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

描述（由申请人提供）：为了了解人类HIV感染的神经系统并发症的需要，我们基于一种新的嵌合HIV， EcoHIV构建了一种全身HIV感染的小鼠模型。小鼠的EcoHIV感染特别适合研究病毒在大脑中的复制和疾病，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，诱导脑细胞基因表达的变化，并在一些小鼠中引起神经病理。在这一修订后的应用中，我们的主要目标是利用小鼠的EcoHIV感染来确定HIV-1是否通过感染单核细胞的进入或通过其他机制在大脑中建立感染。具体目的是：1)优化单核细胞系中不同细胞类型的EcoHIV感染；2)比较无细胞病毒与感染单核/巨噬细胞在脑内建立EcoHIV感染的效率；3)检测T细胞缺陷和单核细胞缺失小鼠的EcoHIV在大脑中的进入和负荷。这些方法将包括单核细胞分化和小胶质细胞分离的高效系统，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物的定量PCR，明确识别感染细胞的双色免疫荧光显微镜，通过接种感染的等基因单核细胞感染小鼠；以及利用QPCR作为一种独特的细胞标记物，定量测定单核细胞向大脑的迁移。这些研究通过在动物模型中测试HIV-1神经侵袭性的关键假设来满足PAS 05078的挑战。明确确定病毒进入大脑的传播模式将为一项重大研究奠定基础，该研究旨在通过耗尽HIV细胞储存库或通过控制游离病毒的总体负担来探索抑制大脑感染的最佳途径。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病毒在大脑中的感染。这里提出的研究将使用实验室小鼠和一种新形式的艾滋病毒来清楚地确定病毒如何进入大脑建立感染，以开发更好的方法来阻止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.