A three-dimensional vessel-on-chip model to study Puumala orthohantavirus pathogenesis

Puumala orthohantavirus (PUUV) infection in humans can result in hemorrhagic fever with renal syndrome. Endothelial cells (ECs) are primarily infected with increased vascular permeability as a central aspect of pathogenesis. Historically, most studies included ECs cultured under static two-dimensional (2D) conditions, thereby not recapitulating the physiological environment due to their lack of flow and inherent pro-inflammatory state. Here, we present a high-throughput method for culturing primary human umbilical vein ECs in 3D vessels-on-chip in which we compared host responses of these ECs to those of static 2D-cultured ECs on a transcriptional level. The phenotype of ECs in vessels-on-chip more closely resembled the in vivo situation due to higher similarity in expression of genes encoding described markers for disease severity and coagulopathy, including IDO1, LGALS3BP, IL6 and PLAT, and more diverse endothelial-leukocyte interactions in the context of PUUV infection. In these vessels-on-chip, PUUV infection did not directly increase vascular permeability, but increased monocyte adhesion. This platform can be used for studying pathogenesis and assessment of possible therapeutics for other endotheliotropic viruses even in high biocontainment facilities. Overall design: To investigate endothelial cell responses in the context of Puumala orthohantavirus in a physiologically relevant model, we infected primary human umbilical vein endothelial cells, cultured either in three-dimensional (3D) endothelial vessels-on-chip or static 2D in 96-wells plates, in triplicate and performed bulk RNA-Sequencing. Additionally, to demonstrate the added value of the vessel-on-chip system, we included parallel conditions where ECs were treated with tumor necrosis factor alpha (TNFa) in both culture systems. We then compared differential gene expressions and performed pathway enrichment analyses between virus-, mock-infected and TNFa-treated samples.