鸭坦布苏病毒细胞适应毒免疫原性及致弱分子基础研究

发布时间：2018-02-25 22:05

本文关键词： 鸭坦布苏病毒灭活疫苗准种致弱细胞适应 E蛋白　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：坦布苏病毒(Tembusuvirus,TMUV)属于黄病毒科(Flaviviridae),黄病毒属(Flavivirus),主要存在于东南亚地区的蚊虫媒介中。病毒感染家禽可引起严重的产蛋下降和中枢神经系统症状,自2010年4月在中国南方部分地区养鸭场爆发以来,已蔓延至我国大部分家禽养殖区域,严重威胁着水禽养殖业。及时了解和掌握该病的发生和流行情况,开展疫苗的研究对有效防治该病具有重要的意义。本研究首先以浓缩的全病毒免疫BALB/c小鼠,利用细胞融合技术获得两株能够稳定分泌抗鸭坦布苏病毒特异性单克隆抗体的杂交瘤细胞株,产生的单克隆抗体可特异性地识别病毒E蛋白白。以此为基础,建立了阻断ELISA方法用于抗坦布苏病毒抗体的检测。该方法特异性高、敏感性强,与蚀斑减数中和试验符合率高达98.28%,同时具有良好的相关性(r2=0.7998,P0.001)。对2009～2015年间我国部分地区水禽养殖场的血清样本进行抗体检测和病原分离鉴定,结果表明近年来部分养殖场仍然存在不同程度的病毒感染。为进一步开展病毒疫苗研究,本研究以坦布苏病毒JXSP株为对象,通过连续传代使其能稳定适应BHK-21细胞,以低代次毒株为抗原制备了矿物油乳剂灭活疫苗。雏鸭首次免疫后对强毒感染的保护指数可达87%,组织脏器载毒量显著低于未免疫鸭;二次免疫则能完全抵抗病毒感染。产蛋鸭经二次免疫后可有效抵抗强毒的攻击。阻断ELISA检测结果显示,蛋鸭群首免21d后抗体转阳率为97%(39/40),二免后抗体水平显著上升,并能持续4个月以上。随着传代次数的增加,病毒对细胞的适应性明显增强,表现为感染细胞病变进程逐渐加快,病毒滴度不断升高;另一方面,病毒对试验鸭的致病性明显下降。动物感染试验结果表明,第150代细胞适应毒感染雏鸭后不引发明显的临床症状,感染雏鸭各组织脏器载毒量显著低于强毒组;高代次毒株JXSP2-310已充分致弱,仅在感染雏鸭的脾脏组织中检测到少量病毒,易感雏鸭体内连续传代无毒力返强现象。以105PFU//只的剂量免疫蛋鸭10d后攻击强毒,保护率可达100%,表明弱毒株JXSP2-310 具备非常好的免疫原性,可以作为弱毒疫苗的候选株。基因组序列分析显示,与强毒株JXSP2-4相比,高代次弱毒株JXSP2-310全基因组共产生了 41个核苷酸位点的变异,导致23个氨基酸突变。为深入探讨病毒细胞适应及其致弱的分子机制,本研究对坦布苏病毒原代毒株JXSP2-1以及高代次细胞适应毒株(JXSP2-150、JXSP2-280及JXSP2-310)的全基因组进行了深度测序并对其单核苷酸变异多态性进行分析。结果表明,JXSP2-1具备准种特征,但SNP位点数目较少,且变异频率低。而高代次适应毒株的SNP位点数目显著增加,变异频率也显著升高。准种多样性指数统计学分析结果显示,高代次毒株的香农熵与辛普森指数均显著高于原代毒株,表明鸭坦布苏病毒在细胞上传代适应过程中,其准种多样性不断增加。通过蚀斑克隆技术,挑选获得了病毒准种优势突变株,为后续致弱机制的研究奠定基础。为进一步揭示病毒基因突变与毒力变化的相关性,本研究采用反向遗传操作技术获得了鸭坦布苏病毒亲本拯救毒株JXSP-RV,并以构建的JXSP2-4感染性cDNA克隆为骨架,将弱毒株JXSP2_310E蛋白基因置换到JXSP2-4相应区域,拯救获得嵌合毒株JXSP-310E。细胞感染试验表明,嵌合毒株在BHK-21细胞上的增殖能力显著高于亲本拯救毒株。动物感染试验表明,嵌合毒株接种雏鸭与3周龄BALB/c小鼠后不能引起明显的临床症状,且感染雏鸭血液、脑及心脏组织中病毒含量显著低于亲本拯救毒株组。由此推测,鸭坦布苏病毒E蛋白基因的氨基酸变异是影响病毒增殖能力和病毒毒力的关键因素。综上所述,本研究建立了鸭坦布苏病毒阻断ELISA抗体检测方法,成功制备了病毒细胞灭活苗,筛选获得了完全致弱的高代次细胞适应毒株,同时发现病毒细胞适应过程中准种多样性的显著增加并确定E蛋白上氨基酸位点的变异与病毒的适应性及致病性密切相关。这些结果为鸭坦苏病毒的诊断预防提供了有力工具,为病毒致病分子机制的研究提供参考。
[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.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65

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