Identification of Human Cytomegalovirus Pathogenic Genes

描述（申请人提供）：人类巨细胞病毒（HCMV）是艾滋病患者中最常见的机会性感染之一。我的长期研究目标是了解HCMV的发病机制；特别是HCMV如何与宿主细胞相互作用，复制并引起疾病。该建议旨在确定病毒在体内复制所需的HCMV基因。由于没有研究HCMV复制和体内发病机制的动物模型，植入人胎儿组织的严重联合免疫缺陷小鼠（SCID）为这些研究提供了另一种有价值的模型。HCMV临床分离株Toledo株在SCID小鼠植入人体组织中高滴度复制；然而，减毒菌株AD169完全丧失了这种能力。这两种毒株之间的差异在于一个15 kb的基因组片段，该片段编码19个病毒基因，在所有临床分离株（包括Toledo）中仍然存在，但从AD169基因组中删除。我们已经证明HCMV体内复制所需的关键基因确实定位在该区域。本研究将利用托莱多细菌人工染色体（BAG）克隆和高效重组系统产生缺失突变病毒。这将大大简化和促进重组HCMV病毒的构建。这些重组病毒将在SCID小鼠植入的人体组织、内皮细胞和巨噬细胞中进行生长测试。最后，我们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HCMV体内复制和发病机制所必需的基因。这将有助于更好地了解HCMV发病机制的分子基础，并有可能帮助预防和治疗HCMV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.