Search Results (252)

Coxsackieviruses B are single-stranded RNA viruses belonging to the Enterovirus genus and are associated with various clinical outcomes, ranging from acute infections to chronic diseases, such as type 1 diabetes (T1D). It was previously shown that inoculation of Swiss albino mice with CVB4 by the intraperitoneal route induced both anti-CVB4 neutralizing and enhancing activities of serum. This study aimed to investigate the humoral immune response of mice inoculated with CVB4 by the mucosal route. Mice were inoculated orally or intranasally with CVB4, and the anti-CVB4 neutralizing activity of serum and saliva was assessed by a cell culture neutralization assay. Anti-enterovirus (EV) IgG and IgA antibodies were detected in serum and saliva, respectively, by ELISA. The serum-dependent enhancement of CVB4 infection in cultures of murine splenocytes was evaluated by detecting intracellular viral RNA using RT-qPCR. At day 45 post-inoculation, an anti-CVB4 neutralizing activity, the extent of which depends on the amount of inoculated infectious particles, was detected in the serum of mice exposed orally or intranasally. An increase in anti-CVB4 neutralizing activity was observed in the saliva of mice inoculated orally or intranasally during the follow-up. Oral or intranasal inoculation of CVB4 induced a systemic IgG and mucosal IgA response. In addition, serum from these mice harbored an anti-CVB4 enhancing activity in vitro. These data indicate that Swiss albino mice exposed to CVB4 via the mucosal route constitute a potentially useful model for testing strategies to promote the production of protective mucosal and systemic anti-CVB4 antibodies and for verifying whether or not enhanced antibodies are produced. Full article

Oncolytic coxsackievirus B3 (oCVB3) strain PD-H has shown potent oncolytic efficacy and a remarkable safety profile in the treatment of colorectal cancer in vivo after intratumoral (i.t.) injection. In this study, we investigated the safety and efficiency of PD-H following intravenous (i.v.) virus administration. When injected i.v. into Balb/C mice bearing subcutaneous Colon-26 tumors, PD-H led to slightly reduced tumor progression and a significant increase in animal survival, but it also caused multi-organ infection and tissue damage. To improve the safety profile of PD-H, we inserted microRNA target sites (miR-TS) of the heart-specific miR-1, pancreas-specific miR-375, liver-specific miR-122, and brain-specific miR-124 or the tumor-suppressor miR-145 into the genome of PD-H and generated the viruses PD-622TS and PD-145TS. Both viruses replicated similarly and induced cytotoxicity comparable to that of PD-H in the colorectal carcinoma cell lines Colon-26 and CT-26Luc. Their replication was inhibited in HEK293T cells transiently transfected with the cognate microRNAs. In vivo, i.v. administration of PD-145TS and PD-622TS to healthy Balb/C mouse resulted in significantly lower viral titers in the organs of mice and led to significantly less-intense pathological alterations compared to PD-H. PD-622TS injected i.v. into Balb/C mice with CT-26Luc-induced peritoneal carcinomatosis did not induce off-target alterations in normal organs, but it failed to induce a therapeutic effect. These data indicate that PD-H or microRNA-regulated PD derivatives exhibit only limited therapeutic efficacy following i.v. injection in colorectal tumor-bearing mice. However, the newly engineered microRNA-regulated PD-H variants demonstrate improved safety profiles. Full article

Coxsackieviruses possess two proteases that are engaged in cleaving viral polyprotein and hijacking host cell processes such as RNA biosynthesis. Integrator subunit 10 (INTS10), a subunit of the integrator complex, facilitates the processing of small nuclear RNAs (U1 and U2 snRNAs) to regulate cellular transcription. We found that INST10 can be cleaved by Coxsackievirus B (CVB). Hence, we hypothesized that INST10 may play a role in CVB infection. In this study, INTS10 is identified as the substrate of CVB3 protease 3C (3C^pro). The cleavage occurs at the residue Q221 and yields a fragment. Depletion of INTS10 enhanced CVB3 replication and blocked snRNA processing. Overexpression of U1 snRNA inhibited CVB3 infection, whereas its knockdown conversely enhanced it. Similarly, knockdown of U2 snRNA was found to promote CVB3 replication. Taken together, the 3C^pro-mediated cleavage of INTS10 disrupts U snRNA processing, which in turn counteracts the inhibitory effect of snRNA U1 and U2 on virus replication and subverts host defenses. Full article

Coxsackievirus B3 (CVB3) is a picornavirus that causes systemic inflammatory diseases including myocarditis, pericarditis, pancreatitis, and meningoencephalitis. We have previously reported that CVB3 induces mitochondrial fission and mitophagy while inhibiting lysosomal degradation by blocking autophagosome-lysosome fusion. This promotes the release of virus-laden mitophagosomes from host cells as infectious extracellular vesicles (EVs), enabling non-lytic viral egress. Transient receptor potential vanilloid 1 (TRPV1), a heat and capsaicin-sensitive cation channel, regulates mitochondrial dynamics by inducing mitochondrial membrane depolarization and fission. In this study, we found that TRPV1 activation by capsaicin dramatically enhances CVB3 egress from host cells via EVs. Released EVs revealed increased levels of viral capsid protein VP1, mitochondrial protein TOM70, and fission protein phospho-DRP1. Moreover, these EVs were enriched in heat shock protein HSP70, suggesting its role in facilitating infectious EV release from cells. Furthermore, TRPV1 inhibition with capsazepine and SB-366791 significantly reduced viral infection in vitro. Our in vivo studies also found that SB-366791 significantly mitigates pancreatic damage and reduces viral titers in a mouse model of CVB3 pancreatitis. Given the lack of understanding regarding factors that contribute to diverse clinical manifestations of CVB3, our study highlights capsaicin and TRPV1 as potential exacerbating factors that facilitate CVB3 dissemination via mitophagy-derived EVs. Full article

Background: Inactivated enterovirus 71 (EV71) vaccines play a vital role in preventing severe cases of hand, foot, and mouth disease, with their quality and stability determined by the degree of viral particle aggregation. Objective: This study aimed to use dynamic light scattering (DLS) for monitoring EV71 particle size, comprehensively evaluate the effects of environmental stresses on viral aggregation, and identify suitable stabilizing agents. Methods: The DLS technique was validated. Using this method, the effects of pH, ionic strength, freeze–thaw cycles, temperature, and mechanical stresses on viral particle size were assessed. Additionally, the ability of different buffer salts and stabilizers to inhibit stress-induced aggregation was systematically evaluated. Results: The DLS method exhibited robust performance. EV71 particles were stable at pH 7.0–7.5. Exposure to 47 °C and magnetic stirring promoted viral aggregation. Phosphate buffer and citrate buffer exhibited the highest inhibitory effects on heat-induced aggregation and stirring-induced aggregation, respectively. M199 and Tween 80 efficiently mitigated heat-induced particle aggregation and shear stress-induced particle aggregation, respectively. Conclusions: This study demonstrated the performance of DLS in viral aggregation monitoring. Additionally, this study revealed tailored stabilization strategies, providing key insights for vaccine formulation and quality control. Full article

Background/Objectives: Coxsackievirus B3 (CVB3), a neurotropic enterovirus, is a major causative agent of viral encephalitis and myocarditis, yet no protective vaccine or effective antiviral therapy is currently available. Autophagy plays a dual role in viral infections, acting as both an antiviral defense and a process that can be exploited by certain viruses. Although CVB3 has been proposed to utilize autophagosomes as replication platforms, the underlying mechanisms remain controversial. Methods: In this study, we investigated the relationship between CVB3 replication and autophagosome formation under starvation-induced conditions and in ATG5 knockout cells. Results: While nutrient deprivation robustly induced autophagy, CVB3 infection did not trigger autophagosome formation. Moreover, viral replication proceeded efficiently in ATG5-deficient cells lacking autophagosomes. Pharmacological modulation of autophagy using rapamycin, a potent autophagy inducer, did not alter intracellular viral titers or protein expression, although extracellular viral release was modestly reduced. These results indicate that CVB3 replication occurs independently of autophagosome formation, suggesting that pharmacological targeting of autophagy provides limited therapeutic benefit. Conclusions: This study refines our understanding of autophagy as an antiviral target and highlights the need to identify alternative host-directed pathways for antiviral drug development. Full article

Coxsackievirus B3 (CVB3) is a primary causative agent of viral myocarditis (VMC), which can lead to both acute and chronic cardiac inflammation accompanied by progressive heart failure and arrhythmias. Although CVB3 has been implicated in various forms of programmed cell death, whether it triggers necroptosis and the underlying mechanisms remains unclear. This study aimed to investigate the role and mechanism of CVB3-induced necroptosis and its effect on viral replication. Using both in vitro and in vivo models, we demonstrated that CVB3 infection significantly upregulates the expression of key necroptotic markers RIP1 and RIP3 in HeLa cells and mouse myocardial tissues. This upregulation was accompanied by elevated intracellular reactive oxygen species (ROS) levels and suppression of the Nrf2/HO-1 antioxidant pathway. Intervention with the necroptosis inhibitor Necrostatin-1 (Nec-1) or the ROS scavenger N-acetylcysteine (NAC) markedly attenuated cell death, suppressed viral replication, and ameliorated myocardial injury and inflammatory responses in infected mice. Mechanistically, CVB3 inhibits the Nrf2/HO-1 pathway, thereby inducing substantial ROS accumulation that promotes necroptosis. This effect can be reversed by NAC treatment. Our study reveals a novel mechanism through which CVB3 induces ROS-dependent necroptosis via the suppression of the Nrf2/HO-1 pathway, providing new insights into the pathogenesis of viral myocarditis and suggesting potential therapeutic strategies. Full article

Enteroviruses (EVs), particularly those within the species Enterovirus A and B, represent a significant global public health burden, especially in infants and young children. While often causing self-limiting hand, foot, and mouth disease (HFMD), certain serotypes can lead to severe neurological and cardiopulmonary complications. This comprehensive review focuses on the major pathogenic serotypes, including enterovirus A71 (EV-A71), coxsackievirus A16 (CV-A16), coxsackievirus A6 (CV-A6), coxsackievirus B3 (CV-B3), and enterovirus D68 (EV-D68). We began by reconstructing a phylogenetic tree based on VP1 protein sequences, elucidating the genetic relationships and evolutionary patterns among these serotypes, which underpin their diverse antigenicity and epidemiology. Building upon this genetic foundation, the review then provides a detailed synthesis of their distinct pathogenesis, highlighting the five-phase clinical progression from exanthematous phase to convalescence, and their unique tropisms for target organs such as the central nervous system and heart. Progressing to the molecular mechanisms, a critical component of this work is a systematic summary of the specific host receptors that mediate viral entry, including SCARB2 for EV-A71 and CV-A16, sialic acid and ICAM-5 for EV-D68, and CAR/CD55 for CV-B3, explaining the mechanistic basis for their tissue specificity and pathogenicity. Finally, to translate these insights into clinical applications, we critically evaluate the current landscape of vaccine development, noting the high efficacy (~90%) of inactivated EV-A71 vaccines in Asia and the significant global success of poliovirus vaccines, while also addressing the stark lack of cross-protective or licensed vaccines for other prevalent serotypes like CV-A16, CV-A6, and EV-D68. The review concludes that the high genetic diversity and serotype-specific immunity of enteroviruses pose a major challenge, necessitating a concerted shift towards the development of broad-spectrum vaccines and therapeutics informed by an integrated understanding of viral evolution, receptor usage, and pathogenesis. Full article

Nuclear factor of activated T cells 5 (NFAT5), an osmosensitive transcription factor, has been shown to protect against coxsackievirus B3 (CVB3)-induced myocarditis but is susceptible to cleavage by viral proteases. Identifying agents that upregulate NFAT5 may offer a novel antiviral strategy. Cyclodextrins, cyclic oligosaccharides that influence cellular osmolality, are promising candidates. In this study, we demonstrate that NFAT5 is critical for maintaining desmosomal integrity in cardiomyocytes. Cardiac-specific Nfat5-knockout mice showed a significant reduction in desmosomes, as observed by transmission electron microscopy. Furthermore, we identified desmoplakin (DSP), a structural desmosomal protein, as a direct transcriptional target of NFAT5, with reduced expression in Nfat5-knockout mouse hearts and NFAT5-knockdown HeLa cells. Notably, treatment with 5 mM cyclodextrin significantly upregulated NFAT5 expression with minimal cytotoxicity, restored DSP expression, and suppressed CVB3 replication by inhibiting viral RNA transcription, protein synthesis, and virion production. Additionally, cyclodextrin reduced mRNA levels of proinflammatory cytokines interleukin-1 beta and interleukin-8, indicating its potential role as an alleviator of excessive cytokine production. These findings identify NFAT5 as a key regulator of desmoplakin expression and prove cyclodextrin as a dual-functioning agent in counteracting cardiac damage through NFAT5-DSP-mediated protection of desmosome integrity and suppressing proinflammatory cytokine expression in CVB3-induced myocarditis. Full article

Background/Objectives: Enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) are major causative agents of hand, foot and mouth disease (HFMD), often co-circulating and occasionally undergoing genetic recombination. While natural recombinants often involve genomic regions encoding non-structural proteins, their effects on replication and pathogenesis remain unclear. Methods: To address this, four chimera viruses (Chi-CCE, Chi-ECE, Chi-EEC, and Chi-CEC) were constructed with 5′UTR, capsid P1, and non-structural P2 and P3 genes, from CVA16 (denoted as C) or EV-A71 (denoted as E). These chimeras were tested for replication kinetics and cytopathic effects in rhabdomyosarcoma cells while in vivo virulence and protection efficacy were evaluated using a newborn BALB/c mouse model. Results: All chimeric viruses remained viable and exhibited higher replication than CVA16. In vivo, all chimeric viruses were avirulent except Chi-CCE and CVA16, which showed high virulence and viral titres in the brains and limbs of infected newborn mice. This suggests that 5′UTR and capsid P1 genes of CVA16 are critical genetic determinants of virulence. Notably, only the anti-inflammatory cytokine IL-10 was elevated, suggesting potential immune modulation during infection. Inactivated Chi-CCE immunisation conferred 100% protection against lethal CVA16 or mouse-adapted EV-A71 challenge revealing its potential as a bivalent vaccine candidate. Conclusions: Our study demonstrates that recombination between CVA16 and EV-A71 influences viral virulence and protection efficacy with implications for future development of multivalent vaccines. Full article

As part of the WHO’s Global Polio Eradication Initiative, acute flaccid paralysis (AFP) surveillance in children under 15 years old is crucial for monitoring the emergence of polioviruses and tracking Non-Polio Enteroviruses (NPEVs). This study outlines the past 17 years of AFP surveillance in Greece from 2008 to 2024, during which a total of 256 AFP cases were recorded. Stool samples from these cases were analyzed using virus isolation in cell cultures (RD/L20B) and sequencing of NPEV-positive samples. The Attica region reported the highest number of cases with 81 (31%), followed by Central Macedonia and Crete, each with 29 cases (11%). The overall analysis of fecal specimens identified the etiological agent in 18 (7%) specimens, with 13 (4.7%) classified as NPEVs, 4 (1.5%) as adenoviruses, and 1 (0.4%) as a parechovirus. Coxsackievirus A, Coxsackievirus B, and various Echoviruses were the most frequently detected NPEV types. Notably, more than half of these positive specimens (10/18) were from the Attica region, which has the highest population density. These findings highlight the ongoing relevance of AFP surveillance in polio-free settings for broader pathogen monitoring and public health preparedness. Continued vigilance and investment in AFP surveillance are critical to sustaining Greece’s polio-free status and detecting emerging viral threats. Full article

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

The enterovirus Coxsackievirus B3 causes a range of serious health problems, including aseptic meningitis, myocarditis, and pancreatitis. Currently, Coxsackievirus B3 has no targeted antiviral treatments or vaccines, leaving supportive care as the primary management option. Understanding how Coxsackievirus B3 interacts with and alters the blood–brain barrier may help identify new therapies to combat this often-devastating infection. We reanalyzed a previously published RNA sequencing dataset for Coxsackievirus B3-infected human-induced pluripotent stem-cell-derived brain endothelial cells (iBECs) to examine how Coxsackievirus B3 altered mRNA expression. By integrating GSEA, EnrichR, and iLINCs-based perturbagen analysis, we present a novel, systems-level approach to uncover potential drug repurposing candidates for CVB3 infection. We found dynamic changes in host transcriptomic response to Coxsackievirus B3 infection at 2- and 5-day infection time points. Downregulated pathways included ribosomal biogenesis and protein synthesis, while upregulated pathways included a defense response to viruses, and interferon production. Using iLINCs transcriptomic analysis, MEK, PDGFR, and VEGF inhibitors were identified as possible novel antiviral therapeutics. Our findings further elucidate Coxsackievirus B3-associated pathways in (iBECs) and highlight potential drug repurposing candidates, including pelitinib and neratinib, which may disrupt Coxsackievirus B3 pathology at the blood–brain barrier (BBB). Full article

Background: Enterovirus A71 (EV-A71) is considered as the primary causative agent of hand, foot, and mouth disease (HFMD) in young children, leading to severe neurological complications and contributing to substantial mortalities in recent HFMD outbreaks across Asia. Despite this, there is currently no effective antiviral treatment available for EV-A71. RNA interference (RNAi) is a powerful mechanism of post-transcriptional gene regulation that utilizes small interfering RNA (siRNA) to target and degrade specific RNA sequences. Objectives: The aim of this study was to design various siRNAs targeting EV-A71 genomic regions and evaluate the RNAi efficacy against a novel, previously genetically uncharacterized EV-A71 strain. Methods: A novel EV-A71 strain was first sequenced to design target-specific siRNAs. The viral titers, viral protein expression, cytopathic effects, and cell viability of EV-A71-infected HeLa cells were examined to evaluate the specific viral inhibition by the siRNAs. Results: A substantial reduction in viral titers and viral protein synthesis was observed in EV-A71-infected HeLa cells treated with specific siRNAs targeting the VP4, VP3, 2B, and 3A genes. siRNAs delayed cytopathic effects and increased cell viability of EV-A71-infected HeLa cells. Nonspecific interferon induction caused by siRNAs was not observed in this study. In contrast, replication of coxsackievirus B3, another important member of the Enterovirus genus, remained unaffected. Conclusions: Overall, the findings demonstrate that RNAi targeting genomic regions of EV-A71 VP4, VP3, 2B, or 3A could become a potential strategy for controlling EV-A71 infection, and this promising result can be integrated into future anti-EV-A71 therapy developments. Full article

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer characterized by a dense desmoplastic stroma and a highly immunosuppressive tumor microenvironment (TME). The focal adhesion kinase (FAK), a non-receptor tyrosine kinase, is considered a critical regulator of various cellular processes involved in cancer development. FAK inhibitors (FAKi) have proven to be promising therapeutics for cancer treatment including for pancreatic cancer. As monotherapy, however, FAKi showed only a modest effect in clinical studies. In this study, we investigated the cytotoxicity of six FAKi (Defactinib, CEP-37440, VS-4718, VS-6062, Ifebemtinib and GSK2256098) used in clinical trials on five pancreatic tumor cell lines. We further examined whether their anti-tumor activity can be enhanced by combination with the oncolytic coxsackievirus B3 (CVB3) strain PD-H. IC₅₀ analyses identified Defactinib and CEP-37440 as the most potent inhibitors of tumor cell growth. VS-4718, VS-6062, and Ifebemtinib showed slightly lower activity, while GSK2256098 was largely ineffective. The combination of Defactinib, CEP-37440, VS-4718, and VS-6062 with PD-H resulted in varying effects on cytotoxicity, depending on the cell line and the specific FAKi, ranging from no enhancement to a pronounced increase. Using the Chou–Talalay method, we determined combination indices (CI), revealing synergistic, additive, but also antagonistic interactions between the respective FAKi and PD-H. Considering both oncolytic efficacy and the CI, the greatest enhancement in oncolytic activity was achieved when VS-4718 or CEP-37440 was combined with PD-H. These findings indicate that co-treatment with PD-H can potentiate the therapeutic activity of the selected FAKi and may represent a novel strategy to improve treatment outcomes in PDAC. Full article