scholar 引用：6
頁數：15
發表時間：11 August 2017
發表刊物：frontiers in Immunology
作者：Daniel Enosi Tuipulotu, Natalie E. Netzler, Jennifer H. Lun, Jason M. Mackenzie and Peter A. White
摘要：
Viruses inherently exploit normal cellular functions to promote replication and survival. One mechanism involves transcriptional control of the host, and knowledge of the genes modified and their molecular function can aid in understanding viral-host interactions. Norovirus pathogenesis, despite the recent advances in cell cultivation, remains largely uncharacterized. Several studies have utilized the related murine norovirus (MNV,諾如病毒) to identify innate response, antigen presentation, and cellular recognition components that are activated during infection. In this study, we have used next-generation sequencing to probe the transcriptomic changes of MNV-infected mouse macrophages(巨噬細胞). Our in-depth analysis has revealed that MNV is a potent stimulator of the innate response including genes involved in interferon(干擾素) and cytokine(細胞因子) production pathways. We observed that genes involved in viral recognition, namely IFIH1, DDX58, and DHX58 were significantly upregulated with infection, whereas we observed significant downregulation of cytokine receptors (Il17rc, Il1rl1, Cxcr3, and Cxcr5) and TLR7. Furthermore, we identified that pathways involved in protein degradation (including genes Psmb3, Psmb4, Psmb5, Psmb9, and Psme2), antigen(抗原) presentation, and lymphocyte(淋巴細胞) activation are downregulated by MNV infection. Thus, our findings illustrate that MNV induces perturbations in the innate immune transcriptome, particularly in MHC maturation and viral recognition that can contribute to disease pathogenesis.
Keywords: murine norovirus, norovirus infection, transcriptome, innate immunity, host response, antigen presentation, viral recognition
結論：
- 應該是可以參考這一篇paper來進行RNAseq數據的分析，什麼基因上調，pathway等等。
- We present a summary of the host gene expression changes induced by MNV infection in mouse macrophage cells.
- We show that a robust innate immune response is induced by MNV, which coincides with the disease manifestation known to accompany infection.
- Moreover, we have discovered that several elements of the host biology important to innate stimulation and immune recognition are directly affected by MNV infection.
- Overall, this study provides a novel source of global expression changes following MNV infection in an in vitro setting.
- However, given the richness of NGS, our findings represent only a small proportion of the possible analyses with significant potential for further bioinformatic mining. 分析可能只展示了NGS的一小部分信息
- Our findings will likely benefit subsequent research into host–pathogen interactions, not just for MNV, but NoV in general.
Introduction：
- 介紹了諾如病毒的情況，發病機制，易感染人羣等。
- Innate immunity is an essential part of the host response to limit viral replication and prevent disease manifestation.
- Viral activation of PRRs causes a powerful stimulation of several signaling pathways (29) involved in the type I interferon (IFN) response, including the mitogen activating protein kinase (MAPK) (30), nuclear factor kappa B(NFκB) (31), interferon regulatory factor (IRF) (32), and Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways. 這些pathways後續分析需要關注一下
- we analyzed transcriptomic profiles of RAW264.7 cells treated with the TLR7 agonist loxoribine and compared these with MNV-infected cells to reveal the cellular responses induced solely by viral infection. 這個病毒也跟TLR7有關
正文組織架構：
1. Introduction
2. Materials and methods
2.1 Cell Maintenance, Stimulation, and Virus Infections
2.2 RNA Extraction and Quality Control
2.3 Library Preparation and Sequencing
2.4 Sequence Analysis
2.5 Gene Enrichment Analysis
2.6 Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)
2.7 Protein Extraction and Immunoblotting
2.8 Statistical Analyses
3. Results
3.1 Productive MNV Infection in RAW264.7 Cells
3.2 MNV Infection Induces Pronounced Innate Host Expression Changes with Time
3.3 qPCR Validates MNV-Induced Innate Gene Expression Changes
3.4 MNV Infection and Loxoribine Treatment Induce Distinct Expression Profiles in RAW264.7 Cells
3.5 A Robust Innate Response Is Mounted following MNV Infection for 12 h
3.6 MNV-Specific Effects on the Host Response
3.7 MNV Affects Genes Involved in Immune Recognition
4. Discussion
5. Conclusion
正文部分內容摘錄：
- 這個sequence analysis流程完全可以借鑑。
- Bioinformatic analysis of RNA sequencing was performed using the Cufflinks tool suite (47) on the Galaxy server at the University of Queensland, Australia 用的是Galaxy分析
- Following quality control (removal of adapter sequences and trimming), reads were mapped to the 10 mm genome (UCSC) using TopHat (v0.9) with default parameters (51).
- Mapped reads were then assembled into transcripts, normalized and quantified using Cufflinks (v2.2.1.0)
- Assembled transcripts of all replicates, within all conditions, were merged into a single cataloged transcriptome.
- Thereafter, transcript abundance was compared between mock and treated or infected samples using Cuffdiff (v2.2.1.2) to identify differentially expressed genes (DEGs)
- Genes with a fourfold or greater expression change with a FPKM value greater than 1 in at least one sample were considered differentially expressed (DE) for the longitudinal infection analysis.
- Genes with a q-value of <0.05, twofold or greater expression change, and a FPKM value of 1 in at least one sample were considered DE for the analysis of cells infected with MNV or treated with loxoribine for 12 h.
- To confirm the presence or absence of viral replication, sequencing reads were mapped using Bowtie2 (53) to MNV-1 (GenBank accession number DQ285629), Abelson Murine Leukemia virus (Mu-LV) (GenBank accession number: NC_001499), and Moloney Mu-LV (GenBank accession number: NC_001501). The total number of reads at each position across the genome was used to quantify genomic coverage.
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Gene Enrichment Analysis
Enrichment analysis was performed to identify the functional role that the DEGs, identified from MNV infection and loxoribine treatment, play within the host. Gene lists were analyzed on the online servers DAVID (54) and GOrilla (55) for gene ontology and/or KEGG pathway analysis, and the most significant outputs were collated. -
Statistical Analyses
All bar and linear regression graphs were generated in PRISM v.6.0h and error bars are plotted with the SD of either triplicate or quadruplicate experiments of a condition. Correlation analysis was performed using the Pearson method on linear regression analysis. -
對應這些分析的圖，到時候應該需要再一邊運行，一邊仔細看吧。
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然後得到了結果圖以後，也可以參考discussion部分的內容進行分析。