Impact of 5’terminally deleted RNA forms of group-B enterovirus on their pathogenicity and on type 1 interferon response in cardiomyocytes and pancreatic beta cells in vivo and in vitro

Des ARN génomiques d’entérovirus-B tronqués en région 5’terminale non codante (EVB-TD) ont été associés à des myocardites aigues, des cardiomyopathies dilatées chroniques et au diabète de type 1. La pathogenèse n’est pas élucidée. Nous avons étudié chez la souris DBA/2J et dans des cultures de cardiomyocytes et de cellules bêta pancréatiques, l’impact de ces EVB-TD sur les activités de réplication virale, la réponse interféron de type 1 (IFN-1) et les fonctions des cellules cibles. Au cours de l’infection par le virus Coxsackie B3/28 (CVB3) nous avons montré l’émergence et le maintien d’une population virale majoritaire tronquée de 50 nucléotides en région 5’terminale (CVB3-TD50) aux phases aigue puis persistante, dans le cœur et le pancréas, avec respectivement une dégradation de la dystrophine et une diminution de l’expression d’insuline. La transfection d’ARN viraux synthétiques CVB3-TD50 chez la souris et dans des cardiomyocytes humains nous a permis de démontrer leur capacité à moduler l’activation de la voie IFN-1. De plus, leur transfection dans les cellules bêta pancréatiques a montré leur rôle dans la diminution du métabolisme de l’insuline. En conclusion, nos résultats montrent que l’émergence précoce et le maintien de formes majoritaires et réplicatives de CVB3-TD50 module l’activation de la voie IFN-1, favorisant le développement d’une infection persistante associée à un dysfonctionnement des cellules cardiaques et bêta pancréatiques.Enterovirus-B genomic RNA deleted in the 5'terminal non-coding region (EVB-TD) have been associated with acute myocarditis, chronic dilated cardiomyopathy and type 1 diabetes. The pathogenesis is not elucidated. In DBA/2J mice and in cultured cardiomyocytes and pancreatic beta cells, we studied the impact of these EVB-TD on viral replication activities, type 1 interferon (IFN-1) response and target cell functions. During infection with a Coxsackievirus B3/28 (CVB3), we showed the emergence and maintenance of a dominant viral population deleted of 50 nucleotides in the 5'terminal region (CVB3-TD50) at acute and persistent phases, in the heart and pancreas, with respectively a degradation of dystrophin and a decrease in insulin expression. The transfection of synthetic CVB3-TD50 viral RNA in mice and in human cardiomyocytes demonstrated their capacity to modulate the activation of the IFN-1 pathway. In addition, their transfection into pancreatic beta cells showed their role in decreasing insulin metabolism. In conclusion, our results show that the early emergence and maintenance of dominant and replicative forms of CVB3-TD50 modulates the activation of the IFN-1 pathway, leading to the development of a persistent infection associated with cardiac and pancreatic beta cell dysfunctions

Coinfection of Parvovirus B19 with Influenza A/H1N1 Causes Fulminant Myocarditis and Pneumonia. An Autopsy Case Report

Parvovirus-B19 (PVB19) is a frequent causative agent of myocarditis. For unclear reasons, viral reactivation can cause acute myocarditis, a leading cause of sudden death in the young. Influenza A/H1N1(2009) virus (IAV/H1N1) is known for causing flu/pneumonia, but the heart is rarely involved. Co-infections of cardiotropic viruses are rarely reported and the mechanisms of viral interactions remain unknown. A 5-year old girl had a flu-like syndrome, when she suddenly presented with a respiratory distress and cardiac arrest. At autopsy, the lungs were found haemorrhagic. Lungs’ histology showed severe bronchiolitis, diffuse haemorrhagic necrosis, and mononuclear inflammation. In the heart, a moderate inflammation was found with no necrosis. IAV/H1N1 was detected in nasal and tracheal swabs, lungs, and the heart. The viral load was high in the lungs, but low in the heart. PVB19 was detected in the heart with a high viral load. Viral co-infection increases the risk of severe outcome but the mechanisms of interaction between viruses are poorly understood. In our case, viral loads suggested a reactivated PVB19-induced acute myocarditis during an IAV/H1N1 pneumonia. Viral interactions may involve an IAV/H1N1-induced cytokine storm, with a fulminant fatal outcome. Clinically, our case shows the importance of investigating inflammatory pathways as therapeutic targets

Fulminant Myocardial Involvement in Neonatal Echovirus-induced Sepsis. Two Autopsy Cases

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Early Emergence of 5′ Terminally Deleted Coxsackievirus-B3 RNA Forms Is Associated with Acute and Persistent Infections in Mouse Target Tissues

Major EV-B populations characterized by 5′ terminal deletions (5′TD) have been shown to be associated with the development of myocarditis and type 1 diabetes in mice or humans. To date, the dynamics of EV-B 5′TD-RNA forms’ emergence during the course of infection and their impact on cellular functions remain unclear. Using a RACE-PCR approach in CVB3/28-infected mouse organs, we showed an early (3 days post infection, DPI) emergence of major 5′TD populations associated with minor full-length RNA forms. Viral replication activities with infectious particle production were associated with heart, liver, and pancreas acute inflammatory lesions, whereas clearance of viral RNA without organ lesions was observed in the brain, lung, intestines, and muscles from 3 to 7 DPI. At 28 DPI, low viral RNA levels, +/-RNA ratios < 5 associated with viral protein 1 expression revealed a persistent infection in the heart and pancreas. This persistent infection was characterized by molecular detection of only 5′TD RNA forms that were associated with dystrophin cleavage in the heart and insulin production impairment in beta-pancreatic cells. These results demonstrated that major EV-B 5′TD RNA forms can be early selected during systemic infection and that their maintenance may drive EV-induced acute and persistent infections with target cell dysfunctions

Replication Activities of Major 5′ Terminally Deleted Group-B Coxsackievirus RNA Forms Decrease PCSK2 mRNA Expression Impairing Insulin Maturation in Pancreatic Beta Cells

Emergence of 5′ terminally deleted coxsackievirus-B RNA forms (CVB-TD) have been associated with the development of human diseases. These CVB-TD RNA forms have been detected in mouse pancreas during acute or persistent experimental infections. To date, the impact of the replication activities of CVB-TD RNA forms on insulin metabolism remains unexplored. Using an immunocompetent mouse model of CVB3/28 infection, acute and persistent infections of major CVB-TD populations were evidenced in the pancreas. The inoculation of mice with homogenized pancreases containing major CVB-TD populations induced acute and chronic pancreatic infections with pancreatitis. In the mouse pancreas, viral capsid protein 1 (VP1) expression colocalized with a decrease in beta cells insulin content. Moreover, in infected mouse pancreases, we showed a decrease in pro-hormone convertase 2 (PCSK2) mRNA, associated with a decrease in insulin plasmatic concentration. Finally, transfection of synthetic CVB-TD50 RNA forms into cultured rodent pancreatic beta cells demonstrated that viral replication with protein synthesis activities decreased the PCSK2 mRNA expression levels, impairing insulin secretion. In conclusion, our results show that the emergence and maintenance of major CVB-TD RNA replicative forms in pancreatic beta cells can play a direct, key role in the pathophysiological mechanisms leading to the development of type 1 diabetes

Brain exposure to SARS-CoV-2 virions perturbs synaptic homeostasis

International audienceSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with short- and long-term neurological complications. The variety of symptoms makes it difficult to unravel molecular mechanisms underlying neurological sequalae after coronavirus disease 2019 (COVID-19). Here we show that SARS-CoV-2 triggers the up-regulation of synaptic components and perturbs local electrical field potential. Using cerebral organoids, organotypic culture of human brain explants from individuals without COVID-19 and post-mortem brain samples from individuals with COVID-19, we find that neural cells are permissive to SARS-CoV-2 to a low extent. SARS-CoV-2 induces aberrant presynaptic morphology and increases expression of the synaptic components Bassoon, latrophilin-3 (LPHN3) and fibronectin leucine-rich transmembrane protein-3 (FLRT3). Furthermore, we find that LPHN3-agonist treatment with Stachel partially restored organoid electrical activity and reverted SARS-CoV-2-induced aberrant presynaptic morphology. Finally, we observe accumulation of relatively static virions at LPHN3-FLRT3 synapses, suggesting that local hindrance can contribute to synaptic perturbations. Together, our study provides molecular insights into SARS-CoV-2-brain interactions, which may contribute to COVID-19-related neurological disorders

Critical contribution of mitochondria in the development of cardiomyopathy linked to desmin mutation

International audienceAbstract Background Beyond the observed alterations in cellular structure and mitochondria, the mechanisms linking rare genetic mutations to the development of heart failure in patients affected by desmin mutations remain unclear due in part, to the lack of relevant human cardiomyocyte models. Methods To shed light on the role of mitochondria in these mechanisms, we investigated cardiomyocytes derived from human induced pluripotent stem cells carrying the heterozygous DES E439K mutation that were either isolated from a patient or generated by gene editing. To increase physiological relevance, cardiomyocytes were either cultured on an anisotropic micropatterned surface to obtain elongated and aligned cardiomyocytes, or as a cardiac spheroid to create a micro-tissue. Moreover, when applicable, results from cardiomyocytes were confirmed with heart biopsies of suddenly died patient of the same family harboring DES E439K mutation, and post-mortem heart samples from five control healthy donors. Results The heterozygous DES E439K mutation leads to dramatic changes in the overall cytoarchitecture of cardiomyocytes, including cell size and morphology. Most importantly, mutant cardiomyocytes display altered mitochondrial architecture, mitochondrial respiratory capacity and metabolic activity reminiscent of defects observed in patient’s heart tissue. Finally, to challenge the pathological mechanism, we transferred normal mitochondria inside the mutant cardiomyocytes and demonstrated that this treatment was able to restore mitochondrial and contractile functions of cardiomyocytes. Conclusions This work highlights the deleterious effects of DES E439K mutation, demonstrates the crucial role of mitochondrial abnormalities in the pathophysiology of desmin-related cardiomyopathy, and opens up new potential therapeutic perspectives for this disease