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-c), tumour necrosis factor alpha (TNF-a), 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-c and TNF-a. 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. INTRODUCTIONDengue virus (DENV) has become a mosquito-borne virus of immense global health importance, as the disease caused by DENV is now endemic in more than 100 countries, particularly in Asia and Latin America, and about 50 million cases of DENV infection are estimated to occur annually worldwide (Ligon, 2005). DENVs, members of the family Flaviviridae, have positive, single-stranded RNA and belong to four related serotypes, known as DENV-1, -2, -3 and -4. DENV induces a wide range of clinical manifestations and the majority of infected patients experience uncomplicated dengue fever (DF), which is an acute febrile illness typically lasting 3-7 days. Dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) are plasma-leakage syndromes that represent life-threatening manifestations of DENV infection. DHF/DSS can be distinguished from DF by three primary criteria: namely, defects in vascular permeability, haemostatic abnormalities with marked thrombocytopenia and bleeding diathesis (da Fonseca & Fonseca, 2002;Rothman, 2004).Determinants that predispose infected patients to develop DHF have been partially identified (Lei et al., 2001). Various types of cell, such as monocytes and macrophages, dendritic cells, mast cells and hepatocytes, are able to support the replication of DENV (Jindadamrongwech et al., 2004;Kliks e...

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In the present study, 10 clinical isolates of dengue virus were selected according to their susceptibility to the inhibitory effect of nitric oxide radical, NO. Five of them are nitric oxide-susceptible viruses while the other five are nitric oxide-resistant viruses. These isolates were investigated to identify genetic factors that are responsible for the different phenotypes. Due to the evidence showing that NO suppresses DENV RNA polymerase activity, we, therefore, hypothesized that the RdRp domain of NS5 may responsible for NO inhibition. To answer this question, sequences of NS5 gene of NO-susceptible viruses and NO-resistant viruses were compared. We found that these two groups of viruses contain different amino acid sequence at position 621 to 646 in the active site of NS5. These data suggested that response to the inhibitory effect of nitric oxide radical may, at least in part, be regulated by NS5. The effect of these two different phenotypes of viruses on host cells was studied using cDNA array screening. The cDNA array analysis demonstrated that the nitric oxide-resistant group had a stronger influence on host cells since it induced changes in the expression of a greater number of genes than did the nitric oxide-susceptible group, 97 genes versus 71 genes, respectively. The NO-resistant virus also stimulated cytokines known to be virulent factors, such as IL 6, IL 8, RANTES, and the inflammatory factors. In conclusion, our data demonstrated that dengue viruses isolated from patients show genotypic and phenotypic differences which may correlate with virulence.

MYCN amplification is the strongest predictor of high-risk neuroblastoma (NB). The standard procedure to detect MYCN status requires invasive procedures. Extracellular vesicles (EVs) contain molecular signatures of originated cells, present in biofluids, and serve as an invaluable source for cancer liquid biopsies. This study aimed to establish an EV-based method to detect the MYCN status of NB. Two EV subtypes, i.e., microvesicles (MVs) and exosomes, were sequentially isolated from the culture supernatant by step-wise centrifugation, ultrafiltration, and size-exclusion chromatography. Quantitative RT-PCR was performed to detect MYCN mRNA. As a result, MYCN mRNA was detectable in the MVs, but not exosomes, of MYCN-amplified NB cells. MYCN mRNA-containing MVs (MYCN-MV) were successfully detected in three distinct MYCN-amplified NB cell lines but absent in three MYCN non-amplification cells. The simulated samples were prepared by pulsing MVs into human serum. MYCN–MV detection in the simulated samples showed a less interfering effect from the human blood matrix. Validation using clinical specimens (2 mL bone marrow plasma) obtained from patients at various disease stages showed a promising result. Five out of six specimens of MYCN-amplified patients showed positive results, while there were no false positives in four plasma samples of the MYCN non-amplification group. This study communicated a novel EV-based method for detecting the MYCN status of pediatric NB based on MYCN mRNA contents in MVs. Future studies should be pursued in a prospective cohort to determine its true diagnostic performance.