Phylogenetic relationships of Pakistan DENV-1.

<p>Phylogenetic relationships of Dengue viruses type 1 isolated from severe cases in 2011 in Lahore, Pakistan. Numbers at branch nodes indicate maximum likelihood bootstrap values. Underlined virus names represent newly generated sequences. Analyses were based on 505 nts of the envelope gene region. Trees are midpoint rooted. Scale bars indicate nucleotide substitutions per site.</p

Phylogenetic relationships of Pakistan DENV-2.

<p>Phylogenetic relationships of Dengue viruses type 2 isolated from severe cases in 2011 in Lahore, Pakistan. Numbers at branch nodes indicate maximum likelihood bootstrap values. Underlined virus names represent newly generated sequences. Analyses were based on 2373 nts of the capsid, pre-membrane, and envelope gene region. Trees are midpoint rooted. Scale bars indicate nucleotide substitutions per site.</p

Serologic and virologic confirmation of dengue virus infection in Pakistan patients by age group.

1<p>IgM and IgG specific dengue antibody detected using an in-house Luminex platform based microsphere-bead immunoassay. ²Virus isolation was done by intra-thoracic circulation of mosquitoes. ³Combined IgM, PCR and virus isolation results</p

2′-<i>O</i>-MTase mutant DENV-2 has altered sensitivity to IFN, which is partially mediated by IFIT1.

<p>(A) Cells were seeded in a 24-well plate, treated for 24 h with increasing amount of IFN-β and infected with WT or E217A DENV-2. At 72 h post-infection, cells were harvested and analyzed by flow cytometry using 4G2 antibody (against viral envelope protein). (B) Viral titers in culture fluids were measured by plaque assay. Data are representative of three experiments. Means and SD are shown. Statistical analysis was performed using Student's t-test (***, p<0.001; *, p<0.05). (C) HEK293-DC-SIGN cells were transiently transfected with vector alone, human IFIT-1 (ISG56), IFIT-2 (ISG54), IFIT-3 (ISG60), or IFIT-5 (ISG58). On day 2 post-transfection, cells were infected with WT or E217A DENV-2 at an MOI of 5. The cells were analyzed for viral envelope protein expression by flow cytometry at 72 h post-infection. Results represent the mean ± SEM of six independent experiments. Percentage of infected cells was normalized to cells transfected with empty vector. (D) Virus output from transfected cells was determined in the supernatant by plaque assay. The transfection efficiency was 30–50%, (determined by parallel experiments with a GFP expression plasmid (data not shown)). (E) growth kinetics of E217A and WT DENV-2 in HEK293-DC-SIGN cells. Statistical analysis was performed using one-way ANOVA Bonferroni's multiple comparison test (**, p<0.01).</p

Incidence and Risk Factors for Developing Dengue-Associated Hemophagocytic Lymphohistiocytosis in Puerto Rico, 2008 - 2013

<div><p>Background</p><p>Hemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal disorder characterized by fever, pancytopenia, hepatosplenomegaly, and increased serum ferritin. HLH is being increasingly reported as a complication of dengue, a common tropical acute febrile illness.</p><p>Methodology/Principal Findings</p><p>After a cluster of pediatric dengue-associated HLH patients was identified during the 2012–2013 dengue epidemic in Puerto Rico, active surveillance and a case-control investigation was conducted at four referral hospitals to determine the incidence of HLH in children and identify risk factors for HLH following dengue. Patients with dengue-associated HLH (cases) were matched by month of illness onset and admission hospital to dengue patients that did not develop HLH (controls). During 2008–2013, a total of 33 HLH patients were identified, of which 22 (67%) were associated with dengue and 1 died (dengue-associated HLH case-fatality rate: 4.5%). Two patients with dengue-associated HLH had illness onset in 2009, none had illness onset during the 2010 dengue epidemic, and 20 had illness onset during the 2012–2013 epidemic. Frequency of infection with either dengue virus (DENV)-1 or DENV-4 did not differ between cases and controls. Cases were younger than controls (median age: 1 vs. 13 years, p < 0.01), were hospitalized longer (18 vs. 5 days, p < 0.01), and were admitted more frequently to pediatric intensive care units (100% vs. 16%, p < 0.01). Cases had co-infection (18.2% vs. 4.5%, p = 0.04), recent influenza-like illness (54.5% vs. 25.0%, p = 0.01), and longer duration of fever (7 vs. 5 days; p < 0.01). Cases were more likely to have lymphadenopathy, hepatomegaly, splenomegaly, anemia, and elevated liver transaminases (p ≤ 0.02).</p><p>Conclusions/Significance</p><p>During this cluster of dengue-associated HLH cases that was temporally associated with the 2012–2013 epidemic, most patients with dengue-associated HLH were infants and had higher morbidity than dengue inpatients. Physicians throughout the tropics should be aware of HLH as a potential complication of dengue, particularly in patients with anemia and severe liver injury.</p></div

<i>Ae. aegypti</i> susceptibility according to virus type and titer.

*<p>Infected: presence of virus in abdomen.</p>#<p>Disseminated: presence of virus in thorax.</p

Dengue MTase mutants are attenuated and immunogenic.

<p>(A) Viremia kinetics of WT DENV-1 (strain West Pacific 74), DENV-1 K61A, and DENV-1-E216A or a combination of DENV-1 E216A and DENV-2 E217A <i>in vivo</i>. Mice were infected i.p. with 2.75×10⁵ PFU of the indicated virus. Viral titers in the plasma were measured at indicated time points by real-time RT-PCR. (B) Viral titers in plasma of mice immunized i.p. with 2.75×10⁵ PFU DENV-2 WT, DENV-2 E217A (strain TSV01) alone or in combination with DENV-1 E216A (2.75×10⁵ PFU DENV-1 E216A plus 2.75×10⁵ PFU DENV-2 E217A). Blood was taken at indicated time points and viral titers were measured by real-time PCR. (C) Viral titers in the plasma of mice immunized with 2′-<i>O</i>-MTase mutant and challenged as indicated. Numbers in gray boxes indicate WT virus, whereas numbers in white boxes indicate 2′-<i>O</i>-MTase mutant virus. Mice were immunized i.p. with 2.75×10⁵ PFU of the indicated 2′-<i>O</i>-MTase mutant serotype and challenged 30 days later with 5×10⁵ PFU WT DENV-1 strain (strain 05K3126 used for challenge due to its high virulence in mice, unpublished data) or 3×10⁶ PFU WT DENV-2. Blood was taken at indicated time points and viral titers were measured by plaque assay. ND: not detected. (D, E) IgG titers of mice immunized and challenged as described above. Blood was taken at indicated time points post challenge and IgG antibody titers against DENV-1 (D) and DENV-2 (E) were measured by ELISA. Data are representative of two experiments with three to four mice per group in each experiment (A, B) or two pooled experiments (C–E) with a total of 9 mice per group. Bars represent means with SD (A) or means with SEM (B–E).</p

Viremia in E217A-immunized RMs after challenge with wild-type DENV-2^*.

*<p>Animals were challenged with 1×10⁵ PFU of WT DENV-2 on day 64 post-immunization.</p

2′-<i>O</i>-MTase mutant protects against challenge with an aggressive mouse-adapted DENV strain.

<p>(A) Mice were immunized i.p. with 2.75×10⁵ DENV-1 WT, DENV-1 E216A, DENV-2 WT, DENV-2 E217A (strain TSV01) alone or in combination with DENV-1 E216A (2.75×10⁵ PFU DENV-1 E216A plus 2.75×10⁵ PFU DENV-2 E217A). 30 days post immunization mice were challenged i.p. with 10⁷ PFU of D2Y98P <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003521#ppat.1003521-Grant1" target="_blank">[34]</a>. Health status of mice were monitored twice daily. (B) Blood was taken at indicated time points and viral titers were measured by real-time PCR. (C) TNF-α level in plasma of mice was measured at day three post challenge according to the manufacturer's protocol (eBioscience) (C). Data represent means ±SEM from 3 experiments with a total of 7–10 mice (A) or means ±SEM from two experiments with a total of 6–8 mice (B–C). Statistical analysis was performed using 1-way ANOVA Tukey's multiple comparison test (***P<.001).</p

Neutralization and antibody-dependent enhancement of infection (ADE) in immunized AG129 mice.

<p>NT50 values are means ±SD of six to seven mice from two independent experiments.</p><p>Max. ADE values are normalized against 4G2, which was used as an internal standard for infection efficiency per experiment. Values are means ±SD from six to seven mice from two independent experiments.</p><p>Kruskal Wallis test with multiple comparisons:</p>*<p>: p<0.05 compared to PBS.</p>#<p>: p<0.05 compared to DENV-2.</p