Project description: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.
Project description:Numerous studies have described the efficacy of heat inactivation, gamma irradiation, or treatment with guanidium-based chaotropic salts (e.g. TRIzol®) for pathogen inactivation of biological samples to ensure biosafety and biosecurity. However, the effect of these methods on the greater serum proteome are less studied. Here we sought to comprehensively measure the effects of three routinely used pathogen inactivation methods on the serum proteome of Rhesus macaques by characterizing the serum proteome pre-and-post inactivation treatment. Using data independent aquisition-based shotgun LCMS/MS, we evaluated total peptide/protein detection and individual protein abundances (e.g. ALB, APOA1, and CRP) across inactivation methods and compared to their untreated controls. Specifically, we observed improved quantitative reproducibility in gamma-irradiated samples across biological, technical, and experimental replicates compared to chemical inactivation and different heat combinations. These findings represent the first direct, experimental comparisons of effective pathogen inactivation methods on the serologic profiles of non-human primates and provide useful criteria for evaluating methods for biological specimen inactivation prior to proteomic analysis.
Project description: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.
Project description:This study first evaluated the inactivation efficacy of PCDP using spring viremia of carp virus (SVCV) as models for viruses in water. As for the mechanism of virus inactivation, we used molecular tools and high-resolution mass spectrometry to determine reactions of viral proteins and genome after inactivation by PCDP.
Project description:In this project, we investigated the effets of heat inactivation for different time points to evaluate the biosafety and proteome changes.
Project description:We observed that heat shock of Caenorhabditis elegans leads to the formation of nuclear double-stranded RNA (dsRNA) foci, detectable with a dsRNA-specific monoclonal antibody. These foci significantly overlap with nuclear HSF-1 granules. To investigate the molecular mechanism(s) underlying dsRNA foci formation, we used RNA-seq to globally characterize total RNA and immunoprecipitated dsRNA from control and heat-shocked worms. We find antisense transcripts are generally increased after heat shock, and a subset of both sense and antisense transcripts enriched in the dsRNA pool by heat shock overlap with dsRNA transcripts enriched by deletion of tdp-1, which encodes the C. elegans ortholog of TDP-43. Interestingly, transcripts involved in translation are over-represented in the dsRNAs induced by either heat shock or deletion of tdp-1. Also enriched in the dsRNA transcripts are sequences downstream of annotated genes (DoGs), which we globally quantified with a new algorithm. To validate these observations, we used fluorescence in situ hypridization (FISH) to confirm both antisense and downstream of gene transcription for eif-3.B, one of the affected loci we identified.
Project description:Nipah virus (NiV) is a highly pathogenic, negative strand RNA paramyxovirus that has recently emerged from flying foxes to cause serious human disease. To study the poorly-understood role of nonstructural NiV proteins in NiV pathogenesis, we generated recombinant viruse lacking the expression of accesory NiV C protein (NiV∆C).
Project description:Nipah virus (NiV) is a recently emerged zoonotic Paramyxovirus that causes regular outbreaks in East Asia with mortality rate exceeding 75%. Major cellular targets of NiV infection are endothelial cells and neurons. To better understand virus-host interaction, we analysed the transcriptome profile of NiV infection in primary human umbilical vein endothelial cells. We found that NiV infection strongly induces genes involved in interferon response in endothelial cells. Among the top ten upregulated genes, we identified the chemokine CXCL10 (interferon-induced protein 10, IP-10), an important chemoattractant involved in the generation of inflammatory immune response and neurotoxicity. We performed microarray gene expression profiling of NiV infected HUVEC cell (2 replicates) and of uninfected HUVEC cell (2 replicates).
Project description:This explorative study investigated the transcriptional response of C. elegans wild types N2 and CB4856 after Orsay virus infection and Heat shock (n=1). Each strain had a sample that was heat-shocked and infected, one that was heat-shocked and mock-infected, one that was only infected and one that only underwent a mock infection. Age synchronized worms were treated separately according to different treatments mentioned before. After flash freezing, RNA was isolated, labeled and hybridized on oligo microarray (Agilent) slides.