Unique Immune Blood Markers Between Severe Dengue and Sepsis in Children.

BACKGROUND: Pediatric dengue and sepsis share clinical and pathophysiologic aspects. Multiple inflammatory and regulatory cytokines, decoy receptors and vascular permeability factors have been implicated in the pathogenesis of both diseases. The differential pattern and dynamic of these soluble factors, and the relationship with clinical severity between pediatric dengue and sepsis could offer new diagnosis and therapeutic strategies. METHODS: We evaluated the concentration levels of 11 soluble factors with proinflammatory, regulatory and vascular permeability involvement, in plasma from children with dengue or sepsis, both clinically ranging from mild to severe, in the early, late and convalescence phases of the disease. RESULTS: During early acute infection, children with sepsis exhibited specific higher concentration levels of IL-6, vascular endothelial growth factor (VEGF), and its soluble decoy receptor II (sVEGFR2) and lower concentration levels of IL-10 and the soluble tumor necrosis factor receptor 2 (sTNFR2), in comparison with children with severe dengue. In addition, the circulating amounts of soluble ST2, and VEGF/sVEGFR2 were widely associated with clinical and laboratory indicators of dengue severity, whereas secondary dengue virus infections were characterized by an enhanced cytokine response, relative to primary infections. In severe forms of dengue, or sepsis, the kinetics and the cytokines response during the late and convalescence phases of the disease also differentiate. CONCLUSIONS: Dengue virus infection and septic processes in children are characterized by cytokine responses of a specific magnitude, pattern and kinetics, which are implicated in the pathophysiology and clinical outcome of these diseases.

Integrative systems biology characterizes immune-mediated neurodevelopmental changes in murine Zika virus microcephaly.

Characterizing perturbation of molecular pathways in congenital Zika virus (ZIKV) infection is critical for improved therapeutic approaches. Leveraging integrative systems biology, proteomics, and RNA-seq, we analyzed embryonic brain tissues from an immunocompetent, wild-type congenital ZIKV infection mouse model. ZIKV induced a robust immune response accompanied by the downregulation of critical neurodevelopmental gene programs. We identified a negative correlation between ZIKV polyprotein abundance and host cell cycle-inducing proteins. We further captured the downregulation of genes/proteins, many of which are known to be causative for human microcephaly, including Eomesodermin/T-box Brain Protein 2 (EOMES/TBR2) and Neuronal Differentiation 2 (NEUROD2). Disturbances of distinct molecular pathways in neural progenitors and post-mitotic neurons may contribute to complex brain phenotype of congenital ZIKV infection. Overall, this report on protein- and transcript-level dynamics enhances understanding of the ZIKV immunopathological landscape through characterization of fetal immune response in the developing brain.

Development and Validation of a Rapid Lateral Flow E1/E2-Antigen Test and ELISA in Patients Infected with Emerging Asian Strain of Chikungunya Virus in the Americas.

Since its 2013 emergence in the Americas, Chikungunya virus (CHIKV) has posed a serious threat to public health. Early and accurate diagnosis of the disease, though currently lacking in clinics, is integral to enable timely care and epidemiological response. We developed a dual detection system: a CHIKV antigen E1/E2-based enzyme-linked immunosorbent assay (ELISA) and a lateral flow test using high-affinity anti-CHIKV antibodies. The ELISA was validated with 100 PCR-tested acute Chikungunya fever samples from Honduras. The assay had an overall sensitivity and specificity of 51% and 96.67%, respectively, with accuracy reaching 95.45% sensitivity and 92.03% specificity at a cycle threshold (Ct) cutoff of 22. As the Ct value decreased from 35 to 22, the ELISA sensitivity increased. We then developed and validated two lateral flow tests using independent antibody pairs. The sensitivity and specificity reached 100% for both lateral flow tests using 39 samples from Colombia and Honduras at Ct cutoffs of 20 and 27, respectively. For both lateral flow tests, sensitivity decreased as the Ct increased after 27. Because CHIKV E1/E2 are exposed in the virion surfaces in serum during the acute infection phase, these sensitive and specific assays demonstrate opportunities for early detection of this emerging human pathogen.

Importancia de las características clínicas y de laboratorio en el diagnóstico de las infecciones concomitantes por dengue y chikungunya: reporte de un caso probable / Importance of clinical and laboratory characteristics in the diagnosis of dengue and chikungunya co-infection: Probable case report

Se presenta el caso de una mujer de 38 años que consultó inicialmente por fiebre indiferenciada. A pesar de que el cuadro clínico evolucionó con manifestaciones clínicas de dengue con signos de alarma y de que la detección de IgM antidengue en una sola muestra indicaba que se trataba de un caso probable que había podido ocurrir durante los tres meses anteriores, la paciente consultó de forma reiterada, pues no presentaba una mejoría significativa. En el décimo día del inicio de los síntomas, se observó edema simétrico en múltiples articulaciones acompañado de dolor, así como lesiones hiperpigmentadas en el surco nasogeniano. Se confirmó el diagnóstico de chikungunya por la presencia de anticuerpos IgM. Aunque puede pasar desapercibida, en los países endémicos para dengue y chikungunya existe la posibilidad de la infección concomitante, la cual puede agravar la evolución clínica de cada una de estas enfermedades. Por ello, es necesario que el médico considere las características clínicas y de laboratorio de ambas enfermedades para diagnosticar su presencia simultánea, garantizar un manejo adecuado y minimizar las complicaciones.

We report the case of a 38-year-old woman who initially consulted for an undifferentiated fever. Although her clinical condition evolved with signs and symptoms compatible with dengue with alarm signs and that the anti-dengue IgM detection in a single sample indicated it was a probable case that could have happened during the previous three months, the patient kept consulting due to little improvement. On the tenth day after the onset of symptoms, she presented with painful polyarticular symmetric edema, as well as hyperpigmented lesions in the nasolabial fold. Chikungunya diagnosis was confirmed by the presence of IgM antibodies. In endemic countries for dengue and chikungunya, the possibility of co-infection exists, but it may go unnoticed. On the other hand, the co-infection may worsen the clinical course of these diseases. Therefore, physicians should evaluate the clinical and laboratory characteristics of both infections to be able to diagnose the coinfection for adequate management and to minimize complications.

AHR is a Zika virus host factor and a candidate target for antiviral therapy.

Zika virus (ZIKV) is a flavivirus linked to multiple birth defects including microcephaly, known as congenital ZIKV syndrome. The identification of host factors involved in ZIKV replication may guide efficacious therapeutic interventions. In genome-wide transcriptional studies, we found that ZIKV infection triggers aryl hydrocarbon receptor (AHR) activation. Specifically, ZIKV infection induces kynurenine (Kyn) production, which activates AHR, limiting the production of type I interferons (IFN-I) involved in antiviral immunity. Moreover, ZIKV-triggered AHR activation suppresses intrinsic immunity driven by the promyelocytic leukemia (PML) protein, which limits ZIKV replication. AHR inhibition suppressed the replication of multiple ZIKV strains in vitro and also suppressed replication of the related flavivirus dengue. Finally, AHR inhibition with a nanoparticle-delivered AHR antagonist or an inhibitor developed for human use limited ZIKV replication and ameliorated newborn microcephaly in a murine model. In summary, we identified AHR as a host factor for ZIKV replication and PML protein as a driver of anti-ZIKV intrinsic immunity.

Clinical, laboratory and immune aspects of Zika virus-associated encephalitis in children.

OBJECTIVE: To evaluate the clinical, laboratory, and immune characteristics of Zika virus (ZIKV)-associated encephalitis in pediatric patients after the epidemic in Huila, southern Colombia. METHODS: A pediatric neuro-surveillance hospital study was conducted in a referral health center in southern Colombia, from October 2016 to October 2017. Cases of encephalitis were confirmed by nucleic acid amplification tests and serological methods in cerebrospinal fluid (CSF), plasma, and/or urine. Levels of six cytokines were evaluated by flow cytometry. Patients underwent daily clinical and laboratory follow-up. RESULTS: Twenty children with probable encephalitis were included for further studies and 16 of them were confirmed. Four cases of bacterial meningoencephalitis (Streptococcus pneumoniae, group B Streptococcus, Staphylococcus epidermidis, and Escherichia coli) and 12 cases of viral encephalitis were identified, six of them associated with ZIKV infection. Other viral encephalitis cases were caused by herpes viruses (n=3), enterovirus (n=2), and dengue virus type 2 (DENV-2; n=1) infections. ZIKV-associated encephalitis symptoms subsided faster than those of patients with encephalitis caused by other agents. CSF analysis revealed lymphocytic pleocytosis. Compared to healthy controls, children with ZIKV-associated encephalitis presented modest plasma interleukin (IL)-10 but not IL-2, IL-4, IL-6, interferon gamma (IFN-γ), or tumor necrosis factor alpha (TNF-α). Cytokine expression was differentially regulated, as dramatically elevated IL-6, IL-10, and IFN-γ levels were observed in CSF but not in paired plasma samples in one of the patients with ZIKV detectable in CSF. CONCLUSIONS: This study provides evidence that ZIKV is responsible for pediatric encephalitis in endemic areas, and the local presence of the virus may induce cephalic but not systemic expression of cytokines.

Molecular characterization of dengue virus reveals regional diversification of serotype 2 in Colombia.

Dengue is hyperendemic in Colombia, where a cyclic behavior of serotype replacement leading to periodic epidemics has been observed for decades. This level of endemicity favors accumulation of dengue virus genetic diversity and could be linked to disease outcome. To assess the genetic diversity of dengue virus type 2 in Colombia, we sequenced the envelope gene of 24 virus isolates from acute cases of dengue or severe dengue fever during the period 2013-2016. The phylogenetic analysis revealed the circulation of the Asian-American genotype of dengue virus type 2 in Colombia during that period, the intra-genotype variability leading to divergence in two recently circulating lineages with differential geographic distribution, as well as the presence of nonsynonymous substitutions accompanying their emergence and diversification.

Actividad en suero de CK y CK-MB en pacientes con infección por el virus dengue / Serum activity of CK and CK-MB in patients with dengue virus infection

RESUMEN Objetivo Determinar la actividad en suero de CK y CK-MB en pacientes con dengue. Métodos Se realizó un estudio de corte en el Departamento de Antioquia. La población de estudio estuvo constituida por 54 pacientes con diagnóstico de dengue y por 10 controles sanos. A todos los participantes se les tomó muestra de suero para confirmar la infección por dengue y para hacer la medición de la actividad de CK y CK-MB. Resultados La mediana de la edad de los casos de dengue fue 18 años y la de los controles fue 28,5 años. La mitad de los pacientes con dengue (50,9 %) y ninguno del grupo control presentaron CK-MB elevada. Ningún paciente presentó miocarditis, sin embargo, se observó CK-MB elevada en 33,3 %, 44,4 % y 40 % de los casos con bradicardia, taquicardia e hipotensión respectivamente. En 29,6 % de los pacientes con dengue se detectó CK elevada, en contraste con 10 % en el grupo control. Se observó actividad de CK en pacientes con dengue con presencia de síntomas como vómito, hematemesis y dolor abdominal, 87,5 %, 60 % y 50 % respectivamente. Conclusiones En este estudio ningún paciente con dengue presentó cardiopatía o miositis, sin embargo, el hecho de encontrar mayor frecuencia de CK y CK-MB elevadas en los pacientes con dengue con respecto al grupo control, sugiere compromiso del músculo estriado y cardíaco en este grupo. Por esta razón sería pertinente el monitoreo de estas enzimas en pacientes con dengue como parte de la evolución de la enfermedad.(AU)

ABSTRACT Objective To determine the serum activity of CK and CK-MB in patients with dengue infection. Methods A cross section study was conducted in the State of Antioquia, Colombia. The study population consisted in 54 patients with diagnosis of dengue infection and 10 healthy controls. A blood sample was taken from all participants to confirm dengue infection and to measure the activity of CK and CK-MB. Results The median age of dengue cases was 18 years and the median age of healthy controls was 28.5 years. Half of dengue patients (50.9 %) had elevated levels of CK-MB, in contrast with the healthy controls in which none presented increase of this enzyme. No patient presented myocarditis; however, elevated CK-MB was observed in 33.3 %, 44.4 % and 40 % of cases with bradycardia, tachycardia and hypotension respectively. In 29.6 % of the dengue patients, high level of CK was detected, in contrast to 10 % in the control group. Activity of CK elevated was observed in dengue patients with symptoms such as vomiting, hematemesis and abdominal pain, 87.5 %, 60 % and 50 %, respectively. Conclusions In this study, no patient with dengue infection had heart disease or myositis; however, the finding of a higher frequency of elevated level CK and CK-MB in the dengue patients compared to the control group suggests the involvement of the striated muscle and of the cardiac muscle in this group. For this reason, the monitoring of these enzymes should be considered as part of the monitoring of patients with dengue.(AU)

Induction of Expansion and Folding in Human Cerebral Organoids.

An expansion of the cerebral neocortex is thought to be the foundation for the unique intellectual abilities of humans. It has been suggested that an increase in the proliferative potential of neural progenitors (NPs) underlies the expansion of the cortex and its convoluted appearance. Here we show that increasing NP proliferation induces expansion and folding in an in vitro model of human corticogenesis. Deletion of PTEN stimulates proliferation and generates significantly larger and substantially folded cerebral organoids. This genetic modification allows sustained cell cycle re-entry, expansion of the progenitor population, and delayed neuronal differentiation, all key features of the developing human cortex. In contrast, Pten deletion in mouse organoids does not lead to folding. Finally, we utilized the expanded cerebral organoids to show that infection with Zika virus impairs cortical growth and folding. Our study provides new insights into the mechanisms regulating the structure and organization of the human cortex.

Biomarcadores pronósticos de gravedad del dengue / Biomarkers for the prognosis of severe dengue

.

Introduction: There are very few strategies for the early detection of the patients who might develop the severe form of the illness. Objective: To evaluate the utility of serum levels of some immune response mediators as early biomarkers for the severe dengue prognosis during the early phase of the illness. Materials and methods: Using a case-control design nested in a multicenter cohort from the AEDES network (a Colombian multicenter study), we compared TNF a, ST2, TRAIL and IDO levels in samples which were obtained during the early phase of the illness. Results: ST2, TRAIL and TNF a levels were higher in severe dengue patients compared with uncomplicated patients (p<0.0001), as follows: OR=24.8, CI95%= 6.1- 98.0; OR=18.0, CI95%= 4.6-69.1; OR=NC, CI95%= NC, respectively. We did not find statistically significant differences between IDO levels in severe dengue and uncomplicated dengue (p=1.000, OR=1.0, CI95%= 0.2-6.1). Conclusions: In the early phase of the dengue infection (96 hours), ST2, TRAIL and TNF a quantifications could contribute to the prediction of complications of the illness.

Dengue virus type 3 adaptive changes during epidemics in São Jose de Rio Preto, Brazil, 2006-2007.

Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of São José de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak.

High plasma levels of soluble ST2 but not its ligand IL-33 is associated with severe forms of pediatric dengue.

Identification of early determinants of dengue disease progression, which could potentially enable individualized patient care are needed at present times. Soluble ST2 (sST2) has been recently reported to be elevated in the serum of children older than 2 years old and adults with dengue infection and it was correlated with secondary infections as well as with severe presentations of the disease. The mechanism by which secreted ST2 is linked to severe dengue and plasma leakage remains unclear. One possibility is that IL-33 ligand may be elevated, contributing to membrane bound ST2 as part of the immune activation in dengue infection. We determined plasma levels of sST2 and the ligand IL-33 in 66 children with acute secondary dengue infections clinically classified using the guidelines of the World Health Organization, 2009. Dengue infection showed significant increases in cytokines IL-12p70, IL-10, IL-8, IL-6, IL-1ß and TNFα measured by flow cytometry based assay compared to uninfected individuals. In contrast, IL-33 levels remained unchanged between infected and uninfected individuals. The levels of sST2 positively correlated with values of IL-6 and IL-8 and inversely correlated with number of median value of platelet levels. In addition to circulating cytokine positive correlations we found that sST2 and isoenzyme creatine kinase-MB (CK-MB), a marker of myocardial muscle damage present in severe dengue cases were associated. Our pediatric study concluded that in dengue infections sST2 elevation does not involve concomitant changes of IL-33 ligand. We propose a study to assess its value as a predictor factor of disease severity.

[Biomarkers for the prognosis of severe dengue]. / Biomarcadores pronósticos de gravedad del dengue.

INTRODUCTION: There are very few strategies for the early detection of the patients who might develop the severe form of the illness. OBJECTIVE: To evaluate the utility of serum levels of some immune response mediators as early biomarkers for the severe dengue prognosis during the early phase of the illness. MATERIALS AND METHODS: Using a case-control design nested in a multicenter cohort from the AEDES network (a Colombian multicenter study), we compared TNF a, ST2, TRAIL and IDO levels in samples which were obtained during the early phase of the illness. RESULTS: ST2, TRAIL and TNF a levels were higher in severe dengue patients compared with uncomplicated patients (p<0.0001), as follows: OR=24.8, CI95%= 6.1- 98.0; OR=18.0, CI95%= 4.6-69.1; OR=NC, CI95%= NC, respectively. We did not find statistically significant differences between IDO levels in severe dengue and uncomplicated dengue (p=1.000, OR=1.0, CI95%= 0.2-6.1). CONCLUSIONS: In the early phase of the dengue infection (96 hours), ST2, TRAIL and TNF a quantifications could contribute to the prediction of complications of the illness.

Serological detection of West Nile virus in horses and chicken from Pantanal, Brazil

In an effort to detect West Nile virus (WNV) in Brazil, we sampled serum from horses and chickens from the Pantanal region of the state of Mato Grosso and tested for flavivirus-reactive antibodies by blocking ELISA. The positive samples were further confirmed for serological evidence of WNV infection in three (8%) of the 38 horses and one (3.2%) of the 31 chickens using an 80% plaque-reduction neutralisation test (PRNT80). These results provide evidence of the circulation of WNV in chickens and horses in Pantanal.

CD8+ T cells use TRAIL to restrict West Nile virus pathogenesis by controlling infection in neurons.

Previous studies of mice have demonstrated that an orchestrated sequence of innate and adaptive immune responses is required to control West Nile virus (WNV) infection in peripheral and central nervous system (CNS) tissues. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; also known as CD253) has been reported to inhibit infection with dengue virus, a closely related flavivirus, in cell culture. To determine the physiological function of TRAIL in the context of flavivirus infection, we compared the pathogenesis of WNV in wild-type and TRAIL(-/-) mice. Mice lacking TRAIL showed increased vulnerability and death after subcutaneous WNV infection. Although no difference in viral burden was detected in peripheral tissues, greater viral infection was detected in the brain and spinal cord at late times after infection, and this was associated with delayed viral clearance in the few surviving TRAIL(-/-) mice. While priming of adaptive B and T cell responses and trafficking of immune and antigen-specific cells to the brain were undistinguishable from those in normal mice, in TRAIL(-/-) mice, CD8(+) T cells showed qualitative defects in the ability to clear WNV infection. Adoptive transfer of WNV-primed wild-type but not TRAIL(-/-) CD8(+) T cells to recipient CD8(-/-) mice efficiently limited infection in the brain and spinal cord, and analogous results were obtained when wild-type or TRAIL(-/-) CD8(+) T cells were added to WNV-infected primary cortical neuron cultures ex vivo. Collectively, our results suggest that TRAIL produced by CD8(+) T cells contributes to disease resolution by helping to clear WNV infection from neurons in the central nervous system.

Comparación de los perfiles de transcripción de pacientes con fiebre de dengue y fiebre hemorrágica por dengue que muestra diferencias en la respuesta inmunitaria y claves en la inmunopatogénesis / Comparison of the transcriptional profiles of patients with dengue fever and dengue hemorrhagic fever reveals differences in the immune response and clues in immunopathogenesis

Introducción. El dengue puede manifestarse como una enfermedad leve o evolucionar hasta una enfermedad grave, llamada fiebre hemorrágica por dengue, cuyos mecanismos de inmunopatogénesis no son claros. Objetivo. Utilizar un análisis de microarreglos para identificar los genes de la respuesta inmunitaria diferencialmente expresados en niños colombianos con dengue leve y grave. Materiales y métodos. Se evaluaron los cambios de la expresión génica de células mononucleares de sangre periférica de niños con fiebre de dengue y fiebre hemorrágica por dengue en fase aguda, mediante el microarreglo de Affymetrix HG-U133_Plus_2. Resultados. Los pacientes con fiebre hemorrágica por dengue expresaron transcritos para interleucina 6, quimiocinas, complemento y pentraxina 3, al igual que inhibidores de la actividad de linfocitos (gen 3 de activación de linfocitos y catepsina L1). Un modelo de interacción desarrollado para estos genes mostró al factor tisular como central en la red generada. Por el contrario, los pacientes con fiebre de dengue expresaron inhibidores de la actividad de citocinas, complemento y leucotrienos lactotransferrina, inhibidor peptidasa serpina del complemento C1, leucotrieno B (4-omega hidroxilasa 2). Conclusiones. Los resultados podrían indicar que durante la fiebre de dengue, los inhibidores de citocinas y del complemento logran controlar el daño al endotelio y el aumento de la permeabilidad vascular, mientras que, en los pacientes con fiebre hemorrágica por dengue, la disfunción de las células inmunitarias y la acción no regulada del complemento y de las citocinas, conducen a un estado de “hipercoagulacion” y daño endotelial. La identificación del papel patógeno de las moléculas encontradas podría contribuir a la interpretación de la patogenia y al desarrollo de fármacos terapéuticos. Palabras clave: dengue, transcripción genética, análisis de micromatrices, fiebre hemorrágica del dengue, proteínas del sistema del complemento, citocinas.

Levels of soluble ST2 in serum associated with severity of dengue due to tumour necrosis factor alpha stimulation.

The interleukin-1 receptor-like-1 protein (IL1RL1), also known as ST2, has been shown previously to regulate T-cell function and is produced by T cells and endothelial cells. It was reported recently to be elevated in mild dengue patients during acute disease. The ST2 gene encodes several splice products: L (long), V (short) and s (soluble). A cohort of 38 patients with dengue haemorrhagic fever (DHF) and mild dengue fever (DF) were evaluated using a secreted soluble ST2 (sST2) ELISA. The RNA expression of ST2 was evaluated by real-time quantitative RT-PCR using patients' peripheral blood mononuclear cells (PBMCs) and in vitro using human umbilical vein endothelial cells (HUVECs) exposed to sera from dengue patients. DHF patients had higher levels of serum sST2, tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-8 and IL-10 compared with DF patients and normal healthy control individuals. However, viraemia was indistinguishable between mild and severe cases. No changes in ST2 mRNA expression were found in PBMCs from these two groups of dengue patients. In vitro, sST2 was elevated in HUVECs treated with patient sera. Neutralization of TNF-alpha in patient sera by pre-treatment with a TNF-alpha antibody inhibited the upregulation of sST2 expression in HUVECs. These results implicate serum TNF-alpha in the modulation of expression of sST2 in an in vitro system, and indicate that sST2 could be associated with the severity of disease. Further studies to determine whether sST2 levels are predictive of the severe form of the disease and the role of sST2 in immune regulation are warranted.

Gene expression profiling of dengue infected human primary cells identifies secreted mediators in vivo.

We used gene expression profiling of human primary cells infected in vitro with dengue virus (DENV) as a tool to identify secreted mediators induced in response to the infection. Affymetrix GeneChip analysis of human primary monocytes, B cells and dendritic cells infected with DENV in vitro showed strong induction of monocyte chemotactic protein 2 (MCP-2/CCL8), interferon gamma-induced protein 10 (IP-10/CXCL10) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10). The expression of these genes was confirmed in dendritic cells infected with DENV in vitro at mRNA and protein levels. A prospectively enrolled cohort of DENV-infected Venezuelan patients was used to measure the levels of these proteins in serum during three different periods of the disease. Results showed significant increase of MCP-2, IP-10, and TRAIL levels in patients infected with DENV during the febrile period, when compared to healthy donors and patients with other febrile illnesses. MCP-2 and IP-10 levels were still elevated during the post-febrile period while TRAIL levels dropped close to normal after defervescense. Patients with primary infections had higher TRAIL levels than patients with secondary infections during the febrile period of the disease. Increased levels of IP-10, TRAIL and MCP-2 in acute DENV infections suggest a role for these mediators in the immune response to the infection. MCP-2 was identified in this work as a new unreported and important dengue-related protein and IP-10 was confirmed as a novel and strong pro-inflammatory marker in acute disease.

Efficient dengue virus (DENV) infection of human muscle satellite cells upregulates type I interferon response genes and differentially modulates MHC I expression on bystander and DENV-infected cells.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes an acute febrile disease in humans, characterized by musculoskeletal pain, headache, rash and leukopenia. The cause of myalgia during DENV infection is still unknown. To determine whether DENV can infect primary muscle cells, human muscle satellite cells were exposed to DENV in vitro. The results demonstrated for the first time high-efficiency infection and replication of DENV in human primary muscle satellite cells. Changes in global gene expression were also examined in these cells following DENV infection using Affymetrix GeneChip analysis. The differentially regulated genes belonged to two main functional categories: cell growth and development, and antiviral type I interferon (IFN) response genes. Increased expression of the type I IFN response genes for tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), melanoma-derived antigen 5 (MDA-5), IFN-gamma-inducible protein 10 (IP-10), galectin 3 soluble binding protein (LGals3BP) and IFN response factor 7 (IRF7) was confirmed by quantitative RT-PCR. Furthermore, higher levels of cell-surface-bound intracellular adhesion molecule-1 (ICAM-1) and soluble ICAM-1 in the cell-culture medium were detected following DENV infection. However, DENV infection impaired the ability of the infected cells in the culture medium to upregulate cell-surface expression of MHC I molecules, suggesting a possible mechanism of immune evasion by DENV. The findings of this study warrant further clinical research to identify whether muscle cells are targets for DENV infection during the acute stage of the disease in vivo.