IL-22 modulates IL-17A production and controls inflammation and tissue damage in experimental dengue infection.

Dengue virus (DENV), a mosquito-borne flavivirus, is a public health problem in many tropical countries. IL-22 and IL-17A are key cytokines in several infectious and inflammatory diseases. We have assessed the contribution of IL-22 and IL-17A in the pathogenesis of experimental dengue infection using a mouse-adapted DENV serotype 2 strain (P23085) that causes a disease that resembles severe dengue in humans. We show that IL-22 and IL-17A are produced upon DENV-2 infection in immune-competent mice. Infected IL-22(-/-) mice had increased lethality, neutrophil accumulation and pro-inflammatory cytokines in tissues, notably IL-17A. Viral load was increased in spleen and liver of infected IL-22(-/-) mice. There was also more severe liver injury, as seen by increased transaminases levels and tissue histopathology. γδ T cells and NK cells are sources of IL-17A and IL-22, respectively, in liver and spleen. We also show that DENV-infected HepG2 cells treated with rhIL-22 had reduced cell death and decreased IL-6 production. IL-17RA(-/-) mice were protected upon infection and IL-17A-neutralizing-Ab-treatment partially reversed the phenotype observed in IL-22(-/-) -infected mice. We suggest that disrupting the balance between IL-22 and IL-17A levels may represent an important strategy to reduce inflammation and tissue injury associated with severe dengue infection.

Receptors and routes of dengue virus entry into the host cells.

Dengue is the most prevalent arthropod-borne viral disease, caused by dengue virus, a member of the Flaviviridae family. Its worldwide incidence is now a major health problem, with 2.5 billion people living in risk areas. In this review, we integrate the structural rearrangements of each viral protein and their functions in all the steps of virus entry into the host cells. We describe in detail the putative receptors and attachment factors in mammalian and mosquito cells, and the recognition of viral immunocomplexes via Fcγ receptor in immune cells. We also discuss that virus internalization might occur through distinct entry pathways, including clathrin-mediated or non-classical clathrin-independent endocytosis, depending on the host cell and virus serotype or strain. The implications of viral maturation in virus entry are also explored. Finally, we discuss the mechanisms of viral genome access to the cytoplasm. This includes the role of low pH-induced conformational changes in the envelope protein that mediate membrane fusion, and original insights raised by our recent work that supports the hypothesis that capsid protein would also be an active player in this process, acting on viral genome translocation into the cytoplasm.

Intracellular nucleic acid delivery by the supercharged dengue virus capsid protein.

Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.

A real-time PCR procedure for detection of dengue virus serotypes 1, 2, and 3, and their quantitation in clinical and laboratory samples.

Infectious diseases are a major cause of mortality in the world and, among them, dengue is considered the main human arbovirus. No effective vaccines or antiviral drugs are available for this illness, and it is estimated that 2.5 billion people live at risk, leading to millions of dengue cases annually. The most common method for dengue virus (DENV) quantitation is the plaque assay, but there are DENV strains that do not form plaques. For this reason, a PCR protocol able to detect and quantify DENV in the different kinds of samples employed for DENV study is of great value. In this study, a real-time PCR method suitable not only for clinical objectives but also for laboratory routine is described. Sequences from several strains of DENV comprising the four serotypes were aligned. A fragment located at the 5'UTR region of the virus genome was used to generate the primers and the probe for real-time PCR. This method was successfully used to identify and quantify distinct dengue virus strains and serotypes in clinical samples, in sera from patients infected with dengue virus, and in the mosquito Aedes aegypti, as well as to study virus replication in different cell lines.

Gene expression analysis during dengue virus infection in HepG2 cells reveals virus control of innate immune response.

OBJECTIVES: Liver damage occurs during Dengue Virus infection and constitutes a characteristic of severe forms of the disease. The present study was focused on the modulation of gene expression in a human hepatic cell lineage, HepG2, in response to Dengue Virus infection. METHODS: The global gene expression changes in HepG2 cells after 6, 24 and 48h of infection with Dengue Virus were investigated using a new tool of microarray data analysis and real-time PCR. RESULTS: HepG2 cells infected with Dengue Virus showed alterations in several signaling pathways involved in innate immune response. The analysis of pattern recognition pathways genes demonstrated that TLR3, TLR8, RIG-I and MDA5 mRNAs were up-regulated during Dengue Virus infection along with an increase in the expression of the type I interferon, IFN-beta and pro-inflammatory cytokines IL-6, IL-8 and RANTES genes. CONCLUSIONS: Our results suggest that innate immune pathways are involved in the recognition of Dengue Virus by HepG2 cells. These observations may contribute to the understanding of the inflammatory responses induced by Dengue Virus-hepatocytes interaction during dengue diseases.