Lymphatic vessel dysfunction contributes to severe dengue pathogenesis

Dengue virus (DENV) infection is a major global health threat, affecting more than half of the world’s population. Severe dengue is a life-threatening condition characterised by systemic bleeding, vascular leakage, and interstitial fluid accumulation that can progress to hypovolaemic shock. Circulating DENV non-structural protein 1 (NS1) has long been implicated in driving vascular hyperpermeability through its disruptive effects on endothelial cell junctions and the glycocalyx. The lymphatic system, which runs alongside the vascular network, plays a critical role in resorbing and recirculating interstitial fluid and immune cells extravasated from blood vessels. Despite its importance in maintaining tissue fluid homeostasis, the impact of dengue disease on lymphatic vessels has not previously been explored. Here, we present the first evidence that DENV-2 NS1 induces marked hyperpermeability in lymphatic endothelial cells, as measured by transendothelial electrical resistance, and impairs lymphangiogenesis in vitro. These effects were not attributable to changes in cell viability, morphology, or metabolic activity, as assessed by live/dead and metabolic assays and image analysis. Instead, we observed a defect in lymphatic endothelial cell migration, measured by scratch assay, which may underlie the reduced lymphangiogenic potential. Bulk RNA-seq, immunocytochemistry, and advanced image analysis further demonstrated pronounced reorganisation of cell–cell junctions, the cytoskeleton, and focal adhesions. Notably, junctional proteins including VE-cadherin, ZO-1, and Claudin-5 were not downregulated but instead displayed disorganised distribution along the cell junctions or aberrant cytoplasmic localisation. These structural disruptions became even more pronounced under flow conditions produced using a microfluidic system. Together, these findings demonstrate for the first time that DENV-2 NS1 directly disrupts lymphatic endothelial cell function, leading to junctional disorganisation and hyperpermeability. Such impairment of lymphatic drainage may contribute to the pathophysiology of severe dengue. ### Competing Interest Statement The authors have declared no competing interest.