Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers

Abstract

Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, including in organoid or animal studies. While protocols for these analyses are typically developed with biosafety level 2 (BSL-2) considerations in mind, such analyses are equally useful for the study of viruses that require higher biosafety containment levels. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories ensuring virus inactivation and must therefore be modified. Here we show that TCL buffer (Qiagen), which was developed for bulk sequencing of small numbers of cells and also facilitates scRNA-seq, inactivates both Ebola virus (EBOV) and SARS-CoV-2, BSL-4 and BSL-3 viruses, respectively. We show that additional heat treatment, necessary for the more stringent biosafety concerns for BSL-4-derived samples, was additionally sufficient to inactivate EBOV-containing samples. Critically, this heat treatment had minimal effects on extracted RNA quality and downstream sequencing results.

Art of the Kill: Designing and Testing Viral Inactivation Procedures for Highly Pathogenic Negative Sense RNA Viruses

“…Each inactivation study comes with its own challenges. Chemical inactivation often requires the removal of cytotoxic reagents that interfere with testing the viability of inactivated viruses in cell culture [ 8 , 9 , 10 , 11 , 12 , 13 ]. Physical inactivation, such as gamma irradiation and heat, relies on highly specific parameters, including the sample volume, number of cells or viral particles, protein content, temperature, and inactivation time [ 5 , 14 , 15 , 16 ].…”

“…To determine the cell number of the T175 flasks at the time of inactivation, an extra flask with cells was incubated for the same time and used to count the cells. The PBS/DMEM and TRIzol samples were vortexed, incubated for 10 min at room temperature, and purified using size exclusion columns (Amicon Ultra-0.5 Centrifugal Filter Unit 10 kDa; Millipore-Sigma, Burlington, MA, USA), as previously described [ 8 ]. The samples were eluted in 0.5 mL of PBS.…”

“…The ratio of TRIzol LS to the sample was 3:1 ( v / v ), as recommended by the manufacturer. The samples were vortexed, incubated for 10 min at room temperature, and purified using size exclusion columns (Amicon Ultra-0.5 Centrifugal Filter Unit 10 kDa) as previously described [ 8 ]. The samples were eluted in 0.5 mL of PBS.…”

“…To examine formalin inactivation of EBOV particles, 100 µL of concentrated EBOV-ZsGreen stock (see Table S1 for the conditions) in PBS or 100 µL of PBS were mixed with 100 µL of 20% formalin (Richard-Allan Scientific) for a final formalin concentration of 10% ( v/v ). The samples were incubated for 1 or 6 h at 4 °C and purified using Amicon size exclusion columns as previously described [ 8 ]. The samples were eluted in 0.5 mL of PBS.…”

Single-cell RNA sequencing reveals the immune features and viral tropism in the central nervous system of mice infected with Japanese encephalitis virus

“…This technique has been successfully utilized to gain insights into the diversity of various tissue cells, including those in the brain [ 32 – 34 ]. Furthermore, scRNA-seq has been applied to study a range of viral infections, such as SARS-CoV-2, RABV, influenza virus, human immunodeficiency virus (HIV), hepatitis B virus (HBV), Ebola virus, and Zika virus (ZIKV) [ 35 – 41 ]. These studies have provided unbiased and comprehensive visualizations of the specific cells targeted by these viruses, as well as a holistic understanding of the host's immune responses.…”