The continuous threats posed by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, including the emergence of potentially more infectious and deadly variants, necessitate ongoing studies to uncover novel and detailed mechanisms driving disease severity. Using single-cell transcriptomics, we conducted a secondary data analysis of bronchoalveolar lavage fluid (BALF) from COVID-19 patients of varying severities and healthy controls to comprehensively examine immune responses. We observed significant immune cell alterations correlating with disease severity. In severe cases, macrophages showed upregulation of pro-inflammatory genes TNFα and IL1β, contributing to severe inflammation and tissue damage. Neutrophils exhibited increased activation, marked by S100A8, CXCL8, and IL1β expression, with extended viability and reduced phagocytosis. Genes such as MCL1 and HIF1α supported extended viability, while MSR1 and MRC1 indicated reduced phagocytosis. Enhanced formation of neutrophil extracellular traps (NETs) and reduced clearance, indicated by NET-associated markers, were linked to thrombo-inflammation and organ damage. Both macrophages and neutrophils in severe cases showed impaired efferocytosis, indicated by decreased expression of MSR1 and TREM2 in macrophages and downregulation of FCGR3B in neutrophils, leading to the accumulation of apoptotic cells and exacerbating inflammation. Severe cases were characterized by M1 macrophages with high TNFα and IL1β, while milder cases had M2 macrophages with elevated PPARγ. Dendritic cells (DCs) in severe cases exhibited reduced proportions and attenuated expression of MHC class I genes (HLA-A, HLA-B, HLA-C) and co-stimulatory molecules (CD80, CD86), alongside increased cytochrome c expression, indicating impaired antigen presentation and enhanced apoptosis. NK and T cells in severe cases demonstrated altered receptor and gene expression, with increased activation markers IFNγ and ISG15, suggesting a paradoxical state of activation and exhaustion. This analysis highlights the critical role of dysregulated neutrophil, macrophage, dendritic cell, NK, and T cell responses in severe COVID-19, identifying potential therapeutic targets and providing novel insights into the disease.

The presence of an exopolysaccharide (EPS) layer surrounding bacterial cells, termed a “glycocalyx”, confers protection against toxic molecules. However, the effect of glycocalyx integrity on the tolerance to such agents is poorly understood. Using a modified disc-diffusion assay, we tested the susceptibility to a panel of antibiotics and oxidative stress-inducing compounds of various mutant strains of the social predatory Gram-negative soil bacterium Myxococcus xanthus; the selected mutants were those that manifest different physical states of their respective EPS glycocalyces. While the overall presence of an EPS layer was indeed beneficial for tolerance, the integrity of this layer was also found to affect the susceptibility of the bacterium to killing; however, this finding was not universal, and instead was dependent on the specific compound tested. Thus, the integrity of the cell-surface EPS glycocalyx plays an important role in the tolerance of M. xanthus to harmful compounds.

The dengue virus (DENV) exploits host cell exosome pathways to disseminate and evade immunity. However, the host factors enabling this process remain poorly defined. Here, we demonstrate that DENV infection robustly induces expression of the short isoform of Reticulon 3 (RTN3S) in hepatic (Huh7) and monocytic cells, and that RTN3S is a critical driver of infectious exosome biogenesis. RTN3S physically associates with double-stranded viral RNA and the DENV non-structural protein 3 (NS3) in infected cells, indicating its integration into the viral replication complex. Loss of RTN3 markedly reduced exosome production and the exosomal export of viral RNA and proteins, demonstrating that RTN3S is required for efficient exosome-mediated viral release. Conversely, overexpression of full-length RTN3S dramatically increased the release of infectious virus-containing exosomes; truncation of the RTN3S C-terminal domain abolished this enhancement, confirming the essential role of the C-terminus in RTN3S’s pro-viral exosomal function. In DENV-infected monocytes, we observed a shift toward a CD16-positive intermediate phenotype, accompanied by the upregulation of genes involved in vesicle biogenesis and stress response. These infected monocytes also secreted higher levels of inflammatory cytokines. Similarly, monocytes from Dengue patients exhibited high RTN3 expression, which correlated with an expansion of intermediate (CD16⁺) subsets and enriched expression of vesicle trafficking machinery genes. These findings reveal a previously unrecognized mechanism by which DENV hijacks RTN3S to promote the formation of infectious exosomes, thereby facilitating viral dissemination and immune evasion. RTN3S thus represents a novel element of the Dengue pathogenesis and a potential target for host-directed antiviral strategies.

Neutrophils are among the early responders of the innate immune system and play a key role in host defense against viral infections. Beyond their classical antimicrobial functions, neutrophils can engage in a specialized defense mechanism by releasing web-like extracellular DNA known as neutrophil extracellular traps (NETs). These extracellular traps are a mesh-like network of chromatin DNA decorated with cellular components, including histones, proteases, and antimicrobial enzymes, that function to contain and limit the spread of pathogens. While NET formation contributes to antiviral immunity, accumulating evidence indicates that excessive or dysregulated NET formation can significantly contribute to immunopathology during viral infections. Thus, depending on the context and outcome, NET formation may be viewed as a double-edged sword. Therefore, understanding the regulatory mechanisms governing NET formation and its harmful effects is critical for developing therapeutic strategies that enhance antiviral defense while minimizing tissue damage. In this review, we provide a comprehensive overview of the molecular mechanisms that drive NET formation and clearance, with a particular focus on how viruses modulate these processes to influence disease outcome. We also discuss the pathways underlying NET formation and subsequent neutrophil cell death (NETosis), including canonical and non-canonical pathways, and highlight key signaling axes involving SYK, MAPKs, and NF-κB. Using SARS-CoV-2 and hepatitis B virus as representative models, we examine how different viral components trigger, exploit, or evade NET targeting and how persistent accumulation of NETs can contribute to hyperinflammation, progressive tissue injury, and post-viral syndromes. We further explore emerging evidence linking impaired NET clearance and neutrophil heterogeneity, particularly low-density neutrophils (LDNs), to chronic inflammation and post-viral sequelae such as long COVID and autoimmune hepatitis. Finally, we summarize current and emerging therapeutic strategies aimed at modulating NET formation or enhancing NET clearance. Altogether, this review underscores the dual nature of NETs in viral infections, highlighting their potential roles in antiviral defense and tissue injury, and provides a framework for the development of targeted interventions to limit virus-induced immunopathology.

Dengue virus (DENV) continues to pose a major global health burden, yet therapeutic options remain limited due to the virus's capacity for immune evasion, serotype variability, and persistence. While exosomes have been implicated as vehicles for viral dissemination and immune evasion, the cellular mechanisms underlying their generation during DENV infection remain poorly defined. Here, we identify the endoplasmic reticulum (ER)-associated host protein Reticulon 3 (RTN3), particularly its short isoform RTN3S, as a critical regulator of replication-competent viral cargo loading during infectious exosome biogenesis in DENV infection. Using hepatic and monocytic cell models, we revealed that RTN3S expression is induced upon infection and that RTN3S directly associates with DENV replication complexes, facilitating the packaging of replication-competent viral RNA and host proteins into infectious exosomes. Loss of RTN3 function via CRISPR-Cas9 markedly attenuated exosome production and reduced the transfer of infectious viral components to recipient naïve cells. Mutational analyses of RTN3S further revealed that both its N-terminal amphipathic and C-terminal domains are essential for exosomal loading of viral material. Single-cell RNA-sequencing of peripheral blood mononuclear cells (PBMCs) from DENV-infected individuals confirmed RTN3 upregulation in monocytes, particularly in those displaying intermediate/classical phenotypes, and revealed a transcriptional signature linking RTN3 to ER stress, vesicle trafficking, and impaired antiviral responses. These findings uncover a previously unrecognized RTN3-centered mechanism by which DENV hijacks the host exosomal machinery to propagate infection and potentially escape immune surveillance. Thus, our findings demonstrate a novel function for RTN3 in orchestrating the biogenesis of infectious exosomes, providing mechanistic insight and identifying a new therapeutic axis for combating flavivirus infections through host-directed approaches.

Extracellular vesicles (EVs) can disseminate replication-competent viral genomes complexed with selected host proteins, enabling stealth cell-to-cell transfer within lipid membrane-enclosed bubbles. In addition to complementing free-virion spread, EV-associated genomes can be protected from neutralizing antibodies and persist under conditions in which classical virion production decreases. Here, we propose a route-resolved framework in which interconnected cellular secretory pathways, including endoplasmic reticulum (ER) remodeling, multivesicular body (MVB) biogenesis, secretory autophagy, and plasma-membrane budding, jointly generate EV heterogeneity and create discrete opportunities for the capture, protection, and export of infectious cargo. We highlight reticulon-3 (RTN3), an ER-shaping protein, as an upstream regulator that can couple infection-induced ER microdomains to endosomal docking and autophagy-linked trafficking decisions that bias intermediates toward secretion rather than degradation. Supporting this view, transmission electron microscopy of dengue virus-infected cells reveals extensive vesicular remodeling, including irregular MVBs adjacent to the plasma membrane and autophagosome-like double-membrane structures, consistent with altered vesicular routing following RTN3 perturbation. Collectively, these route-resolved, spatially organized spatio-organelle changes support a pathomechanistic model in which RTN3-mediated ER remodeling reshapes ER-endosome-autophagy trafficking interfaces, creating regulated decision points that can be leveraged to stratify infectious EV subsets (with infectivity-linked single-vesicle and quantitative proteomics approaches) and to inform host-directed strategies that curb non-lytic viral dissemination.