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Dengue laboratory diagnosis is essentially based on detection of the virus, its components or antibodies directed against the virus in blood samples. Blood, however, may be difficult to draw in some patients, especially in children, and sampling during outbreak investigations or epidemiological studies may face logistical challenges or limited compliance to invasive procedures from subjects. The aim of this study was to assess the possibility of using saliva and urine samples instead of blood for dengue diagnosis. Serial plasma, urine and saliva samples were collected at several time-points between the day of admission to hospital until three months after the onset of fever in children with confirmed dengue disease. Quantitative RT-PCR, NS1 antigen capture and ELISA serology for anti-DENV antibody (IgG, IgM and IgA) detection were performed in parallel on the three body fluids. RT-PCR and NS1 tests demonstrated an overall sensitivity of 85.4%/63.4%, 41.6%/14.5% and 39%/28.3%, in plasma, urine and saliva specimens, respectively. When urine and saliva samples were collected at the same time-points and tested concurrently, the diagnostic sensitivity of RNA and NS1 detection assays was 69.1% and 34.4%, respectively. IgG/IgA detection assays had an overall sensitivity of 54.4%/37.4%, 38.5%/26.8% and 52.9%/28.6% in plasma, urine and saliva specimens, respectively. IgM were detected in 38.1% and 36% of the plasma and saliva samples but never in urine. Although the performances of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood specimens is not possible.

Background Dengue is a global disease, transmitted by the Aedes vectors. In 2018, there were 80 615 dengue cases with 147 deaths in Malaysia. Currently, the nationwide surveillance programs are dependent on Aedes larval surveys and notifications of lab-confirmed human infections. The existing, reactive programs appear to lack sensitivity and proactivity. More efficient dengue vector surveillance/control methods are needed. Methods A parallel, cluster, randomized controlled, interventional trial is being conducted for 18 months in Damansara Damai, Selangor, Malaysia, to determine the efficacy of using gravid oviposition sticky (GOS) trap and dengue non-structural 1 (NS1) antigen test for early surveillance of dengue among Aedes mosquitoes to reduce dengue outbreaks. Eight residential apartments were randomly assigned into intervention and control arms. GOS traps are set at the apartments to collect Aedes weekly, following which dengue NS1 antigen is detected in these mosquitoes. When a dengue-positive mosquito is detected, the community will be advised to execute vector search-and-destroy and protective measures. The primary outcome concerns the the percentage change in the (i) number of dengue cases and (ii) durations of dengue outbreaks. Whereas other outcome measures include the change in density threshold of Aedes and changes in dengue-related knowledge, attitude and practice among cluster inhabitants. Discussion This is a proactive and early dengue surveillance in the mosquito vector that does not rely on notification of dengue cases. Surveillance using the GOS traps should be able to efficiently provide sufficient coverage for multistorey dwellings where population per unit area is likely to be higher. Furthermore, trapping dengue-infected mosquitoes using the GOS trap, helps to halt the dengue transmission carried by the mosquito. It is envisaged that the results of this randomized controlled trial will provide a new proactive, cheap and targeted surveillance tool for the prevention and control of dengue outbreaks. Trial registration This is a parallel-cluster, randomized controlled, interventional trial, registered at ClinicalTrials.gov (ID: NCT03799237), on 8th January 2019 (retrospectively registered).

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. The outbreak of CHIKV infection has been seen in many tropical and subtropical regions of the biosphere. Current reports evidenced that after outbreaks in 2005–06, the fitness of this virus propagating in Aedes albopictus enhanced due to the epistatic mutational changes in its envelope protein. In our study, we evaluated the prevalence of intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) in CHIKV proteome. IDPs/IDPRs are known as members of a ‘Dark Proteome’ that defined as a set of polypeptide segments or whole protein without unique three-dimensional structure within the cellular milieu but with significant biological functions, such as cell cycle regulation, control of signaling pathways, and maintenance of viral proteomes. However, the intrinsically disordered aspects of CHIKV proteome and roles of IDPs/IDPRs in the pathogenic mechanism of this important virus have not been evaluated as of yet. There are no existing reports on the analysis of intrinsic disorder status of CHIKV. To fulfil this goal, we have analyzed the abundance and functionality of IDPs/IDPRs in CHIKV proteins, involved in the replication and maturation. It is likely that these IDPs/IDPRs can serve as novel targets for disorder based drug design.

RIG-I is a cytosolic pathogen recognition receptor that engages viral RNA in infected cells to trigger innate immune defenses through its adaptor protein MAVS. MAVS resides on mitochondria and peroxisomes, but how its signaling is coordinated among these organelles has not been defined. Here we show that a major site of MAVS signaling is the mitochondrial-associated membrane (MAM), a distinct membrane compartment that links the endoplasmic reticulum to mitochondria. During RNA virus infection, RIG-I is recruited to the MAM to bind MAVS. Dynamic MAM tethering to mitochondria and peroxisomes then coordinates MAVS localization to form a signaling synapse between membranes. Importantly, the hepatitis C virus NS3/4A protease, which cleaves MAVS to support persistent infection, targets this synapse for MAVS proteolysis from the MAM, but not from mitochondria, to ablate RIG-I signaling of immune defenses. Thus, the MAM mediates an intracellular immune synapse that directs antiviral innate immunity.

A straightforward in situ detection method for dengue infection was demonstrated through the molecular imprinting of a dengue nonstructural protein 1 (NS1) epitope into an electropolymerized molecularly imprinted polyterthiophene (E-MIP) film sensor. The key enabling step in the sensor fabrication is based on an epitope imprinting strategy, in which short peptide sequences derived from the original target molecules were employed as the main template for detection and analysis. The formation of the E-MIP sensor films was facilitated using cyclic voltammetry (CV) and monitored in situ by electrochemical quartz crystal microbalance (EC-QCM). Surface properties were analyzed using different techniques including atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and polarization modulation-infrared reflection-adsorption (PM-IRRAS). The standard calibration curve (R = 0.9830) was generated for the detection of the epitope, Ac-VHTWTEQYKFQ-NH2, with a linear range of 0.2 to 30 μg/mL and detection limit of 0.073 μg/mL. A separate calibration curve (R = 0.9786) was obtained using spiked buffered solutions of dengue NS1 protein, which resulted in a linear range of 0.2 to 10 μg/mL and a detection limit of 0.056 μg/mL. The fabricated E-MIP sensor exhibited long-term stability, high sensitivity, and good selectivity towards the targeted molecules. These results indicated that the formation of the exact and stable cavity imprints in terms of size, shape, and functionalities was successful. In our future work, we aim to use our E-MIP sensors for NS1 detection in real-life samples such as serum and blood.

Early diagnosis of dengue virus (DENV) infection can improve clinical outcomes by ensuring close follow-up, initiating appropriate supportive therapies and raising awareness to the potential of hemorrhage or shock. Non-structural glycoprotein-1 (NS1) has proven to be a useful biomarker for early diagnosis of dengue. A number of rapid diagnostic tests (RDTs) and enzyme-linked immunosorbent assays (ELISAs) targeting NS1 antigen (Ag) are now commercially available. Here we evaluated these tests using a well-characterized panel of clinical samples to determine their effectiveness for early diagnosis. Retrospective samples from South America were used to evaluate the following tests: (i) \"Dengue NS1 Ag STRIP\" and (ii) \"Platelia Dengue NS1 Ag ELISA\" (Bio-Rad, France), (iii) \"Dengue NS1 Detect Rapid Test (1st Generation)\" and (iv) \"DENV Detect NS1 ELISA\" (InBios International, United States), (v) \"Panbio Dengue Early Rapid (1st generation)\" (vi) \"Panbio Dengue Early ELISA (2nd generation)\" and (vii) \"SD Bioline Dengue NS1 Ag Rapid Test\" (Alere, United States). Overall, the sensitivity of the RDTs ranged from 71.9%-79.1% while the sensitivity of the ELISAs varied between 85.6-95.9%, using virus isolation as the reference method. Most tests had lower sensitivity for DENV-4 relative to the other three serotypes, were less sensitive in detecting secondary infections, and appeared to be most sensitive on Day 3-4 post symptom onset. The specificity of all evaluated tests ranged from 95%-100%. ELISAs had greater overall sensitivity than RDTs. In conjunction with other parameters, the performance data can help determine which dengue diagnostics should be used during the first few days of illness, when the patients are most likely to present to a clinic seeking care.

Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 105infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-α and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.

BackgroundVascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown MethodsThe major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patients with DSS were also analyzed ResultsSoluble NS1 activated complement to completion, and activation was enhanced by polyclonal and monoclonal antibodies against NS1. Complement was also activated by cell-associated NS1 in the presence of specific antibodies. Plasma levels of NS1 and terminal SC5b-9 complexes correlated with disease severity. Large amounts of NS1, complement anaphylatoxin C5a, and the terminal complement complex SC5b-9 were present in pleural fluids from patients with DSS ConclusionsComplement activation mediated by NS1 leads to local and systemic generation of anaphylatoxins and SC5b-9, which may contribute to the pathogenesis of the vascular leakage that occurs in patients with DHF/DSS

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

Dengue virus (DENV) nonstructural 1 (NS1) protein is a specific and sensitive biomarker for the diagnosis of dengue. In this study, an efficient electrochemical biosensor that uses chemically modified affinity peptides was developed for the detection of dengue virus NS1. A series of amino acid-substituted synthetic peptides was rationally designed, chemically synthesized and covalently immobilized to a gold sensor surface. The sensor performance was monitored via square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS). Potential affinity peptides specific for NS1 were chosen according to the dynamic current decrease in SWV experiments. Using circular dichroism, the molar ellipticity of peptides (DGV BP1-BP5) was determined, indicating that they had a mostly similar in random coil structure, not totally identical. Using SWV, DGV BP1 was selected as a promising recognition peptide and limit of detection for NS1 was found to be 1.49 μg/mL by the 3-sigma rule. DGV BP1 showed good specificity and stability for NS1, with low signal interference. The validation of the sensor to detect NS1 proteins was confirmed with four dengue virus culture broth (from serotype 1 to 4) as proof-of-concept. The detection performance of our sensor incorporating DGV BP1 peptides showed a statistically significant difference. These results indicate that this strategy can potentially be used to detect the dengue virus antigen, NS1, and to diagnosis dengue fever within a miniaturized portable device in point-of-care testing.