一种新的水貂致死性圆环病毒感染特性及Cap蛋白功能研究

Members of the family Circoviridae are non-enveloped, icosahedrally shaped viruses with diameters between 16 and 26 nm and with a small, circular, single-stranded DNA genome-the smallest known autonomously replicating viral genome. The family Circoviridae contains two genera, Gyrovirus and Circovirus, both of which have been associated with diseases in animals, including birds, pigs and dogs. Avian circoviruses have been associated with beak abnormalities, lethargy, and anorexia in pigeons, and production losses and mortality in geese. Porcine circovirus 2 causes respiratory and enteric diseases, dermatitis, and reproductive problems in pigs, resulting in major economic losses to the pork industry. Genomes of cycloviruses, a proposed new genus within the family Circoviridae, have been identified in human and chimpanzee feces and human cerebrospinal fluids. Recently, circoviruses have been found in bats and have shown a large genetic diversity. However, to date no circovirus infection has been reported in mustelids. Recently, we report the discovery and characterization of a novel circovirus, MiCV, in the liver, digestive tract and fecal specimens from mink with diarrhea as the main clinic sign. All mink in diseased farms appeared to be affected, with 80% showing clinical signs, about 7% of the affected mink died. Circoviruses have an ambisense genome organization containing two major inversely arranged open reading frames, encoding the rolling circle replication initiator protein gene (rep) and capsid protein gene (cap).Cap protein is the main antigenic determinant of the virus and a recent finding links pathogenicity with certain alterations of the sequence of the cap protein. To determine the pathogenesis of MiCV, on the one hand, we are planning to infect the different cells in vitro and mice and mink in vivo, detect the growth,replication and dissemination of mink circoviruses in vitro and in vivo. On the other hand, the yeast two-hybrid system will be used to identify interactions of cap with host proteins,cap with the binding properties of the host protein will be analyze by co-immunoprecipitation combined with confocal laser technology. We will also investigate whether the host protein influences MiCV replication and pathogenicity by the over-expression and knockdown technology.

动物圆环病毒主要包括哺乳动物和禽类的病原，虽然近年来陆续从人和黑猩猩粪便中分离到多株圆环病毒，但其致病性和公共卫生意义并不清楚。迄今，国内外尚无毛皮动物感染圆环病毒的报道。本研究前期从免疫过水貂病毒性肠炎疫苗的腹泻病貂样本中分离到一株水貂圆环病毒（MiCV），该病毒在发病水貂群中的感染率约80%，致死率达7%，严重影响水貂养殖业的经济。衣壳蛋白Cap是动物圆环病毒主要免疫原性蛋白，最近研究还发现，Cap蛋白与病毒毒力密切相关。为了深入研究MiCV感染机制及Cap蛋白功能，一方面通过体内、体外病毒感染实验，明确MiCV在感染、复制、移行和分布上的特性及其免疫分子机制；另一方面利用双杂交系统筛选与衣壳蛋白Cap互作的宿主蛋白，再通过免疫共沉淀结合激光共聚焦技术，明确Cap与宿主蛋白的结合特性，进一步采用过表达和Knockdown技术揭示Cap蛋白调控MiCV感染及致病的分子机制。

水貂黄白痢又称秋痢，水貂顽固性痢疾，本研究负责人首次发现其病原为水貂圆环病毒，该病由于发病率高达70%，对种貂生产具有明显的影响。本研究先后尝试了病毒分离，建立了水貂圆环病毒人工感染模型，针对感染动物组织进行了病理学观察和分析，成功表达了病毒Cap蛋白，并利用该Cap蛋白开展了免疫研究。结果表明，经系列筛选和连续适应，未获得对病毒敏感的细胞，也没能使病毒适应任何一种现有细胞系；通过口服感染途径，建立了黄白痢水貂感染模型，成功复制了本病。针对肠、肝、肾、肺、淋巴结和脾的组织学观察，发现本病毒可引起肠、肝、肾、肺等严重炎症反应，出现炎性淋巴细胞浸润和细胞变性，但在淋巴结和脾脏未见明显病理学变化。说明该圆环病毒主要感染非免疫期系统器官并导致这些器官损害，对于免疫器官的危害较为轻微或无。通过对Cap蛋白基因密码子优化，采用杆状病毒特异性早、晚期双启动子，构建了Cap蛋白的杆状病毒表达载体，实现了Cap蛋白高效表达，表达量在100微克/ml以上。针对水貂实验免疫Cap并攻毒的结果表明，免疫Cap蛋白可防止机体攻毒感染水貂圆环病毒对各相关组织的损伤，田间针对大连庄河和山东诸城的1000只水貂的免疫结果表明，重组Cap蛋白对水貂具有明显的免疫保护效果，未免疫对照貂群约70%自然发病，免疫貂群无一发病，说明水貂圆环病毒Cap蛋白具有良好的免疫原性，具有阻止水貂黄白痢发生的作用。本研究采用杆状病毒表达的cap蛋白亚单位疫苗由于具有良好的临床保护效果，具有重要的应用价值。