Small extracellular vesicles from the human endothelial cell line EA.hy 926 exert a self-cell activation and modulate DENV-2 genome replication and infection in naïve endothelial cells.

Extracellular vesicles (EVs) play crucial roles in cell signaling and communication, transporting molecules that convey a message to target cells. During infectious diseases, EVs can also carry viral molecules that may contribute to viral spread, as previously reported for dengue virus (DENV). EVs from infected endothelial cells (EC) may harbor viral segments and various sets of molecules that could contribute to endothelial dysfunction during severe dengue. However, the effect of these EVs on non-infected EC (NIC) remain unknown. We characterized the EVs produced by the human EC line EA.hy 926 infected with DENV-2 and assessed their functional impact on polarized NIC. Results showed that infection induced an increased in the quantity of produced EVs, which differentially carried proteins mainly involved in proteosome activity, along with a peptide of the NS5 viral protein. Additionally, all types of Y-RNAs were found, accompanied by a set of differentially loaded microRNAs (miRs) that could regulate DENV genome. Pre-treatment of polarized NIC with small EVs (sEVs) from infected EC before DENV-2 infection caused EC activation, a decrease in viral genome replication, and a protective effect against barrier disruption during the first 24h post-infection, suggesting that sEVs could be important in the pathology or resolution of DENV and a promising therapeutic tool for infectious diseases.

Zika Virus: A Neurotropic Warrior against High-Grade Gliomas-Unveiling Its Potential for Oncolytic Virotherapy.

Gliomas account for approximately 75-80% of all malignant primary tumors in the central nervous system (CNS), with glioblastoma multiforme (GBM) considered the deadliest. Despite aggressive treatment involving a combination of chemotherapy, radiotherapy, and surgical intervention, patients with GBM have limited survival rates of 2 to 5 years, accompanied by a significant decline in their quality of life. In recent years, novel management strategies have emerged, such as immunotherapy, which includes the development of vaccines or T cells with chimeric antigen receptors, and oncolytic virotherapy (OVT), wherein wild type (WT) or genetically modified viruses are utilized to selectively lyse tumor cells. In vitro and in vivo studies have shown that the Zika virus (ZIKV) can infect glioma cells and induce a robust oncolytic activity. Consequently, interest in exploring this virus as a potential oncolytic virus (OV) for high-grade gliomas has surged. Given that ZIKV actively circulates in Colombia, evaluating its neurotropic and oncolytic capabilities holds considerable national and international importance, as it may emerge as an alternative for treating highly complex gliomas. Therefore, this literature review outlines the generalities of GBM, the factors determining ZIKV's specific tropism for nervous tissue, and its oncolytic capacity. Additionally, we briefly present the progress in preclinical studies supporting the use of ZIKV as an OVT for gliomas.

Development and Optimization of a Multiplex Real-Time RT-PCR to Detect SARS-CoV-2 in Human Samples.

PCR and its variants (RT-PCR and qRT-PCR) are valuable and innovative molecular techniques for studying nucleic acids. qPCR has proven to be highly sensitive, efficient, and reproducible, generating reliable results that are easy to analyze. During the COVID-19 pandemic, qPCR became the gold standard technique for detecting the SARS-CoV-2 virus that allowed to confirm the infection event, and those asymptomatic ones, and thus save millions of lives. In-house multiplex qPCR tests were developed worldwide to detect different viral targets and ensure results, follow the infections, and favor the containment of a pandemic. Here, we present the detailed fundamentals of the qPCR technique based on fluorogenic probes and processes to develop and optimize a successful multiplex RT-qPCR test for detecting SARS-CoV-2 that could be used to diagnose COVID-19 accurately.

A strategy for entomo-virological surveillance of yellow fever, dengue, zika, and chikungunya viruses in field-collected mosquitoes.

Arboviruses transmitted by Culicidae insects are significant threats to human health, presenting dynamic transmission cycles and involving different vectors and hosts. The surveillance and characterization of the vectors involved in these cycles are crucial for understanding and preventing potential outbreaks. Therefore, we propose a strategy that we used for entomological surveillance of urban, rural, and sylvatic mosquitoes and to characterize natural infection by four major arboviruses.•Immature and adult mosquitoes were collected intra, peri and extradomicilie of urban and rural households, using different collection methodologies.•Mosquitoes were pooled or separated in head-thorax and abdomen, according to the species.•A multiplex nested RT-PCR (Reverse transcription polymerase chain reaction) method was used for the simultaneous detection of dengue virus (DENV), zika virus (ZIKV), chikungunya virus (CHIKV), and yellow fever virus (YFV).Overall, this strategy proved helpful for vectors surveillance at different ecosystems, as well as for implementing a low-cost molecular surveillance system that allows the early detection of potential outbreaks, and identify other potential vectors involved in viral transmission.

Multifunctional magnetoliposomes as drug delivery vehicles for the potential treatment of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. Therefore, development of novel technologies and strategies to treat PD is a global health priority. Current treatments include administration of Levodopa, monoamine oxidase inhibitors, catechol-O-methyltransferase inhibitors, and anticholinergic drugs. However, the effective release of these molecules, due to the limited bioavailability, is a major challenge for the treatment of PD. As a strategy to solve this challenge, in this study we developed a novel multifunctional magnetic and redox-stimuli responsive drug delivery system, based on the magnetite nanoparticles functionalized with the high-performance translocating protein OmpA and encapsulated into soy lecithin liposomes. The obtained multifunctional magnetoliposomes (MLPs) were tested in neuroblastoma, glioblastoma, primary human and rat astrocytes, blood brain barrier rat endothelial cells, primary mouse microvascular endothelial cells, and in a PD-induced cellular model. MLPs demonstrated excellent performance in biocompatibility assays, including hemocompatibility (hemolysis percentages below 1%), platelet aggregation, cytocompatibility (cell viability above 80% in all tested cell lines), mitochondrial membrane potential (non-observed alterations) and intracellular ROS production (negligible impact compared to controls). Additionally, the nanovehicles showed acceptable cell internalization (covered area close to 100% at 30 min and 4 h) and endosomal escape abilities (significant decrease in lysosomal colocalization after 4 h of exposure). Moreover, molecular dynamics simulations were employed to better understand the underlying translocating mechanism of the OmpA protein, showing key findings regarding specific interactions with phospholipids. Overall, the versatility and the notable in vitro performance of this novel nanovehicle make it a suitable and promising drug delivery technology for the potential treatment of PD.

Multidisciplinary approach for surveillance and risk identification of yellow fever and other arboviruses in Colombia.

Arbovirus, a critical threat to human health, have complex and dynamic life cycles. With reports of Yellow fever virus (YFV) causing spillover from sylvatic transmission cycles, and dengue (DENV), chikungunya (CHIKV), and Zika (ZIKV) viruses expanding from urban to rural areas. We explored a multidisciplinary approach to analyze arbovirus transmission through vectors, and identify biological and sociodemographic determinants associated with their transmission risk in urban and rural areas in a Colombian municipality. We visited 178 urban and 97 rural households, registered sociodemographic characteristics and vaccination status for each of these households, collected adult and immature mosquitoes at the intra-, peri-, and extra-domicile, and surveyed forest patches in rural areas. Infections of YFV, DENV, ZIKV, and CHIKV in the mosquitoes collected in the wild were analyzed using a reverse transcriptase PCR. We identified various risk factors of transmission associated with a high Aedes aegypti infestation in urban areas and their presence in rural settlements and Haemagogus janthinomys and other sylvatic mosquitoes near urban areas. The collected Ae. aegypti females from urban areas had a high infection rate of YFV (5.8%) and CHIKV (58.8%), and those from rural settlements had a high infection rate of DENV (33%), CHIKV (16.7%), and ZIKV (16.7%). The infection rates of YFV in the thorax of the sylvatic mosquitoes H. janthinomys and Aedes serratus collected from the forest patches were 14.3 and 42.1%, respectively. We could discern the transmission determinants associated with climatic, socioeconomic, and anthropogenic factors and YFV vaccination status. This multidisciplinary approach for surveillance of arboviral diseases allowed us to independently detect and integrate factors indicating an early risk of rural transmission of DENV, CHIKV, and ZIKV and rural and urban outbreaks of YFV in the study area. This study provides a helpful tool for designing and focalizing prevention strategies.

Endothelial Dysfunction, HMGB1, and Dengue: An Enigma to Solve.

Dengue is a viral infection caused by dengue virus (DENV), which has a significant impact on public health worldwide. Although most infections are asymptomatic, a series of severe clinical manifestations such as hemorrhage and plasma leakage can occur during the severe presentation of the disease. This suggests that the virus or host immune response may affect the protective function of endothelial barriers, ultimately being considered the most relevant event in severe and fatal dengue pathogenesis. The mechanisms that induce these alterations are diverse. It has been suggested that the high mobility group box 1 protein (HMGB1) may be involved in endothelial dysfunction. This non-histone nuclear protein has different immunomodulatory activities and belongs to the alarmin group. High concentrations of HMGB1 have been detected in patients with several infectious diseases, including dengue, and it could be considered as a biomarker for the early diagnosis of dengue and a predictor of complications of the disease. This review summarizes the main features of dengue infection and describes the known causes associated with endothelial dysfunction, highlighting the involvement and possible relationship between HMGB1 and DENV.

Field validation of the performance of paper-based tests for the detection of the Zika and chikungunya viruses in serum samples.

In low-resource settings, resilience to infectious disease outbreaks can be hindered by limited access to diagnostic tests. Here we report the results of double-blinded studies of the performance of paper-based diagnostic tests for the Zika and chikungunya viruses in a field setting in Latin America. The tests involved a cell-free expression system relying on isothermal amplification and toehold-switch reactions, a purpose-built portable reader and onboard software for computer vision-enabled image analysis. In patients suspected of infection, the accuracies and sensitivities of the tests for the Zika and chikungunya viruses were, respectively, 98.5% (95% confidence interval, 96.2-99.6%, 268 serum samples) and 98.5% (95% confidence interval, 91.7-100%, 65 serum samples) and approximately 2 aM and 5 fM (both concentrations are within clinically relevant ranges). The analytical specificities and sensitivities of the tests for cultured samples of the viruses were equivalent to those of the real-time quantitative PCR. Cell-free synthetic biology tools and companion hardware can provide de-centralized, high-capacity and low-cost diagnostics for use in low-resource settings.

Extracellular vesicles of U937 macrophage cell line infected with DENV-2 induce activation in endothelial cells EA.hy926.

Endothelial activation and alteration during dengue virus (DENV) infection are multifactorial events; however, the role of extracellular vesicles (EVs) in these phenomena is not known. In the present study, we characterized the EVs released by DENV-2 infected U937 macrophage cell line and evaluated the changes in the physiology and integrity of the EA.hy926 endothelial cells exposed to them. U937 macrophages were infected, supernatants were collected, and EVs were purified and characterized. Then, polarized endothelial EA.hy926 cells were exposed to the EVs for 24 h, and the transendothelial electrical resistance (TEER), monolayer permeability, and the expression of tight junction and adhesion proteins and cytokines were evaluated. The isolated EVs from infected macrophages corresponded to exosomes and apoptotic bodies, which contained the viral NS3 protein and different miRs, among other products. Exposure of EA.hy926 cells to EVs induced an increase in TEER, as well as changes in the expression of VE-cadherin and ICAM in addition leads to an increase in TNF-α, IP-10, IL-10, RANTES, and MCP-1 secretion. These results suggest that the EVs of infected macrophages transport proteins and miR that induce early changes in the physiology of the endothelium, leading to its activation and eliciting a defense program against damage during first stages of the disease, even in the absence of the virus.

Prevalence of dengue antibodies in healthy children and adults in different Colombian endemic areas.

OBJECTIVES: Colombia is a dengue hyperendemic country; however, the prevalence of antibodies against dengue in the general population including the inhabitants of rural areas is unknown. This study aimed to determine the prevalence of dengue IgM and IgG antibodies in healthy children and adults in urban and rural areas of seven different endemic regions in Colombia between 2013 and 2015. DESIGN OR METHOD: Blood samples from healthy volunteers (1,318) were processed by serology (by indirect IgG and capture IgM and IgG ELISA) and molecular tests to detect viral RNA and circulating serotypes. RESULTS: The seroprevalence of IgG for dengue were 85% in children and over 90% for adults. In addition to the high IgM positive rate (14.9%) and secondary recent infection marker rate (capture IgG, 16%), 8.4% of the healthy volunteers were positive for dengue virus (DENV) RNA. CONCLUSION: This study confirmed the broad and permanent circulation of DENV in Colombia and the high rates of infection and reinfection suffered by its inhabitants. This information can be used by the health authorities to strengthen vector control and vaccine policies and review the algorithms of diagnosis and disease management in children and adults.