West Nile Virus Poster FINAL
Transcription
West Nile Virus Poster FINAL
Understanding West Nile Virus Infection: The QIAGEN Bioinformatics Solution: Biomedical Genomics Workbench + Ingenuity Pathway Analysis (IPA). Jean-Noel Billaud, PhD and Stuart Tugendreich, PhD; QIAGEN Silicon Valley (Redwood City, CA) Top Canonical Pathways and their predicted activation state: No approved therapy for humans • • • • Immune response-related pathways are induced by WNV infection. Note that interferon signaling (right panel) is predicted to be activated, promoting an increase antiviral response and a decrease of replication of Flaviviridae (WNV family). Mosquito-transmitted virus Worldwide public health concern Viral pathogenesis is incompletely understood In 2012: highest number of human WNV cases in the U.S. since 2003 (CDC) West Nile Virus Infection • Infection in healthy humans is asymptomatic, but can be severe in elderly and immunocompromised • Neuroinvasive forms of WNV infection: Characterized both by neuronal death and infiltration of mononuclear effector cells Whose activities promote both viral clearance and neuronal injury Life-threatening neurological disease (meningitis and encephalitis) • Immune Response: Induction of type-1 IFNs and humoral immunity provide front-line defense against WNV pathogenesis, Cellular immunity (γδ T cells, CD4+, and CD8+ αβ T cells) for host recovery from WNV infection. Downstream Effects Analysis: Biological Splicing Variant of interest: The isoforms (yellow processes associated with WNV infection indicate a strong immune response (innate immune response, antibody response, cell movement of dendritic cells). These biological processes are all predicted to be increased and they participate in the antiviral response. halo) are differentially expressed. One of them, ZAPS (short form of ZC3HAV1) is of interest. ZAPS is upregulated in the infected macrophages (red filled circle) and is a stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses. Some Key questions to be asked with transcriptomics using IPA • What are the signaling or metabolic pathways present, are they activated or inhibited? • What are the underlying transcriptional programs? • What biological processes are involved and in what way? • Are there any splicing variants of interest? Splicing Variants are involved in the inhibition of virus: Specific Study GSE40718: Identification of Genes Critical for Resistance to Infection by West Nile Virus using RNA-Seq Analysis. • Infection of primary macrophages from healthy human donors (10 individuals, MOI=1, 24 hrs). • PolyA+ RNA was prepared from uninfected and WNV-infected primary macrophages, fragmented, and subjected to sequencing using the Illumina Genome Analyzer 2. • Processing the RNA-seq data in Biomedical Genomics Workbench isoforms of ZC3HAV1, Causal Network: CLEC7A might be a useful therapeutic target to decrease WNV-associated encephalitis (orange circle). CLEC7A is a master regulator (Upstream Analysis) and has already been described as Host Susceptibility Factor required by WNV to infect cells. Download the FASTQ files from European Nucleotide Archive (or binary files from SRA) Import and process FASTQ files using Biomedical Genomics Workbench (BXWB) o Set up the RNA-seq Analysis in BXWB o Select Reference Genome (human Ensembl) o Select Mapping options o Select Expression Level Options Set Up the Experiment at Gene Level (GE) or Transcripts Level (TE): Infected vs. control SP100, CFLAR, MX1 as well IL27 participate in the antiviral response and lead to inhibition of virus. This Causal Network allows to set a new hypothesis in conjunction with MAP (Molecule Activity Predictor). MAP simulates the inhibition of CLEC7A and the impact on the encephalitis. When CLEC7A is inhibited or downregulated, the encephalitis is decreased (blue circle). This hypothesis is ready to be tested and validated. • Send dataset to IPA using Plugin IPA from BXWB (Fold Change, p-value, FDR) • Analyze the processed dataset in IPA Dataset: 3285 isoforms with >10 RPKM in either mock or infected, |fold change|>1, p<0.05 Analysis: 976 genes with |fold change| >2 and p-value <0.01 Conclusion Using Biomedical Genomics Workbench, we have been able to: Upload RNA-seq data (FASTQ files from SRA). Align to the genome of interest (human Ensembl) Quantitate and obtain differential expression between samples Seamlessly send data directly into IPA for biological interpretation Using IPA, we have been able to: Understand signaling pathways involved in antiviral immune response Discover potential transcriptional program(s) (CLEC7A) Visualize differentially expressed splicing variants (view of ZAPS) Discover biological processes participating in antiviral response Highlight new hypotheses (CLEC7A inhibition).