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

Area of Science:

• Virology
• Genomics
• Molecular Biology

Background:

• Single-cell RNA sequencing (scRNA-seq) offers insights into virus-host interactions by distinguishing infected from bystander cells.
• Biosafety Level 4 (BSL-4) pathogen research requires robust virus inactivation protocols for sample preparation.
• Existing scRNA-seq workflows often lack adequate virus inactivation for BSL-4 agents.

Purpose of the Study:

• To adapt a microfluidic partitioning scRNA-seq platform for safe handling of BSL-4 pathogens.
• To identify effective methods for inactivating Nipah virus (NiV) within the scRNA-seq workflow.
• To ensure downstream scRNA-seq data integrity after virus inactivation.

Main Methods:

• Testing chemical and physical inactivation methods on a commercial microfluidic scRNA-seq platform.
• Evaluating the efficacy of a 5-minute, 85°C incubation for NiV inactivation.
• Implementing an additional heat inactivation step post-cDNA synthesis for enhanced biosafety.

Main Results:

• A 5-minute incubation at 85°C was the only standard protocol step that inactivated NiV.
• An additional heat step after cDNA synthesis successfully inactivated NiV-containing samples.
• The post-cDNA synthesis heat step did not compromise sample quality or scRNA-seq results.

Conclusions:

• The adapted scRNA-seq workflow ensures BSL-4 pathogen inactivation.
• Post-cDNA synthesis heat treatment is a viable strategy for NiV inactivation in scRNA-seq.
• This method enables safe and reliable scRNA-seq analysis of BSL-4 virus infections.