A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection with SARS-CoV-2 in humans causes coronavirus disease 2019 (COVID-19) and ...has been rapidly spreading around the globe
. SARS-CoV-2 shows some similarities to other coronaviruses; however, treatment options and an understanding of how SARS-CoV-2 infects cells are lacking. Here we identify the host cell pathways that are modulated by SARS-CoV-2 and show that inhibition of these pathways prevents viral replication in human cells. We established a human cell-culture model for infection with a clinical isolate of SARS-CoV-2. Using this cell-culture system, we determined the infection profile of SARS-CoV-2 by translatome
and proteome proteomics at different times after infection. These analyses revealed that SARS-CoV-2 reshapes central cellular pathways such as translation, splicing, carbon metabolism, protein homeostasis (proteostasis) and nucleic acid metabolism. Small-molecule inhibitors that target these pathways prevented viral replication in cells. Our results reveal the cellular infection profile of SARS-CoV-2 and have enabled the identification of drugs that inhibit viral replication. We anticipate that our results will guide efforts to understand the molecular mechanisms that underlie the modulation of host cells after infection with SARS-CoV-2. Furthermore, our findings provide insights for the development of therapies for the treatment of COVID-19.
SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell ...signaling events underlying SARS-CoV-2 infection hinders therapy development. We use a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phosphoproteomics. We identify viral protein phosphorylation and define phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways are activated. Drug-protein network analyses revealed GFR signaling as key pathways targetable by approved drugs. The inhibition of GFR downstream signaling by five compounds prevents SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as a central pathway essential for SARS-CoV-2 replication. It provides novel strategies for COVID-19 treatment.
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•Phosphoproteomics of SARS-CoV-2-infected cells reveal the signaling landscape•SARS-CoV-2 proteins are extensively phosphorylated in host cells•Infection leads to the activation of growth factor receptor signaling•Drugs inhibiting growth factor receptor signaling prevent viral replication
In this study, Klann et al. dissected the host cell signaling landscape upon infection with SARS-CoV-2. Mapping differential signaling networks identified a number of pathways activated during infection. Drug-target network analysis revealed potential therapeutic targets. Growth factor receptor signaling was highly activated upon infection and its inhibition prevented SARS-CoV-2 replication in cells.
A group of 126 German nationals was evacuated from Wuhan to Frankfurt after screening for symptoms of Covid-19 and was to undergo a 14-day quarantine and additional screening in Frankfurt. The ...authors discovered evidence of SARS-CoV-2 infection in throat swabs from 2 evacuated persons who had passed the symptoms-based screening.
•The automated immunoassays showed a higher sensitivity than the ELISA based assays.•The assay using the S and N protein as antigens showed the highest sensitivity.•There were differences in the ...immune response (targeting SARS-CoV-2 S/N antigen).•The titers generated with the examined assays correlated well with the PRNT.
Serological SARS-CoV-2 assays are urgently needed for diagnosis, contact tracing and for epidemiological studies. So far, there is limited data on how recently commercially available, high-throughput immunoassays, using different recombinant SARS-CoV-2 antigens, perform with clinical samples. Focusing on IgG and total antibodies, we demonstrate the performance of four automated immunoassays (Abbott Architect™ i2000 (N protein-based)), Roche cobas™ e 411 analyzer (N protein-based, not differentiating between IgA, IgM or IgG antibodies), LIAISON®XL platform (S1 and S2 protein-based), VIRCLIA® automation system (S1 and N protein-based) in comparison to two ELISA assays (Euroimmun SARS-CoV-2 IgG (S1 protein-based) and Virotech SARS-CoV-2 IgG ELISA (N protein-based)) and an in-house developed plaque reduction neutralization test (PRNT). We tested follow up serum/plasma samples of individuals PCR-diagnosed with COVID-19. When calculating the overall sensitivity, in a time frame of 49 days after first PCR-positivity, the PRNT as gold standard, showed the highest sensitivity with 93.3% followed by the dual-target assay for the VIRCLIA® automation system with 89%. The overall sensitivity in the group of N protein-based assays ranged from 66.7 to 77.8% and in the S protein-based-assays from 71.1 to 75.6%. Five follow-up samples of three individuals were only detected in either an S and/or N protein-based assay, indicating an individual different immune response to SARS-CoV-2 and the influence of the used assay in the detection of IgG antibodies. This should be further analysed. The specificity of the examined assays was ≥ 97%. However, because of the low or unknown prevalence of SARS-CoV-2, the examined assays in this study are currently primarily eligible for epidemiological investigations, as they have limited information in individual testing.
Wastewater-based monitoring of the spread of the new SARS-CoV-2 virus, also referred to as wastewater-based epidemiology (WBE), has been suggested as a tool to support epidemiology. An extensive ...sampling campaign, including nine municipal wastewater treatment plants, has been conducted in different cities of the Federal State of North Rhine-Westphalia (Germany) on the same day in April 2020, close to the first peak of the corona crisis. Samples were processed and analysed for a set of SARS-CoV-2-specific genes, as well as pan-genotypic gene sequences also covering other coronavirus types, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, a comprehensive set of chemical reference parameters and bioindicators was analysed to characterize the wastewater quality and composition. Results of the RT-qPCR based gene analysis indicate the presence of SARS-CoV-2 genetic traces in different raw wastewaters. Furthermore, selected samples have been sequenced using Sanger technology to confirm the specificity of the RT-qPCR and the origin of the coronavirus. A comparison of the particle-bound and the dissolved portion of SARS-CoV-2 virus genes shows that quantifications must not neglect the solid-phase reservoir. The infectivity of the raw wastewater has also been assessed by viral outgrowth assay with a potential SARS-CoV-2 host cell line in vitro, which were not infected when exposed to the samples. This first evidence suggests that wastewater might be no major route for transmission to humans. Our findings draw attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater.
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•The first study that reports the detection of SARS-CoV-2 in wastewater in Germany using RT-qPCR.•The presence of SARS-CoV-2 was confirmed by sequencing, but also the risk of false-positive results has been elucidated.•In raw wastewater, 3.0 to 20 gene equivalents/mL was found in raw wastewater.•The replication potential tests were negative for wastewater samples.•Sanger sequencing was required to differentiate the genetic pattern clearly.
•Darunavir showed no in vitro antiviral activity against SARS-CoV-2 (EC50 > 100 μM).•Remdesivir demonstrated potent antiviral activity, confirming validity of the assay.•Overall, the data do not ...support use of darunavir for treatment of COVID-19.
Given the high need and the absence of specific antivirals for treatment of COVID-19 (the disease caused by severe acute respiratory syndrome-associated coronavirus-2 SARS-CoV-2), human immunodeficiency virus (HIV) protease inhibitors are being considered as therapeutic alternatives.
Prezcobix/Rezolsta is a fixed-dose combination of 800 mg of the HIV protease inhibitor darunavir (DRV) and 150 mg cobicistat, a CYP3A4 inhibitor, which is indicated in combination with other antiretroviral agents for the treatment of HIV infection. There are currently no definitive data on the safety and efficacy of DRV/cobicistat for the treatment of COVID-19. The in vitro antiviral activity of darunavir against a clinical isolate from a patient infected with SARS-CoV-2 was assessed.
DRV showed no antiviral activity against SARS-CoV-2 at clinically relevant concentrations (EC50 > 100 μM). Remdesivir, used as a positive control, demonstrated potent antiviral activity (EC50 = 0.38 μM).
Overall, the data do not support the use of DRV for the treatment of COVID-19.
Abstract
Aims
Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as a global pandemic. SARS-CoV-2 infection can lead to elevated ...markers of cardiac injury associated with higher risk of mortality. It is unclear whether cardiac injury is caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here, we investigate whether cardiomyocytes are permissive for SARS-CoV-2 infection.
Methods and results
Two strains of SARS-CoV-2 infected human induced pluripotent stem cell-derived cardiomyocytes as demonstrated by detection of intracellular double-stranded viral RNA and viral spike glycoprotein expression. Increasing concentrations of viral RNA are detected in supernatants of infected cardiomyocytes, which induced infections in Caco-2 cell lines, documenting productive infections. SARS-CoV-2 infection and induced cytotoxic and proapoptotic effects associated with it abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signalling, apoptosis, and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a 3D cardiosphere tissue model. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Coronavirus particles were further observed in cardiomyocytes of a patient with coronavirus disease 2019. Infection of induced pluripotent stem cell-derived cardiomyocytes was dependent on cathepsins and angiotensin-converting enzyme 2, and was blocked by remdesivir.
Conclusion
This study demonstrates that SARS-CoV-2 infects cardiomyocytes in vitro in an angiotensin-converting enzyme 2- and cathepsin-dependent manner. SARS-CoV-2 infection of cardiomyocytes is inhibited by the antiviral drug remdesivir.
Graphical Abstract
Human cytomegalovirus (CMV) is a significant cause of morbidity and mortality in patient groups at risk. We have previously shown that the anti-CMV IgG seroprevalence in an urban region of Germany ...has changed over the last decades. Overall, a decline from 63.7 to 57.25% had been observed between 1988–1997 and 1998–2008 (
p
< 0,001). Here, we continuously follow the trends to the most recent decade 2009 to 2018. In a retrospective analysis, we determined the seroprevalence of CMV IgG antibodies in our patient cohort, stratified by gender and selected groups at risk (e.g., patients with HIV infection; women of childbearing age). The overall prevalence of anti-CMV IgG non-significantly declined further from 57.25% in 1998–2008 to 56.48% in 2009–2018 (
p
= 0.881). Looking at gender differences, overall CMV seroprevalence in males declined to 52.82% (from 55.54% in 1998–2008;
p
= 0.0254), while it non-significantly increased in females to 59.80%. The high seroprevalence in patients with a known HIV infection further increased from 87.46% in 1998–2008 to 92.93% in the current period (
p
= 0.9999). In women of childbearing age, no significant changes over the last three decades could be observed. The CMV seroprevalence in oncological patients was determined to be 60.64%. Overall, the former significant decline of CMV seroprevalence between the decades 1988–1997 and 1998–2008 in this urban region of Germany slowed down to a non-significant decrease of 0.77% (1998–2008 vs. 2009–2018). This might be an indicator that CMV seroprevalence has reached a plateau.
SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic, in which acute respiratory infections are associated with high socio-economic burden. We applied high-content screening to a ...well-defined collection of 5632 compounds including 3488 that have undergone previous clinical investigations across 600 indications. The compounds were screened by microscopy for their ability to inhibit SARS-CoV-2 cytopathicity in the human epithelial colorectal adenocarcinoma cell line, Caco-2. The primary screen identified 258 hits that inhibited cytopathicity by more than 75%, most of which were not previously known to be active against SARS-CoV-2 in vitro. These compounds were tested in an eight-point dose response screen using the same image-based cytopathicity readout. For the 67 most active molecules, cytotoxicity data were generated to confirm activity against SARS-CoV-2. We verified the ability of known inhibitors camostat, nafamostat, lopinavir, mefloquine, papaverine and cetylpyridinium to reduce the cytopathic effects of SARS-CoV-2, providing confidence in the validity of the assay. The high-content screening data are suitable for reanalysis across numerous drug classes and indications and may yield additional insights into SARS-CoV-2 mechanisms and potential therapeutic strategies.
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