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1.  A Prospective Nested Case-Control Study of Dengue in Infants: Rethinking and Refining the Antibody-Dependent Enhancement Dengue Hemorrhagic Fever Model 
PLoS Medicine  2009;6(10):e1000171.
Analyses of a prospective case-control study of infant dengue by Daniel Libraty and colleagues casts doubt on the antibody-dependent enhancement model for dengue hemorrhagic fever.
Dengue hemorrhagic fever (DHF) is the severe and life-threatening syndrome that can develop after infection with any one of the four dengue virus (DENV) serotypes. DHF occurs almost exclusively in individuals with secondary heterologous DENV infections and infants with primary DENV infections born to dengue immune mothers. The widely accepted explanation for the pathogenesis of DHF in these settings, particularly during infancy, is antibody-dependent enhancement (ADE) of DENV infection.
Methods and Findings
We conducted a prospective nested case-control study of DENV infections during infancy. Clinical data and blood samples were collected from 4,441 mothers and infants in up to two pre-illness study visits, and surveillance was performed for symptomatic and inapparent DENV infections. Pre-illness plasma samples were used to measure the associations between maternally derived anti-DENV3 antibody-neutralizing and -enhancing capacities at the time of DENV3 infection and development of infant DHF.
The study captured 60 infants with DENV infections across a wide spectrum of disease severity. DENV3 was the predominant serotype among the infants with symptomatic (35/40) and inapparent (15/20) DENV infections, and 59/60 infants had a primary DENV infection. The estimated in vitro anti-DENV3 neutralizing capacity at birth positively correlated with the age of symptomatic primary DENV3 illness in infants. At the time of symptomatic DENV3 infection, essentially all infants had low anti-DENV3 neutralizing activity (50% plaque reduction neutralizing titers [PRNT50] ≤50) and measurable DENV3 ADE activity. The infants who developed DHF did not have significantly higher frequencies or levels of DENV3 ADE activity compared to symptomatic infants without DHF. A higher weight-for-age in the first 3 mo of life and at illness presentation was associated with a greater risk for DHF from a primary DENV infection during infancy.
This prospective nested case-control study of primarily DENV3 infections during infancy has shown that infants exhibit a full range of disease severity after primary DENV infections. The results support an initial in vivo protective role for maternally derived antibody, and suggest that a DENV3 PRNT50 >50 is associated with protection from symptomatic DENV3 illness. We did not find a significant association between DENV3 ADE activity at illness onset and the development of DHF compared with less severe symptomatic illness. The results of this study should encourage rethinking or refinement of the current ADE pathogenesis model for infant DHF and stimulate new directions of research into mechanisms responsible for the development of DHF during infancy.
Trial registration NCT00377754
Please see later in the article for the Editors' Summary
Editors' Summary
Every year, dengue infects 50–100 million people living in tropical and subtropical areas. The four closely related viruses that cause dengue (DENV1–4) are transmitted to people through the bites of female Aedes aegypti mosquitoes, which acquire the viruses by feeding on the blood of an infected person. Many people who become infected with DENV have no symptoms but some develop dengue fever, a severe, flu-like illness that lasts a few days. Other people—about half a million a year—develop a potentially fatal condition called dengue hemorrhagic fever (DHF). In DHF, which can be caused by any of the DENVs, small blood vessels become leaky and friable. This leakiness causes nose and gum bleeds, bruising and, in the worst cases, failure of the circulatory system and death. There is no vaccine to prevent dengue and no specific treatment for dengue fever or DHF. However, with standard medical care—in particular, replacement of lost fluids—most people can survive DHF.
Why Was This Study Done?
DHF is increasingly common, but why do only some people develop DHF after infection with DENV? The widely accepted explanation for the development of DHF is “antibody-dependent enhancement” (ADE) of DENV infection. DHF occurs almost exclusively in two settings; (i) children and adults who become infected with a second DENV serotype after an initial “primary” DENV infection with a different serotype, and (ii) infants with primary DENV infections whose mothers have some DENV immunity. The ADE model suggests that in individuals who develop DHF, although there are some antibodies (proteins made by the immune system to fight infections) against DENV in their blood (in secondary heterologous infections, antibodies left over from the primary infection; in infants with primary infections, antibodies acquired from their mothers before birth), these antibodies cannot “neutralize” the virus. Instead, they bind to it and enhance its uptake by certain immune system cells, thus increasing viral infectivity and triggering an immunological cascade that results in DHF. In this prospective, nested case-control study, the researchers directly test the ADE model for infant DHF. In a prospective study, a group of people is selected and followed to see if they develop a disease; in a nested case-control study, each case is compared with people in the group who do not develop the disease.
What Did the Researchers Do and Find?
The researchers collected clinical data and blood samples from 4,441 mothers and their babies at up to two pre-illness study visits. They then followed the infants for a year to see which of them developed symptomatic and symptom-free DENV infections. Finally, they used the pre-illness blood samples to estimate the maternally derived anti-DENV antibody-neutralizing and -enhancing capacities in the infants at the time of DENV infection. 60 infants were infected with DENV—mainly DENV3—during the study. All but one infection was a primary infection. The infected infants showed a wide range of disease severity. Infants who had a high DENV3 neutralizing capacity at birth tended to develop symptomatic DENV3 infections later than infants who had a low DENV3 neutralizing capacity at birth. All the infants who developed a symptomatic DENV3 infection had a low estimated DENV3 neutralizing activity at the time of infection, and nearly all had measurable levels of DENV3 ADE activity. Infants who developed DHF did not have significantly higher frequencies or levels of DENV3 ADE activity than DENV3-infected infants with less severe symptoms.
What Do These Findings Mean?
These findings indicate that maternally derived anti-DENV3 antibody initially provides protection against dengue infections. That is, babies born to DENV immune mothers are protected against dengue infections by maternally derived antibodies. Over time, the level of these antibodies declines until eventually the infant becomes susceptible to DENV infections. However, the lack of a significant association between the estimated level of DENV3 ADE activity at illness onset and the development of DHF rather than a less severe illness throws doubt onto (but does not completely rule out) the current ADE pathogenesis model for infant DHF, at least for DENV3 infections. The results of this study, the researchers conclude, should encourage rethinking or refinement of the ADE model for infant DHF and should promote further prospective studies into the development of DHF during infancy.
Additional Information
Please access these Web sites via the online version of this summary at provides review articles, news, opinions, research articles, and reports on dengue (in English)
The US Centers for Disease Control and Prevention provide detailed information about dengue fever and dengue hemorrhagic fever (in English and Spanish)
The World Health Organization provides information on dengue fever and dengue hemorrhagic fever around the world (in several languages)
Links to additional resources about dengue are provided by MedlinePlus (in English and Spanish)
Wikipedia has a page on antibody-dependent enhancement of viral infections (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2762316  PMID: 19859541
2.  High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses 
Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates.
Author Summary
Dengue, a re-emergent human disease that places nearly half of the world's population at risk, threatens to further expand in geographical distribution. The lack of an available effective dengue vaccine has encouraged the search for antiviral drugs as an alternative approach. In recent years, drug discovery through high-throughput screening has become a trend in the search for dengue antivirals. In this study, we developed an image-based dengue high-throughput/high-content assay using prevalent viral strains of three dengue serotypes (DENV1, DENV2 and DENV3) isolated from dengue outbreaks in South America and a laboratory-adapted strain of DENV4. We demonstrated the usefulness of our image-based dengue HT/HCA in identifying potential dengue antivirals by screening a small subset of chemical compounds for inhibition of dengue virus infection in a human-derived host cell line (Huh-7.5), and partially characterized their activities against dengue infection in a mosquito host cell line (C6/36), a distantly-related virus (hepatitis C virus), and an unrelated virus that is transmitted by the same mosquito vector (chikungunya virus).
PMCID: PMC3578765  PMID: 23437413
3.  Preexisting Japanese Encephalitis Virus Neutralizing Antibodies and Increased Symptomatic Dengue Illness in a School-Based Cohort in Thailand 
Dengue viruses (DENVs) and Japanese encephalitis virus (JEV) have significant cross-reactivity in serological assays; the clinical implications of this remain undefined. An improved understanding of whether and how JEV immunity modulates the clinical outcome of DENV infection is important as large-scale DENV vaccine trials will commence in areas where JEV is co-endemic and/or JEV immunization is routine.
Methods and Findings
The association between preexisting JEV neutralizing antibodies (NAbs) and the clinical severity of DENV infection was evaluated in a prospective school-based cohort in Thailand that captured asymptomatic, non-hospitalized, and hospitalized DENV infections. Covariates considered included age, baseline DENV antibody status, school of attendance, epidemic year, and infecting DENV serotype. 942 children experienced at least one DENV infection between 1998 and 2002, out of 3,687 children who were enrolled for at least one full year. In crude analysis, the presence of JEV NAbs was associated with an increased occurrence of symptomatic versus asymptomatic infection (odds ratio [OR] = 1.55, 95% CI: 1.08–2.23) but not hospitalized illness or dengue hemorrhagic fever (DHF). The association was strongest in children with negative DENV serology (DENV-naive) (OR = 2.75, 95% CI: 1.12–6.72), for whom the presence of JEV NAbs was also associated with a symptomatic illness of longer duration (5.4 days for JEV NAb+ versus 2.6 days for JEV NAb-, p = 0.048). JEV NAbs were associated with increased DHF in younger children with multitypic DENV NAb profiles (OR = 4.05, 95% CI: 1.18 to 13.87). Among those with JEV NAbs, the association with symptomatic illness did not vary by antibody titer.
The prior existence of JEV NAbs was associated with an increased probability of symptomatic as compared to asymptomatic DENV illness. These findings are in contrast to previous studies suggesting an attenuating effect of heterologous flavivirus immunity on DENV disease severity.
Author Summary
Dengue viruses (DENVs) and Japanese encephalitis virus (JEV) have significant cross-reactivity in serological assays, but the possible clinical implications of this remain poorly understood. Interactions between these flaviviruses are potentially important for public health because wild-type JEV continues to co-circulate with DENV in Southeast Asia, the area with the highest burden of DENV illness, and JEV vaccination coverage in this region is high. In this study, we examined how preexisting JEV neutralizing antibodies (NAbs) influenced the clinical severity of subsequent DENV infection using data from a prospective school-based cohort study in Thailand that captured a wide range of clinical severities, including asymptomatic, non-hospitalized, and hospitalized DENV infections. We found that the prior existence of JEV NAbs was associated with an increased occurrence of symptomatic versus asymptomatic DENV infection. This association was most notable in DENV-naives, in whom the presence of JEV NAbs was also associated with an illness of longer duration. These findings suggest that the issue of heterologous flavivirus immunity and DENV infection merits renewed attention and interest and that DENV vaccine developers might incorporate detailed assessments of preexisting immunity to non-DENV flaviviruses and histories of vaccination against non-DENV flaviviruses in evaluating DENV vaccine safety and efficacy.
PMCID: PMC3186763  PMID: 21991398
4.  Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2 
Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control.
Methodology/Principal Findings
We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials.
We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.
Author Summary
Dengue virus (DENV) infection remains a serious health threat despite the availability of supportive care in modern medicine. Monoclonal antibodies (mAbs) of DENV would be powerful research tools for antiviral development, diagnosis and pathological investigations. Here we described generation and characterization of seventeen mAbs with high reactivity for E protein of DENV. Four of these mAbs showed high neutralizing activity against DENV-2 infection in mice. The monoclonal antibody mAb DB32-6 showed the strongest neutralizing activity against diverse DENV-2 and protected DENV-2-infected mice against mortality in therapeutic models. We identified neutralizing epitopes of DENV located at residues K310 and E311 of viral envelope protein domain III (E-DIII) through the combination of biological and molecular strategies. Comparing the strong neutralizing activity of mAbs targeting A-strand with mAbs targeting lateral ridge, we found that epitopes located in A-strand induced stronger neutralizing activity than those located on the lateral ridge. DB32-6 humanized version was successfully developed. Humanized DB32-6 variant retained neutralizing activity and prevented DENV infection. Understanding the epitope-based antibody-mediated neutralization is crucial to controlling dengue infection. Additionally, this study also introduces a novel humanized mAb as a candidate for therapy of dengue patients.
PMCID: PMC3341331  PMID: 22563515
5.  Correlation of Serotype-Specific Dengue Virus Infection with Clinical Manifestations 
Disease caused by the dengue virus (DENV) is a significant cause of morbidity throughout the world. Although prior research has focused on the association of specific DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) with the development of severe outcomes such as dengue hemorrhagic fever and dengue shock syndrome, relatively little work has correlated other clinical manifestations with a particular DENV serotype. The goal of this study was to estimate and compare the prevalence of non-hemorrhagic clinical manifestations of DENV infection by serotype.
Methodology and Principal Findings
Between the years 2005–2010, individuals with febrile disease from Peru, Bolivia, Ecuador, and Paraguay were enrolled in an outpatient passive surveillance study. Detailed information regarding clinical signs and symptoms, as well as demographic information, was collected. DENV infection was confirmed in patient sera with polyclonal antibodies in a culture-based immunofluorescence assay, and the infecting serotype was determined by serotype-specific monoclonal antibodies. Differences in the prevalence of individual and organ-system manifestations were compared across DENV serotypes. One thousand seven hundred and sixteen individuals were identified as being infected with DENV-1 (39.8%), DENV-2 (4.3%), DENV-3 (41.5%), or DENV-4 (14.4%). When all four DENV serotypes were compared with each other, individuals infected with DENV-3 had a higher prevalence of musculoskeletal and gastrointestinal manifestations, and individuals infected with DENV-4 had a higher prevalence of respiratory and cutaneous manifestations.
Specific clinical manifestations, as well as groups of clinical manifestations, are often overrepresented by an individual DENV serotype.
Author Summary
Dengue virus (DENV) causes disease in millions of people annually and disproportionately affects those in the developing world. DENVs may be divided into four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) and a geographical region may be affected by one or more DENV serotypes simultaneously. Infection with DENV may cause life-threatening disease such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), but more often causes less severe manifestations affecting a wide range of organs. Although many previous reports have explored the role of the different DENV serotypes in the development of severe manifestations, little attention has focused on the relative role of each DENV serotype in the development of cutaneous, respiratory, gastrointestinal, musculoskeletal, and neurological manifestations. We recruited a large group of participants from four countries in South America to compare the prevalence of more than 30 manifestations among the four different DENV serotypes. We found that certain DENV serotypes were often associated with a higher prevalence of a certain manifestation (e.g., DENV-3 and diarrhea) or manifestation group (e.g., DENV-4 and cutaneous manifestations).
PMCID: PMC3341333  PMID: 22563516
6.  A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease 
Dengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via high-throughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, showing a 50% effective concentration (EC50) of 1.03 ± 0.09 μM. Without detectable cytotoxicity, the compound inhibited replication or viral RNA synthesis in all four serotypes of DENV but not in Japanese encephalitis virus (JEV). Sequencing analyses of several individual clones derived from BP13944-resistant RNAs purified from cells harboring the DENV-2 replicon revealed a consensus amino acid substitution (E66G) in the region of the NS3 protease domain. Introduction of E66G into the DENV replicon, an infectious DENV cDNA clone, and recombinant NS2B/NS3 protease constructs conferred 15.2-, 17.2-, and 3.1-fold resistance to BP13944, respectively. Our results identify an effective small-molecule inhibitor, BP13944, which likely targets the DENV NS3 protease. BP13944 could be considered part of a more effective treatment regime for inhibiting DENV in the future.
PMCID: PMC3910792  PMID: 24145533
7.  Conservation and Variability of Dengue Virus Proteins: Implications for Vaccine Design 
Genetic variation and rapid evolution are hallmarks of RNA viruses, the result of high mutation rates in RNA replication and selection of mutants that enhance viral adaptation, including the escape from host immune responses. Variability is uneven across the genome because mutations resulting in a deleterious effect on viral fitness are restricted. RNA viruses are thus marked by protein sites permissive to multiple mutations and sites critical to viral structure-function that are evolutionarily robust and highly conserved. Identification and characterization of the historical dynamics of the conserved sites have relevance to multiple applications, including potential targets for diagnosis, and prophylactic and therapeutic purposes.
Methodology/Principal Findings
We describe a large-scale identification and analysis of evolutionarily highly conserved amino acid sequences of the entire dengue virus (DENV) proteome, with a focus on sequences of 9 amino acids or more, and thus immune-relevant as potential T-cell determinants. DENV protein sequence data were collected from the NCBI Entrez protein database in 2005 (9,512 sequences) and again in 2007 (12,404 sequences). Forty-four (44) sequences (pan-DENV sequences), mainly those of nonstructural proteins and representing ∼15% of the DENV polyprotein length, were identical in 80% or more of all recorded DENV sequences. Of these 44 sequences, 34 (∼77%) were present in ≥95% of sequences of each DENV type, and 27 (∼61%) were conserved in other Flaviviruses. The frequencies of variants of the pan-DENV sequences were low (0 to ∼5%), as compared to variant frequencies of ∼60 to ∼85% in the non pan-DENV sequence regions. We further showed that the majority of the conserved sequences were immunologically relevant: 34 contained numerous predicted human leukocyte antigen (HLA) supertype-restricted peptide sequences, and 26 contained T-cell determinants identified by studies with HLA-transgenic mice and/or reported to be immunogenic in humans.
Forty-four (44) pan-DENV sequences of at least 9 amino acids were highly conserved and identical in 80% or more of all recorded DENV sequences, and the majority were found to be immune-relevant by their correspondence to known or putative HLA-restricted T-cell determinants. The conservation of these sequences through the entire recorded DENV genetic history supports their possible value for diagnosis, prophylactic and/or therapeutic applications. The combination of bioinformatics and experimental approaches applied herein provides a framework for large-scale and systematic analysis of conserved and variable sequences of other pathogens, in particular, for rapidly mutating viruses, such as influenza A virus and HIV.
Author Summary
Dengue viruses (DENVs) circulate in nature as a population of 4 distinct types, each with multiple genotypes and variants, and represent an increasing global public health issue with no prophylactic and therapeutic formulations currently available. Viral genomes contain sites that are evolutionarily stable and therefore highly conserved, presumably because changes in these sites have deleterious effects on viral fitness and survival. The identification and characterization of the historical dynamics of these sites in DENV have relevance to several applications such as diagnosis and drug and vaccine development. In this study, we have identified sequence fragments that were conserved across the majority of available DENV sequences, analyzed their historical dynamics, and evaluated their relevance as candidate vaccine targets, using various bioinformatics-based methods and immune assay in human leukocyte antigen (HLA) transgenic mice. This approach provides a framework for large-scale and systematic analysis of other human pathogens.
PMCID: PMC2491585  PMID: 18698358
8.  Inhibition of Dengue Virus Polymerase by Blocking of the RNA Tunnel▿  
Journal of Virology  2010;84(11):5678-5686.
Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)20]. Chemical modification of the initial “hit” improved the compound potency to an IC50 (that is, a concentration that inhibits 50% RdRp activity) of 0.7 μM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC50 of 5 μM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC50s of >100 μM against hepatitis C virus RdRp and human DNA polymerase α and β. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.
PMCID: PMC2876596  PMID: 20237086
9.  Novel Benzoxazole Inhibitor of Dengue Virus Replication That Targets the NS3 Helicase 
Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.
PMCID: PMC3623359  PMID: 23403421
10.  Development of an Antigen Capture Immunoassay Based on Monoclonal Antibodies Specific for Dengue Virus Serotype 2 Nonstructural Protein 1 for Early and Rapid Identification of Dengue Virus Serotype 2 Infections▿  
The dengue virus (DENV) has four distinct serotypes (DENV1, DENV2, DENV3, and DENV4) that require differentiation for effective prevention of morbid diseases. The recently developed DENV1-specific NS1 antigen capture enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibodies (MAbs) that recognize distinct epitopes on nonstructural protein 1 (NS1) of a specific DENV serotype is convenient and cost-effective, but assays have not yet been developed for DENV serotypes 2 to 4. This paper describes the development and validation of a DENV2-specific NS1 antigen capture ELISA by selection and optimization of the pair of well-characterized MAbs that recognized epitopes specific for DENV2 NS1 from a large panel of MAbs. The DENV2 NS1 ELISA displayed exclusive sensitivity with the DENV2 serotype and did not cross-react with the other three DENV serotypes. The sensitivity and specificity of the DENV2 NS1 ELISA were 83.3% (25/30) and 100% (504/504) when used to test 30 acute-phase serum samples from patients infected with DENV2 identified by virus isolation or reverse transcription-PCR serotyping and 504 serum samples from healthy individuals, respectively. The specificity of this assay was also evaluated using a panel of serum samples which were positive for DENV1, other flaviviruses, and nonflaviviruses; no cross-reactions were observed in these clinical samples. The DENV2 NS1 ELISA was eightfold more sensitive than a commercially available serotype-cross-reactive NS1 ELISA (Panbio Diagnostics, Brisbane, Australia) when the two assays were used to test the DENV2-infected cell culture supernatants in parallel. The Panbio NS1 ELISA displayed variation in sensitivity between DENV serotypes. The DENV2-specific NS1 antigen capture ELISA can be used as a tool for the rapid identification of DENV2 infections.
PMCID: PMC2620663  PMID: 19020106
11.  Dominant Cross-Reactive B Cell Response during Secondary Acute Dengue Virus Infection in Humans 
The four serotypes of dengue virus (DENV) cause dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Severe disease has been associated with heterotypic secondary DENV infection, mediated by cross-reactive antibodies (Abs) and/or cross-reactive T cells. The role of cross-reactive immunity in mediating enhanced disease versus cross-protection against secondary heterotypic DENV infection is not well defined. A better understanding of the cross-reactive immune response in natural infections is critical for development of safe and effective tetravalent vaccines. We studied the B cell phenotype of circulating B cells in the blood of pediatric patients suspected of dengue during the 2010–2011 dengue season in Managua, Nicaragua (n = 216), which was dominated by the DENV-3 serotype. We found a markedly larger percentage of plasmablast/plasma cells (PB/PCs) circulating in DENV-positive patients as compared to patients with Other Febrile Illnesses (OFIs). The percentage of DENV-specific PB/PCs against DENV-3 represented 10% of the circulating antibody-producing cells (ASCs) in secondary DENV-3 infections. Importantly, the cross-reactive DENV-specific B cell response was higher against a heterotypic serotype, with 46% of circulating PB/PCs specific to DENV-2 and 10% specific to DENV-3 during acute infection. We also observed a higher cross-reactive DENV-specific IgG serum avidity directed against DENV-2 as compared to DENV-3 during acute infection. The neutralization capacity of the serum was broadly cross-reactive against the four DENV serotypes both during the acute phase and at 3 months post-onset of symptoms. Overall, the cross-reactive B cell immune response dominates during secondary DENV infections in humans. These results reflect our recent findings in a mouse model of DENV cross-protection. In addition, this study enabled the development of increased technical and research capacity of Nicaraguan scientists and the implementation of several new immunological assays in the field.
Author Summary
Dengue is the most common mosquito-borne viral infection of humans, with half the world's population at risk for infection. Four different dengue virus serotypes (DENV-1 to -4) can cause the disease, which can be either inapparent or present with flu-like symptoms (Dengue Fever), also known as “breakbone fever”. In a number of cases, the disease can be more severe and sometimes fatal, with signs of bleeding and vascular leakage leading to shock (Dengue Hemorrhagic Fever/Dengue Shock Syndrome). Severe disease has been associated with secondary sequential DENV infections, i.e., infection with a second DENV serotype different from the serotype causing the first infection. No specific treatment or vaccine is available. Understanding how the human immune response develops during a natural infection can be beneficial for future vaccine studies and trials. B cells are a subset of cells that produce antibodies and are thus essential in the response to natural infections and vaccines. We show here that during secondary DENV infections in humans, the B cell immune response to a previous infecting DENV serotype is stronger than the response against the current infecting serotype. In addition, this study allowed the development of research capacity and implementation of new immunological assays in Nicaragua.
PMCID: PMC3308930  PMID: 22448292
12.  Detection of Serotype-Specific Antibodies to the Four Dengue Viruses Using an Immune Complex Binding (ICB) ELISA 
Dengue virus (DENV) infections are preferentially diagnosed by detection of specific IgM antibodies, DENV NS1 antigen assays or by amplification of viral RNA in serum samples of the patients. The type-specific immunity to the four worldwide circulating DENV serotypes can be determined by neutralization assays. An alternative to the complicated neutralization assays would be helpful to study the serotype-specific immune response in people in DENV hyperendemic areas but also in subjects upon DENV vaccination.
In consecutive samples of patients with DENV-1- 4 infection type-specific antibodies were detected using an immune complex binding (ICB) ELISA. During incubation of serum samples and enzyme- labeled recombinant envelope domain III (EDIII) antigens immune complexes (ICs) are formed, which are simultaneously bound to a solid phase coated with an Fc–receptor (CD32). After a single washing procedure the bound labeled ICs can be determined. To further improve type-specific reactions high concentrations of competing heterologous unlabeled ED III proteins were added to the labeled antigens.
Follow-up serum samples of 64 patients with RT-PCR confirmed primary DENV-1, -2, -3 or -4 infections were tested against four enzyme-labeled recombinant DENV EDIII antigens. Antibodies to the EDIII antigens were found in 55 patients (sensitivity 86%). A complete agreement between the serotype detected by PCR in early samples and the serotype-specific antibody in later samples was found. Type-specific anti-EDIII antibodies were first detected 9–20 days after onset of the disease. In 21% of the samples collected from people in Vietnam secondary infections with antibodies to two serotypes could be identified.
The data obtained with the ICB-ELISA show that after primary DENV infection the corresponding type-specific antibodies are detected in almost all samples collected at least two weeks after onset of the disease. The method will be of value to determine the distribution of the various type-specific anti–DENV antibodies in DENV endemic areas.
Author Summary
Infections with four different dengue viruses are threatening 2.5 billion people in tropical countries. Since most antibodies to these four viruses are cross-reacting, a type-specific ELISA would be valuable to study the immune response to the circulating viruses in patients but also in healthy subjects in endemic counties. Therefore a novel DENV immune complex binding (ICB) ELISA was developed to detect serotype-specific antibodies to all four dengue virus serotypes in human serum samples. The tests use labeled recombinant EDIII antigens of the four DENV strains. Numerous samples of patients with RT-PCR confirmed dengue fever were assessed by the new method. In samples of 55 patients with primary dengue fever full agreement between the serotype detected by RT-PCR and the serotype-specific antibody based on the ICB ELISA was obtained. The type-specific antibodies were not observed before the second week of illness. Our data suggest that using the ICB ELISA in healthy adult subjects in an endemic region (Vietnam) both primary and secondary infections can be identified. The method may help to analyze the distribution of the four dengue viruses in the tropics.
PMCID: PMC3873247  PMID: 24386498
13.  Dengue Virus Co-opts UBR4 to Degrade STAT2 and Antagonize Type I Interferon Signaling 
PLoS Pathogens  2013;9(3):e1003265.
An estimated 50 million dengue virus (DENV) infections occur annually and more than forty percent of the human population is currently at risk of developing dengue fever (DF) or dengue hemorrhagic fever (DHF). Despite the prevalence and potential severity of DF and DHF, there are no approved vaccines or antiviral therapeutics available. An improved understanding of DENV immune evasion is pivotal for the rational development of anti-DENV therapeutics. Antagonism of type I interferon (IFN-I) signaling is a crucial mechanism of DENV immune evasion. DENV NS5 protein inhibits IFN-I signaling by mediating proteasome-dependent STAT2 degradation. Only proteolytically-processed NS5 can efficiently mediate STAT2 degradation, though both unprocessed and processed NS5 bind STAT2. Here we identify UBR4, a 600-kDa member of the N-recognin family, as an interacting partner of DENV NS5 that preferentially binds to processed NS5. Our results also demonstrate that DENV NS5 bridges STAT2 and UBR4. Furthermore, we show that UBR4 promotes DENV-mediated STAT2 degradation, and most importantly, that UBR4 is necessary for efficient viral replication in IFN-I competent cells. Our data underscore the importance of NS5-mediated STAT2 degradation in DENV replication and identify UBR4 as a host protein that is specifically exploited by DENV to inhibit IFN-I signaling via STAT2 degradation.
Author Summary
Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. At present, there are no vaccines and no specific antiviral therapeutics to prevent or treat DENV infections. We previously described that the NS5 protein of DENV inhibits type I interferon signaling in virus-infected cells by mediating STAT2 degradation. This property allows DENV to overcome the antiviral effects of type I interferon, contributing to viral replication in the host. We have now obtained new insight into the mechanism by which DENV NS5 induces STAT2 degradation. NS5 bridges STAT2 with the cellular protein UBR4, a member of a family of predicted E3 ligases, resulting in UBR4-mediated STAT2 degradation. Elimination of UBR4 or mutations in NS5 that prevent its binding to UBR4 prevent NS5 from inducing STAT2 degradation. Importantly, UBR4 is required for optimal DENV replication in the presence of a competent type I interferon system. Our data demonstrate the requirement of a host factor, UBR4, for DENV to overcome the antiviral interferon response. This information might be important for the design of specific DENV inhibitors that prevent dengue virus from evading innate immunity.
PMCID: PMC3610674  PMID: 23555265
14.  The development, optimization and validation of an assay for high throughput antiviral drug screening against Dengue virus 
Dengue virus (DENV) is listed as one of the NIAID Category A priority pathogens. Dengue disease is endemic in most tropical countries, with an estimated 2.5 billion people living in areas at risk of DENV infection. Due to the lack of vaccines and antiviral drugs, it is now a huge public health burden around the world. In order to screen large compound libraries for the identification of novel antivirals targeting DENV, it is essential to develop a high throughput screening (HTS) amenable assay. Here, we present the development, optimization and validation of a cytopathic effect-based assay against Dengue virus serotype-2 (DENV-2). The assay conditions, including cell culturing conditions, DMSO tolerance and the multiplicity of infection, were optimized in both 96- and 384-well plates. Assay robustness and reproducibility were determined under the optimized conditions in 96-well plate, including Z'-value of 0.71, signal-to-background ratio of 6.88, coefficient of variation of 6.3% in mock-infected cells and 12.3% in DENV-2 infected cells. This assay was further miniaturized into a 384-well plate format with similar assay robustness and reproducibility comparing with these in the 96-well plate format. This assay was then validated using the LOPAC1280 compound library, demonstrating its repeatability with comparable assay robustness and reproducibility. This fully developed and validated HTS amenable assay could be used in future studies to screen large compound libraries for the identification of novel antivirals against dengue disease.
PMCID: PMC2802053  PMID: 20057980
Dengue virus; high throughput screening; HTS; cytopathic effect; CPE; assay development; assay optimization; assay validation; antiviral
15.  Inhibition of Dengue Virus through Suppression of Host Pyrimidine Biosynthesis▿ 
Journal of Virology  2011;85(13):6548-6556.
Viral replication relies on the host to supply nucleosides. Host enzymes involved in nucleoside biosynthesis are potential targets for antiviral development. Ribavirin (a known antiviral drug) is such an inhibitor that suppresses guanine biosynthesis; depletion of the intracellular GTP pool was shown to be the major mechanism to inhibit flavivirus. Along similar lines, inhibitors of the pyrimidine biosynthesis pathway could be targeted for potential antiviral development. Here we report on a novel antiviral compound (NITD-982) that inhibits host dihydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine biosynthesis. The inhibitor was identified through screening 1.8 million compounds using a dengue virus (DENV) infection assay. The compound contains an isoxazole-pyrazole core structure, and it inhibited DENV with a 50% effective concentration (EC50) of 2.4 nM and a 50% cytotoxic concentration (CC50) of >5 μM. NITD-982 has a broad antiviral spectrum, inhibiting both flaviviruses and nonflaviviruses with nanomolar EC90s. We also show that (i) the compound inhibited the enzymatic activity of recombinant DHODH, (ii) an NITD-982 analogue directly bound to the DHODH protein, (iii) supplementing the culture medium with uridine reversed the compound-mediated antiviral activity, and (iv) DENV type 2 (DENV-2) variants resistant to brequinar (a known DHODH inhibitor) were cross resistant to NITD-982. Collectively, the results demonstrate that the compound inhibits DENV through depleting the intracellular pyrimidine pool. In contrast to the in vitro potency, the compound did not show any efficacy in the DENV-AG129 mouse model. The lack of in vivo efficacy is likely due to the exogenous uptake of pyrimidine from the diet or to a high plasma protein-binding activity of the current compound.
PMCID: PMC3126545  PMID: 21507975
16.  Chimpanzee Fab Fragments and a Derived Humanized Immunoglobulin G1 Antibody That Efficiently Cross-Neutralize Dengue Type 1 and Type 2 Viruses 
Journal of Virology  2004;78(23):12910-12918.
Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative to vaccines for prevention of illness caused by dengue viruses (DENV) and other flaviviruses, including the West Nile virus. In a previous study, repertoire cloning to recover Fab fragments from bone marrow mRNA of chimpanzees infected with all four DENV serotypes (dengue virus serotype 1 [DENV-1] to DENV-4) was described. In that study, a humanized immunoglobulin G1 (IgG1) antibody that efficiently neutralized DENV-4 was recovered and characterized. In this study, the phage library constructed from the chimpanzees was used to recover Fab antibodies against the other three DENV serotypes. Serotype-specific neutralizing Fabs were not identified. Instead, we recovered DENV-neutralizing Fabs that specifically precipitated the envelope protein and were cross-reactive with all four DENV serotypes. Three of the Fabs competed with each other for binding to DENV-1 and DENV-2, although each of these Fabs contained a distinct complementarity determining region 3 (CDR3)-H sequence. Fabs that shared an identical or nearly identical CDR3-H sequences cross-neutralized DENV-1 and DENV-2 at a similar high 50% plaque reduction neutralization test (PRNT50) titer, ranging from 0.26 to 1.33 μg/ml, and neutralized DENV-3 and DENV-4 but at a titer 10- to 20-fold lower. One of these Fabs, 1A5, also neutralized the West Nile virus most efficiently among other flaviviruses tested. Fab 1A5 was converted to a full-length antibody in combination with human sequences for production in mammalian CHO cells. Humanized IgG1 1A5 proved to be as efficient as Fab 1A5 for cross-neutralization of DENV-1 and DENV-2 at a titer of 0.48 and 0.95 μg/ml, respectively. IgG1 1A5 also neutralized DENV-3, DENV-4, and the West Nile virus at a PRNT50 titer of approximately 3.2 to 4.2 μg/ml. This humanized antibody represents an attractive candidate for further development of immunoprophylaxis against DENV and perhaps other flavivirus-associated diseases.
PMCID: PMC525007  PMID: 15542643
17.  Inhibition of Dengue Virus Replication by a Class of Small-Molecule Compounds That Antagonize Dopamine Receptor D4 and Downstream Mitogen-Activated Protein Kinase Signaling 
Journal of Virology  2014;88(10):5533-5542.
Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds—dihydrodibenzothiepines (DHBTs), identified through high-throughput screening—with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds.
IMPORTANCE The dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.
PMCID: PMC4019099  PMID: 24599995
18.  Utility of Humanized BLT Mice for Analysis of Dengue Virus Infection and Antiviral Drug Testing 
Journal of Virology  2014;88(4):2205-2218.
Dengue virus (DENV) is the cause of a potentially life-threatening disease that affects millions of people worldwide. The lack of a small animal model that mimics the symptoms of DENV infection in humans has slowed the understanding of viral pathogenesis and the development of therapies and vaccines. Here, we investigated the use of humanized “bone marrow liver thymus” (BLT) mice as a model for immunological studies and assayed their applicability for preclinical testing of antiviral compounds. Human immune system (HIS) BLT-NOD/SCID mice were inoculated intravenously with a low-passage, clinical isolate of DENV-2, and this resulted in sustained viremia and infection of leukocytes in lymphoid and nonlymphoid organs. In addition, DENV infection increased serum cytokine levels and elicited DENV-2-neutralizing human IgM antibodies. Following restimulation with DENV-infected dendritic cells, in vivo-primed T cells became activated and acquired effector function. An adenosine nucleoside inhibitor of DENV decreased the circulating viral RNA when administered simultaneously or 2 days postinfection, simulating a potential treatment protocol for DENV infection in humans. In summary, we demonstrate that BLT mice are susceptible to infection with clinical DENV isolates, mount virus-specific adaptive immune responses, and respond to antiviral drug treatment. Although additional refinements to the model are required, BLT mice are a suitable platform to study aspects of DENV infection and pathogenesis and for preclinical testing of drug and vaccine candidates.
PMCID: PMC3911540  PMID: 24335303
19.  Antiviral activity of four types of bioflavonoid against dengue virus type-2 
Virology Journal  2011;8:560.
Dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. Effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2) in Vero cell was evaluated. Anti-dengue activity of these compounds was determined at different stages of DENV-2 infection and replication cycle. DENV replication was measured by Foci Forming Unit Reduction Assay (FFURA) and quantitative RT-PCR. Selectivity Index value (SI) was determined as the ratio of cytotoxic concentration 50 (CC50) to inhibitory concentration 50 (IC50) for each compound.
The half maximal inhibitory concentration (IC50) of quercetin against dengue virus was 35.7 μg mL-1 when it was used after virus adsorption to the cells. The IC50 decreased to 28.9 μg mL-1 when the cells were treated continuously for 5 h before virus infection and up to 4 days post-infection. The SI values for quercetin were 7.07 and 8.74 μg mL-1, respectively, the highest compared to all bioflavonoids studied. Naringin only exhibited anti-adsorption effects against DENV-2 with IC50 = 168.2 μg mL-1 and its related SI was 1.3. Daidzein showed a weak anti-dengue activity with IC50 = 142.6 μg mL-1 when the DENV-2 infected cells were treated after virus adsorption. The SI value for this compound was 1.03. Hesperetin did not exhibit any antiviral activity against DENV-2. The findings obtained from Foci Forming Unit Reduction Assay (FFURA) were corroborated by findings of the qRT-PCR assays. Quercetin and daidzein (50 μg mL-1) reduced DENV-2 RNA levels by 67% and 25%, respectively. There was no significant inhibition of DENV-2 RNA levels with naringin and hesperetin.
Results from the study suggest that only quercetin demonstrated significant anti-DENV-2 inhibitory activities. Other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant inhibition of DENV-2 virus replication. These findings, together with those previously reported suggest that select group of bioflavonoids including quercetin and fisetin, exhibited significant inhibitory activities against dengue virus. This group of flavonoids, flavonol, could be investigated further to discover the common mechanisms of inhibition of dengue virus replication.
PMCID: PMC3271998  PMID: 22201648
Antiviral; Dengue virus; Flavonoid; Quercetin; Naringin; Daidzein; Hesperetin
20.  Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes 
Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design.
Author Summary
Although there is an increased recognition of the role of T-cells in both dengue pathogenesis and protection, comprehensive analysis of T-cell activation during dengue infection is hampered by the small repertoire of known human dengue T-cell epitopes. Although dengue serotype 3 (DENV3) is responsible for numerous outbreaks worldwide, most of the known epitopes are from studies of dengue 2 serotype (DENV2). In this study, we identified novel DENV3 T-cell epitopes in HLA transgenic mice that were confirmed by HLA binding assays. A subset of these epitopes activated memory T-cells from subjects who were dengue IgG positive and primed naïve T-cells from dengue IgG negative individuals. Notably, some of HLA class II epitopes bearing highly conserved regions common to all four dengue serotypes could bind to multiple HLAs. We postulate that these highly conserved and HLA promiscuous T-helper epitopes can be important components of a dengue tetravalent vaccine.
PMCID: PMC3794980  PMID: 24130917
21.  Monocyte Recruitment to the Dermis and Differentiation to Dendritic Cells Increases the Targets for Dengue Virus Replication 
PLoS Pathogens  2014;10(12):e1004541.
Dengue virus (DENV) causes the most prevalent arthropod-borne viral disease in humans. Although Aedes mosquitoes transmit DENV when probing for blood in the skin, no information exists on DENV infection and immune response in the dermis, where the blood vessels are found. DENV suppresses the interferon response, replicates, and causes disease in humans but not wild-type mice. Here, we used mice lacking the interferon-α/β receptor (Ifnar–/–), which had normal cell populations in the skin and were susceptible to intradermal DENV infection, to investigate the dynamics of early DENV infection of immune cells in the skin. CD103+ classical dendritic cells (cDCs), Ly6C– CD11b+ cDCs, and macrophages in the steady-state dermis were initial targets of DENV infection 12-24 hours post-inoculation but then decreased in frequency. We demonstrated recruitment of adoptively-transferred Ly6Chigh monocytes from wild-type and Ifnar–/– origin to the DENV-infected dermis and differentiation to Ly6C+ CD11b+ monocyte-derived DCs (moDCs), which became DENV-infected after 48 hours, and were then the major targets for virus replication. Ly6Chigh monocytes that entered the DENV-infected dermis expressed chemokine receptor CCR2, likely mediating recruitment. Further, we show that ∼100-fold more hematopoietic cells in the dermis were DENV-infected compared to Langerhans cells in the epidermis. Overall, these results identify the dermis as the main site of early DENV replication and show that DENV infection in the skin occurs in two waves: initial infection of resident cDCs and macrophages, followed by infection of monocytes and moDCs that are recruited to the dermis. Our study reveals a novel viral strategy of exploiting monocyte recruitment to increase the number of targets for infection at the site of invasion in the skin and highlights the skin as a potential site for therapeutic action or intradermal vaccination.
Author Summary
The skin and its immune cells are an important barrier against invading pathogens. Dengue is a major public health problem worldwide, with no specific therapeutic or vaccine available. Aedes mosquitoes transmit dengue virus (DENV) to humans via the skin when taking a blood meal. Previous studies have examined DENV infection only in the epidermis, the uppermost layer of the skin, but no information existed about DENV infection in the dermis, the layer below that contains blood vessels. We established a model of DENV infection in the skin of mouse ears, as biopsies from naturally-infected human skin are unavailable. The normal dermis contains classical dendritic cells (DCs) and macrophages, which we found to be the initial targets of DENV infection. Monocytes that circulate in the blood were then recruited to the dermis and differentiated to monocyte-derived DCs, an inflammatory DC subset. These newly-recruited monocytes and monocyte-derived DCs became DENV-infected in a second wave and then were the major targets for DENV replication. Our study identifies how DENV exploits the immune response by infecting cells that are recruited to the skin as part of antiviral defense. These results should help future research to develop new strategies for vaccination and therapeutics against dengue.
PMCID: PMC4256458  PMID: 25474197
22.  Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay ▿ †  
Dengue virus (DENV) causes disease globally, with an estimated 25 to 100 million new infections per year. At present, no effective vaccine is available, and treatment is supportive. In this study, we identified BP2109, a potent and selective small-molecule inhibitor of the DENV NS2B/NS3 protease, by a high-throughput screening assay using a recombinant protease complex consisting of the central hydrophilic portion of NS2B and the N terminus of the protease domain. BP2109 inhibited DENV (serotypes 1 to 4), but not Japanese encephalitis virus (JEV), replication and viral RNA synthesis without detectable cytotoxicity. The compound inhibited recombinant DENV-2 NS2B/NS3 protease with a 50% inhibitory concentration (IC50) of 15.43 ± 2.12 μM and reduced the reporter expression of the DENV-2 replicon with a 50% effective concentration (EC50) of 0.17 ± 0.01 μM. Sequencing analyses of several individual clones derived from BP2109-resistant DENV-2 RNAs revealed that two amino acid substitutions (R55K and E80K) are found in the region of NS2B, a cofactor of the NS2B/NS3 protease complex. The introduction of R55K and E80K double mutations into the dengue virus NS2B/NS3 protease and a dengue virus replicon construct conferred 10.3- and 73.8-fold resistance to BP2109, respectively. The E80K mutation was further determined to be the key mutation conferring dengue virus replicon resistance (61.3-fold) to BP2109, whereas the R55K mutation alone did not affect resistance to BP2109. Both the R55K and E80K mutations are located in the central hydrophilic portion of the NS2B cofactor, where extensive interactions with the NS3pro domain exist. Thus, our data provide evidence that BP2109 likely inhibits DENV by a novel mechanism.
PMCID: PMC3019636  PMID: 20937790
23.  DENV Inhibits Type I IFN Production in Infected Cells by Cleaving Human STING 
PLoS Pathogens  2012;8(10):e1002934.
Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.
Author Summary
Dengue virus (DENV) is a pathogen with a high impact in human health that replicates in a wide range of cells of the immune system. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response (IFN). Thus, DENV can inhibit type I IFN signaling (described by several groups), and type I IFN production (described by our group). We documented the inhibition of type I IFN production in human monocyte derived dendritic cells (MDDCs) with an otherwise strong cytokine and chemokine profile in those cells and that the NS2B3 protease complex of DENV functions as an antagonist of type I IFN production, and its proteolytic activity is necessary for this event. Here we identify the human adaptor molecule STING as a target of the NS2B3 protease complex and characterize the mechanism of inhibition of the type I IFN production in primary human MDDCs mediated by this viral factor. We also describe that DENV NS2B3 cannot degrade the mouse version of STING, a phenomenon that strictly restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.
PMCID: PMC3464218  PMID: 23055924
24.  Spatial Dimensions of Dengue Virus Transmission across Interepidemic and Epidemic Periods in Iquitos, Peru (1999–2003) 
Knowledge of spatial patterns of dengue virus (DENV) infection is important for understanding transmission dynamics and guiding effective disease prevention strategies. Because movement of infected humans and mosquito vectors plays a role in the spread and persistence of virus, spatial dimensions of transmission can range from small household foci to large community clusters. Current understanding is limited because past analyses emphasized clinically apparent illness and did not account for the potentially large proportion of inapparent infections. In this study we analyzed both clinically apparent and overall infections to determine the extent of clustering among human DENV infections.
Methodology/Principal Findings
We conducted spatial analyses at global and local scales, using acute case and seroconversion data from a prospective longitudinal cohort in Iquitos, Peru, from 1999–2003. Our study began during a period of interepidemic DENV-1 and DENV-2 transmission and transitioned to epidemic DENV-3 transmission. Infection status was determined by seroconversion based on plaque neutralization testing of sequential blood samples taken at approximately six-month intervals, with date of infection assigned as the middate between paired samples. Each year was divided into three distinct seasonal periods of DENV transmission. Spatial heterogeneity was detected in baseline seroprevalence for DENV-1 and DENV-2. Cumulative DENV-3 seroprevalence calculated by trimester from 2001–2003 was spatially similar to preexisting DENV-1 and DENV-2 seroprevalence. Global clustering (case-control Ripley's K statistic) appeared at radii of ∼200–800 m. Local analyses (Kuldorf spatial scan statistic) identified eight DENV-1 and 15 DENV-3 clusters from 1999–2003. The number of seroconversions per cluster ranged from 3–34 with radii from zero (a single household) to 750 m; 65% of clusters had radii >100 m. No clustering was detected among clinically apparent infections.
Seroprevalence of previously circulating DENV serotypes can be a predictor of transmission risk for a different invading serotype and, thus, identify targets for strategically placed surveillance and intervention. Seroprevalence of a specific serotype is also important, but does not preclude other contributing factors, such as mosquito density, in determining where transmission of that virus will occur. Regardless of the epidemiological context or virus serotype, human movement appears to be an important factor in defining the spatial dimensions of DENV transmission and, thus, should be considered in the design and evaluation of surveillance and intervention strategies.
Author Summary
To target prevention and control strategies for dengue fever, it is essential to understand how the virus travels through the city. We report spatial analyses of dengue infections from a study monitoring school children and adult family members for dengue infection at six-month intervals from 1999–2003, in the Amazonian city of Iquitos, Peru. At the beginning of the study, only DENV serotypes 1 and 2 were circulating. Clusters of infections of these two viruses were concentrated in the northern region of the city, where mosquito indices and previous DENV infection were both high. In 2002, DENV-3 invaded the city, replacing DENV-1 and -2 as the dominant strain. During the invasion process, the virus spread rapidly across the city, at low levels. After this initial phase, clusters of infection appeared first in the northern region of the city, where clusters of DENV-1 and DENV-2 had occurred in prior years. Most of the clusters we identified had radii >100 meters, indicating that targeted or reactive treatment of these high-risk areas might be an effective proactive intervention strategy. Our results also help explain why vector control within 100 m of a dengue case is often not successful for large-scale disease prevention.
PMCID: PMC3283551  PMID: 22363822
25.  The Type-Specific Neutralizing Antibody Response Elicited by a Dengue Vaccine Candidate Is Focused on Two Amino Acids of the Envelope Protein 
PLoS Pathogens  2013;9(12):e1003761.
Dengue viruses are mosquito-borne flaviviruses that circulate in nature as four distinct serotypes (DENV1-4). These emerging pathogens are responsible for more than 100 million human infections annually. Severe clinical manifestations of disease are predominantly associated with a secondary infection by a heterotypic DENV serotype. The increased risk of severe disease in DENV-sensitized populations significantly complicates vaccine development, as a vaccine must simultaneously confer protection against all four DENV serotypes. Eliciting a protective tetravalent neutralizing antibody response is a major goal of ongoing vaccine development efforts. However, a recent large clinical trial of a candidate live-attenuated DENV vaccine revealed low protective efficacy despite eliciting a neutralizing antibody response, highlighting the need for a better understanding of the humoral immune response against dengue infection. In this study, we sought to identify epitopes recognized by serotype-specific neutralizing antibodies elicited by monovalent DENV1 vaccination. We constructed a panel of over 50 DENV1 structural gene variants containing substitutions at surface-accessible residues of the envelope (E) protein to match the corresponding DENV2 sequence. Amino acids that contribute to recognition by serotype-specific neutralizing antibodies were identified as DENV mutants with reduced sensitivity to neutralization by DENV1 immune sera, but not cross-reactive neutralizing antibodies elicited by DENV2 vaccination. We identified two mutations (E126K and E157K) that contribute significantly to type-specific recognition by polyclonal DENV1 immune sera. Longitudinal and cross-sectional analysis of sera from 24 participants of a phase I clinical study revealed a markedly reduced capacity to neutralize a E126K/E157K DENV1 variant. Sera from 77% of subjects recognized the E126K/E157K DENV1 variant and DENV2 equivalently (<3-fold difference). These data indicate the type-specific component of the DENV1 neutralizing antibody response to vaccination is strikingly focused on just two amino acids of the E protein. This study provides an important step towards deconvoluting the functional complexity of DENV serology following vaccination.
Author Summary
Despite decades of research, there remains a critical need for a dengue virus (DENV) vaccine. Vaccine development efforts are complicated by a requirement to protect against four DENV serotypes (DENV1-4), and incomplete immunity as a risk factor for severe disease. Antibodies play a major protective role against DENV. However, they also have been implicated in severe clinical manifestations of DENV infection. The antibody response to DENV is composed of antibodies that neutralize only the infecting DENV serotype (type-specific), as well as those that are cross-reactive. Cross-reactive antibodies are hypothesized to contribute to severe dengue following heterologous infections. Identifying DENV epitopes that are targets of type-specific neutralizing antibodies may facilitate vaccine development and the identification of correlates of protection. In this study, we identified amino acids on DENV1 recognized by type-specific neutralizing antibodies elicited by DENV1 vaccination. Our results indicate that the type-specific DENV1 response is remarkably focused on just two regions of the DENV1 envelope protein. Furthermore, a significant contribution of antibodies with this specificity was a common feature among vaccine recipients. This study identifies targets of neutralizing antibodies elicited by DENV1 vaccination and provides an important first step toward identifying epitopes recognized by each component of a tetravalent vaccine.
PMCID: PMC3857832  PMID: 24348242

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