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

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 622 ◽  
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.

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

2020 ◽  
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.

scholarly journals Improved plaque assay for human coronaviruses 229E and OC43

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10639
Author(s):  
Nicole Bracci ◽  
Han-Chi Pan ◽  
Caitlin Lehman ◽  
Kylene Kehn-Hall ◽  
Shih-Chao Lin
Keyword(s):  
Low Cost ◽  
Plaque Assay ◽  
Lung Epithelial Cells ◽  
Cell Contact ◽  
Lung Epithelial ◽  
Rd Cells ◽  
Level 3 ◽  
Coronavirus Infection ◽  
Human Coronaviruses ◽  
Biosafety Level

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.

scholarly journals Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings

2021 ◽  
Author(s):  
Marek Widera ◽  
Sandra Westhaus ◽  
Holger F. Rabenau ◽  
Sebastian Hoehl ◽  
Denisa Bojkova ◽  
...  
Keyword(s):  
Culture Media ◽  
Vero Cells ◽  
Heat Inactivation ◽  
The Novel ◽  
Safety Issues ◽  
Rapid Spread ◽  
Level 3 ◽  
Infected Cells ◽  
Novel Coronavirus ◽  
Plastic Surfaces

AbstractThe novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In this study, we evaluated lysis buffers that are commonly used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. In addition, viral stability in cell culture media at 4 °C and on display glass and plastic surfaces used in laboratory environment was analyzed. Furthermore, we evaluated chemical and non-chemical inactivation methods including heat inactivation, UV-C light, addition of ethanol, acetone-methanol, and PFA, which might be used as a subsequent inactivation step in the case of insufficient inactivation. We infected susceptible Caco-2 and Vero cells with pre-treated SARS-CoV-2 and determined the tissue culture infection dose 50 (TCID50) using crystal violet staining and microscopy. In addition, lysates of infected cells and virus containing supernatant were subjected to RT-qPCR analysis. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate the virus. In conclusion, careful evaluation of the used inactivation methods is required especially for non-denaturing buffers. Additional inactivation steps might be necessary before removal of lysed viral samples from BSL-3.

scholarly journals Antiviral Activity of the PropylamylatinTM Formula against the Novel Coronavirus SARS-CoV-2 In Vitro Using Direct Injection and Gas Assays in Virus Suspensions

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 415
Author(s):  
Ashley N. Brown ◽  
Gary Strobel ◽  
Kaley C. Hanrahan ◽  
Joe Sears
Keyword(s):  
Propionic Acid ◽  
Infectious Virus ◽  
Direct Injection ◽  
Plaque Assay ◽  
Antiviral Effect ◽  
Dependent Manner ◽  
Global Public Health ◽  
Novel Coronavirus ◽  
The Individual

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of novel coronavirus disease 2019 (COVID-19), has become a severe threat to global public health. There are currently no antiviral therapies approved for the treatment or prevention of mild to moderate COVID-19 as remdesivir is only approved for severe COVID-19 cases. Here, we evaluated the antiviral potential of a Propylamylatin formula, which is a mixture of propionic acid and isoamyl hexanoates. The Propylamylatin formula was investigated in gaseous and liquid phases against 1 mL viral suspensions containing 105 PFU of SARS-CoV-2. Viral suspensions were sampled at various times post-exposure and infectious virus was quantified by plaque assay on Vero E6 cells. Propylamylatin formula vapors were effective at inactivating infectious SARS-CoV-2 to undetectable levels at room temperature and body temperature, but the decline in virus was substantially faster at the higher temperature (15 min versus 24 h). The direct injection of liquid Propylamylatin formula into viral suspensions also completely inactivated SARS-CoV-2 and the rapidity of inactivation occurred in an exposure dependent manner. The overall volume that resulted in 90% viral inactivation over the course of the direct injection experiment (EC90) was 4.28 µls. Further investigation revealed that the majority of the antiviral effect was attributed to the propionic acid which yielded an overall EC90 value of 11.50 µls whereas the isoamyl hexanoates provided at most a 10-fold reduction in infectious virus. The combination of propionic acid and isoamyl hexanoates was much more potent than the individual components alone, suggesting synergy between these components. These findings illustrate the therapeutic promise of the Propylamylatin formula as a potential treatment strategy for COVID-19 and future studies are warranted.

scholarly journals Network Experiences from a Cross-Sector Biosafety Level-3 Laboratory Collaboration: A Swedish Forum for Biopreparedness Diagnostics

2017 ◽  
Vol 15 (4) ◽  
pp. 384-391 ◽  
Author(s):  
Johanna Thelaus ◽  
Anna Lindberg ◽  
Susanne Thisted Lambertz ◽  
Mona Byström ◽  
Mats Forsman ◽  
...  
Keyword(s):  
Level 3 ◽  
Biosafety Level

scholarly journals COVID-19-related anosmia is associated with viral persistence and inflammation in human olfactory epithelium and brain infection in hamsters

2021 ◽  
pp. eabf8396
Author(s):  
Guilherme Dias de Melo ◽  
Françoise Lazarini ◽  
Sylvain Levallois ◽  
Charlotte Hautefort ◽  
Vincent Michel ◽  
...  
Keyword(s):  
Olfactory Epithelium ◽  
Cell Types ◽  
Brain Infection ◽  
Olfactory Neuroepithelium ◽  
Optimal Medical Management ◽  
Infected Cells ◽  
Multiple Cell ◽  
Lung Pathogenesis ◽  
Taste Dysfunction

Whereas recent investigations have revealed viral, inflammatory and vascular factors involved in SARS-CoV-2 lung pathogenesis, the pathophysiology of neurological disorders in COVID-19 remains poorly understood. Olfactory and taste dysfunction are common in COVID-19, especially in mildly symptomatic patients. Here, we conducted a virologic, molecular, and cellular study of the olfactory neuroepithelium of seven patients with COVID-19 presenting with acute loss of smell. We report evidence that the olfactory neuroepithelium may be a major site of SARS-CoV2 infection with multiple cell types, including olfactory sensory neurons, support cells, and immune cells, becoming infected. SARS-CoV-2 replication in the olfactory neuroepithelium was associated with local inflammation. Furthermore, we showed that SARS-CoV-2 induced acute anosmia and ageusia in golden Syrian hamsters, lasting as long as the virus remained in the olfactory epithelium and the olfactory bulb. Finally, olfactory mucosa sampling from patients showing long-term persistence of COVID-19-associated anosmia revealed the presence of virus transcripts and of SARS-CoV-2-infected cells, together with protracted inflammation. SARS-CoV-2 persistence and associated inflammation in the olfactory neuroepithelium may account for prolonged or relapsing symptoms of COVID-19, such as loss of smell, which should be considered for optimal medical management of this disease.

scholarly journals Establishment of Biosafety Level 3 Laboratory Infrastructure to Study with Highly Pathogen Organisms and Principles of Operation: Review

2014 ◽  
Vol 34 (1) ◽  
pp. 120-136
Author(s):  
Fatıma YÜCEL ◽  
Hivda ÜLBEĞİ POLAT ◽  
Esin AKÇAEL ◽  
Taşkın DENİZ
Keyword(s):  
Level 3 ◽  
Biosafety Level
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