Dustin R. Glasner

The four dengue virus serotypes (DENV1 to DENV4) are mosquito-borne flaviviruses that cause up to ~100 million cases of dengue annually worldwide. Severe disease is thought to result from immunopathogenic processes involving serotype cross-reactive antibodies and T cells that together induce vasoactive cytokines, causing vascular leakage that leads to shock. However, no viral proteins have been directly implicated in triggering endothelial permeability, which results in vascular leakage. DENV nonstructural protein 1 (NS1) is secreted and circulates in patients' blood during acute infection; high levels of NS1 are associated with severe disease. We show that inoculation of mice with DENV NS1 alone induces both vascular leakage and production of key inflammatory cytokines. Furthermore, simultaneous administration of NS1 with a sublethal dose of DENV2 results in a lethal vascular leak syndrome. We also demonstrate that NS1 from DENV1, DENV2, DENV3, and DENV4 triggers endothelial barrier dysfunction, causing increased permeability of human endothelial cell monolayers in vitro. These pathogenic effects of physiologically relevant amounts of NS1 in vivo and in vitro were blocked by NS1-immune polyclonal mouse serum or monoclonal antibodies to NS1, and immunization of mice with NS1 from DENV1 to DENV4 protected against lethal DENV2 challenge. These findings add an important and previously overlooked component to the causes of dengue vascular leak, identify a new potential target for dengue therapeutics, and support inclusion of NS1 in dengue vaccines.

Dengue is the most prevalent arboviral disease in humans and a major public health problem worldwide. Systemic plasma leakage, leading to hypovolemic shock and potentially fatal complications, is a critical determinant of dengue severity. Recently, we and others described a novel pathogenic effect of secreted dengue virus (DENV) non-structural protein 1 (NS1) in triggering hyperpermeability of human endothelial cells in vitro and systemic vascular leakage in vivo. NS1 was shown to activate toll-like receptor 4 signaling in primary human myeloid cells, leading to secretion of pro-inflammatory cytokines and vascular leakage. However, distinct endothelial cell-intrinsic mechanisms of NS1-induced hyperpermeability remained to be defined. The endothelial glycocalyx layer (EGL) is a network of membrane-bound proteoglycans and glycoproteins lining the vascular endothelium that plays a key role in regulating endothelial barrier function. Here, we demonstrate that DENV NS1 disrupts the EGL on human pulmonary microvascular endothelial cells, inducing degradation of sialic acid and shedding of heparan sulfate proteoglycans. This effect is mediated by NS1-induced expression of sialidases and heparanase, respectively. NS1 also activates cathepsin L, a lysosomal cysteine proteinase, in endothelial cells, which activates heparanase via enzymatic cleavage. Specific inhibitors of sialidases, heparanase, and cathepsin L prevent DENV NS1-induced EGL disruption and endothelial hyperpermeability. All of these effects are specific to NS1 from DENV1-4 and are not induced by NS1 from West Nile virus, a related flavivirus. Together, our data suggest an important role for EGL disruption in DENV NS1-mediated endothelial dysfunction during severe dengue disease.

Flaviviruses cause systemic or neurotropic-encephalitic pathology in humans. The flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein involved in viral replication, immune evasion, and vascular leakage during dengue virus infection. However, the contribution of secreted NS1 from related flaviviruses to viral pathogenesis remains unknown. Here, we demonstrate that NS1 from dengue, Zika, West Nile, Japanese encephalitis, and yellow fever viruses selectively binds to and alters permeability of human endothelial cells from lung, dermis, umbilical vein, brain, and liver in vitro and causes tissue-specific vascular leakage in mice, reflecting the pathophysiology of each flavivirus. Mechanistically, each flavivirus NS1 leads to differential disruption of endothelial glycocalyx components, resulting in endothelial hyperpermeability. Our findings reveal the capacity of a secreted viral protein to modulate endothelial barrier function in a tissue-specific manner both in vitro and in vivo, potentially influencing virus dissemination and pathogenesis and providing targets for antiviral therapies and vaccine development.

We identified a novel SARS-CoV-2 variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California. Named B.1.427/B.1.429 to denote its 2 lineages, the variant emerged around May 2020 and increased from 0% to >50% of sequenced cases from September 1, 2020 to January 29, 2021, exhibiting an 18.6-24% increase in transmissibility relative to wild-type circulating strains. The variant carries 3 mutations in the spike protein, including an L452R substitution. Our analyses revealed 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation found in the B.1.1.7, B.1.351, and P.1 variants. Antibody neutralization assays showed 4.0 to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California associated with decreased antibody neutralization warrants further investigation.