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Infections with SARS-CoV-2 can be asymptomatic, but they can also be accompanied by a variety of symptoms that result in mild to severe coronavirus disease-19 (COVID-19) and are sometimes associated with systemic symptoms. Although the viral infection originates in the respiratory system, it is unclear how the virus can overcome the alveolar barrier, which is observed in severe COVID-19 disease courses. To elucidate the viral effects on the barrier integrity and immune reactions, we used mono-cell culture systems and a complex human chip model composed of epithelial, endothelial, and mononuclear cells. Our data show that SARS-CoV-2 efficiently infected epithelial cells with high viral loads and inflammatory response, including interferon expression. By contrast, the adjacent endothelial layer was neither infected nor did it show productive virus replication or interferon release. With prolonged infection, both cell types were damaged, and the barrier function was deteriorated, allowing the viral particles to overbear. In our study, we demonstrate that although SARS-CoV-2 is dependent on the epithelium for efficient replication, the neighboring endothelial cells are affected, e.g., by the epithelial cytokines or components induced during infection, which further results in the damage of the epithelial/endothelial barrier function and viral dissemination.IMPORTANCESARS-CoV-2 challenges healthcare systems and societies worldwide in unprecedented ways. Although numerous new studies have been conducted, research to better understand the molecular pathogen-host interactions are urgently needed. For this, experimental models have to be developed and adapted. In the present study we used mono cell-culture systems and we established a complex chip model, where epithelial and endothelial cells are cultured in close proximity. We demonstrate that epithelial cells can be infected with SARS-CoV-2, while the endothelium did not show any infection signs. Since SARS-CoV-2 is able to establish viremia, the link to thromboembolic events in severe COVID-19 courses is evident. However, whether the endothelial layer is damaged by the viral pathogens or whether other endothelial-independent homeostatic factors are induced by the virus is essential for understanding the disease development. Therefore, our study is important as it demonstrates that the endothelial layer could not be infected by SARS-CoV-2 in our in vitro experiments, but we were able to show the destruction of the epithelial-endothelial barrier in our chip model. From our experiments we can assume that virus-induced host factors disturbed the epithelial-endothelial barrier function and thereby promote viral spread.
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The seven signal transducers of transcription (STATs) are cytokine-inducible modular transcription factors. They transmit the stimulation of cells with type I interferons (IFN-α/IFN-ß) and type II interferon (IFN-É£) into altered gene expression patterns. The N-terminal domain (NTD) of STAT1 is a surface for STAT1/STAT1 homodimer and STAT1/STAT2 heterodimer formation and allows the cooperative DNA binding of STAT1. We investigated whether the STAT1 NTD-mediated dimerization affected the IFN-induced tyrosine phosphorylation of STAT1, its nuclear translocation, STAT1-dependent gene expression, and IFN-dependent antiviral defense. We reconstituted human STAT1-negative and STAT2-negative fibrosarcoma cells with STAT1, NTD-mutated STAT1 (STAT1AA), STAT1 with a mutated DNA-binding domain (DBD), or STAT2. We treated these cells with IFN-α and IFN-É£ to assess differences between IFN-α-induced STAT1 homo- and heterodimers and IFN-É£-induced STAT1 homodimers. Our data demonstrate that IFNs induce the phosphorylation of STAT1 and STAT1AA at Y701 and their nuclear accumulation. We further reveal that STAT1AA can be phosphorylated in response to IFN-α in the absence of STAT2 and that IFN-É£-induced STAT1AA can activate gene expression directly. However, STAT1AA largely fails to bind STAT2 and to activate IFN-α-induced expression of endogenous antiviral STAT1/STAT2 target proteins. Congruent herewith, both an intact STAT1 NTD and STAT2 are indispensable to establish an antiviral state with IFN-α. These data provide new insights into the biological importance of the STAT1 NTD.
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Interferón Tipo I/metabolismo , Interferón gamma/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/fisiología , Antivirales/metabolismo , Línea Celular , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Fosforilación/fisiología , Transporte de Proteínas/fisiología , Factor de Transcripción STAT2/metabolismoRESUMEN
The human cytomegalovirus (HCMV) is a common pathogen, which causes severe or even deadly diseases in immunocompromised patients. In addition, congenital HCMV infection represents a major health concern affecting especially the lung tissue of the susceptible individuals. Antivirals are a useful strategy to treat HCMV-caused diseases. However, all approved drugs target viral proteins but significant toxicity and an increasing resistance against these compounds have been observed. In infected cells, numerous host molecules have been identified to play important roles during HCMV replication. Among others, HCMV infection depends on the presence of bioactive sphingolipids. In this study, the role of sphingosine-1-phosphate (S1P) signaling in HCMV-infected human embryonal lung fibroblasts (HELF) was analyzed. Viral replication depended on the functional activity of sphingosine kinases (SK). During SK inhibition, addition of extracellular S1P restored HCMV replication. Moreover, neutralization of extracellular S1P by anti-S1P antibodies decreased HCMV replication as well. While the application of FTY720 as an functional antagonist of S1P receptor (S1PR)1,3-5 signaling did not reduce HCMV replication significantly, JTE-013, an inhibitor of S1PR2, decreased viral replication. Furthermore, inhibition of Rac-1 activity reduced HCMV replication, whereas inhibition of the Rac-1 effector protein Rac-1-activated kinase 1 (PAK1) had no influence. In general, targeting S1P-induced pathways, which are essential for a successful HCMV replication, may represent a valuable strategy to develop new antiviral drugs.
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Citomegalovirus/crecimiento & desarrollo , Fibroblastos/metabolismo , Fibroblastos/virología , Lisofosfolípidos/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Replicación Viral , Células Cultivadas , Humanos , Pulmón/citología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/metabolismoRESUMEN
In this study, approaches were developed to examine the phenotypes of nonviable clinical varicella-zoster virus (VZV) strains with amino acid substitutions in the thymidine kinase (TK) (open reading frame 36 [ORF36]) and/or DNA polymerase (Pol) (ORF28) suspected to cause resistance to antivirals. Initially, recombinant TK proteins containing amino acid substitutions described as known or suspected causes of antiviral resistance were analyzed by measuring the TK activity by applying a modified commercial enzyme immunoassay. To examine the effects of these TK and Pol substitutions on the replication of recombinant virus strains, the method of en passant mutagenesis was used. Targeted mutations within ORF36 and/or ORF28 and an autonomously expressed gene of the monomeric red fluorescent protein for plaque identification were introduced into the European wild-type VZV strain HJO. Plaque reduction assays revealed that the amino acid substitutions with unknown functions in TK, Q303stop, N334stop, A163stop, and the deletion of amino acids 7 to 74 aa (Δaa 7 to 74), were associated with resistance against acyclovir (ACV), penciclovir, or brivudine, whereas the L73I substitution and the Pol substitutions T237K and A955T revealed sensitive viral phenotypes. The results were confirmed by quantitative PCR by measuring the viral load under increasing ACV concentrations. In conclusion, analyzing the enzymatic activities of recombinant TK proteins represent a useful tool for evaluating the significance of amino acid substitutions in the antiviral resistance of clinical VZV strains. However, direct testing of replication-competent viruses by the introduction of nonsynonymous mutations in a VZV bacterial artificial chromosome using en passant mutagenesis led to reliable phenotypic characterization results.
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Antivirales/farmacología , Herpesvirus Humano 3/efectos de los fármacos , Herpesvirus Humano 3/enzimología , Proteínas Recombinantes/metabolismo , Timidina Quinasa/metabolismo , Línea Celular , Farmacorresistencia Viral/genética , Humanos , Proteínas Recombinantes/genética , Timidina Quinasa/genéticaRESUMEN
The administration of drugs to inhibit metabolic pathways not only reduces the risk of obesity-induced diseases in humans but may also hamper the replication of different viral pathogens. In order to investigate the value of the US Food and Drug Administration-approved anti-obesity drug orlistat in view of its anti-viral activity against different human-pathogenic viruses, several anti-viral studies, electron microscopy analyses as well as fatty acid uptake experiments were performed. The results indicate that administrations of non-cytotoxic concentrations of orlistat reduced the replication of coxsackievirus B3 (CVB3) in different cell types significantly. Moreover, orlistat revealed cell protective effects and modified the formation of multi-layered structures in CVB3-infected cells, which are necessary for viral replication. Lowering fatty acid uptake from the extracellular environment by phloretin administrations had only marginal impact on CVB3 replication. Finally, orlistat reduced also the replication of varicella-zoster virus moderately but had no significant influence on the replication of influenza A viruses. The data support further experiments into the value of orlistat as an inhibitor of the fatty acid synthase to develop new anti-viral compounds, which are based on the modulation of cellular metabolic pathways.
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Fármacos Antiobesidad/farmacología , Antivirales/farmacología , Lactonas/farmacología , Animales , Línea Celular , Enterovirus Humano B/efectos de los fármacos , Herpesvirus Humano 3/efectos de los fármacos , Humanos , Virus de la Influenza A/efectos de los fármacos , Orlistat , Replicación Viral/efectos de los fármacosRESUMEN
Introduction: Treatment of influenza A virus infections is currently limited to few direct acting antiviral substances. Repurposing other established pharmaceuticals as antivirals could aid in improving treatment options. Methods: This study investigates the antiviral properties of ProcCluster® and procaine hydrochloride, two derivatives of the local anesthetic procaine, in influenza A virus infection of A549, Calu-3 and MDCK cells. Results: Both substances inhibit replication in all three of these cell lines in multi-cycle experiments. However, cell line-dependent differences in the effects of the substances on viral RNA replication and subsequent protein synthesis, as well as release of progeny viruses in single-cycle experiments can be observed. Both ProcCluster® and procaine hydrochloride delay endosome fusion of the virus early in the replication cycle, possibly due to the alkaline nature of the active component procaine. In A549 and Calu-3 cells an additional effect of the substances can be observed at late stages in the first replication cycle. Interestingly, this effect is absent in MDCK cells. We demonstrate that ProcCluster® and procaine hydrochloride inhibit phospholipase A2 (PLA2) enzymes from A549 but not MDCK cells and confirm that specific inhibition of calcium independent PLA2 but not cytosolic PLA2 has antiviral effects. Discussion: We show that ProcCluster® and procaine hydrochloride inhibit influenza A virus infection at several stages of the replication cycle and have potential as antiviral substances.
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UNLABELLED: Coxsackievirus B3 (CVB3) is a major cause of acute and chronic forms of myocarditis. Previously, direct viral injury and post-infectious autoimmune response were suspected as main pathogenetic mechanisms. However, induction of pro-inflammatory cytokines may be crucial for pathogenesis in spite of host protein shut off caused by CVB3 replication. We investigated the global expression profile of pro-inflammatory genes induced by acute and persistent (carrier state) CVB3 infection in human fibroblast cell cultures with DNA microarrays, quantitative RT-PCR and ELISA. Rapid induction of a typical spectrum of about 30 inflammation-related genes (e.g., PTGS2, CCL2, IL-1ß, IL-6, IL-8, CSF2, MMP-1, MMP-3, and MMP-15) suggested an essential, autocrine role of IL-1. This hypothesis was confirmed by over-expression of IL-1RI, which resulted in a cytokine response upon CVB3 infection in HEK 293 cells otherwise refractory to CVB3-caused gene expression. Blocking IL-1 receptor type I (IL-1RI)-signaling during CVB3 infection with an IL-1 receptor antagonist (IL-1ra) as well as knockdown of IL-1RI using siRNA abrogated cytokine response in human fibroblasts. Both IL-1α and IL-1ß are relevant for the induction of inflammation-related genes during CVB3 infection as shown by neutralization experiments. Paracrine effects of IL-1 on the subset of non-infected cells in carrier state infected fibroblast cultures enhanced induction of inflammation-related genes. CONCLUSIONS: A broad spectrum of inflammatory cytokines was induced by CVB3 replication via a pathway that requires IL-1 signaling. Our results suggest that IL-1ra may be used as a therapeutic agent to limit inflammation and tissue destruction in myocarditis.
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Enterovirus Humano B/inmunología , Fibroblastos/inmunología , Fibroblastos/virología , Interleucina-1/biosíntesis , Interleucina-1/inmunología , Transducción de Señal , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/patología , Perfilación de la Expresión Génica , Humanos , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Herpes simplex virus type 1 (HSV-1) is a widespread contagious pathogen, mostly causing mild symptoms on the mucosal entry side. However, systemic distribution, in particular upon reactivation of the virus in immunocompromised patients, may trigger an innate immune response and induce damage of organs. In these conditions, HSV-1 may infect vascular endothelial cells, but little is known about the regulation of HSV-1 replication and possible defense mechanisms in these cells. The current study addresses the question of whether the host cell protein AMP-activated protein kinase (AMPK), an important metabolic sensor, can control HSV-1 replication in endothelial cells. We show that downregulation of the catalytic subunits AMPKα1 and/or AMPKα2 increased HSV-1 replication as monitored by TCID50 titrations, while a potent AMPK agonist, MK-8722, strongly inhibited it. MK-8722 induced a persistent phosphorylation of the AMPK downstream targets acetyl-CoA carboxylase (ACC) and the rapamycin-sensitive adaptor protein of mTOR (Raptor) and, related to this, impairment of ACC1-mediated lipid synthesis and the mechanistic target of the rapamycin complex-1 (mTORC1) pathway. Since blockade of mTOR by Torin-2 as well as downregulation of ACC1 by siRNA also decreased HSV-1 replication, MK-8722 is likely to exert its anti-viral effect via mTORC1 and ACC1 inhibition. Importantly, MK-8722 was able to reduce virus replication even when added after HSV-1. Together, our data highlight the importance of endothelial cells as host cells for HSV-1 replication upon systemic infection and identify AMPK, a metabolic host cell protein, as a potential target for antiviral strategies against HSV-1 infection and its severe consequences. IMPORTANCE Herpes simplex virus type 1 (HSV-1) is a common pathogen that causes blisters or cold sores in humans. It remains latent in infected individuals and can be reactivated multiple times. In adverse conditions, for instance, in immunocompromised patients, HSV-1 can lead to serious complications such as encephalitis, meningitis, or blindness. In these situations, infection of endothelial cells lining the surface of blood vessels may contribute to the manifestation of disease. Here, we describe the role of AMP-activated protein kinase (AMPK), a potent regulator of cellular energy metabolism, in HSV-1 replication in endothelial cells. While downregulation of AMPK potentiates HSV-1 replication, pharmacological AMPK activation inhibits it by limiting the availability of required host cell macromolecules such as proteins or fatty acids. These data highlight the role of metabolic host cell proteins as antiviral targets and reveal activation of endothelial AMPK as a potential strategy to protect from severe consequences of HSV-1 infection.
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Coxsackievirus B3 (CVB3) is a human pathogen that causes acute and chronic infections, but an antiviral drug to treat these diseases has not yet been developed for clinical use. Several intracellular pathways are altered to assist viral transcription, RNA replication, and progeny release. Among these, fatty acid synthase (FAS) expression is increased. In order to test the potential of FAS inhibition as an anti-CVB3 strategy, several experiments were performed, including studies on the correlation of CVB3 replication and FAS expression in human Raji cells and an analysis of the time and dose dependence of the antiviral effect of FAS inhibition due to treatment with amentoflavone. The results demonstrate that CVB3 infection induces an up-regulation of FAS expression already at 1 h postinfection (p.i.). Incubation with increasing concentrations of amentoflavone inhibited CVB3 replication significantly up to 8 h p.i. In addition, suppression of p38 MAP kinase activity by treatment with SB239063 decreased FAS expression as well as viral replication. These data provide evidence that FAS inhibition via amentoflavone administration might present a target for anti-CVB3 therapy.
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Biflavonoides/farmacología , Infecciones por Coxsackievirus/enzimología , Regulación hacia Abajo/efectos de los fármacos , Enterovirus Humano B/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ácido Graso Sintasas/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Línea Celular , Infecciones por Coxsackievirus/tratamiento farmacológico , Infecciones por Coxsackievirus/virología , Enterovirus Humano B/genética , Enterovirus Humano B/fisiología , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Humanos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Many viruses modify cellular processes for their own benefit. The enterovirus 3A protein inhibits endoplasmic reticulum (ER)-to-Golgi transport, a function previously suggested to be important for viral suppression of immune responses. Here, we show that a virus carrying a 3A protein defective in inhibiting ER-to-Golgi transport is indeed less virulent in mice, and we unravel the mechanism by which 3A inhibits this trafficking step. Evidence is provided that 3A inhibits the activation of the GTPase ADP-ribosylation factor 1 (Arf1), which regulates the recruitment of the COP-I coat complex to membranes. 3A specifically inhibits the function of GBF1, a guanine nucleotide exchange factor for Arf1, by interacting with its N terminus. By specifically interfering with GBF1-mediated Arf1 activation, 3A may prove a valuable tool in dissecting the early steps of the secretory pathway.
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Factor 1 de Ribosilacion-ADP/antagonistas & inhibidores , Proteína Coat de Complejo I/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Proteínas Virales/farmacología , Factor 1 de Ribosilacion-ADP/metabolismo , Animales , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Chlorocebus aethiops , Proteína Coat de Complejo I/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/fisiología , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/fisiología , Factores de Intercambio de Guanina Nucleótido/biosíntesis , Factores de Intercambio de Guanina Nucleótido/fisiología , Ratones , Modelos Animales , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiologíaRESUMEN
The search for new antiviral strategies to treat influenza A virus (IAV) infections is one major international health care activity. Hereby, the IAV-caused misuse of cellular nuclear factor kappa B (NF-κB) signaling pathways in infected cells represents one target for antiviral therapy. In the present study, pyrrolidine dithiocarbamate (PDTC), which is known as an antioxidant and as an inhibitor of IAV-induced NF-κB activation, was studied in vivo. After the antiviral activity of PDTC was confirmed in MDCK cells, mice-infected with the mouse-adapted strain of IAV A/PR/8/34 (H1N1)-were treated intraperitoneally simultaneously with PDTC (75, 150, 200 mg/kg body weight). The influence of PDTC administrations was evaluated on viral replication and inflammatory reactions in lung tissue up to 14 days postinfection (p. i.). This therapy increased survival up to 80% and reduced IAV-caused weight loss and viral replication in lung tissue in a dose-dependent manner. Protective effects were less pronounced, if the therapy started later on during an ongoing IAV infection. In addition, simultaneous PDTC treatment also limited IAV-caused infiltration of immune cells as well as local interferon-γ expression in lung tissue. These results imply that PDTC decreases IAV-caused disease in mice significantly. Therefore, the development of drugs like PDTC that interfere with NF-κB signaling may represent a modern focus of anti-IAV therapy.
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Antivirales/uso terapéutico , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Pirrolidinas/uso terapéutico , Tiocarbamatos/uso terapéutico , Animales , Antivirales/administración & dosificación , Antivirales/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H1N1 del Virus de la Influenza A/fisiología , Pulmón/efectos de los fármacos , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/virología , Pirrolidinas/administración & dosificación , Pirrolidinas/farmacología , Tiocarbamatos/administración & dosificación , Tiocarbamatos/farmacología , Resultado del Tratamiento , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Diagnostic assays for severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) that are easy to perform and produce fast results are essential for timely decision making regarding the isolation of contagious individuals. OBJECTIVE: We evaluated the CE-approved eazyplex® SARS-CoV-2, a ready-to-use real time RT-LAMP assay for identification of the SARS-CoV-2â¯N and ORF8 genes from swabs in less than 30â¯min without RNA extraction. STUDY DESIGN: Oropharyngeal and nasal swabs from 100 positive and 50 negative patients were inoculated into 0.9 % saline and tested by NeuMoDx™ RT-PCR. An aliquot was diluted fivefold in Copan sputum liquefying (SL) solution and directly analyzed by eazyplex® SARS-CoV-2. In addition, 130 patient swabs were prospectively tested with both methods in parallel. Analytical sensitivity of the assay was determined using virus stock dilutions. RESULTS: Positive percent agreement (PPA) between the eazyplex® SARS-CoV-2 and RT-PCR was 74 % for samples with Ct values < 35. When using a Ct cut-off ≤ 28 the PPA increased to 97.4 %. In the prospective part of the study overall PPA of the eazyplex® kit was 66.7 % but increased to 100 % when only Ct values ≤ 28 were considered. There were no false positive results. The median time to positivity was 12.5â¯min for the N gene and 16.75â¯min for ORF8. Analytical sensitivity was 3.75 TCID50/mL. 105 virus copies/mL were reproducibly detected. CONCLUSION: The eazyplex® SARS-CoV-2 is a rapid assay that accurately identifies samples with high viral loads. It may be useful for near-patient testing outside of a molecular diagnostic laboratory.
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Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Nasofaringe/virología , Orofaringe/virología , ARN Viral/aislamiento & purificación , SARS-CoV-2/aislamiento & purificación , Pruebas Diagnósticas de Rutina , Humanos , Pruebas en el Punto de AtenciónRESUMEN
Clinical observations indicate that COVID-19 is a systemic disease. An investigation of the viral distribution within the human body and its correlation with tissue damage can aid in understanding the pathophysiology of SARS-CoV-2 infection. We present a detailed mapping of the viral RNA in 61 tissues and organs of 11 deceased patients with COVID-19. The autopsies were performed within the early postmortem interval (between 1.5 and 15 hr, mean: 5.6 hr) to minimize the bias due to viral RNA and tissue degradation. Very high viral loads (>104copies/ml) were detected in most patients' lungs, and the presence of intact viral particles in the lung tissue could be verified by transmission electron microscopy. Interestingly, viral RNA was detected throughout various extrapulmonary tissues and organs without visible tissue damage. The dissemination of SARS-CoV-2-RNA throughout the body supports the hypothesis that there is a maladaptive host response with viremia and multiorgan dysfunction.
Since the discovery of the new coronavirus that causes COVID-19, scientists have been scrambling to understand the different features of the virus. While a lot more is now known about SARS-CoV-2, several key questions have proved more difficult to answer. For example, it remained unclear where the virus travels to in the body and causes the most harm. To help answer this question, Deinhardt-Emmer, Wittschieber et al. performed postmortem examinations on 11 patients who had recently died of COVID-19. After sampling 61 different organs and tissues from each patient, several tests were used to detect traces of SARS-CoV-2. The experiments showed that the largest pool of SARS-CoV-2 was present in the lungs, where it had caused severe damage to the alveolae, the delicate air sacs at the end of the lungs' main air tubes. Small amounts of the virus were also detected in other organs and tissues, but no severe tissue damage was seen. In addition, Deinhardt-Emmer, Wittschieber et al. found that each patient had increased levels of some of the proteins involved in inflammation and blood clotting circulating their bloodstream. This suggests that the inflammation caused by SARS-CoV-2 leads to an excessive immune reaction throughout the entire body. This research provides important new insights into which areas of the body are most impacted by SARS-CoV-2. These findings may help to design more effective drug treatments that target the places SARS-CoV-2 is most likely to accumulate and help patients fight off the infection at these regions.
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Autopsia , COVID-19/patología , SARS-CoV-2/aislamiento & purificación , Anciano , Anciano de 80 o más Años , COVID-19/sangre , COVID-19/inmunología , COVID-19/terapia , Causas de Muerte , Comorbilidad , Femenino , Humanos , Inflamación/sangre , Inflamación/inmunología , Pulmón/patología , Pulmón/virología , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , ARN Viral/sangre , Carga ViralRESUMEN
Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase. This enzyme is involved in a plethora of cellular processes, including apoptosis, autophagy, cell proliferation, and DNA repair. Remarkably, PP2A can act as a context-dependent tumor suppressor or promoter. Active PP2A complexes consist of structural (PP2A-A), regulatory (PP2A-B), and catalytic (PP2A-C) subunits. The regulatory subunits define the substrate specificity and the subcellular localization of the holoenzyme. Here we condense the increasing evidence that the PP2A B-type subunit PR130 is a critical regulator of cell identity and oncogenic transformation. We summarize knowledge on the biological functions of PR130 in normal and transformed cells, targets of the PP2A-PR130 complex, and how diverse extra- and intracellular stimuli control the expression and activity of PR130. We additionally review the impact of PP2A-PR130 on cardiac functions, neuronal processes, and anti-viral defense and how this might affect cancer development and therapy.
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Transformación Celular Neoplásica/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Animales , Apoptosis , Autofagia , Proliferación Celular , Transformación Celular Neoplásica/genética , Reparación del ADN , Pruebas de Función Cardíaca , Humanos , Especificidad por SustratoRESUMEN
Infections with the herpes simplex virus type 1 (HSV-1) are common and widespread. Most infections remain undetected but severe forms may develop in newborns and in immunocompromised patients. Moreover, HSV-1 might be involved in the pathogenesis of atherosclerosis, which may include viral infection of the endothelium. Antiviral therapy is efficient to treat symptomatic patients. However, an increasing accumulation of resistance-associated mutations has been observed in the viral genome. Thus, new antiviral strategies are focused on host factors. Among others, signaling of bioactive sphingolipids seems to be important in mediating HSV-1 replication. With the present study, regulation and function of sphingosine-1-phosphate (S1P)-based signaling were analyzed in HSV-1-infected human umbilical vein endothelial cells (HUVEC). Our data indicate that viral replication in endothelial cells relies on sphingosine kinase (SK) activity and S1P receptor (S1PR)1,3-5 signaling, which involves the activation of phosphatidylinositol-3-kinase (PI3K) and the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac-1). Inhibitor- or siRNA-meditated reduction of Rac-1 activity decreased HSV-1 replication. In general, targeting S1P-related signaling may be a successful strategy to establish new anti-HSV-1 therapies.
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Herpesvirus Humano 1/fisiología , Interacciones Microbiota-Huesped , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/virología , Lisofosfolípidos/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Células Cultivadas , Herpesvirus Humano 1/genética , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingolípidos/metabolismo , Esfingosina/metabolismo , Replicación ViralRESUMEN
BACKGROUND: Molecular assays based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) may be useful for rapid diagnosis of the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) because of the easy performance and the option to bypass RNA extraction. OBJECTIVES: This study was designed to evaluate the clinical performance of the CE-labeled variplexTM real time SARS-CoV-2 RT-LAMP assay in comparison to commercial RT-PCRs. STUDY DESIGN: RNA extracted from pharyngeal swabs was tested by variplex™ RT-LAMP and Corman's LightMix™ E gene RT-PCR as reference. Samples of respiratory secretions from Coronavirus infection disease (COVID-19) and negative control patients were analyzed by variplex™ without RNA extraction and tested in parallel with the Allplex™ and VIASURE BD MAX RT-PCRs. RESULTS: Using isolated RNA variplex™ RT-LAMP showed a sensitivity of 75 % compared to LightMix E gene RT-PCR but contrary to the latter it produced no false-positive results. For the evaluation of samples from respiratory secretions concordance analysis showed only a moderate agreement between the variplex™ RT-LAMP conducted on unprocessed samples and Allplex™ and VIASURE RT-PCRs (Cohen's κ ranging from 0.52-0.56). Using the approach to define a sample as true-positive when at least two assays gave a positive result the clinical sensitivities were as follows: 76.3 % for variplex™, 84.2 % for Allplex™ and 68.4 % for VIASURE. However, when results of RT-PCR and RT-LAMP were combined diagnostic sensitivity was increased to 92-100 %. CONCLUSION: The variplex RT-LAMP may serve as a rapid test to be combined with a RT-PCR assay to increase the diagnostic accuracy in patients with suspected COVID-19 infection.
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Prueba de COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , SARS-CoV-2/genética , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y EspecificidadRESUMEN
Cardiovascular diseases are the major cause of human death and have been linked to many different risk factors. Among them, coxsackievirus B3 (CVB3), as a member of the enterovirus group, is one of the most important infectious agents of virus-induced myocarditis. Despite the fact that the molecular structure of this pathogen has been characterized very precisely, there is no virus-specific preventive or therapeutic procedure against CVB3-induced heart disease in clinical use today. A promising approach to prevent CVB3-caused myocarditis represents the mutation of the viral genome in a way that coding sequences of cytokines are integrated into the viral RNA. Recombinant cytokine-expressing CVB3 variants were established to increase the local cytokine concentration and to modulate TH1-/TH2-specific immune responses. Especially protective against CVB3-induced murine myocarditis is the application of an interferon-gamma (IFN-gamma)-expressing recombinant coxsackievirus variant. The local and simultaneous expression of an immuno-relevant cytokine by the virus itself induces a strong and long-lasting immune response which protects laboratory animals against lethal infections.
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Infecciones por Coxsackievirus/inmunología , Infecciones por Coxsackievirus/terapia , Enterovirus Humano B/inmunología , Miocarditis/inmunología , Miocarditis/terapia , Vacunas Virales/inmunología , Animales , Infecciones por Coxsackievirus/prevención & control , Infecciones por Coxsackievirus/virología , Enterovirus Humano B/genética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Miocarditis/prevención & control , Miocarditis/virología , Páncreas/inmunología , Páncreas/virología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéutico , Vacunas Virales/uso terapéuticoRESUMEN
Several different procedures have been developed to deliver essential genes to an organism by viral vectors. Some reports have already been published demonstrating the potential to use enteroviruses as transfer vehicles. One application of these viral vectors is the organ-specific expression of immunoregulatory cytokines. It has been shown previously that local expression of interferon-gamma (IFN-gamma) by the recombinant coxsackievirus CVB3/IFN-gamma conferred protection against virus-caused disease via direct and indirect mechanisms. Using a murine model of CVB3-induced myocarditis, other aspects of the CVB3/IFN-gamma application as a vaccine were studied concerning route of administration, age, and presence of a pre-existing immune response.
Asunto(s)
Infecciones por Coxsackievirus/prevención & control , Enterovirus Humano B/inmunología , Interferón gamma/inmunología , Vacunas Virales/inmunología , Animales , Enterovirus Humano B/genética , Inyecciones Intramusculares , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Interferón gamma/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Miocarditis/inmunología , Miocarditis/prevención & control , Páncreas/virología , Suero/inmunología , Suero/virología , Bazo/virología , Análisis de Supervivencia , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Coxsackievirus is linked to a large variety of severe human and animal diseases such as myocarditis. The interplay between host factors and virus components is crucial for the fate of the infected cells. However, host proteins which may play a role in coxsackievirus-induced diseases are ill-defined. Two-dimensional gel electrophoresis of protein extracts obtained from coxsackievirus B3 (CVB3)-infected and uninfected HeLa or HepG2 cells combined with spot analysis revealed several proteins which are exclusively up-regulated in infected cells. One of these proteins was identified as the fatty acid synthase (FAS). By using cerulenin and C75, two known inhibitors of FAS we were able to significantly block CVB3 replication. FAS appears to be directly involved in CVB3-caused pathology and is therefore suitable as a therapeutic target in CVB3-induced diseases.
Asunto(s)
Enterovirus Humano B/crecimiento & desarrollo , Ácido Graso Sintasas/biosíntesis , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Secuencia de Aminoácidos , Antivirales/farmacología , Línea Celular , Cerulenina/farmacología , Electroforesis en Gel Bidimensional , Inhibidores Enzimáticos , Ácido Graso Sintasas/antagonistas & inhibidores , Ácido Graso Sintasas/química , Humanos , Datos de Secuencia Molecular , Regulación hacia Arriba , Replicación Viral/efectos de los fármacosRESUMEN
Infection by Enterovirus A71 (EV-A71) is an important cause of hand, foot, and mouth disease (HFMD). Outbreaks including severe cases with neurological and cardiopulmonary complications have been reported particularly from Southeast Asia. In Europe, the epidemiology of EV-A71 is not well understood. In summer 2015, a two-year-old girl from Thuringia, Germany, presented with rhombencephalitis/brainstem encephalitis associated with severe neurological and cardiopulmonary complications. EV-A71 was detected in stool and almost the entire viral genome was amplified and sequenced. While the capsid protein VP1-encoding region belongs to the EV-A71 subgenogroup C1, the 3D polymerase encoding region represents a unique lineage. Thus, the data suggest that the Thuringian EV-A71 sequence likely represents a recombinant. The case underlines the importance of intensified EV-A71 surveillance in Germany and Europe including analysis of full-genome data.