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

Abstract

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.

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms

“…Wild-type and CRISPR-edited Caco-2 cells were grown at 37°C, 5% CO 2 in DMEM, 10% FBS. SARS-CoV-2 stocks were grown and titered on Vero E6 cells as described previously ( 61 ). Wild-type and CRISPR-edited Caco-2 cell lines were infected with SARS-CoV-2 at a MOI of 0.01 in DMEM supplemented with 2% FBS.…”

Inactivation and Elimination of SARS-CoV-2 in Biosamples Using Simple Fixatives and Ultrafiltration

“…Ultrafiltration methods using membrane-filter devices have been reported previously to concentrate viruses, including SARS-CoV-2 various sample types [ 8 , 9 , 14 , 15 , 16 ]. In those studies, Amicon ® Ultra-15 with a 30 kDa MWCO centrifugal filter device was used to concentrate the virus by centrifugation at 4500× g for 10 min at 4 °C.…”

Pulsed broad-spectrum UV light effectively inactivates SARS-CoV-2 on multiple surfaces

“…Ten-fold serial dilutions of SARS-CoV-2 containing samples were added onto the cells (100 µL) and virus was adsorbed for 1 hour with shaking at 15-minute intervals. After the adsorption period, 1 mL of 0.6% microcrystalline cellulose in DMEM supplemented with 2% fetal bovine serum and 1x antibioticantimycotic was overlaid onto to the cells and plates were incubated at 37C/5% CO2 for 72 hours, as described previously [30]. After incubation, the microcrystalline cellulose overlay was aspirated from the well, and cells were fixed with 10% neutral buffered formalin for 1 hour at room temperature.…”