scholarly journals Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mart M Lamers ◽  
Anna Z Mykytyn ◽  
Tim I Breugem ◽  
Yiquan Wang ◽  
Douglas C Wu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Culture Conditions ◽  
Virus Propagation ◽  
Human Airway ◽  
Culture Adaptation ◽  
Propagation Methods ◽  
Transformed Cell Lines ◽  
Airway Cells

Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 – that expresses serine proteases – prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

scholarly journals Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

2021 ◽  
Author(s):  
Mart M. Lamers ◽  
Anna Z. Mykytyn ◽  
Tim I. Breugem ◽  
Yiquan Wang ◽  
Douglas C. Wu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Culture Conditions ◽  
Virus Propagation ◽  
Human Airway ◽  
Culture Adaptation ◽  
Propagation Methods ◽  
Transformed Cell Lines ◽  
Airway Cells

AbstractVirus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein that facilitates serine protease-mediated entry into human airway cells. We report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents MBCS mutations. Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

scholarly journals The SARS-CoV-2 multibasic cleavage site facilitates early serine protease-mediated entry into organoid-derived human airway cells

2020 ◽  
Author(s):  
Anna Z. Mykytyn ◽  
Tim I. Breugem ◽  
Samra Riesebosch ◽  
Debby Schipper ◽  
Petra B. van den Doel ◽  
...  
Keyword(s):  
Serine Protease ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Highly Pathogenic ◽  
Remarkable Feature ◽  
Health Crisis ◽  
Human Airway ◽  
Cell Culture Systems ◽  
Serine Protease Activity ◽  
Airway Cells

AbstractAfter the SARS-CoV outbreak in 2003, a second zoonotic coronavirus named SARS-CoV-2, emerged late 2019 in China and rapidly caused the COVID-19 pandemic leading to a public health crisis of an unprecedented scale. Despite the fact that SARS-CoV-2 uses the same receptor as SARS-CoV, transmission and pathogenesis of both viruses seem to be quite distinct. A remarkable feature of the SARS-CoV-2 spike is the presence of a multibasic cleavage site, which is absent in the SARS-CoV spike. The viral spike protein not only attaches to the entry receptor, but also mediates fusion after cleavage by host proteases. Here, we report that the SARS-CoV-2 spike multibasic cleavage site increases infectivity on differentiated organoid-derived human airway cells. Compared with SARS-CoV, SARS-CoV-2 entered faster into the lung cell line Calu-3, and more frequently formed syncytial cells in differentiated organoid-derived human airway cells. Moreover, the multibasic cleavage site increased entry speed and plasma membrane serine protease usage relative to endosomal entry using cathepsins. Blocking serine protease activity using the clinically approved drug camostat mesylate effectively inhibited SARS-CoV-2 entry and replication in differentiated organoid-derived human airway cells. Our findings provide novel information on how SARS-CoV-2 enters relevant airway cells and highlight serine proteases as an attractive antiviral target.Significance StatementHighly pathogenic coronaviruses have spilled from animals to humans three times in the past two decades. Late 2019, SARS-CoV-2 emerged in China and was declared a pandemic by March 2020. The other two highly pathogenic coronaviruses, SARS-CoV and MERS-CoV, emerged in 2002 and 2012, respectively, but did not attain sustained human-to-human transmission. Given the high diversity of coronaviruses in animals, urbanization and increased air travel, future coronavirus pandemics are likely to occur intermittently. Identifying which factors determine pandemic potential and pathogenicity are therefore of key importance to global health. Additionally, there is an urgent need to rapidly translate fundamental knowledge to the clinic, a process that is expedited through the use of relevant cell culture systems.

scholarly journals Increased transmission of SARS-CoV-2 lineage B.1.1.7 (VOC 2020212/01) is not accounted for by a replicative advantage in primary airway cells or antibody escape

2021 ◽  
Author(s):  
Jonathan C. Brown ◽  
Daniel H. Goldhill ◽  
Jie Zhou ◽  
Thomas P. Peacock ◽  
Rebecca Frise ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cleavage Site ◽  
Vero Cells ◽  
Spike Protein ◽  
Airway Epithelial ◽  
Post Vaccination ◽  
Human Airway ◽  
Rapid Spread ◽  
The Uk ◽  
Airway Cells

AbstractLineage B.1.1.7 (Variant of Concern 202012/01) is a new SARS-CoV-2 variant which was first sequenced in the UK in September 2020 before becoming the majority strain in the UK and spreading worldwide. The rapid spread of the B.1.1.7 variant results from increased transmissibility but the virological characteristics which underpin this advantage over other circulating strains remain unknown. Here, we demonstrate that there is no difference in viral replication between B.1.1.7 and other contemporaneous SARS-CoV-2 strains in primary human airway epithelial (HAE) cells. However, B.1.1.7 replication is disadvantaged in Vero cells potentially due to increased furin-mediated cleavage of its spike protein as a result of a P681H mutation directly adjacent to the S1/S2 cleavage site. In addition, we show that B.1.1.7 does not escape neutralisation by convalescent or post-vaccination sera. Thus, increased transmission of B.1.1.7 is not caused by increased replication, as measured on HAE cells, or escape from serological immunity.

scholarly journals SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Anna Z Mykytyn ◽  
Tim I Breugem ◽  
Samra Riesebosch ◽  
Debby Schipper ◽  
Petra B van den Doel ◽  
...  
Keyword(s):  
Serine Protease ◽  
Viral Entry ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Spike Protein ◽  
Remarkable Feature ◽  
Human Airway ◽  
Entry Strategy ◽  
Entry Speed

Coronavirus entry is mediated by the spike protein which binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the SARS-CoV-2 spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells, and more frequently formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy.

Serum IL-22 level in asthmatic patients and expression of IL-22R1 in human airway cells

2011 ◽  
Vol 30 (12) ◽  
pp. 1291-1295
Author(s):  
Zhan ZHANG ◽  
Ya-ling LUO ◽  
Li-li ZHOU ◽  
Wen-yan LAI ◽  
Jian XU ◽  
...  
Keyword(s):  
Asthmatic Patients ◽  
Human Airway ◽  
Airway Cells

scholarly journals Electrochemical Stability of Poly(3,4-Ethylenedioxythiophene) Derivatives Under Cell Culture Conditions

2021 ◽  
Vol 1885 (3) ◽  
pp. 032004
Author(s):  
Qichao Pan ◽  
Zuwei Zhang ◽  
Yaqiong Zhang ◽  
Yaopeng Zhang ◽  
Bo Zhu
Keyword(s):  
Cell Culture ◽  
Culture Conditions ◽  
Electrochemical Stability ◽  
Cell Culture Conditions

scholarly journals Self-organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nathan Jeger-Madiot ◽  
Lousineh Arakelian ◽  
Niclas Setterblad ◽  
Patrick Bruneval ◽  
Mauricio Hoyos ◽  
...  
Keyword(s):  
Cell Culture ◽  
3D Cell Culture ◽  
Culture Conditions ◽  
Acoustic Levitation ◽  
Cell Sheets ◽  
Cell Therapies ◽  
Cellular Models ◽  
Cell Spheroids

AbstractIn recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells.

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