Identification, cloning, and in vitro characterization of transmitted/founder vir

HIV/SIV疫苗研究中最重要的知识差距之一与生物学有关 负责粘膜传播的病毒的特性。在这个HIVRAD财团中， 该项目将利用我们实验室最近发现的一种新的 识别经粘膜传播的HIV-1和SIV病毒基因组的策略(Salazar等人，J Virol，2008； Keele，PNAS，2008；Salazar，J Exp Med，2009；Keele，J Exp Med，2009)。这一战略是基于 模型背景下血浆病毒RNA的单基因组扩增、测序和分析 随机病毒进化，识别那些实际负责传播和生产的病毒 感染。这项创新反过来又使分子克隆和生物学首次成为可能 这种传播/创始病毒基因组的特征。在这个项目中，我们建议将这一点延长 使SIV-猕猴感染模型的技术能够检验以下假设： 传播性/创建者SIV将揭示细胞嗜性、受体和共受体使用的特定属性， 和中和表型，这是通过直肠传播成功的必要条件和充分条件， 经阴道和静脉感染途径。具体目标包括： 1.鉴定和计数下列印度恒河猴传播/创始病毒 新的遗传多样性SIVsmm菌株的直肠内、阴道内和静脉感染。 分子克隆传播型/方块型病毒的全长基因组 经直肠、阴道和静脉途径感染。 目的：确定分子克隆传播型/方正病毒的体外生物学特性。 准备在印度恒河猴体内进行体内测试的传播/创始病毒。 这些研究的结果将为了解粘膜HIV/SIV的分子机制提供新的线索 传播，识别疫苗引发的免疫反应的新靶点，并产生急需的一组 新的遗传多样性的SIV挑战毒株用于传播、发病机制和疫苗研究。

One of the most important knowledge gaps in HIV/SIV vaccine research relates to the biological properties of viruses that are responsible for mucosal transmission. Within this HIVRAD consortium, this project will address this priority area by taking advantage of our laboratory's recent discovery of a novel strategy for identifying mucosally transmitted HIV-1 and SIV viral genomes (Salazar et al., J Virol, 2008; Keele, PNAS, 2008; Salazar, J Exp Med, 2009; Keele, J Exp Med, 2009). This strategy, which is based on single genome amplification, sequencing and analysis of plasma viral RNA within the context of a model of random virus evolution, identifies those viruses that are actually responsible for transmission and productive infection. This innovation, in turn, makes possible for the first time the molecular cloning and biological characterization of such transmitted/founder virus genomes. In this project, we propose to extend this enabling technology to the SIV-macaque infection model to test the following hypothesis: Molecular clones of transmitted/founder SIVs will reveal the particular properties of cell tropism, receptor and co-receptor usage, and neutralization phenotype that are necessary and sufficient for successful transmission via intrarectal, intravaginal and intravenous infection routes. Specific aims include: 1. To identify and enumerate transmitted/founder viruses in Indian rhesus macaques following intrarectal, intravaginal and intravenous infection with new genetically diverse SIVsmm strains. To moleculariy clone full-length genomes of transmitted/founder viruses responsible for productive infection by intrarectal, intravaginal and intravenous routes. To determine the in vitro biological properties of molecular clone-derived transmitted/founder viruses. To prepare stocks of transmitted/founder viruses for in vivo testing in Indian rhesus macaques. Results from these studies will shed new light on the molecular mechanisms underiying mucosal HIV/SIV transmission, identify new targets for vaccine-elicited immune responses, and generate a much needed set of new genetically-diverse SIV challenge strains for transmission, pathogenesis and vaccine research.