STRUCTURE AND FUNCTION OF THE ADV GENOME

这个项目的目的是研究基因组结构的各个方面 水貂阿留申病细小病毒（ADV）的研究进展 一个主要的努力是确定ADV基因组的哪些片段支配 各种病毒分离株的致病性和复制能力 允许在细胞培养中。 通过一系列的片段交换 非致病性ADV-G的全长感染性分子克隆 菌株和致病性ADV的部分（15-88个图位单位）克隆，我们 发现含有致病性ADV的53-65 μ片段的克隆 在细胞培养物中表达病毒抗原但不产生感染性病毒 在细胞培养中。 这个片段是衣壳的编码区的一部分 ADV-G和ADV-G之间仅存在4个氨基酸差异， 致病性ADV;该片段不含高变区。 使用基于聚合酶链反应的诱变策略，我们得到了 在这4种氨基酸中的e上发生突变，并发现从这种突变中拯救的病毒 克隆保留了亲本ADV-G的特征。 几个嵌合克隆产生的ADV可以在细胞中被拯救， 培养，并测定这些病毒诱导疾病的能力， 成年或新生水貂。 在细胞培养中复制的病毒不会引起任何 成年或新生水貂的重大疾病。 我们继续研究ADV蛋白在真核细胞中的表达， 表达系统。 我们发现空ADV颗粒的合成 是由一种重组牛痘病毒引导的， 两种ADV衣壳蛋白（VV-IL 1）的序列。 这些粒子可能是 纯化和带状的密度约。1.33 g/ml的氯化铯溶液， 与标准ADV感染中产生的空ADV颗粒相同。 VV- 将IL 1注射到小鼠中; 8/8只小鼠产生可检测的抗体， 间接免疫荧光法检测，但只有2/8产生中和 抗体的 成年水貂VV-IL-1和WR疫苗免疫试验 病毒感染后，水貂体内ADV的形成速度加快， 低剂量的ADV

The purpose of this project is to study various aspects of genome structure and function of the Aleutian mink disease parvovirus (ADV). A major effort is to determine which segments of the ADV genome govern pathogenicity and the ability of various virus isolates to replicate permissively in cell culture. Using a series of segmental exchanges between a full-length infectious molecular clone of the nonpathogenic ADV-G strain and a partial (15-88 map unit) clone of pathogenic ADVs, we have found that clones containing the 53-65 mu segment of pathogenic ADVs express viral antigen in cell culture but do not produce infectious virus in cell culture. This segment is part of the coding region for the capsid proteins and only 4 amino acid differences occur between ADV-G and the pathogenic ADVs; this segment does not contain the hypervariable region. Using a mutagenesis strategy based on the polymerase chain reaction we have mutated on e of these 4 amino acids and found that virus rescued from this clone retains the characteristics of the parent ADV-G. Several of the chimeric clones yielded ADV that could be rescued in cell culture and these viruses were assayed for the ability to induce disease in adult or newborn mink. No virus that replicated in cell culture caused any significant disease in either adult or newborn mink. We have continued to study the expression of ADV proteins in eukaryotic expression systems. We have found that synthesis of empty ADV particles was directed by a recombinant vaccinia virus that contained the coding sequences for both ADV capsid proteins (VV-IL1). These particles could be purified and banded at a density of ca. 1.33 g/ml in cesium chloride, identical to empty ADV particles produced in standard ADV infections. VV- IL1 was injected into mice; 8/8 mice produced detectable antibody as assayed by indirect immunofluorescence, but only 2/8 produced neutralizing antibody. Vaccination of adult mink with either VV-IL1 or WR vaccinia virus led to an accelerated from of ADV when the mink were challenged with a low dose of ADV.