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Background and Aims: Viral infections are the leading cause of myocarditis. Besides acute cardiac complications, late-stage sequelae such as myocardial fibrosis may develop, importantly impacting the prognosis. Coxsackievirus B3 (CVB)-induced myocarditis in mice is the most commonly used translational model to study viral myocarditis and has provided the majority of our current understanding of the disease pathophysiology. Nevertheless, the late stages of disease, encompassing fibrogenesis and arrhythmogenesis, have been underappreciated in viral myocarditis research to date. The present study investigated the natural history of CVB-induced myocarditis in C57BL/6J mice, expanding the focus beyond the acute phase of disease. In addition, we studied the impact of sex and inoculation dose on the disease course., Methods and Results: C57BL/6J mice (12 weeks old; n=154) received a single intraperitoneal injection with CVB to induce viral myocarditis, or vehicle (PBS) as control. Male mice (n=92) were injected with 5 × 10 5 (regular dose) (RD) or 5 × 10 6 (high dose) (HD) plaque-forming units of CVB, whereas female mice received the RD only. Animals were sacrificed 1, 2, 4, 8, and 11 weeks after CVB or PBS injection. Virally inoculated mice developed viral disease with a temporary decline in general condition and weight loss, which was less pronounced in female animals (P<.001). In male CVB mice, premature mortality occurred between days 8 and 23 after inoculation (RD: 21%, HD: 20%), whereas all female animals survived. Over the course of disease, cardiac inflammation progressively subsided, with faster resolution in female mice. There were no substantial group differences in the composition of the inflammatory cell infiltrates: predominance of cytotoxic T cells at day 7 and 14, and a switch from arginase1-reactive macrophages to iNOS-reactive macrophages from day 7 to 14 were the main findings. There was concomitant development and maturation of different patterns of myocardial fibrosis, with enhanced fibrogenesis in male mice. Virus was almost completely cleared from the heart by day 14. Serum biomarkers of cardiac damage and cardiac expression of remodeling genes were temporarily elevated during the acute phase of disease. Cardiac CTGF gene upregulation was less prolonged in female CVB animals. In vivo electrophysiology studies at weeks 8 and 11 demonstrated that under baseline conditions (i.e. in the absence of proarrhythmogenic drugs), ventricular arrhythmias could only be induced in CVB animals. The cumulative arrhythmia burden throughout the entire stimulation protocol was not significantly different between CVB and control groups., Conclusion: CVB inoculation in C57BL/6J mice represents a model of acute self-limiting viral myocarditis, with progression to different patterns of myocardial fibrosis. Sex, but not inoculation dose, seems to modulate the course of disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Viral myocarditis (VMC) is an inflammatory disease of the myocardium caused by cardioviral infection, especially coxsackievirus B3 (CVB3), and is a major contributor to acute heart failure and sudden cardiac death in children and adolescents. LncRNA MALAT1 knockdown reportedly inhibits the differentiation of Th17 cells to attenuate CVB3-induced VMC in mice. Moreover, long non-coding RNAs (lncRNAs) interact with RNA-binding proteins (RBPs) to regulate UPF1-mediated mRNA decay. However, it remains unclear whether MALAT1 can bind to UPF1 to mediate the mRNA decay of its target genes in VMC. Herein, we aimed to explore the effect of lncRNA MALAT1 on UPF1-mediated SIRT6 mRNA decay in VMC using in vivo and in vitro experiments. CVB3-infected BABL/C mice were used as VMC models, and MALAT1 interfering adenovirus was injected to achieve MALAT1 knockdown. The heart function of the VMC mice was assessed using echocardiography. Pathological changes in myocardial tissues were assessed after hematoxylin-eosin staining. Myocardial injury and inflammation were evaluated by measuring creatine kinase isoenzyme B, cardiac troponin T, interleukin (IL)-1β, and IL-18. TUNEL staining was performed to assess apoptosis in myocardial tissues. In vitro experiments were performed using H9c2 cells after transfection and CVB3 infection. The lactic dehydrogenase release, caspase-1 activity, and IL-1β and IL-18 levels in the cellular supernatant were detected. Western blotting was performed to determine the expression of pyroptosis-related proteins (GSDMD-N, NLRP3, ASC, and Cleaved-Caspase-1) and Wnt/β-catenin signal pathway-related proteins (Wnt1, β-catenin, and p-GSK-3β). RNA immunoprecipitation and RNA stability assays assessed the relationship between MALAT1, UPF1, and SIRT6. CVB3-infected mice and H9c2 cells exhibited elevated MALAT1 and reduced SIRT6 expression. MALAT1 knockdown or SIRT6 overexpression suppressed inflammation and pyroptosis and inhibited the activation of the Wnt/β-catenin signal pathway in myocardial tissues and cells. MALAT1 enhanced the enrichment of SIRT6 mRNA by UPF1 and disturbed the stability of SIRT6 mRNA to promote the development of VMC. MALAT1 can bind UPF1 to mediate SIRT6 mRNA decay and activate the Wnt/β-catenin signal pathway in VMC., Competing Interests: Declarations Conflict of interest The authors declare that they have no competing interests. Ethical Approval and Consent to Participate The experimental design was approved by the Ethics Committee of Hunan Children’s Hospital (Approval no. KYSQ-2022–304). All experiments involving mice were conducted based on the instructions on the Guide for Care and Use of Laboratory Animals issued by the National Institutes of Health. Consent for Publication Not applicable., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Acute pancreatitis (AP) is an inflammatory disease initiated by the death of exocrine acinar cells, but its pathogenesis remains unclear. Signal transducer and activator of transcription 3 (STAT3) is a multifunctional factor that regulates immunity and the inflammatory response. The protective role of STAT3 is reported in Coxsackievirus B3 (CVB3)-induced cardiac fibrosis, yet the exact role of STAT3 in modulating viral-induced STAT1 activation and type I interferon (IFN)-stimulated gene (ISG) transcription in the pancreas remains unclarified. In this study, we tested whether STAT3 regulated viral-induced STAT1 translocation. We found that CVB3, particularly capsid VP1 protein, markedly upregulated the phosphorylation and nuclear import of STAT3 (p-STAT3) while it significantly impeded the nuclear translocation of p-STAT1 in the pancreases and hearts of mice on day 3 postinfection (p.i.). Immunoblotting and an immunofluorescent assay demonstrated the increased expression and nuclear translocation of p-STAT3 but a blunted p-STAT1 nuclear translocation in CVB3-infected acinar 266-6 cells. STAT3 shRNA knockdown or STAT3 inhibitors reduced viral replication via the rescue of STAT1 nuclear translocation and increasing the ISRE activity and ISG transcription in vitro. The knockdown of STAT1 blocked the antiviral effect of the STAT3 inhibitor. STAT3 inhibits STAT1 activation by virally inducing a potent inhibitor of IFN signaling, the suppressor of cytokine signaling-3 ((SOCS)-3). Sustained pSTAT1 and the elevated expression of ISGs were induced in SOCS3 knockdown cells. The in vivo administration of HJC0152, a pharmaceutical STAT3 inhibitor, mitigated the viral-induced AP and myocarditis pathology via increasing the IFNβ as well as ISG expression on day 3 p.i. and reducing the viral load in multi-organs. These findings define STAT3 as a negative regulator of the type I IFN response via impeding the nuclear STAT1 translocation that otherwise triggers ISG induction in infected pancreases and hearts. Our findings identify STAT3 as an antagonizing factor of the IFN-STAT1 signaling pathway and provide a potential therapeutic target for viral-induced AP and myocarditis.

Viral myocarditis (VMC) is the major reason for sudden cardiac death among both children and young adults. Of these, coxsackievirus B3 (CVB3) is the most common causative agent of myocarditis. Recently, the role of signaling pathways in the pathogenesis of VMC has been evaluated in several studies, which has provided a new perspective on identifying potential therapeutic targets for this hitherto incurable disease. In the present study, in vivo and in vitro experiments showed that CVB3 infection leads to increased Bim expression and triggers apoptosis. In addition, by knocking down Bim using RNAi, we further confirmed the biological function of Bim in apoptosis induced by CVB3 infection. We additionally found that Bim and forkhead box O1 class (FOXO1) inhibition significantly increased the viability of CVB3-infected cells while blocking viral replication and viral release. Moreover, CVB3-induced Bim expression was directly dependent on FOXO1 acetylation, which is catalyzed by the co-regulation of CBP and SirTs. Furthermore, the acetylation of FOXO1 was an important step in Bim activation and apoptosis induced by CVB3 infection. The findings of this study suggest that CVB3 infection induces apoptosis through the FOXO1 acetylation-Bim pathway, thus providing new insights for developing potential therapeutic targets for enteroviral myocarditis., (© 2023. The Author(s).)

Nonischaemic myocardial fibrosis is associated with cardiac dysfunction, malignant arrhythmias and sudden cardiac death. In the absence of a specific aetiology, its finding as late gadolinium enhancement (LGE) on cardiac magnetic resonance imaging is often attributed to preceding viral myocarditis. Athletes presenting with ventricular arrhythmias often have nonischaemic LGE. Previous studies have demonstrated an adverse effect of exercise on the course of acute viral myocarditis. In this study, we have investigated, for the first time, the impact of endurance training on longer-term outcomes such as myocardial fibrosis and arrhythmogenicity in a murine coxsackievirus B3 (CVB)-induced myocarditis model. Male C57BL/6J mice (n = 72) were randomly assigned to 8 weeks of forced treadmill running (EEX) or no exercise (SED). Myocarditis was induced 2 weeks later by a single intraperitoneal injection with CVB, versus vehicle in the controls (PBS). In a separate study, mice (n = 30) were subjected to pretraining for 13 weeks (preEEX), without continuation of exercise during myocarditis. Overall, continuation of exercise resulted in a milder clinical course of viral disease, with less weight loss and better preserved running capacity. CVB-EEX and preEEX-CVB mice tended to have a lower mortality rate. At sacrifice (i.e. 6 weeks after inoculation), the majority of virus was cleared from the heart. Histological assessment demonstrated prominent myocardial inflammatory infiltration and cardiomyocyte loss in both CVB groups. Inflammatory lesions in the CVB-EEX group contained higher numbers of pro-inflammatory cells (iNOS-reactive macrophages and CD8 + T lymphocytes) compared to these in CVB-SED. Treadmill running during myocarditis increased interstitial fibrosis [82.4% (CVB-EEX) vs. 56.3% (CVB-SED); P = 0.049]. Additionally, perivascular and/or interstitial fibrosis with extensive distribution was more likely to occur with exercise [64.7% and 64.7% (CVB-EEX) vs. 50% and 31.3% (CVB-SED); P = 0.048]. There was a numerical, but not significant, increase in the number of scars per cross-section (1.9 vs. 1.2; P = 0.195), with similar scar distribution and histological appearance in CVB-EEX and CVB-SED. In vivo electrophysiology studies did not induce sustained monomorphic ventricular tachycardia, only nonsustained (usually polymorphic) runs. Their cumulative beat count and duration paralleled the increased fibrosis between CVB-EEX and CVB-SED, but the difference was not significant (P = 0.084 for each). Interestingly, in mice that were subjected to pretraining only without continuation of exercise during myocarditis, no differences between pretrained and sedentary mice were observed at sacrifice (i.e. 6 weeks after inoculation and training cessation) with regard to myocardial inflammation, fibrosis, and ventricular arrhythmogenicity. In conclusion, endurance exercise during viral myocarditis modulates the inflammatory process with more pro-inflammatory cells and enhances perivascular and interstitial fibrosis development. The impact on ventricular arrhythmogenesis requires further exploration., (© 2024. The Author(s).)

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to be aberrantly activated in viral myocarditis (VMC), but the role of its subtype CaMKIIδ in VMC remains unclear.VMC mice and cardiomyocytes models were induced by Coxsackievirus B3 (CVB3) treatment. Mice that underwent sham surgery and saline-treated cardiomyocytes served as controls. Body weight, survival, left ventricular ejection fraction (LVEF), and fractional shortening (LVFS) were measured, and HE staining was performed to evaluate heart function in VMC mice model and sham control. Inflammation factors in serum or cell supernatant were detected by ELISA. Expressions of CaMKIIδ, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), nuclear factor NF-kappaB (NF-κB) signals, and inflammation factors were examined by quantitative real time polymerase chain reaction (qRT-PCR) or western blot. CCK-8, EdU, and flow cytometry were used to evaluate cell behaviors. Co-immunoprecipitation (Co-IP), RNA immunoprecipitation (RIP), and RNA pull-down were utilized to validate molecule interaction. Methylated RNA immunoprecipitation (MeRIP) was performed to measure N6-methyladenosine (m6A) level of specific molecule.CaMKIIδ was upregulated in VMC mice and CVB3-treated primary cardiomyocytes, of which knockdown improved cell viability, proliferation, and suppressed cell apoptosis in vitro, thereby alleviating myocarditis in vivo. The stability of CaMKIIδ was attributed to the presence of IGF2BP2 through m6A modification. Loss of CaMKIIδ repressed NF-κB pathway via negatively and directly regulating TIRAP to be involved in inflammatory damage.CaMKIIδ, stabilized by m6A reader IGF2BP2, modulated NF-κB pathway via interacting with TIRAP to alter cell viability, proliferation, and apoptosis, thereby affecting VMC outcome., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Viral myocarditis (VMC) is one of the most common acquired heart diseases in children and teenagers. However, its pathogenesis is still unclear, and effective treatments are lacking. This study aimed to investigate the regulatory pathway by which exosomes alleviate ferroptosis in cardiomyocytes (CMCs) induced by coxsackievirus B3 (CVB3). CVB3 was utilized for inducing the VMC mouse model and cellular model. Cardiac echocardiography, left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were implemented to assess the cardiac function. In CVB3-induced VMC mice, cardiac insufficiency was observed, as well as the altered levels of ferroptosis-related indicators (glutathione peroxidase 4 (GPX4), glutathione (GSH), and malondialdehyde (MDA)). However, exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) could restore the changes caused by CVB3 stimulation. Let-7a-5p was enriched in hucMSCs-exo, and the inhibitory effect of hucMSCs-exo let-7a-5p mimic on CVB3-induced ferroptosis was higher than that of hucMSCs-exo mimic NC (NC: negative control). Mothers against decapentaplegic homolog 2 (SMAD2) increased in the VMC group, while the expression of zinc-finger protein 36 (ZFP36) decreased. Let-7a-5p was confirmed to interact with SMAD2 messenger RNA (mRNA), and the SMAD2 protein interacted directly with the ZFP36 protein. Silencing SMAD2 and overexpressing ZFP36 inhibited the expression of ferroptosis-related indicators. Meanwhile, the levels of GPX4, solute carrier family 7, member 11 (SLC7A11), and GSH were lower in the SMAD2 overexpression plasmid (oe- SMAD2 )+ let-7a-5p mimic group than in the oe-NC+ let-7a-5p mimic group, while those of MDA, reactive oxygen species (ROS), and Fe 2+ increased. In conclusion, these data showed that ferroptosis could be regulated by mediating SMAD2 expression. Exo- let-7a-5p derived from hucMSCs could mediate SMAD2 to promote the expression of ZFP36, which further inhibited the ferroptosis of CMCs to alleviate CVB3-induced VMC.

Ethnopharmacological Relevance: Jingfang Granules (JFG) originate from the traditional herbal formula Jingfang Baidu powder. It has the effects of inducing sweating and dispelling wind. It is a classic medication used for treating external pathogenic factors and viral diseases. However, the therapeutic mechanism of JFG for viral myocarditis needs further clarification., Aim of the Study: This study aimed to explore the therapeutic efficacy of JFG on coxsackievirus B3-induced viral myocarditis (VMC), along with the elucidation of its underlying mechanisms., Materials and Methods: C57 BL/6JNifdc mice were divided randomly into several groups: control, model, Jingfang Granule groups (0.23, 0.46, and 0.69 g/20g, respectively), and a positive group (oseltamivir, 19.33 mg/kg). Following the establishment of the VMC model, the mice underwent an 8 -week treatment regimen. Pathological alterations in cardiac tissues and inflammatory protein expression were monitored. Differential gene analysis was conducted utilizing transcriptomic techniques. The differential gene mucolipin 1 (Mcoln1) was knocked down by transfection with siRNA in H9C2 cell, and investigative techniques such as immunoblotting, qRT-PCR, immunofluorescence, JC-1 staining, reactive oxygen species (ROS) detection, and mitochondrial stress testing were employed to examine its mechanism of action., Results: JFG significantly mitigates the pathological damage observed in the cardiac tissues of CVB3-induced VMC mice and attenuates the expression of inflammatory genes. Subsequently, differentially expressed genes are identified through transcriptomic analysis and validated via PCR. Among these, the upregulation of Mcoln1 promotes autophagy, facilitating the clearance of damaged mitochondria and excessive ROS. This has been substantiated through in vitro experiments. Excessive ROS precipitates a reduction in mitochondrial membrane potential, instigating cell apoptosis. In accordance with TUNEL staining results, JFG acts to inhibit cell apoptosis. To ascertain whether Mcoln1 is a crucial target for JFG in treating VMC, Mcoln1 was suppressed in H9C2 cells. The suppression of Mcoln1 hinders the elevation in autophagy levels post-JFG treatment, obstructs the enhancement of mitochondrial function, and impedes the clearance of ROS. Furthermore, the inhibitory effect of JFG on cell apoptosis is attenuated., Conclusion: The research findings indicate that JFG has a protective effect on CVB3-induced H9C2 cell injury. JFG may exert its effects in VMC treatment by enhancing autophagy to suppress cell apoptosis through the mitochondrial pathway, thereby counteracting cell damage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Viral myocarditis is an inflammatory disease of the myocardium, often leads to cardiac dysfunction and death. PARKIN (PRKN) and PINK1, well known as Parkinson's disease-associated genes, have been reported to be involved in innate immunity and mitochondrial damage control. Therefore, we investigated the role of parkin and PINK1 in coxsackievirus B3 (CVB3)-induced viral myocarditis because the etiology of myocarditis is related to abnormal immune response to viral infection and mitochondrial damage. After viral infection, the survival was significantly lower and myocardial damage was more severe in parkin knockout (KO) and PINK1 KO mice compared to wild-type (WT) mice. Parkin KO and PINK1 KO showed defective immune cell recruitment and impaired production of antiviral cytokines such as interferon-gamma, allowing increased viral replication. In addition, parkin KO and PINK1 KO mice were more susceptible to CVB3-induced mitochondrial damage than WT mice, resulting in susceptibility to viral-induced cardiac damage. Finally, using publicly available RNA-seq data, we found that pathogenic mutants of the PRKN gene are more common in patients with dilated cardiomyopathy and myocarditis than in controls or the general population. This study will help elucidate the molecular mechanism of CVB3-induced viral myocarditis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Increasing evidence has shown that extracellular vesicles or exosomes released from virus-infected cells contain viral particles, genomes, or other pathogenic factors that move to neighbor cells, contributing to virus dissemination and productive infection. Our recent study demonstrated that exosomes carrying CVB3 virions exhibited greater infection efficiency than free virions because they accessed various entry routes, overcoming restrictions to viral tropism. However, the pathogenicity of exosomes carried CVB3 and their effect on immunological properties have not yet been completely explained. In the current study, we sought to explore whether exosomes exert their effect on the CVB3-induced pathogenesis or evade the immune attack. Our results showed that exosomes-carried CVB3 could effectively infect viral receptor-negative immune cells in vivo, resulting in inducing immune system loss. Importantly, the exosomes-carried CVB3 had the ability to escape the neutralizing antibodies activity resulting in inducing the severe onset of myocarditis. Using the genetically engineered mouse with deficiency of exosomes, we observed that the exosomes-carried CVB3 reinforced an aggravated pathogenesis. By understanding how exosomes promote the course of viral disease, clinical applications of exosomes can be developed., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Picornaviruses infect a wide variety of cell types in vitro , with rapid replication kinetics and pronounced cytopathic effect. Coxsackievirus B3 (CVB3) can also establish a persistent infection in vivo that can lead to pathology, including dilated cardiomyopathy and myocarditis. One model system to study persistent infection is the pancreatic ductal cell line PANC-1, which CVB3 infects and is maintained indefinitely. We have characterized this model for CVB3 infection to study persistent infection for over 6 months. We find that CVB3 rapidly replicates within PANC-1 cells without robust cytopathic effect, and after 1 month in culture, titers stabilize. We find that infection does not significantly affect cellular viability. Persistent virus reverts to lytic infection when transferred to Huh7 or Vero cells. We find that persistent CVB3 adapts to PANC-1 cells via mutation of its capsid proteins and, curiously, the viral polymerase (3Dpol) to generate a high-fidelity polymerase. Persistent infection is associated with reduced cleavage of eIF4G, reduced plaque size, and decreasing particle infectivity. We further find that polyamine metabolism is altered in persistently infected cells, with the rate-limiting enzyme ornithine decarboxylase (ODC1) reduced in translation. We further find that targeting polyamine synthesis reduces persistent infection without affecting the viability of the PANC-1 cells. Finally, we find that viral fidelity is essential to maintaining CVB3 infection, and targeting viral fidelity reduces persistent virus infection. Together, these data highlight a novel role for polyamines and fidelity in persistent CVB3 infection and suggest avenues for therapeutic development to target persistent infection. IMPORTANCE Enteroviruses are significant human pathogens that can cause severe disease, including cardiomyopathies. Viruses like coxsackievirus B3 (CVB3) can cause tissue damage by lytically infecting cells; however, CVB3 can also persistently infect, which has been associated with several pathologies. Studying persistent infection in vitro is challenging, as CVB3 lytically infects most cellular model systems. Here, we show that CVB3 establishes persistent infection in pancreatic ductal cells in vitro , similar to prior studies on other coxsackieviruses. We also show that this infection results in adaptation of the virus to these cells, as well as changes to cellular metabolism of polyamines., Competing Interests: The authors declare no conflict of interest.

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.

Background: Different serotypes of coxsackievirus B (CVB), which is the most common cause of viral myocarditis, target cardiomyocytes through Coxsackie and Adenovirus Receptor and Decay-Accelerating Factor. Both receptors are expressed in the fetal heart. We hypothesized that infection with different serotypes of CVB during early pregnancy plays a role in pathogenesis of congenital heart defect (CHD)., Methods: In this study, we use a murine model to infect with CVB1, CVB4, and combination of CVB3 + CVB4 during a critical period in gestation. We examined offspring of pregnant mice for fetal death and heart defects following viral infection., Result: Fetuses from uninfected control dams showed normal heart development, while maternal CVB infection precipitates CHD: majorly ventricular septal defects (VSD) and non-compaction of ventricular myocardium (NC), with some infrequent cases of double outlet right ventricle, left ventricle wall rupture, right ventricle hypertrophy, and thickened/dysplastic semilunar valves. Infection of pregnant dams with CVB1 leads to 44% VSD and 41.2% NC cases, while with CVB4 leads to 31.7% VSD and 13.3% NC cases. Co-infection with CVB3 + CVB4 increases fetal pathology to 51.3% VSD and 41% NC cases. Infection can also result in fetal death, with higher incidences with CVB3 + CVB4 with 46.2% cases, compared to 33.3% by CVB1 and 21.7% by CVB4. Male fetuses were more susceptible to all phenotypes., Conclusion: Our report shows that prenatal CVB infections can lead to pathogenesis of certain heart defects in mouse model, particularly exacerbated with co-infections. This data confirms a link between prenatal CVB infection and CHD development and highlights it is not unique to just one serotype of CVB., (© 2022 Wiley Periodicals LLC.)

Background: Myocardial inflammation and apoptosis are key processes in coxsackievirus B3 (CVB3)-induced acute viral myocarditis (AVMC). Accumulating evidence reveals the essential roles of long noncoding RNAs (lncRNAs) in the pathogenesis of AVMC. Here, we aimed to evaluate the biological functions of a novel lncRNA guanylate-binding protein 9 (lncGBP9) in AVMC progression and further explore its underlying mechanisms., Methods: Initially, mouse models of AVMC were constructed by CVB3 infection. The expression and localization of lncGBP9 in heart tissues were analyzed using RT-qPCR and FISH. Adeno-associated virus serotype 9 (AAV9)-mediated lncGBP9 knockdown was then employed to clarify its roles in survival, cardiac function, and myocardial inflammation and apoptosis. Moreover, the mRNA and protein levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) were detected by RT-qPCR and ELISA, and the regulation of lncGBP9 knockdown on the NF-κB signaling pathway was investigated by Western blotting. Using an in vitro model of HL-1 cardiomyocytes exposed to CVB3 infection, the effects of lncGBP9 knockdown on cell viability, inflammation, and apoptosis were further evaluated in vitro., Results: Increased lncGBP9 expression was detected in the heart tissues of AVMC mice and CVB3-infected HL-1 cells, and was mainly located in the cytoplasm. Knockdown of lncGBP9 remarkably alleviated the severity of AVMC in CVB3-infected mice, as verified by improved cardiac function, and reduced myocardial inflammation and apoptosis. Additionally, lncGBP9 knockdown suppressed the NF-κB signaling pathway and consequently reduced productions of pro-inflammatory cytokines in vivo. In vitro functional assays further confirmed that lncGBP9 knockdown promoted cell viability, inhibited cell apoptosis, and reduced pro-inflammatory cytokines release in CVB3-infected HL-1 cells through suppressing NF-κB activation., Conclusions: Collectively, lncGBP9 knockdown exerts anti-inflammatory and anti-apoptotic effects in CVB3-induced AVMC, which may be mediated in part via NF-κB signaling pathway. These findings highlight lncGBP9 as an attractive target for therapeutic interventions., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Viral myocarditis (VMC), which is defined as inflammation of the myocardium with consequent myocardial injury, may develop chronic disease eventually leading to dilated cardiomyopathy (DCM). Molecular mechanisms underlying the progression from acute VMC (aVMC), to chronic VMC (cVMC) and finally to DCM, are still unclear. Here, we established mouse models of VMC and DCM with Coxsackievirus B3 infection and conducted NMR-based metabolomic analysis of aqueous metabolites extracted from cardiac tissues of three histologically classified groups including aVMC, cVMC and DCM. We showed that these three pathological groups were metabolically distinct from their normal counterparts and identified three impaired metabolic pathways shared by these pathological groups relative to normal controls, including nicotinate and nicotinamide metabolism; alanine, aspartate and glutamate metabolism; and D-glutamine and D-glutamate metabolism. We also identified two extra impaired metabolic pathways in the aVMC group, including glycine, serine and threonine metabolism; and taurine and hypotaurine metabolism Furthermore, we identified potential cardiac biomarkers for metabolically distinguishing these three pathological stages from normal controls. Our results indicate that the metabolomic analysis of cardiac tissues can provide valuable insights into the molecular mechanisms underlying the progression from acute VMC to DCM.

Atrial dysfunction is a relatively common complication of acute myocarditis, although its pathophysiology is unclear. There is limited information on myocarditis-associated histological changes in the atria and how they develop in time. The aim of this study therefore was to investigate inflammation, fibrosis and viral genome in the atria in time after mild CVB3-induced viral myocarditis (VM) in mice. C3H mice (n = 68) were infected with 10 5 PFU of Coxsackievirus B3 (CVB3) and were compared with uninfected mice (n = 10). Atrial tissue was obtained at days 4, 7, 10, 21, 35 or 49 post-infection. Cellular infiltration of CD45+ lymphocytes, MAC3+ macrophages, Ly6G+ neutrophils and mast cells was quantified by (immuno)histochemical staining. The CVB3 RNA was determined by in situ hybridization, and fibrosis was evaluated by elastic van Gieson (EvG) staining. In the atria of VM mice, the numbers of lymphocytes on days 4 and 7 (p < .05) and days 10 (p < .01); macrophages on days 7 (p < .01) and 10 (p < .05); neutrophils on days 4 (p < .05); and mast cells on days 4 and 7 (p < .05) increased significantly compared with control mice and decreased thereafter to basal levels. No cardiomyocyte death was observed, and the CVB3 genome was detected in only one infected mouse on Day 4 post-infection. No significant changes in the amount of atrial fibrosis were found between VM and control mice. A temporary increase in inflammation is induced in the atria in the acute phase of CVB3-induced mild VM, which may facilitate the development of atrial arrhythmia and contractile dysfunction., (© 2022 The Authors. International Journal of Experimental Pathology published by John Wiley & Sons Ltd on behalf of Company of the International Journal of Experimental Pathology (CIJEP).)

Cardiomyocyte apoptosis is critical for the development of viral myocarditis (VMC), which is one of the leading causes of cardiac sudden death in young adults. Our previous studies have demonstrated that elevated calpain activity is involved in the pathogenesis of VMC. This study aimed to further explore the underlying mechanisms. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin were infected with coxsackievirus B3 (CVB3) to establish a VMC model. Apoptosis was detected with flow cytometry, TUNEL staining, and western blotting. Cardiac function was measured using echocardiography. Mitochondrial function was measured using ATP assays, JC-1, and MitoSOX. Mitochondrial morphology was observed using MitoTracker staining and transmission electron microscopy. Colocalization of dynamin-related protein 1 (Drp-1) in mitochondria was examined using immunofluorescence. Phosphorylation levels of Drp-1 at Ser637 site were determined using western blotting analysis. We found that CVB3 infection impaired mitochondrial function as evidenced by increased mitochondrial ROS production, decreased ATP production and mitochondrial membrane potential, induced myocardial apoptosis and damage, and decreased myocardial function. These effects of CVB3 infection were attenuated by inhibition of calpain both by PD150606 treatment and calpastatin overexpression. Furthermore, CVB3-induced mitochondrial dysfunction was associated with the accumulation of Drp-1 in the outer membrane of mitochondria and subsequent increase in mitochondrial fission. Mechanistically, calpain cleaved and activated calcineurin A, which dephosphorylated Drp-1 at Ser637 site and promoted its accumulation in the mitochondria, leading to mitochondrial fission and dysfunction. In summary, calpain inhibition attenuated CVB3-induced myocarditis by reducing mitochondrial fission, thereby inhibiting cardiomyocyte apoptosis. Calpain is activated by CVB3 infection. Activated calpain cleaves calcineurin A and converts it to active form which could dephosphorylate Drp-1 at Ser637 site. Then, the active Drp-1 translocates from the cytoplasm to mitochondria and triggers excessive mitochondrial fission. Eventually, the balance of mitochondrial dynamics is broken, and apoptosis occurs., (© 2021. The Author(s).)

Gestational viral infection has been associated with congenital heart disease (CHD). Few studies, however, have studied the potential role of gestational Coxsackievirus B (CVB) exposure in the pathogenesis of CHD. We prospectively enrolled women with pregnancies affected by CHD to explore possible associations with in utero CVB exposure. Serum samples were obtained from 122 women referred for fetal echocardiography between 2006 and 2018. We quantified CVB IgG and IgM levels, with titers ≥ 15.0 U/mL considered positive and measured neutralizing antibodies for three CVB serotypes: CVB1, CVB3, and CVB4. Using data from the national enterovirus surveillance system, we compared the annual exposure rates for each serotype in our cohort to infections reported across the United States. 98 pregnancies with no genetic defects were included. Overall, 29.6% (29/98) had positive IgG and 4.1% (4/98) of women had positive CVB IgM titers. To explore first-trimester CVB exposure, we focused exclusively on the 26 women with positive IgG and negative IgM titers. 61.5% (16/26) had neutralizing antibodies against a single serotype and 38.5% (10/26) against multiple CVB serotypes. CVB4 neutralizing antibodies were the most common (65.4%, 17/26), followed by CVB3 (53.9%, 14/26) and CVB1 (30.8%, 8/26). Among these, 30.8% of babies presented pulmonary valve anomalies: 19.2% (5/26) pulmonary atresia, and 11.5% (3/26) pulmonary stenosis. 23.1% (6/26) of babies had coronary sinusoids. CVB exposure in our cohort mirrored that of reported infections in the United States. Our results suggest a possible association between gestational CVB exposure and specific CHD, particularly pulmonary valve anomalies and coronary sinusoids., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Coxsackievirus A2 (CVA2) has recently been constantly detected, and is associated with viral myocarditis in children. Our previous study demonstrated that CVA2 led to heart damage in a neonatal murine model. However, the molecular mechanism of heart injury caused by CVA2 remains largely unknown. Emerging evidence suggests the significant functions of miRNAs in Coxsackievirus infection. To investigate potential miRNAs involved in heart injury caused by CVA2, our study, for the first time, conducted a RNA-seq in vivo employing infected mice hearts. In total, 87, 101 and 76 differentially expressed miRNAs were identified at 3 days post infection (dpi), 7 dpi and 7 dpi vs 3 dpi. Importantly, above 3 comparison strategies shared 34 differentially expressed miRNAs. These results were confirmed by quantitative PCR (qPCR). Next, we did GO, KEGG, and miRNA-mRNA integrated analysis of differential miRNAs. The dual-luciferase reporter assay confirmed the miRNA-mRNA pairs. To further confirm the above enriched pathways and processes, we did Western blotting and immunofluorescence staining. Our results suggest that inflammatory responses, T cell activation, apoptosis, autophagy, antiviral immunity, NK cell infiltration, and the disruption of tight junctions are involved in the pathogenesis of heart injury caused by CVA2. The dysregulated miRNAs and pathways recognized in the current study can improve the understanding of the intricate interactions between CVA2 and the heart injury, opening a novel avenue for the future study of CVA2 pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wu, Zhu, Qian, Hu, Ji, Li, Zhu, Liang and Jin.)

Viral myocarditis (VMC) is an inflammatory heart condition which can induce dilated cardiomyopathy (DCM). However, molecular mechanisms underlying the progression of VMC into DCM remain exclusive. Here, we established mouse models of VMC and DCM by infecting male BALB/c mice with Coxsackievirus B3 (CVB3), and performed NMR-based metabonomic analyses of mouse sera. The mouse models covered three pathological stages including: acute VMC (aVMC), chronic VMC (cVMC) and DCM. We recorded 1 D 1 H-NMR spectra on serum samples and conducted multivariate statistical analysis on the NMR data. We found that metabolic profiles of these three pathological stages were distinct from their normal controls (CON), and identified significant metabolites primarily responsible for the metabolic distinctions. We identified significantly disturbed metabolic pathways in the aVMC, cVMC and DCM stages relative to CON, including: taurine and hypotaurine metabolism; pyruvate metabolism; glycine, serine and threonine metabolism; glycerolipid metabolism. Additionally, we identified potential biomarkers for discriminating a VMC, cVMC and DCM from CON including: taurine, valine and acetate for aVMC; glycerol, valine and leucine for cVMC; citrate, glycine and isoleucine for DCM. This work lays the basis for mechanistically understanding the progression from acute VMC to DCM, and is beneficial to exploitation of potential biomarkers for prognosis and diagnosis of heart diseases.

Purpose: We evaluated the activity of intratumoral Coxsackievirus A21 (V937) in 57 patients with unresectable stage IIIC or IV melanoma., Patients and Methods: In this multicenter, open-label, phase II study, patients received up to a total V937 dose of 3 × 10 8 TCID 50 (50% tissue culture infectious dose) in a maximum 4.0-mL volume by intratumoral injection. Ten sets of V937 injections were administered between days 1 and 127 (NCT01227551). Patients who had stable disease or were responding could continue treatment in an extension study (NCT01636882). Response and progression status were based on contrast-enhanced computed tomography, magnetic resonance imaging, or caliper measurement and were categorized using immune-related Response Evaluation Criteria in Solid Tumors (irRECIST). Other evaluations included monitoring of adverse events and serum levels of V937 and anti-V937 antibody titers. The primary efficacy end point was 6-month progression-free survival (PFS) rate per irRECIST., Results: The primary efficacy end point, 6-month PFS rate per irRECIST, was 38.6% (95% CI, 26.0 to 52.4). Durable response rate (partial or complete response for ≥ 6 months) was 21.1% per irRECIST. Best overall response rate (complete plus partial response) was 38.6% (unconfirmed) and 28.1% (confirmed) per irRECIST. Regression of melanoma was observed in noninjected lesions. Based on Kaplan-Meier estimation, 12-month PFS was 32.9% (95% CI, 19.5 to 46.9) per irRECIST and 12-month overall survival was 75.4% (95% CI, 62.1 to 84.7). No treatment-related grade ≥ 3 adverse events occurred. Viral RNA was detected in serum within 30 minutes of administration. Neutralizing antibody titers increased to > 1:16 in all patients after day 22, without effect on clinical or immunologic response., Conclusion: V937 was well tolerated and warrants further investigation for treatment of patients with unresectable melanoma. Studies of combination approaches with V937 and immune checkpoint inhibitors are ongoing., Competing Interests: Robert H. I. AndtbackaEmployment: Novartis, Merck Sharp & Dohme, Seven and Eight BiopharmaceuticalsStock and Other Ownership Interests: Novartis, Merck Sharp & Dohme Brendan CurtiHonoraria: Clinigen Group, NektarConsulting or Advisory Role: MerckResearch Funding: Bristol Myers Squibb, Galectin Therapeutics, Clinigen GroupPatents, Royalties, Other Intellectual Property: Biomarkers for OX40 responseTravel, Accommodations, Expenses: Agonox Gregory A. DanielsHonoraria: Sanofi/RegeneronConsulting or Advisory Role: Sanofi/RegeneronSpeakers' Bureau: Regeneron, Array BioPharma, Sanofi/RegeneronResearch Funding: Bristol Myers Squibb, Amgen, Viralytics, Nektar, Merck Sigrun HallmeyerLeadership: Association of Community Cancer Centers (ACCC)Honoraria: Cardinal HealthConsulting or Advisory Role: Bristol Myers Squibb, Cardinal Health, Array PharamceuticalSpeakers' Bureau: Bristol Myers SquibbTravel, Accommodations, Expenses: Cardinal Health, Bristol Myers SquibbUncompensated Relationships: Society for Immunotherapy of Cancer Eric D. WhitmanConsulting or Advisory Role: Merck Sharp & Dohme, Bristol Myers Squibb, Castle Biosciences, Novartis, Eisai, PfizerSpeakers' Bureau: Bristol Myers Squibb, Merck Sharp & Dohme, Castle Biosciences, Sanofi/RegeneronResearch Funding: Bristol Myers Squibb, Merck Sharp & Dohme, Castle Biosciences, Genentech/Roche, Amgen, TRACON Pharma, AstraZeneca/MedImmune, Provectus, Oncolys BioPharma, Iovance Biotherapeutics, Dynavax Technologies, OncoSec, Toray Industries, Array BioPharmaPatents, Royalties, Other Intellectual Property: Nerve monitoring dissection device, Lighted Polyhedral surgical retractor Jose LutzkyConsulting or Advisory Role: Castle Biosciences, Iovance Biotherapeutics, Replimune, RegeneronResearch Funding: Bristol Myers Squibb, Novartis, Iovance Biotherapeutics, Immunocore, Regeneron, Replimune, Vyriad Lynn E. SpitlerLeadership: Nuclear RNA Networks IncStock and Other Ownership Interests: Nuclear RNA Networks Inc Praveen K. BommareddyEmployment: ReplimuneStock and Other Ownership Interests: Replimune Mark GroseConsulting or Advisory Role: ImmVirX Meihua WangEmployment: MerckStock and Other Ownership Interests: Merck Cai WuEmployment: Merck (at the time the work was conducted)Stock and Other Ownership Interests: Merck Sharp & Dohme Howard L. KaufmanEmployment: ImmuneeringLeadership: ImmuneeringStock and Other Ownership Interests: ImmuneeringHonoraria: Society for Immunotherapy of CancerNo other potential conflicts of interest were reported.

Viral diseases are a major threat to modern society and the global health system. It is therefore of utter relevance to understand the way viruses affect the host as a basis to find new treatment solutions. The understanding of viral myocarditis (VMC) is incomplete and effective treatment options are lacking. This review will discuss the mechanism, effects, and treatment options of the most frequent myocarditis-causing viruses namely enteroviruses such as Coxsackievirus B3 (CVB3) and Parvovirus B19 (PVB19) on the human heart. Thereby, we focus on: 1. Viral entry: CVB3 use Coxsackievirus-Adenovirus-Receptor (CAR) and Decay Accelerating Factor (DAF) to enter cardiac myocytes while PVB19 use the receptor globoside (Gb4) to enter cardiac endothelial cells. 2. Immune system responses: The innate immune system mediated by activated cardiac toll-like receptors (TLRs) worsen inflammation in CVB3-infected mouse hearts. Different types of cells of the adaptive immune system are recruited to the site of inflammation that have either protective or adverse effects during VMC. 3. Autophagy: CVB3 evades autophagosomal degradation and misuses the autophasomal pathway for viral replication and release. 4. Viral replication sites: CVB3 promotes the formation of double membrane vesicles (DMVs), which it uses as replication sites. PVB19 uses the host cell nucleus as the replication site and uses the host cell DNA replication system. 5. Cell cycle manipulation: CVB3 attenuates the cell cycle at the G1/S phase, which promotes viral transcription and replication. PVB19 exerts cell cycle arrest in the S phase using its viral endonuclease activity. 6. Regulation of apoptosis: Enteroviruses prevent apoptosis during early stages of infection and promote cell death during later stages by using the viral proteases 2A and 3C, and viroporin 2B. PVB19 promotes apoptosis using the non-structural proteins NS1 and the 11 kDa protein. 7. Energy metabolism: Dysregulation of respiratory chain complex expression, activity and ROS production may be altered in CVB3- and PVB19-mediated myocarditis. 8. Ion channel modulation: CVB3-expression was indicated to alter calcium and potassium currents in Xenopus laevis oocytes and rodent cardiomyocytes. The phospholipase 2-like activity of PVB19 may alter several calcium, potassium and sodium channels. By understanding the general pathophysiological mechanisms of well-studied myocarditis-linked viruses, we might be provided with a guideline to handle other less-studied human viruses., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Sporadic occurrences and outbreaks of hand, foot, and mouth disease (HFMD) caused by Coxsackievirus A2 (CVA2) have frequently reported worldwide recently, which pose a great challenge to public health. Epidemiological studies have suggested that the main cause of death in critical patients is pulmonary edema. However, the pathogenesis of this underlying comorbidity remains unclear. In this study, we utilized the 5-day-old BALB/c mouse model of lethal CVA2 infection to evaluate lung damage. We found that the permeability of lung microvascular was significantly increased after CVA2 infection. We also observed the direct infection and apoptosis of lung endothelial cells as well as the destruction of tight junctions between endothelial cells. CVA2 infection led to the degradation of tight junction proteins (e.g., ZO-1, claudin-5, and occludin). The gene transcription levels of von Willebrand factor (vWF), endothelin (ET), thrombomodulin (THBD), granular membrane protein 140 (GMP140), and intercellular cell adhesion molecule-1 (ICAM-1) related to endothelial dysfunction were all significantly increased. Additionally, CVA2 infection induced the increased expression of inflammatory cytokines (IL-6, IL-1β, and MCP-1) and the activation of p38 mitogen-activated protein kinase (MAPK). In conclusion, the disruption of the endothelial barrier contributes to acute lung injury induced by CVA2 infection; targeting p38-MAPK signaling may provide a therapeutic approach for pulmonary edema in critical infections of HFMD.

Viral myocarditis (VMC) is a cardiac disease associated with myocardial inflammation and injury induced by virus infection. Cardiomyocytes have recently been regarded as key players in eliciting and modulating inflammation within the myocardium. Kruppel-like factor 10 (KLF10) is a crucial regulator of various pathological processes and plays different roles in a variety of diseases. However, its role in VMC induced by coxsackievirus B3 (CVB3) infection remains unknown. In this study, we report that cardiac KLF10 confers enhanced protection against viral myocarditis. We found that KLF10 expression was downregulated upon CVB3 infection. KLF10 deficiency enhanced cardiac viral replication and aggravated VMC progress. Bone marrow chimera experiments indicated that KLF10 expression in nonhematopoietic cells was involved in the pathogenesis of VMC. We further identified MCP-1 as a novel target of KLF10 in cardiomyocytes, and KLF10 cooperated with histone deacetylase 1 (HDAC1) to negatively regulate MCP-1 expression by binding its promoter, leading to activation of MCP-1 transcription and recruitment of Ly6C high monocytes/macrophages into the myocardium. This novel mechanism of MCP-1 regulation by KLF10 might provide new insights into the pathogenesis of VMC and a potential therapeutic target for VMC., (© 2020. CSI and USTC.)

Type B coxsackieviruses (CV-B) frequently infect the central nervous system (CNS) causing neurological diseases notably meningitis and encephalitis. These infections occur principally among newborns and children. Epidemiological studies of patients with nervous system disorders demonstrate the presence of infectious virus, its components, or anti-CV-B antibodies. Some experimental studies conducted in vitro and in vivo support the potential association between CV-B and idiopathic neurodegenerative diseases such as amyotrophic lateral sclerosis and psychiatric illness such as schizophrenia. However, mechanisms explaining how CV-B infections may contribute to the genesis of CNS disorders remain unclear. The proposed mechanisms focus on the immune response following the viral infection as a contributor to pathogenesis. This review describes these epidemiological and experimental studies, the modes of transmission of CV-B with an emphasis on congenital transmission, the routes used by CV-B to reach the brain parenchyma, and plausible mechanisms by which CV-B may induce CNS diseases, with a focus on potential immunopathogenesis., (© 2020 John Wiley & Sons Ltd.)

Enteroviruses are suspected to contribute to insulin-producing β cell loss and hyperglycemia-induced diabetes. However, mechanisms are not fully defined. Here, we show that coxsackievirus B type 4 (CVB4) infection in human islet-engrafted mice and in rat insulinoma cells displays loss of unconventional prefoldin RPB5 interactor (URI) and PDX1, affecting β cell function and identity. Genetic URI ablation in the mouse pancreas causes PDX1 depletion in β cells. Importantly, diabetic PDX1 heterozygous mice overexpressing URI in β cells are more glucose tolerant. Mechanistically, URI loss triggers estrogen receptor nuclear translocation leading to DNA methyltransferase 1 (DNMT1) expression, which induces Pdx1 promoter hypermethylation and silencing. Consequently, demethylating agent procainamide-mediated DNMT1 inhibition reinstates PDX1 expression and protects against diabetes in pancreatic URI-depleted mice . Finally, the β cells of human diabetes patients show correlations between viral protein 1 and URI, PDX1, and DNMT1 levels. URI and DNMT1 expression and PDX1 silencing provide a causal link between enterovirus infection and diabetes., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Previous research suggests that hepatocytes catabolize chemical toxins but do not remove microbial agents, which are filtered out by other liver cells (Kupffer cells and endothelial cells). Here we show that, contrary to current understanding, hepatocytes trap and rapidly silence type B coxsackieviruses (CVBs). In genetically wildtype mice, this activity causes hepatocyte damage, which is alleviated in mice carrying a hepatocyte-specific deletion of the coxsackievirus-adenovirus receptor. However, in these mutant mice, there is a dramatic early rise in blood-borne virus, followed by accelerated systemic disease and increased mortality. Thus, wild type hepatocytes act similarly to a sponge for CVBs, protecting against systemic illness at the expense of their own survival. We speculate that hepatocytes may play a similar role in other viral infections as well, thereby explaining why hepatocytes have evolved their remarkable regenerative capacity. Our data also suggest that, in addition to their many other functions, hepatocytes might be considered an integral part of the innate immune system.

Coxsackievirus A24 variant (CVA24v) is a major causative agent of acute hemorrhagic conjunctivitis outbreaks worldwide, yet the evolutionary and transmission dynamics of the virus remain unclear. To address this, we analyzed and compared the 3C and partial VP1 gene regions of CVA24v isolates obtained from five outbreaks in Cuba between 1986 and 2009 and strains isolated worldwide. Here we show that Cuban strains were homologous to those isolated in Africa, the Americas and Asia during the same time period. Two genotypes of CVA24v (GIII and GIV) were repeatedly introduced into Cuba and they arose about two years before the epidemic was detected. The two genotypes co-evolved with a population size that is stable over time. However, nucleotide substitution rates peaked during pandemics with 4.39 × 10 -3 and 5.80 × 10 -3 substitutions per site per year for the 3C and VP1 region, respectively. The phylogeographic analysis identified 25 and 19 viral transmission routes based on 3C and VP1 regions, respectively. Pandemic viruses usually originated in Asia, and both China and Brazil were the major hub for the global dispersal of the virus. Together, these data provide novel insight into the epidemiological dynamics of this virus and possibly other pandemic viruses.

Aims: The coxsackievirus B3 (CVB3) mouse myocarditis model is the standard model for investigation of virus-induced myocarditis but the pancreas, rather than the heart, is the most susceptible organ in mouse. The aim of this study was to develop a CVB3 mouse myocarditis model in which animals develop myocarditis while attenuating viral infection of the pancreas and the development of severe pancreatitis., Methods and Results: We developed the recombinant CVB3 variant H3N-375TS by inserting target sites (TS) of miR-375, which is specifically expressed in the pancreas, into the 3'UTR of the genome of the pancreo- and cardiotropic CVB3 variant H3. In vitro evaluation showed that H3N-375TS was suppressed in pancreatic miR-375-expressing EndoC-βH1 cells >5 log10, whereas its replication was not suppressed in isolated primary embryonic mouse cardiomyocytes. In vivo, intraperitoneal (i.p.) administration of H3N-375TS to NMRI mice did not result in pancreatic or cardiac infection. In contrast, intravenous (i.v.) administration of H3N-375TS to NMRI and Balb/C mice resulted in myocardial infection and acute and chronic myocarditis, whereas the virus was not detected in the pancreas and the pancreatic tissue was not damaged. Acute myocarditis was characterized by myocardial injury, inflammation with mononuclear cells, induction of proinflammatory cytokines, and detection of replicating H3N-375TS in the heart. Mice with chronic myocarditis showed myocardial fibrosis and persistence of H3N-375TS genomic RNA but no replicating virus in the heart. Moreover, H3N-375TS infected mice showed distinctly less suffering compared with mice that developed pancreatitis and myocarditis after i.p. or i.v application of control virus., Conclusion: In this study, we demonstrate that by use of the miR-375-sensitive CVB3 variant H3N-375TS, CVB3 myocarditis can be established without the animals developing severe systemic infection and pancreatitis. As the H3N-375TS myocarditis model depends on pancreas-attenuated H3N-375TS, it can easily be used in different mouse strains and for various applications., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Viral myocarditis (VMC) is a widely studied but poorly understood inflammatory cardiomyopathy which mainly affects children and young adults and results in adverse outcomes. Cardiomyocyte apoptosis was reported important in the progress of coxsackievirus B3 (CVB3)-induced VMC and the blocking of this process may contribute to the therapeutic effect towards VMC. Therefore, this study was designed to explore whether survivin, one of the strongest antiapoptotic proteins, can protect cardiomyocytes from apoptosis in VMC and to discover its related mechanisms. Here, the cultured neonatal mouse cardiomyocytes (NMCs) were exposed to CVB3 to establish the cell model of VMC and the results of Western Blot showed that the protein expression of survivin in CVB3-infected NMCs varied at different post-infection time. Lentivirus was next used to examine the function of survivin in CVB3-infected NMCs. TUNEL assay demonstrated that the overexpression of survivin interrupted CVB3-induced apoptosis. It was next examined whether caspase-3 and -9 were involved in the antiapoptotic pathway initiated by survivin via Western Blot. The results showed a reverse relationship between the protein expression of survivin and that of cleaved caspase-3 and cleaved caspase-9, suggesting that survivin may attenuate apoptosis through restraining the activity of caspase-3 and -9. Moreover, the supernatant fluid of cultured NMCs was extracted to detect the quantitation of released lactate dehydrogenase (LDH) and a sharp decrease was discovered in the survivin-overexpressed group compared to the CVB3-infected group, indicating a protective role of survivin in the cell model of CVB3-induced myocarditis. This study demonstrated that survivin was triggered by CVB3 infection in NMCs and survivin executed its antiapoptotic effects via caspase-3- and caspase-9-dependent signaling pathway.

Coxsackievirus B (CVB) is a common human enterovirus that causes systemic infection but specifically replicates to high titers in the pancreas. It was reported that certain viruses induce mitochondrial fission to support infection. We documented that CVB triggers mitochondrial fission and blocking mitochondrial fission limits infection. The transient receptor potential channels have been implicated in regulating mitochondrial dynamics; namely, the heat and capsaicin receptor transient receptor potential cation channel subfamily V member 1 (TRPV1) contributes to mitochondrial depolarization and fission. When we transiently warmed HeLa cells to 39 °C prior to CVB exposure, infection was heightened, whereas cooling cells to 25 °C reduced infection. Inducing "cold" by stimulating transient receptor potential cation channel subfamily M member 8 (TRPM8) with menthol led to reduced infection and also resulted in lower levels of mitochondrial fission during infection. Additionally, menthol stabilized levels of mitochondrial antiviral signaling (MAVS) which is known to be tied to mitochondrial dynamics. Taken together, this highlights a novel pathway wherein CVB relies on TRPV1 to initiate proviral mitochondrial fission, which may contribute to the disruption of antiviral immunity. TRPM8 has been shown to antagonize TRPV1, and thus we hypothesize that stimulating TRPM8 blocks TRPV1-mediated mitochondrial fragmentation following CVB exposure and attenuates infection.

Coxsackie B viruses (CV-B) are associated with several central nervous system (CNS) disorders. These viruses are predominantly transmitted by fecal-oral route but vertical transmission can also occur. This work attempted to study the immune response ensuing vertical transmission of CV-B to the brain, and its eventual implementation in the brain pathogenesis. To this end, pregnant Swiss albino mice were inoculated with CV-B4 E2 or with sterile medium for control animals. At different ages after birth, brains were collected and analyzed for virus infection, histopathological changes and immune response. Infectious particles were detected in offspring's brain which demonstrates vertical transmission of the virus. This infection is persistent since the long lasting detection of viral RNA in offspring's brain. Some pathological signs including meningitis, edema and accumulation of inflammatory cells within and surrounding the inflammatory areas were observed. Immunoflorescence staining unveiled the presence of T lymphocytes and microgliosis in the sites of lesion for a long period after birth. Multiplex cytokines measurement upon supernatants of in vitro mixed brain cells and extracted mononuclear cells from offspring's brain has demonstrated an elevated secretion of the pro-inflammatory cytokines TNFα, IL-6 and IFNα and the chemokines RANTES and MCP-1. Hence, vertical transmission of CV-B4 and its persistence within offspring's brain can lead to pathological features linked to increased and sustained immune response., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Coxsackievirus B (CVB) causes severe morbidity and mortality in neonates and is sometimes associated with severe brain damage resulting from acute severe viral encephalomyelitis. However, the neuropathology of CVB infection remains unclear. A prototype strain of coxsackievirus B2 (Ohio-1) induces brain lesions in neonatal mice, resulting in dome-shaped heads, ventriculomegaly, and loss of the cerebral cortex. Here, we characterized the glial pathology in this mouse model. Magnetic resonance imaging revealed an absence of the cerebral cortex within 2 weeks after inoculation. Histopathology showed that virus replication triggered activation of microglia and astrocytes, and induced apoptosis in the cortex, with severe necrosis and lateral ventricular dilation. In contrast, the brainstem and cerebellum remained morphologically intact. Immunohistochemistry revealed high expression of the coxsackievirus and adenovirus receptor (a primary receptor for CVB) in mature neurons of the cortex, hippocampus, thalamus, and midbrain, demonstrating CVB2 infection of mature neurons in these areas. However, apoptosis and neuroinflammation from activated microglia and astrocytes differed in thalamic and cortical areas. Viral antigens were retained in the brains of animals in the convalescence phase with seroconversion. This animal model will contribute to a better understanding of the neuropathology of CVB infection., (© 2019 American Association of Neuropathologists, Inc. All rights reserved.)

Calpains are calcium-activated neutral cysteine proteases. The dysregulation of calpain activity has been found to be related to cardiovascular diseases, for which calpain inhibition is used as a treatment. Viral myocarditis (VMC) is primarily caused by Coxsackievirus group B3 virus infection (CVB3). CVB3 virus infection induces autophagy and hijacks this process to facilitate its replication. In this study, we found that calpain was significantly activated in hearts affected by VMC. However, pharmacologically inhibiting calpain aggravated VMC symptoms in mice due to myocardial inflammation and cardiac dysfunction. The inhibition of calpain activity in vitro led to the accumulation of LC3-II and increased levels of p62/SQSTM1 protein expression, suggesting that autophagic flux was impaired by calpain inhibition. These effects of calpain inhibition were also observed in capn4-specific myocardial knockout mice in vivo. Furthermore, our results provided evidence that calpain inhibition in VMC, unlike other cardiovascular diseases, exacerbated the disease symptom by impairing CVB3-induced autophagic flux, which may subsequently reduce virus autolysosome degradation. Our findings indicated that calpain inhibition may not be a good treatment for VMC disease in a clinical setting., (Copyright © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: Coxsackie-B-viruses (CVB) are frequent causes of acute myocarditis and dilated cardiomyopathy, but an effective antiviral therapy is still not available. Previously, we and others have demonstrated that treatment with an engineered sCAR-Fc (soluble coxsackievirus-adenovirus receptor fused to the carboxyl-terminus of human IgG) efficiently neutralizes CVB3 and inhibits the development of cardiac dysfunction in mice with acute CVB3-induced myocarditis. In this study, we analyzed the potential of sCAR-Fc for treatment of chronic CVB3-induced myocarditis in an outbred NMRI mouse model., Methods: NMRI mice were infected with the CVB3 strain 31-1-93 and treated with a sCAR-Fc expressing adeno-associated virus 9 vector 1, 3, and 7 days after CVB3 infection. Chronic myocarditis was analyzed on day 28 after infection., Results: Initial investigations showed that NMRI mice develop pronounced chronic myocarditis between day 18 and day 28 after infection with the CVB3 strain 31-1-93. Chronic cardiac infection was characterized by inflammation and fibrosis as well as persistence of viral genomes in the heart tissue and by cardiac dysfunction. Treatment of NMRI mice resulted in a distinct reduction of cardiac inflammation and fibrosis and almost complete elimination of virus RNA from the heart by day 28 after infection. Moreover, hemodynamic measurement revealed improved cardiac contractility and diastolic relaxation in treated mice compared with mice treated with a control vector (mean±SD; maximal pressure, 81.9±9.2 versus 69.4±8.6 mm Hg, P =0.02; left ventricular ejection fraction, 68.9±8.5 versus 54.2±11.5%, P =0.02; dP/dt max , 7275.2±1674 versus 4432.6±1107 mm Hg/s, P =0.004; dP/dt min , -4046.9±776 versus -3146.3±642 mm Hg/s, P =0.046). The therapeutic potential of sCAR-Fc is limited, however, since postponed start of sCAR-Fc treatment either 3 or 7 days after infection could not attenuate myocardial injury., Conclusions: Early therapeutic employment of sCAR-Fc, initiated at the beginning of the primary viremia, inhibits the development of chronic CVB3-induced myocarditis and improves the cardiac function to a level equivalent to that of uninfected animals.

Type B coxsackieviruses (CVB) are enteroviruses responsible for a common infectious myocarditis and pancreatitis. DCs and regulatory T cells (Tregs) are key players in controlling virus replication and regulating the immune response and tissue damage, respectively. However, the mechanisms underlying cellular migration to target tissues remain unclear. In the present study, we found that CVB5 infection induced CCL17 production and controlled the migration of CCR4 + DCs and CCR4 + Tregs to the pancreatic lymph nodes (pLN). CVB5 infection of CCR4 -/- mice reduced the migration of the CD8α + DC subset and reduced DC activation and production of IFN-β and IL-12. Consequently, CCR4 -/- mice presented decreased IFN-γ-producing CD4 + and CD8 + T cells, an increased viral load and more severe pancreatitis. In addition, CCR4 -/- mice had impaired Treg accumulation in pLN as well as increased T lymphocyte activation. Adoptive transfer of CCR4 + Tregs but not CCR4 - Tregs was able to regulate T lymphocyte activation upon CVB5 infection. The present data reveal a previously unknown role for CCR4 in coordinating immune cell migration to CVB-infected tissues and in controlling subsequent pancreatitis. These new insights may contribute to the design of future therapies for acute and chronic infection of non-polio enteroviruses.

Viral myocarditis is potentially fatal and lacking a specific treatment. Exosomes secreted by cardiac progenitor cells (CPCs) have emerged as a promising tool for cardioprotection and repair. In this study, we investigated whether CPCs-derived exosomes (CPCs-Ex) could utilize the mTOR signal pathway to reduce the apoptosis in viral myocarditis. In vitro, exosomes were, respectively, added to H9C2 cells after CVB3 infection to detect the anti-apoptosis effect of CPCs-Ex. Compared with the controls, the apoptosis rate was reduced, accompanied with the depressed expression of viral capsid protein 1 (VP1) and pro-apoptosis factors of Bim/caspase families. Meanwhile, the phosphorylation of Akt, mTOR, and p70S6K were promoted, but that of 4EBP1 was suppressed. In vivo, the results of apoptosis, expression of CVB3 and pro-apoptosis factors, and phosphorylation of Akt/mTOR factors of CVB3-infected cardiomyocytes were consistent with that of vitro. Following that, we use Rapamycin and MK-2206 to inhibit the Akt/mTOR signaling pathway, meanwhile, Rattus 4EBP1, p70S6K, Akt1 and Akt2 were transfected to H9C2 cells to establish the stably transfected cell lines. In the group with Rapamycin or MK-2206 pretreatment, CPCs-Ex also could decrease the apoptosis of H9C2 cells and expression of CVB3 mRNA, followed by decreased expression of apoptosis factors. In Akt2, p70S6K and 4EBP1 overexpression groups, CPCs-Ex promoted CVB3-induced apoptosis, VP1 expression and cleavage of caspase-3. Our results therefore identify CPCs-Ex exerts an anti-apoptosis effect in CVB3-infected cells by abrogating the proliferation of CVB3 and modulating the mTOR signaling pathways as well as the expression of Bcl-2 and caspase families. Viral myocarditis, mainly caused by CVB3 infection, is lacking a specific treatment. Our study identified an anti-apoptosis role of CPCs-Ex in CVB3-infected cells and rats, which shown that CPCs-Ex may be an effective tool to treat viral myocarditis. We believe that with more in-depth research on the functionality of CPCs-Ex, there will be a breakthrough in the treatment of viral myocarditis.

Fingolimod (FTY720) after phosphorylation, as the ligand of sphingosine 1-phosphate receptors (S1PRs), plays an important role in cell proliferation and differentiation. In this article, FTY720 in the treatment of coxsackievirus B3 (CVB3)-induced viral myocarditis was closely related to apoptosis and AKT/caspase-3 apoptotic pathways. We found that CVB3 inhibited myocardial apoptosis at the early stage with upregulating p-AKT level and downregulating activated caspase-3 level for replication of virus progeny, whereas it promoted apoptosis at a late stage with downregulating p-AKT and upregulating activated caspase-3 for releasing the newly synthesized virus to spread. Interestingly, FTY720 could reverse this trend; it promoted apoptosis at an early stage and inhibited apoptosis at the late stage in vivo and vitro, which proved the antiviral effect. We also found that S1PR1, S1PR4, and S1PR5, rather than S1PR2 and S1PR3, were regulated by FTY720 in this process. The results confirmed that FTY720 alleviates CVB3-induced myocarditis and inhibits viral replication through regulating S1PRs and AKT/caspase-3 pathways with a bidirectional regulation of apoptosis., (© 2019 Wiley Periodicals, Inc.)

Myocarditis, inflammation of the heart muscle, affects all demographics and is a major cause of sudden and unexpected death in young people. It is most commonly caused by viral infections of the heart, with coxsackievirus B3 (CVB3) being among the most prevalent pathogens. To understand the molecular pathogenesis of CVB3 infection and provide strategies for developing treatments, we examined the role of a key nuclear pore protein 98 (NUP98) in the setting of viral myocarditis. NUP98 was cleaved as early as 2 h post-CVB3 infection. This cleavage was further verified through both the ectopic expression of viral proteases and in vitro using purified recombinant CVB3 proteases (2A and 3C), which demonstrated that CVB3 2A but not 3C is responsible for this cleavage. By immunostaining and confocal imaging, we observed that cleavage resulted in the redistribution of NUP98 to punctate structures in the cytoplasm. Targeted siRNA knockdown of NUP98 during infection further increased viral protein expression and viral titer, and reduced cell viability, suggesting a potential antiviral role of NUP98. Moreover, we discovered that expression levels of neuregulin-1 (NRG1), a cardioprotective gene, and presenilin-1 (PSEN1), a cellular protease processing the tyrosine kinase receptor ERBB4 of NRG1, were reliant upon NUP98 and were downregulated during CVB3 infection. In addition, expression of these NUP98 target genes in myocardium tissue not only occurred at an earlier phase of infection, but also appeared in areas away from the initial inflammatory regions. Collectively, CVB3-induced cleavage of NUP98 and subsequent impairment of the cardioprotective NRG1-ERBB4/PSEN1 signaling cascade may contribute to increased myocardial damage in the context of CVB3-induced myocarditis. To our knowledge, this is the first study to demonstrate the link between NUP98 and the NRG1 signaling pathway in viral myocarditis.

Coxsackieviruses B (CV-B) belong to the EV-B species. CV-B and particularly CV-B4 are thought to be involved in the development of chronic diseases like type 1 diabetes (T1D). The mechanisms of the enteroviral pathogenesis of T1D are not well known, yet. The in vitro studies are rich with information but in vivo infection models are needed to investigate the impact of viruses onto organs. Our objective was to study the impact of CV-B4E2 combined with a single sub-diabetogenic dose of streptozotocin (STZ) on the pancreas of mice. The infection with CV-B4E2 of CD1 outbred mice treated with a sub-diabetogenic dose of STZ induced hyperglycemia and hypoinsulinemia. Along with the chemokine IP-10, viral RNA and infectious particles were detected in the pancreas. The pancreas of these animals was also marked with insulitis and other histological alterations. The model combining STZ and CV-B4E2 opens the door to new perspectives to better understand the interactions between virus and host, and the role of environmental factors capable, like STZ, to predispose the host to the diabetogenic effects of enteroviruses.

The role of B cells in viral myocarditis (VMC) remains controversial. In order to establish a role and mechanism of action for B cells in acute VMC, we established an acute VMC mouse model by intraperitoneal injection of Coxsackie virus group B type 3 (CVB3). At day 7, mice were analyzed using myocardial histopathology, and the presence of M2 macrophages in spleen and heart. Mice were divided into four groups, all having a C57BL/6 background: control group; wild-type (WT) VMC; mMt/mMt (-/-) VMC (BKO), and BKO + B cell VMC. A role for B cells was demonstrated by a significant reduction in myocardial pathological score and an increase in the frequency of M2 macrophages in the BKO group, when compared to the WT group. Once BKO mice underwent B cell reconstitution with isolated WT B cells, the myocardial pathological score was increased significantly, while the frequency of M2 macrophages decrease. Our findings demonstrate that B cells increase myocardial inflammation by suppressing M2 polarization in acute VMC in vivo.

Latency associated peptide (LAP) is a protein expressed on the membrane of some regulatory T cells (Treg). LAP + Treg have a greater immunomodulatory effect than that of their negative counterparts. In this study, we presented the data on the proportion of LAP + Treg out of CD4 + cells in mice with viral myocarditis, which we believed was more sensitive and specific than that of the ratio of total Treg in CD4 + cells. Comparing with the previously recognized total Treg, LAP + Treg was a better biomarker on myocardial inflammation., (© 2018 Wiley Periodicals, Inc.)

Objective: To determine the effects and mechanism of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1, CC1)-mediated regulation of the Coxsackie and Adenovirus Receptor (CAR) after Coxsackievirus B3 (CVB3) infection., Methods: A mouse CC1 overexpression recombinant virus was constructed, followed by insertion of a pLVX-CEACAM 1-zsgreen-puro (rLV-CEACAM 1) plasmid into the recombinant retrovirus. Cardiac myocytes were assigned into different groups according to various treatments. The apoptosis rate and cell activity in each group were observed. Further, CAR expression and SYK, IL-1β, and p-SYK levels were measured., Results: The recombinant retrovirus titer was measured as 1.5 × 10 TUs/ml. The apoptosis rate of cardiac myocytes in the CC1 overexpression plus CVB3 group was significantly elevated, and the relative expression of the CAR gene was the highest in the CC1 overexpression plus CVB3 group. TNF-α and IL-1β levels increased due to CC1 overexpression and further increased after CVB3 infection. CAR protein expression also changed along with the levels of CC1, SYK, and TNF-α after infection., Conclusion: CC1 may promote CAR expression after CVB3 infection and regulate CAR protein expression by activating the CC1-SYK-TNF-α signaling axis during the infection process.

Current approaches to influenza control rely on vaccines matched to viruses in circulation. Universal influenza vaccines would offer the advantage of providing broad protection against diverse strains of influenza virus. Candidate universal vaccines are developed using model systems, often testing in naïve animals. Yet the human population is not naïve, having varied immune histories that include exposure to viruses. We studied a candidate universal influenza vaccine (replication deficient adenoviruses expressing the conserved influenza A antigens NP and M2 [A/NP+M2-rAd]) given intranasally, the route previously shown to be most effective. To model recipients exposed to viruses, we used mice given rhinovirus (RV1B), respiratory syncytial virus (RSV-A2), influenza B virus, or influenza A virus before or after universal influenza vaccine. Vaccine performance was assessed by measuring immune responses to NP and M2, and monitoring weight loss and survival following influenza A challenge. Prior influenza A virus infection enhanced the response to the vaccine by priming to conserved influenza A antigens. RSV-A2 or RV1B had no effect on antibody responses to NP and M2 in serum. None of the viruses inhibited the ability of the vaccine to protect against influenza A virus challenge. The study demonstrates that the usefulness of this universal vaccine is not confined to the immunologically naïve and supports possible use in a human population with a varied history of respiratory infections., Competing Interests: The authors have declared that no competing interests exist.

Overwhelming cardiac inflammation has been reported to be the pathogenic mechanism of Coxsackievirus B3 (CVB3)-induced viral myocarditis (VMC), while the detailed molecular mechanisms remain unknown. Membrane-bound dipeptidases (MBD, also known as Dpep) have been shown to be involved in inflammatory diseases. However, the clear and direct evidence of their impacts on inflammation is still lacking. In this study, our results revealed that Dpep2 expression was remarkably increased during CVB3 infection, and primarily produced by the cardiac tissue-infiltrating macrophages instead of constitutive cardiomyocytes. Macrophages have been reported to play an important pathological role in driving VMC. Interestingly, macrophage-specific Dpep2 deletion robustly aggravated CVB3-induced cardiac inflammation, evidenced by augmented expression of TNF-α, IL-6, and MCP-1 in heart tissue. In addition, Dpep2-deficient bone-marrow derived macrophages (BMDMs) generated more TNF-α, IL-6, and MCP-1 after CVB3 stimulation compared with the control BMDMs. Moreover, this suppressive effect of Dpep2 on macrophages relied on its repression on NF-κB signaling pathway, but not on its conventional hydrolysate LTE4. Taken together, this study revealed that Dpep2 could protect against CVB3-induced VMC by acting as a suppressor of macrophage inflammation. Better understanding how macrophage Dpep2 dampened the cardiac inflammation would provide us with insights for the efficient control of CVB3-induced VMC.

Coxsackieviruses of group B (CVB) are well-known causes of acute and chronic myocarditis. Chronic myocarditis can evolve into dilated cardiomyopathy (DCM) characterized by fibrosis and cardiac remodeling. Interleukin-1β (IL-1β) plays a decisive role in the induction of the inflammatory response as a consequence of viral replication. In this study, we analyzed the effects of IL-1β neutralization on the transition of acute to chronic myocarditis in a mouse model of CVB3 myocarditis. Mice were treated with an anti-murine IL-1β antibody as a surrogate for Canakinumab at different time points post CVB3 infection. Treatment was performed in the early phase (day 1-14 pi, day 3-14 pi) or at a later stage of myocarditis (day 14-28 pi). Subsequently, the hearts were examined histologically, immunohistochemically and by molecular biology. A significant reduction of viral replication, cardiac damage and inflammation was found after administration of the antibody in the early phase and in the later phase of infection. Furthermore, less collagen I deposition and a considerable reduction of fibrosis were found in antibody-treated mice. Using microarray analysis, a significant upregulation of various extracellular matrix and fibrosis-associated molecules was found in CVB3-infected mice, including TGF-β, TIMP-1 and MMP12, as well as diverse matricellular proteins, whereas, these molecules were significantly downregulated in all IL-1β antibody-treated infected mice. Neutralization of IL-1β at different stages of enteroviral infection prevents the development of chronic viral myocarditis by reducing inflammation, interstitial fibrosis and adverse cardiac remodeling. These findings are relevant for the treatment of patients with acute and chronic myocarditis.

Adenosine deaminases acting on RNA (ADAR) are enzymes that regulate RNA metabolism through post-transcriptional mechanisms. ADAR1 is involved in a variety of pathological conditions including inflammation, cancer, and the host defense against viral infections. However, the role of ADAR1p150 in vascular disease remains unclear. In this study, we examined the expression of ADAR1p150 and its role in viral myocarditis (VMC) in a mouse model. VMC mouse cardiomyocytes showed significantly higher expression of ADAR1p150 compared to the control samples. Coimmunoprecipitation verified that ADAR1p150 forms a complex with Dicer in VMC. miRNA-222, which is involved in many cardiac diseases, is highly expressed in cardiomyocytes in VMC. In addition, the expression of miRNA-222 was promoted by ADAR1p150/Dicer. Among the target genes of miRNA-222, the expression of phosphatase-and-tensin (PTEN) protein was significantly reduced in VMC. By using a bioinformatics tool, we found a potential binding site of miRNA-222 on the PTEN gene's 3'-UTR, suggesting that miRNA-222 might play a regulatory role. In cultured cells, miR-222 suppressed PTEN expression. Our findings suggest that ADAR1p150 plays a key role in complexing with Dicer and promoting the expression of miRNA-222, the latter of which suppresses the expression of the target gene PTEN during VMC. Our work reveals a previously unknown role of ADAR1p150 in gene expression in VMC.

Infections with Coxsackievirus B3 and other members of the enterovirus genus are a common reason for myocarditis and sudden cardiac death in modern society. Despite intensive scientific efforts to cure enterovirus infections, there is still no standardized treatment option. The complexity of Coxsackievirus B3´s effects on the host cell make well defined studies on this topic very challenging. However, recent publications report newly found effects of CVB3´s structural and non-structural proteins on infected cells. For the first time, the viral capsid protein VP1 was shown to have direct influence on the viral life-cycle. By shortening the G0 and the G2 phase and simultaneously prolonging the G1 and G1-S phase, the translation of viral proteins is enhanced and the production of viable CVB3 particles is promoted. Coxsackievirus B3´s viroporin, protein 2B, was recently studied in more detail as well. Structural and physiological analyses identified two hydrophilic α-helices in the structure of 2B, enabling it to insert into cellular membranes of host cells. As main target of 2B the endoplasmatic reticulum was identified. The insertion of 2B into the ER membranes leads to an uncontrolled calcium outflow into the cytoplasm. Additional insertion of 2B into the cell membrane leads to host cell destabilization and in the end to release of viral progeny. The importance of the Coxsackievirus B3´s proteases 2A and 3C in pathogenicity is observed since years. Recently, DAP5 and eIf4G were identified as new cleavage targets for protease 2A. Cleavage of DAP-5 into DAP5-N and DAP5-C changes the gene expression of the host cell and promotes cell death. Additionally, protease 3C targets and cleaves procaspase 8 promoting the mitochondrial apoptosis pathway and cell death. Recent studies identified significant effects of CVB3 on mitochondria of infected cells. Mouse cardiomyocytes showed decreased activities of respiratory chain complexes I-III and changed transcription of important subunits of the complexes I-IV. A disrupted energy metabolism may be one of the main causes of cardiac insufficiency and death in CVB3 infected patients. In addition to a modified energy metabolism, CVB3 affects cardiac ion channels, KCNQ1 in particular. SGK1, which is an important mediator in KCNQ1 membrane insertions, is highly upregulated during CVB3 infections. This results in an increased insertion of KCNQ1 into the cell membrane of cardiac cells. Under stress conditions, this KCNQ1 overshoot may lead to a disturbed cardiac action potential and therefore to sudden cardiac death, as it is often observed in CVB3 infected persons., Competing Interests: No conflicts of interest exist., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Enteroviruses and Coxsackie viruses are common causes of aseptic meningitis and encephalitis in children. These infections usually have a benign, self-limited course. However, they can have a florid presentation in immunocompromised patients, such as neonates, patients exposed to immunosuppressive drugs, such as transplant recipients and patients with agammaglobulinemia. We present a rare case of rapidly progressive acute encephalopathy caused by Coxsackie meningoencephalitis and complicated by refractory status epilepticus in an immunocompetent adult male. Our case highlights the importance of having a broad differential in patients presenting with rapidly progressive acute encephalopathy. Although rare, an enterovirus infection caused by a Coxsackie virus subtype can have a severe presentation causing significant morbidity. This case, also underscores the importance of searching for underlying immunodeficiency in malignant presentations of common viral infections. Hence, rapidly progressive acute encephalopathy due to coxsackievirus can occur in immunocompetent individuals. Aggressive and systematic diagnostic and therapeutic approach in such severe cases can influence overall outcomes.