The four divergent serotypes of dengue virus are the causative agents of dengue fever, dengue hemorrhagic fever and dengue shock syndrome. About two-fifths of the world's population live in areas where dengue is prevalent, and thousands of deaths are caused by the viruses every year. Dengue virus is transmitted from one person to another primarily by the yellow fever mosquito, Aedes aegypti. Recent studies have begun to define how the dengue viral proteins interact with host proteins to mediate viral replication and pathogenesis. A combined analysis of these studies, however, suggests that many virus-host protein interactions remain to be identified, especially for the mosquito host. In this study, we used high-throughput yeast two-hybrid screening to identify mosquito and human proteins that physically interact with dengue proteins. We tested each identified host protein against the proteins from all four serotypes of dengue to identify interactions that are conserved across serotypes. We further confirmed many of the interactions using co-affinity purification assays. As in other large-scale screens, we identified some previously detected interactions and many new ones, moving us closer to a complete host – dengue protein interactome. To help summarize and prioritize the data for further study, we combined our interactions with other published data and identified a subset of the host-dengue interactions that are now supported by multiple forms of evidence. These data should be useful for understanding the interplay between dengue and its hosts and may provide candidates for drug targets and vector control strategies.
Shedding of microparticles (MPs) is a consequence of apoptotic cell death and cellular activation. Low levels of circulating MPs in blood help maintain homeostasis, whereas increased MP generation is linked to many pathological conditions. Herein, we investigated the role of MPs in dengue virus (DENV) infection. Infection of various susceptible cells by DENV led to apoptotic death and MP release. These MPs harbored a viral envelope protein and a nonstructural protein 1 (NS1) on their surfaces. Ex vivo analysis of clinical specimens from patients with infections of different degrees of severity at multiple time points revealed that MPs generated from erythrocytes and platelets are two major MP populations in the circulation of DENV-infected patients. Elevated levels of red blood cell-derived MPs (RMPs) directly correlated with DENV disease severity, whereas a significant decrease in platelet-derived MPs was associated with a bleeding tendency. Removal by mononuclear cells of complement-opsonized NS1-anti-NS1 immune complexes bound to erythrocytes via complement receptor type 1 triggered MP shedding in vitro, a process that could explain the increased levels of RMPs in severe dengue. These findings point to the multiple roles of MPs in dengue pathogenesis. They offer a potential novel biomarker candidate capable of differentiating dengue fever from the more serious dengue hemorrhagic fever. IMPORTANCEDengue is the most important mosquito-transmitted viral disease in the world. No vaccines or specific treatments are available. Rapid diagnosis and immediate treatment are the keys to achieve a positive outcome. Dengue virus (DENV) infection, like some other medical conditions, changes the level and composition of microparticles (MPs), tiny bag-like structures which are normally present at low levels in the blood of healthy individuals. This study investigated how MPs in culture and patients' blood are changed in response to DENV infection. Infection of cells led to programmed cell death and MP release. In patients' blood, the majority of MPs originated from red blood cells and platelets. Decreased platelet-derived MPs were associated with a bleeding tendency, while increased levels of red blood cell-derived MPs (RMPs) correlated with more severe disease. Importantly, the level of RMPs during the early acute phase could serve as a biomarker to identify patients with potentially severe disease who require immediate care. E ach year, up to 390 million people are infected by dengue virus (DENV), the most significant mosquito-borne virus of the Flaviviridae family, posing serious socioeconomic and health burdens globally (1). Four serotypes of viruses (DENV serotype 1 [DENV-1], DENV-2, DENV-3, and DENV-4) are transmitted to humans by Aedes mosquitoes to establish both endemic and epidemic transmission cycles primarily in tropical and subtropical countries (1). Although the majority of DENV infections in humans are subclinical, a small fraction of infected individuals develops clinical symptoms ranging from a self-...
Background Dengue is the most significant mosquito-borne viral disease; there are no specific therapeutics. The antiparasitic drug ivermectin efficiently inhibits the replication of all 4 dengue virus serotypes in vitro. Methods We conducted 2 consecutive randomized, double-blind, placebo-controlled trials in adult dengue patients to evaluate safety and virological and clinical efficacies of ivermectin. After a phase 2 trial with 2 or 3 days of 1 daily dose of 400 µg/kg ivermectin, we continued with a phase 3, placebo-controlled trial with 3 days of 400 µg/kg ivermectin. Results The phase 2 trial showed a trend in reduction of plasma nonstructural protein 1 (NS1) clearance time in the 3-day ivermectin group compared with placebo. Combining phase 2 and 3 trials, 203 patients were included in the intention to treat analysis (100 and 103 patients receiving ivermectin and placebo, respectively). Dengue hemorrhagic fever occurred in 24 (24.0%) of ivermectin-treated patients and 32 (31.1%) patients receiving placebo (P = .260). The median (95% confidence interval [CI]) clearance time of NS1 antigenemia was shorter in the ivermectin group (71.5 [95% CI 59.9–84.0] hours vs 95.8 [95% CI 83.9–120.0] hours, P = .014). At discharge, 72.0% and 47.6% of patients in the ivermectin and placebo groups, respectively had undetectable plasma NS1 (P = .001). There were no differences in the viremia clearance time and incidence of adverse events between the 2 groups. Conclusions A 3-day 1 daily dose of 400 µg/kg oral ivermectin was safe and accelerated NS1 antigenemia clearance in dengue patients. However, clinical efficacy of ivermectin was not observed at this dosage regimen.
Dengue hemorrhagic fever (DHF) is caused by infection with dengue virus (DENV). Four different serotypes (DENV1-4) co-circulate in dengue endemic areas. The viral RNA genome-based reverse-transcription PCR (RT-PCR) is the most widely used method to identify DENV serotypes in patient specimens. However, the non-structural protein 1 (NS1) antigen as a biomarker for DENV serotyping is an emerging alternative method. We modified the serotyping-NS1-enzyme linked immunosorbent assay (stNS1-ELISA) from the originally established assay which had limited sensitivity overall and poor specificity for the DENV2 serotype. Here, four biotinylated serotype-specific antibodies were applied, including an entirely new design for detection of DENV2. Prediction of the infecting serotype of retrospective acute-phase plasma from dengue patients revealed 100% concordance with the standard RT-PCR method for all four serotypes and 78% overall sensitivity (156/200). The sensitivity of DENV1 NS1 detection was greatly improved (from 62% to 90%) by the addition of a DENV1/DENV3 sub-complex antibody pair. Inclusive of five antibody pairs, the stNS1-ELISA (plus) method showed an overall increased sensitivity to 85.5% (171/200). With the same clinical specimens, a commercial NS1 rapid diagnostic test (NS1-RDT) showed 72% sensitivity (147/200), significantly lower than the stNS1-ELISA (plus) performance. In conclusion, the stNS1-ELISA (plus) is an improved method for prediction of DENV serotype and for overall sensitivity. It could be an alternative assay not only for early dengue diagnosis, but also for serotype identification especially in remote resource-limited dengue endemic areas.
Detection and quantification of viruses in laboratory and clinical samples are standard assays in dengue virus (DENV) studies. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered to be the standard for DENV detection and quantification due to its high sensitivity. However, qRT-PCR offers only quantification relative to a standard curve and consists of several “in-house” components resulting in interlaboratory variations. We developed and optimized a protocol for applying one-step RT-droplet digital PCR (RT-ddPCR) for DENV detection and quantification. The lower limit of detection (LLOD95) and the lower limit of quantification (LLOQ) for RT-ddPCR were estimated to be 1.851 log10-copies/reaction and 2.337 log10-copies/reaction, respectively. The sensitivity of RT-ddPCR was found to be superior to qRT-PCR (94.87% vs. 90.38%, p = 0.039) while no false positives were detected. Quantification of DENV in clinical samples was independently performed in three laboratories showing interlaboratory variations with biases <0.5 log10-copies/mL. The RT-ddPCR protocol presented here could help harmonize DENV quantification results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity.
Dengue virus (DENV) infection is a significant global health problem. There are no specific therapeutics or widely available vaccines. Early diagnosis is critical for patient management. Viral RNA detection by multiplex RT-PCR using multiple pairs of primers/probes allowing the simultaneous detection of all four DENV serotypes is commonly used. However, increasing the number of primers in the RT-PCR reaction reduces the sensitivity of detection due to the increased possibility of primer dimer formation. Here, a one tube, singleplex real-time RT-PCR specific to DENV 3′-UTR was developed for the detection and quantification of pan-DENV with no cross reactivity to other flaviviruses. The sensitivity of DENV detection was as high as 96.9% in clinical specimens collected at the first day of hospitalization. Our assay provided equivalent PCR efficiency and RNA quantification among each DENV serotype. The assay’s performance was comparable with previously established real-time RT-PCR targeting coding sequences. Using both assays on the same specimens, our results indicate the presence of defective virus particles in the circulation of patients infected with all serotypes. Dual regions targeting RT-PCR enhanced the sensitivity of viral genome detection especially during the late acute phase when viremia rapidly decline and an incomplete viral genome was clinically evident.
Dengue prototype strains are widely used for virological study. The strains presented here have been cultured under different laboratory environments, resulting in accumulating genetic variations.
To understand how proteins function to control cellular processes, their interactions with other proteins must be identified and characterised. The yeast two‐hybrid system is a simple and efficient assay for protein interactions. In a yeast two‐hybrid assay, the two proteins to be tested are expressed in a yeast nucleus with each protein fused to one‐half of a transcription activator. If the two‐hybrid proteins interact, the transcription activator is reconstituted and turns on reporter genes that can be easily detected. This assay has been used to identify tens of thousands of protein interactions, to map protein interaction domains and to characterise mutant variants of proteins. A variety of related assays have been developed, all based on the ability of two interacting hybrid proteins to activate a reporter system. These assays along with the original yeast two‐hybrid assay contribute to the characterisation of the protein interactions – or protein interactome – for humans and a wide range of other organisms. Key Concepts The function of most proteins involves interacting with one or more other proteins. A binary interaction is a direct physical interaction between two proteins. Understanding a protein's function requires charting its binary interactions. The interactome is all of the protein interactions for a particular cell or an entire organism. Two‐hybrid assays detect binary protein interactions by expressing the two test proteins in cells as hybrids fused to protein moieties that when brought into proximity via the protein interaction produce a detectable signal. In a yeast two‐hybrid assay, the two proteins to be tested for interaction are fused to the two halves of a transcription factor in yeast. Two‐hybrid assays, like all protein interaction assays, can produce false positives, which are interactions that are detected in the assay even though they do not occur under normal conditions in vivo . Two‐hybrid and other protein interaction assays can also result in missed interactions or false negatives. Use of multiple different protein interaction assays can reduce the number of false negatives and provide cross‐validation to rule out false positives.
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