Project description:To analyse the host responses of Bama mini-pigs to ASFV infection, we chose the spleen and inguinal lymph nodes from Bama mini-pigs infected with ASFV at a dose of 103 HAD50 and the mock group for transcriptomic analysis.
Project description:The malaria parasite Plasmodium replicates and differentiates in red blood cells of its host. The erythropoietic niches (spleen and bone marrow) are important but poorly understood reservoirs of asexual replication and sexual development. We aimed to understand how the parasite adapts to its host organ and a host cell level. For this, we performed single-cell RNA-seq (scRNA-seq) analysis on host and parasite cells derived from spleen, blood and bone marrow. Organs were harvested from two infected and one uninfected mice and parasite cells were enriched to about 50% by flow sorting (infected samples only). To identify host cells by surface expression (CITE-seq), cells were stained with barcoded antibodies targeting CD44 and CD71. Droplet-based scRNA-seq of these samples was performed using 10X genomics technology and cDNA was sequenced on Illumina.
Project description:African swine fever virus (ASFV) is the causative agent of African swine fever, a highly contagious and usually fatal disease in pigs. The pathogenesis of ASFV infection has not been clearly elucidated. Here, we used single-cell RNA-sequencing technology to survey the transcriptomic landscape of ASFV-infected primary porcine alveolar macrophages. The temporal dynamic analysis of viral genes revealed increased expression of viral transmembrane genes. Molecular characteristics in the ASFV-exposed cells exhibited the activation of antiviral signaling pathways with increased expression levels of interferon-stimulated genes and inflammatory- and cytokine-related genes. By comparing infected cells with unexposed cells, we showed that the unfolded protein response (UPR) pathway was activated in low viral load cells, while the expression level of UPR-related genes in high viral load cells was less than that in unexposed cells. Cells infected with various viral loads showed signature transcriptomic changes at the median progression of infection. Within the infected cells, differential expression analysis and coregulated virus–host analysis both demonstrated ASFV promoted metabolic pathways but inhibited interferon and UPR signaling, implying the regulation pathway of viral replication in host cells. Furthermore, our results revealed that the cell apoptosis pathway was activated upon ASFV infection. Mechanistically, the production of tumor necrosis factor alpha (TNF-α) induced by ASFV infection is necessary for cell apoptosis, highlighting the importance of TNF-α in ASFV pathogenesis. Collectively, the data provide insights into the comprehensive host responses and complex virus–host interactions during ASFV infection, which may instruct future research on antiviral strategies.
Project description:Although phagocytic cells are documented targets of Leishmania parasites, it is unclear whether other cell types can be infected. Here, we use unbiased scRNA-seq to simultaneously analyze host cell and Leishmania donovani transcriptomes to identify and annotate parasitized cells in spleen and bone marrow in chronically infected mice. Our dual-scRNA-seq methodology allows the detection of heterogenous parasitized populations. In the spleen, monocytes and macrophages are the dominant parasitized cells, while megakaryocytes, basophils and NK cells are found unexpectedly infected. In the bone marrow, the Hematopoietic Stem Cells (HSCs) expressing phagocytic receptors FcγR and CD93 are the main parasitized cells. Additionally, we also detect parasitized cycling basal cells, eosinophils, and macrophages in chronically infected mice. Flow cytometric analysis confirms the presence of parasitized HSCs. Ourunbiased dual scRNA-seq method identifies rare, parasitized cells, possibly implicated in pathogenesis, persistence, and protective immunity using a non-targeted approach.
Project description:African swine fever virus (ASFV) is one of the most devastating swine pathogens characterized by nearly 100% mortality in naive herds and was recently emerged the in China. In this study, we generated the expression profile of porcine alveolar macrophages (PAMs) infected with a high pathogenic ASFV (Pig/Heilongjiang/2018 (Pig/HLJ/18) ASFV). Our data indicated that ASFV infection lead to a strong inhibition of host immunity but promote chemokine-mediated signaling pathway and neutrophil chemotaxis. Moreover, ASFV infection can modulate the host miRNA involved regulation network, leading to a significant increase of host metabolism related genes and acceleration of virus replication. Furthermore, ASFV-derived viral small RNAs (vsRNAs) can target some host immune response related genes. In conclusion, our transcriptome-wide data provide some insights into the regulatory mechanism during ASFV infection.
Project description:Transcriptional profiling of porcine macrophages infected with Afraican swine fever virus (ASFV) infected cells and mock infection was conducted in this experiment.
Project description:We describe the recall response to African swine fever virus (ASFV) by applying single-cell RNA-sequencing (scRNA-seq) of submandibular lymph node (LN) cells from a control and a vaccinated animal with the live attenuated BA71ΔCD2 vaccine prototype. Cell suspensions obtained from the digested tissues were in vitro stimulated for 16 hours with BA71ΔCD2 ASFV, and methanol-fixed prior sequencing using the 10x Genomics scRNA-seq platform. We identified 22 transcriptionally distinctive clusters which were assigned to different cell subsets based on canonical lineage markers. Interferon-stimulated genes (ISG) were significantly upregulated in several cell subsets from the vaccinated pig when compared to the control one. Several populations were overrepresented in LN cells from the vaccinated pig, suggesting their contribution to the ASFV-specific recall response. These included several B cell subsets such as plasmablasts and plasma cells, an undefined CD4 T cell subset, and γδ T cells. The proinflammatory CXCL10 chemokine was strongly upregulated in plasmablasts, in the undefined CD4 T cell subset, and in crosspresenting DCs, thus showing the induction of a Th1-biased recall response. Of particular interest was the presence of a robust vaccine-specific cytotoxic response characterized by elevated numbers of responding CD8 T cells. Indeed, these subset significantly upregulated GZMA.1, confirming their activated status, while it was similarly expressed in NK cells from both samples. Overall, scRNA-seq allowed deciphering the complex protective immune response against ASFV infection, demonstrating a major role of an early Th1-mediated inflammatory response concomitant with a rapid expansion of cytotoxic CD8 T cells.
Project description:Long noncoding RNAs (lncRNAs) participate in regulating many biological processes. However, their roles in African swine fever virus(ASFV) pathogenicity are largely unknown. Here, we analyzed the expression profile of lncRNAs and mRNAs in the ASFV-infected or uninfected PAMs by high-throughput sequencing
Project description:Large DNA viruses are known to manipulate and modify host miRNAs during infection. Therefore the aim of this study was to investigate the impact of infection with the large DNA virus; African swine fever virus (ASFV) on host miRNAs. Small RNA sequencing libraries were prepared from RNA extracted from ASFV (Benin 97/1) infected primary porcine macrophages at 0, 6 and 16 hours post infection. Libraries were pooled and sequenced on 1 lane of an Illumina HiSeq, yielding sequences aligning to a total of 247 different mature Sus scrofa miRNAs. On average, 3779095 (± 1911525) miRNA reads were obtained per sample. The results revealed no widespread modification to host miRNAs, though a number of specific miRNAs were differentially expressed during ASFV infection. Notably, a small number of miRNAs (Ssc-miR-10b, Ssc-miR-144 and Ssc-miR-486) were rapidly upregulated 2-6 fold within the first hour of infection.
Project description:Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy and understanding disease progression. Redox factor-1 (Ref-1), a redox signaling protein, regulates the DNA binding activity of several transcription factors, including HIF-1. The conversion of HIF-1 from an oxidized to reduced state leads to enhancement of its DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia.Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions. Experimental Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model. Matched samples were also collected for bulk proteomic analysis of the four conditions. scRNA-seq data was validated using proteomics and qRT-PCR. Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays and qRT-PCR. Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions.