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Heat inactivation of Nipah virus for downstream single cell RNA sequencing

ABSTRACT: Single-cell RNA sequencing (scRNA-seq) has aided greatly in the study of viruses to distinguish responses from infected versus bystander cells in complex systems. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories. Here we show that TCL buffer (Qiagen), inactivates both Ebola virus (EBOV) and SARS-CoV-2, representative BSL-4 and BSL-3 viruses. We show that additional heat treatment was additionally sufficient to inactivate EBOV-containing samples, and had minimal effects on extracted RNA quality and downstream sequencing results.

ORGANISM(S): Homo sapiens

PROVIDER: GSE247406 | GEO | 2024/01/20

REPOSITORIES: GEO

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Project description:Ebola (EBOV) virus causes severe and often lethal hemorrhagic fever in humans and nonhuman primates (NHP), and has been classified as a Category A bioweapon agent. There are currently no approved preventive vaccines or postexposure treatments for EBOV hemorrhagic fever. The mechanisms of EBOV pathogenesis are only partially understood, but the dysregulation of normal host immune responses (including destruction of lymphocytes, increases in levels of circulating proinflammatory cytokines, and development of coagulation abnormalities) is thought to play a major role. Accumulating evidence suggests that much of the observed pathology is not the direct result of virus-induced structural damage but rather is due to the release of soluble immune mediators from EBOV-infected cells. It is therefore essential to understand how the candidate therapeutic may be interrupting the disease process and/or targeting the infectious agent. Identification of effective treatment strategies may greatly benefit based on identification of molecular features of the host response to infection and treatment. In order to identify these gene signatures related to correlates of protection, we used a DNA microarray-based approach to compare the host genome-wide responses of EBOV-infected NHP responding to candidate therapeutics. With this approach, we have identified genes that appear to correlate with survival, including chemokine ligand 8 (CCL8/MCP-2), and revealed a subset of distinctly differently expressed genes that may provide possible targets for future diagnostics or therapeutics. These analyses will assist us in understanding the pathogenic mechanisms of EBOV infection as well as identify improved therapeutic strategies. Transcriptional analysis of global gene expression changes in Zaire Ebola Virus (ZEBOV)-infected rhesus macaques that were treated with either recombinant nematode anticoagulant protein c2 (rNAPc2) or recombinant human activated protein C (rhAPC). Animals were infected with 1000pfu ZEBOV, then subsequently treated with rNAPc2 or rhAPC. Four animals were left untreated for controls. Blood samples were taken at specified days post-infection and PBMCs were isolated from the samples and inactivated in TRIzol reagent. Total RNA was isolated from the samples, then linearly amplified and hybridized to a whole genome long-oligonucleotide microarray in a two color comparative format with a commercially available human reference RNA from Stratagene as a consistent control in dataset comparisons.

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Heat inactivation of Nipah virus for downstream single cell RNA sequencing

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