Dengue Virus Type 2 Infection Research Articles

Antiviral activity of pimecrolimus against dengue virus type 2 infection in vitro and in vivo

Dengue virus (DENV) infection is a public health concern in several countries and is associated with severe diseases, such as dengue hemorrhagic fever and dengue shock syndrome. DENVs are transmitted to humans via the bites of infected Aedes mosquitoes, and no antiviral therapeutics are currently available. In this work, we aimed to identify antiviral drugs against DENV type 2 (DENV2) infections and selected pimecrolimus as a potential antiviral drug candidate. Pimecrolimus significantly inhibited DENV2-mediated cell death and replication in vitro. We also confirmed a decrease in the number of plaques formed as well as in the envelope protein levels of DENV2. The time-of-addition and course experiments revealed that pimecrolimus inhibited DENV2 infection during the early stages of the virus replication cycle. In an experimental mouse model, orally administered pimecrolimus alleviated body weight loss and lethality caused by DENV2 infection, which we used as readouts of the drug’s antiviral potency. Furthermore, pimecrolimus significantly inhibited the DENV2 load and ameliorated focal necrosis in the liver and spleen. Taken together, our in vitro and in vivo findings suggest that pimecrolimus is a promising antiviral drug candidate for the treatment of DENV2 infection.

In-vitro antiviral activity of Carica papaya formulations against dengue virus type 2 and chikungunya virus

Dengue and chikungunya are diseases of global health significance and currently, no antivirals are available to treat these arboviral diseases. Carica papaya leaves extract is traditionally used to treat thrombocytopenia in patients infected with the dengue virus. The current study was undertaken to study the antiviral activity of commercially available Carica papaya leaves extract (CPLE) based products and CPLE prepared in four formulations against dengue virus type 2 (DENV-2) and chikungunya virus (CHIKV). Maximum nontoxic concentrations of the commercially available CPLE based products and CPLE based formulations (Carica papaya leaves in powder form, Carica papaya leaves in lyophilized form, Carica papaya leaves based silver nanoparticles and supercritical fluid extract of Carica papaya leaves) were used for screening the antiviral activity. The antiviral activity against DENV-2 and CHIKV were assessed post infection using focus forming unit assay. Effective formulations were tested under different conditions i.e. pretreatment, cotreatment and posttreatment. The virus output after treatment was assessed by real-time RT-PCR, immunofluorescence assay and focus forming unit assay. The results revealed Carica papaya leaves based silver nanoparticles and supercritical fluid extract of Carica papaya leaves formulations showed significant inhibition in case of DENV while papaya leaves in powder form showed significant reduction in case of CHIKV. This study demonstrates the antiviral activity of CPLE formulations against DENV-2 and CHIKV infection in in-vitro system and needs further validation in in-vivo models.

The efficacy of the traditional Thai remedy “Ya-Ha-Rak” against dengue virus type 2

BackgroundYa-Ha-Rak (YHR) or Bencha-Loga-Wichian is a traditional Thai medicine of polyherbal formulation, comprising five roots, commonly used as an antipyretic and antiseptic. Dengue virus infection exhibits high fever, which is the most common sign of febrile diseases, not just dengue infection. However, thus far, no study has reported the efficacy of YHR against this virus. Therefore, this study aimed to screen in vitro the efficacy of YHR in killing dengue virus type 2 (DENV-2). MethodWater (aqueous-YHR) and ethanol (EtOH-YHR) were used for studying phenolic and flavonoid contents, antioxidant scavenging activity (DDPH and ABTS), and cytotoxicity. In addition, the in vitro DENV-2 killing efficacy of YHR extracts was examined by the quantity of the DENV-2 focus-forming units and copies of RNA in cell culture supernatants. ResultsThe total phenolic content and the antioxidant scavenging activity (DDPH and ABTS) of EtOH-YHR were significantly higher than the aqueous-YHR. However, the aqueous-YHR was lower in cellular cytotoxicity than the EtOH-YHR. Quercetin showed the highest concentration among the 10 polyphenolic contents in the aqueous-YHR and EtOH-YHR extracts. In addition, the concentration of quercetin in EtOH-YHR was found to be almost 2.5-fold higher than that of aqueous-YHR. The EtOH-YHR was found to have higher inhibitory efficacy for DENV-2 infection than aqueous-YHR. ConclusionThe results obtained show that EtOH-YHR is a potential traditional Thai medicinal plant used as an alternative therapeutic agent against the deadly Dengue virus.

ML-SA1 and SN-2 inhibit endocytosed viruses through regulating TRPML channel expression and activity

Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3), as key channels in the endosomal-lysosomal system, are associated with many different cellular processes, including ion release, membrane trafficking and autophagy. In particular, they can also facilitate viral entry into host cells and enhance viral infection. We previously identified that two selective TRPML agonists, ML-SA1 and SN-2, that showed antiviral activities against dengue virus type 2 (DENV2) and Zika virus (ZIKV) in vitro, but their antiviral mechanisms are still elusive. Here, we reported that ML-SA1 could inhibit DENV2 replication by downregulating the expression of both TRPML2 and TRPML3, while the other TRPML activator, SN-2, suppressed DENV2 infection by reducing only TRPML3 expression. Consistently, the channel activities of both TRPML2 and TRPML3 were also found to be associated with the antiviral activity of ML-SA1 on DENV2 and ZIKV, but SN-2 relied only on TRPML3 channel activity. Further mechanistic experiments revealed that ML-SA1 and SN-2 decreased the expression of the late endosomal marker Rab7, dependent on TRPML2 and TRPML3, indicating that these two compounds likely inhibit viral infection by promoting vesicular trafficking from late endosomes to lysosomes and then accelerating lysosomal degradation of the virus. As expected, neither ML-SA1 nor SN-2 inhibited herpes simplex virus type I (HSV-1), whose entry is independent of the endolysosomal network. Together, our work reveals the antiviral mechanisms of ML-SA1 and SN-2 in targeting TRPML channels, possibly leading to the discovery of new drug candidates to inhibit endocytosed viruses.

Identification of quinolone derivatives as effective anti-Dengue virus agents

Dengue virus (DENV) infection is one of the most important infectious diseases in tropical and subtropical regions around the world. Previously, we performed an initial phenotypic screening of 7000 compounds using DENV type 2 (DENV2)-infected BHK-21 cells to identify small molecules which could inhibit virus replication. In this study, we describe two novel compounds with anti-DENV2 activity, tentatively named Compound-X and Compound-Y. Both compounds possess a quinolone skeleton, and the EC50s of Compound-X and Compound-Y against DENV2 were 3.9 μM and 9.2 μM, respectively. Based on a DENV replicon assay, it was suggested that these compounds have anti-DENV2 activity by inhibition of a step in virus replication. Furthermore, using mutational analysis we obtained compounds-resistant to DENV2 infection and identified a mutation, V130A in the NS5 methyltransferase (MTase) domain. However, these compounds did not inhibit MTase activity. In addition, incorporation of an additional NS1 N246D mutation with the NS5 V130A mutation in DENV2 resulted in recovery of viral replication and a further reduction of the sensitivity to the quinolone compounds by an unknown mechanism. Therefore further investigations are required to clarify the antiviral mechanisms of these quinolone compounds.

Glycyrrhizic acid derivatives as Dengue virus inhibitors.

Dengue virus (DENV) is one of the most geographically distributed pathogenic flaviviruses transmitted by mosquitoes Aedes sps. In this study, the structure-antiviral activity relationships of Glycyrrhizic acid (GL) derivatives was evaluated by the inhibitory assays on the cytopathic effect (CPE) and viral infectivity of DENV type 2 (DENV2) in Vero E6 cells. GL (96% purity) had a low cytotoxicity to Vero E6 cells, inhibited DENV2-induced CPE, and reduced the DENV-2 infectivity with the IC50 of 8.1 μM. Conjugation of GL with amino acids or their methyl esters and the introduction of aromatic acylhydrazide residues into the carbohydrate part strongly influenced on the antiviral activity. Among compounds tested GL conjugates with isoleucine 13 and 11-aminoundecanoic acid 17 were found as potent anti-DENV2 inhibitors (IC50 1.2-1.3 μM). Therefore, modification of GL is a perspective way in the search of new antivirals against DENV2 infection.

A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication.

Host cells infected with dengue virus (DENV) often trigger endoplasmic reticulum (ER) stress, a key process that allows viral reproduction, without killing the host cells until the late stage of the virus life-cycle. However, little is known regarding which DENV viral proteins interact with the ER machinery to support viral replication. In this study, we identified and characterized a novel host factor, stress-associated ER protein 1 (SERP1), which interacts with the DENV type 2 (DENV-2) NS4B protein by several assays, for example, yeast two-hybrid, subcellular localization, NanoBiT complementation, and co-immunoprecipitation. A drastic increase (34.5-fold) in the SERP1 gene expression was observed in the DENV-2-infected or replicon-transfected Huh7.5 cells. The SERP1 overexpression inhibited viral yields (37-fold) in the DENV-2-infected Huh7.5 cells. In contrast, shRNAi-knockdown and the knockout of SERP1 increased the viral yields (3.4- and 16-fold, respectively) in DENV-2-infected HEK-293 and Huh7.5 cells, respectively. DENV-2 viral RNA replication was severely reduced in stable SERP1-expressing Huh7.5 cells transfected with DENV-2 replicon plasmids. The overexpression of DENV-2 NS4B alleviated the inhibitory effect of SERP1 on DENV-2 RNA replication. Taking these results together, we hypothesized that SERP1 may serve as an antiviral player during ER stress to restrict DENV-2 infection. Our studies revealed novel anti-DENV drug targets that may facilitate anti-DENV drug discovery.

Influences of interaction between dengue virus type 2-infected human umbilical vein endothelial cells and macrophages on major inflammatory cytokines

Objective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells (HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor (M-CSF) to prepare macrophages. The purity of CD14+ CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-infected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pretreating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2.66±0.53, P<0.05) and then decreased. The purity of macrophages detected by flow cytometry was (89.16±2.07) %. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16.10±0.17 and 29.76±0.58, while the expression levels at 24 h in the uninfected group were 1.46±0.67 and 1.60±0.54, respectively. Expression of IL-6, IL-8, TNF-α and IL-1β at mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-α and IL-1β expression at 24 h were 45.82±3.72, 52.34±1.69 (12 h), 8.94±1.75 and 30.96±1.44 in the infected macrophages, and 1.16±0.22, 1.15±0.21, 1.11±0.09 and 1.47±0.31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infected HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-culture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0.01) and that of IL-6, IL-8, TNF-α and IL-1β in macrophages (P<0.01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-α and IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent. Key words: Dengue virus type 2 (DENV-2); Human umbilical vein endothelial cell (HUVEC); Macrophage; Cytokine; Immunoregulation

Antivirus effectiveness of ivermectin on dengue virus type 2 in Aedes albopictus.

BackgroundDengue fever is the most rapidly spreading mosquito-borne viral disease over the past 50 years, with a 30-fold increase in global incidence. Dengue vector control is a key component for the dengue control strategy, since no absolutely effective vaccine or drug is available yet. However, the rapid rise and spread of mosquito insecticide resistance have become major threats to the efficiency of insecticide-based vector control activities. Thus, innovative vector control tools are badly needed. This study aims to confirm the antivirus effectiveness of ivermectin on dengue virus type 2 (DENV-2) in Aedes albopictus (Skuse, 1894), then to explore its potential use in the combating to the dengue epidemics.MethodsAedes albopictus were first infected with DENV-2 in human whole blood, and at the fourth day after infectious blood feeding, they were divided into eight groups. Seven of them were held for six days with access to 0, 2, 4, 8, 16, 32 and 64 ng/ml ivermectin, respectively, and the last one was set as a historical control group, which was stored at -80°C until being detected at the same time with the other groups. Each mosquito was detected using real-time fluorescent RT-PCR kit. DENV-2 RNA concentration (copies/ml) and infection rate in each group were compared.ResultsBoth of quantitatively and qualitatively inhibiting effects of ivermectin have been detected in this study. Generally, DENV-2 replicated well in Aedes albopictus without ivermectin intervention, whose virus loads exhibited significantly higher when the mosquitoes were holding from 4 days to 10 days after infectious blood feeding. In contrast, with the treatment of ivermectin, the infection rate was reduced by as much as 49.63%. The regression equation between infection rates (Y2) and ivermectin concentration log2 values (X2) was obtained as Y2 = 91.41–7.21*X2 with R2 = 0.89.ConclusionIvermectin can directly or indirectly inhibit DENV-2 multiplication in Aedes albopictus. Moreover, the actual concentration for application in zooprophylaxis needs to be confirmed in the further field trials.

Stearoly-CoA desaturase 1 differentiates early and advanced dengue virus infections and determines virus particle infectivity.

Positive strand RNA viruses, such as dengue virus type 2 (DENV2) expand and structurally alter ER membranes to optimize cellular communication pathways that promote viral replicative needs. These complex rearrangements require significant protein scaffolding as well as changes to the ER chemical composition to support these structures. We have previously shown that the lipid abundance and repertoire of host cells are significantly altered during infection with these viruses. Specifically, enzymes in the lipid biosynthesis pathway such as fatty acid synthase (FAS) are recruited to viral replication sites by interaction with viral proteins and displayed enhanced activities during infection. We have now identified that events downstream of FAS (fatty acid desaturation) are critical for virus replication. In this study we screened enzymes in the unsaturated fatty acid (UFA) biosynthetic pathway and found that the rate-limiting enzyme in monounsaturated fatty acid biosynthesis, stearoyl-CoA desaturase 1 (SCD1), is indispensable for DENV2 replication. The enzymatic activity of SCD1, was required for viral genome replication and particle release, and it was regulated in a time-dependent manner with a stringent requirement early during viral infection. As infection progressed, SCD1 protein expression levels were inversely correlated with the concentration of viral dsRNA in the cell. This modulation of SCD1, coinciding with the stage of viral replication, highlighted its function as a trigger of early infection and an enzyme that controlled alternate lipid requirements during early versus advanced infections. Loss of function of this enzyme disrupted structural alterations of assembled viral particles rendering them non-infectious and immature and defective in viral entry. This study identifies the complex involvement of SCD1 in DENV2 infection and demonstrates that these viruses alter ER lipid composition to increase infectivity of the virus particles.

Influences of interaction between DENV-2-infected HUVECs and regulatory T cells on major inflammatory cytokines

Objective To investigate the influences on major inflammatory cytokines after co-culturing regulatory T cells (Treg) with human umbilical vein endothelial cells (HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Peripheral blood mononuclear cells (PBMC) were extracted from concentrated human leukocytes by density gradient centrifugation.Treg cells were sorted by immunomagnetic beads.Expression of CD4, CD25 and CD127 molecules on the membrane of Treg cells was detected by flow cytometry to identify the purity of Treg cells.HUVECs pretreated with or without sphingosine-1-phosphate S1P type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h were first infected with DENV-2 and then co-cultured with Treg cells.Expression of IL-6, IL-8, TNF-α, IL-10 and TGF-β at mRNA level was detected by real-time RT-PCR.Levels of IL-6, IL-8, IL-10 and TGF-β in the culture supernatants were detected by a double-antibody sandwich ELISA. Results The purity of Treg cells was (84.3±0.5)%.Expression of NS1 at mRNA level in DENV-2-infected HUVECs first gradually increased and then decreased after reaching the peak at 24 h (3.03±0.26, P<0.01). Enhanced expression of IL-6, IL-8 and TNF-α at mRNA level in HUVECs was observed after DENV-2 infection (P<0.01). Expression of these cytokines at every time point was decreased after co-culturing DENV-2-infected HUVECs with Treg cells (P<0.05), but was still higher than that before infection.CYM-5442 pretreatment decreased the expression of IL-6, IL-8 and TNF-α at mRNA level in DENV-2-infected HUVECs and inhibited the secretion of IL-10 and TGF-β by Treg cells that were co-cultured with DENV-2-infected HUVECs. Conclusion Primary HUVECs infected by DENV-2 can enhance the secretion of IL-10 and TGF-β by Treg cells, and the suppressive cytokines produced by Treg cells can reduce the production of inflammatory cytokines by DENV-2-infected HUVECs. Key words: DENV-2; HUVEC; Treg cell; Cytokine; Immunomodulation

Geraniin extracted from the rind of Nephelium lappaceum binds to dengue virus type-2 envelope protein and inhibits early stage of virus replication

BackgroundThe rapid rise and spread in dengue cases, together with the unavailability of safe vaccines and effective antiviral drugs, warrant the need to discover and develop novel anti-dengue treatments. In this study the antiviral activity of geraniin, extracted from the rind of Nephelium lappaceum, against dengue virus type-2 (DENV-2) was investigated.MethodsGeraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC50 value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays’. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay.ResultsCytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC50 of 1.75 μM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin’s interaction with rE-DIII with high affinity.ConclusionsGeraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.

PERK Signal-Modulated Protein Translation Promotes the Survivability of Dengue 2 Virus-Infected Mosquito Cells and Extends Viral Replication.

Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5′-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells. This suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. When the PERK signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. This suggested that ER stress response was alleviated through the PERK-mediated shutdown of global proteins in DENV2-infected C6/36 cells. In the meantime, the activities of caspases-9 and -3 and the apoptosis-related cell death rate increased in C6/36 cells with PERK inhibition. This reflected that the PERK-signaling pathway is involved in determining cell survival, presumably by reducing DENV2-induced ER stress. Looking at the PERK downstream target, α-subunit of eukaryotic initiation factor 2 (eIF2α), an increased phosphorylation status was only shown in infected C6/36 cells. This indicated that recruitment of ribosome binding to the mRNA 5′-cap structure could have been impaired in cap-dependent translation. It turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to DENV2 infection. As synthesis of viral proteins was not affected by the PERK signal pathway, an alternate mode other than cap-dependent translation may be utilized. This finding provides insights into elucidating how the PERK signal pathway modulates dynamic translation of proteins and helps mosquito cells survive continuous replication of the DENV2. It was ecologically important for virus amplification in mosquitoes and transmission to humans.

Dengue virus type 2 infection inhibits the expression of toll-like receptors 4 and 7 in murine bone marrow-derived dendritic cells

Objective To analyze the changes in the expression of Toll-like receptors 4 (TLR4) and 7 on the surface of murine bone marrow-derived dendritic cells following dengue virus type 2 (DEN2) infection. Methods DEN2 NGC strain was infected into BALB/c suckling mice through intracranial injection and injected into C6/36 cells to induce the in vivo and in vitro proliferation of DEN2, respectively. RT-PCR was performed to identify DEN2 RNA. Reed-Muench method was used to determine the 50% tissue culture infective dose (TCID50) of DEN2. Dendritic cells (DCs) were prepared by stimulating bone marrow cells isolated form C57BL/6 mice with IL-4 and GM-CSF and then identified by flow cytometry. The prepared murine bone marrow-derived DCs were infected with DEN2 and observed with direct immunofluorescence assay. Dynamic changes in the expression of CD11c, CD86 and I-A/I-E molecules on DCs after DEN2 infection were detected by flow cytometry. Levels of DEN2 RNA and the expression of TLR4 and TLR7 at mRNA level were dynamically detected by real-time quantitative PCR. Results The TCID50 of DEN2 to C6/36 cells was 10-5.8. Murine bone marrow-derived DCs were acquired with a purity of 70% and could be infected with DEN2 in vitro. The percentages of CD86 and I-A/I-E molecules on the surface of DCs infected with 1×105 TCID50 of DEN2 were statistically different from those of the negative control group. Neither of the two groups showed a significant difference in the percentages of membrane molecules over time. However, the percentages of membrane molecules on DCs increased with increasing viral load. Compared with the negative control group, the levels of DEN2 RNA in infection groups were increased with increasing virus load, while the expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level was decreased with increasing viral load. Conclusion DEN2 infection promotes the maturation of DCs. Expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level decreases with increasing viral load, which suggests that TLR4 and TLR7 are closely related to viral infection and play a certain role in the pathogenesis of DEN infection. Key words: Dengue virus type 2; DC; TLR4; TLR7

Effects of interaction between dengue virus type 2-infected HUVECs and human CD4+ T cells on the expression of adhesion molecules and immunosuppressive factors

Objective To investigate the effects of interaction between human umbilical vein endothelial cells (HUVECs) which were infected with dengue virus type 2 (DENV-2) and CD4+ T cells on the expression of ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), IL-10 and TGF-β1 at mRNA level for further understanding the immunological mechanism of DENV infection. Methods HUVECs were treated with CYM-5442, a selective agonist for sphingosine-1-phosphate receptor 1 (S1P1), for 24 hours and then infected with 103 TCID50 (50% tissue culture infective dose) of DENV-2 before co-culturing with CD4+ T cells. Changes in the expression of NS1 (DENV-2 nonstructural protein), SPHK1 (sphingosine kinase 1, phosphorylating sphingosine to S1P), ICAM-1, VCAM-1, IL-10 and TGF-β1 at mRNA level were detected by real-time PCR after 4, 8, 12, 24, 48 and 72 hours of co-culturing. Results There was a certain timeliness in the expression of NS1 at mRNA level after infecting HUVECs with DENV-2 and the expression reached a peak at 24 h. Treating HUVECs with or without CYM-5442 had no significant influence on the expression of DENV-2 NS1 at mRNA level. The expression of SPHK1 at mRNA level was significantly increased after treating HUVECs with CYM-5442 and DENV-2 (P<0.05). Compared with DENV-2-infected or untreated HUVECs, Co-culturing DENV-2-infected HUVECs with CD4+ T cells increased the expression of ICAM-1 and VCAM-1 in HUVECs at mRNA level (P<0.01) as well as the expression of IL-10 in CD4+ T cells at mRNA level (P<0.05), but had no significant influence on the expression of TGF-β1 in CD4+ T cells at mRNA level. Conclusion This study shows that DENV-2 can replicate and proliferate in HUVECs, but CD4+ T cells inhibit the replication and proliferation. CD4+ T cells play an important role in promoting the expression of VCAM-1 and ICAM-1 in DENV-2-infected HUVECs at mRNA level, activating HUVECs and increasing inflammation, which may be associated with increased vascular permeability induced by DENV-2 infection. Co-culturing CD4+ T cells with DENV-2-infected HUVECs promotes the expression of IL-10 in CD4+ T cells at mRNA level, but has no significant effect on TGF-β1. Key words: HUVEC; CD4+ T cell; DENV-2; Cytokine; Immunoregulation

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a host factor required for dengue virus and Junín virus multiplication

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are cellular factors involved in the replication of several viruses. In this study we analyzed the expression and intracellular localization of hnRNP A2 and hnRNP K in cell cultures infected with two viruses that cause human hemorrhagic fevers: dengue virus type 2 (DENV-2) and Junín virus (JUNV). We determined that DENV-2 promoted the cytoplasmic translocation of hnRNP K and to a lesser extent of hnRNP A2, meanwhile, JUNV infection induced an increase in hnRNP K cytoplasmic localization whereas hnRNP A2 remained mainly in the nucleus of infected cells. Both hnRNP K and hnRNP A2 were localized predominantly in the nucleus of JUNV persistently-infected cells even after superinfection with JUNV indicating that persistent infection does not alter nucleo-cytoplasmic transport of these hnRNPs. Total levels of hnRNP K expression were unaffected by DENV-2 or JUNV infection. In addition we determined, using small interfering RNAs, that hnRNP K knockout inhibits DENV-2 and JUNV multiplication. Our results indicate that DENV-2 and JUNV induce hnRNP K cytoplasmic translocation to favor viral multiplication.

Olive baboons: a non-human primate model for testing dengue virus type 2 replication

This study evaluated the use of a non-human primate, the olive baboon (Papio anubis), as a model of dengue infection. Olive baboons closely resemble humans genetically and physiologically and have been used extensively for assessing novel vaccine formulations. Two doses of dengue virus type 2 (DENV-2) were tested in baboons: 10(3) and 10(4) pfu. Similarly, African green monkeys received the same quantity of virus and acted as positive controls. Following exposure, high levels of viremia were detected in both animal species. There was a trend to detect more days of viremia and more homogeneous viral titers in animals receiving the low viral dose. In addition, baboons infected with the virus generally exhibited positive virus isolation 1 day later than African green monkeys. Humoral responses consisting of antiviral and neutralizing antibodies were detected in all animals after infection. We conclude that baboons provide an alternative non-human primate species for experimental DENV-2 infection and we recommend their use for further tests of vaccines, administering the lowest dose assayed: 10(3) pfu.

Domain Ⅲ of Dengue Virus Serotype 2 Envelope: Expression at High Levels in Escherichia coli and Competitive Inhibition of Virus Entry

Abstract Obejective The domain Ⅲ of dengue virus type 2 envelope was cloned and expressed in Escherichia coli and the recombinant protein inhibited virus effect was tested. Methods In this study, the domain Ⅲ (DⅢ) protein of the dengue virus type-2 (DENV-2) envelope (E) antigen was expressed in Escherichia coli by fusion with a carrier protein. The protein was purified using enzymatic cleavage and affinity purification. Rabbit immunization and antibody detection was carried out. Inhibition of DENV-2 infection was observed by DENV-2 EDⅢ protein and its immunity rabbits serum. Results The recombinant expression DENV-2 EDⅢ protein plasmid was constructed successfully. After isopropyl thiogalactoside induction, a specific soluble 29 kD protein was obtained, and the expression product accounted for 68.87% of the total protein of the cell lysate. Western blotting demonstrated the reactivity of the recombinant protein with his-tag and DENV (Ⅰ-Ⅳ) monoclonal antibodies. The protein was purified using enzymatic cleavage and affinity purification. The purified recombinant EDⅢ protein inhibited the entry of DENV-2 into BHK-21 cells. DENV-2 plaque neutralization assays were carried out using serially diluted antibodies against EDⅢ protein. At a 1:16 dilution, the antibodies produced at least 90% neutralization of the DENV-2 virus. Furthermore, the antibodies continued to exhibit high neutralization effects (approximately 80%) until the anti-EDⅢ antibody titer reached 1:1 024. Conclusions DENV-2 EDⅢ was cloned and expressed successfully. DENV-2 EDⅢ protein could be useful in the development of inexpensive dengue vaccine. The data also suggested that DENV-2 employed an attachment molecule or receptor for its entry into C6/36 mosquito cells.

Reduced sphingosine kinase 1 activity in dengue virus type-2 infected cells can be mediated by the 3′ untranslated region of dengue virus type-2 RNA

Sphingosine kinase 1 (SphK1) is a lipid kinase with important roles including regulation of cell survival. We have previously shown reduced SphK1 activity in cells with an established dengue virus type-2 (DENV-2) infection. In this study, we examined the effect of alterations in SphK1 activity on DENV-2 replication and cell death and determined the mechanisms of the reduction in SphK1 activity. Chemical inhibition or overexpression of SphK1 after established DENV-2 infection had no effect on infectious DENV-2 production, although inhibition of SphK1 resulted in enhanced DENV-2-induced cell death. Reduced SphK1 activity was observed in multiple cell types, regardless of the ability of DENV-2 infection to be cytopathic, and was mediated by a post-translational mechanism. Unlike bovine viral diarrhea virus, where SphK1 activity is decreased by the NS3 protein, SphK1 activity was not affected by DENV-2 NS3 but, instead, was reduced by expression of the terminal 396 bases of the 3' UTR of DENV-2 RNA. We have previously shown that eukaryotic elongation factor 1A (eEF1A) is a direct activator of SphK1 and here DENV-2 RNA co-localized and co-precipitated with eEF1A from infected cells. We propose that the reduction in SphK1 activity late in DENV-2-infected cells is a consequence of DENV-2 out-competing SphK1 for eEF1A binding and hijacking cellular eEF1A for its own replication strategy, rather than a specific host or virus-induced change in SphK1 to modulate viral replication. Nonetheless, reduced SphK1 activity may have important consequences for survival or death of the infected cell.

Viperin Is Induced following Dengue Virus Type-2 (DENV-2) Infection and Has Anti-viral Actions Requiring the C-terminal End of Viperin

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.