Dengue virus (DENV) causes dengue fever and dengue haemorrhagic fever/dengue shock syndrome, both considered major public-health problems worldwide. We generated a lethal DENV-2 strain (D220) by 10 additional cycles of subcutaneous inoculation of mice with supernatant from mosquito cells infected with the previously characterized strain D2S10, followed by harvesting of serum. D220 induces mortality at ten-fold lower doses than D2S10 in mice lacking only the alpha/beta interferon (IFN-a/b) receptor in C57BL/6 or 129 backgrounds under both non-enhanced and antibody-enhanced conditions. Sequence analysis of the complete viral genome revealed five amino acid changes between D220 and D2S10, of which two (K122I in envelope and V115A in NS4B) appear to account for the observed phenotypic differences between the viruses. By causing mortality at lower doses in C57BL/6 mice lacking only the IFNa/b receptor, D220 constitutes an improved tool for study of DENV-induced pathogenesis, as well as for testing potential vaccines and antiviral drugs against DENV.The four serotypes of dengue virus (DENV-1-4) are mosquitoborne and cause dengue fever (DF) and dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). Found in tropical and subtropical regions of the world, DENV causes an estimated 50-100 million infections annually and places over 3 billion people at risk of infection (WHO, 2009). Most primary DENV infections with any serotype are asymptomatic or lead to the self-limited febrile illness DF; however, secondary infection with a different DENV serotype leads to increased risk of developing severe dengue disease (Halstead, 2007). This increase in severity upon secondary infection is thought to be mediated in part via antibody-dependent enhancement (ADE), whereby interaction between antibodies generated during a prior infection and the current infecting serotype can lead to increased uptake of virus via Fc receptors expressed on susceptible myeloid cells (Halstead, 2003).Establishing a small-animal model is an important step in understanding the mechanisms underlying dengue pathogenesis and immunity. The first mouse models used high doses of neurovirulent DENV strains delivered intracranially into immunocompetent mice; this caused neurotropic disease and paralysis in infected mice, which is typically not observed in human dengue (Raut et al., 1996;Schlesinger, 1977). One of the second-generation models used a neurotropic mouseadapted DENV2 strain in mice of the 129/Sv mouse background deficient in alpha/beta interferon (IFN-a/b) and IFN-c receptors (AG129 mice) (Johnson & Roehrig, 1999;Zompi & Harris, 2012). In more recent work, a peripherally adapted DENV-2 virus, designated D2S10, was generated by alternately passaging the Taiwanese DENV-2 isolate PL046 between Aedes albopictus C6/36 cells and AG129 mouse serum. The D2S10 virus was lethal in AG129 mice at 10 7 p.f.u. and induced a vascular leak phenotype mediated in part by tumour necrosis factor alpha (TNF-a) (Shresta et al., 2006), which is similar to observations in s...

A major challenge in dengue vaccine development is that cross-reactive anti-DENV antibodies (Abs) can be protective or potentially enhance disease via antibody-dependent enhancement (ADE). We recently showed that immunization with adjuvanted recombinant NS1 from all four DENV serotypes protects against DENV2 challenge in a mouse model of lethal vascular leak syndrome. Conversely, we found that DENV NS1 by itself triggers endothelial permeability in vitro and vascular leak in vivo. Here, we evaluated survival to lethal DENV challenge in mice immunized with (1) NS1+alum; (2) NS1+Monophosphoryl Lipid A and AddaVax (MA); (3) NS1+Sigma adjuvant system and CpG DNA (SCpG); (4) ovalbumin+adjuvant (OVA); or (5) a sublethal dose of DENV2 strain PLO46.NS1. OVA or NS1+alum immunized mice were not protected, whereas immunization with NS1+MA or NS1+SCpG or DENV2-PL046 resulted in 100% survival. We characterized Ab responses to NS1 from DENV1-3 using an antigen microarray tiled with 20-mer peptides overlapping by 15 amino acids and identified 5 regions with significant levels of antibody reactivity in the NS1+MA group. We also profiled Ab responses to NS1 in humans naturally infected with DENV-2 or -3 in longitudinal serum samples from Nicaragua. One region in the NS1 “wing” domain was immunodominant in both mouse and human studies, and two regions were identified only in NS1-immunized mice; thus, vaccination can generate Abs to regions that are not targeted in natural infection and could provide additional protection against a lethal DENV infection. Overall, we identified several immunodominant regions, which were in functionally important locations on the DENV NS1 protein and are potential correlates of protection.

Dengue virus (DENV), consisting of serotypes 1–4, is the most medically important flavivirus, causing an estimated 50–100 million dengue cases globally every year. Most symptomatic DENV infections present as an acute febrile illness; however, severe complications include vascular leakage leading to hemorrhage and hypovolemic shock. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis, activating immune cells and acting directly on endothelial barriers, causing them to become pathologically hyperpermeable. While recent work has delved into the mechanisms behind the endothelial cell-intrinsic pathway of DENV NS1-induced vascular leak, much less is known about how DENV NS1 interacts with immune cells and what role this activation plays in DENV infection. We have discovered that DENV NS1 can trigger activation of the inflammasome, a family of cytosolic innate immune sensors that react to danger-associated molecular patterns. DENV NS1 induces the release of the pro-inflammatory cytokine IL-1β in human and murine monocytes in a caspase-1 dependent manner that is independent of NLRP3. Additionally, we found that caspase-1-deficient, but not NLRP3-deficient, mice are more susceptible to infection in a murine model of DENV infection. These results indicate that the inflammasome acts as a sensor of the DENV viral toxin NS1 and plays a protective role during DENV infections This work was supported by NIAID/NIH grant R01 AI24493 (E.H.)