concanavalin-a and Dengue

Arboviruses have been emerging as a significant global health problem due to the recurrent epidemics. Arboviruses require the development of new diagnostic devices due to the nonspecific clinical manifestations. Herein, we report a biosensor based on cysteine (Cys), zinc oxide nanoparticles (ZnONp), and Concanavalin A (ConA) lectin to differentiate between arboviruses infections. ConA is capable of interacting with the saccharide components of the viral capsid. In this study, we evaluated the reproducibility, sensitivity, and specificity of the sensor for the virus of Dengue type 2 (DENV2), Zika (ZIKV), Chikungunya (CHIKV), and Yellow fever (YFV). Atomic force microscopy measurements confirmed the electrode surface modification and revealed a heterogeneous topography during the biorecognition process. Cyclic voltammetry (CV) and impedance spectroscopy (EIS) were used to characterize the biosensor. The blockage of the oxidation-reduction process is related to the formation of Cys-ZnONp-ConA system on the electroactive area and its subsequent interaction with viral glycoproteins. The sensor exhibited a linear response to different concentrations of the studied arboviruses. Our study demonstrates that ConA lectin recognizes the structural glycoproteins of the DENV2, ZIKV, CHIKV, and YFV. DENV2 is the most structurally similar to ZIKV. Our results have shown that the impedimetric response correlates with the structural glycoproteins, as follow: DENV2 (18.6 kΩ) > ZIKV (14.6 kΩ) > CHIKV (6.86 kΩ) > YFV (5.98 kΩ). The homologous structural regions contribute to ConA-arboviruses recognition. Our results demonstrate the use of the proposed system for the development of biosensors for arboviruses infections.

Topics: Arbovirus Infections; Arboviruses; Biosensing Techniques; Chikungunya Fever; Chikungunya virus; Concanavalin A; Cysteine; Dengue; Dengue Virus; Diagnosis, Differential; Electrochemistry; Electrodes; Glucose; Humans; Mannose; Metal Nanoparticles; Yellow Fever; Yellow fever virus; Zika Virus; Zika Virus Infection; Zinc Oxide

In this work, we developed a biosystem based on Concanavalin A (ConA) and lipid membranes to recognize glycoproteins from the serum of patients contaminated with dengue serotypes 1, 2 and 3 (DENV1, DENV2 and DENV3). The modified gold electrode was characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and atomic force microscopy. Morphological analyses of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC-ConA, DPPC-ConA-DENV1, DPPC-ConA-DENV2 and DPPC-ConA-DENV3 revealed the existence of a non-uniform covering and large globules. EIS and CV measurements have shown that redox probe reactions on the modified gold electrodes were partially blocked due to the adsorption of lipid-ConA system and reveal the interaction response of the immobilized ConA to the presence of glycoproteins of dengue serum. The biosystem exhibited a wide linear response to different concentrations of sera of dengue serotypes 1, 2 and 3. A higher impedimetric response to glycoproteins present in dengue serotype 3 was observed. Our results demonstrate the applicability of lectin and lipid membranes to the development of biosensors for dengue infections.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Biosensing Techniques; Cell Membrane; Concanavalin A; Dengue; Electrochemistry; Electrodes; Glycoproteins; Gold; Humans; Liposomes; Point-of-Care Systems

Formation of stable thin films of mixed xyloglucan (XG) and alginate (ALG) onto Si/SiO(2) wafers was achieved under pH 11.6, 50mM CaCl(2), and at 70 degrees C. XG-ALG films presented mean thickness of (16+/-2)nm and globules rich surface, as evidenced by means of ellipsometry and atomic force microscopy (AFM), respectively. The adsorption of two glucose/mannose-binding seed (Canavalia ensiformis and Dioclea altissima) lectins, coded here as ConA and DAlt, onto XG-ALG surfaces took place under pH 5. Under this condition both lectins present positive net charge. ConA and DAlt adsorbed irreversibly onto XG-ALG forming homogenous monolayers approximately (4+/-1)nm thick. Lectins adsorption was mainly driven by electrostatic interaction between lectins positively charged residues and carboxylated (negatively charged) ALG groups. Adhesion of four serotypes of dengue virus, DENV (1-4), particles to XG-ALG surfaces were observed by ellipsometry and AFM. The attachment of dengue particles onto XG-ALG films might be mediated by (i) H bonding between E protein (located at virus particle surface) polar residues and hydroxyl groups present on XG-ALG surfaces and (ii) electrostatic interaction between E protein positively charged residues and ALG carboxylic groups. DENV-4 serotype presented the weakest adsorption onto XG-ALG surfaces, indicating that E protein on DENV-4 surface presents net charge (amino acid sequence) different from E proteins of other serotypes. All four DENV particles serotypes adsorbed similarly onto lectin films adsorbed. Nevertheless, the addition of 0.005mol/L of mannose prevented dengue particles from adsorbing onto lectin films. XG-ALG and lectin layers serve as potential materials for the development of diagnostic methods for dengue.

Topics: Alginates; Concanavalin A; Dengue; Dengue Virus; Glucans; Glucuronic Acid; Hexuronic Acids; Hydrogen-Ion Concentration; Lectins; Microscopy, Atomic Force; Silicon Dioxide; Spectroscopy, Fourier Transform Infrared; Virion; Xylans

It is believed that the pathogenesis of dengue is generated by a deregulation of the immunological response. Dengue virus-infected monocytes/macrophages are likely to secrete monokines, which play a role in clinical features observed in patients with dengue haemorrhagic fever or dengue shock syndrome. This is a report on a study on 45 individuals presenting clinical and laboratory characteristics of dengue virus infection. During the acute phase of infection, immunophenotyping of peripheral mononuclear leukocytes was carried out in 19 patients and demonstrated a reduced frequency of CD2+ lymphocytes and their CD4+ and CD8+ subsets. Normal ratios were recovered during convalescence. Also, during the acute phase, mononuclear cells proliferated poorly in response to mitogens and dengue antigens as detected by incorporation of radiolabeled thymidine. During convalescence the lymphoproliferative response was re-established. In addition, the presence of circulating cytokines was investigated in the plasma of the same 45 patients. Concentrations of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and soluble tumor necrosis factor receptor (sTNF-Rp75) were found to be significantly elevated in patients when compared to normal controls. The increase in TNF-alpha was correlated with haemorrhagic manifestations and the increase in IL-10 with platelet decay. The data demonstrate that during the acute phase of dengue infection subsets of T lymphocytes are depressed in terms of both rate and function and provide evidence that circulating pro-inflammatory cytokines, such as TNF-alpha, are important in the pathogenesis and severity of dengue. IL-10 may be downregulating lymphocyte and platelet function.

Topics: Adult; Aged; Aged, 80 and over; Cell Division; Concanavalin A; Cytokines; Dengue; Female; Humans; Immunophenotyping; Interferon-gamma; Interleukin-10; Male; Middle Aged; T-Lymphocyte Subsets; T-Lymphocytes; Tumor Necrosis Factor-alpha