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Hepatitis B virus (HBV) reactivation in immunosuppressed patients can cause considerable morbidity and mortality. The aim of our study was to evaluate factors associated with acute liver failure (ALF) in HBV reactivation. Clinical, laboratory, and virological data of 87 patients with HBV reactivation were analyzed retrospectively. Teno torque virus (TTV) plasma loads were measured as a measure of immune competence. HBV genomes isolated from 47 patients were analyzed by next-generation sequencing. A functional analysis of identified HBsAg mutants was performed. In patients with ALF the diagnosis was significantly later confirmed than in the non-ALF group. Patients diagnosed during immunosuppression had a milder clinical course compared to later diagnosed patients (p = 0.018, OR = 4.17). TTV viral loads did not differ significantly between the two groups. The HBV genomes isolated from ALF patients had higher viral complexity. A mutation in C-region of HBsAg (L216*), was associated with reduced HBsAg production and secretion. Patients diagnosed with HBV reactivation during immunosuppression had a milder clinical course compared to patients diagnosed during immune reconstitution. ALF was associated with higher viral complexity. An HBsAg mutation (L216*) was found to be more frequent in ALF patients and was associated with reduced HBsAg production and secretion.
Introduction: Reliable and cost-effective diagnostics for hepatitis E virus (HEV) infection are necessary. The aim of our study was to investigate which diagnostic test is most accurate to detect HEV infection in immunocompetent and immunosuppressed patients in a real world setting. Patients and Methods: We performed a retrospective analysis of 1165 patients tested for HEV antibodies and HEV PCR at the same time point. Clinical, laboratory and virological data were taken from patient charts. HEV IgA was measured in a subgroup of 185 patients. Results: HEV RNA was detectable in 61 patients (5.2%); most of them (n = 49, 80.3%/n = 43, 70.5%) were HEV IgM+ and IgG+; however, 12 patients (19.6%) were HEV RNA positive/HEV IgM negative and 17 patients (27.8%) were HEV RNA positive/HEV IgG negative. Ten HEV RNA positive patients (16.4%) had neither HEV IgG nor IgM antibodies. Importantly, all of them were immunosuppressed. HEV IgA testing was less sensitive than HEV IgM for HEV diagnosis. Conclusions: HEV infection can be overlooked in patients without HEV specific antibodies. Performing PCR is necessary to diagnose or exclude HEV infection in immunocompromised hosts. In immunocompetent patients, a screening based on HEV antibodies (IgG/IgM) is sufficient.
Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air–liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease.
Recent findings in permanent cell lines suggested that SARS-CoV-2 Omicron BA.1 induces a stronger interferon response than Delta. Here, we show that BA.1 and BA.5 but not Delta induce an antiviral state in air-liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells and primary human monocytes. Both Omicron subvariants caused the production of biologically active type I (α/β) and III (λ) interferons and protected cells from super-infection with influenza A viruses. Notably, abortive Omicron infection of monocytes was sufficient to protect monocytes from influenza A virus infection. Interestingly, while influenza-like illnesses surged during the Delta wave in England, their spread rapidly declined upon the emergence of Omicron. Mechanistically, Omicron-induced interferon signalling was mediated via double-stranded RNA recognition by MDA5, as MDA5 knock-out prevented it. The JAK/ STAT inhibitor baricitinib inhibited the Omicron-mediated antiviral response, suggesting it is caused by MDA5-mediated interferon production, which activates interferon receptors that then trigger JAK/ STAT signalling. In conclusion, our study 1) demonstrates that only Omicron but not Delta induces a substantial interferon response in physiologically relevant models, 2) shows that Omicron infection protects cells from influenza A virus super-infection, and 3) indicates that BA.1 and BA.5 induce comparable antiviral states.
It becomes more and more obvious that deregulation of host metabolism play an important role in SARS-CoV-2 pathogenesis with implication for increased risk of severe course of COVID-19. Furthermore, it is expected that COVID-19 patients recovered from severe disease may experience long-term metabolic disorders. Thereby understanding the consequences of SARS-CoV-2 infection on host metabolism can facilitate efforts for effective treatment option. We have previously shown that SARS-CoV-2-infected cells undergo a shift towards glycolysis and that 2-deoxy-D-glucose (2DG) inhibits SARS-CoV-2 replication. Here, we show that also pentose phosphate pathway (PPP) is remarkably deregulated. Since PPP supplies ribonucleotides for SARS-CoV-2 replication, this could represent an attractive target for an intervention. On that account, we employed the transketolase inhibitor benfooxythiamine and showed dose-dependent inhibition of SARS-CoV-2 in non-toxic concentrations. Importantly, the antiviral efficacy of benfooxythiamine was further increased in combination with 2DG.
SARS-CoV-2 is causing the coronavirus disease 2019 (COVID-19) pandemic, for which effective pharmacological therapies are needed. SARS-CoV-2 induces a shift of the host cell metabolism towards glycolysis, and the glycolysis inhibitor 2-deoxy-d-glucose (2DG), which interferes with SARS-CoV-2 infection, is under development for the treatment of COVID-19 patients. The glycolytic pathway generates intermediates that supply the non-oxidative branch of the pentose phosphate pathway (PPP). In this study, the analysis of proteomics data indicated increased transketolase (TKT) levels in SARS-CoV-2-infected cells, suggesting that a role is played by the non-oxidative PPP. In agreement, the TKT inhibitor benfooxythiamine (BOT) inhibited SARS-CoV-2 replication and increased the anti-SARS-CoV-2 activity of 2DG. In conclusion, SARS-CoV-2 infection is associated with changes in the regulation of the PPP. The TKT inhibitor BOT inhibited SARS-CoV-2 replication and increased the activity of the glycolysis inhibitor 2DG. Notably, metabolic drugs like BOT and 2DG may also interfere with COVID-19-associated immunopathology by modifying the metabolism of immune cells in addition to inhibiting SARS-CoV-2 replication. Hence, they may improve COVID-19 therapy outcomes by exerting antiviral and immunomodulatory effects.
SARS-CoV-2 is a novel coronavirus currently causing a pandemic. We show that the majority of amino acid positions, which differ between SARS-CoV-2 and the closely related SARS-CoV, are differentially conserved suggesting differences in biological behaviour. In agreement, novel cell culture models revealed differences between the tropism of SARS-CoV-2 and SARS-CoV. Moreover, cellular ACE2 (SARS-CoV-2 receptor) and TMPRSS2 (enables virus entry via S protein cleavage) levels did not reliably indicate cell susceptibility to SARS-CoV-2. SARS-CoV-2 and SARS-CoV further differed in their drug sensitivity profiles. Thus, only drug testing using SARS-CoV-2 reliably identifies therapy candidates. Therapeutic concentrations of the approved protease inhibitor aprotinin displayed anti-SARS-CoV-2 activity. The efficacy of aprotinin and of remdesivir (currently under clinical investigation against SARS-CoV-2) were further enhanced by therapeutic concentrations of the proton pump inhibitor omeprazole (aprotinin 2.7-fold, remdesivir 10-fold). Hence, our study has also identified anti-SARS-CoV-2 therapy candidates that can be readily tested in patients.
Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits.
Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a read-out for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in a broad range of cell culture models, independently of cytopathogenic effect formation. Compared to other cell culture models, the Caco-2 subline Caco-2-F03 displayed superior performance, as it possesses a stable SARS-CoV-2 susceptible phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of PHGDH, CLK-1, and CSF1R. The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the HK2 inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false positive hits.
Recently, we have shown that SARS-CoV-2 Omicron virus isolates are less effective at inhibiting the host cell interferon response than Delta viruses. Here, we present further evidence that reduced interferon-antagonising activity explains at least in part why Omicron variant infections are inherently less severe than infections with other SARS-CoV-2 variants. Most importantly, we here also show that Omicron variant viruses display enhanced sensitivity to interferon treatment, which makes interferons promising therapy candidates for Omicron patients, in particular in combination with other antiviral agents.