Prior swine influenza virus infection of differentiated airway epithelial cells enhances secondary infection by avian influenza virus and facilitates the generation of reassortant viruses

猪在甲型流感病毒（IAV）的流行病学中发挥着重要作用，特别是在传播到世界各地并与发病率和死亡率增加相关的大流行病毒的产生方面。这类病毒通常是含有来自猪、禽和/或人IAV的基因片段的重组体。相应的重配事件需要来自不同物种的病毒共同感染气道细胞。猪被认为是最有可能产生能够感染人气道细胞的抵抗病毒的宿主。由于猪在禽IAV向人类的种间传播中的这种作用，猪被指定为“混合容器”。以前，猪细胞被认为含有猪/人IAV（α 2，6-连接的唾液酸）和禽IAV（α 2，3-连接的唾液酸）的结合位点，因此比人细胞更适合禽和人/猪病毒的共感染。然而，人和猪气道中的唾液酸分布已被证明非常相似。我们假设，从猪IAV感染中恢复的猪气道细胞是IAV共感染的最有希望的候选细胞。在再生阶段，当基底细胞分化成特化细胞时，这些细胞缺乏纤毛一段时间，因此其粘膜纤毛清除功能受损。此外，唾液酸的表达介于基底细胞（α 2，3-linkage型）和分化良好的上皮细胞（α 2，6-linkage型）之间，即它们包含两种连接类型。为了证实这一假设，我将应用气-液界面培养来比较猪（SW）和禽IAV对分化良好的猪气道上皮细胞的单一感染。在第二步中，将表征从swIAV感染中恢复的呼吸道上皮细胞，特别关注唾液酸。除凝集素染色外，还将进行不同IAV的结合试验，以确定再生期细胞与分化良好的细胞在人、猪和禽类IAV结合位点方面是否不同。在第三部分中，将分析猪和禽IAV对猪气道细胞的共感染。我们将比较两种合并感染的情况：（i）猪和禽病毒伴侣同时加入到分化良好的气道上皮细胞中;（ii）禽IAV合并感染发生在从swIAV感染中恢复的气道细胞中。在项目的最后一部分，我将确定两种合并感染情况下的重组率。此外，将分析选择的重组病毒感染人分化的气道上皮细胞的能力。该项目的结果将为禽流感病毒和猪/人流感病毒共同感染气道上皮细胞提供新的见解，从而更好地了解大流行性流感病毒的产生。

Pigs play an important role in the epidemiology of influenza A viruses (IAV), especially in the generation of pandemic viruses that spread all over the world and are associated with increased morbidity and mortality. Such viruses are usually reassortants containing gene segments from swine, avian, and/or human IAV. The respective reassortment events require the co-infection of airway cells by viruses from different species. Pigs are considered the most likely host for generating reassortant viruses that are able to infect human airway cells. Because of this role in the interspecies transmission of avian IAV to humans, pigs have been designated as "mixing vessel". Previously, porcine cells have been thought to contain binding sites for both swine/human IAV (alpha2,6-linked sialic acids) and avian IAV (alpha2,3-linked sialic acids) and therefore to be more suited than human cells for co-infection by avian and human/swine viruses. However, the sialic acid distributions in the human and swine airways have been shown to be very similar. We hypothesize, that swine airway cells recovering from a swine IAV infection are most promising candidate cells for co-infection by IAV. During the regeneration phase, when basal cells are differentiating into specialized cells, these cells lack for some time cilia and therefore are impaired in their mucociliary clearance function. Furthermore, the sialic acid expression is intermediate between that of basal cells (alpha2,3-linkeage type) and well-differentiated epithelial cells (alpha2,6-linkage type), i.e, they contain both linkage types.To confirm this hypothesis, I will apply air-liquid interface cultures to compare the mono-infection of well-differentiated swine airway epithelial cells by swine (sw) and avian IAV. In a second step, respiratory epithelial cells recovering from a swIAV infection will be characterized, with a special focus on the sialic acids. In addition to lectin staining, binding assays with different IAV will be performed to find out whether cells in the regeneration phase differ from well-differentiated cells with respect to binding sites for human, swine and avian IAV. In the third section, co-infection of swine airway cells by swine and avian IAV will be analyzed. We will compare two co-infection scenarios: (i) the swine and the avian virus partners are added at the same time to well-differentiated airway epithelial cells; (ii) co-infection by avian IAV occurs with airway cells recocvering from a swIAV infection. In the final part of the project, I will determine the reassortment rates of the two co-infection scenarios. Furthermore, selected reassortant viruses will be analyzed for their ability to infect human differentiated airway epithelial cells. The results of this project will provide new insights into the co-infection of airway epithelial cells by avian and swine/human viruses and thus provide a better understanding of the generation of pandemic influenza viruses.