- 學位論文
錦鯉疱疹病毒IL-10對細胞免疫調節激素的影響
Effect of Recombinant Cyhv3IL-10 on Cell-Mediated Immuno-Cytokine Regulation in CyHV-3 Infection.
摘要
錦鯉疱疹病毒 （cyprinid herpesvirus 3, CyHV-3, KHV） 為Cyprinivirus屬，Alloherpesviridae科，疱疹病毒目（ Herpesvirales）的成員。其為一帶有封套的雙股 DNA病毒，病毒大小約為 167-200 nm，核酸序列長度為 295 kbp (Michel et al., 2010a; Rakus et al., 2013)。 CyHV-3對鯉魚(Cyprinus carpio )、錦鯉(Cyprinus carpio haematopterus )以及鯉魚之雜交品種具有高傳染性、高致死性(Ouyang et al., 2013)，對鯉魚養殖業造成重大的危害(Ouyang et al., 2013)。 如同其他疱疹病毒，CyHV-3也會有潛伏感染，而潛伏感染通常與免疫逃脫有關，Aoki等人曾在2007年對CyHV-3進行全基因分析，發現了五個與免疫逃脫有關的蛋白，分別為G-protein coupled receptor (encoded by ORF16)，TNF receptor homologues (ORF4 and ORF12)，E3L like protein(encoded by ORF112) 及 cyhv3IL-10 (ORF134) (Aoki et al., 2007)。Ouyang等人於2013年透過蛋白質體分析，發現感染CyHV-3魚隻體內的病毒蛋白以ORF12與ORF134蛋白表現量最大(Ouyang et al., 2013)，加上已有多篇論文證實IL-10與病毒潛伏有關(Ouyang et al., 2014)。因此本研究的目的為評估cyhv3IL-10在魚體感染疱疹病毒後，是否參與發炎反應的調控以及與病毒潛伏的關係。 本實驗以重組蛋白cyhv3IL-10（ rvIL-10） 分別進行體內(In vivo)以及體外（ In vitro） 的實驗。在in vitro實驗中，以錦鯉巨噬細胞所產生的IL-12基因以及IL-10下游基因socs-3來評估rvIL-10蛋白的活性。在in vivo實驗中以naïve 及carrier魚感染錦鯉疱疹病毒72hr ，給予rvIL-10蛋白，每天觀察魚隻及其臨床症狀，並利用即時定量聚合酶鏈鎖反應(Real-Time PCR)檢測鰓、腎臟、脾臟的病毒量以及脾臟的免疫相關細胞激素的基因表現。 研究發現，給予具有活性的rvIL-10蛋白後，魚隻的臨床症狀較不明顯，病毒的複製也受到限制。在免疫的調節上，Th1免疫反應以及發炎相關免疫調控皆有延後的現象，推測是因 rvIL-10重組蛋白具有免疫抑制功能，導致錦鯉免疫反應延後，病毒複製受到的抑制與免疫功能的抑制可能使得病毒進入潛伏感染，而讓施打rvIL-10的錦鯉症狀減緩。魚隻感染CyHV-3後給予rvIL-10可有效緩解錦鯉疱疹病毒所造成的症狀，這是本研究新的發現，然而，rvIL-10造成此現象的詳細機制仍需更進一步研究。
並列摘要
The cyprinid herpesvirus 3 (CyHV-3, KHV) is taxonomically grouped within the genus of cyprius, the family Alloherpesviridae and the order of herpesvirales. The genome size of CyHV-3 is 295 kb and mature viral particles are 167 to 200 nm in diameter and are surrounded by double strand DNA envelope. CyHV-3 is highly contagious and highly lethal to Cyprinus carpio (Cyprinus carpio), koi (Cyprinus carpio haematopterus) and the common carp hybrids that cause serious impact to aquaculture. CyHV-3, as the same as other herpesvirus, may cause inapparent infection which is associated with immune evasion. There are five proteins related to immune evasion discovery through genome analysis of CyHV-3 in 2007 by Aoki., et al. These proteins are G-protein coupled receptor (encoded by ORF16), TNFR homologues (encoded by ORF4 and ORF12), E3L like protein (encoded by ORF112) and an interleukine-10 (IL-10) homologue (encoded by ORF134). Furthermore, IL-10 is one of the most important anti-inflammatory cytokines with a key role in the termination of inflammation and restoration of homeostasis, suggesting that CyHV-3 IL-10 might have functions on host immunosuppression, resulting that virus can’t be completely removed by host. The important immunomodulatory functions that ensure viral persistence in the host, then became carriers, cause persistent infection. Persistent infection is a major risk for CyHV-3 prevention, thus causing the virus to spread quickly, resulting in a serious lethality. Therefore, this is mainly to study cyhv3IL-10 protein. In this study, cyhv3IL-10 recombinant protein (rvIL-10) is applied in the in vivo experiment to verify the function of rvIL-10 in koi inflammatory response and anti-viral pathways immune mechanism regulation. The in vitro study, IL-12 gene and IL-10 accessory gene socs-3 produced by macrophage are measured to evaluate the bioactivity of cyhv3IL-10 proteins. The results showed that after the administration of rvIL-10 protein, the death number of koi was less than the control group, and the virus replication is also limited. In addition, the regulations of cell-mediated immune response present a delayed phase. In conclusion, this study provided a new insight that rvIL-10 may have the potential to be used to reduce the symptoms produced after CyHV-3 infection. However, the mechanism of rvIL-10 regulations still needs further studies.