水貂阿留申病病毒强毒株致弱后毒力和免疫原性变化规律研究

The epidemiological surey showed that Aleutian mink disease is one of the three epideminc disease to the mink farming industry. In spite of several attepmts to provide an efficient protective protein based vaccine,experiment have failed so far. The study showed that inactived vaccine against Aleutian mink disease provided partial protection. On the basis of isolation and identification of Aleutian minke disease virus, the aim of this work to cultured, domestication and attenuated Aleutian mink disease virus in vitro,observe the variation of virulence for TCID50 and LD50,compare and analysis the genomics before and after attenuated,find the gene which caused the changes in virulence and adaptation to cell culture.The mink were immuned with attenuated ADV,observe the variation of humoral and cellar immunity,determine the protective immunity after virus challenge,clarify the variation of virulence and immunogenicity of virulent strain of Aleutian mink disease virus after attenuated.The aim of this study to constuct the attenuated vaccine against Aleutian mink disease,solve the problem of mink farming industry "none of vaccine provide all protection against Aleutian mink disease ",control and extinguish the Aleutian mink disease,play an important role on development of mink farming industry.

流行病学调查表明，水貂阿留申病是严重危害水貂养殖业的三大疫病之首。研究表明，水貂阿留申病细胞灭活疫苗具有一定的免疫保护效果，但由于水貂阿留申病的致病机理和免疫机理十分复杂，迄今为止尚未开发出有效防制水貂阿留申病的疫苗。为此，本课题在完成水貂阿留申病病毒分离与鉴定的基础上，拟对水貂阿留申病毒强毒株体外培养、驯化和致弱；通过测定TCID50和LD50观察毒力变化规律；通过致弱前后的基因组学比较分析，寻找引起毒力变化和适应细胞培养的变异基因；用致弱后的ADV免疫动物，系统观察其体液免疫和细胞免疫水平变化规律并进行攻毒试验测定其免疫保护力；阐明水貂阿留申病病毒强毒株致弱后毒力和免疫原性变化规律。这将为研制出有效防制水貂阿留申病的弱毒疫苗，破解长期困扰水貂养殖业的难题-"水貂阿留申病尚无有效防制疫苗"做出有益的探索和尝试，为控制和扑灭水貂阿留申病的传播与流行，促进水貂养殖业健康发展产生深远的影响。

水貂阿留申病又称浆细胞增多症，是由自主复制型细小病毒水貂阿留申病毒引起的一种主要侵害水貂免疫细胞，导致自身免疫系统的紊乱并逐渐衰竭的慢性传染性疾病。研究表明水貂阿留申病细胞灭活疫苗具有一定的免疫保护效果，但由于水貂阿留申病毒感染水貂后，水貂体内不产生或只产生少量的中和抗体，而产生的多数抗体不仅不能中和病毒的毒力，反而通过介导抗体依赖性增强作用，进一步帮助水貂阿留申病毒对靶细胞的侵染。再者，由于病毒抗原与抗体形成的复合物特有的理化特点，可导致抗原抗体复合物沉积并引发肾小球肾炎和动脉炎。正是由于水貂阿留申病这种复杂的致病机理和免疫机理，迄今为止尚未开发出有效防制水貂阿留申病的疫苗。为此，本课题组首先对因水貂阿留申病致死的水貂内脏进行ADV野毒株的分离，并对分离的毒株进行生物学特性分析和PCR鉴定，成功的分离并鉴定出5株ADV毒株；根据Genbank上公布的不同ADV毒株的基因序列，设计并合成了12对引物，对所分离的病毒进行全基因扩增；对分离的病毒进行全基因测序，并将结果与ADV参考毒株以及细小病毒亚科各属代表种的全基因序列进行核苷酸序列分析及同源性比较，构建了相应的系统发育进化树，分析了ADV与其它各属成员的进化关系；采用CRFK传代细胞、鼠L细胞株、水貂肺传代细胞等多种细胞，对ADV分离毒株进行体外培养和驯化，并对培养条件进行优化。这将为研制出有效防制水貂阿留申病的弱毒疫苗，破解长期困扰水貂养殖业的难题-"水貂阿留申病尚无有效防制疫苗"做出有益的探索和尝试，为控制和扑灭水貂阿留申 病的传播与流行，促进水貂养殖业健康发展产生深远的影响。

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