key: cord-0996269-d8ufzkys authors: Jureka, Alexander S.; Silvas, Jesus A.; Basler, Christopher F. title: Propagation, inactivation, and safety testing of SARS-CoV-2 date: 2020-05-13 journal: bioRxiv DOI: 10.1101/2020.05.13.094482 sha: 326c4ae68551fc3cea0e16210f33bb840eab9b2a doc_id: 996269 cord_uid: d8ufzkys 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. 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. The novel coronavirus SARS-CoV-2, the causative agent of Coronavirus disease 2019 (CoVID- The expanded interest in studying SARS-CoV-2 to address the current pandemic requires 33 that many laboratories acquire the capacity to work with the virus. However, despite the rapidly To help address these needs and to facilitate SARS-CoV-2 research efforts, we describe here 49 methods for the propagation of SARS-CoV-2 in multiple cell lines. We have also determined a more 50 efficient method for quantifying virus by plaque assay and have developed a SARS-CoV-2-specific 51 focus forming assay which can enhance throughput of assays requiring quantification of viral titers. Additionally, we describe validation if methods for the inactivation of SARS-CoV-2 through the use 53 of TRIzol, 10% neutral buffered formalin, beta-propiolactone, and heat. Taken together, the data 54 presented here will serve to provide researchers with a helpful basis of information to aid in their 55 work on SARS-CoV-2. To validate heat treatment as method to inactivate SARS-CoV-2, SARS-CoV-2 virus was 154 separated into the following validation groups: non-infected control, room temperature control 155 (1x10 6 pfu per replicate), 100C for 5 minutes (1x10 6 pfu per replicate), 100C for 10 minutes (1x10 6 pfu 156 per replicate), and 100C for 15 minutes (1x10 6 pfu per replicate). 1.5 ml microcentrifuge 157 polypropylene tubes containing the virus (500 µL total volume) were exposed to direct heat in a heat together these data demonstrate that using MCC as an overlay media is an effective and far more 233 efficient method than the use of traditional agarose overlays. For viruses like SARS-CoV-2 which produce significant CPE in permissive cell lines, traditional 238 plaque assays are the standard for virus quantification. However, traditional plaque assays require 239 waiting for a particular virus to produce significant enough CPE for quantifiable plaque formation. As described above, SARS-CoV-2 is most readily quantifiable by plaque assay at 3 days post-241 infection. Here, we set out to develop and immunohistochemical assay to reliably determine SARS- Taken together, these data demonstrate that SARS-CoV-2 containing samples can be accurately 247 quantified within 24 hours, and in a higher throughput manner than traditional plaque assays. Currently, there is limited data published on methods that successfully inactivate SARS-CoV-2. To help fill this gap in knowledge, we tested the ability of TRIzol, formalin, beta-propiolactone, and 256 direct heat to successfully inactivate SARS-CoV-2. TRIzol is a well-known and widely used reagent for the isolation of nucleic acids and in some inactivating SARS-CoV-2 at concentrations ranging from 0.5% to 2% (total formaldehyde 275 concentration) after 1 hour at room temperature (Table 1) Given that 10% neutral buffered formalin (NBF) contains 4% formaldehyde, treatment of SARS-CoV-282 2 containing samples with 10% NBF for 1 hour at room temperature is more than adequate for SARS- CoV-2 inactivation. Our data also indicates that lower concentrations (down to 0.5% total 284 formaldehyde concentration) will effectively inactivate SARS-CoV-2 for the purposes of processing 285 of liquid samples, such as isolation of whole virions. Given its use in the preparation of vaccines, we wanted to determine if BPL could provide a 297 rapid method for the purification of inactivated viral particles. Our results indicate that after 298 purification of BPL treated SARS-CoV-2 stocks over a 20% sucrose gradient ( Figure 4A ) that intact 299 viral particles are readily apparent by electron microscopy ( Figure 4B ). We also identified that both The ability to grow and accurately quantify infectious virus is critical for virological studies. Here, we sought to determine the growth kinetics of SARS-CoV-2 in several commonly used cell lines Formaldehyde is a ubiquitously used reagent in life sciences and has numerous applications 374 such as the fixation of cells for microscopy studies such as indirect immunofluorescence of infected 375 cells and as a disinfectant for scientific equipment. Here, we demonstrate that treatment of SARS- CoV-2 infected cells with formaldehyde at a concentration at or above 0.5% for one hour at room 377 temperature effectively inactivates SARs-CoV-2. Given the wide ranges of uses for formaldehyde, we 378 hope this data will assist in the safe processing of probable or confirmed SARS-CoV-2 containing Taken together, we hope the methods and data reported here will serve to expedite the much-387 needed research required to address this unprecedented pandemic. Funding: This research was funded by NIH grants to CFB, including P01AI120943 and R01AI143292. 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