本文選題：鴨坦布蘇病毒　切入點(diǎn)：天然免疫反應(yīng)　出處：《山東農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：鴨坦布蘇病毒(duck Tembusu virus,DTMUV)是一種有囊膜的,單股正鏈RNA病毒,屬于黃病毒科黃病毒屬。DTMUV于2010年首先在福建、浙江等地的發(fā)病鴨場(chǎng)中分離到,主要引起產(chǎn)蛋鴨群產(chǎn)蛋下降、商品肉鴨生長(zhǎng)遲緩和神經(jīng)癥狀。該病傳播迅速,給我國(guó)養(yǎng)鴨業(yè)造成了巨大的經(jīng)濟(jì)損失。宿主天然免疫反應(yīng)在病原微生物感染早期發(fā)揮重要作用,模式識(shí)別受體(pattern recognition receptors,PRRs)作為宿主天然免疫系統(tǒng)的關(guān)鍵組成元件,主要識(shí)別病原微生物的保守成分。RIG-I樣受體(RIG-I like receptor,RLR)是主要分布于非免疫細(xì)胞中的一類(lèi)胞漿PRRs,包含維甲酸誘導(dǎo)基因I(retinoic acid induced gene-I,RIG-I)、黑色素瘤分化相關(guān)基因(melanoma differentiation associated gene 5,MDA5)以及遺傳和生理學(xué)實(shí)驗(yàn)基因2(laboratory of genetics and physiology-2,LGP2),主要識(shí)別RNA病毒,通過(guò)接頭分子線粒體抗病毒信號(hào)蛋白(mitochondrial antiviral signaling protein,MAVS),啟動(dòng)下游信號(hào)級(jí)聯(lián)反應(yīng),合成一系列的細(xì)胞因子,如I型干擾素和炎性細(xì)胞因子等,使宿主建立抗病毒天然免疫狀態(tài)。目前,對(duì)DTMUV的致病性還不清楚,DTMUV感染后誘導(dǎo)的宿主天然免疫反應(yīng)以及RLR在病毒感染過(guò)程中的作用更是缺乏了解。本研究以櫻桃谷雛鴨為實(shí)驗(yàn)動(dòng)物,通過(guò)病理組織學(xué)分析、熒光定量PCR等方法,首先觀察DTMUV對(duì)不同日齡櫻桃谷鴨的致病性差異,以及感染后誘導(dǎo)的天然免疫反應(yīng),然后使用過(guò)表達(dá)、RNA干擾和雙熒光素酶報(bào)告基因檢測(cè)等方法對(duì)RIG-I、MDA5和MAVS在病毒感染過(guò)程中的作用進(jìn)行分析,初步探討了DTMUV對(duì)RLR信號(hào)通路的抑制作用。研究?jī)?nèi)容主要分為以下四個(gè)方面:1.DTMUV對(duì)不同日齡櫻桃谷鴨的致病性研究1、3和7周齡的櫻桃谷鴨通過(guò)腿部肌肉注射途徑感染DTMUV,然后觀察感染鴨的臨床癥狀、病理組織學(xué)變化、組織含毒量以及血清中和抗體等變化規(guī)律。結(jié)果發(fā)現(xiàn),1周齡雛鴨在感染后第2天出現(xiàn)明顯的以神經(jīng)紊亂為主的臨床癥狀,在感染后第5-7天,部分感染鴨死亡。3周齡感染鴨表現(xiàn)類(lèi)似但程度較輕的臨床癥狀,且未見(jiàn)死亡。而7周齡感染鴨僅出現(xiàn)一過(guò)性的食欲減退。實(shí)驗(yàn)鴨主要組織的病變程度隨著感染日齡的增大而逐漸減輕。1周齡感染鴨出現(xiàn)明顯的心內(nèi)膜出血、脾腫大,回腸淋巴組織集中處黏膜腫脹,肝、腎腫脹且有出血,腦膜充血。3周齡感染鴨的病變程度較輕微,7周齡鴨則未見(jiàn)明顯的眼觀病變。但是,病理組織學(xué)分析顯示,三組感染鴨均呈現(xiàn)典型的病毒性腦炎病變。組織中病毒含量檢測(cè)發(fā)現(xiàn),感染后第1天,DTMUV即能在1周齡感染鴨的大腦中檢測(cè)到,感染后第3-5天,1周齡鴨的主要組織含毒量均顯著高于另外兩組感染鴨對(duì)應(yīng)組織的含毒量(P0.05)。感染鴨血清中IFN-γ、IL-2及中和抗體的含量呈相似性變化,但1周齡鴨與另外兩組鴨存在顯著性差異(P0.05)。更重要的是,盡管各組實(shí)驗(yàn)鴨的血清中和抗體在DTMUV感染后第7-19天無(wú)顯著差異,但是1周齡鴨的病毒清除速度卻顯著慢于3、7周齡鴨,表明與鴨日齡相關(guān)的自身免疫系統(tǒng)即天然免疫在感染早期清除病毒的過(guò)程中發(fā)揮關(guān)鍵作用�？偟膩�(lái)說(shuō),本部分研究證實(shí)DTMUV對(duì)不同日齡鴨的致病性不同,日齡越小致病性越強(qiáng),與日齡相關(guān)的免疫反應(yīng)能力對(duì)DTMUV的致病性具有重要影響。2.DTMUV感染雛鴨和鴨胚成纖維細(xì)胞(DEFs)后誘導(dǎo)的天然免疫反應(yīng)為確定DTMUV感染后誘導(dǎo)的天然免疫反應(yīng),我們通過(guò)相對(duì)熒光定量PCR檢測(cè)了病毒感染1周齡雛鴨脾和腦組織中天然免疫相關(guān)基因的變化。結(jié)果顯示,在DTMUV感染的早期階段,病毒能顯著上調(diào)RIG-I、MDA5和TLR3的表達(dá)(P0.05),說(shuō)明這些PRRs參與DTMUV感染后誘導(dǎo)的天然免疫反應(yīng)。同時(shí),炎性細(xì)胞因子(IL-1β、L-2、IL-6和IL-8)和抗病毒蛋白(Mx、OAS等)也大量表達(dá),其中IL-6表達(dá)量的增加最為顯著。在感染鴨的脾和腦中,I型和II型IFNs的變化不同。在脾中,兩種類(lèi)型IFNs均呈現(xiàn)不同程度的上調(diào),但是在腦中,二者變化趨勢(shì)差異較大。除此之外,我們還檢測(cè)了DTMUV體外感染DEFs后RIG-I、MDA5、I型IFNs和部分促炎細(xì)胞因子的表達(dá)變化,結(jié)果與體內(nèi)實(shí)驗(yàn)類(lèi)似,RIG-I和MDA5被病毒激活而過(guò)量表達(dá),導(dǎo)致IFN-α和IFN-β、抗病毒蛋白OAS以及IL-6、IL-8等的大量表達(dá)。上述研究結(jié)果說(shuō)明,DTMUV感染雛鴨和DEFs后其抗病毒天然免疫反應(yīng)被迅速激活,但仍不足以抵抗病毒短時(shí)間內(nèi)快速、大量的增殖,同時(shí),促炎細(xì)胞因子如IL-6等的大量產(chǎn)生亦能對(duì)宿主自身造成一定的免疫損傷,從而加重病情。3.RIG-I和MDA5參與宿主的抗DTMUV感染為了進(jìn)一步明確RIG-I和MDA5在抗DTMUV感染過(guò)程中的作用,使用過(guò)表達(dá)和雙熒光素酶報(bào)告基因檢測(cè)等技術(shù)分析了病毒感染DEFs后二者介導(dǎo)的IFN-β的產(chǎn)生情況。首先通過(guò)前期的實(shí)驗(yàn),我們發(fā)現(xiàn)DTMUV能誘導(dǎo)RIG-I、MDA5、MAVS和IFN-β在m RNA水平上顯著上調(diào)。進(jìn)一步地,先在DEFs上分別過(guò)表達(dá)RIG-I和MDA5的全長(zhǎng)及其效應(yīng)結(jié)構(gòu)域,再感染DTMUV,發(fā)現(xiàn)兩個(gè)受體及其效應(yīng)結(jié)構(gòu)域均能抑制DTMUV增殖。繼而,我們克隆并表達(dá)了鴨MAVS基因(du MAVS),通過(guò)過(guò)表達(dá)和RNA干擾證實(shí),du MAVS在DTMUV感染過(guò)程中是必須的。此外,DTMUV感染后能進(jìn)一步刺激RIG-I和MDA5誘導(dǎo)的轉(zhuǎn)錄因子NF-κB和IRF-7的產(chǎn)量,說(shuō)明過(guò)表達(dá)的RIG-I和MDA5識(shí)別DTMUV核酸后能通過(guò)NF-κB和IRF-7信號(hào)通路激活下游IFN-β。本部分研究初步確定RIG-I/MDA5-MAVS-IRF-7/NF-κB信號(hào)通路介導(dǎo)IFN-β參與抗DTMUV的天然免疫反應(yīng)。4.DTMUV的非結(jié)構(gòu)蛋白NS1抑制RIG-I和MDA5介導(dǎo)的信號(hào)通路前期發(fā)現(xiàn)預(yù)先感染了DTMUV的細(xì)胞,再次經(jīng)RIG-I或MDA5刺激后,其誘導(dǎo)的IFN-β表達(dá)量顯著低于對(duì)照組細(xì)胞,提示DTMUV對(duì)RIG-I和MDA5介導(dǎo)的IFN-β的產(chǎn)生存在一定的抑制作用。為了進(jìn)一步明確DTMUV通過(guò)何種方式拮抗宿主IFN-β的產(chǎn)生,我們分別克隆并構(gòu)建了病毒7個(gè)非結(jié)構(gòu)蛋白NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5的真核表達(dá)質(zhì)粒,實(shí)驗(yàn)發(fā)現(xiàn),過(guò)表達(dá)有NS1蛋白的細(xì)胞中IFN-β含量較對(duì)照組顯著下降,表明DTMUV的NS1蛋白在拮抗RIG-I和MDA5的信號(hào)通路中起主要作用。進(jìn)一步地,將NS1和du MAVS的真核表達(dá)質(zhì)粒共轉(zhuǎn)染DEFs,轉(zhuǎn)染后24 h,通過(guò)激光共聚焦觀察發(fā)現(xiàn),DTMUV的NS1和du MAVS在細(xì)胞中存在共定位現(xiàn)象,初步確定DTMUV的NS1蛋白可能是通過(guò)與du MAVS的相互作用抑制了RLR信號(hào)通路。但是,此部分結(jié)果還需要通過(guò)免疫共沉淀等試驗(yàn)進(jìn)行進(jìn)一步的驗(yàn)證。
[Abstract]:Duck Tembusu virus (duck Tembusu, virus, DTMUV) is an enveloped, single stranded RNA virus belonging to the Flaviviridae flavivirus.DTMUV in 2010 first in Fujian, isolated from diseased duck field in Zhejiang and other places, mainly caused by the laying ducks egg drop, duck growth retardation and neurological symptoms. The disease spread rapidly, causing huge economic losses to our country. One of the most natural host immune responses in the early infection of pathogenic microorganisms play an important role in pattern recognition receptors (pattern recognition, receptors, PRRs) is the key of the host innate immune system components, conservative constituents of.RIG-I like receptor recognition the main pathogenic microorganisms (RIG-I like receptor, RLR) is a kind of PRRs was mainly distributed in the cytoplasm of non immune cells, including retinoic acid inducible gene I (retinoic acid induced gene-I, RIG-I), melanoma differentiation associated gene (M Elanoma differentiation associated gene 5, MDA5) and the genetic and physiological experiment gene 2 (Laboratory of genetics and physiology-2, LGP2), mainly through the joint identification of RNA virus, molecular mitochondrial antiviral signaling protein (mitochondrial antiviral signaling protein, MAVS), and activate the downstream signaling cascade of cytokine synthesis a series, such as type I interferon and inflammatory cytokines, the host innate immune antiviral state established. At present, the pathogenicity of DTMUV is unclear, DTMUV induced after infection of host innate immune reaction and RLR virus infection in the process is the lack of understanding. In this study, Yingtao Gu ducklings were used as the experimental animal, by histopathological analysis, fluorescence quantitative PCR methods, the first observation of pathogenicity of DTMUV to different ducks, as well as natural immunity after infection induced reaction, however After over expression, RNA interference and dual luciferase reporter gene assay method to analyze the role of MDA5 RIG-I, and MAVS in virus infection, investigate the inhibitory effects of DTMUV on RLR signal pathway. The main research contents are divided into the following four aspects: 1.DTMUV pathogenicity to ducks of different 1,3 and 7 week old Cherry Valley Duck through the legs intramuscular injection of DTMUV infection, clinical symptoms, and then observe the pathological changes of tissues of infected duck, virus content and serum neutralizing antibody were obtained. The results showed that 1 week old ducklings in infection occurred second days after clinical symptoms of neurological disorders mainly in the obvious. 5-7 days after infection, some infected duck deaths.3 weeks old infected duck were similar to clinical symptoms but to a lesser extent, and no death. At the age of 7 weeks and only infected ducks had transient experimental anorexia. The severity of the main organization with the increase of duck infection age decreased.1 week old infected duck appeared endocardial hemorrhage, splenomegaly, ileal lymphoid tissue concentration at the mucosal swelling, liver, kidney swelling and bleeding, the severity of congestion in.3 week old ducks infected meninges mild, 7 week old duck is not clear eye view lesions. However, histopathological analysis showed that three groups of infected ducks showed typical viral disease. Detection of virus content in tissues, first days after infection, DTMUV can be detected in the 1 week old duck infection in the brain, the 3-5 day after infection, the main organization of 1 week old ducks the virus content was significantly higher than that of the other two groups the amount of drug containing infected duck tissues (P0.05). The infection of duck serum levels of IFN-, IL-2 content and neutralization antibody showed a similar change, but there is a significant difference between the 1 week old duck and duck in the other two groups (P0.05). The more important Is that although serum neutralizing antibody in experimental duck DTMUV infection after 7-19 days of no significant difference, but the virus clearance rate of 1 week old ducks were significantly slower than the 3,7 week old duck, duck and indicate the age's own immune system that innate immunity plays a key role in the process of clearing the virus in the early stage of infection in general, this part of the study confirmed that the DTMUV of different pathogenicity of different day old duck, the smaller age of pathogenic stronger pathogenic immune response associated with the age of DTMUV has an important influence on.2.DTMUV Infected Ducklings and duck embryo fibroblast (DEFs) innate immune response to natural immune response after in order to determine the DTMUV induced after infection induced, we examined the change of virus infection natural immunity 1 week old ducklings of spleen and brain related genes by relative quantitative PCR. The results showed that in the early stage of DTMUV infection, virus Could increase the expression of MDA5 and RIG-I, TLR3 (P0.05), to explain the natural immune response induced by the PRRs in DTMUV after infection. At the same time, inflammatory cytokines (L-2, IL-6 and IL-1 beta, IL-8) and antiviral proteins (Mx, OAS) is widely expressed, the expression of IL-6 increased the most significant. In infected duck spleen and brain, the change of I type and II type of IFNs. In the spleen, two types of IFNs showed different degrees of increase, but in the brain, the change trend of the two big difference. In addition, we also examined DTMUV in vitro after infection with DEFs RIG-I, MDA5 I, IFNs and proinflammatory cytokines part expression results similar to the in vivo experiment, RIG-I and MDA5 are activated by viruses and excessive expression, leading to IFN- alpha and IFN- beta, antiviral protein OAS and IL-6, a large number of IL-8. The results showed that, DTMUV Infected Ducklings and DEFs after its antiviral innate immunity Immune response is activated rapidly, but still not enough to resist the virus quickly in a short time, a large number of proliferation, at the same time, a large number of proinflammatory cytokines such as IL-6 can also cause certain immune injury to host itself, thus aggravating the condition of.3.RIG-I and MDA5 in the host anti DTMUV infection in order to further clarify the role of RIG-I and MDA5 in the anti DTMUV infection, using overexpression and dual luciferase reporter gene assay technique to analyze the virus infection DEFs two mediated IFN- beta. Firstly, through the experiment, we found that DTMUV can induce RIG-I, MDA5, MAVS and IFN- increased significantly in M beta RNA level further. First, in DEFs respectively and the effect of overexpression of full-length domains RIG-I and MDA5, and then infected with DTMUV, found two receptor and effector domain could inhibit the proliferation of DTMUV. Then, we cloned and expressed the duck MAV S gene (DU MAVS), the expression of RNA and Du confirmed that the interference of MAVS is necessary in the process of DTMUV infection. In addition, DTMUV infection can stimulate RIG-I and MDA5 induced NF- transcription factor kappa B and IRF-7 yield, that over expression of RIG-I and MDA5 to identify DTMUV nucleic acid can activate the downstream IFN- Beta Kappa B via NF- and IRF-7 signaling pathways. This part of the study identified RIG-I/MDA5-MAVS-IRF-7/NF- signaling pathway B signaling pathway mediated by IFN- is involved in the innate immune response against.4.DTMUV DTMUV non structural protein NS1 inhibits RIG-I and MDA5 mediated early discovery of pre infected DTMUV cells, again after RIG-I or MDA5 stimulation. The induced expression of IFN- was significantly lower than the control group cells, suggesting that DTMUV of RIG-I and MDA5 mediated IFN- beta had certain inhibitory effect. In order to further clarify the means by which DTMUV host IFN- beta antagonists We have cloned and constructed 7 virus non structural protein NS1, NS2A, NS2B, NS3, NS4A, eukaryotic expression plasmid, NS4B and NS5 found that over expression of NS1 protein in cells of IFN- beta content significantly decreased compared to the control group, DTMUV showed that NS1 protein play a major role in the signaling pathway antagonism of RIG-I and MDA5. Furthermore, NS1 and Du MAVS eukaryotic expression plasmids were transfected into DEFs, 24 h after transfection by laser confocal observation, DTMUV NS1 and Du MAVS were colocalized in cells, initially identified DTMUV NS1 protein may be through interaction with Du MAVS the inhibition of RLR signaling pathway. However, the results of this part are validated by CO immunoprecipitation experiments.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.65

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