本文關(guān)鍵詞： 鴨 坦布蘇病毒 滅活疫苗 準(zhǔn)種 致弱 細(xì)胞適應(yīng) E蛋白　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：坦布蘇病毒(Tembusuvirus,TMUV)屬于黃病毒科(Flaviviridae),黃病毒屬(Flavivirus),主要存在于東南亞地區(qū)的蚊蟲(chóng)媒介中。病毒感染家禽可引起嚴(yán)重的產(chǎn)蛋下降和中樞神經(jīng)系統(tǒng)癥狀,自2010年4月在中國(guó)南方部分地區(qū)養(yǎng)鴨場(chǎng)爆發(fā)以來(lái),已蔓延至我國(guó)大部分家禽養(yǎng)殖區(qū)域,嚴(yán)重威脅著水禽養(yǎng)殖業(yè)。及時(shí)了解和掌握該病的發(fā)生和流行情況,開(kāi)展疫苗的研究對(duì)有效防治該病具有重要的意義。本研究首先以濃縮的全病毒免疫BALB/c小鼠,利用細(xì)胞融合技術(shù)獲得兩株能夠穩(wěn)定分泌抗鴨坦布蘇病毒特異性單克隆抗體的雜交瘤細(xì)胞株,產(chǎn)生的單克隆抗體可特異性地識(shí)別病毒E蛋白白。以此為基礎(chǔ),建立了阻斷ELISA方法用于抗坦布蘇病毒抗體的檢測(cè)。該方法特異性高、敏感性強(qiáng),與蝕斑減數(shù)中和試驗(yàn)符合率高達(dá)98.28%,同時(shí)具有良好的相關(guān)性(r2=0.7998,P0.001)。對(duì)2009～2015年間我國(guó)部分地區(qū)水禽養(yǎng)殖場(chǎng)的血清樣本進(jìn)行抗體檢測(cè)和病原分離鑒定,結(jié)果表明近年來(lái)部分養(yǎng)殖場(chǎng)仍然存在不同程度的病毒感染。為進(jìn)一步開(kāi)展病毒疫苗研究,本研究以坦布蘇病毒JXSP株為對(duì)象,通過(guò)連續(xù)傳代使其能穩(wěn)定適應(yīng)BHK-21細(xì)胞,以低代次毒株為抗原制備了礦物油乳劑滅活疫苗。雛鴨首次免疫后對(duì)強(qiáng)毒感染的保護(hù)指數(shù)可達(dá)87%,組織臟器載毒量顯著低于未免疫鴨;二次免疫則能完全抵抗病毒感染。產(chǎn)蛋鴨經(jīng)二次免疫后可有效抵抗強(qiáng)毒的攻擊。阻斷ELISA檢測(cè)結(jié)果顯示,蛋鴨群首免21d后抗體轉(zhuǎn)陽(yáng)率為97%(39/40),二免后抗體水平顯著上升,并能持續(xù)4個(gè)月以上。隨著傳代次數(shù)的增加,病毒對(duì)細(xì)胞的適應(yīng)性明顯增強(qiáng),表現(xiàn)為感染細(xì)胞病變進(jìn)程逐漸加快,病毒滴度不斷升高;另一方面,病毒對(duì)試驗(yàn)鴨的致病性明顯下降。動(dòng)物感染試驗(yàn)結(jié)果表明,第150代細(xì)胞適應(yīng)毒感染雛鴨后不引發(fā)明顯的臨床癥狀,感染雛鴨各組織臟器載毒量顯著低于強(qiáng)毒組;高代次毒株JXSP2-310已充分致弱,僅在感染雛鴨的脾臟組織中檢測(cè)到少量病毒,易感雛鴨體內(nèi)連續(xù)傳代無(wú)毒力返強(qiáng)現(xiàn)象。以105PFU//只的劑量免疫蛋鴨10d后攻擊強(qiáng)毒,保護(hù)率可達(dá)100%,表明弱毒株JXSP2-310 具備非常好的免疫原性,可以作為弱毒疫苗的候選株�；蚪M序列分析顯示,與強(qiáng)毒株JXSP2-4相比,高代次弱毒株JXSP2-310全基因組共產(chǎn)生了 41個(gè)核苷酸位點(diǎn)的變異,導(dǎo)致23個(gè)氨基酸突變。為深入探討病毒細(xì)胞適應(yīng)及其致弱的分子機(jī)制,本研究對(duì)坦布蘇病毒原代毒株JXSP2-1以及高代次細(xì)胞適應(yīng)毒株(JXSP2-150、JXSP2-280及JXSP2-310)的全基因組進(jìn)行了深度測(cè)序并對(duì)其單核苷酸變異多態(tài)性進(jìn)行分析。結(jié)果表明,JXSP2-1具備準(zhǔn)種特征,但SNP位點(diǎn)數(shù)目較少,且變異頻率低。而高代次適應(yīng)毒株的SNP位點(diǎn)數(shù)目顯著增加,變異頻率也顯著升高。準(zhǔn)種多樣性指數(shù)統(tǒng)計(jì)學(xué)分析結(jié)果顯示,高代次毒株的香農(nóng)熵與辛普森指數(shù)均顯著高于原代毒株,表明鴨坦布蘇病毒在細(xì)胞上傳代適應(yīng)過(guò)程中,其準(zhǔn)種多樣性不斷增加。通過(guò)蝕斑克隆技術(shù),挑選獲得了病毒準(zhǔn)種優(yōu)勢(shì)突變株,為后續(xù)致弱機(jī)制的研究奠定基礎(chǔ)。為進(jìn)一步揭示病毒基因突變與毒力變化的相關(guān)性,本研究采用反向遺傳操作技術(shù)獲得了鴨坦布蘇病毒親本拯救毒株JXSP-RV,并以構(gòu)建的JXSP2-4感染性cDNA克隆為骨架,將弱毒株JXSP2_310E蛋白基因置換到JXSP2-4相應(yīng)區(qū)域,拯救獲得嵌合毒株JXSP-310E。細(xì)胞感染試驗(yàn)表明,嵌合毒株在BHK-21細(xì)胞上的增殖能力顯著高于親本拯救毒株。動(dòng)物感染試驗(yàn)表明,嵌合毒株接種雛鴨與3周齡BALB/c小鼠后不能引起明顯的臨床癥狀,且感染雛鴨血液、腦及心臟組織中病毒含量顯著低于親本拯救毒株組。由此推測(cè),鴨坦布蘇病毒E蛋白基因的氨基酸變異是影響病毒增殖能力和病毒毒力的關(guān)鍵因素。綜上所述,本研究建立了鴨坦布蘇病毒阻斷ELISA抗體檢測(cè)方法,成功制備了病毒細(xì)胞滅活苗,篩選獲得了完全致弱的高代次細(xì)胞適應(yīng)毒株,同時(shí)發(fā)現(xiàn)病毒細(xì)胞適應(yīng)過(guò)程中準(zhǔn)種多樣性的顯著增加并確定E蛋白上氨基酸位點(diǎn)的變異與病毒的適應(yīng)性及致病性密切相關(guān)。這些結(jié)果為鴨坦蘇病毒的診斷預(yù)防提供了有力工具,為病毒致病分子機(jī)制的研究提供參考。
[Abstract]:Tembusu virus (Tembusuvirus, TMUV) belongs to the family Flaviviridae flavivirus (Flaviviridae), (Flavivirus), mainly in Southeast Asia. Mosquito poultry can cause severe egg drop and central nervous system symptoms of virus infection, since April 2010 in parts of the South China duckery outbreak has spread to most of China poultry farming area, a serious threat to waterfowl breeding. Understand and grasp the occurrence of the disease and the epidemic situation of vaccine research has an important significance for effective prevention and treatment of this disease. In this study, the concentration of virus immune BALB/c mice by cell fusion technique to obtain two strains could stably secrete monoclonal antibody against duck Tanzania Busu virus specific hybridoma cell lines and monoclonal antibody can specifically identify the virus E protein. On this basis, established the blockade of ELISA Method for the detection of antibody against Tembusu virus. This method has high specificity, sensitivity, and plaque reduction neutralization test with rate of 98.28%, and has a good correlation (r2=0.7998, P0.001). The serum samples of 2009~2015 years in some areas of China waterfowl breeding field were detected and antibody pathogen isolation and identification results in recent years show that part of the farm still exists different degree of virus infection. For further research on virus vaccine, in this study, Tembusu virus strain JXSP as the object, through continuous passage so that it can adapt to the BHK-21 stable cells, antigen preparation of mineral oil emulsion inactivated vaccine with low passage strain. After the first immunization of ducklings the virulent infection protection index reached 87%, the amount of drug loaded organs was significantly lower than that of non immunized duck; two immunization can completely resist virus infection. Laying ducks after two times of immunization can be effective against Resistance to virulent virus attack. Blocking ELISA detection results showed that the laying ducks first free after 21d antibody positive rate was 97% (39/40), increased significantly after two free antibody level, and can last more than 4 months. With the increase in the number of passages, the adaptability of virus on cells significantly enhanced performance for infected cell lesions the process accelerates, the virus titer increased; on the other hand, pathogenic viruses of ducks decreased significantly. Animal infection test results show that the 150th generation of cell adapted Virus Infected Ducklings after causes no obvious clinical symptoms, the infection of organ tissues of ducklings viral loads were significantly lower than that of virulent group; high passage strain JXSP2-310 has fully attenuated only detectable virus in Ducklings Infected with spleen tissue in susceptible ducklings passaged avirulent strong virulent attack. Returning to 105PFU// only the ducks after 10d dose immunization, the protection rate was 100%, showed attenuated Strain JXSP2-310 has excellent immunogenicity, can be used as a vaccine candidate strain. Genome sequence analysis showed that, compared with JXSP2-4 virulent strain, high passage attenuated strain JXSP2-310 genome mutation produced a total of 41 nucleotide sites, resulting in 23 amino acid mutations. In order to further explore the virus cell and its molecular mechanism to adapt to weak, the research on the adaptive strain of Tembusu virus strain JXSP2-1 and primary high passage cells (JXSP2-150, JXSP2-280 and JXSP2-310) of the whole genome of deep sequencing and analyzed the single nucleotide polymorphism in the variation. The results show that JXSP2-1 has the characteristics of quasi SNP, but the number is less, and the frequency of variation low and high passage. The number of SNP sites to strain increased significantly, the variation frequency was significantly increased. The quasispecies diversity index statistical analysis showed that the Shannon entropy generation with high strain The Simpson index was significantly higher than that of primary strains indicated that the duck Tembusu virus in cell passage adaptation process, the quasi species diversity increased. Through the plaque cloning technology, selection of obtained virus quasispecies dominant mutant, lay the foundation for the follow-up study of attenuated mechanism. In order to further reveal the virus gene mutations associated with the virulence change, this study used reverse genetics technology has obtained the duck Tembusu virus strain JXSP-RV and to save the parents, the JXSP2-4 infectious cDNA clone for the skeleton, the attenuated JXSP2_310E protein gene replacement to JXSP2-4 corresponding area, save the chimeric strain JXSP-310E. cell infection test showed that the proliferation ability of chimeric strains in BHK-21 cells the parents save that was significantly higher than that of strains. Animal infection test, cause obvious clinical strains inoculated ducklings cannot be fitted with 3 week old BALB/c mice The symptoms, and the ducklings infected blood, virus content in brain and heart tissues were significantly lower than the parental strain group. Save the inferred amino acid mutation dtmuv virus E protein gene is a key factor affecting the proliferation ability and virulence of the virus virus. In summary, this study established a method for detection of ELISA antibody against duck Tembusu virus was successfully blocked preparation of cell virus inactivated vaccine, was completely caused by high passaged weak adapted strain screening, also found that the virus cell adapted significantly increased quasispecies diversity and determine the process of adaptation and variation of pathogenicity of virus and amino acid sites of E protein are closely related. These results provide a powerful tool for diagnosis and prevention duck Tansu virus, and provide reference for the study of molecular mechanism of pathogenicity of the virus.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65

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