【作者】 李建文；

【导师】 王红宁；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2006， 博士

【摘要】 兔病毒性出血症是由兔出血症病毒（rabbit hemorrhagic disease virus，RHDV）引起兔的一种急性、烈性、高致死性传染病，是一种毁灭性传染病，给养兔业造成巨大的经济损失。1984年我国首先爆发兔病毒性出血症，目前呈全国性分布，2004年GenBank登录了第一株中国株RHDV的全基因组序列，我国RHDV的分子流行病学资料非常缺乏，且迄今国内仍未见RHDV基因组序列测定的详细报道。目前国内外还没有RHDV反向遗传学和核酸疫苗的相关研究报道。鉴于此，本研究主要包括以下几个方面： 1．兔出血症病毒WHNRH株的分离鉴定：采用病死兔的肝脏组织进行病原的分离，将其经兔体传3代后接种兔呈规律性死亡和病理变化，其特征与兔病毒性出血症相同，采用氯仿处理，聚乙二醇沉淀法对第4代死亡兔肝脏组织进行处理，所得产物具有血凝特性，其血凝效价可达2¹⁶，该血凝作用能够被抗RHDV的高免血清所抑制，该产物能够与抗RHDV的高免血清在琼脂扩散实验中形成清晰可见的沉淀线，将该液体经磷钨酸染色后进行电镜观察，结果发现该产物中存在一种大小约为30-40nm、无囊膜，符合RHDV形态特征的病毒粒子。参考GenBank中登录的兔出血症病毒的基因组序列，在保守区设计一对RHDV特异的PCR引物对该分离病毒进行RT-PCR扩增，结果扩增出与预期片段大小相符的PCR条带，对产物的克隆测序结果表明，该株病毒与已经报道的RHDV的对应区域的同源率达90.2％-97.2％，从分子水平证实本研究分离毒株为兔病毒性出血症病毒（RHDV），将其命名为WHNRH株。 2．兔出血症病毒WHNRH株基因组序列的测定与分析：采用片段重叠的更多还原

【Abstract】 Rabbit hemorrhagic disease was an acute and fatal contagious disease for rabbits caused by rabbit hemorrhagic disease virus(RHDV) and caused heavy economy losses.The disease first broke out in China in 1984 and until now it existed all over China. The molecular epidemiology of RHD in China was in deficiency and there only one genome sequence of RHDV in GenBank from China until 2004. The sequencing progress had not been reported in details before in China. The reverse genetics and DNA vaccine research had not beeen reported in the world.So the research included the following contents:1.Isolation and identification of RHDV WHNRH strain: Liver tissues from animals that were suspected to have died of rabbit hemorrhagic disease (RHD) were used for isolation and identification of the causative agent. Since there is no cell culture system to grow RHDV, animals were used for virus isolation in the study. The suspected causative agent was inoculated and went through rabbits three times,then the inoculated rabbits died regularly and the pathological changes were identical to RHD after third passage in rabbitsA method purifying RHDV with chloroform and polyethylene glycol 6000 was applied to treat the liver tissues of died rabbits. The causative agent isolated as a preparation reacted positively by hemagglutination (HA) assays with 216 hemagglutination units and the hemagglutination was inhibited by serum against RHDV specially.In agar diffusion there form clear precipitate lines between the preparation and positive serum against RHDV.The preparation was negative-stained with phosphotungstic acid and then observed with electron microscope, many virion without peplos measured approximately 30-40 nm in diameter was obtained. Morphologically, the isolated virus were identical to RHDV.A pair of更多还原