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. IMPORTANCE SARS-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.
Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopore's adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective ‘human host depletion’ resulted in a 1.70 fold (± 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.
In response to the SARS-CoV-2 pandemic, a highly increased sequencing effort has been established worldwide to track and trace ongoing viral evolution. Technologies, such as nanopore sequencing via the ARTIC protocol are used to reliably generate genomes from raw sequencing data as a crucial base for molecular surveillance. However, for many labs that perform SARS-CoV-2 sequencing, bioinformatics is still a major bottleneck, especially if hundreds of samples need to be processed in a recurring fashion. Pipelines developed for short-read data cannot be applied to nanopore data. Therefore, specific long-read tools and parameter settings need to be orchestrated to enable accurate genotyping and robust reference-based genome reconstruction of SARS-CoV-2 genomes from nanopore data. Here we present poreCov, a highly parallel workflow written in Nextflow, using containers to wrap all the tools necessary for a routine SARS-CoV-2 sequencing lab into one program. The ease of installation, combined with concise summary reports that clearly highlight all relevant information, enables rapid and reliable analysis of hundreds of SARS-CoV-2 raw sequence data sets or genomes. poreCov is freely available on GitHub under the GNUv3 license: github.com/replikation/poreCov.
The SHV β-lactamases (BLs) have undergone strong allele diversification that has changed their substrate specificities. Based on 147 NCBI entries for SHV alleles, in silico mathematical models predicted 5 positions as relevant for the β-lactamase inhibitor (BLI)-resistant (2br) phenotype, 12 positions as relevant for the extended-spectrum BL (ESBL) (2be) phenotype, and 2 positions as related solely to the narrow-spectrum (2b) phenotype. These positions and six additional positions described in other studies (including one promoter mutation) were systematically substituted and investigated for their substrate specificities in a BL-free Escherichia coli background, representing, to our knowledge, the most comprehensive substrate and substitution analysis for SHV alleles to date. An in vitro analysis confirmed the essentiality of positions 238 and 179 for the 2be phenotype and of position 69 for the 2br phenotype. The E240K and E240R substitutions, which do not occur alone in known 2br SHV variants, led to a 2br phenotype, indicating a latent BLI resistance potential of these substitutions. The M129V, A234G, S271I, and R292Q substitutions conferred latent resistance to cefotaxime. In addition, seven positions that were found not always to be associated with the ESBL phenotype resulted in increased resistance to ceftaroline. We also observed that coupling of a strong promoter (IS26) to an A146V mutant with the 2b phenotype resulted in highly increased resistance to BLIs, cefepime, and ceftaroline but not to third-generation cephalosporins, indicating that SHV enzymes represent an underestimated risk for empirical therapies that use piperacillin-tazobactam or cefepime to treat different infectious diseases caused by Gram-negative bacteria.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.