PATHOGENESIS OF ALEUTIAN DISEASE VIRUS INFECTION

阿留申水貂病细小病毒(ADV)持续感染水貂 引起免疫调节紊乱，包括多克隆 高丙种球蛋白血症，浆细胞增多症，免疫复合体疾病， 间质性和肾小球肾炎。这一系列发现表明 一种细胞因子紊乱。本项目的一个重点是评估角色 阿尔茨海默病中的细胞因子。几种水貂细胞因子和两种细胞因子的独立克隆 管家基因已经被开发出来了。一种非放射性核糖核酸酶 对这些基因的保护分析(RPA)也已经发展起来。 水貂细小病毒AdV和水貂肠炎病毒的转录 (MEV)在同步的CRFK细胞中进行比较。明显不同的是 不同信使核糖核酸的总转录水平和相对比率 都被注意到了。ADV的总转录速率约为1% 那就是MEV。编码VP的R3转录本占MEV的80% MRNA，但只有8%的ADV基因。R1，NS1的编码在5%至10%之间 MEV和ADV的病毒RNA的同源性。R2和R2‘m RNA，编码NS2， MEV约为10%，ADV约为80%。序列分析表明， ADV R2还可以为VP和NS2编码，尽管它将 使用P3启动子而不是P36启动子通过泄漏扫描启动。 RPA法检测ADV在体内的转录。成绩单是 很容易在感染后10天被检测到。总体水平 转录是体外情况的1%，但相对比例 在初步实验中，各种adv mRNAs中似乎有 体内实验与体外实验相同。 为了比较ADV蛋白和ADV蛋白的亚细胞定位 允许性和限制性感染、特异性抗血清中的核酸 对于非结构蛋白(NS)和衣壳蛋白(VP)已被制备和 与荧光染料结合。聚合酶链式反应已被用来制作探针 荧光原位杂交(FISH)。这些试剂已用于 在多通道共聚焦显微镜中表征允许性ADV CRFK细胞感染。ADV DNA、NS和VP存在于特定的 在感染过程中不同的细胞内位置。 此外，复制中间体的空间分离是明显的。 对水貂淋巴结的初步显微镜分析表明， 生发中心亮区与暗区的比值随时间的延长而降低 ADV感染。紧随其后的是卵泡退缩，变薄 小叶周围和髓索增厚。 淋巴组织结构的功能紊乱可能提供线索 ADV诱导免疫失调的机制，就像它在HIV中的作用一样 感染。

Persistent infection of mink with Aleutian mink disease parvovirus (ADV) causes disturbances of immune regulation, including polyclonal hypergammaglobulinemia, plasmacytosis, immune complex disease, interstitial and glomerulonephritis. The spectrum of findings suggests a cytokine disorder. One focus of this project is to evaluate the role of cytokines in AD. Independent clones of several mink cytokines and two housekeeping genes have been developed. A non-radioactive RNAse protection assay (RPA) for these genes has also been developed. Transcription of ADV and another mink parvovirus, mink enteritis virus (MEV) was compared in synchronized CRFK cells. Pronounced differences in total transcript levels and relative ratios of the various mRNA species were noted. The overall rate of ADV transcription was approximately 1% that of MEV. The R3 transcript, coding for VPs, comprised 80% of MEV mRNA, but only 8% of ADV mRNA. R1, coding for NS1 was between 5 and 10% of the viral RNA for both MEV and ADV. R2 and R2' mRNA, coding for NS2, was about 10% for MEV, but 80% for ADV. Sequence analysis indicates that the ADV R2 could also code for VP as well as NS2, although it would have to initiate by leaky scanning using the P3 rather than the P36 promoter. ADV transcription in vivo was also examined by RPA. Transcripts were readily detected ten days after infection. The overall level of transcription is <1% of the in vitro situation, but the relative ratios of the various ADV mRNAs appeared, in preliminary experiments, to be the same in vivo as in vitro. In order to compare the subcellular localization of ADV proteins and nucleic acids in permissive and restricted infection, specific antisera for nonstructural (NS) and capsid proteins (VP) have been prepared and conjugated to fluorochromes. PCR has been used to make probes for fluorescence in situ hybridization (FISH). These reagents have been used in multi-channel confocal microscopy to characterize permissive ADV infection in CRFK cells. ADV DNA, NS and VP are found in specific intracellular locations that differ during the time course of infection. Furthermore, spatial segregation of replicative intermediates is evident. Preliminary microscopic analysis of mink lymph nodes suggests that the ratio of germinal center light zones to dark zones decreases following ADV infection. This is followed by involution of follicles, thinning of the perifollicular region and thickening of the medullary cords. Functional disruption of lymphoid structure may provide clues to the mechanism of ADV induced immune dysregulation, as it has in HIV infections.