Modeling influenza virus replication in primary human lung cells

描述(申请人提供)：描述对高致病性禽流感(H5N1)人类大流行的关注集中在几个相关问题上：了解甲型流感感染的发病机制和传播，大流行前对潜在大流行毒株的识别，以及大流行前疫苗的开发。虽然分子生物学已经提供了对永生化细胞中复制周期的详细了解，但人类呼吸道不同表型上皮细胞之间完整组织水平上的流感复制仍然知之甚少。我们缺少人类呼吸道的定量动力学解释，以及一种菌株(但不是密切相关的菌株)如何启动人与人之间的传播。我们建议对人类呼吸道上皮细胞的早期流感病毒粒子生产力进行建模。该体外模型使用在气液界面(ALI)培养中生长的原代人类分化的气管、支气管和细支气管肺泡上皮细胞来记录人类病原体(H3N2株和H5N1禽流感株)的病毒粒子生成动力学。基于代理的计算机模型是由我们中的一个人(CB)设计和实现的二维元胞自动机风格的模拟。从体外模型收集的动力学数据将用于验证计算机模型的细节和参数值，然后计算机模型将用于研究体外模型中无法测量的变量和结果。该项目的目标是利用实验获得的流感病毒在原代人肺上皮细胞中复制的动力学数据来计算病毒生长的指标，特别是Ro(繁殖率，一个原代细胞产生的平均二次感染细胞数)，以获得与人体组织相关的毒力和传播力指标，能够在不建立昂贵的体内模型的情况下区分毒株。使用体外模型的数据，我们将确定计算机模型中的以下参数：单位细胞病毒产量、病毒粒子扩散率和上皮细胞吸收病毒粒子的单位速率。H3N2型人类致病性甲型流感毒株的动力学将与选定的人类致病性H5N1禽流感毒株在实验和计算机模型中进行比较。我们将实验性地操纵ALI培养的环境，以增强或减少固有宿主反应的成分，包括粘蛋白和表面活性物质收集素。将在计算机模型中实施类似的操作，并对所有不同的菌株在两种模型之间的结果进行比较。然后，我们将使用基于代理的模型来系统地研究每个固有粘膜防御元件对每个流感毒株的R0的个人和协同影响的相对贡献。该项目开发了一种测试，旨在区分具有威胁性的禽流感病毒株和非威胁性禽流感病毒株。该测试测量流感病毒在培养的成熟人肺呼吸道细胞中的生长，并使用数学分析来计算病毒通过培养物的传播。禽流感被许多人认为是世界上对公共卫生的下一个最大威胁。

DESCRIPTION (provided by applicant): Description Concern about a human pandemic of highly pathogenic avian (H5N1) influenza has focused attention on several related problems: understanding the pathogenesis and transmission of influenza A infections, pre-pandemic recognition of potential pandemic strains, and pre-pandemic vaccine development. Although molecular biology has provided a detailed understanding of the replication cycle in immortalized cells, influenza replication at the intact tissue level among phenotypically diverse epithelial cells of the human respiratory tract remains poorly understood. We are missing the quantitative kinetics accounting in the human airway and how one strain, but not a closely related strain, can initiate person-to-person transmission. We propose to model early influenza virion productivity in human airway epithelial cells. The in vitro model uses primary human differentiated tracheobronchial and bronchiolo-alveolar epithelial cells grown in air-liquid interface (ALI) culture to document the kinetics of virion productivity of human pathogens, both H3N2 strains and H5N1 avian influenza strains. The agent-based computer model is a two-dimensional cellular automata style simulation, designed and implemented by one of us (CB). Kinetic data collected from the in vitro model will be used validate the details and parameter values of the computer model, and then the computer model will be used to study unmeasurable variables and outcomes in the in vitro model. The goal of the project is to use experimentally derived kinetic data of influenza virus replication in primary human lung epithelial cells to calculate metrics of viral growth, particularly the Ro (reproductive rate, average number of secondary infected cells produced by one primary cell), in order to obtain a human tissue-relevant indicator of virulence and transmissibility, capable of distinguishing strains without expensive in vivo models. Using data from the in vitro model, we will determine the following parameters in the computer model: unit rate of viral production per cell, virion diffusion rate, and the unit rate of virion absorption by epithelial cells. The kinetics of strains of H3N2 human pathogenic influenza A will be compared to those of selected human pathogenic H5N1 avian influenza strains, both experimentally and in the computer model. We will manipulate the environment of the ALI culture experimentally to augment or diminish components of the innate host response including mucins and surfactant collectins. Analogous manipulations will be implemented in the computer model and results compared between the two models for all the different strains. We will then use the agent-based model to systematically study the relative contribution of each innate mucosal defense element, both in terms of individual and collaborative impacts on the R0 for each influenza strain. This project develops a test designed to distinguish threatening bird flu strains from non-threatening strains. The test measures influenza virus growth in cultured mature human lung airway cells and uses mathematical analysis to calculate the spread of the virus through the culture. Bird flu is considered by many to be potentially the next greatest threat to public health in the world.