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

Abstract

The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) causes Coronavirus disease-2019 (COVID-19), which is an ongoing pandemic that has significantly affected the health, economy, and socio-economic status of individuals worldwide. Laboratory research using in vitro, ex vivo and in vivo models has been accelerated to understand the pathogenesis of SARS-CoV-2 infection. However, such experimental research involving SARS-CoV-2 is restricted to biocontainment/safety level-3 (BSL-3) settings, due to the high pathogenicity of this virus. Since many of the downstream analyses of SARS-CoV-2-infected biological samples need to be conducted in a non-BSL3 setting, it is important to ensure that the samples are fully decontaminated and safe for subsequent analysis. Here, we report the effectiveness of standard procedures used to fix cells and tissues for pathological analysis, including 2% or 4% paraformaldehyde, 50%–70% ethanol, 10% neutral buffered formalin and ultrafiltration using membranes with a molecular weight cut-off (MWCO) ranging from 3 to 30 kDa, for inactivating or eliminating SARS-CoV-2. We validated these methods in experimental laboratory samples, such as viral inoculum in cell culture media, SARS-CoV-2 infected host cells and animal tissue lysates. We found that 15 minutes’ treatment of viral inoculum (105 plaque-forming units; PFU) or SARS-CoV-2 infected cells with paraformaldehyde or 70% ethanol resulted in complete inactivation of the virus. The treatment of infected hamster lung tissues with 10% neutral buffered formalin also fully inactivated the virus. However, only 3 kDa ultracentrifuge filter was effective in eliminating the virus to an undetectable limit in the filtrate. Our validated methods are useful for decontaminating biological samples to reduce infection risk and safe handling in BSL2 facilities.

An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy

“…Then the wells were replenished with 2 mL of DMEM + FBS. At 24 h post-infection, the cell culture supernatants were removed and the cells were fixed with 4% paraformaldehyde (Cat# 19943-k2, Thermo Scientific, USA) as described previously 71 .…”

Preclinical coronavirus studies and pathology: Challenges of the high-containment laboratory

“…8 SARS-CoV-2 can be inactivated in different tissue and fluid samples using several methods, including ultraviolet irradiation, formalin fixation, and heat inactivation. 18 , 19 , 24 , 36 Federal regulations require that institutions have internally validated inactivation data for all work with Select Agents, 7 where each proposed inactivation method must be validated in each specific tissue or fluid specimen that will potentially be removed from the BSL3 facility and approved by an internal regulatory group, usually the IBC. 25 Published and well-established protocols for inactivating nonselect BSL3 agents, such as SARS-CoV-2 and M. tuberculosis , can be used to remove samples from the BSL3 facility, but institutions may still require their high-containment laboratories to perform internal inactivation validation studies for certain tissues or when using specific pathogen strains.…”

An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy

“…Then the wells were replenished with 2 mL of DMEM + FBS. At 24 hours post-infection, the cell culture supernatants were removed and the cells were xed with 4% paraformaldehyde (Cat# 19943-k2, Thermo Scienti c, USA) as described previously 69 .…”