Methods of Inactivation of SARS-CoV-2 for Downstream Biological Assays

Abstract

The scientific community has responded to the coronavirus disease 2019 (COVID-19) pandemic by rapidly undertaking research to find effective strategies to reduce the burden of this disease. Encouragingly, researchers from a diverse array of fields are collectively working towards this goal. Research with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undertaken in high-containment laboratories; however, it is often desirable to work with samples at lower-containment levels. To facilitate the transfer of infectious samples from high-containment laboratories, we have tested methods commonly used to inactivate virus and prepare the sample for additional experiments. Incubation at 80°C, a range of detergents, Trizol reagents, and UV energies were successful at inactivating a high titer of SARS-CoV-2. Methanol and paraformaldehyde incubation of infected cells also inactivated the virus. These protocols can provide a framework for in-house inactivation of SARS-CoV-2 in other laboratories, ensuring the safe use of samples in lower-containment levels.

Keywords: SARS-Cov-2; Trizol; detergents; inactivation; methanol; paraformaldehyde; temperature.

Figure 1.

Limit of detection of SARS-CoV-2 by different methods. A, Assay to quantify the limits of detection. Known titers of virus were prepared with (right) or without (left) concentrating the sample in a centrifugal column. Quantification of controls was performed using TCID₅₀. Lower limit of detection was 3.16 TCID₅₀/mL: (n = 3; mean ± SEM). B, Cytotoxicity from uninfected control samples diluted in a TCID₅₀ assay (n = 3; mean ± SEM). Abbreviations: PFU, plaque-forming unit; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; TCID₅₀, 50% tissue culture infectious dose.

Figure 2.

The proportion of SARS-CoV-2 inactivation assays with CPE. Samples were either diluted for assays (left) or inactivation agent removed using centrifugal columns (right). Control samples with 10⁰, 10¹, and 10² PFU of SARS-CoV-2 were used as positive controls and to determine the limit of detection for each method. Abbreviations: CPE, cytopathic effect; PFU, plaque-forming unit; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SDS, sodium dodecyl sulfate.

Figure 3.

Effect of fixation on infectious virus in cells. A, TCID₅₀ assay comparing untreated infected cells with fixed infected cells. Lower limit of detection was 0.5 TCID₅₀ (log₁₀)/mL (n = 3; mean ± SEM). B, The proportion of inactivation assays with CPE. Abbreviations: CPE, cytopathic effect; SEM, standard error of the mean; TCID₅₀, 50% tissue culture infectious dose.

Figure 4.

Quantification of SARS-CoV-2 following exposure to different energies of UV light. A, The concentration of viable SARS-CoV-2 following exposure to UV light measured by TCID₅₀ assay. Lower limit of detection was 0.5 TCID₅₀(log₁₀)/mL. B, The concentration of viable SARS-CoV-2 following exposure to UV measured by plaque assay. Both assays confirmed the complete inactivation of the sample after exposure to 0.04 J/cm2 or higher UV energy. Results are expressed as mean ± SEM (n = 3). Abbreviations: PFU, plaque-forming unit; SEM, standard error of the mean; TCID₅₀, 50% tissue culture infectious dose.