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 在过去的一年里，我们开始了全长嵌合克隆的研究， 该战略一直是取代感染性病毒的片段， 非致病性ADV-G株的分子克隆， 来自致病性ADV的分子克隆的片段直接来源于 被感染的动物器官迄今为止测定的3种构建体中有2种未能 在细胞中转染并连续传代后产生感染性ADV 这些是15 - 88或43 - 65图谱嵌合的克隆 单元段。65 - 73图谱单位片段的嵌合克隆能够 复制与亲本ADV-G相似。这些结果表明，在 在细胞培养物中允许复制的至少一个决定因子映射到 基因组的43 - 65图单位部分，其包含氨基末端 在病毒体蛋白中，非结构蛋白的羧基末端 以及与其中一个剪接相关的序列。 我们还开始研究ADV蛋白在牛痘中的表达， 真核表达系统。代表拼接的R3的片段 编码两种ADV结构蛋白的mRNA已经重组到 一种传染性牛痘病毒。该重组痘苗病毒（VV-1L1） VP1（p85）和VP2（p75）的合成。病毒抗原定位于 到细胞核。此外，可以使用VV-IL 1感染细胞的裂解物 作为抗ADV试验抗原。最后，细小病毒 通过电子显微镜观察颗粒。所有必要的信号 用于VP1/VP2以及那些 所需的颗粒组装。

The purpose of this project is to study various aspects of genome structure and function of the Aleutian mink disease parvovirus (ADV). In the past year we have initiated studies on full-length chimeric clones of ADV. The strategy has been to replace segments of an infectious molecular clone of the nonpathogenic ADV-G strain with corresponding fragments of molecular clones from pathogenic ADVs derived directly from infected animal organs. Two of the 3 constructs assayed to date failed to produce infectious ADV after transfection and serial passage in cell culture; these were clones chimeric for either the 15-88 or the 43-65 map unit segments. A clone chimeric for the 65-73 map unit segment was able to replicate similar to the parent ADV-G. These results suggested that at least one determinant for permissive replication in cell culture maps to the 43-65 map unit portion of the genome, which contains the amino terminus of the virion proteins, the carboxy terminus of the non-structural proteins and sequences associated with one of the splices. We have also begun to study the expression of ADV proteins in a vaccinia based eukaryotic expression system. A segment representing the spliced R3 mRNA that codes for both ADV structural proteins has been recombined into an infectious vaccinia virus. This recombinant vaccinia (VV-lL1) directed synthesis of both VP1 (p85) and VP2 (p75). The viral antigen was localized to the nucleus. Furthermore, lysates of VV-IL1 infected cells could be used as an ADV test antigen in counterimmunoelectrophoresis. Finally, parvovirus particles were observed by electron microscopy. Thus, all signals necessary for production and nuclear transport of both VPl/VP2 as well as those required for particle assembly were present.