CMV PATHOGENESIS AND CELLULAR TROPISM

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 人巨细胞病毒（HCMV）是一种普遍存在的疱疹病毒，感染50-90%的人群。 HCMV通过唾液、尿液和母乳等身体分泌物传播。一旦进入体内，病毒感染上皮细胞和内皮细胞，并最终感染白细胞，主要是单核细胞和中性粒细胞，然后将病毒全身传播到多个器官，包括唾液腺，膀胱和乳腺。 虽然在大多数情况下没有症状，但HCMV可在移植后免疫功能低下的患者、艾滋病患者和胎儿中引起致命疾病。 尽管强调了对CMV疫苗的需求，但迄今为止还没有成功的疫苗被开发出来。这部分受到巨细胞病毒的种属特异性和缺乏精确模拟HCMV的代表性动物模型的限制。 所有临床HCMV病例的关键步骤是内皮细胞中的病毒复制和白细胞介导的相关病毒传播。 HCMV通过正常白细胞-内皮细胞粘附机制的病毒发作、微融合的形成以及允许病毒通过直接细胞间运输在内皮细胞和白细胞之间双向传播。 防止病毒在宿主中传播可以降低CMV疾病的严重程度和病毒向他人的传播。 体外研究表明，HCMV UL 131 -128基因对于病毒在内皮细胞中复制和转移到白细胞是必需的。 此外，UL 146 -147中的病毒编码的趋化因子IL-8和GRO？在体外强烈增强病毒向嗜中性粒细胞的传递。 迄今为止的体内研究有限。 β-疱疹病毒具有高度的物种特异性。与HCMV最接近的是黑猩猩CMV（CCMV），但这不是一个实用的动物模型。 恒河猴是一种应用较为广泛的实验动物，而恒河猴巨细胞病毒（rhesusCMV，RhCMV）是HCMV基因家族中的主要成员。 最近有多个报道表征了RhCMV中代表HCMV UL 131 -128和UL 146 -147基因同源物的基因。 在最近的一份报告中，Lilja et.等人在BAC衍生的感染性病毒中修复了RhCMV株68-1的缺失的UL 131-UL 128基因座，并观察到修复的衍生物在恒河猴上皮细胞和培养的人上皮细胞和内皮细胞中的复制比亲本68-1病毒有效得多。 这些最新的成果将有助于检查CMV细胞的嗜性和传播，提高我们对恒河猴和人类CMV发病机制的理解。 在本研究中，我们将研究UL 131 -128和UL 146 -147基因对RhCMV的细胞嗜性和传播的影响。 我们将比较恒河猴体内感染的细胞嗜性与天然野生型RhCMV、RhCMV毒株68-1（缺乏UL 131 -128基因区域并具有反向UL 146 -147基因区域）和RhCMV毒株180.92（缺乏UL 146 -147基因区域）。 我们假设感染了缺乏UL 128复合物的菌株68-1的动物不会表现出内皮感染。 我们进一步预期，在我们的体内模型中缺乏完整的UL 128复合物将阻碍病毒传播到唾液腺和膀胱，并因此减少唾液和尿液中的病毒脱落。 此外，我们假设感染了缺乏UL 146 -147的菌株180.92的动物不会表现出嗜中性趋化性。 由于我们相信中性粒细胞也在系统性病毒传播中起作用，我们预期与野生型RhCMV相比，感染缺乏功能性UL 146 -147的CMV株的动物将表现出减少的病毒传播。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Human cytomegalovirus (HCMV) is a ubiquitous ¿-herpesvirus infecting 50-90% of the population. HCMV is transmitted through bodily secretions including saliva, urine, and breast milk. Once inside the body the virus infects epithelial and endothelial cells and eventually infects leukocytes, primarily monocytes and neutrophils, which then disseminate the virus systemically to multiple organs including salivary glands, urinary bladder, and mammary gland. Although asymptomatic in most cases, HCMV can cause fatal disease in immunocompromised patients after transplantation, in AIDS patients, and in the fetus. Although a need for a CMV vaccine has been emphasized, no successful vaccine has been developed to date. This is partially limited by the species specificity of cytomegaloviruses and the lack of a representative animal model that precisely mimics HCMV. A crucial step in all clinical HCMV cases is viral replication in endothelial cells and the associated viral dissemination mediated by leukocytes. HCMV is transmitted bidirectionally between endothelial cells and leukocytes through viral seizure of normal leukocyte-endothelial cell adhesion mechanisms, formation of microfusion, and permitting virus passage through direct intercellular transport. Preventing the dissemination of the virus in a host may reduce the severity of CMV disease and transmission of the virus to others. In vitro studies indicate that HCMV UL131-128 genes are essential for viral replication in endothelial cells and transfer to leukocytes. In addition, virally encoded chemotactic factors IL-8 and GRO¿ within UL146-147 strongly enhance virus passage to neutrophils in vitro. In vivo studies to date are limited. Beta-herpesviruses are highly species-specific. The closest relative to HCMV is chimpanzee CMV (CCMV), but this is not a practical animal model. Rhesus macaques are a more widely used experimental animal species and rhesus CMV (RhCMV) contains most of the HCMV gene families. Multiple reports have recently characterized genes in RhCMV that represent the HCMV UL131-128 and UL146-147 gene homologues. In a recent report, Lilja et. al. repaired the deleted UL131-UL128 locus of RhCMV strain 68-1 in a BAC-derived infectious virus and observed that the repaired derivative replicates much more efficiently than parental 68-1 virus in rhesus epithelial cells and in cultured human epithelial cells and endothelial cells. These recent achievements will facilitate examination of CMV cellular tropism and dissemination improving our understanding of the pathogenesis of CMV in both rhesus monkeys and humans. In this study we will examine the effects of UL131-128 and UL146-147 genes on the cellular tropism and dissemination of RhCMV. We will compare cellular tropism from in vivo infections in rhesus macaques with naturally occurring wild-type RhCMV, RhCMV strain 68-1 (lacking the UL131-128 gene region and having inverted UL146-147 gene region), and RhCMV strain 180.92 (lacking the UL146-147 gene region). We hypothesize that animals infected with strain 68-1, which lacks UL128 complex, will not exhibit endothelial infection. We further expect that lack of intact UL128 complex in our in vivo model will hamper viral dissemination to the salivary glands and urinary bladder, and consequently reduce viral shedding in saliva and urine. In addition, we hypothesize that animals infected with strain180.92, which lacks UL146-147, will not exhibit neutrophilic chemotaxis. Since we believe that neutrophils also play a role in systemic viral dissemination, we expect that animals infected with CMV strains lacking functional UL146-147 will exhibit reduced viral dissemination compared to the wild-type RhCMV.