Overcoming the airway epithelium barrier in the early phase of bovine viral diarrhea virus (BVDV) infection

牛病毒性腹泻/粘液性腹泻病（BVD/ MD）是一种重要的牛传染病。病原体BVD病毒（BVDV）是黄病毒科瘟病毒属的一种正链RNA病毒。已知BVDV经口鼻和呼吸道进入，并从呼吸道传播至各种器官和组织。对感染的初始阶段知之甚少。在拟定研究项目的初步工作背景下，表明未分化极化呼吸道上皮细胞对BVDV的顶侧和基底侧感染高度敏感，但病毒释放仅通过细胞的顶侧发生。因此，瘟病毒如何穿过气道上皮屏障仍是未知的。然而，已经确定BVDV和其他瘟病毒对免疫细胞具有很强的嗜性。因此，本项目的一个重要目标是探索BVDV通过呼吸道上皮屏障并从那里传播到免疫细胞的途径，在项目的第一部分，将研究气道上皮细胞（气管/支气管上皮细胞）的感染。在病毒学研究所建立的两种细胞培养系统可用于终末分化细胞的分析：气液界面（ALI）培养和精密肺切片（PCLS）。随后，将检查尚未完成分化的细胞或上皮损伤后处于再生阶段的细胞。最后，我们将分析BVDV是否可以通过利用细胞间的渗漏连接以细胞旁方式克服上皮屏障。为了确定不同培养系统和不同感染条件下的不同感染特征（取决于分化程度）是否与细胞受体的存在相关或解释，将分析BVDV受体CD 46的表达。为了检测CD 46，可获得病毒学研究所生产的单克隆抗体。另一个重点将是对巨噬细胞感染的研究。首先，将研究巨噬细胞单一培养物的感染。随后，将建立巨噬细胞和气道上皮细胞的共培养物，以发现巨噬细胞感染是否可用于克服上皮屏障。未来项目的其他有趣观点包括细胞先天免疫在呼吸道上皮感染BVDV和病毒-病毒或病毒-细菌共感染中的作用。

Bovine Viral Diarrhea/ Mucosal Disease (BVD/ MD) is an economically important notifiable disease of cattle. The causative agent, BVD virus (BVDV), is a plus strand RNA virus of the family Flaviviridae, genus Pestivirus. BVDV is known to enter oronasally and through the respiratory tract, from where it spreads to various organs and tissues. The initial stage of infection is poorly understood. In the context of preliminary work for the proposed research project it was shown that non-differentiated polarized respiratory epithelial cells are highly susceptible to apical and basolateral infection with BVDV, but virus release occurs only via the apical side of the cells. Thus, it remains unknown how pestiviruses cross the barrier of the airway epithelium. However, it is well established that BVDV and other pestiviruses have a strong tropism for immune cells. Therefore, an important goal of this project is to explore the pathway by which BVDV crosses the barrier of the respiratory epithelium and spreads from there to immune cells.In the first part of the project, the infection of airway epithelial cells (tracheal/bronchial epithelial cells) will be investigated. Two cell culture systems established at the Institute of Virology are available for the analysis of end-differentiated cells: Air-liquid-interface (ALI) cultures and precision lung slices (PCLS). Subsequently, cells that have not yet completed differentiation or cells that are in the regeneration phase after epithelial injury will be examined. Finally, we will analyze whether BVDV can overcome the epithelial barrier in a paracellular manner by exploiting leaky junctions between cells. To find out whether the different infection characteristics in the different culture systems and under the various infection conditions depending on the degree of differentiation can be correlated or explained with the presence of the cellular receptor, the expression of the receptor for BVDV, CD46, will be analyzed. For the detection of CD46 a monoclonal antibody is available, which was produced at the Institute of Virology. Another focus will be studies on the infection of macrophages. First, the infection of macrophage monocultures will be investigated. Subsequently, co-cultures of macrophages and airway epithelial cells will be established to find out whether macrophage infection can be used to overcome the epithelial barrier. Other interesting perspectives for future projects include the role of cellular innate immunity for respiratory epithelial infection with BVDV, and viral-viral or viral-bacterial co-infections.