Viral co-infection of differentiated airway cells pre-infected by influenza viruses

在呼吸道感染后，在人和动物的标本中，经常检测到不止一种感染源。继发感染可能导致更严重的病程。共感染过程中病原菌与寄主之间的分子相互作用尚未得到很好的描述。为了应用尽可能接近体内情况的体外模型，我们建立了猪呼吸道上皮细胞的气液界面培养体系，并利用该体系分析了流感病毒对分化的呼吸道上皮细胞的感染。感染导致纤毛细胞丢失。这种缺失被分化成特化细胞的基底细胞所弥补。这些细胞维持上皮的屏障功能；然而，在较长的时间内，它们不含纤毛，在此期间，它们不能促进粘液纤毛清除系统。此外，这些细胞在表面蛋白的表达模式上不同于纤毛细胞。基于这些发现，我们假设在再生过程中，感染灶对继发性病原体感染的易感性增加。在拟议的项目中，我们将分析被流感病毒感染的呼吸道表面细胞是否显示出对继发性病原体感染的易感性增加。关于二次病原体，我们将重点放在病毒上。在项目的第一阶段，我们将应用不同的方法来表征流感病毒感染后再生过程中表面蛋白表达模式的变化。我们将特别关注一种病毒受体，猪氨肽酶N(PAPN)，猪呼吸道冠状病毒(PRCoV)的细胞受体。在下一章中，我们将分析先前感染流感病毒对随后感染PRCoV的影响。第二种模式病毒是猪繁殖与呼吸综合征病毒(PRRSV)。这种病毒以巨噬细胞为主要靶细胞。我们将确定感染PRRSV的巨噬细胞在感染流感病毒后对呼吸道上皮细胞的黏附增加到什么程度；此外，我们将分析黏附巨噬细胞是否能够将PRRSV感染传播到呼吸道上皮屏障。最后，我们将确定用猪呼吸道细胞获得的结果是否也适用于人类呼吸道上皮细胞。该项目的结果将揭示以前感染流感病毒在多大程度上促进了二次病毒感染。通过这种方式，我们将增加关于病原体与宿主在联合感染过程中相互作用的知识。

After infection of the airways, in both human and animal specimen, often more than one infectious agent is detected. Secondary infections may result in a more severe course of the disease. The molecular interactions between the pathogens and the host during co-infection are not well characterized. To apply an in vitro model which mimics the in vivo situation as closely as possible, we have established an air-liquid interface culture system for porcine airway epithelial cells and used it to analyze the infection of differentiated respiratory epithelial cells by influenza viruses. Infection resulted in the loss of ciliated cells. The loss was compensated by basal cells that differentiated into specialized cells. These cells maintain the barrier function of the epithelium; however, for a longer time period they do not contain cilia and during this time, they cannot contribute to the mucociliary clearance system. Furhermore, these cells differ from ciliated cells in the expression pattern of surface proteins. Based on these findings we hypothesize that during the regeneration, the foci of infection have an increased susceptibility to infection by secondary pathogens. In the proposed project we will analyse whether airway eptihelial cells infected by influenza viruses show an increased susceptibility to infection by secondary pathogens. With respect to secondary pathogens, we will focus on viruses. In the first stage of the project, we will apply different methods to characterize changes in the expression pattern of surface proteins during regeneration after an infection by influenza viruses. Our special focus will be on a virus receptor, porcine amonopeptidase N (pAPN), the cellular receptor for porcine respiratory coronavirus (PRCoV). In the following chapter, we will analyse how a prior infection by influenza viruses affects the subsequent infection by PRCoV. The second model virus is porcine reproductive and respiratory syndrome virus (PRRSV). This virus uses macrophages as primary target cells. We will determine to what extent the adherence of PRRSV-infected macrophages to the airway epithelium is increased after an infection by influenza viruses; furthermore, we will analyse whether the adhering macrophages are able to transmit the PRRSV infection across the barrier of the airway epithelium. Finally, we will determine whether the results obtained with porcine airway cells are valid also for human respiratory epithelial cells. The results of this project will reveal to what extent prior infection by influenza viruses facilitates infection by secondary viral infection. In this way, we will increase the knowledge about the interactions between pathogens and the host during co-infections.