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As the MHC-I-pathway is key to antigen presentation to cytotoxic T-cells and, therefore, recognition by the host adaptive immune system, we hypothesized that SARS-CoV-2 including its Variants of Concern (VOCs), influences MHC-I expression on epithelial cell surfaces as an immune evasion strategy. We conducted an in vitro time course experiment with the human airway epithelial cell line Calu-3 and the human colorectal adenocarcinoma cell line Caco-2. Cells were infected with SARS-CoV-2 strains non-VOC/B.1.1, Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, and Delta/B.1.617.2. At 2, 24, 48 and 72 h post-infection we performed RT-qPCR to track viral replication. Simultaneously, we performed intracellular staining with a serum of a double-vaccinated healthy adult containing a high amount of spike protein antibody. In flow cytometry experiments, we differentiated between infected (spike protein positive) and bystander (spike protein negative) cells. To compare their HLA expression levels, cells were stained extracellularly with anti-HLA-A-IgG and anti-HLA-B,C-IgG. While HLA-A expression was stable on infected Calu-3 cells for all variants, it increased to different degrees on bystander cells in samples infected with VOCs Beta, Gamma, Delta, or non-VOC over the time course analyzed. In contrast, HLA-A levels were stable in bystander Calu-3 cells in samples infected with the Alpha variant. The upregulation of MHC-I on spike protein negative bystander cells in Calu-3 cell cultures infected with Beta, Gamma, Delta, and partly non-VOC might suggest that infected cells are still capable of secreting inflammatory cytokines like type-I interferons stimulating the MHC-I expression on bystander cells. In comparison, there was no distinct effect on HLA expression level on Caco-2 cells of any of the VOCs or non-VOC. Further investigations of the full range of immune evasion strategies of SARS-CoV-2 variants are warranted. [ABSTRACT FROM AUTHOR]

Background: During the ongoing Covid-19 pandemic caused by the emerging virus SARS-CoV-2, research in the field of coronaviruses has expanded tremendously. The genome of SARS-CoV-2 has rapidly acquired numerous mutations, giving rise to several Variants of Concern (VOCs) with altered epidemiological, immunological, and pathogenic properties. Methods: As cell culture models are important tools to study viruses, we investigated replication kinetics and infectivity of SARS-CoV-2 in the African Green Monkey-derived Vero E6 kidney cell line and the two human cell lines Caco-2, a colon epithelial carcinoma cell line, and the airway epithelial carcinoma cell line Calu-3. We assessed viral RNA copy numbers and infectivity of viral particles in cell culture supernatants at different time points ranging from 2 to 96 h post-infection. Results: We here describe a systematic comparison of growth kinetics of the five SARS-CoV-2 VOCs Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/B.1.617.2, and Omicron/B.1.1.529 and a non-VOC/B.1.1 strain on three different cell lines to provide profound information on the differential behaviour of VOCs in different cell lines for researchers worldwide. We show distinct differences in viral replication kinetics of the SARS-CoV-2 non-VOC and five VOCs on the three cell culture models Vero E6, Caco-2, and Calu-3. Conclusion: This is the first systematic comparison of all SARS-CoV-2 VOCs on three different cell culture models. This data provides support for researchers worldwide in their experimental design for work on SARS-CoV-2. It is recommended to perform virus isolation and propagation on Vero E6 while infection studies or drug screening and antibody-based assays should rather be conducted on the human cell lines Caco-2 and Calu-3. [ABSTRACT FROM AUTHOR]

Background: Fast, reliable and easy to handle methods are required to facilitate urgently needed point-of-care testing (POCT) in the current coronavirus pandemic. Life-threatening severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread all over the world, infecting more than 33,500,000 people and killing over 1 million of them as of October 2020. Infected individuals without any symptoms might still transfer the virus to others underlining the extraordinary transmissibility of this new coronavirus. In order to identify early infections effectively, treat patients on time and control disease spreading, rapid, accurate and onsite testing methods are urgently required. Results: Here we report the development of a loop-mediated isothermal amplification (LAMP) based method to detect SARS-CoV-2 genes ORF8 and N directly from pharyngeal swab samples. The established reverse transcription LAMP (RT-LAMP) assay detects SARS-CoV-2 directly from pharyngeal swab samples without previous time-consuming and laborious RNA extraction. The assay is sensitive and highly specific for SARS-CoV-2 detection, showing no cross reactivity when tested on 20 other respiratory pathogens. The assay is 12 times faster and 10 times cheaper than routine reverse transcription real-time polymerase chain reaction, depending on the assay used. Conclusion: The fast and easy to handle RT-LAMP assay amplifying specifically the genomic regions ORF8 and N of SARS-CoV-2 is ideally suited for POCT at e.g. railway stations, airports or hospitals. Given the current pandemic situation, rapid, cost efficient and onsite methods like the here presented RT-LAMP assay are urgently needed to contain the viral spread. [ABSTRACT FROM AUTHOR]

Senescence was recently linked to neurodegeneration and astrocytes are one of the major cell types to turn senescent under neurodegenerative conditions. Senescent astrocytes were detected in Parkinson's disease (PD) patients' brains besides reactive astrocytes, yet the difference between senescent and reactive astrocytes is unclear. We aimed to characterize senescent astrocytes in comparison to reactive astrocytes and investigate differences and similarities. In a cell culture model of human fetal astrocytes, we determined a unique senescent transcriptome distinct from reactive astrocytes, which comprises dysregulated pathways. Both, senescent and reactive human astrocytes activated a proinflammatory pattern. Astrocyte senescence was at least partially depending on active mechanistic-target-of-rapamycin (mTOR) and DNA-damage response signaling, both drivers of senescence. To further investigate how PD and senescence connect to each other, we asked if a PD-linked environmental factor induces senescence and if senescence impairs midbrain neurons. We could show that the PD-linked pesticide rotenone causes astrocyte senescence. We further delineate, that the senescent secretome exaggerates rotenone-induced neurodegeneration in midbrain neurons differentiated from human induced pluripotent stem cells (hiPSC) of PD patients with alpha-synuclein gene (SNCA) locus duplication. Unlabelled Image • Senescent astrocytes express a unique transcriptome pattern. • A proinflammatory state is activated in both senescent and reactive astrocytes. • Stress-induced senescence in astrocytes depends at least in part on active mTOR and DDR signaling. • The PD-linked pesticide rotenone induces astrocyte senescence. • Human SNCA duplication neurons exhibit a rotenone dependent vulnerability to senescent stimuli. [ABSTRACT FROM AUTHOR]