Search Results (17)

Coxsackievirus B3 (CV-B3) infection causes inflammatory conditions such as viral myocarditis and meningitis, and incidence rates are rising annually. While children are more likely to be affected by severe manifestations, the molecular basis of this age-dependent susceptibility is poorly understood. In this study, we used young Balb/c mice at three developmental stages (7-, 14-, and 30-day-old mice) to investigate CV-B3 pathogenesis. Our findings revealed that 7-day-old mice exhibited substantial infection susceptibility and pathological severity compared to older mice. Critically, an age-dependent analysis showed a progressive decline in the expression of CV-B3-binding Coxsackievirus and Adenovirus Receptor (CAR) splice variants (CAR1 and CAR2) at both the transcriptional and translational levels as the mice matured from 7 to 30 days. These receptor isoforms demonstrated a direct correlation with viral replication efficiency in younger hosts. Concurrently, aging was associated with a rise in non-binding CAR variants (CAR3 and CAR4). During CV-B3 infection, the abundance of CAR1/CAR2 in young mice facilitated accelerated viral proliferation, coupled with the hyperactivation of the NLRP3 inflammasome and the expansion of IL-17-producing γδT cells (γδT17 cells). This cascade triggered excessive production of proinflammatory cytokines (IL-1β, IL-18, and IL-17), culminating in pronounced inflammatory infiltrates within cardiac and cerebral tissues. These findings establish NLRP3 inflammasome dysregulation as a critical determinant of CV-B3-induced tissue damage and provide novel insights into the heightened susceptibility to CV-B infection during early life and its associated severe disease rates. Full article

Enteroviruses are a genus of small RNA viruses that are responsible for approximately one billion global infections annually. These infections range in severity from the common cold and flu-like symptoms to more severe diseases, such as viral myocarditis, pancreatitis, and neurological disorders, that continue to pose a global health challenge with limited therapeutic strategies currently available. In the current study, we sought to understand the interaction between coxsackievirus B3 (CVB3), which is a model enterovirus, and macrophage cells, as there is limited understanding of how this virus interacts with macrophage innate immune cells. Our study demonstrated that CVB3 can robustly activate macrophages without apparent viral replication in these cells. We also showed that myeloid cells lacked the viral entry receptor coxsackievirus and adenovirus receptor (CAR). However, the expression of exogenous CAR in RAW264.7 macrophages was unable to overcome the viral replication deficit. Interestingly, the CAR expression was associated with altered inflammatory responses during prolonged infection. Additionally, we identified the autophagy protein LC3 as a novel stimulus for macrophage activation. These findings provide new insights into the mechanisms of CVB3-induced macrophage activation and its implications for viral pathogenesis. Full article

Human adenovirus (HAdV) is a common pathogen, which can lead to various clinical symptoms and—in some cases—central nervous system (CNS) dysfunctions, such as encephalitis and meningitis. Although the initial events of virus entry have already been identified in various cell types, the mechanism of neuronal uptake of adenoviruses is relatively little understood. The aim of this study was to investigate early events during adenoviral infection, in particular to determine the connection between cellular coxsackievirus and adenovirus receptor (CAR), clathrin, caveolin, and early endosomal proteins (EEA1 and Rab5) with the entry of HAdVs into primary murine neurons in vitro. An immunofluorescence assay and confocal microscopy analysis were carried out to determine HAdV4, 5, and 7 correlation with CAR, clathrin, caveolin, and early endosomal proteins in neurons. The quantification of Pearson’s coefficient between CAR and HAdVs indicated that the HAdV4 and HAdV5 types correlated with CAR and that the correlation was more substantial for HAdV5. Inhibition of clathrin-mediated endocytosis using chlorpromazine limited the infection with HAdV, whereas inhibition of caveolin-mediated endocytosis did not affect virus entry. Thus, the entry of tested HAdV types into neurons was most likely associated with clathrin but not caveolin. It was also demonstrated that HAdVs correlate with the Rab proteins (EEA1, Rab5) present in early vesicles, and the observed differences in the manner of correlation depended on the serotype of the virus. With our research, we strove to expand knowledge regarding the mechanism of HAdV entry into neurons, which may be beneficial for developing potential therapeutics in the future. Full article

The coxsackievirus and adenovirus receptor (CAR) is very well known as an epithelial tight junction and cardiac intercalated disc protein; it mediates attachment and infection via the coxsackievirus B3 (CVB3) and type 5 adenovirus. Macrophages play important roles in early immunity during viral infections. However, the role of CAR in macrophages is not well studied in relation to CVB3 infection. In this study, the function of CAR was observed in the Raw264.7 mouse macrophage cell line. CAR expression was stimulated by treatment with lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α). In thioglycollate-induced peritonitis, the peritoneal macrophage was activated and CAR expression was increased. The macrophage-specific CAR conditional knockout mice (KO) were generated from lysozyme Cre mice. The expression of inflammatory cytokine (IL-1β and TNF-α) was attenuated in the KO mice’s peritoneal macrophage after LPS treatment. In addition, the virus was not replicated in CAR-deleted macrophages. The organ virus replication was not significantly different in both wild-type (WT) and KO mice at days three and seven post-infection (p.i). However, the inflammatory M1 polarity genes (IL-1β, IL-6, TNF-α and MCP-1) were significantly increased, with increased rates of myocarditis in the heart of KO mice compared to those of WT mice. In contrast, type1 interferon (IFN-α and β) was significantly decreased in the heart of KO mice. Serum chemokine CXCL-11 was increased in the KO mice at day three p.i. compared to the WT mice. The attenuation of IFN-α and β in macrophage CAR deletion induced higher levels of CXCL-11 and more increased CD4 and CD8 T cells in KO mice hearts compared to those of WT mice at day seven p.i. These results demonstrate that macrophage-specific CAR deletion increased the macrophage M1 polarity and myocarditis in CVB3 infection. In addition, chemokine CXCL-11 expression was increased, and stimulated CD4 and CD8 T cell activity. Macrophage CAR may be important for the regulation of innate-immunity-induced local inflammation in CVB3 infection. Full article

As a tumor-targeting oncolytic adenovirus (Ad), conditionally replicating adenovirus (CRAd) can access the cell interior by binding to coxsackievirus-Ad receptors (CARs) and specifically replicate and destroy cancer cells without lethal effects on normal cells. The transduction efficiency of CRAd is highly dependent on the number of CARs on the cell membrane. However, not all tumor cells highly express CARs; therefore, improving the transduction efficiency of CRAd is beneficial for improving its antitumor effect. In this study, 6-cyclohexyl methyl-β-D-maltoside (6-β-D), as maltoside transfection agent, showed several advantages, including high transfection efficiency, low toxicity, and potential for intensive use and easy operation. With pretreatment of cancer cells with low concentration of 6-β-D (≤5 μg/mL), the transduction efficiency of “model” Ad (eGFP-Ad) was improved 18-fold compared to eGFP-Ad alone. 6-β-D improved the antitumor effect of CRAd while being safe for normal cells, in which treatment with 6-β-D helped the lethal effects of CRAd at a multiplicity-of-infection ratio of 10 (MOI 10) achieve the oncolytic outcomes of MOI 50. This means that if CRAd is combined with 6-β-D, the amount of CRAd used in clinical practice could be greatly reduced without diminishing its curative effect or exposing patients to the potential side effects of high-titer CRAd. Finally, the underlying mechanism of antitumor effect of CRAd + 6-β-D was primarily investigated, and we found that 6-β-D increased the virus’s replication in cancer cells at the early stage of infection and activated the apoptosis signaling pathway at the late stage of the cell cycle. This research will provide an effective technical reference for further improving Ad-mediated cancer gene therapy in clinical practice. Full article

Background: in this report, we describe the case of an 83-year-old woman vaccinated with ChadOx1 nCoV-19 who developed a so-called vaccine-induced thrombosis with thrombocytopenia syndrome and who did not develop any antibodies against the spike protein of SARS-CoV-2 at 30 days following the administration of her first dose of ChadOx1 nCoV-19. Experimental section: two serum samples from the patient and 5 serum samples from 5 control individuals having received the two-dose regimen vaccination with ChadOx1 nCoV-19 were evaluated. In order to investigate the lack of response to the vaccination, a cell model was developed. This model permits to evaluate the interaction between responsive cells (A549) possessing the Coxsackievirus and Adenovirus Receptor (CAR), a defined concentration of ChadOx1 nCoV-19 and serial dilution of the patient or the control serum. The aim was to assess the impact of these sera on the production of the spike (S) protein induced by the transfection of the genetic material of ChadOx1 nCoV-19 into the A549 cells. The S protein is measured in the supernatant using an ELISA technique. Results: interestingly, the serum from the patient who developed the vaccine-induced thrombosis with thrombocytopenia syndrome impaired the production of S protein by the A549 cells transfected with ChadOx1 nCoV-19. This was not observed with the controls who did not interfere with the transfection of ChadOx1 nCoV-19 into A549 cells since the S protein is retrieved in the supernatant fraction. Conclusion: based on the data coming from the clinical and the cell model information, we found a possible explanation on the absence of antibody response in our patient. She has, or has developed, characteristics that prevent the production of the S protein in contrast to control subjects. We were not able to investigate the entire mechanism behind this resistance which deserve further investigations. A link between this resistance and the development of the thrombosis with thrombocytopenia syndrome following vaccination with ChadOx1 nCoV-19 cannot be excluded. Full article

Adenovirus (Ad) is a widely studied viral vector for cancer therapy as it can be engineered to cause selective lysis of cancer cells. However, Ad delivery is limited in treating cancers that do not have coxsackievirus and adenovirus receptors (CAR). To overcome this challenge, Ad-encapsulated liposomes were developed that enhance the delivery of Ads and increase therapeutic efficacy. Cationic empty liposomes were manufactured first, to which an anionic Ad were added, which resulted in encapsulated Ad liposomes through charge interaction. Optimization of the liposome formula was carried out with series of formulation variables experiments using an extrusion process, which is ideal for laboratory-scale small batches. Later, the optimized formulation was manufactured with a homogenization technique—A high shear rotor-stator blending, that is ideal for large-scale manufacturing and is in compliance with Good Manufacturing Practices (GMP). Comparative in vitro transduction, physicochemical characterization, long-term storage stability at different temperature conditions, and in vivo animal studies were performed. Ad encapsulated liposomes transduced CAR deficient cells 100-fold more efficiently than the unencapsulated Ad (p ≤ 0.0001) in vitro, and 4-fold higher in tumors injected in nude mice in vivo. Both extrusion and homogenization performed similarly–with equivalent in vitro and in vivo transduction efficiencies, physicochemical characterization, and long-term storage stability. Thus, two Ad encapsulated liposomes preparation methods used herein, i.e., extrusion vs. homogenization were equivalent in terms of enhanced Ad performance and long-term storage stability; this will, hopefully, facilitate translation to the clinic. Full article

The coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell adhesion, cell signaling, and viral infection. The 8-exon encoded isoform (CAR^Ex8) resides at the apical surface of polarized epithelia, where it is accessible as a receptor for adenovirus entering the airway lumen. Given its pivotal role in viral infection, it is a target for antiviral strategies. To understand the regulation of CAR^Ex8 and determine the feasibility of receptor downregulation, the half-life of total and apical localized CAR^Ex8 was determined and correlated with adenovirus transduction. Total and apical CAR^Ex8 has a relatively short half-life of approximately 2 h. The half-life of apical CAR^Ex8 correlates well with adenovirus transduction. These results suggest that antiviral strategies that aim to degrade the primary receptor for apical adenovirus infection will be effective within a relatively short time frame after application. Full article

Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAR^Ex8), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAR^Ex8 under a doxycycline inducible promoter (MDCK-CAR^Ex8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAR^Ex8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAR^Ex8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections. Full article

Human multipotent mesenchymal stromal cells (hMSCs) are currently developed as cell therapeutics for different applications, including regenerative medicine, immune modulation, and cancer treatment. The biological properties of hMSCs can be further modulated by genetic engineering. Viral vectors based on human adenovirus type 5 (HAdV-5) belong to the most frequently used vector types for genetic modification of human cells in vitro and in vivo. However, due to a lack of the primary attachment receptor coxsackievirus and adenovirus receptor (CAR) in hMSCs, HAdV-5 vectors are currently not suitable for transduction of this cell type without capsid modification. Here we present several transduction enhancers that strongly enhance HAdV-5-mediated gene transfer into both bone marrow- and adipose tissue-derived hMSCs. Polybrene, poly-l-lysine, human lactoferrin, human blood coagulation factor X, spermine, and spermidine enabled high eGFP expression levels in hMSCs. Importantly, hMSCs treated with enhancers were not affected in their migration behavior, which is a key requisite for many therapeutic applications. Exemplary, strongly increased expression of tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) (a secreted model therapeutic protein) was achieved by enhancer-facilitated HAdV-5 transduction. Thus, enhancer-mediated HAdV-5 vector transduction is a valuable method for the engineering of hMSCs, which can be further exploited for the development of innovative hMSC therapeutics. Full article

Oncolytic virotherapy represents one of the most advanced strategies to treat otherwise untreatable types of cancer. Despite encouraging developments in recent years, the limited fraction of patients responding to therapy has demonstrated the need to search for new suitable viruses. Coxsackievirus B3 (CVB3) is a promising novel candidate with particularly valuable features. Its entry receptor, the coxsackievirus and adenovirus receptor (CAR), and heparan sulfate, which is used for cellular entry by some CVB3 variants, are highly expressed on various cancer types. Consequently, CVB3 has broad anti-tumor activity, as shown in various xenograft and syngeneic mouse tumor models. In addition to direct tumor cell killing the virus induces a strong immune response against the tumor, which contributes to a substantial increase in the efficiency of the treatment. The toxicity of oncolytic CVB3 in healthy tissues is variable and depends on the virus strain. It can be abrogated by genetic engineering the virus with target sites of microRNAs. In this review, we present an overview of the current status of the development of CVB3 as an oncolytic virus and outline which steps still need to be accomplished to develop CVB3 as a therapeutic agent for clinical use in cancer treatment. Full article

Coxsackievirus and adenovirus receptor (CAR) is present in epithelial and vascular endothelial cell junctions. We have previously shown a hemorrhagic phenotype in germ-line CAR knock-out mouse embryos; we have also found that CAR interacts with ZO-1 and β-catenin. However, the role of CAR in vascular endothelial junction permeability has not been proven. To understand the roles of CAR in the vascular endothelial junctions, we generated endothelium-specific CAR knockout (CAR-eKO) mice. In the absence of CAR, the endothelial cell layer showed an increase in transmembrane electrical resistance (TER, Ω) and coxsackievirus permeability. Evans blue dye and 70 kDa dextran-FITC were delivered by tail vein injection. We observed increased vascular permeability in the hearts of adult CAR-eKO mice compare with wild-type (WT) mice. There was a marked increase in monocyte and macrophage penetration into the peritoneal cavity caused by thioglycolate-induced peritonitis. We found that CAR ablation in endothelial cells was not significantly increased coxsackievirus B3 (CVB3) induced myocarditis in murine model. However, tissue virus titers were significantly higher in CAR-eKO mice compared with WT. Moreover, CVB3 was detected in the brain of CAR-eKO mice. Endothelial CAR deletion affects the expression of major endothelial junction proteins, such as cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1) in the cultured endothelial cells as well as liver vessel. We suggest that CAR expression is required for normal vascular permeability and endothelial tight junction homeostasis. Furthermore, CVB3 organ penetration and myocarditis severities were dependent on the endothelial CAR level. Full article

Human adenovirus (HAdV)-F40 and -F41 are leading causes of diarrhea and diarrhea-associated mortality in children under the age of five, but the mechanisms by which they infect host cells are poorly understood. HAdVs initiate infection through interactions between the knob domain of the fiber capsid protein and host cell receptors. Unlike most other HAdVs, HAdV-F40 and -F41 possess two different fiber proteins—a long fiber and a short fiber. Whereas the long fiber binds to the Coxsackievirus and adenovirus receptor (CAR), no binding partners have been identified for the short fiber. In this study, we identified heparan sulfate (HS) as an interaction partner for the short fiber of enteric HAdVs. We demonstrate that exposure to acidic pH, which mimics the environment of the stomach, inactivates the interaction of enteric adenovirus with CAR. However, the short fiber:HS interaction is resistant to and even enhanced by acidic pH, which allows attachment to host cells. Our results suggest a switch in receptor usage of enteric HAdVs after exposure to acidic pH and add to the understanding of the function of the short fibers. These results may also be useful for antiviral drug development and the utilization of enteric HAdVs for clinical applications such as vaccine development. Full article

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence. Full article

Introduction: Coxsackievirus/adenovirus receptors (CARs) and desmoglein-2 (DSG2) are similar molecules to adenovirus-based vectors in the cell membrane. They have been found to be associated with lung epithelial cell tumorigenesis and can be useful markers in predicting survival outcome in lung adenocarcinoma (LUAD). Methods: A gene ontology enrichment analysis disclosed that DSG2 was highly correlated with CAR. Survival analysis was then performed on 262 samples from the Cancer Genome Atlas, forming “Stage 1A” or “Stage 1B”. We therefore analyzed a tissue microarray (TMA) comprised of 108 lung samples and an immunohistochemical assay. Computer counting software was used to calculate the H-score of the immune intensity. Cox regression and Kaplan–Meier analyses were used to determine the prognostic value. Results: CAR and DSG2 genes are highly co-expressed in early stage LUAD and associated with significantly poorer survival (p = 0.0046). TMA also showed that CAR/DSG2 expressions were altered in lung cancer tissue. CAR in the TMA was correlated with proliferation, apoptosis, and epithelial–mesenchymal transition (EMT), while DSG2 was associated with proliferation only. The Kaplan–Meier survival analysis revealed that CAR, DSG2, or a co-expression of CAR/DSG2 was associated with poorer overall survival. Conclusions: The co-expression of CAR/DSG2 predicted a worse overall survival in LUAD. CAR combined with DSG2 expression can predict prognosis. Full article