular endothelial cells from different vascular beds promotes both common and specific functional changes.

Dengue shock syndrome (DSS), the major life threatening outcome of severe dengue disease, which occurs in some patients in the course of dengue infection, is the consequence of plasma leakage in the microvascular territories. Data from clinical and in vitro studies suggest that an inadequate immunological response is partly responsible for the pathophysiology of DSS, but few is known concerning the consequences of direct infection of endothelial cells by dengue virus per se. In this study, an attempt was made to study the response of two microvascular human cell lines originating, respectively, from liver and dermis to infection by a dengue type 2 virus, by analyzing the virus-induced modulation of functional markers. It is shown that the two microvascular cell lines exhibit both common and specific behaviors upon infection. In particular, LSEC and HMEC-1 replicate efficiently the low-passage virus and respond to infection by over-producing inflammatory mediators involved in the cross talk with circulating immune cells. However, direct infection modulates differently the cell surface expression of molecules critically involved in the interactions between endothelial and inflammatory cells. ICAM-1 and HLA-I are up regulated as a consequence of infection in LSEC whereas direct infection results in downregulation of ICAM-1 in HMEC-1. The present results show that infection of human microvascular cells by unadapted dengue virus results in both common and specific activation patterns depending likely on the tissue origin of the cells, thus suggesting that endothelia from different territories may contribute differently to the pathophysiological events in the course of dengue infection.

Dengue type 3 virus, Saint Martin, 2003-2004.

We describe the spread of a dengue virus during an outbreak in Saint Martin island (French West Indies) during winter 2003-2004. Dengue type 3 viruses were isolated from 6 patients exhibiting clinical symptoms. This serotype had not been detected on the island during the preceding 3 years. Genome sequence determinations and analyses showed a common origin with dengue type 3 viruses isolated in Martinique 2 years earlier.

Genetic characterization of newly reintroduced dengue virus type 3 in Martinique (French West Indies).

Dengue type 3 viruses were isolated from patients in Martinique between 1999 and 2002. This serotype had not been detected on the island in the last 20 years. Genomic sequence determination and analysis showed great stability of the virus during the period studied.

[Dengue fever: outbreak in southern Europe?]. / La dengue: bientôt en Europe du Sud?

Epidemiology of dengue fever is changing dramatically. The worldwide incidence is rising and clinical symptoms are worsening. Reports describing forms associated with haemorrhages or shock syndrome involving both children and adults are increasingly frequent in regions beyond Southeast Asia where the first cases were observed. Many mechanisms could be implicated in these changes, including modifications of the virus, host, vector, or socio-economic factors. The new facilities in the laboratory diagnostic (MAC-ELISA; molecular biology), the commercialization of these assays allow not only assessment of morbidity and mortality in endemic areas and early detection of epidemic outbreaks but also evaluation of socio-economic impact and effectiveness of control measures. Now, the efficiency of the fight must be better, otherwise dengue will grow up during this new century.

Evidence for in vitro falsely-primed cDNAs that prevent specific detection of virus negative strand RNAs in dengue-infected cells: improvement by tagged RT-PCR.

The identification of cell types replicating dengue viruses is an important step towards the understanding of the pathophysiology of dengue severe forms. Since the detection of negative strand viral RNAs is the more reliable marker of active replication for single-strand positive sense RNA viruses, we reassessed the specificity of RT-PCR assays already developed to detect dengue negative strand RNAs. Studying mammalian Vero cells infected by a dengue-2 strain, it was shown that falsely-primed cDNAs are generated in vitro during the reverse transcription step and are amplified subsequently by PCR. Since this may compromise the specificity of existing RT-PCR systems, we developed a tagged RT-PCR assay and addressed the role of some critical factors in such a system. Optimization of the negative strand-specific tagged RT-PCR allowed to resolve the problems due to the PCR amplification of falsely-primed cDNAs. Using this assay it was possible to detect specifically negative strand RNAs as soon as 3h after Vero cells have been exposed to the dengue-2 strain and we showed that this system is highly specific. Thus, the present dengue negative strand-specific tagged RT-PCR assay may help to reassess viral replication in the context of dengue pathophysiology.