Direct effects on endothelial cells are essential for perivascular cell (pericyte)-dependent amplification of orthoflavivirus NS1-mediated microvascular leakage

The emerging tick-borne flaviviruses Alkhumra haemorrhagic fever virus (AHFV) and Kyasanur Forest disease virus (KFDV) can cause severe haemorrhagic disease, yet the mechanisms driving vascular leakage remain unclear. The microvascular capillaries and post-capillary venues affected during orthoflavivirus-related haemorrhagic disease consist of an endothelial cell barrier ensheathed in and supported by perivascular cells (pericytes), and we recently identified a critical role for pericytes in amplifying dengue microvascular dysfunction. The orthoflavivirus non-structural protein 1 (NS1) has been implicated in endothelial dysfunction and vascular leakage for several tick- and mosquito-borne flaviviruses. Here, we examined whether NS1 from AHFV and KFDV disrupts microvascular barrier integrity in pericyte-endothelial cell cocultures. Under our experimental conditions, we found AHFV and KFDV NS1 to disrupt the ability of pericytes to support endothelial cell function, which is required for maintenance of the microvascular barrier, but saw no endothelial hyperpermeability when NS1-treated endothelial cells were cultured alone or in combination with pericytes. Our findings suggest that a direct effect of NS1 on endothelial dysfunction is required for pericyte-driven amplification of hyperpermeability, and that effects on pericytes alone are insufficient to trigger microvascular leakage. Our work highlights the synergistic effect of microvascular cells during orthoflavivirus haemorrhage and emphasises the need for further work into the mechanisms of vascular dysfunction during AHFV and KFDV infection.