Dengue Virus Type 3 Isolated from a Fatal Case with Visceral Complications Induces Enhanced Proinflammatory Responses and Apoptosis of Human Dendritic Cells

Abstract

. Strain DENV3/5532 was found to display significantly higher replicative ability than DENV3/ 290 in monocyte-derived dendritic cells (mdDCs). In addition, compared to DENV3/290 results, mdDCs exposed to DENV3/5532 showed increased production of proinflammatory cytokines associated with higher rates of programmed cell death, as shown by annexin V staining. The observed phenotype was due to viral replication, and tumor necrosis factor alpha (TNF-␣) appears to exert a protective effect on virus-induced mdDC apoptosis. These results suggest that the DENV3/5532 strain isolated from the fatal case replicates within human dendritic cells, modulating cell survival and synthesis of inflammatory mediators.

Immature Dendritic Cells Generated from Cryopreserved Human Monocytes Show Impaired Ability to Respond to LPS and to Induce Allogeneic Lymphocyte Proliferation

“…Peripheral blood was obtained from healthy donors who gave written informed consent to participate. CD14 + cells were purified and induced to differentiate into imdDCs as previously described [15].…”

“…CD14 + cells were thawed by incubation for 1 to 2 min at 37°C in a water bath, and washed with iFBS by centrifugation at 300× g for 10 min. Fresh or cryopreserved CD14 + cells (5×10 5 viable cells due to Trypan Blue exclusion assay) were incubated in RPMI medium (Lonza, Walkersville, USA) supplemented with 10% iFBS, 100 IU/mL of penicillin, 100 µg/mL of streptomycin, 2 mM of L-Glutamine, 2.5 µg/mL of amphotericin (Gibco-BRL, Grand Island, NY), 100 ng/mL IL-4 and 50 ng/mL GM-CSF (PeproTech, Rocky Hill, NJ) for 6 to 7 days, as previously described [15]. The expression of imdDCs markers (CD11c and HLA-DR) and CD14 (PE, PercP and FITC conjugated antibodies, respectively; BD Pharmigen™, USA) were evaluated in frh- and crp-imdDCs by flow cytometry in a FACSAria II (Becton & Dickinson, San Jose, CA), as described by Silveira et al (2011).…”

Cytokines and Viral Hemorrhagic Fever: Potential for Therapeutic Intervention

“…The genotype of the strain has been shown to be important in disease severity. The more virulent strains of dengue are able to replicate faster and to higher titers, thus increasing the rate of cell death and production of the associated pro-inflammatory cytokines [50]. Host genetics have also been shown to play a role in disease severity.…”

Single point mutations in the helicase domain of the NS3 protein enhance dengue virus replicative capacity in human monocyte-derived dendritic cells and circumvent the type I interferon response

“…Viral RNA was purified from the supernatant of the infected cells using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA). After the viral inocula were removed, the cells were cultured in RPMI-1640 plus 10% FBS, 100 IU/ml penicillin, 100 mg/ml streptomycin, 0Á25 mg/ml amphotericin B and 2 mM L-glutamine (GIBCO-BRL) [36]. The cells were then collected at 8, 24 and 48 hpi, and total RNA was extracted using the RNeasy Mini Kit (Qiagen), according to the manufacturer's recommendations.…”

“…Melting curves were used to verify product specificity. The housekeeping gene 18S was also included to normalize the amplification reaction [36]. Gene modulation was calculated by adapting the 2 2DDCT method described by Livak et al in…”