Mode of virus transmission into the brain in a mouse model of HIV-infecion

描述(申请人提供)：为了了解人类感染HIV的神经系统并发症的需要，我们基于一种新的嵌合HIV，EcoHIV构建了一个系统性HIV感染的小鼠模型。小鼠的EcoHIV感染特别适合于研究病毒在脑内的复制和疾病，因为病毒感染巨噬细胞和淋巴细胞，在接种后早期进入大脑，导致脑细胞基因表达的变化，以及一些小鼠的神经病理。在这项修订的应用中，我们的主要目标是利用小鼠的EcoHIV感染来最终确定HIV-1是通过感染的单核细胞进入大脑还是通过其他机制在大脑中建立感染。具体目标是1)优化单核细胞谱系中不同细胞类型的EcoHIV感染；2)比较无细胞病毒和感染的单核/巨噬细胞在脑中建立EcoHIV感染的效率；以及3)测试T细胞缺陷和单核细胞耗尽小鼠的EcoHIV进入大脑和负载。这些方法将包括有效的单核细胞分化和小胶质细胞分离系统，从大脑中分离DNA和RNA的优化方法，病毒DNA和选定的脑细胞转录本的定量聚合酶链式反应，明确识别感染细胞的两种彩色免疫荧光显微镜，以及通过接种受感染的同基因单核细胞对小鼠的感染；以及使用定量聚合酶链式反应对单核细胞向大脑迁移的定量测定。这些研究通过在动物模型中测试艾滋病毒-1神经侵袭性的关键假说，满足了PAS 05078的挑战。明确病毒进入大脑的传播模式(S)将为一项重大研究奠定基础，该研究旨在通过耗尽艾滋病毒的细胞储存库或通过控制总的游离病毒负担来研究抑制脑感染的最佳途径。由于HIV-1感染者在运动和记忆方面继续出现问题，因此有必要控制并最终阻止大脑中的病毒感染。这里提出的研究将利用实验室小鼠和一种新型艾滋病毒来明确确定病毒如何进入大脑以建立感染，以开发更好的方法来阻断艾滋病毒疾病的这一方面，并改善受感染人群的健康。

DESCRIPTION (provided by applicant): In response to the need to understand the neurological complications of HIV infection of human beings, we have constructed a mouse model of systemic HIV infection based upon a new chimeric HIV, EcoHIV. EcoHIV infection of mice is particularly appropriate to study of virus replication and disease in the brain because the virus infects macrophages as well as lymphocytes, enters the brain early after inoculation, induces changes in brain cell gene expression, and neuropathology in some mice. Our primary goal in this revised application is to employ EcoHIV infection of mice to determine conclusively whether HIV-1 establishes infection in the brain through entry of infected monocytes or through other mechanisms. The Specific Aims are 1) To optimize EcoHIV infection in different cell types in the monocyte lineage; 2) To compare cell-free virus to infected monocyte/macrophages for the efficiency of establishment of EcoHIV infection in the brain; and 3) To test T cell-deficient and monocyte-depleted mice for EcoHIV entry and load in the brain. The methods will include efficient systems for monocyte differentiation and microglial cell isolation, optimized approaches for isolation of DNA and RNA from the brain, quantitative PCR for viral DNA and selected brain cell transcripts, two color immunofluorescence microscopy to unambiguously identify infected cells, infection of mice by inoculation of infected isogenic monocytes; and quantitative determination of monocyte migration into the brain using QPCR for a unique cellular marker. These studies meet the challenge of PAS 05078 by testing a key hypothesis of HIV-1 neuroinvasiveness in an animal model. Clear identification of the mode(s) of virus transmission into the brain will build the foundation for a major study to investigate the optimal route to inhibit brain infection either by depletion of cellular reservoirs of HIV or by control of overall burden of free virus. Because HIV-1 infected people continue to develop problems in movement and memory there is a need to control and ultimately block virus infection in the brain. The study proposed here will employ laboratory mice and a new form of HIV to clearly determine how the virus enters the brain to establish infection to develop better methods to block this facet of HIV disease and improve health for the infected population.