Identification of Human Cytomegalovirus Pathogenic Genes

描述(申请人提供)：人类巨细胞病毒(HCMV)是艾滋病患者最常见的机会性感染之一。我的长期研究目标是了解巨细胞病毒的发病机制，特别是巨细胞病毒如何与宿主细胞相互作用、复制和致病。这项建议旨在定义病毒在体内复制所需的HCMV基因。由于目前尚无研究人巨细胞病毒体内复制和致病机制的动物模型，因此植入人胎儿组织的严重联合免疫缺陷小鼠(SCID)为这方面的研究提供了一种新的有价值的动物模型。人巨细胞病毒临床分离株托莱多株在SCID小鼠体内的移植人体组织中以高滴度复制；然而，减毒株AD169完全失去了这一能力。这两个毒株之间的差异是一个15kb的基因组片段，编码19个病毒基因，保留在包括托莱多在内的所有临床分离株中，但从AD169基因组中删除。我们已经证明了人巨细胞病毒体内复制所需的关键基因确实定位于该区域。在这个方案中，一个托莱多细菌人工染色体(BAG)克隆和一个高效的重组系统将被用来产生缺失突变病毒。这将大大简化和促进重组人巨细胞病毒病毒的构建。这些重组病毒将在植入SCID小鼠的人体组织以及内皮细胞和巨噬细胞中进行生长测试。最后，将绘制和分析这两个系统中病毒复制所需的病毒基因。这项工作将使我们能够确定在体内复制和致病过程中对巨细胞病毒至关重要的基因。这将有助于更好地了解巨细胞病毒致病的分子基础，并有可能有助于预防和治疗巨细胞病毒相关疾病，特别是艾滋病患者。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is one of the most common opportunistic infections in AIDS patients. My long-term research goal is to understand HCMV pathogenesis; specifically how HCMV interacts with host cells, replicates and causes disease. This proposal is designed to define the HCMV genes required for viral replication in vivo. Since animal models for studying HCMV replication and pathogenesis in vivo are not available, the severe combined immunodeficient mice (SCID) implanted with human fetal tissues provide us an alternative and valuable model for these studies. The HCMV clinical isolate Toledo strain replicates with high titers in the implanted human tissues in SCID mice; however, attenuated strain AD169 has completely lost this ability. The difference between these two strains is a 15-kb genomic segment, encoding 19 viral genes, that remains in all clinical isolates tested, including Toledo, but is deleted from the AD169 genome. We have demonstrated that the crucial genes required for HCMV in vivo replication are indeed localized in the region. In this proposal, a Toledo bacterial artificial chromosome (BAG) clone and a highly efficient recombination system will be used to generate deletion mutant viruses. This will significantly simplify and facilitate the construction of recombinant HCMV viruses. These recombinant viruses will be tested for their growth in the implanted human tissues in SCID mice and endothelial cells and macrophages. Finally, the viral genes needed for viral replication in these two systems will be mapped and analyzed. This work will allow us to identify genes essential for HCMV in vivo replication and pathogenesis. It should lead to a better understanding of the molecular basis of HCMV pathogenesis, and has the potential to help in the prevention and treatment of HCMV related diseases, especially in AIDS patients.