Dengue Virus Infection of Primary Endothelial Cells Induces Innate Immune Responses, Changes in Endothelial Cells Function and Is Restricted by Interferon-Stimulated Responses

Abstract

1 Although endothelial cell (EC) infection is not widespread during dengue virus (DENV) infection in 2vivo, the endothelium is the site of the pathogenic effects seen in severe DENV disease. In this study 3 we investigated DENV infection of primary EC and defined factors that influence infection in this cell 4 type. 5Consistent with in vivo findings where EC infection is infrequent, only 3-15% of EC became 6 productively DENV-2-infected in vitro. This low level infection could not be attributed to inhibition 7 by heparin, EC donor variation, heterogeneity, or biological source. DENV-infection of EC was 8 associated with induction of innate immune responses, including increased STAT1 protein, STAT1-9 phosphorylation, and IFN-β, OAS-1, IFIT-1/ISG56 and viperin mRNA. Antibody blocking of IFN-β 10 inhibited the induction of OAS1, IFIT1/ISG56 and viperin while shRNA knockdown of viperin 11 enhanced DENV-infection in EC. DENV-infection of EC resulted in increased activity of 12 sphingosine kinase 1, a factor important in maintaining vascular integrity, and altered basal and 13 stimulated changes in barrier integrity of DENV-infected EC monolayers. 14 Thus, DENV productively infects only a small percentage of primary EC but this has a major 15 influence on induction of IFN-β driven innate immune responses that can restrict infection while the 16 EC themselves are functionally altered. These changes may have important consequences for the 17 endothelium and are reflective of pathogenic changes associated with vascular leakage, as seen in 18 DENV disease.

Reduction in sphingosine kinase 1 influences the susceptibility to dengue virus infection by altering antiviral responses

“…Close analysis of the time-course of the reduction in SK1 in DENV-infected cells demonstrates a correlation with high levels of the DENV 39 UTR, which can compete for the SK1 activator eEF1A (Carr et al, 2013a;Leclercq et al, 2008Leclercq et al, , 2011, which is utilized by flaviviruses to assist replication (Blackwell & Brinton, 1997;Davis et al, 2007). Additionally, our previous data (Carr et al, 2013a;Calvert et al, 2015), as well as that of Yamane et al (2009), suggest an increase in SK1 activity in the early hours following infection. Thus the roles of SK1 in viral infection may vary in relation to the time post-infection (p.i.…”

“…In the study herein, we have more closely examined the first 24 h following DENV infection, but failed to demonstrate any additional changes in SK1 activity. In contrast, we have recently described an increase in SK1 activity in DENV-infected human umbilical vein endothelial cells (HUVECs) within 24 h of infection (Calvert et al, 2015). In this HUVEC infection model there is only a low percentage of DENVinfected cells and we believe the induction of SK1 is secondary to the induction of host cell responses in these cells, such as production of TNF-a.…”

Investigation of sphingosine kinase 1 in interferon responses during dengue virus infection

“…Concurrent with this increase in infectious virus release, DENV infection induced p‐STAT1 in both WT and SK1 −/− pMEF, with a reproducible reduction in SK1 −/− compared with WT pMEF (Figures 2b and c). Total STAT1 increased at 48 h pi in both WT and SK1 −/− cells, as we have previously described in DENV‐infected endothelial cells 29 . Total STAT1 levels tended to be lower in uninfected SK1 −/− compared with WT pMEF (see also Figure 3b), although this signal was low and varied between donor MEF preparations.…”

Immune-mediated cytokine storm and its role in severe dengue

“…Transcriptional analysis of DENV-infected endothelial cells showed activation of transcriptional programs related to cell death, type I IFN, angiogenesis, cytokines and chemokines, complement, and coagulation [76,77]. Cultures of human umbilical vein endothelial cells showed altered membrane integrity even though the frequencies of infected endothelial cells were low [78,79].…”