Search

BACKGROUND: Yellow fever virus (YFV) infections are a major global disease concern with high mortality in humans, and as such it is critical to identify clinical correlates of disease severity. While nonstructural protein 1 (NS1) of the related dengue virus is implicated in contributing to vascular leak, little is known about the role of YFV NS1 in severe YF and mechanisms of vascular dysfunction in YFV infections. METHODS: Using serum samples from laboratory-confirmed YF patients with severe (n = 39) or non-severe (n = 18) disease in a well-defined hospital observational cohort in Brazil, plus samples from healthy uninfected controls (n = 11), we investigated factors associated with disease severity and endothelial dysfunction. FINDINGS: We found significantly increased levels of NS1, as well as syndecan-1, a marker of vascular leak, in serum from severe YF as compared to non-severe YF or control groups. We also showed that hyperpermeability of endothelial cell monolayers treated with serum from severe YF patients was significantly higher compared to non-severe YF and control groups, as measured by transendothelial electrical resistance (TEER). Further, we demonstrated that YFV NS1 induces shedding of syndecan-1 from the surface of human endothelial cells. Notably, YFV NS1 serum levels significantly correlated with syndecan-1 serum levels, TEER values, and signs of disease severity. Syndecan-1 levels also significantly correlated with clinical laboratory parameters of disease severity, viral load, hospitalization, and death. INTERPRETATION: This study provides further evidence for endothelial dysfunction as a mechanism of YF pathogenesis in humans and suggests serum quantification of YFV NS1 and syndecan-1 as valuable tools for disease diagnosis and/or prognosis. FUNDING: This work was supported by the US NIH and FAPESP.

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of vascular leak are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to induce barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-β signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-β signaling axis are required for S-mediated barrier dysfunction. Notably, we show that SARS-CoV-2 infection caused leak in vivo, which was reduced by inhibiting integrins. Our findings offer mechanistic insight into SARS-CoV-2-triggered vascular leak, providing a starting point for development of therapies targeting COVID-19.

The four dengue virus serotypes (DENV1-4) cause major public health problems worldwide. Highly neutralizing type-specific human monoclonal antibodies (hmAbs) target conformation-dependent epitopes on the DENV envelope protein, including 1F4, a DENV1 type-specific hmAb. Using a recombinant DENV2 virus displaying the DENV1 1F4 epitope (rDENV2/1), we measured the proportion and kinetics of DENV1 neutralizing antibodies targeting the 1F4 epitope in individuals living in Asia and the Americas where different DENV1 genotypes were circulating. Samples from 20 individuals were analyzed 3 and 18 months post-primary DENV1 infection, alongside samples from 4 individuals collected annually for four years post-primary DENV1 infection, from two studies in Nicaragua. We also analyzed convalescent post-primary DENV1 plasma samples from Sri Lankan individuals. We found that neutralizing antibodies recognizing the 1F4 epitope vary in prevalence across both populations and were detected from 20 days to four years post-infection. Additionally, both populations displayed substantial variability, with a range of high to low proportions of DENV1 type-specific neutralizing antibodies recognizing the 1F4 epitope seen across individuals. Thus, the 1F4 epitope is a major but not exclusive target of type-specific neutralizing antibodies post-primary infection with different DENV1 genotypes in Asia and Latin America, and additional epitopes likely contribute to type-specific neutralization of DENV1.

The Flaviviridae are a family of viruses that include the important arthropod-borne human pathogens dengue virus (DENV), West Nile virus, Zika virus, Japanese encephalitis virus, and yellow fever virus. Flavivirus nonstructural protein 1 (NS1) is essential for virus replication but is also secreted from virus-infected cells. Extracellular NS1 acts as a virulence factor during flavivirus infection in multiple ways, including triggering endothelial dysfunction and vascular leak via interaction with endothelial cells. While the role of NS1 in inducing vascular leak and exacerbating pathogenesis is well appreciated, if and how NS1-triggered endothelial dysfunction promotes virus infection remains obscure. Flaviviruses have a common need to disseminate from circulation into specific tissues where virus-permissive cells reside. Tissue-specific dissemination is associated with disease manifestations of a given flavivirus, but mechanisms dictating virus dissemination are unclear. Here we show that NS1-mediated endothelial dysfunction promotes virus dissemination in vitro and in vivo . In mouse models of DENV infection, we show that anti-NS1 antibodies decrease virus dissemination, while the addition of exogenous NS1 promotes virus dissemination. Using an in vitro system, we show that NS1 promotes virus dissemination in two distinct ways: (1) promoting crossing of barriers and (2) increasing infectivity of target cells in a tissue- and virus-specific manner. The capacity of NS1 to modulate infectivity correlates with a physical association between virions and NS1, suggesting a potential NS1-virion interaction. Taken together, our study indicates that flavivirus NS1 promotes virus dissemination across endothelial barriers, providing an evolutionary basis for virus-triggered vascular leak., Author Summary: The Flaviviridae contain numerous medically important human pathogens that cause potentially life-threatening infections. Over half of the world's population is at risk of flavivirus infection, and this number is expected to increase as climate change expands the habitats of the arthropod vectors that transmit these flaviviruses. There are few effective vaccines and no therapeutics approved for prevention or treatment of flavivirus infection, respectively. Given these challenges, understanding how and why flaviviruses cause pathogenesis is critical for identifying targets for therapeutic intervention. The secreted nonstructural protein 1 (NS1) of flaviviruses is a conserved virulence factor that triggers endothelial dysfunction in a tissue-specific manner. It is unknown if this endothelial dysfunction provides any benefit for virus infection. Here we provide evidence that NS1-triggered endothelial dysfunction facilitates virus crossing of endothelial barriers and augments infection of target cells in vitro and promotes virus dissemination in vivo . This study provides an evolutionary explanation for flaviviruses evolving the capacity to trigger barrier dysfunction and highlights NS1 and the pathways governing endothelial dysfunction, as therapeutic targets to prevent flavivirus dissemination.

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo , independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-β signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-β signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.