Exploring the interplay between miRNAs, apoptosis and viral load, in Dengue virus infection.

Dengue virus (DENV) is a major global health concern, causing millions of infections annually. Understanding the cellular response to DENV infection is crucial for developing effective therapies. This study provides an in-depth analysis of the cellular response to Dengue virus (DENV) infection, with a specific focus on the interplay between microRNAs (miRNAs), apoptosis, and viral load across different DENV serotypes. Utilizing a variety of cell lines infected with four DENV serotypes, the research methodically quantifies viral load, and the expression levels of miRNA-15, miRNA-16, and BCL2 protein, alongside measuring apoptosis markers. Methodologically, the study employs quantitative PCR for viral load and miRNA expression analysis, and Western blot for apoptosis and BCL2 detection, with a statistical framework that includes ANOVA and correlation analysis to discern significant differences and relationships. The findings reveal that despite similar viral loads across DENV serotypes, DENV-2 exhibits a marginally higher load. A notable upregulation of miRNA-15 and miRNA-16 correlates positively with increased viral load, suggesting their potential role in modulating viral replication. Concurrently, a marked activation of caspases 3 and 7, along with changes in BCL2 protein levels, underscores the role of apoptosis in the cellular response to DENV infection. Conclusively, the study enhances the understanding of miRNA involvement in DENV pathogenesis, highlighting miRNA-15 and miRNA-16 as potential regulatory agents in viral replication and apoptosis. These findings pave the way for further exploration into miRNA-based therapeutic strategies against DENV infection.

Experimental Dengue Virus Type 4 Infection Increases the Expression of MicroRNAs-15/16, Triggering a Caspase-Induced Apoptosis Pathway.

The World Health Organization has estimated the annual occurrence of approximately 392 million dengue virus (DENV) infections in more than 100 countries where the virus is endemic, which represents a serious threat to humanity. DENV is a serologic group with four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) belonging to the genus Flavivirus, in the family Flaviviridae. Dengue is the most widespread mosquito-borne disease in the world. The ~10.7 kb DENV genome encodes three structural proteins (capsid (C), pre-membrane (prM), and envelope (E)) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS1 protein is a membrane-associated dimer and a secreted, lipid-associated hexamer. Dimeric NS1 is found on membranes both in cellular compartments and cell surfaces. Secreted NS1 (sNS1) is often present in patient serum at very high levels, which correlates with severe dengue symptoms. This study was conducted to discover how the NS1 protein, microRNAs-15/16 (miRNAs-15/16), and apoptosis are related during DENV-4 infection in human liver cell lines. Huh 7.5 and HepG2 cells were infected with DENV-4, and miRNAs-15/16, viral load, NS1 protein, and caspases-3/7 were quantified after different durations of infection. This study demonstrated that miRNAs-15/16 were overexpressed during the infection of HepG2 and Huh 7.5 cells with DENV-4 and had a relationship with NS1 protein expression, viral load, and the activity of caspases-3/7, thus making these miRNAs potential injury markers during DENV infection in human hepatocytes.

Replication of Rocio virus in primary cultures of mouse neural cells.

OBJECTIVE: This study verified the replication efficiency of the Rocio virus in a primary culture of mouse neural cells. METHODS: Mixed primary cultures (neurons/glia) obtained from the brains of newborn isogenic BALB/c mice were inoculated with Rocio virus on the 7 th day of culture, and the development of cytopathogenic effects was monitored. The infection was confirmed via immunocytochemistry (anti-ROCV), while viral replication was quantified in infected primary cultures. The titration method used depended on the infection period. RESULTS: Rocio virus efficiently infected primary cultured neural cells, with the highest viral titer causing cytopathic changes was observed at 2 days post infection. The virus-infected primary culture survived for up to 7 days post infection, and viral load quantitation showed viral replication kinetics compatible with the cell death kinetics of cultures. CONCLUSION: The findings of this study suggest that mouse neural cell primary cultures support Rocio virus replication and could be used as an alternative system for studying Flavivirus infection in the central nervous system.

Comparative Analysis of Human Hepatic Lesions in Dengue, Yellow Fever, and Chikungunya: Revisiting Histopathological Changes in the Light of Modern Knowledge of Cell Pathology.

Arboviruses, such as yellow fever virus (YFV), dengue virus (DENV), and chikungunya virus (CHIKV), present wide global dissemination and a pathogenic profile developed in infected individuals, from non-specific clinical conditions to severe forms, characterised by the promotion of significant lesions in different organs of the harbourer, culminating in multiple organ dysfunction. An analytical cross-sectional study was carried out via the histopathological analysis of 70 samples of liver patients, collected between 2000 and 2017, with confirmed laboratory diagnoses, who died due to infection and complications due to yellow fever (YF), dengue fever (DF), and chikungunya fever (CF), to characterise, quantify, and compare the patterns of histopathological alterations in the liver between the samples. Of the histopathological findings in the human liver samples, there was a significant difference between the control and infection groups, with a predominance of alterations in the midzonal area of the three cases analysed. Hepatic involvement in cases of YF showed a greater intensity of histopathological changes. Among the alterations evaluated, cell swelling, microvesicular steatosis, and apoptosis were classified according to the degree of tissue damage from severe to very severe. Pathological abnormalities associated with YFV, DENV, and CHIKV infections showed a predominance of changes in the midzonal area. We also noted that, among the arboviruses studied, liver involvement in cases of YFV infection was more intense.

Replication of Rocio virus in primary cultures of mouse neural cells

ABSTRACT Objective This study verified the replication efficiency of the Rocio virus in a primary culture of mouse neural cells. Methods Mixed primary cultures (neurons/glia) obtained from the brains of newborn isogenic BALB/c mice were inoculated with Rocio virus on the 7 th day of culture, and the development of cytopathogenic effects was monitored. The infection was confirmed via immunocytochemistry (anti-ROCV), while viral replication was quantified in infected primary cultures. The titration method used depended on the infection period. Results Rocio virus efficiently infected primary cultured neural cells, with the highest viral titer causing cytopathic changes was observed at 2 days post infection. The virus-infected primary culture survived for up to 7 days post infection, and viral load quantitation showed viral replication kinetics compatible with the cell death kinetics of cultures. Conclusion The findings of this study suggest that mouse neural cell primary cultures support Rocio virus replication and could be used as an alternative system for studying Flavivirus infection in the central nervous system.

New Insights into the Mechanism of Immune-Mediated Tissue Injury in Yellow Fever: The Role of Immunopathological and Endothelial Alterations in the Human Lung Parenchyma.

Yellow fever (YF) may cause lesions in different organs. There are no studies regarding the in situ immune response in the human lung and investigating immunopathological aspects in fatal cases can help to better understand the evolution of the infection. Lung tissue samples were collected from 10 fatal cases of human yellow fever and three flavivirus-negative controls who died of other causes and whose lung parenchymal architecture was preserved. In YFV-positive fatal cases, the main histopathological changes included the massive presence of diffuse alveolar inflammatory infiltrate, in addition to congestion and severe hemorrhage. The immunohistochemical analysis of tissues in the lung parenchyma showed significantly higher expression of E-selectin, P-selectin, ICAM-1, VCAM-1 in addition to cytokines such as IL-4, IL-10, IL-13, TNF- α, IFN-γ and TGF-ß compared to the negative control. The increase in immunoglobulins ICAM-1 and VCAM-1 results in strengthening of tissue transmigration signaling. E-selectin and P-selectin actively participate in this process of cell migration and formation of the inflammatory infiltrate. IFN-γ and TNF-α participate in the process of cell injury and viral clearance. The cytokines IL-4 and TGF-ß, acting in synergism, participate in the process of tissue regeneration and breakdown. The anti-inflammatory cytokines IL-4, IL-10 and IL-13 also act in the reduction of inflammation and tissue repair. Our study indicates that the activation of the endothelium aggravates the inflammatory response by inducing the expression of adhesion molecules and cytokines that contribute to the rolling, recruitment, migration and eliciting of the inflammatory process in the lung parenchyma, contributing to the fatal outcome of the disease.

Emergence of New Immunopathogenic Factors in Human Yellow Fever: Polarisation of the M1/M2 Macrophage Response in the Renal Parenchyma.

Macrophages in the kidney play a pathogenic role in inflammation and fibrosis. Our study aimed to understand the polarisation of the M1 and M2 phenotypic profiles of macrophages in injured kidney tissue retrieved from fatal cases of yellow fever virus (YFV). A total of 11 renal tissue biopsies obtained from patients who died of yellow fever (YF) were analysed. To detect antibodies that promote the classical and alternative pathways of macrophage activation, immunohistochemical analysis was performed to detect CD163, CD68, inducible nitric oxide synthase (iNOS), arginase 1, interleukin (IL)-4, IL-10, interferon (IFN)-γ, IFN-ß, tumour necrosis factor (TNF)-α, IL-13, and transforming growth factor (TGF)-ß. There was a difference in the marker expression between fatal cases of YFV and control samples, with increased expression in the cortical region of the renal parenchyma. The immunoexpression of CD68 and CD163 receptors suggests the presence of activated macrophages migrating to infectious foci. The rise in IL-10, IL-4, and IL-13 indicated their potential role in the inactivation of the inflammatory macrophage response and phenotypic modulation of M2 macrophages. The altered expression of IFN-γ and IFN-ß demonstrates the importance of the innate immune response in combating microorganisms. Our findings indicate that the polarisation of M1 and M2 macrophages plays a vital role in the renal immune response to YFV.