Core 2: Virology, Molecular, and Histology Core

摘要-病毒学、分子和组织学核心(核心2) 众所周知，尽管严格遵守协议，但隐藏或微妙的因素在 实验室可以使与病毒繁殖和qPCR相关的技术产生不同的 结果。因此，为了确保跨项目的一致性，最大限度地减少混淆，并最大限度地提高成果的影响，a 提出了病毒学、分子和组织学核心。这一核心将由彼得·巴里博士领导，他为 现有和成熟的病毒库繁殖和病毒参数分析技术 感染(包括病毒载量、组织病理学和抗原表达的量化)。这些方法 是严格测试该项目涉及免疫学(项目1)和病毒学的假设的中心 (项目2)先天性巨细胞病毒感染(CCMV)的决定因素，以及为NHP核心产生病毒储备， 用于评估病毒变种的出现/选择的纯化DAN(核心3)，以及病毒的可量化测量 用于严格统计分析的复制和疾病(核心4)。病毒学、分子、 和组织学核心如下：特征良好的、高滴度的上皮嗜性类群 (目标1)，对接种动物的组织和体液进行灵敏分析以量化RhCMV基因组复制 通过聚合酶链式反应(QPCR)(AIM 2)和组织病理学/免疫组织化学(IHC)分析来表征 病毒病理学和抗原表达(目标3)。提供单一来源的病毒和敏感和 对RhCMV基因组拷贝数和抗原表达的特定分析将使统一的起始材料 和分析技术--对评估该计划的总体目标至关重要的能力： 明确影响CCMV传播的母体免疫反应和病毒特征 胎儿感染的结局。

ABSTRACT – Virology, Molecular, and Histology Core (Core 2) It is well known that, despite strict adherence to protocols, hidden or subtle factors that vary among laboratories can cause techniques such as those concerning virus propagation and qPCR to produce differing outcomes. Thus, to ensure cross-project consistency, minimize confounding, and maximize impact of results, a Virology, Molecular, and Histology Core is proposed. This Core will be led by Dr. Peter Barry, who brings to the Program extant and well established techniques in virus stock propagation and analyses of viral parameters of infection (including quantification of viral loads, histopathology, and antigen expression). These approaches are central to rigorous testing of the project's hypotheses addressing the immunologic (Project 1) and virologic (Project 2) determinants of congenital CMV infection (cCMV), and generating virus stocks for the NHP Core, purified DAN to assess for emergence/selection of viral variants (Core 3), and quantifiable measures of viral replication and disease for rigorous statistical analyses (Core 4). Specific outputs of the Virology, Molecular, and Histology Core are as follows: well-characterized, high titer stocks of epithelial-tropic stocks of RhCMV (Aim 1), sensitive analyses of tissues and fluids from inoculated animals to quantify RhCMV genome copy numbers by PCR (qPCR) (Aim 2), and histopathologic/immunohistochemical (IHC) analyses to characterize viral pathology and antigen expression (Aim 3). Providing singly sourced stocks of virus and sensitive and specific assays for RhCMV genome copy numbers and antigen expression will enable uniform starting material and analytic techniques—capabilities that are critical for evaluating the overall objective of this Program: to define the maternal immune responses and viral characteristics that impact cCMV transmission and the outcome of fetal infection.