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: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.
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.
Project description:Considering global climate changes, incidences of combined drought and heat stress are likely to increase in the future and will considerably influence plant-pathogen interactions. Until now, little is known about plants exposed to simultaneously occurring abiotic and biotic stresses. To shed some light on molecular plant responses to multiple stress factors, a versatile multi-factorial test system, allowing simultaneous application of heat, drought and virus stress, was developed. Comparative analysis of single, double and triple stress responses by transcriptome and metabolome analysis revealed that gene expression under multi-factorial stress is not predictable from single stress treatments. Hierarchical cluster and principal component analysis identified heat as the major stress factor clearly separating heat-stressed from non-heat stressed plants. We identified 11 genes differentially regulated in all stress combinations as well as 23 genes specifically-regulated under triple stress. Furthermore, we showed that virus treated plants displayed enhanced expression of defense genes, which was abolished in plants additionally subjected to heat and drought stress. Triple stress also reduced expression of genes involved in the R-mediated disease response and increased the cytoplasmic protein response which was not seen under single stress conditions. These observations suggested that abiotic stress factors significantly altered TuMV-specific signaling networks which lead to a deactivation of defense responses and a higher susceptibility of plants. Collectively, our transcriptome and metabolome data provide a powerful resource to study plant responses during multi-factorial stress and allows identifying metabolic processes and functional networks involved in tripartite interactions of plants with their environment. Stress induced gene expression in Arabidopsis leaves was measured after exposure to single and combined abiotic and biotic stress. Plants were grown on soil for 21 days till virus infection. Eight days later controlled drought stress was applied. At the end of the treatments heat was applied for three days. Four biological replicates have been hybridized for each treatment. Furthermore, Arabidopsis plants were exposed to a single severe heat stress (37°C day/33°C night) to mimic the severity of the triple stress experiment.