71 research outputs found
Cloning, sequencing and characterization of nusG gene from Streptomyces coelicolor A3 (2)
Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E
Dengue virus (DENV) nonstructural protein-1 (NS1) is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection. Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection. However, it has remained unclear how NS1 becomes associated with the plasma membrane, as it contains no membrane-spanning sequence motif. Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans, we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E. DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells. Moreover, DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells. This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues. Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection
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Smartphone multiplex microcapillary diagnostics using Cygnus: development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples
Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device–termed Cygnus–with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-μl plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (κ = 0.68, 95%CI 0.58–0.77) and NS1-LFT (κ = 0.71, 95%CI 0.63–0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction
Antagonism of the complement component C4 by flavivirus nonstructural protein NS1
The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection
DNA immunization as a technology platform for monoclonal antibody induction
To combat the threat of many emerging infectious diseases, DNA immunization offers a unique and powerful approach to the production of high-quality monoclonal antibodies (mAbs) against various pathogens. Compared with traditional protein-based immunization approaches, DNA immunization is efficient for testing novel immunogen designs, does not require the production or purification of proteins from a pathogen or the use of recombinant protein technology and is effective at generating mAbs against conformation-sensitive targets. Although significant progress in the use of DNA immunization to generate mAbs has been made over the last two decades, the literature does not contain an updated summary of this experience. The current review provides a comprehensive analysis of the literature, including our own work, describing the use of DNA immunization to produce highly functional mAbs, in particular, those against emerging infectious diseases. Critical factors such as immunogen design, delivery approach, immunization schedule, use of immune modulators and the role of final boost immunization are discussed in detail
Bacterial Transmembrane Proteins that Lack N-Terminal Signal Sequences
Tail-anchored membrane proteins (TAMPs), a class of proteins characterized by their lack of N-terminal signal sequence and Sec-independent membrane targeting, play critical roles in apoptosis, vesicle trafficking and other vital processes in eukaryotic organisms. Until recently, this class of membrane proteins has been unknown in bacteria. Here we present the results of bioinformatic analysis revealing proteins that are superficially similar to eukaryotic TAMPs in the bacterium Streptomyces coelicolor. We demonstrate that at least four of these proteins are bona fide membrane-spanning proteins capable of targeting to the membrane in the absence of their N-terminus and the C-terminal membrane-spanning domain is sufficient for membrane targeting. Several of these proteins, including a serine/threonine kinase and the SecE component of the Sec translocon, are widely conserved in bacteria
A human PrM antibody that recognizes a novel cryptic epitope on dengue E glycoprotein
10.1371/journal.pone.0033451PLoS ONE74
A Simplified Positive-Sense-RNA Virus Construction Approach That Enhances Analysis Throughput
Here we present an approach that advances the throughput of a genetic analysis of a positive-sense RNA virus by simplifying virus construction. It enabled comprehensive dissection of a complex, multigene phenotype through rapid derivation of a large number of chimeric viruses and construction of a mutant library directly from a virus pool. The versatility of the approach described here expands the applicability of diverse genetic approaches to study these viruses
Correction: Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication
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