Negative regulation of type I interferon signaling by integrin-linked kinase permits dengue virus replication.

Dengue virus (DENV) infection can induce life-threatening dengue hemorrhagic fever/dengue shock syndrome in infected patients. DENV is a threat to global health due to its growing numbers and incidence of infection in the last 50 years. During infection, DENV expresses ten structural and nonstructural proteins modulating cell responses to benefit viral replication. However, the lack of knowledge regarding the cellular proteins and their functions in enhancing DENV pathogenesis impedes the development of antiviral drugs and therapies against fatal DENV infection. Here, we identified that integrin-linked kinase (ILK) is a novel enhancing factor for DENV infection by suppressing type I interferon (IFN) responses. Mechanistically, ILK binds DENV NS1 and NS3, activates Akt and Erk, and induces NF-κB-driven suppressor of cytokine signaling 3 (SOCS3) expression. Elevated SOCS3 in DENV-infected cells inhibits phosphorylation of STAT1/2 and expression of interferon-stimulated genes (ISGs). Inhibiting ILK, Akt, or Erk activation abrogates SOCS3 expression. In DENV-infected mice, the treatment of an ILK inhibitor significantly reduces viral loads in the brains, disease severity, and mortality rate. Collectively, our results show that ILK is a potential therapeutic target against DENV infection.

A novel chimeric dengue vaccine candidate composed of consensus envelope protein domain III fused to C-terminal-modified NS1 protein.

There is an urgent need for a safe and effective vaccine against dengue virus (DENV) which infects about 390 million humans per year. In the present study we combined modifications of two DENV proteins, the nonstructural protein 1 (NS1) and the envelope (E) protein, to produce a DENV vaccine candidate with enhanced features. One of these modified proteins was a C-terminal-deleted fragment of NS1 called ΔC NS1 which we have shown previously to be protective without the potentially harmful effects of cross-reactive epitopes common to surface antigens on platelets and endothelial cells. The other modified protein was an envelope protein domain III (cEDIII) containing a consensus amino acid sequence among the four serotypes of DENV, which induces neutralizing antibody against all four DENV serotypes. The cEDIII and ΔC NS1 were expressed as a fusion protein cEDIII-ΔC NS1 and its protective effects against DENV were evaluated in a mouse model. C3H/HeN mice were immunized three times with cEDIII-ΔC NS1 fusion protein mixed with alum as adjuvant. Sera collected from cEDIII-ΔC NS1-immunized mice neutralized four serotypes of DENV and also caused complement-mediated cytolysis of HMEC-1 cells infected with each of the four different DENV serotypes. Mice immunized with cEDIII-ΔC NS1 and challenged with DENV showed reduced serum virus titer, soluble NS1 and bleeding time, compared with mice infected with DENV alone. The results reveal that antibodies induced by cEDIII-ΔC NS1 not only show anti-viral efficacy by in vitro assays but also provide protective effects against DENV infection in a mouse model. The cEDIII-ΔC NS1 thus represents a novel, effective DENV vaccine candidate.

Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage.

Dengue virus (DENV) infection, the most common mosquito-transmitted viral infection, can cause a range of diseases from self-limiting dengue fever to life-threatening dengue hemorrhagic fever and shock syndrome. Thrombocytopenia is a major characteristic observed in both mild and severe dengue disease and is significantly correlated with the progression of dengue severity. Previous studies have shown that DENV nonstructural protein 1 (NS1), which can be secreted into patients' blood, can stimulate immune cells via Toll-like receptor 4 (TLR4) and can cause endothelial leakage. However, it is unclear whether DENV NS1 can directly induce platelet activation or cause thrombocytopenia during DENV infection. In this study, we first demonstrated that DENV but not Zika virus cell culture supernatant could induce P-selectin expression and phosphatidylserine (PS) exposure in human platelets, both of which were abolished when NS1 was depleted from the DENV supernatant. Similar results were found using recombinant NS1 from all four serotypes of DENV, and those effects were blocked in the presence of anti-NS1 F(ab')2, anti-TLR4 antibody, a TLR4 antagonist (Rhodobacter sphaeroides lipopolysaccharide, LPS-Rs) and a TLR4 signaling inhibitor (TAK242), but not polymyxin B (an LPS inhibitor). Moreover, the activation of platelets by DENV NS1 promoted subthreshold concentrations of adenosine diphosphate (ADP)-induced platelet aggregation and enhanced platelet adhesion to endothelial cells and phagocytosis by macrophages. Finally, we demonstrated that DENV-induced thrombocytopenia and hemorrhage were attenuated in TLR4 knockout and wild-type mice when NS1 was depleted from DENV supernatant. Taken together, these results suggest that the binding of DENV NS1 to TLR4 on platelets can trigger its activation, which may contribute to thrombocytopenia and hemorrhage during dengue infection.

Minocycline suppresses dengue virus replication by down-regulation of macrophage migration inhibitory factor-induced autophagy.

Dengue virus (DENV) infection is the most prevalent mosquito-borne viral infection of which there is no licensed therapeutic drug available. Previous studies have shown that minocycline, an antibiotic, can inhibit DENV infection in vitro. However, the mechanism is not fully understood. It is known that macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is involved in dengue disease development; MIF can induce autophagy, and autophagy can facilitate DENV replication. Therefore, we tested the hypothesis that MIF-induced autophagy is involved in minocycline treatment against DENV infection. We first showed that DENV infection induced MIF secretion and autophagy flux in HuH-7 cells. Suppression of endogenous MIF by short hairpin RNA (shRNA) and inhibition of MIF by its inhibitors attenuated DENV replication and autophagy formation. In addition, minocycline treatment suppressed DENV-induced MIF secretion and autophagy in vitro. Finally, we demonstrated that minocycline treatment attenuated viral load, MIF secretion, autophagy and increase survival in DENV-infected mice. These results suggest that inhibition of MIF-induced autophagy by minocycline might represent an alternative therapeutic approach against DENV infection.

Dengue virus-induced ER stress is required for autophagy activation, viral replication, and pathogenesis both in vitro and in vivo.

Dengue virus (DENV) utilizes the endoplasmic reticulum (ER) for replication and assembling. Accumulation of unfolded proteins in the ER lumen leads to ER stress and unfolded protein response (UPR). Three branches of UPRs temporally modulated DENV infection. Moreover, ER stress can also induce autophagy. DENV infection induces autophagy which plays a promotive role in viral replication has been reported. However, the role of ER stress in DENV-induced autophagy, viral titer, and pathogenesis remain unclear. Here, we reveal that ER stress and its downstream UPRs are indispensable for DENV-induced autophagy in various human cells. We demonstrate that PERK-eIF2α and IRE1α-JNK signaling pathways increased autophagy and viral load after DENV infection. However, ATF6-related pathway showed no effect on autophagy and viral replication. IRE1α-JNK downstream molecule Bcl-2 was phosphorylated by activated JNK and dissociated from Beclin 1, which playing a critical role in autophagy activation. These findings were confirmed as decreased viral titer, attenuated disease symptoms, and prolonged survival rate in the presence of JNK inhibitor in vivo. In summary, we are the first to reveal that DENV2-induced ER stress increases autophagy activity, DENV replication, and pathogenesis through two UPR signaling pathways both in vitro and in vivo.

Honeysuckle aqueous extract and induced let-7a suppress dengue virus type 2 replication and pathogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Honeysuckle (Lonicera japonica Thunb.), a traditional Chinese herb, has widely been used to treat pathogen infection. However, the underlying-mechanism remains elusive. AIMS OF THE STUDY: To reveal the host microRNA (miRNA) profile with the anti-viral activity after honeysuckle treatment. MATERIALS AND METHODS: Here we reveal the differentially expressed miRNAs by Solexa® deep sequencing from the blood of human and mice after the aqueous extract treatment. Among these overexpressed innate miRNAs both in human and mice, let-7a is able to target the NS1 region (nt 3313-3330) of dengue virus (DENV) serotypes 1, 2 and 4 predicated by the target predication software. RESULTS: We confirmed that let-7a could target DENV2 at the predicated NS1 sequence and suppress DENV2 replication demonstrated by luciferase-reporter activity, RT-PCR, real-time PCR, Western blotting and plaque assay. ICR-suckling mice consumed honeysuckle aqueous extract either before or after intracranial injection with DENV2 showed decreased levels of NS1 RNA and protein expression accompanied with alleviated disease symptoms, decreased virus load, and prolonged survival time. Similar results were observed when DENV2-infected mice were intracranially injected with let-7a. CONCLUSION: We reveal that honeysuckle attenuates DENV replication and related pathogenesis in vivo through induction of let-7a expression. This study opens a new direction for prevention and treatment of DENV infection through induction of the innate miRNA let-7a by honeysuckle.

In vitro Assays for Measuring Endothelial Permeability byTranswells and Electrical Impedance Systems.

Vascular leakage is an important feature in several diseases, such as septic shock, viral hemorrhagic fever, cancer metastasis and ischemia-reperfusion injuries. Thus establishing assays for measuring endothelial permeability will provide insight into the establishment or progression of such diseases. Here, we provide transwell permeability assay and electrical impedance sensing assay for studying endothelial permeability in vitro. With these methods, the effect of a molecule on endothelial permeability could be defined.

Anti-dengue virus nonstructural protein 1 antibodies contribute to platelet phagocytosis by macrophages.

Thrombocytopenia is an important clinical manifestation of dengue disease. The hypotheses concerning the pathogenesis of thrombocytopenia include decreased production and increased destruction or consumption of platelets. We previously suggested a mechanism of molecular mimicry in which antibodies (Abs) directed against dengue virus (DENV) nonstructural protein 1 (NS1) cross-react with platelets. Furthermore, several lines of evidence show activation of endothelial cells (ECs) and macrophages are related to dengue disease severity. Previous studies also suggested that Ab-opsonised platelets facilitate the engulfment of platelets by macrophages. Here we show that TNF-α-activated ECs upregulate adhesion molecule expression to enhance the binding of platelets and macrophages and lead to anti-DENV NS1 Ab-mediated platelet phagocytosis. We further demonstrate that the interaction between macrophages and TNF-α-activated ECs requires binding of FcγR with the Fc region of platelet-bound anti-DENV NS1 Abs. Importantly, the binding of anti-DENV NS1 Abs to platelets did not interfere with platelet adhesion to ECs. The adhesion molecules ICAM-1 and ß3 integrin expressed on ECs as well as the FcγR expressed on macrophages were critical in anti-DENV NS1 Ab-mediated platelet phagocytosis on activated ECs. Moreover, anti-DENV NS1 Abs dramatically enhanced platelet engulfment by macrophages in a murine model of DENV infection. Our study provides evidence for a novel role for anti-DENV NS1 Abs in the pathogenesis of thrombocytopenia in dengue disease by enhancing platelet phagocytosis by macrophages.

Macrophage migration inhibitory factor triggers chemotaxis of CD74+CXCR2+ NKT cells in chemically induced IFN-γ-mediated skin inflammation.

IFN-γ mediates chemically induced skin inflammation; however, the mechanism by which IFN-γ-producing cells are recruited to the sites of inflammation remains undefined. Secretion of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, from damaged cells may promote immune cell recruitment. We hypothesized that MIF triggers an initial step in the chemotaxis of IFN-γ-producing cells in chemically induced skin inflammation. Using acute and chronic models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mouse ears, MIF expression was examined, and its role in this process was investigated pharmacologically. The cell populations targeted by MIF, their receptor expression patterns, and the effects of MIF on cell migration were examined. TPA directly caused cytotoxicity accompanied by MIF release in mouse ear epidermal keratinocytes, as well as in human keratinocytic HaCaT cells. Treatment with the MIF antagonist (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester considerably attenuated TPA-induced ear swelling, leukocyte infiltration, epidermal cell proliferation, and dermal angiogenesis. Inhibition of MIF greatly diminished the dermal infiltration of IFN-γ(+) NKT cells, whereas the addition of exogenous TPA and MIF to NKT cells promoted their IFN-γ production and migration, respectively. MIF specifically triggered the chemotaxis of NKT cells via CD74 and CXCR2, and the resulting depletion of NKT cells abolished TPA-induced skin inflammation. In TPA-induced skin inflammation, MIF is released from damaged keratinocytes and then triggers the chemotaxis of CD74(+)CXCR2(+) NKT cells for IFN-γ production.

Propolis inhibits TGF-ß1-induced epithelial-mesenchymal transition in human alveolar epithelial cells via PPARγ activation.

Emerging evidence suggests that the transforming growth factor (TGF)-ß1-induced epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to airway remodeling in severe asthma and fibrotic lung diseases. Studies have shown that extracts from propolis protect chemical-induced cardiac and liver fibrosis in animals. This study assesses the inhibitory effect of propolis on TGF-ß1-induced EMT in serum-deprived A549 cells (human AECs). Experimental results show progressive cell morphological changes, decreased E-cadherin, increased N-cadherin production, intracellular F-actin rearrangement, increased reactive oxygen species (ROS) production, and increased cell motility with increasing TGF-ß1 concentration. A549 cells pretreated with propolis and then treated with TGF-ß1 for 24 h regained epithelial cell morphology, decreased the production of N-cadherin and ROS, and had reduced motility. Propolis prevents the effects of TGF-ß1-induced Smad2 and AKT activation pathways and Snail expression. Moreover, propolis pretreatment may prevent the TGF-ß1-induced down-regulation of nuclear hormone receptors and peroxisome proliferator-activated receptor gamma (PPARγ) protein in A549 cells, whose effect was blocked by adding PPARγ antagonist, GW9662. Two active components of propolis, caffeic acid phenethyl ester (CAPE) and pinocembrin (PIN), only had partial effects on TGF-ß1-induced EMT in A549 cells. The results of this study suggest that natural propolis extracts may prevent TGF-ß1-induced EMT in immortalized type II AECs via multiple inhibitory pathways, which may be clinically applied in the prevention and/or treatment of EMT-related fibrotic diseases as well as airway remodeling in chronic asthma.

Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation.

Autophagy is an evolutionarily conserved catabolic process that maintains cellular homeostasis under stress conditions such as starvation and pathogen infection. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that plays important roles in inflammation and tumorigenesis. Cytokines such as IL-1ß and TNF-α that are induced by MIF have been shown to be involved in the induction of autophagy. However, the actual role of MIF in autophagy remains unclear. Here, we have demonstrated that incubation of human hepatoma cell line HuH-7 cells with recombinant MIF (rMIF) induced reactive oxygen species (ROS) production and autophagy formation, including LC3-II expression, LC3 punctae formation, autophagic flux, and mitochondria membrane potential loss. The autophagy induced by rMIF was inhibited in the presence of MIF inhibitor, ISO-1 as well as ROS scavenger N-acetyl-L-cysteine (NAC). In addition, serum starvation-induced MIF release and autophagy of HuH-7 cells were partly blocked in the presence of NAC. Moreover, diminished MIF expression by shRNA transfection or inhibition of MIF by ISO-1 decreased serum starvation-induced autophagy of HuH-7 cells. Taken together, these data suggest that cell autophagy was induced by MIF under stress conditions such as inflammation and starvation through ROS generation.

Macrophage migration inhibitory factor induced by dengue virus infection increases vascular permeability.

Dengue virus (DENV) infection can cause mild dengue fever or severe dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Serum levels of the macrophage migration inhibitory factor (MIF) have been shown to be correlated with severity and mortality in DENV patients, but the pathogenic roles of MIF in DHF/DSS are still unclear. Increase in vascular permeability is an important hallmark of DHF/DSS. In this study, we found that DENV infection of the human hepatoma cell line (Huh 7) induced MIF production. Conditioned medium collected from DENV-infected Huh 7 cells enhanced the permeability of the human endothelial cell line (HMEC-1) which was reduced in the presence of a MIF inhibitor, ISO-1 or medium from DENV-infected MIF knockdown Huh 7 cells. To further identify whether MIF can alter vascular permeability, we cloned and expressed both human and murine recombinant MIF (rMIF) and tested their effects on vascular permeability both in vitro and in vivo. Indirect immunofluorescent staining showed that the tight junction protein ZO-1 of HMEC-1 was disarrayed in the presence of rMIF and partially recovered when cells were treated with ISO-1 or PI3K/MEK-ERK/JNK signaling pathway inhibitors such as Ly294002, U0126, and SP600215. In addition, ZO-1 disarray induced by MIF was also recovered when CD74 or CXCR2/4 expression of HMEC-1 were inhibited. Last but not least, the vascular permeabilities of the peritoneal cavity and dorsal cutaneous capillary were also increased in mice treated with rMIF. Taken together; these results suggest that MIF induced by DENV infection may contribute to the increase of vascular permeability during DHF/DSS. Therapeutic intervention of MIF by its inhibitor or neutralizing antibodies may prevent DENV-induced lethality.

The dengue virus envelope protein induced PAI-1 gene expression via MEK/ERK pathways.

Dengue virus (DV) infections cause mild dengue fever or severe life-threatening dengue haemorrhagic fever (DHF)/ dengue shock syndrome (DSS). DV-infected patients have high plasma concentrations of plasminogen activator inhibitor type I (PAI-1). However, the mechanism to cause haemorrhage in DV infections remains poorly understood. In this study, investigation was carried out on the purified recombinant domain III of the envelope glycoprotein of DV serotypes 2 (EIII) and the signalling pathways of EIII leading to PAI-1 gene expression were measured by RT-PCR, Western blot, and immunofluorescence stain. Reporter gene constructs containing serially 5'-deleted sequences of the proximal human PAI-1 promoter region were constructed and then transfected to Huh7 cells, a human hepatoma cell line, prior to EIII treatment. EIII increased the PAI-1 mRNA and protein levels in a dose-dependent manner in Huh7 cells. Results showed that U0126, an inhibitor of extracellular signal-regulated kinase (ERK) kinase (MEK), almost completely suppressed EIII-induced PAI-1 expression. The results suggest that the MEK/ERK signalling pathways mediate the EIII-dependent induction of PAI-1 gene expression via the proximal promoter region.

The envelope glycoprotein domain III of Dengue virus type 2 induced the expression of anticoagulant molecules in endothelial cells.

Dengue virus (DV) causes a non-specific febrile illness known as Dengue fever (DF), and a severe life-threatening illness, Dengue hemorrhagic fever/Dengue shock syndrome (DHF/DSS). Hemostatic changes induced by this virus involve three main factors: thrombocytopenia, endothelial cell damage, and significant abnormalities of the coagulation and fibrinolysis systems. The pathogenesis of bleeding in DV infections remains unknown. In this article, we focused on the DV activating endothelial cells and altering the parameters of hemostasis system. The expression of hemostasis-related factors, Thrombomodulin, TF, TFPI, t-PA, and PAI-1, in DV-infected cells were determined by RT-PCR. Flow cytometry analysis and immunofluorescence staining confirmed that the expression levels of TM in the DV-infected HMEC-1 and THP-1 cells were increased. In addition, the purified recombinant domain III of the envelope glycoprotein of DV (EIII) could induce the expression of TM in the HMEC-1 cells and THP-1 cells. The TM expression induced by DV or EIII in the endothelial cells and monocytic cells suggests that the EIII of DV plays an important role in the pathogenesis of DHF/DSS.

Annexin A2 on lung epithelial cell surface is recognized by severe acute respiratory syndrome-associated coronavirus spike domain 2 antibodies.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection causes lung failure characterized by atypical pneumonia. We previously showed that antibodies against SARS-CoV spike domain 2 (S2) in the patient sera can cross-react with human lung epithelial cells; however, the autoantigen is not yet identified. In this study, we performed proteomic studies and identified several candidate autoantigens recognized by SARS patient sera in human lung type II epithelial cell A549. Among the candidate proteins, annexin A2, which was identified by mass spectrometry analysis and had the highest score by Mascot data search, was further characterized and investigated for its role as an autoantigen. By confocal microscopic observation, SARS patient sera and anti-S2 antibodies were co-localized on A549 cells and both of them were co-localized with anti-annexin A2 antibodies. Anti-annexin A2 antibodies bound to purified S2 proteins, and anti-S2 bound to immunoprecipitated annexin A2 from A549 cell lysate in a dose-dependent manner. Furthermore, an increased surface expression and raft-structure distribution of annexin A2 was present in A549 cells after stimulation with SARS-induced cytokines interleukin-6 and interferon-gamma. Cytokine stimulation increased the binding capability of anti-S2 antibodies to human lung epithelial cells. Together, the upregulated expression of annexin A2 by SARS-associated cytokines and the cross-reactivity of anti-SARS-CoV S2 antibodies to annexin A2 may have implications in SARS disease pathogenesis.

Anti-dengue virus nonstructural protein 1 antibodies recognize protein disulfide isomerase on platelets and inhibit platelet aggregation.

Hemorrhagic syndrome is a hallmark of severe dengue diseases. We previously suggested a mechanism of molecular mimicry in which antibodies against dengue virus (DV) nonstructural protein 1 (NS1) cross-react with platelets. In the present study, we demonstrate that protein disulfide isomerase (PDI) on the platelet surface is recognized by anti-DV NS1 antibodies. Anti-DV NS1 obtained from hyperimmunized mouse sera inhibited PDI activity and platelet aggregation, and both inhibitory effects were prevented when anti-DV NS1 antibodies were preabsorbed with PDI. Anti-PDI antibodies bound to a peptide consisting of amino acid residues 311-330 (P311-330) of NS1. This peptide was a predicted epitope analyzed by homologous sequence alignments between DV NS1 and PDI. The platelet binding activities of anti-PDI and anti-DV NS1 antibodies were both reduced by P311-330 preabsorption. Similar to the findings using anti-DV NS1, antibodies against P311-330 bound to PDI and platelets, followed by inhibition of PDI activity and platelet aggregation. Furthermore, the cross-reactivity of dengue hemorrhagic fever patient sera with platelets was reduced when patient sera were preabsorbed with PDI or P311-330. Dengue hemorrhagic fever patient sera also inhibited platelet aggregation, while PDI or P311-330 reduced this inhibitory effect. In conclusion, anti-DV NS1 antibodies cross-react with PDI on platelet surface causing inhibition of platelet aggregation, which may provide implications in dengue disease pathogenesis.

Dengue virus induces thrombomodulin expression in human endothelial cells and monocytes in vitro.

OBJECTIVES: Dengue virus (DV) infections can cause severe life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). However, the mechanism to cause hemorrhage in DV infections remains poorly understood. Thrombomodulin (TM), expressed on the surface of endothelial cells and monocytes, is very important in regulation of coagulation and inflammation. Therefore, the effect of DV on the TM expression was studied in vitro using both endothelial cells and monocytes. METHODS AND RESULTS: The expression of TM in human endothelial cell line, HMEC-1, monocytic cell line THP-1 and peripheral blood mononuclear cells derived from human blood was increased after DV infection, UV-inactivated DV or recombinant DV envelop protein domain III stimulation as demonstrated by flow cytometry and immunofluorescent staining. Western blot analysis further confirmed only DV but not enterovirus 71 infection of HMEC-1 cells increased TM protein expression. In addition, RT-PCR analysis showed the increase of TM mRNA as well as other protein C activation-related molecules in DV stimulated HMEC-1 in a dose-dependent manner. CONCLUSION: These results suggest that DV stimulation of human endothelial cells and monocytes can increase the expression of TM, which may contribute to the anticoagulant properties of cells during DV infection.

Autophagic machinery activated by dengue virus enhances virus replication.

Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that is favorable for viral replication.

Dengue virus infection induces passive release of high mobility group box 1 protein by epithelial cells.

OBJECTIVES: Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are severe complications of secondary dengue virus (DV) infection. In the current study, we provide the first evidence of induction of cellular necrosis by DV type 2 (DV-2). METHODS AND RESULTS: The epithelial cell line A549 can support replication of dengue virus as demonstrated by expression of viral NS1 antigen and virus plaque assay. DV-2 infection of cells induced cell death in approximately half of the cells that were actively infected. Using sodium 3'-[1-(phenylaminocarbonyl)-3, 4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate [XTT]-based cell viability assays, we found that DV-2 infection at a multiplicity of infection (MOI) of 10 resulted in significant death of cells as well as high extracellular lactate dehydrogenase (LDH) activity and leakage of the high mobility group 1 (HMGB1) protein into the extracellular space. CONCLUSIONS: These results suggest that HMGB1 may be a signal of tissue or cellular injury by DV-2, which in turn is likely to induce and/or enhance an immune reaction.

Dengue viruses can infect human primary lung epithelia as well as lung carcinoma cells, and can also induce the secretion of IL-6 and RANTES.

Dengue viruses (DENV) are herein demonstrated for the first time as being able to infect and replicate in human primary lung epithelium and various lung cancer cell lines. The detection of dengue virus particles and viral negative strand RNA synthesis in the cell, in conjunction with the release of viral progenies in culture supernatants, support the notion that lung cells are susceptible to dengue virus infection. The replication efficiency of DENV in lung cancer cells from high to low is: DEN-2 (dengue virus type-2), DEN-3, DEN-4 and DEN-1. Moreover, the susceptibility of the six lung cancer cell lines to DEN-2 infection is: SW1573>A549>H1435; H23; H520; Bes2B. DEN-2 infection significantly increased the expression levels of IL-6 and RANTES in four of the six lung cancer cell lines, which is consistent with the high expression levels of these molecules in DHF/DSS patients. IL-6 expression induced by DEN-2 infection was NF-kappaB dependent. In summary, our results indicate that lung epithelial cell is a possible target of dengue viruses and IL-6 and RANTES may play pivotal roles in lung related immuno-pathogenesis.