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

牛病毒性腹泻/黏膜病（BVD/ MD）是牛的一种重要的经济通报疾病。BVD病毒（BVDV）是一种黄病毒科鼠疫病毒属的正链RNA病毒。已知BVDV通过口鼻和呼吸道进入，从那里扩散到各种器官和组织。人们对感染的初始阶段知之甚少。在该研究项目的前期工作中，研究人员发现未分化的极化呼吸道上皮细胞对BVDV的根尖和基底侧感染高度敏感，但病毒仅通过细胞的根尖侧释放。因此，尚不清楚鼠疫病毒是如何穿过气道上皮屏障的。然而，已经确定BVDV和其他鼠疫病毒对免疫细胞有很强的趋向性。因此，本项目的一个重要目标是探索BVDV穿过呼吸道上皮屏障并从那里扩散到免疫细胞的途径。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。病毒学研究所建立的两种细胞培养系统可用于分析终末分化细胞：空气-液界面（ALI）培养和精确肺切片（PCLS）。随后，对尚未完成分化的细胞或上皮损伤后处于再生阶段的细胞进行检查。最后，我们将分析BVDV是否能够通过利用细胞间的渗漏连接以细胞旁方式克服上皮屏障。为了了解在不同的培养体系和不同的感染条件下，不同的感染特征取决于分化程度，是否可以与细胞受体的存在相关或解释，我们将分析BVDV受体CD46的表达。用于检测CD46的单克隆抗体是可用的，它是由病毒学研究所生产的。另一个重点将是研究巨噬细胞的感染。首先，研究单核巨噬细胞的感染情况。随后，我们将建立巨噬细胞与气道上皮细胞共培养，研究是否可以利用巨噬细胞感染来克服上皮屏障。未来项目的其他有趣观点包括细胞先天免疫在BVDV呼吸道上皮感染中的作用，以及病毒-病毒或病毒-细菌共感染。

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.