Transcriptomic analysis in head kidney of rainbow trout, Oncorhynchus mykiss, infected by infectious hematopoietic necrosis virus
- 발행기관 강릉원주대학교 일반대학원
- 발행년도 2019
- 학위수여년월 2020. 8
- 학위명 석사
- 학과 및 전공 도움말 일반대학원 해양생물공학과
- 실제URI http://www.dcollection.net/handler/kangnung/000000010710
- UCI I804:42001-000000010710
- 본문언어 영어
초록/요약 도움말
Infectious hematopoietic necrosis virus (IHNV) is an important pathogen causing clinical diseases and mass mortalities in salmonid fishes and 2 genotypes of high virulent IHNV-Shizuoka and low virulent IHNV-Nagano are identified in Korea. To understand pathogenic process and defense strategies during IHNV infection in rainbow trout, RNA seq-based transcriptome analysis was performed in head kidney at day 1, 3 and 5 post-infection. For identifying difference in their infection, we tested the virulence of two genotypes of IHNV. Consequently, infection with IHNV-Shizuoka strain showed a higher mortality of 85 % than infection with IHNV-Nagano of 40 %. In RNA-seq analysis, total numbers of differentially ex-pressed genes (DEGs) in IHNV-Nagano infected fish were 464, 2360 and 1599 and in IHNV-Shizuoka were 618, 2626 and 774 at day 1, 3 and 5, respectively. In en-richment analysis of gene ontology (GO) annotations, 4 terms including ‘negative regulation of type I interferon production’ and ‘positive regulation of interleukin-1 beta secretion’ were commonly identified in all infected samples and time points, and other GO terms were differentially involved depending genotypes and time. In KEGG pathway analysis, PRR signaling pathways like RIG-I-like receptor signal-ing pathway and Toll-like receptor signaling pathway were commonly identified in IHNV-Nagano at all time points while metabolism-related pathways like Glycolysis / Gluconeogenesis and Glyoxylate and dicarboxylate metabolism identified in IHNV-Shizuoka at day 1. Protein-protein interaction (PPI) network and centrality analysis revealed that immune system and signaling molecules and interaction pathways are up-regulated with the highest centrality of TNF in IHNV-Nagano at day 1 and 3, while metabolism-related pathways are up-regulated with the highest centrality of GAPDH in IHNV-Shizuoka at day 1. In addition, IHNV-Nagano and IHNV-Shizuoka seems to lead to death and interfere with wound recovery caused by apoptosis by down-regulating fibronectin and Matrix metalloproteinase 9 (MMP9), respectively, at day 5. Interestingly, IHNV-Nagano appears to regulate the amount of viral protein by using ubiquitin–proteasome system (UPS) at 1 and 3 day. The reproducibility and repeatability of transcriptome analysis were validated by RT-qPCR. Taken together, IHNV infection dynamically changed gene expression patterns in a major immune organ of rainbow trout during the first week and in-duced defense mechanism of infected fish by regulating immune and inflammatory pathways through PRR signaling at early stage of infection. IHNV-Shizuoka infec-tion activated the energy metabolic pathway and consumed the energy for viral rep-lication and showed a greater host cell shutoff response. On the other hand, IHNV-Nagano infection activated immune response at all time points. However, both IHNV-Shizuoka and IHNV-Nagano infection may have failed to recover the wound by apoptosis caused after IHNV infection and appear to lead to death.
more목차 도움말
Abstract.......................................................................................................................1
1. Introduction.............................................................................................................3
2. Materials and Methods.............................................................................................7
2.1. Ethics statement.....................................................................................................7
2.2. Preparation of fish..................................................................................................7
2.3. Preparation of virus ................................................................................................8
2.4. Virulence test by intraperitoneal (i.p.) challenge ......................................................8
2.5. Experimental challenge with IHNV RtCc0517c and RtYw0717b...............................9
2.6. RNA isolation and RNA sequencing.........................................................................9
2.7. Data analysis...........................................................................................................11
2.7.1. Transcriptome annotation....................................................................................11
2.7.2. Differentially expressed gene analysis...................................................................12
2.7.3. Gene Ontology (GO) analysis of DEGs..................................................................12
2.7.4. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs.....12
2.7.5. Functional and protein-protein interaction (PPI) analysis of DEGs...........................13
2.8. cDNA synthesis and real-time quantitative PCR (RT-qPCR) analy-sis...........................13
3. Results........................................................................................................................17
3.1. Mortality in two genotype IHNV challenges .............................................................17
3.2. Overview of RNA-seq data.......................................................................................19
3.3. Differential expression profiles.................................................................................24
3.4. GO analysis.............................................................................................................30
3.5. KEGG analysis.........................................................................................................39
3.6. Protein-protein interaction network (PPI) analysis of DEGs ......................................47
3.7. Verification of RNA seq data by RT-qPCR.................................................................56
4. Discus-sion.................................................................................................................59
5. 국문요약......................................................................................................................68
6. Refer-ences.................................................................................................................71
7. 감사의 글...........................................................................................................80
