RESUMEN
The Zika virus (ZIKV) is a global health threat due to its rapid spread and severe health implications, including congenital abnormalities and neurological complications. Differentiating ZIKV from other arboviruses such as dengue virus (DENV) is crucial for effective diagnosis and treatment. This study presents the development of a biosensor for detecting the ZIKV non-structural protein 1 (NS1) using gold nanoparticles (AuNPs) functionalized with monoclonal antibodies employing dynamic light scattering (DLS). The biosensor named ZINS1-mAb-AuNP exhibited specific binding to the ZIKV NS1 protein, demonstrating high colloidal stability indicated by a hydrodynamic diameter (DH) of 140â nm, detectable via DLS. In the absence of the protein, the high ionic strength medium caused particle aggregation. This detection method showed good sensitivity and specificity, with a limit of detection (LOD) of 0.96â µg mL-1, and avoided cross-reactivity with DENV2 NS1 and SARS-CoV-2 spike proteins. The ZINS1-mAb-AuNP biosensor represents a promising tool for the early and accurate detection of ZIKV, facilitating diagnostic and treatment capabilities for arboviral infections.
RESUMEN
This chapter describes the methods of propagation and titration for DENV-1 to -4, which are required for most of the experiments using live viruses. DENV does not reach titers as high as those of other viruses or as high as desired for their use in biological assays. Although DENV grows in many different cell lines derived from both vertebrate and invertebrate cells, the most common cell lines used for virus isolation and propagation are mosquito cells C6/36 from Aedes albopictus. Amongst several methods for virus quantification, plaque assay stands out as being very practical and inexpensive. This technique is carried out essentially to estimate virion density in a particular material, being extremely important when evaluating the effect of an antiviral treatment or antibody neutralization capacity, for example. In this assay, viral particles are serially diluted and added onto confluent cell monolayers immersed in a semisolid medium, which is responsible for limiting virus spread throughout the culture. Therefore, regarding the medium consistency, once a virion successfully infects a cell, the newly produced particles can only infect neighboring cells, eventually leading them to death. This phenomenon can be observed as round gaps or plaques in the culture after staining live cells with a crystal violet solution. Then, plaques are counted and used to determine plaque-forming units per milliliter. Here, we describe a protocol established by our group for dengue virus (DENV) titration with porcine kidney (PS) cells.
Asunto(s)
Virus del Dengue , Dengue , Aedes , Animales , Antivirales , Bioensayo , Línea Celular , PorcinosRESUMEN
Dengue is a mosquito-borne infectious disease that is highly endemic in tropical and subtropical countries. Symptomatic patients can rapidly progress to severe conditions of hemorrhage, plasma extravasation, and hypovolemic shock, which leads to death. The blood tests of patients with severe dengue typically reveal low levels of high-density lipoprotein (HDL), which is responsible for reverse cholesterol transport (RCT) and regulation of the lipid composition in peripheral tissues. It is well known that dengue virus (DENV) depends on membrane cholesterol rafts to infect and to replicate in mammalian cells. Here, we describe the interaction of DENV nonstructural protein 1 (NS1) with apolipoprotein A1 (ApoA1), which is the major protein component of HDL. NS1 is secreted by infected cells and can be found circulating in the serum of patients with the onset of symptoms. NS1 concentrations in plasma are related to dengue severity, which is attributed to immune evasion and an acute inflammatory response. Our data show that the DENV NS1 protein induces an increase of lipid rafts in noninfected cell membranes and enhances further DENV infection. We also show that ApoA1-mediated lipid raft depletion inhibits DENV attachment to the cell surface. In addition, ApoA1 is able to neutralize NS1-induced cell activation and to prevent NS1-mediated enhancement of DENV infection. Furthermore, we demonstrate that the ApoA1 mimetic peptide 4F is also capable of mediating lipid raft depletion to control DENV infection. Taken together, our results suggest the potential of RCT-based therapies for dengue treatment. These results should motivate studies to assess the importance of RCT in DENV infection in vivo. IMPORTANCE DENV is one of the most relevant mosquito-transmitted viruses worldwide, infecting more than 390 million people every year and leading to more than 20 thousand deaths. Although a DENV vaccine has already been approved, its potential side effects have hampered its use in large-scale immunizations. Therefore, new treatment options are urgently needed to prevent disease worsening or to improve current clinical management of severe cases. In this study, we describe a new interaction of the NS1 protein, one of the major viral components, with a key component of HDL, ApoA1. This interaction seems to alter membrane susceptibility to virus infection and modulates the mechanisms triggered by DENV to evade the immune response. We also propose the use of a mimetic peptide named 4F, which was originally developed for atherosclerosis, as a potential therapy for relieving DENV symptoms.