Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality

Abstract

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus–host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

Preservation of scRNA-Seq Libraries Using Existing Inactivation Protocols

“…The additional steps of library prep are relatively short ( Figure 2 ), often taking less than an hour, and there are multiple stopping points in every protocol that can allow for overnight incubations or time between the steps. In addition, in a companion article by Dr. Mühlberger and colleagues also published in Pathogens , the authors demonstrate how two consecutive heat steps after cDNA generation can inactivate the Nipah virus as a viable alternative to allow for an earlier exit from the BSL-4 [ 24 ]. However, this would need to be demonstrated at each lab individually and across all viral families, resulting in a high cost.…”