Improved plaque assay for human coronaviruses 229E and OC43

In light of the COVID-19 pandemic, studies that work to understand SARS-CoV-2 are urgently needed. In turn, the less severe human coronaviruses such as HCoV-229E and OC43 are drawing newfound attention. These less severe coronaviruses can be used as a model to facilitate our understanding of the host immune response to coronavirus infection. SARS-CoV-2 must be handled under biosafety level 3 (BSL-3) conditions. Therefore, HCoV-229E and OC43, which can be handled at BSL-2 provide an alternative to SARS-CoV-2 for preclinical screening and designing of antivirals. However, to date, there is no published effective and efficient method to titrate HCoVs other than expensive indirect immunostaining. Here we present an improved approach using an agarose-based conventional plaque assay to titrate HCoV 229E and OC43 with mink lung epithelial cells, Mv1Lu. Our results indicate that titration of HCoV 229E and OC43 with Mv1Lu is consistent and reproducible. The titers produced are also comparable to those produced using human rhabdomyosarcoma (RD) cells. More importantly, Mv1Lu cells display a higher tolerance for cell-cell contact stress, decreased temperature sensitivity, and a faster growth rate. We believe that our improved low-cost plaque assay can serve as an easy tool for researchers conducting HCoV research.

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Propagation, Inactivation, and Safety Testing of SARS-CoV-2

Viruses ◽

10.3390/v12060622 ◽

2020 ◽

Vol 12 (6) ◽

pp. 622 ◽

Cited By ~ 11

Author(s):

Alexander S. Jureka ◽

Jesus A. Silvas ◽

Christopher F. Basler

Keyword(s):

Plaque Assay ◽

Virus Infectivity ◽

Safety Testing ◽

Level 3 ◽

Chinese Province ◽

Infected Cells ◽

Multiple Cell ◽

Novel Coronavirus ◽

Effective Procedures ◽

Biosafety Level

In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.

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Propagation, inactivation, and safety testing of SARS-CoV-2

10.1101/2020.05.13.094482 ◽

2020 ◽

Cited By ~ 1

Author(s):

Alexander S. Jureka ◽

Jesus A. Silvas ◽

Christopher F. Basler

Keyword(s):

Plaque Assay ◽

Virus Infectivity ◽

Safety Testing ◽

Level 3 ◽

Chinese Province ◽

Infected Cells ◽

Multiple Cell ◽

Novel Coronavirus ◽

Effective Procedures ◽

Biosafety Level

AbstractIn late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. Because work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable safe study of RNA, DNA and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.

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Microtubule dynamics and Rac-1 signaling independently regulate barrier function in lung epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00443.2006 ◽

2007 ◽

Vol 293 (5) ◽

pp. L1321-L1331 ◽

Cited By ~ 11

Author(s):

Magdalena J. Lorenowicz ◽

Mar Fernandez-Borja ◽

Anne-Marieke D. van Stalborch ◽

Marian A. J. A. van Sterkenburg ◽

Pieter S. Hiemstra ◽

...

Keyword(s):

Cell Adhesion ◽

Epithelial Cells ◽

Barrier Function ◽

Lung Epithelial Cells ◽

Cell Junctions ◽

Epithelial Barrier ◽

Cell Contact ◽

Epithelial Barrier Function ◽

Lung Epithelial ◽

Cell Cell

Cadherin-mediated cell-cell adhesion controls the morphology and function of epithelial cells and is a critical component of the pathology of chronic inflammatory disorders. Dynamic interactions between cadherins and the actin cytoskeleton are required for stable cell-cell contact. Besides actin, microtubules also target intercellular, cadherin-based junctions and contribute to their formation and stability. Here, we studied the role of microtubules in conjunction with Rho-like GTPases in the regulation of lung epithelial barrier function using real-time monitoring of transepithelial electrical resistance. Unexpectedly, we found that disruption of microtubules promotes epithelial cell-cell adhesion. This increase in epithelial barrier function is accompanied by the accumulation of β-catenin at cell-cell junctions, as detected by immunofluorescence. Moreover, we found that the increase in cell-cell contact, induced by microtubule depolymerization, requires signaling through a RhoA/Rho kinase pathway. The Rac-1 GTPase counteracts this pathway, because inhibition of Rac-1 signaling rapidly promotes epithelial barrier function, in a microtubule- and RhoA-independent fashion. Together, our data suggest that microtubule-RhoA-mediated signaling and Rac-1 control lung epithelial integrity through counteracting independent pathways.

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Anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component Andrographolide in human lung epithelial cells and cytotoxicity evaluation in major organ cell representatives

10.1101/2020.12.08.415836 ◽

2020 ◽

Author(s):

Khanit Sa-ngiamsuntorn ◽

Ampa Suksatu ◽

Yongyut Pewkliang ◽

Piyanoot Thongsri ◽

Phongthon Kanjanasirirat ◽

...

Keyword(s):

Epithelial Cells ◽

Human Lung ◽

Plaque Assay ◽

Andrographis Paniculata ◽

Lung Epithelial Cells ◽

Major Health Problem ◽

Lung Epithelial ◽

Novel Coronavirus ◽

Effective Drugs ◽

Human Lung Epithelial Cells

ABSTRACTThe coronavirus disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem affecting more than fifty million cases with over one million deaths globally. The effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine anti-SARS-CoV–2 activity of Andrographis paniculata extract and its major component andrographolide. SARS-CoV-2 at 25TCID50 was able to reach the maximal infectivity of 95% in Calu-3 cells. Post-infection treatment of A. paniculata and andrographolide in SARS-CoV–2 infected Calu-3 cells significantly inhibited the production of infectious virions with the IC50 of 0.036 μg/mL and 0.034 μM, respectively, as determined by plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3) and brain (SH-SY5Y), showed the CC50 of >100 μg/mL for A. paniculata extract and 13.2-81.5 μM for andrographolide, respectively, corresponding to the selectivity index over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV–2 infection.

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