Coxsackievirus B3 Research Articles

Abstract Mo077: Cardiac Troponin I3 Kinase in Viral Myocarditis

Myocarditis is a leading cause of heart failure and sudden cardiac death. Often caused by viral infection, myocarditis is defined histopathologically as mononuclear immune infiltration in the heart muscle. While most patients recover with no long-term effects, some deteriorate rapidly. It is unknown what controls this variable susceptibility to severe disease. Without this knowledge, the ability to develop targeted therapies for acute myocarditis is limited. Previous studies utilizing mouse genetics identified a locus that controls susceptibility to viral myocarditis. Cardiomyocyte-specific kinase TNNI3K is one candidate gene within this locus, but its role in myocarditis has not been established. Importantly, millions of people throughout the world carry natural variants of TNNI3K. My overall hypothesis is that TNNI3K confers resistance to viral myocarditis in a kinase-dependent manner. In an initial study, we infected strain background-matched wild-type, Tnni3k knockout, and Tnni3k kinase-dead mice with heart-passaged coxsackievirus B3. After 10 days, during the peak of acute myocarditis, mice missing functional Tnni3k protein were found to have higher viral genomes and greater presence of immune cell markers (CD45+), clearly demonstrating the role of Tnni3k in the response to viral myocarditis. H&E-stained heart sections show large clusters of hematoxylin-stained nuclei, indicating infiltration of ventricular tissue by immune cells, with more of these clusters in Tnni3k knockout hearts. Because kinase-dead mutants are similarly compromised as fully null mice, this indicates that the kinase activity of TNNI3K is critical in the antiviral response. These results are the first demonstration of the in vivo activity of a specific gene that modifies viral myocarditis sensitivity. Future work will expand on this study by examining the effects of coxsackievirus B3 infection of Tnni3k mutant mice at early and late time points. Additionally, efforts are currently underway to identify the specific mechanisms by which TNNI3K regulates the immune response to viral infection.

1mΨ influences the performance of various positive-stranded RNA virus-based replicons

Self-amplifying RNAs (saRNAs) are versatile vaccine platforms that take advantage of a viral RNA-dependent RNA polymerase (RdRp) to amplify the messenger RNA (mRNA) of an antigen of interest encoded within the backbone of the viral genome once inside the target cell. In recent years, more saRNA vaccines have been clinically tested with the hope of reducing the vaccination dose compared to the conventional mRNA approach. The use of N1-methyl-pseudouridine (1mΨ), which enhances RNA stability and reduces the innate immune response triggered by RNAs, is among the improvements included in the current mRNA vaccines. In the present study, we evaluated the effects of this modified nucleoside on various saRNA platforms based on different viruses. The results showed that different stages of the replication process were affected depending on the backbone virus. For TNCL, an insect virus of the Alphanodavirus genus, replication was impaired by poor recognition of viral RNA by RdRp. In contrast, the translation step was severely abrogated in coxsackievirus B3 (CVB3), a member of the Picornaviridae family. Finally, the effects of 1mΨ on Semliki forest virus (SFV), were not detrimental in in vitro studies, but no advantages were observed when immunogenicity was tested in vivo.

An intranasal attenuated Coxsackievirus B3 vaccine induces strong systemic and mucosal immunity against CVB3 lethal challenge.

Coxsackievirus B3 (CVB3) triggers viral myocarditis, with no effective vaccine yet. This fecal-oral transmitted pathogen has prompted interest in mucosal immunization strategies to impede CVB3 spread. We developed a new attenuated vaccine strain, named CVB3(mu). The potential of CVB3(mu) to stimulate mucosal immune protection remains to be elucidated. This study evaluates the attenuation characteristics of CVB3(mu) via a rapid evolution cellular model and RNA sequencing. Its temperature sensitivity and safety were evaluated through in vitro and in vivo experiments. The mucosal immunity protection of CVB3(mu) was assessed via intranasal immunization in Balb/c mice. The results indicate that CVB3(mu) exhibits temperature sensitivity and forms smaller plaques. It sustains fewer genetic mutations and still possesses certain attenuated traits up to the 25th passage, in comparison to CVB3(WT). Intranasal immunization elicited a significant serum neutralizing antibodies, and a substantial sIgA response in nasal washes. In vivo trials revealed CVB3(mu) protection in adult mice and passive protection in suckling mice against lethal CVB3(WT) challenges. In conclusion, CVB3(mu), a live attenuated intranasal vaccine, provides protection involving humoral and mucosal immunity, making it a promising candidate to control CVB3 spread and infection.

Crystal structures of the 3C proteases from Coxsackievirus B3 and B4.

Enteroviruses cause a wide range of disorders with varying presentations and severities, and some enteroviruses have emerged as serious public health concerns. These include Coxsackievirus B3 (CVB3), an active causative agent of viral myocarditis, and Coxsackievirus B4 (CVB4), which may accelerate the progression of type 1 diabetes. The 3C proteases from CVB3 and CVB4 play important roles in the propagation of these viruses. In this study, the 3C proteases from CVB3 and CVB4 were expressed in Escherichia coli and purified by affinity chromatography and gel-filtration chromatography. The crystals of the CVB3 and CVB4 3C proteases diffracted to 2.10 and 2.01 Å resolution, respectively. The crystal structures were solved by the molecular-replacement method and contained a typical chymotrypsin-like fold and a conserved His40-Glu71-Cys147 catalytic triad. Comparison with the structures of 3C proteases from other enteroviruses revealed high similarity with minor differences, which will guide the design of 3C-targeting inhibitors with broad-spectrum properties.

Coxsackievirus B infection invokes unique cell-type specific responses in primary human pancreatic islets.

Coxsackievirus B (CVB) infection has long been considered an environmental factor precipitating Type 1 diabetes (T1D), an autoimmune disease marked by loss of insulin-producing β cells within pancreatic islets. Previous studies have shown CVB infection negatively impacts islet function and viability but do not report on how virus infection individually affects the multiple cell types present in human primary islets. Therefore, we hypothesized that the various islet cell populations have unique transcriptional responses to CVB infection. Here, we performed single-cell RNA sequencing on human cadaveric islets treated with either CVB or poly(I:C), a viral mimic, for 24 and 48 hours. Our global analysis reveals CVB differentially induces dynamic transcriptional changes associated with multiple cell processes and functions over time whereas poly(I:C) promotes an immune response that progressively increases with treatment duration. At the single-cell resolution, we find CVB infects all islet cell types at similar rates yet induces unique cell-type specific transcriptional responses with β, α, and ductal cells having the strongest response. Sequencing and functional data suggest that CVB negatively impacts mitochondrial respiration and morphology in distinct ways in β and α cells, while also promoting the generation of reactive oxygen species. We also observe an increase in the expression of the long-noncoding RNA MIR7-3HG in β cells with high viral titers and reveal its knockdown reduces gene expression of viral proteins as well as apoptosis in stem cell-derived islets. Together, these findings demonstrate a cell-specific transcriptional, temporal, and functional response to CVB infection and provide new insights into the relationship between CVB infection and T1D.

Enterovirus 3A protein disrupts endoplasmic reticulum homeostasis through interaction with GBF1.

Enteroviruses are single-stranded, positive-sense RNA viruses causing endoplasmic reticulum (ER) stress to induce or modulate downstream signaling pathways known as the unfolded protein responses (UPR). However, viral and host factors involved in the UPR related to viral pathogenesis remain unclear. In the present study, we aimed to identify the major regulator of enterovirus-induced UPR and elucidate the underlying molecular mechanisms. We showed that host Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1), which supports enteroviruses replication, was a major regulator of the UPR caused by infection with enteroviruses. In addition, we found that severe UPR was induced by the expression of 3A proteins encoded in human pathogenic enteroviruses, such as enterovirus A71, coxsackievirus B3, poliovirus, and enterovirus D68. The N-terminal-conserved residues of 3A protein interact with the GBF1 and induce UPR through inhibition of ADP-ribosylation factor 1 (ARF1) activation via GBF1 sequestration. Remodeling and expansion of ER and accumulation of ER-resident proteins were observed in cells infected with enteroviruses. Finally, 3A induced apoptosis in cells infected with enteroviruses via activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/C/EBP homologous protein (CHOP) pathway of UPR. Pharmaceutical inhibition of PERK suppressed the cell death caused by infection with enteroviruses, suggesting the UPR pathway is a therapeutic target for treating diseases caused by infection with enteroviruses.IMPORTANCEInfection caused by several plus-stranded RNA viruses leads to dysregulated ER homeostasis in the host cells. The mechanisms underlying the disruption and impairment of ER homeostasis and its significance in pathogenesis upon enteroviral infection remain unclear. Our findings suggested that the 3A protein encoded in human pathogenic enteroviruses disrupts ER homeostasis by interacting with GBF1, a major regulator of UPR. Enterovirus-mediated infections drive ER into pathogenic conditions, where ER-resident proteins are accumulated. Furthermore, in such scenarios, the PERK/CHOP signaling pathway induced by an unresolved imbalance of ER homeostasis essentially drives apoptosis. Therefore, elucidating the mechanisms underlying the virus-induced disruption of ER homeostasis might be a potential target to mitigate the pathogenesis of enteroviruses.

Structural basis for a highly conserved RNA-mediated enteroviral genome replication.

Enteroviruses contain conserved RNA structures at the extreme 5' end of their genomes that recruit essential proteins 3CD and PCBP2 to promote genome replication. However, the high-resolution structures and mechanisms of these replication-linked RNAs (REPLRs) are limited. Here, we determined the crystal structures of the coxsackievirus B3 and rhinoviruses B14 and C15 REPLRs at 1.54, 2.2 and 2.54 Å resolution, revealing a highly conserved H-type four-way junction fold with co-axially stacked sA-sD and sB-sC helices that are stabilized by a long-range A•C•U base-triple. Such conserved features observed in the crystal structures also allowed us to predict the models of several other enteroviral REPLRs using homology modeling, which generated models almost identical to the experimentally determined structures. Moreover, our structure-guided binding studies with recombinantly purified full-length human PCBP2 showed that two previously proposed binding sites, the sB-loop and 3' spacer, reside proximally and bind a single PCBP2. Additionally, the DNA oligos complementary to the 3' spacer, the high-affinity PCBP2 binding site, abrogated its interactions with enteroviral REPLRs, suggesting the critical roles of this single-stranded region in recruiting PCBP2 for enteroviral genome replication and illuminating the promising prospects of developing therapeutics against enteroviral infections targeting this replication platform.

Mapping mutational fitness effects across the coxsackievirus B3 proteome reveals distinct profiles of mutation tolerability.

RNA viruses have notoriously high mutation rates due to error-prone replication by their RNA polymerase. However, natural selection concentrates variability in a few key viral proteins. To test whether this stems from different mutation tolerance profiles among viral proteins, we measured the effect of >40,000 non-synonymous mutations across the full proteome of coxsackievirus B3 as well as >97% of all possible codon deletions in the nonstructural proteins. We find significant variation in mutational tolerance within and between individual viral proteins, which correlated with both general and protein-specific structural and functional attributes. Furthermore, mutational fitness effects remained stable across cell lines, suggesting selection pressures are mostly conserved across environments. In addition to providing a rich dataset for understanding virus biology and evolution, our results illustrate that incorporation of mutational tolerance data into druggable pocket discovery can aid in selecting targets with high barriers to drug resistance.

Etiology of Enteroviral Meningitis in Ekaterinburg Before and the After Pandemic of COVID‑19

Introduction. The incidence of enterovirus meningitis in Ekaterinburg has long exceeded the average in Russia. As a result of sanitary, anti-epidemic and restrictive measures related to the COVID-19 pandemic, there was a decrease in morbidity, which led to a reduction in the circulation of non-polio enteroviruses and created risks after lifting of restrictions.The purpose of the study is to conduct a comparative analysis of non-polio enteroviruses isolated in Ekaterinburg from patients with enterovirus before and after the COVID-19 pandemic.Materials and methods. Genotyping positive samples by sequencing VP1 gene (cerebrospinal fluid, nasopharyngeal, faeces) from patients with aseptic meningitis was performed using the BLAST service. Multiple alignment and phylogenetic analysis were performed using the MEGA software.Results. In 2022, after lifting restrictive measures, the incidence of enteroviruses in Ekaterinburg increased 10 times compared to the previous year (3,390 per 100 thousand inhabitants), but remained 6.5 times lower than the long-term average (22,035 per 100 thousand population). 7 types of non-polio enteroviruses were detected: 2 species A (Coxsackievirus A4, Enterovirus A71) and 5 species B (Coxsackievirus A9 and B2, Echovirus 6, 7, 9). Coxsackievirus A9 dominated, followed by Echovirus 6.Discussion. In 2022, Coxsackievirus A9 prevailed as the predominant type in enterovirus, was not epidemiologically significant and was rarely found in the cerebrospinal fluid samples. Echovirus 6, the second most frequently detected, was characteristic of the Ural Federal District. The remaining viruses (Coxsackievirus A4 and B2, Echovirus 7 and 9, Enterovirus A71) were also detected in patients with enterovirus before the COVID-19 pandemic.Conclusion. After the cancellation of prophylactic measures after the COVID-19 pandemic, the incidence of enteroviruses in Ekaterinburg during 2022 increased due to the restoration of circulation of non-polio enteroviruses.

Genome-Driven Discovery of Antiviral Atralabdans A-C from the Soil-Dwelling Streptomyces atratus.

Heterologous expression of an atr terpenoid gene cluster derived from Streptomyces atratus Gö66 in S. albus J1074 led to the discovery of three novel labdane diterpenoids featuring an unprecedented 6/6/5-fused tricyclic skeleton, designated as atralabdans A-C (1-3), along with a known compound, labdanmycin A. Compounds 1-3 were identified through extensive spectroscopic analysis, including NMR calculations with DP4+ probability analysis, and a comparative assessment of experimental and theoretical electronic circular dichroism (ECD) spectra. A plausible biosynthetic pathway for these compounds was proposed. Compounds 1-3 exhibited inhibitory activity against the human neurotropic coxsackievirus B3 (CVB3); 1 was the most potent, surpassing the positive control ribavirin with a higher therapeutic index.

Molecular epidemiology of enteroviruses from Guatemalan wastewater isolated from human lung fibroblasts.

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

Robust Systemic and Mucosal Immune Responses to Coxsackievirus B3 Elicited by Spider Silk Protein Based Nanovaccines via Subcutaneous Immunization

AbstractCoxsackievirus B3 (CVB3) is a member of the enterovirus genus and linked to several diseases, including myocarditis, which can progress to dilated cardiomyopathy. Despite ongoing preclinical efforts, no clinically approved vaccines against CVB3 are currently available, highlighting the urgent need for effective prophylactic solutions. In this study, a nanovaccine platform based on spider minor ampullate silk protein (MiSp) is introduced. This platform utilizes protein nanoparticles engineered from chimeric proteins that incorporate CVB3 antigenic peptides into customized MiSp, subsequently loaded with all‐trans retinoic acid (RA). These functional nanovaccines are capable of eliciting both mucosal and systemic immune responses following subcutaneous administration and demonstrate significant protective effects against CVB3 infection in mice. This study signifies an approach in peptide‐based parenteral vaccine strategies, utilizing engineered MiSp nanoparticles combined with RA. This methodology represents a promising pathway for preventing enterovirus infections by leveraging the unique immunomodulatory properties of spidroins and RA to combat these pathogens effectively.

Comparison of structure and immunogenicity of CVB1-VLP and inactivated CVB1 vaccine candidates.

Coxsackievirus B1 (CVB1) is a common cause of acute and chronic myocarditis, dilated cardiomyopathy and aseptic meningitis. However, no CVB-vaccines are available for human use. In this study, we investigated the immunogenicity of virus-like particle (VLP) and inactivated whole-virus vaccines for CVB1 when administrated to mice via either subcutaneous or intranasal routes formulated with and without commercial and experimental adjuvants. Here, the potential of utilizing epigallocatechin-3-gallate (EGCG) as a mucosal adjuvant synergistically with its ability to inactivate the virus were investigated. EGCG had promising adjuvant properties for CVB1-VLP when administered via the parenteral route but limited efficacy via intranasal administration. However, intranasal administration of the formalin-inactivated virus induced high CVB1-specific humoral, cellular, and mucosal immune responses. Also, based on CVB1-specific IgG-antibody responses, we conclude that CVB1-VLP can be taken up by immune cells when administrated intranasally and further structural engineering for the VLP may increase the mucosal immunogenicity. The preparations contained mixtures of compact and expanded A particles with 85% expanded in the formalin-inactivated virus, but only 52% in the VLP observed by cryogenic electron microscopy. To correlate the structure to immunogenicity, we solved the structures of the CVB1-VLP and the formalin-inactivated CVB1 virus at resolutions ranging from 2.15 A to 4.1 A for the expanded and compact VLP and virus particles by image reconstruction. These structures can be used in designing mutations increasing the stability and immunogenicity of CVB1-VLP in the future. Overall, our results highlight the potential of using formalin inactivated CVB1 vaccine in mucosal immunization programs and provide important information for future development of VLP-based vaccines against all enteroviruses.

Investigating TIP30-Mediated regulation of mTORC1 signaling as a therapeutic strategy for coxsackievirus B3-Induced viral myocarditis

This study aims to elucidate the role of TIP30 (30 KDa HIV-1 TAT-Interacting Protein) in the progression of coxsackievirus B3 (CVB3)-induced viral myocarditis. TIP30 knockout and wildtype mice were intraperitoneally infected with CVB3 and evaluated at day 7 post-infection. HeLa cells were transfected with TIP30 lentiviral particles and subsequently infected with CVB3 to evaluate viral replication, cellular pathogenesis, and mechanistic target of rapamycin complex 1 (mTORC1) signaling. Deletion of the TIP30 gene heightened heart virus titers and mortality rates in mice with CVB3-induced myocarditis, exacerbating cardiac damage and fibrosis, and elevating pro-inflammatory factors level. In vitro experiments demonstrated the modulation of mTORC1 signaling by TIP30 during CVB3 infection in HeLa cells. TIP30 overexpression mitigated CVB3-induced cellular pathogenesis and VP1 expression, with rapamycin, an mTOR1 inhibitor, reversing these effects. These findings suggest TIP30 plays a critical protective role against CVB3-induced myocarditis by regulating mTORC1 signaling.

Transcription factor EB (TFEB) interaction with RagC is disrupted during enterovirus D68 infection.

Enteroviruses are among the most significant causes of human disease. Some enteroviruses are responsible for severe paralytic diseases such as poliomyelitis or acute flaccid myelitis. The latter disease is associated with multiple non-polio enterovirus species, including enterovirus D68 (EV-D68), enterovirus 71, and coxsackievirus B3 (CVB3). Here, we demonstrate that EV-D68 interacts with a host transcription factor, transcription factor EB (TFEB), to promote viral RNA(vRNA) replication and regulate the egress of virions from cells. TFEB was previously implicated in the viral egress of CVB3, and the viral protease 3C cleaves TFEB during infection. Here, we show that EV-D68 3C protease also cleaves TFEB after the peak of vRNA replication. This cleavage disrupts TFEB interaction with the host protein RagC, which changes the localization and regulation of TFEB. TFEB lacking a RagC-binding domain inhibits autophagic flux and promotes virus egress. These mechanistic insights highlight how common host factors affect closely related, medically important viruses differently.

Metabolomics combined with network pharmacology reveals a role for astragaloside IV in inhibiting enterovirus 71 replication via PI3K-AKT signaling

BackgroundAstragaloside IV (AST-IV), as an effective active ingredient of Astragalus membranaceus (Fisch.) Bunge. It has been found that AST-IV inhibits the replication of dengue virus, hepatitis B virus, adenovirus, and coxsackievirus B3. Enterovirus 71 (EV71) serves as the main pathogen in severe hand-foot-mouth disease (HFMD), but there are no specific drugs available. In this study, we focus on investigating whether AST-IV can inhibit EV71 replication and explore the potential underlying mechanisms.MethodsThe GES-1 or RD cells were infected with EV71, treated with AST-IV, or co-treated with both EV71 and AST-IV. The EV71 structural protein VP1 levels, the viral titers in the supernatant were measured using western blot and 50% tissue culture infective dose (TCID50), respectively. Network pharmacology was used to predict possible pathways and targets for AST-IV to inhibit EV71 replication. Additionally, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to investigate the potential targeted metabolites of AST-IV. Associations between metabolites and apparent indicators were performed via Spearman’s algorithm.ResultsThis study illustrated that AST-IV effectively inhibited EV71 replication. Network pharmacology suggested that AST-IV inhibits EV71 replication by targeting PI3K-AKT. Metabolomics results showed that AST-IV achieved these effects by elevating the levels of hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-hydroxy-1 H-indole-3- acetamide, oxypurinol, while reducing the levels of PC (14:0/15:0). Furthermore, AST-IV also mitigated EV71-induced oxidative stress by reducing the levels of MDA, ROS, while increasing the activity of T-AOC, CAT, GSH-Px. The inhibition of EV71 replication was also observed when using the ROS inhibitor N-Acetylcysteine (NAC). Additionally, AST-IV exhibited the ability to activate the PI3K-AKT signaling pathway and suppress EV71-induced apoptosis.ConclusionThis study suggests that AST-IV may activate the cAMP and the antioxidant stress response by targeting eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-Hydroxy-1 H-indole-3-acetamide, oxypurinol and PC (14:0/15:0). This activation can further stimulate the PI3K-AKT signaling to inhibit EV71-induced apoptosis and EV71 replication.Graphical

Heart-specific NFAT5 knockout suppresses type I interferon signaling and aggravates coxsackievirus-induced myocarditis.

Nuclear factor of activated T cells 5 (NFAT5) is an osmosensitive transcription factor that is well-studied in renal but rarely explored in cardiac diseases. Although the association of Coxsackievirus B3 (CVB3) with viral myocarditis is well-established, the role of NFAT5 in this disease remains largely unexplored. Previous research has demonstrated that NFAT5 restricts CVB3 replication yet is susceptible to cleavage by CVB3 proteases. Using an inducible cardiac-specific Nfat5-knockout mouse model, we uncovered that NFAT5-deficiency exacerbates cardiac pathology, worsens cardiac function, elevates viral load, and reduces survival rates. RNA-seq analysis of CVB3-infected mouse hearts revealed the significant impact of NFAT5-deficiency on gene pathways associated with cytokine signaling and inflammation. Subsequent in vitro and in vivo investigation validated the disruption of the cytokine signaling pathway in response to CVB3 infection, evidenced by reduced expression of key cytokines such as interferon β1 (IFNβ1), C-X-C motif chemokine ligand 10 (CXCL10), interleukin 6 (IL6), among others. Furthermore, NFAT5-deficiency hindered the formation of stress granules, leading to a reduction of important stress granule components, including plakophilin-2, a pivotal protein within the intercalated disc, thereby impacting cardiomyocyte structure and function. These findings unveil a novel mechanism by which NFAT5 inhibits CVB3 replication and pathogenesis through the promotion of antiviral type I interferon signaling and the formation of cytoplasmic stress granules, collectively identifying NFAT5 as a new cardio protective protein.

The influence of endurance exercise training on myocardial fibrosis and arrhythmogenesis in a coxsackievirus B3 myocarditis mouse model

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.

A 3-month-old neonatal rhesus macaque HFMD model caused by coxsackievirus B1 infection and viral tissue tropism.

Coxsackievirus B1 (CVB1), an enterovirus with multiple clinical presentations, has been associated with potential long-term consequences, including hand, foot, and mouth disease (HFMD), in some patients. However, the related animal models, transmission dynamics, and long-term tissue tropism of CVB1 have not been systematically characterized. In this study, we established a model of CVB1 respiratory infection in rhesus macaques and evaluated the clinical symptoms, viral load, and immune levels during the acute phase (0-14 days) and long-term recovery phase (15-30 days). We also investigated the distribution, viral clearance, and pathology during the long-term recovery period using 35 postmortem rhesus macaque tissue samples collected at 30 days postinfection (d.p.i.). The results showed that the infected rhesus macaques were susceptible to CVB1 and exhibited HFMD symptoms, viral clearance, altered cytokine levels, and the presence of neutralizing antibodies. Autopsy revealed positive viral loads in the heart, spleen, pancreas, soft palate, and olfactory bulb tissues. HE staining demonstrated pathological damage to the liver, spleen, lung, soft palate, and tracheal epithelium. At 30 d.p.i., viral antigens were detected in visceral, immune, respiratory, and muscle tissues but not in intestinal or neural tissues. Brain tissue examination revealed viral meningitis-like changes, and CVB1 antigen expression was detected in occipital, pontine, cerebellar, and spinal cord tissues at 30 d.p.i. This study provides the first insights into CVB1 pathogenesis in a nonhuman primate model of HFMD and confirms that CVB1 exhibits tissue tropism following long-term infection.

GPR15-dependent T cell recruitment in the acute phase of viral myocarditis is associated with improved virus elimination and outcome

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): DZHK (German Centre for Cardiovascular Research) Background Myocarditis is an inflammatory heart disease that is primarily caused by viral pathogens such as Coxsackievirus B3 (CVB3). The clinical course of myocarditis has a wide spectrum of outcomes, ranging from complete recovery to cardiac dysfunction and dilated cardiomyopathy. The G protein-coupled receptor 15 (GPR15) has been identified as a T cell homing receptor associated with inflammatory diseases. Purpose The aim of this study was to address the following questions: Does GPR15 deficiency (i) lead to differences in immune cell infiltration or inflammation in the acute phase of myocarditis; (ii) does it affect elimination of CVB3 and thus outcome; (iii) does it affect immune cell chemotaxis or functionality? Therefore, GPR15-deficient (Gpr15gfp/gfp) and WT mice were infected with CVB3 to study the acute and subacute phase of myocarditis. Results In the subacute phase of myocarditis 16 days p.i., virus persistence was observed in more than 85% of Gpr15gfp/gfp mice, while more than 70% of WT mice successfully cleared the virus. In addition, Gpr15gfp/gfp mice showed reduced cardiac function accompanied by increased fibrosis compared to WT mice. Based on these findings, we analysed the acute phase of myocarditis in more detail. Comparing the infected genotypes, no significant differences were found at 6 days p.i. in contrast to 5 and 7 days p.i.. After 5 days p.i., Gpr15gfp/gfp mice showed significantly lower gene expression of inflammatory cytokines and immune cell markers, especially regulatory T cells. In contrast, after 7 days p.i., inflammatory cytokines and markers for regulatory and cytotoxic T cells were significantly increased in infected Gpr15gfp/gfp mice compared to infected WT mice. RNASeq confirmed that the response to the virus did not decrease from day 6 to 7 in infected Gpr15gfp/gfp mice, as observed in infected WT mice. Furthermore, gene ontology (GO) analyses revealed altered chemotaxis and increased cytotoxic T cell-related GO terms in Gpr15gfp/gfp mice 7 days p.i.. In addition, we demonstrated in vitro that chemotaxis of both regulatory and cytotoxic T cells depends on the interaction between GPR15 and its ligand GPR15L and that loss of GPR15 abrogates chemotaxis of T cells towards GPR15L. Conclusion Here we show that GPR15 deficiency leads to reduced virus elimination in the heart and thus to adverse remodelling and impaired cardiac function. Delayed recruitment of regulatory T cells was associated with prolonged persistence of cytotoxic and regulatory T cells in Gpr15gfp/gfp mice. In addition, RNASeq revealed a prolonged inflammatory response and altered chemotaxis in Gpr15gfp/gfp mice. Through in vitro studies, we identified GPR15L as an important chemokine for the recruitment of regulatory and cytotoxic T cells. In conclusion, insufficient virus elimination could be caused by delayed recruitment of T cells, leading to a prolonged inflammatory response and an adverse outcome in GPR15-deficient mice.