The dynamics of mucosal cytomegalovirus replication and barriers to dissemination during acquisition of infection

获得感染期间粘膜巨细胞病毒复制的动态和传播障碍

• 批准号: RGPIN-2018-05666
• 负责人: Gantt, Soren
• 金额: $ 4.66万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761979
• 关键词: dynamics; mucosal; cytomegalovirus; replication; barriers

Cytomegalovirus (CMV) infects more than 90% of humans, and is primarily acquired at mucosal surfaces. Upon exposure, CMV initially replicates in mucosal epithelium and then spreads to establish life-long infection in distant anatomic sites. However, we have found that CMV acquisition is highly inefficient in humans, and infection is frequently transient without spread beyond the mucosa. We have determined that these natural CMV transmission dynamics are best explained by weak cell-to-cell spread from only 1 or very few infected mucosal cells at the start of infection. Thus, although life-long CMV infection eventually becomes established in most people, this typically occurs only after large numbers of exposures, most of which many are rapidly extinguished.This project aims to better define the basis of this severe transmission bottleneck, to understand why CMV infection does not occur. We will elucidate the earliest events of CMV acquisition, using in vitro, murine, and mathematical models to define the viral and host conditions that determine whether mucosal CMV replication persists and spreads, or terminates following exposure to a range of viral inoculum sizes. We hypothesize that CMV dynamics in mucosal epithelium are influenced by stochastic as well as host immune factors, and that infection terminates when viral replication does not reach a threshold required for dissemination. We also predict that innate immune barriers can be overcome by larger viral inocula. We will address these questions following specific aims: Aim 1: Define the dynamics of CMV mucosal replication and spread in cultured cells and mice. We will use wild-type and recombinant CMV strains to measure viral expansion in cultured epithelial cells and in the mucosa of mice by live bioimaging, PCR, and histopathology. We will also determine the frequency of self-limited mucosal infections vs. disseminated infections over a range of inoculum sizes.Aim 2: Identify the role of host innate immunity in determining successful CMV infection. We will identify the immune responses that constrain CMV spread in mucosal cells, by inducing or blocking antiviral factors as well as transcriptomics using in vitro and murine systems, including knock-out mice.Aim 3: Construct mathematical models to elucidate determinants of successful CMV infection. Detailed in vitro and murine data will be used to supplement our extensive human data, and allow refinement and expansion of our mathematical models of CMV acquisition. Model predictions will represent hypotheses to be tested in further rounds of experiments.This work will be conducted by pre- and postdoctoral trainees, in a highly collaborative interdisciplinary learning environment at UBC. By integrating state-of-the-art virology, immunology, and mathematical modeling data, we will gain fundamental insights into the determinants of whether a natural CMV infection succeeds or fails.

巨细胞病毒（CMV）感染超过90%的人类，并且主要在粘膜表面获得。暴露后，巨细胞病毒最初在粘膜上皮中复制，然后扩散到远处的解剖部位建立终身感染。然而，我们发现巨细胞病毒在人体内的获得效率非常低，感染通常是短暂的，不会扩散到粘膜之外。我们已经确定，这些自然的巨细胞病毒传播动力学最好的解释是，在感染开始时，只有1个或极少数被感染的粘膜细胞在细胞间传播弱。因此，尽管终生巨细胞病毒感染最终会在大多数人身上建立起来，但这通常只发生在大量暴露之后，其中许多暴露很快就消失了。该项目旨在更好地定义这种严重传播瓶颈的基础，以了解为什么巨细胞病毒感染没有发生。我们将阐明CMV获得的最早事件，使用体外、小鼠和数学模型来定义病毒和宿主条件，这些条件决定了粘膜CMV复制是否持续和扩散，或在暴露于一系列病毒接种量后终止。我们假设粘膜上皮中的巨细胞病毒动力学受到随机因素和宿主免疫因素的影响，并且当病毒复制未达到传播所需的阈值时感染终止。我们还预测，先天免疫屏障可以克服更大的病毒接种。我们将解决这些问题以下具体目标：目标1：定义巨细胞病毒粘膜复制和扩散的动态在培养细胞和小鼠。我们将使用野生型和重组CMV菌株，通过活体生物成像、PCR和组织病理学来测量病毒在培养的上皮细胞和小鼠粘膜中的扩增情况。我们还将在接种量范围内确定自限性粘膜感染与播散性感染的频率。目的2：确定宿主先天免疫在确定巨细胞病毒感染成功中的作用。我们将通过在体外和小鼠系统（包括敲除小鼠）中诱导或阻断抗病毒因子以及转录组学，确定抑制巨细胞病毒在粘膜细胞中传播的免疫反应。目的3：构建数学模型来阐明巨细胞病毒感染成功的决定因素。详细的体外和小鼠数据将用于补充我们广泛的人体数据，并允许改进和扩展我们的CMV获取的数学模型。模型预测将代表在进一步的实验中验证的假设。这项工作将在UBC高度协作的跨学科学习环境中由博士前和博士后学员进行。通过整合最先进的病毒学、免疫学和数学建模数据，我们将获得自然巨细胞病毒感染成功或失败的决定因素的基本见解。