Dengue Virus Production Research Articles

Dengue virus (DENV) antibody-dependent enhancement of infection upregulates the production of anti-inflammatory cytokines, but suppresses anti-DENV free radical and pro-inflammatory cytokine production, in THP-1 cells

The immunopathogenesis of dengue haemorrhagic fever and dengue shock syndrome is thought to be mediated by a variety of host factors. Enhancing antibodies are one of the key regulating molecules. These antibodies, via antibody-dependent enhancement (ADE) of infection, are able to facilitate dengue virus (DENV) growth in Fc-bearing host cells. The mechanism of ADE-enhanced DENV production is believed to be mediated through increasing the infected-cell mass. In the present work, the effect of ADE infection was explored further, focusing on the post-entry events of ADE infection. It was hypothesized that the higher virus production in ADE infection compared with DENV infection may be due to the ability of this infection pathway to suppress key antiviral molecules. Therefore, the influence of ADE infection on pro- and anti-inflammatory cytokines, including interleukin-12 (IL-12), gamma interferon (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), IL-6 and IL-10, was investigated and it was found that DENV infection via the Fc receptor-mediated pathway was able to suppress the transcription and translation of IL-12, IFN-gamma and TNF-alpha. In contrast, infection via this route facilitated expression and synthesis of the anti-inflammatory cytokines IL-6 and IL-10. Moreover, this study demonstrates that the ADE infection pathway also suppresses an innate anti-DENV mediator, nitric oxide radicals, by disrupting the transcription of the iNOS gene transcription factor, IRF-1, and blocking the activation of STAT-1. In conclusion, ADE infection not only facilitates the entry process, but also modifies innate and adaptive intracellular antiviral mechanisms, resulting in unrestricted DENV replication in THP-1 cells.

The Secreted Form of Dengue Virus Nonstructural Protein NS1 Is Endocytosed by Hepatocytes and Accumulates in Late Endosomes: Implications for Viral Infectivity

The flavivirus nonstructural protein NS1 is expressed as three discrete species in infected mammalian cells: an intracellular, membrane-associated form essential for viral replication, a cell surface-associated form that may be involved in signal transduction, and a secreted form (sNS1), the biological properties of which remain elusive. To determine the distribution of the dengue virus (DEN) sNS1 protein in vivo, we have analyzed by immunohistological means the tissue tropism of purified DEN sNS1 injected intravenously into adult mice. The sNS1 protein was found predominantly associated with the liver, where hepatocytes appeared to represent a major target cell. We further showed that sNS1 could be efficiently endocytosed by human Huh7 and HepG2 hepatocytes in vitro. After its internalization, the protein was detected intracellularly for at least 48 h without being substantially degraded. Colocalization studies of sNS1 with markers of the endolysosomal compartments revealed that the protein was specifically targeted to lysobisphosphatidic acid-rich structures reminiscent of late endosomes, as confirmed by electron microscopy. Intracellular accumulation of sNS1 in Huh7 cells enhanced the fluid phase uptake of rhodamine-labeled dextran. Furthermore, preincubation of Huh7 cells with sNS1 increased dengue virus production after infection with the homologous strain of DEN-1 virus. Our results demonstrate that the accumulation of DEN sNS1 in the late endosomal compartment of hepatocytes potentializes subsequent dengue virus infection in vitro, raising the possibility that sNS1 may contribute to viral propagation in vivo.

Alpha-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum.

We report that endoplasmic reticulum alpha-glucosidase inhibitors have antiviral effects on dengue (DEN) virus. We found that glucosidase inhibition strongly affects productive folding pathways of the envelope glycoproteins prM (the intracellular glycosylated precursor of M [membrane protein]) and E (envelope protein): the proper folding of prM bearing unprocessed N-linked oligosaccharide is inefficient, and this causes delayed formation of prME heterodimer. The complexes formed between incompletely folded prM and E appear to be unstable, leading to a nonproductive pathway. Inhibition of alpha-glucosidase-mediated N-linked oligosaccharide trimming may thus prevent the assembly of DEN virus by affecting the early stages of envelope glycoprotein processing.

Antibody-enhanced dengue virus infection in primate leukocytes

DENGUE viruses, types 1–4, are arthropod-borne flaviviruses which cause dengue shock syndrome (DSS) in humans possessing pre-infection antibody, passively acquired or derived from heterotypic infection1. Although the immuno-pathological mechanism of DSS is not fully understood, experimental studies suggest that dengue virus production is immunologically regulated. Monkeys with monotypic immunity to dengue types 1, 3 or 4 viruses, when challenged with dengue 2, had significantly higher levels of circulating virus than did similarly infected susceptible animals2. This may be attributable to the replication of virus in leukocytes. In infected monkeys, virus was frequently recovered from buffy coat cells and from lymphatic tissues3. The role of leukocytes in enhanced infection is further supported by the observation that dengue replicates readily in cultures of peripheral blood leukocytes (PBL) prepared from immune simian or human donors, but poorly or not at all in leukocytes from non-immune hosts4–6. Studies on the immunological specificity of this phenomenon have been hindered by the requirement either for expensive experimental hosts (monkeys) or for human donors with chronologically defined dengue infections. Here we describe the in vitro enhancement of dengue infection in PBL by antibody. This system provides a provisional model for DSS in young infants during primary dengue infections7,8.