Dengue-infected Cells Research Articles

Alkyne-Tagged Apigenin, a Chemical Tool to Navigate Potential Targets of Flavonoid Anti-Dengue Leads.

A flavonoid is a versatile core structure with various cellular, immunological, and pharmacological effects. Recently, flavones have shown anti-dengue activities by interfering with viral translation and replication. However, the molecular target is still elusive. Here we chemically modified apigenin by adding an alkyne moiety into the B-ring hydroxyl group. The alkyne serves as a chemical tag for the alkyne-azide cycloaddition reaction for subcellular visualization. The compound located at the perinuclear region at 1 and 6 h after infection. Interestingly, the compound signal started shifting to vesicle-like structures at 6 h and accumulated at 24 and 48 h after infection. Moreover, the compound treatment in dengue-infected cells showed that the compound restricted the viral protein inside the vesicles, especially at 48 h. As a result, the dengue envelope proteins spread throughout the cells. The alkyne-tagged apigenin showed a more potent efficacy at the EC50 of 2.36 ± 0.22, and 10.55 ± 3.37 µM, respectively, while the cytotoxicities were similar to the original apigenin at the CC50 of 70.34 ± 11.79, and 82.82 ± 11.68 µM, respectively. Molecular docking confirmed the apigenin binding to the previously reported target, ribosomal protein S9, at two binding sites. The network analysis, homopharma, and molecular docking revealed that the estrogen receptor 1 and viral NS1 were potential targets at the late infection stage. The interactions could attenuate dengue productivity by interfering with viral translation and suppressing the viral proteins from trafficking to the cell surface.

A proposed molecular mechanism for pathogenesis of severe RNA-viral pulmonary infections.

Background: Certain riboviruses can cause severe pulmonary complications leading to death in some infected patients. We propose that DNA damage induced-apoptosis accelerates viral release, triggered by depletion of host RNA binding proteins (RBPs) from nuclear RNA bound to replicating viral sequences. Methods: Information theory-based analysis of interactions between RBPs and individual sequences in the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), Influenza A (H3N2), HIV-1, and Dengue genomes identifies strong RBP binding sites in these viral genomes. Replication and expression of viral sequences is expected to increasingly sequester RBPs - SRSF1 and RNPS1. Ordinarily, RBPs bound to nascent host transcripts prevents their annealing to complementary DNA. Their depletion induces destabilizing R-loops. Chromosomal breakage occurs when an excess of unresolved R-loops collide with incoming replication forks, overwhelming the DNA repair machinery. We estimated stoichiometry of inhibition of RBPs in host nuclear RNA by counting competing binding sites in replicating viral genomes and host RNA. Results: Host RBP binding sites are frequent and conserved among different strains of RNA viral genomes. Similar binding motifs of SRSF1 and RNPS1 explain why DNA damage resulting from SRSF1 depletion is complemented by expression of RNPS1. Clustering of strong RBP binding sites coincides with the distribution of RNA-DNA hybridization sites across the genome. SARS-CoV-2 replication is estimated to require 32.5-41.8 hours to effectively compete for binding of an equal proportion of SRSF1 binding sites in host encoded nuclear RNAs. Significant changes in expression of transcripts encoding DNA repair and apoptotic proteins were found in an analysis of influenza A and Dengue-infected cells in some individuals. Conclusions: R-loop-induced apoptosis indirectly resulting from viral replication could release significant quantities of membrane-associated virions into neighboring alveoli. These could infect adjacent pneumocytes and other tissues, rapidly compromising lung function, causing multiorgan system failure and other described symptoms.

A proposed molecular mechanism for pathogenesis of severe RNA-viral pulmonary infections

Background: Certain riboviruses can cause severe pulmonary complications leading to death in some infected patients. We propose that DNA damage induced-apoptosis accelerates viral release, triggered by depletion of host RNA binding proteins (RBPs) from nuclear RNA bound to replicating viral sequences. Methods: Information theory-based analysis of interactions between RBPs and individual sequences in the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), Influenza A (H3N2), HIV-1, and Dengue genomes identifies strong RBP binding sites in these viral genomes. Replication and expression of viral sequences is expected to increasingly sequester RBPs - SRSF1 and RNPS1. Ordinarily, RBPs bound to nascent host transcripts prevents their annealing to complementary DNA. Their depletion induces destabilizing R-loops. Chromosomal breakage occurs when an excess of unresolved R-loops collide with incoming replication forks, overwhelming the DNA repair machinery. We estimated stoichiometry of inhibition of RBPs in host nuclear RNA by counting competing binding sites in replicating viral genomes and host RNA. Results: Host RBP binding sites are frequent and conserved among different strains of RNA viral genomes. Similar binding motifs of SRSF1 and RNPS1 explain why DNA damage resulting from SRSF1 depletion is complemented by expression of RNPS1. Clustering of strong RBP binding sites coincides with the distribution of RNA-DNA hybridization sites across the genome. SARS-CoV-2 replication is estimated to require 32.5-41.8 hours to effectively compete for binding of an equal proportion of SRSF1 binding sites in host encoded nuclear RNAs. Significant changes in expression of transcripts encoding DNA repair and apoptotic proteins were found in an analysis of influenza A and Dengue-infected cells in some individuals. Conclusions: R-loop-induced apoptosis indirectly resulting from viral replication could release significant quantities of membrane-associated virions into neighboring alveoli. These could infect adjacent pneumocytes and other tissues, rapidly compromising lung function, causing multiorgan system failure and other described symptoms.

A proposed molecular mechanism for pathogenesis of severe RNA-viral pulmonary infections.

Background: Certain riboviruses can cause severe pulmonary complications leading to death in some infected patients. We propose that DNA damage induced-apoptosis accelerates viral release, triggered by depletion of host RNA binding proteins (RBPs) from nuclear RNA bound to replicating viral sequences. Methods: Information theory-based analysis of interactions between RBPs and individual sequences in the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), Influenza A (H3N1), HIV-1, and Dengue genomes identifies strong RBP binding sites in these viral genomes. Replication and expression of viral sequences is expected to increasingly sequester RBPs - SRSF1 and RNPS1. Ordinarily, RBPs bound to nascent host transcripts prevents their annealing to complementary DNA. Their depletion induces destabilizing R-loops. Chromosomal breakage occurs when an excess of unresolved R-loops collide with incoming replication forks, overwhelming the DNA repair machinery. We estimated stoichiometry of inhibition of RBPs in host nuclear RNA by counting competing binding sites in replicating viral genomes and host RNA. Results: Host RBP binding sites are frequent and conserved among different strains of RNA viral genomes. Similar binding motifs of SRSF1 and RNPS1 explain why DNA damage resulting from SRSF1 depletion is complemented by expression of RNPS1. Clustering of strong RBP binding sites coincides with the distribution of RNA-DNA hybridization sites across the genome. SARS-CoV-2 replication is estimated to require 32.5-41.8 hours to effectively compete for binding of an equal proportion of SRSF1 binding sites in host encoded nuclear RNAs. Significant changes in expression of transcripts encoding DNA repair and apoptotic proteins were found in an analysis of influenza A and Dengue-infected cells in some individuals. Conclusions: R-loop-induced apoptosis indirectly resulting from viral replication could release significant quantities of membrane-associated virions into neighboring alveoli. These could infect adjacent pneumocytes and other tissues, rapidly compromising lung function, causing multiorgan system failure and other described symptoms.

Dengue virus targets RBM10 deregulating host cell splicing and innate immune response.

RNA-seq experiments previously performed by our laboratories showed enrichment in intronic sequences and alterations in alternative splicing in dengue-infected human cells. The transcript of the SAT1 gene, of well-known antiviral action, displayed higher inclusion of exon 4 in infected cells, leading to an mRNA isoform that is degraded by non-sense mediated decay. SAT1 is a spermidine/spermine acetyl-transferase enzyme that decreases the reservoir of cellular polyamines, limiting viral replication. Delving into the molecular mechanism underlying SAT1 pre-mRNA splicing changes upon viral infection, we observed lower protein levels of RBM10, a splicing factor responsible for SAT1 exon 4 skipping. We found that the dengue polymerase NS5 interacts with RBM10 and its sole expression triggers RBM10 proteasome-mediated degradation. RBM10 over-expression in infected cells prevents SAT1 splicing changes and limits viral replication, while its knock-down enhances the splicing switch and also benefits viral replication, revealing an anti-viral role for RBM10. Consistently, RBM10 depletion attenuates expression of interferon and pro-inflammatory cytokines. In particular, we found that RBM10 interacts with viral RNA and RIG-I, and even promotes the ubiquitination of the latter, a crucial step for its activation. We propose RBM10 fulfills diverse pro-inflammatory, anti-viral tasks, besides its well-documented role in splicing regulation of apoptotic genes.

Impaired production of immune mediators in dengue virus type 2-infected mononuclear cells of adults with end stage renal disease

Chronic kidney disease is an epidemiologically identified risk factor for development of severe dengue in dengue-affected patients. However, available data on the immune pathogenesis in end stage renal disease (ESRD) patients affected by dengue is insufficient. We performed an in vitro study to evaluate the sequential immunological reactions and viral load in dengue virus type 2-infected mononuclear cells of patients with ESRD (n = 34) and in healthy controls (n = 30). The concentrations of interleukins (IL)-1 receptor antagonist (Ra), IL-2, IL-6, IL-8, IL-10, IL-12p40, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1b (MIP-1b), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α and viral load cycle threshold (Ct) were measured in the dengue virus type 2-infected mononuclear cells at 6 h, 24 h, 48 h, and 72 h post-infection. We found in the ESRD group significantly higher GM-CSF and IL-2 levels at 6 h post-infection. However, IL-8, IL-10, IL-12p40, TNF-α, MCP-1, and MIP-1b levels were found significantly lower than in the control group. At 24 h, 48 h, and 72 h post-infection, significantly lower levels of IL-1Ra, IL-6, IL-8, IL-10, IL-12p40, TNF-α, MCP-1, and MIP-1b were detected in ESRD group. Concentration of VEGF at 24 h and 48 h, and of GM-CSF at 48 h and 72 h were also found to be lower in ESRD group than in control group. Compared with controls, the viral load Ct values were significantly lower in ESRD group at 6 h and 24 h post-infection No significant difference in viral load Ct values between two groups was found at 48 h and 72 h post-infection. Our study discloses that the expression of immune mediators of dengue-infected mononuclear cells is impaired in ESRD patients.

Mitochondrial selective autophagy (mitophagy) during Dengue infection

Mitophagy (mitochondrial‐selective autophagy) and mitochondrial dynamics co‐ordinate to maintain mitochondrial homeostasis and quality control. Mutation in the proteins involved in mitophagy such as PARKIN and PINK1 have been linked to neurodegenerative disorders like Parkinson's and Alzheimer's. Mitophagy plays a key role in eliminating damaged mitochondria and keeps the accumulation of mitochondrial garbage in check. Damaged mitochondria can trigger a vicious cycle of mitochondrial dysregulation and damage hence defective clearance of damaged mitochondria further exacerbates the mitochondrial injury. Mitochondrial injury can lead to the release of mitochondrial damage associated molecular patterns (MTDs) into the cellular cytoplasm and extracellular milieu. Defective mitophagy has been implicated in hyperactivation of inflammasomes and inflammatory signalling. We observe that Dengue virus (DENV) promotes substantial mitochondrial injury in hepatic cells. During the initial phase of infection Dengue‐infected cells display tubular mitochondrial network, however as the infection proceeds, we observe accumulation of abnormally swollen mitochondria suggestive of severe mitochondrial injury. Scoring for mitochondrial injury by quantitating the average number of abnormally swollen mitochondria in Dengue infected cells suggested a time‐dependent increase in mitochondrial injury in infected cells. Monitoring autophagy flux with mRFP‐GFP‐LC3 tandem construct, we observed that Dengue promotes global autophagy. Further, we confirmed the same by monitoring LC3‐II levels via immunoblot analysis. Subsequently, we also monitored mitophagy levels in Dengue‐infected cells using a novel mitochondria‐specific traffic light reporter mito‐mRFP‐GFP. In contrast to the global autophagy, we observed that Dengue inhibits mitochondrial‐selective autophagy. CCCP‐induced mitophagy was also attenuated in Dengue‐infected cells. Interestingly, PINK‐1 mediated Parkin phosphorylation increased in a time‐dependent fashion suggesting that PINK‐1 and Parkin effectively coordinate to flag the damaged mitochondria for elimination via mitophagy in Dengue‐infected cells. In agreement, we also observed the colocalization of the autophagosomal marker LC3‐GFP with mitochondria in Dengue‐infected cells. However, we did not observe significant colocalization of mitochondria with lysosomes in DENV‐infected cells. Overall our observations suggest a defect in the downstream events of mitophagy in Dengue infection. A defect in mitophagy inhibits the rapid turnover of damaged mitochondria leading to their accumulation in Dengue‐infected cells. Hence, we speculate that defective mitophagy plays a major role in the exponential increase of inflammation during dengue pathogenesis. Currently, we are exploring the underlying mechanism involved in the attenuation of mitophagy in Dengue‐infected cells. Our studies will enhance knowledge on molecular mechanisms that underlie Dengue pathogenesis and provide new avenues for development of potential therapeutic strategies.Support or Funding InformationWellcome Trust‐ DBT India Alliance Intermediate fellowship ‐ IA/I/15/1/501826 awarded to Dr G H Syed.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of cell viability dyes in antiviral assays with RNA viruses that exhibit different cytopathogenic properties

Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue® measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80–90, and 50% maximal cytopathic effect (CPE), respectively, in Vero or Vero 76 cells Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC50) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10–20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC50) assays with dyes in uninfected proliferating cells produced similar CC50 values for EICAR (1.5–8.9μM) and MPA (0.8–2.5μM). 6-Azauridine toxicity was 6.1–17.5μM with NR, VTG, and WST-1, compared to 48–92μM with ALB-A and ALB-F (P<0.001). Curiously, the CC50 values for 3-deazaguanine were 83–93μM with ALB-F versus 2.4–7.0μM with all other dyes including ALB-A (P<0.001). Overall, ALB minimized the toxicities detected with these two inhibitors. Because the choice of dyes affected CC50 values, this impacted on the resulting in vitro selectivity indexes (calculated as CC50/EC50 ratio).

Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation.

A virus that reproduces in a host without killing cells can easily establish a successful infection. Previously, we showed that dengue-2, a virus that threatens 40% of the world, induces autophagy, enabling dengue to reproduce in cells without triggering cell death. Autophagy further protects the virus-laden cells from further insults. In this study, we evaluate how it does so; we show that dengue upregulates host pathways that increase autophagy, namely endoplasmic reticulum (ER) stress and ataxia telangiectasia mutated (ATM) signaling followed by production of reactive oxygen species (ROS). Inhibition of ER stress or ATM signaling abrogates the dengue-conferred protection against other cell stressors. Direct inhibition of ER stress response in infected cells decreases autophagosome turnover, reduces ROS production and limits reproduction of dengue virus. Blocking ATM activation, which is an early response to infection, decreases transcription of ER stress response proteins, but ATM has limited impact on production of ROS and virus titers. Production of ROS determines only late-onset autophagy in infected cells and is not necessary for dengue-induced protection from stressors. Collectively, these results demonstrate that among the multiple autophagy-inducing pathways during infection, ER stress signaling is more important to viral replication and protection of cells than either ATM or ROS-mediated signaling. To limit virus production and survival of dengue-infected cells, one must address the earliest phase of autophagy, induced by ER stress.

Discriminating dengue-infected hepatic cells (WRL-68) using dielectrophoresis.

Dielectrophoresis (DEP), the induced movement of dielectric particles placed in a nonuniform electric field, has been used as a potential technique for manipulation and separation of many biological samples without destructive consequences to the cell. Cells of the same genotype in different physiological and pathological states have unique morphological and structural features, therefore, it is possible to differentiate between them using their DEP responses. This paper reports the experimental discrimination of normal and dengue-infected human hepatic fetal epithelial cells (WRL-68 cells) based on their DEP crossover frequency, at which no resultant movement occurs in the cells in response to the DEP force. A microarray dot electrode was used to conduct the DEP experiments. The DEP forces applied to the cells were quantified by analyzing the light intensity shift within the electrode's dot region based on the Cumulative Modal Intensity Shift image analysis technique. The differences in dielectric properties between infected and uninfected cells were exploited by plotting a unique DEP spectrum for each set of cells. We observed that the crossover frequency decreased from 220 kHz for the normal WRL-68 cells to 140 kHz after infection with the dengue virus in a medium conductivity of 100 μS/cm. We conclude that the change in the DEP crossover frequency between dengue-infected cells and their healthy counterparts should allow direct characterization of these cell types by exploiting their electrophysiological properties.

The combination of type I IFN, TNF-α, and cell surface receptor engagement with dendritic cells enables NK cells to overcome immune evasion by dengue virus.

Clinical studies have suggested the importance of the NK cell response against dengue virus (DenV), an arboviral infection that afflicts >50 million individuals each year. However, a comprehensive understanding of the NK cell response against dengue-infected cells is lacking. To characterize cell-contact mechanisms and soluble factors that contribute to the antidengue response, primary human NK cells were cocultured with autologous DenV-infected monocyte-derived dendritic cells (DC). NK cells responded by cytokine production and the lysis of target cells. Notably, in the absence of significant monokine production by DenV-infected DC, it was the combination of type I IFNs and TNF-α produced by DenV-infected DC that was important for stimulating the IFN-γ and cytotoxic responses of NK cells. Cell-bound factors enhanced NK cell IFN-γ production. In particular, reduced HLA class I expression was observed on DenV-infected DC, and IFN-γ production was enhanced in licensed/educated NK cell subsets. NK-DC cell contact was also identified as a requirement for a cytotoxic response, and there was evidence for both perforin/granzyme as well as Fas/Fas ligand-dependent pathways of killing by NK cells. In summary, our results have uncovered a previously unappreciated role for the combined effect of type I IFNs, TNF-α, and cell surface receptor-ligand interactions in triggering the antidengue response of primary human NK cells.

Effect of Hippophae rhamnoides leaf extract against Dengue virus infection in human blood-derived macrophages

Dengue virus occurs as four distinct serotypes, called Dengue 1, 2, 3, and 4. Symptomatic dengue virus infection ranges from a self limited febrile illness, dengue fever (DF), to a more severe disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The anti-Dengue treatment is severely hampered as no specific therapeutic agents are available. Even present treatment strategies for Dengue are more supportive than curative. In the present study anti-dengue activity of Hippophae rhamnoides (Seabuckthorn, SBT) leaf extract was evaluated in Dengue virus type-2 infected blood-derived human macrophages as macrophages are the primary target of Dengue virus infection. Infected cells were treated with SBT leaf extract and compared with commercially available anti-viral drug, Ribavirin. The extract was able to maintain the cell viability of Dengue-infected cells at par with Ribavirin along with the decrease and increase in TNF-alpha and IFN-gamma respectively. Anti-dengue activity of SBT extract was further determined by the traditional plaque assay. These observations suggest that the SBT leaf extract has a significant anti-dengue activity and has the potential for the treatment of Dengue.

The Dual-Specific Binding of Dengue Virus and Target Cells for the Antibody-Dependent Enhancement of Dengue Virus Infection

Using flow cytometric assay and monoclonal anti-dengue Ab, we observed that both anti-E and anti-prM Abs could enhance dengue virus infection in a concentration-dependent but serotype-independent manner. Increases were found in both the percentage of dengue-infected cells and the expression of dengue E and NS1 protein per cell. Dengue virion binding and infection were enhanced on FcR-bearing cells via the Fc-FcgammaRII pathway. Furthermore, anti-prM Ab also enhanced dengue virion binding and infection on cells lacking FcR, such as BHK-21 or A549 cells, by the mechanism of peptide (CPFLKQNEPEDIDCW)-specific binding. Anti-prM Ab cross-reacted with BHK-21 or A549 cells and recognized self-Ags such as heat shock protein 60. In summary, a novel mechanism of anti-prM Ab-mediated enhancement on dengue virus infection was found to be mediated by dual specific binding to dengue virion and to target cells, in addition to the traditional enhancement on FcR-bearing cells.

Therapeutic effects of dengue 2 virus capsid protein and staphylococcal nuclease fusion protein on dengue-infected cell cultures

Dengue infection poses a serious public health problem in most tropical and subtropical areas. No effective antiviral drugs or vaccines are currently available against dengue infection. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against dengue infection, we constructed a plasmid expressing a fusion protein consisting of staphylococcal nuclease (SN) fused to dengue 2 virus capsid protein (D2C), and investigated its effects on the production of infectious virions when introduced into BHK cells infected with dengue virus. The results indicated that D2C-SN can be expressed and tolerated in this mammalian cell culture. The enzymatically active SN moiety was incorporated into nascent virions during the process of viral assembly. By comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of wild-type SN, on the infectivity of progeny virions, we clearly demonstrated that nucleolytic activity was the major antiviral mechanism. Expression of D2C-SN fusion protein as a therapeutic agent resulted in a reduction in infectious titers of 12- to 60-fold. Therefore, dengue virus may be particularly vulnerable to a CTVI therapeutic approach.

Quantitation of dengue virus specific CD4+ T cells by intracellular cytokine staining

We developed an intracellular cytokine staining assay to quantify dengue specific memory T cells elicited by a primary dengue virus (DEN) infection. Peripheral blood mononuclear cells (PBMC) of volunteers who received experimental live attenuated monovalent DEN vaccines were stimulated with glutaraldehyde-inactivated dengue-infected Vero cell culture lysates from all four DEN serotypes. CD4+ T cell frequencies to previously identified MHC class II peptides were equivalent to 40–70% of the responses using virus infected cell lysates in two donors. IFN-γ responses to DEN were detected from 0.04% to 0.45% CD4+ T cells. The highest IFN-γ response was elicited by antigens from the homologous serotype. We detected serotype-specific and -cross reactive CD4+ T cell cytokine responses from all donors. This assay is suitable for measuring DEN specific CD4+ T cells in human PBMC from large population studies where donor haplotype is unknown or highly variant.

Evidence for in vitro falsely-primed cDNAs that prevent specific detection of virus negative strand RNAs in dengue-infected cells: improvement by tagged RT-PCR

The identification of cell types replicating dengue viruses is an important step towards the understanding of the pathophysiology of dengue severe forms. Since the detection of negative strand viral RNAs is the more reliable marker of active replication for single-strand positive sense RNA viruses, we reassessed the specificity of RT-PCR assays already developed to detect dengue negative strand RNAs. Studying mammalian Vero cells infected by a dengue-2 strain, it was shown that falsely-primed cDNAs are generated in vitro during the reverse transcription step and are amplified subsequently by PCR. Since this may compromise the specificity of existing RT-PCR systems, we developed a tagged RT-PCR assay and addressed the role of some critical factors in such a system. Optimization of the negative strand-specific tagged RT-PCR allowed to resolve the problems due to the PCR amplification of falsely-primed cDNAs. Using this assay it was possible to detect specifically negative strand RNAs as soon as 3 h after Vero cells have been exposed to the dengue-2 strain and we showed that this system is highly specific. Thus, the present dengue negative strand-specific tagged RT-PCR assay may help to reassess viral replication in the context of dengue pathophysiology.

T cell-mediated cytotoxicity against dengue-infected target cells.

A cytotoxic T lymphocyte (CTL) response to dengue virus-infected target cells is described. Effector cells were generated in an in vitro secondary culture and appeared to be T cells possessing both the Lyt 1.1 and Lyt 2.1 surface antigens. A stronger CTL response was noted with the H-2k haplotype compared to H-2d, and H-2 compatibility was required between CTL and target cells. CTL generated showed some cross-reactivity with target cells infected with Japanese encephalitis virus (JEV), another flavivirus, but not with target cells infected with an alphavirus, Sindbis. The significance and importance of these findings are discussed.

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity.

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.

Detection of dengue cell-surface antigens by peroxidase-labeled antibodies and immune cytolysis.

The appearance of dengue-specific plasma membrane (DSPM) antigens in infected LLC-MK(2) cell cultures was studied by (51)Cr release in immune cytolysis and at an ultrastructural level using peroxidase-labeled antibodies. DSPM antigen was first detected at 36 h with electron microscopy in approximately 30% of the cells, and this percentage did not increase with time. However, both surface staining with peroxidase-labeled antibodies and (51)Cr release indicated that the amount of DSPM antigen per cell increases with time. The appearance of (51)Cr release in immune cytolysis experiments with dengue-infected cells occurred much later than the peak of infectious virus release. This was in sharp contrast to immune cytolysis with a group A arbovirus, Eastern equine encephalitis, in which the kinetics of release of infectious virus and (51)Cr release were identical. This suggests different mechanisms of insertion of viral plasma membrane antigens in Eastern equine encephalitis and dengue-infected LLC-MK(2) cells.