Inactivation of Material from SARS-CoV-2-Infected Primary Airway Epithelial Cell Cultures

Abstract

Given that the airway epithelium is the initial site of infection, study of primary human airway epithelial cells (AEC) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be crucial to improved understanding of viral entry factors and innate immune responses to the virus. Centers for Disease Control and Prevention (CDC) guidance recommends work with live SARS-CoV-2 in cell culture be conducted in a Biosafety Level 3 (BSL-3) laboratory. To facilitate downstream assays of materials from experiments there is a need for validated protocols for SARS-CoV-2 inactivation to facilitate safe transfer of material out of a BSL-3 laboratory. We propagated stocks of SARS-CoV-2, then evaluated the effectiveness of heat (65 °C) or ultraviolet (UV) light inactivation. We infected differentiated human primary AECs with SARS-CoV-2, then tested protocols designed to inactivate SARS-CoV-2 in supernatant, protein isolate, RNA, and cells fixed for immunohistochemistry by exposing Vero E6 cells to materials isolated/treated using these protocols. Heating to 65 °C for 10 min or exposing to UV light fully inactivated SARS-CoV-2. Furthermore, we found in SARS-CoV-2-infected primary AEC cultures that treatment of supernatant with UV light, isolation of RNA with Trizol^®, isolation of protein using a protocol including sodium dodecyl sulfate (SDS) 0.1% and Triton X100 1%, and fixation of AECs using 10% formalin and Triton X100 1%, each fully inactivated SARS-CoV-2.

Keywords: COVID-19; RNA; SARS-CoV-2; Vero E6; airway; epithelial; heat; inactivation; protein.

Figure 1

Each experimental condition, and dilution within each condition, were completed in triplicate. Results were consistent within each dilution, therefore a single well per dilution is presented for simplicity. Panel (A) presents crystal violet stained uninfected Vero E6 cells, Vero E6 cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at multiplicity of infection of multiplicity of infection (MOI) = 0.5, Vero E6 cells exposed to SARS-CoV-2 that had been exposed to heat (65 °C) for 10 min, and Vero E6 cells exposed to SARS-CoV-2 that had been treated with UV light (200,000 microjoules over a 5-min). Panel (B) presents light microscopy images of uninfected Vero E6 cells, Panel (C) presents light microscopy images of Vero E6 cells infected with SARS-CoV-2, and Panel (D) presents light microscopy images of Vero E6 cells exposed to heat-inactivated SARS-CoV-2.

Figure 2

Confocal images of uninfected differentiated airway epithelial cells (AECs) and AECs following infection with SARS-CoV-2 at a MOI of 0.5 for 96 h labeled for spike protein (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Panel (A) presents an image obtained from the apical portion of the differentiated uninfected AEC layer. Panel (B) presents a 3-dimensional reconstruction of the uninfected AEC layer. Panel (C) presents an image obtained from the apical portion of a SARS-CoV-2-infected AEC layer. Panel (D) presents a 3-dimensional reconstruction of the SARS-CoV-2-infected AEC layer. Panel (E) presents crystal violet stained Vero cells exposed to cell layer harvested from positive control SARS-CoV-2-infected AECs (Neat concentration represents all cell layer material scrapped from an AEC transwell suspended in 100 µL of phosphate buffered saline) and supernatant from SARS-CoV-2 infected AECs. Positive control SARS-CoV-2 infected AEC experiments were completed using 3 separate human primary AEC lines (3 human donors) with consistent results from all three AEC lines.

Figure 3

Viral replication in SARS-CoV-2-infected organotypic primary human airway epithelial cells (AEC). Viral copy number was measured by performing quantitative polymerase chain reaction (qPCR) using SARS-CoV-2 inactivated RNA isolated 96 h following inoculation of AECs with SARS-CoV-2 at a MOI of 0.5 or UV-inactivated SARS-CoV-2. Panel (A). presents unadjusted copy number and Panel (B). presents copy number adjusted for RNA concentration.

Figure 4

Each experimental condition, and dilution within each condition, were completed in triplicate using 3 separate human primary AEC lines (3 human donors). Results were consistent within each dilution, therefore a single dilution series from one of the 3 primary AEC lines is presented for simplicity. Panel (A) presents crystal violet stained uninfected Vero E6 cells exposed to harvested differentiated airway epithelial cell (AEC) cultures treated with Triton X100 1% and sodium dodecyl sulfate (SDS) 0.1% (protein isolation method), or 10% formalin and Triton X100 1% (immunohistochemistry fixation method). AEC material treated with SDS 0.1% resulted in lysis of Vero cells at NEAT and 10⁻¹ dilution, and exposure of Vero cells to NEAT material from AECs fixed with 10% formalin and Triton X100 1% also resulted in cell death. Panel (B) presents crystal violet stained Vero cells exposed to protein isolated from SARS-CoV-2-infected AECs, material from SARS-CoV-2-infected AECs fixed for immunohistochemistry (IHC), ultraviolet (UV)-light treated supernatant from SARS-CoV-2-infected AECs, and RNA isolated with Trizol^® from SARS-CoV-2-infected AECs. The 10⁻¹ dilution of exposure to protein isolated from SARS-CoV-2-infected AECs was not scored as positive given that the same dilution of uninfected AECs treated with SDS 0.1% was toxic to Vero cells. Panel (C) presents SARS-CoV-2 viral copy number by quantitative PCR of inactivated material isolated for protein analysis using Radioimmunoprecipitation assay buffer (containing Triton X100 1% and SDS 0.1%) and of UV-inactivated supernatant. Using extracted protein material, RNA was secondarily isolated using Trizol^® before conducting PCR. Similarly, following UV-inactivation, RNA was isolated from the supernatant using Trizol^® before conducting PCR.

Figure 5

Summary of viral titers from 50% tissue culture infectious dose (TCID₅₀) assays Panel (A) and observed cytopathic effect (CPE) Panel (B) in SARS-CoV-2 infected Vero E6 cells, Vero cells exposed to SARS-CoV-2 infected AEC cell layer, SARS-CoV-2 exposed to heat (65 °C) for 10 min, SARS-CoV-2 treated with UV light, RNA harvested with Trizol^® from SARS-CoV-2 infected AECs, and SARS-CoV-2 infected AECs fixed for immunohistochemistry (IHC) from SARS-CoV-2-infected AECs. Given that the protein isolation protocol tested (Triton X and SDS) resulted in direct toxicity with complete CPE at 10⁻¹ dilution without SARS-CoV2 (see Figure 4A), this inactivation method is not included in this figure.