The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most pressing medical and socioeconomic challenge. Constituting important correlates of protection, the determination of virus-neutralizing antibodies (NAbs) is indispensable for convalescent plasma selection, vaccine candidate evaluation, and immunity certificates. In contrast to standard serological ELISAs, plaque reduction neutralization tests (PRNTs) are laborious, time-consuming, expensive, and restricted to specialized laboratories. To replace microscopic counting-based SARS-CoV-2 PRNTs by a novel assay exempt from genetically modified viruses, which are inapplicable in most diagnostics departments, we established a simple, rapid, and automated SARS-CoV-2 neutralization assay employing an in-cell ELISA (icELISA) approach. After optimization of various parameters such as virus-specific antibodies, cell lines, virus doses, and duration of infection, SARS-CoV-2-infected cells became amenable as direct antigen source for quantitative icELISA. Antiviral agents such as human sera containing NAbs or antiviral interferons dose dependently reduced the SARS-CoV-2-specific signal. Applying increased infectious doses, the icELISA-based neutralization test (icNT) was superior to PRNT in discriminating convalescent sera with high from those with intermediate neutralizing capacities. In addition, the icNT was found to be specific, discriminating between SARS-CoV-2-specific NAbs and those raised against other coronaviruses. Altogether, the SARS-CoV-2 icELISA test allows rapid (<48 h in total, read-out in seconds) and automated quantification of virus infection in cell culture to evaluate the efficacy of NAbs and antiviral drugs using reagents and equipment present in most routine diagnostics departments.

BackgroundThe bioconversion of lignocellulosic biomass in various industrial processes, such as the production of biofuels, requires the degradation of hemicellulose. Clostridium stercorarium is a thermophilic bacterium, well known for its outstanding hemicellulose-degrading capability. Its genome comprises about 50 genes for partially still uncharacterised thermostable hemicellulolytic enzymes. These are promising candidates for industrial applications.ResultsTo reveal the hemicellulose-degrading potential of 50 glycoside hydrolases, they were recombinantly produced and characterised. 46 of them were identified in the secretome of C. stercorarium cultivated on cellobiose. Xylanases Xyn11A, Xyn10B, Xyn10C, and cellulase Cel9Z were among the most abundant proteins. The secretome of C. stercorarium was active on xylan, β-glucan, xyloglucan, galactan, and glucomannan. In addition, the recombinant enzymes hydrolysed arabinan, mannan, and galactomannan. 20 enzymes are newly described, degrading xylan, galactan, arabinan, mannan, and aryl-glycosides of β-d-xylose, β-d-glucose, β-d-galactose, α-l-arabinofuranose, α-l-rhamnose, β-d-glucuronic acid, and N-acetyl-β-d-glucosamine. The activities of three enzymes with non-classified glycoside hydrolase (GH) family modules were determined. Xylanase Xyn105F and β-d-xylosidase Bxl31D showed activities not described so far for their GH families. 11 of the 13 polysaccharide-degrading enzymes were most active at pH 5.0 to pH 6.5 and at temperatures of 57–76 °C. Investigation of the substrate and product specificity of arabinoxylan-degrading enzymes revealed that only the GH10 xylanases were able to degrade arabinoxylooligosaccharides. While Xyn10C was inhibited by α-(1,2)-arabinosylations, Xyn10D showed a degradation pattern different to Xyn10B and Xyn10C. Xyn11A released longer degradation products than Xyn10B. Both tested arabinose-releasing enzymes, Arf51B and Axh43A, were able to hydrolyse single- as well as double-arabinosylated xylooligosaccharides.ConclusionsThe obtained results lead to a better understanding of the hemicellulose-degrading capacity of C. stercorarium and its involved enzyme systems. Despite similar average activities measured by depolymerisation tests, a closer look revealed distinctive differences in the activities and specificities within an enzyme class. This may lead to synergistic effects and influence the enzyme choice for biotechnological applications. The newly characterised glycoside hydrolases can now serve as components of an enzyme platform for industrial applications in order to reconstitute synthetic enzyme systems for complete and optimised degradation of defined polysaccharides and hemicellulose.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1228-3) contains supplementary material, which is available to authorized users.

Coronavirus disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has set off a global pandemic. There is an urgent unmet need for safe, affordable, and effective therapeutics against COVID-19. In this regard, drug repurposing is considered as a promising approach. We assessed the compounds that affect the endosomal acidic environment by applying human angiotensin-converting enzyme 2 (hACE2)- expressing cells infected with a SARS-CoV-2 spike (S) protein-pseudotyped HIV reporter virus and identified that obatoclax resulted in the strongest inhibition of S protein-mediated virus entry. The potent antiviral activity of obatoclax at nanomolar concentrations was confirmed in different human lung and intestinal cells infected with the SARS-CoV-2 pseudotype system as well as clinical virus isolates. Furthermore, we uncovered that obatoclax executes a double-strike against SARS-CoV-2. It prevented SARS-CoV-2 entry by blocking endocytosis of virions through diminished endosomal acidification and the corresponding inhibition of the enzymatic activity of the endosomal cysteine protease cathepsin L. Additionally, obatoclax impaired the SARS-CoV-2 S-mediated membrane fusion by targeting the MCL-1 protein and reducing furin protease activity. In accordance with these overarching mechanisms, obatoclax blocked the virus entry mediated by different S proteins derived from several SARS-CoV-2 variants of concern such as, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Taken together, our results identified obatoclax as a novel effective antiviral compound that keeps SARS-CoV-2 at bay by blocking both endocytosis and membrane fusion. Our data suggested that obatoclax should be further explored as a clinical drug for the treatment of COVID-19.

The current SARS-CoV-2/COVID-19 pandemic represents an unprecedented medical and socioeconomic crisis. Highly efficient treatment options preventing morbidity and mortality are not broadly available and approved drugs are hardly affordable in developing countries. Even after vaccine approvals, it will take several months until the vaccinated and convalescent individuals establish herd immunity. Meanwhile, non-pharmaceutical interventions and antiviral treatments are indispensable to curb the death toll of the pandemic. To identify cost-effective and ubiquitously available options, we tested common herbs consumed worldwide as herbal teas. We found that aqueous infusions prepared by boiling leaves of the Lamiaceae plants perilla and sage elicit potent antiviral activity against SARS-CoV-2 in human cells. Sustained antiviral activity was evident even when cells were treated for only half an hour, and in therapeutic as well as prophylactic regimens. Given the urgency, such inexpensive and broadly available substances might provide help during the pandemic - especially in low-income regions.

Brief summary: Knowledge concerning SARS-CoV-2-neutralizing antibodies (NAbs) is 14 indispensable for COVID-19 convalescent plasma selection, evaluation of vaccines, and immunity 15 certificates. Our in-cell ELISA (icELISA) test allows rapid (<48h) and high-throughput detection 16 and quantification of SARS-CoV-2-specific NAbs and antiviral activity of drug candidates. 17 18 # Correspondence to 19 Mirko Trilling; Mirko.Trilling@uk-essen.de; ORCID: M Trilling Abstract 28The coronavirus disease 2019 caused by the severe acute respiratory syndrome 29 coronavirus 2 (SARS-CoV-2) is currently the most pressing medical and socioeconomic challenge. 30 Constituting important correlates of protection, determination of virus-neutralizing antibodies 31 (NAbs) is indispensable for convalescent plasma selection, vaccine candidate evaluation, and 32 immunity certificates. In contrast to standard serology ELISAs, plaque reduction neutralization 33 tests (PRNTs) are laborious, time-consuming, expensive, and restricted to specialized laboratories. 34To replace microscopic counting-based SARS-CoV-2 PRNTs by a novel assay exempt from 35 genetically modified viruses, which are inapplicable in most diagnostics departments, we 36 established a simple, rapid, and automated SARS-CoV-2 neutralization assay employing an in-cell 37 ELISA (icELISA) approach. 38 After optimization of various parameters such as virus-specific antibodies, cell lines, virus doses, 39 and duration of infection, SARS-CoV-2-infected cells became amenable as direct antigen source 40 for quantitative icELISA. Using commercially available nucleocapsid protein-specific antibodies, 41 viral infection could easily be quantified in human and highly permissive Vero E6 cells by 42 icELISA. Antiviral agents such as human sera containing NAbs or antiviral interferons dose-43 dependently reduced the SARS-CoV-2-specific signal. Applying increased infectious doses, the 44 icNT was superior to PRNT in discriminating convalescent sera with high from those with 45 intermediate neutralizing capacities.46The SARS-CoV-2 icELISA test allows rapid (<48h in total, read-out in seconds) and automated 47 quantification of virus infection in cell culture to evaluate the efficacy of NAbs as well as antiviral 48 drugs, using reagents and equipment present in most routine diagnostics departments. We propose 49 the icELISA and the icNT for COVID-19 research and diagnostics. 50 65 the outbreak a global pandemic. Since its beginning, the centre of the pandemic shifted from China, 66 via Europe and Northern Americas to Central and Southern Americas. This dynamic nature of the 67 pandemic poses an inherent danger of repetitive local and temporal reintroduction circles. Thus, 68 even countries which coped relatively well with the first wave must prepare in terms of diagnostics 69 capacities for potential future re-emergences.70Most SARS-CoV-2 infections lead to mild or moderate illnesses. However, a considerable fraction 71 of cases proceeds to severe pneumonia or life-threatening acute respir...