Several cellular molecules and components, specifically, cholesterol and lipid rafts have been described as necessary elements for dengue virus entry and signaling in several human cells. Thus, changes in lipid rafts formation and cholesterol levels were evaluated. Here we report that the amount of total cholesterol and lipid rafts formation increase early after infection of Huh-7 cells. This augment correlates with an increase in the amount of low density lipoprotein receptor (LDLr) on the surface of infected cells and also with a lower phosphorylation level of the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). None of the changes were observed in Huh 7 cells infected with VSV used as a control. These results suggest that dengue virus infection increases intracellular cholesterol levels at early times post infection by triggering the modulation of LDL particles uptake and the increase in the enzymatic activity of HMG-CoA reductase.

The endocytic pathway followed by dengue virus to infect the mosquito cells C6/36 HT was analyzed. Using DIL-labeled virions and real-time imaging it was determined that viral entry into C6/36 HT takes approximately 5 to 7 min. Pretreatment of C6/36 HT cells with sucrose and bafilomycin A, but not filipin, inhibited dengue virus infection up to 80%. Furthermore, the overexpression of dominant-negative mutants of Eps15, a molecule required for the formation of clathrin-coated vesicles, reduced dengue infection up to 50%, indicating that dengue virus entry is through clathrin-mediated endocytosis and is pH-dependent. By double-immunofluorescence assays, DIL-labeled particles were colocalized with early endosomes at 5 min and with lysosomes mainly at 30 min post-infection. Finally, disruption of the microtubule and microfilaments by nocodazole and by cytochalasin D reduced viral infection by more than 80%. Taken together these results indicate that dengue virions enter into C6/36 HT cells by clathrin-mediated endocytosis, using the endosomal pathway from early endosomes to acidic lysosomes before viral RNA is released into the cytoplasm.

Dengue virus (DENV) is transmitted to humans by mosquitoes of the genus Aedes. Although several molecules have been described as part of DENV receptor complex in mosquito cells, none of them have been identified. Our group characterized two glycoproteins (40 and 45 kD) as part of the DENV receptor complex in C6/36 cells. Because identification of the mosquito cell receptor has been unsuccessful and some cell receptors described for DENV in mammalian cells are heat-shock proteins (HSPs), the role of HSPs in DENV binding and infection in C6/36 cells was evaluated. Our results indicate that gp45 and a 74-kD molecule (p74), which interact with DENV envelope protein, are immunologically related to HSP90. Although p74 is induced by heat shock, gp45 apparently is not. However, these proteins are relocated to the cell surface after heat-shock treatment, causing an increase in virus binding without any effect on virus yield.

Dengue virus (DENV) is the causative agent of dengue fever and the more severe forms of the infection known as dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS). Secondary infections with a serotype different from the primary infection are considered a risk factor for the development of DHF/DSS. One explanation for the increased risk of DHF/DSS development after heterologous secondary infections is the antibody-dependent enhancement (ADE) hypothesis. This hypothesis postulates that pre-existing non-neutralizing antibodies will form immune complexes with the new serotype-infecting virus that in turn will have enhanced capacity to infect macrophages and other Fcc receptor (FccR)-bearing cells. Despite the evidence supporting the ADE hypothesis, the molecular mechanisms of ADE are not fully understood. In this work, we present evidence which indicates that intact lipid rafts are required for the ADE infection of U937 cells with DENV. Flow cytometry analysis to measure the percentage of infected cells showed that treatment of differentiated U937 cells with nystatin (30 mg ml "1 ), filipin (10 mg ml "1 ) or b-methyl cyclodextrin (30 mM) significantly reduces (P,0.05) the ADE of DENV-4 infection in vitro without any effect on viability or the number of FccR-bearing cells. Later cholesterol replenishment by supplementing treated cell cultures with bovine fetal serum for 24 h re-established lipid raft integrity and reversed the alteration of the ADE in vitro (P,0.05). Our results suggest that ADE of U937 infection by DENV requires the presence of cholesterol and cholesterol-rich membrane microdomains. INTRODUCTIONDengue virus (DENV) belongs to the genus Flavivirus within the family Flaviviridae and comprises four serotypes (DENV1-DENV4). The virion is composed of three structural proteins: the C protein, which forms the nucleocapsid containing the viral genome, and proteins M and E, which are inserted into the lipid membrane surrounding the nucleocapsid. The genome of DENV is composed of a single-stranded positive-strand RNA of approximately 11 kb. The E protein is exposed on the virion surface and is responsible for the binding and entry of virus into the cell. In addition, the E protein is the virion neutralization antigen and is responsible for the genetic variability that leads to the four serotypes. The DENV genome additionally encodes for seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5), which, along with the virion structural proteins, are all derived from the proteolytic processing of a polyprotein (Lindenbach & Rice, 2003).Nowadays, DENV is the most important causative agent of human viral disease transmitted by mosquitoes. The disease caused by DENV presents in three different clinical forms: dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). DF is characterized by high fever, headache, retro-orbital pain, myalgia and arthralgia among other symptoms. It is a self-limited illness and most of those affected recover after 5 or 6 days. However, i...

Current methods for dengue virus quantitation are either time consuming, technically demanding or costly. As an alternative, the commercial enzyme immunoassay Platelia™ Dengue NS1 AG (BioRad Laboratories) was used to monitor semiquantitatively dengue virus replication in cultured cells. The presence of NS1 protein was evaluated in supernatants from Vero and C6/36 HT cells infected with dengue virus. The amount of NS1 detected in the supernatants of infected cells was proportional to the initial MOI used and to the time of post infection harvest. This immunoassay was also able to detect the presence of NS1 in the supernatants of infected human macrophages. Inhibition of dengue virus replication in C6/36 HT cells treated with lysosomotropic drugs was readily monitored with the use of this assay. These results suggest that the Platelia™ Dengue NS1 AG kit can be used as a fast and reliable surrogate method for the relative quantitation of dengue virus replication in cultured cells.

Insect cells adapt to numerous environmental stressors, including chemicals and invasion of pathogenic microorganisms among others, coordinating cellular and organismal responses. Individual cells sense the environment using receptors that trigger signaling pathways that regulate expression of specific effector proteins and/or cellular responses as movement or secretion. In the coordination of responses to stress, scaffold proteins are pivotal molecules that recruit other proteins forming active complexes. The Receptor for Activated C Kinase 1 (RACK1) is the best studied member of the conserved tryptophan-aspartate (WD) repeat family. RACK1 folds in a seven-bladed β-propeller structure and it could be activated during stress, participating in different signaling pathways. The presence and activities of RACK1 in mosquitoes had not been documented before, in this work the molecule is demonstrated in an Aedes albopictus-derived cell line and its reaction to stress is observed under the effect of serum deprivation and the presence of glucocorticoid analog dexamethasone, a chemical used to cause stress in vitro.

Background The novel coronavirus disease (COVID-19) pandemic is the second global health emergency the world has faced in less than two decades, after the H1N1 Influenza pandemic in 2009–2010. Spread of pandemics is frequently associated with increased population size and population density. The geographical scales (national, regional or local scale) are key elements in determining the correlation between demographic factors and the spread of outbreaks. The aims of this study were: (a) to collect the Mexican data related to the two pandemics; (b) to create thematic maps using federal and municipal geographic scales; (c) to investigate the correlations between the pandemics indicators (numbers of contagious and deaths) and demographic patterns (population size and density). Methods The demographic patterns of all Mexican Federal Entities and all municipalities were taken from the database of “Instituto Nacional de Estadística y Geografía” (INEGI). The data of “Centro Nacional de Programas Preventivos y Control de Enfermedades” (CENAPRECE) and the geoportal of Mexico Government were also used in our analysis. The results are presented by means of tables, graphs and thematic maps. A Spearman correlation was used to assess the associations between the pandemics indicators and the demographic patterns. Correlations with a p value < 0.05 were considered significant. Results The confirmed cases (ccH1N1) and deaths (dH1N1) registered during the H1N1 Influenza pandemic were 72.4 thousand and 1.2 thousand respectively. Mexico City (CDMX) was the most affected area by the pandemic with 8,502 ccH1N1 and 152 dH1N1. The ccH1N1 and dH1N1 were positively correlated to demographic patterns; p-values higher than the level of marginal significance were found analyzing the % ccH1N1 and the % dH1N1 vs the population density. The COVID-19 pandemic data indicated 75.0 million confirmed cases (ccCOVID-19) and 1.6 million deaths (dCOVID-19) worldwide, as of date. The CDMX, where 264,330 infections were recorded, is the national epicenter of the pandemic. The federal scale did not allow to observe the correlation between demographic data and pandemic indicators; hence the next step was to choose a more detailed geographical scale (municipal basis). The ccCOVID-19 and dCOVID-19 (municipal basis) were highly correlated with demographic patterns; also the % ccCOVID-19 and % dCOVID-19 were moderately correlated with demographic patterns. Conclusion The magnitude of COVID-19 pandemic is much greater than the H1N1 Influenza pandemic. The CDMX was the national epicenter in both pandemics. The federal scale did not allow to evaluate the correlation between exanimated demographic variables and the spread of infections, but the municipal basis allowed the identification of local variations and “red zones” such as the delegation of Iztapalapa and Gustavo A. Madero in CDMX.