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1.  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
2.  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.
doi:10.1128/AAC.02251-12
PMCID: PMC3623359  PMID: 23403421
3.  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.
doi:10.1128/JVI.02451-09
PMCID: PMC2876596  PMID: 20237086
4.  A novel reporter system for neutralizing and enhancing antibody assay against dengue virus 
BMC Microbiology  2014;14:44.
Background
Dengue virus (DENV) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. Antibody plays distinct roles in controlling DENV infections. Neutralizing antibody is protective against DENV infection, whereas sub-neutralizing concentration of antibody can increase DENV infection, termed antibody-dependent enhancement (ADE). Plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies.
Results
In this study, a novel reporter virus-based system was developed for measuring neutralization and ADE activity. A stable Renilla luciferase reporter DENV (Luc-DENV) that can produce robust luciferase signals in BHK-21 and K562 cells were used to establish the assay and validated against traditional plaque-based assay. Luciferase value analysis using various known DENV-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. The newly developed assay was finally validated with clinical samples from infected animals and individuals.
Conclusions
This reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for DENV antibodies.
doi:10.1186/1471-2180-14-44
PMCID: PMC3930823  PMID: 24548533
Dengue virus; Neutralizing antibody; Enhancing antibody; Luciferase assay
5.  Dengue Virus Entry as Target for Antiviral Therapy 
Journal of Tropical Medicine  2012;2012:628475.
Dengue virus (DENV) infections are expanding worldwide and, because of the lack of a vaccine, the search for antiviral products is imperative. Four serotypes of DENV are described and they all cause a similar disease outcome. It would be interesting to develop an antiviral product that can interact with all four serotypes, prevent host cell infection and subsequent immune activation. DENV entry is thus an interesting target for antiviral therapy. DENV enters the host cell through receptor-mediated endocytosis. Several cellular receptors have been proposed, and DC-SIGN, present on dendritic cells, is considered as the most important DENV receptor until now. Because DENV entry is a target for antiviral therapy, various classes of compounds have been investigated to inhibit this process. In this paper, an overview is given of all the putative DENV receptors, and the most promising DENV entry inhibitors are discussed.
doi:10.1155/2012/628475
PMCID: PMC3317058  PMID: 22529868
6.  An Immunogenic and Protective Alphavirus Replicon Particle-Based Dengue Vaccine Overcomes Maternal Antibody Interference in Weanling Mice▿  
Journal of Virology  2007;81(19):10329-10339.
A candidate pediatric dengue virus (DENV) vaccine based on nonpropagating Venezuelan equine encephalitis virus replicon particles (VRP) was tested for immunogenicity and protective efficacy in weanling mice in the presence and absence of potentially interfering maternal antibodies. A gene cassette encoding envelope proteins prM and E from mouse-adapted DENV type 2 (DENV2) strain NGC was cloned into a VEE replicon vector and packaged into VRP, which programmed proper in vitro expression and processing of DENV2 envelope proteins upon infection of Vero cells. Primary immunization of 3-week-old weanling BALB/c mice in the footpad with DENV2 VRP resulted in high levels of DENV-specific serum immunoglobulin G antibodies and significant titers of neutralizing antibodies in all vaccinates. A booster immunization 12 weeks after the prime immunization resulted in increased neutralizing antibodies that were sustained for at least 30 weeks. Immunization at a range of doses of DENV2 VRP protected mice from an otherwise-lethal intracranial DENV2 challenge. To model vaccination in the presence of maternal antibodies, weanling pups born to DENV2-immune or DENV2-naïve dams were immunized with either DENV2 VRP or live DENV2 given peripherally. The DENV2 VRP vaccine induced neutralizing-antibody responses in young mice regardless of the maternal immune status. In contrast, live-DENV2 vaccination performed poorly in the presence of preexisting anti-DENV2 antibodies. This study demonstrates the feasibility of a VRP vaccine approach as an early-life DENV vaccine in populations with high levels of circulating DENV antibodies and suggests the utility of VRP-based vaccines in other instances where maternal antibodies make early vaccination problematic.
doi:10.1128/JVI.00512-07
PMCID: PMC2045445  PMID: 17652394
7.  Antiviral activity of four types of bioflavonoid against dengue virus type-2 
Virology Journal  2011;8:560.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1743-422X-8-560
PMCID: PMC3271998  PMID: 22201648
Antiviral; Dengue virus; Flavonoid; Quercetin; Naringin; Daidzein; Hesperetin
8.  Co-circulation of two genotypes of dengue virus serotype 3 in Guangzhou, China, 2009 
Virology Journal  2012;9:125.
Dengue is emerging as the most important mosquito borne viral disease in the world. In mainland China, sporadic and large outbreaks of dengue illness caused by the four serotypes of dengue virus (DENV-1 to DENV-4) have been well documented. Guangdong province is the major affected area in China, and DENV-1 has dominantly circulated in Guangdong for a long time. In this study, a family cluster of DENV-3 infection in Guangzhou was described. Three cases were diagnosed as dengue fever based on clinical manifestation, serological and RT-PCR assays. Two DENV-3 strains were isolated in C6/36 cells and the complete genome sequences were determined. Phylogenetic analysis revealed that the new DENV-3 isolates from the family cluster were grouped within genotype III. Considering the fact that several DENV-3 strains within genotype V were also identified in Guangzhou in 2009, at least two genotypes of DENV-3 co-circulated in Guangzhou. Careful investigation and virological analysis should be warranted in the future.
doi:10.1186/1743-422X-9-125
PMCID: PMC3463466  PMID: 22721418
Dengue virus type 3; Family cluster; Genotype; Co-circulation
9.  Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2 
Background
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.
Conclusions/Significance
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.
doi:10.1371/journal.pntd.0001636
PMCID: PMC3341331  PMID: 22563515
10.  T-Cell Immunity to Infection with Dengue Virus in Humans 
Dengue virus (DENV) is the etiologic agent of dengue fever, the most significant mosquito-borne viral disease in humans. Up to 400 million DENV infections occur every year, and severity can range from asymptomatic to an acute self-limiting febrile illness. In a small proportion of patients, the disease can exacerbate and progress to dengue hemorrhagic fever and/or dengue shock syndrome, characterized by severe vascular leakage, thrombocytopenia, and hemorrhagic manifestations. A unique challenge in vaccine development against DENV is the high degree of sequence variation, characteristically associated with RNA viruses. This is of particular relevance in the case of DENV since infection with one DENV serotype (primary infection) presumably affords life-long serotype-specific immunity but only partial and temporary immunity to other serotypes in secondary infection settings. The role of T cells in DENV infection and subsequent disease manifestations is not fully understood. According to the original antigenic sin theory, skewing of T-cell responses induced by primary infection with one serotype causes less effective response upon secondary infection with a different serotype, predisposing to severe disease. Our recent study has suggested an HLA-linked protective role for T cells. Herein, we will discuss the role of T cells in protection and pathogenesis from severe disease as well as the implications for vaccine design.
doi:10.3389/fimmu.2014.00093
PMCID: PMC3945531  PMID: 24639680
DENV; T cells; protection; pathogenesis; HLA; vaccines
11.  Inhibition of Dengue Virus by an Ester Prodrug of an Adenosine Analog▿ † 
Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen that infects humans. Neither a vaccine nor an antiviral therapy is currently available for DENV. Here, we report an adenosine nucleoside prodrug that potently inhibits DENV replication both in cell culture and in a DENV mouse model. NITD449 (2′-C-acetylene-7-deaza-7-carbamoyladenosine) was initially identified as a parental compound that inhibits all four serotypes of DENV with low cytotoxicity. However, in vivo pharmacokinetic studies indicated that NITD449 had a low level of exposure in plasma when dosed orally. To increase the oral bioavailability, we covalently linked isobutyric acids to the 3′- and 5′-hydroxyl groups of ribose via ester linkage to NITD449, leading to the prodrug NITD203 (3′,5′-O-diisobutyryl-2′-C-acetylene-7-deaza-7-carbamoyl-adenosin). Pharmacokinetic analysis showed that upon oral dosing of the prodrug, NITD203 was readily converted to NITD449, resulting in improved exposure of the parental compound in plasma in both mouse and rat. In DENV-infected AG129 mice, oral dosing of the prodrug at 25 mg/kg of body weight reduced peak viremia by 30-fold. Antiviral spectrum analysis showed that NITD203 inhibited various flaviviruses (DENV, yellow fever virus, and West Nile virus) and hepatitis C virus but not Chikungunya virus (an alphavirus). Mode-of-action analysis, using a luciferase-reporting replicon, indicated that NITD203 inhibited DENV RNA synthesis. Although NITD203 exhibited potent in vitro and in vivo efficacies, the compound could not reach a satisfactory no-observable-adverse-effect level (NOAEL) in a 2-week in vivo toxicity study. Nevertheless, our results demonstrate that a prodrug approach using a nucleoside analog could potentially be developed for flavivirus antiviral therapy.
doi:10.1128/AAC.00397-10
PMCID: PMC2916325  PMID: 20516277
12.  Limited cross-reactivity of mouse monoclonal antibodies against Dengue virus capsid protein among four serotypes 
Background
Dengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. Four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4) are classified in the Flavivirus genus of the family Flaviviridae. We prepared monoclonal antibodies against DENV capsid protein from mice immunized with DENV-2 and determined the cross-reactivity with each serotype of DENV and Japanese encephalitis virus.
Methods and results
To clarify the relationship between the cross-reactivity of monoclonal antibodies and the diversity of these viruses, we examined the situations of flaviviruses by analyses of phylogenetic trees. Among a total of 60 prepared monoclonal antibodies specific for DENV, five monoclonal antibodies stained the nuclei of infected cells and were found to be specific to the capsid protein. Three were specific to DENV-2, while the other two were cross-reactive with DENV-2 and DENV-4. No monoclonal antibodies were cross-reactive with all four serotypes. Phylogenetic analysis of DENV amino acid sequences of the capsid protein revealed that DENV-2 and DENV-4 were clustered in the same branch, while DENV-1 and DENV-3 were clustered in the other branch. However, these classifications of the capsid protein were different from those of the envelope and nonstructural 1 proteins. Phylogenetic distances between the four serotypes of DENV were as different as those of other flaviviruses, such as Japanese encephalitis virus and West Nile virus. Large variations in the DENV serotypes were comparable with the differences between species of flavivirus. Furthermore, the diversity of flavivirus capsid protein was much greater than that of envelope and nonstructural 1 proteins.
Conclusion
In this study, we produced specific monoclonal antibodies that can be used to detect DENV-2 capsid protein, but not a cross-reactive one with all serotypes of DENV capsid protein. The high diversity of the DENV capsid protein sequence by phylogenetic analysis supported the low cross-reactivity of monoclonal antibodies against DENV capsid protein.
doi:10.2147/BTT.S37792
PMCID: PMC3512249  PMID: 23209363
Dengue virus; capsid protein; monoclonal antibody; cross-reactivity
13.  Dengue Virus Infection Induces Upregulation of GRP78, Which Acts To Chaperone Viral Antigen Production▿  
Journal of Virology  2009;83(24):12871-12880.
Dengue virus (DENV) pathogenesis is related to the host responses to viral infection within target cells, and therefore, this study assessed intracellular changes in host proteins following DENV infection. Two-dimensional gel electrophoresis and mass spectrometry identified upregulation of the host endoplasmic reticulum (ER) chaperone GRP78 in K562 cells following DENV infection, in the absence of virus-induced cell death. Upregulation of GRP78 in DENV-infected cells was confirmed by immunostaining and confocal microscopy and by Western blot analysis and was also observed in DENV-infected primary monocyte-derived macrophages, a natural target cell type for DENV infection. GRP78 was upregulated in both DENV antigen-positive and -negative cells in the DENV-infected culture, suggesting a bystander effect, with the highest GRP78 levels coincident with high-level DENV antigen production and infectious-virus release. Transfection of target cells to express GRP78 prior to DENV challenge did not affect subsequent DENV infection, but cleavage of GRP78 with the SubAB toxin, during an established DENV infection, yielded a 10- to 100-fold decrease in infectious-virus release, loss of intracellular DENV particles, and a dramatic decrease in intracellular DENV antigen. However, DENV RNA levels were unchanged, indicating normal DENV RNA replication but altered DENV antigen levels in the absence of GRP78. Thus, GRP78 is upregulated by DENV infection and is necessary for DENV antigen production and/or accumulation. This may be a common requirement for viruses such as flaviviruses that depend heavily on the ER for coordinated protein production and processing.
doi:10.1128/JVI.01419-09
PMCID: PMC2786853  PMID: 19793816
14.  Dengue Virus Inhibits Immune Responses in Aedes aegypti Cells 
PLoS ONE  2010;5(5):e10678.
The ability of many viruses to manipulate the host antiviral immune response often results in complex host-pathogen interactions. In order to study the interaction of dengue virus (DENV) with the Aedes aegypti immune response, we have characterized the DENV infection-responsive transcriptome of the immune-competent A. aegypti cell line Aag2. As in mosquitoes, DENV infection transcriptionally activated the cell line Toll pathway and a variety of cellular physiological systems. Most notably, however, DENV infection down-regulated the expression levels of numerous immune signaling molecules and antimicrobial peptides (AMPs). Functional assays showed that transcriptional induction of AMPs from the Toll and IMD pathways in response to bacterial challenge is impaired in DENV-infected cells. In addition, Escherichia coli, a Gram-negative bacteria species, grew better when co-cultured with DENV-infected cells than with uninfected cells, suggesting a decreased production of AMPs from the IMD pathway in virus-infected cells. Pre-stimulation of the cell line with Gram-positive bacteria prior to DENV infection had no effect on DENV titers, while pre-stimulation with Gram-negative bacteria resulted in an increase in DENV titers. These results indicate that DENV is capable of actively suppressing immune responses in the cells it infects, a phenomenon that may have important consequences for virus transmission and insect physiology.
doi:10.1371/journal.pone.0010678
PMCID: PMC2872661  PMID: 20502529
15.  Analysis of Human Monoclonal Antibodies Generated by Dengue Virus-Specific Memory B Cells 
Viral Immunology  2012;25(5):348-359.
Abstract
Dengue, caused by the four serotypes of dengue virus (DENV), represents an expanding global health challenge. The potential for serotype-cross-reactive antibodies to exacerbate disease during a secondary infection with a heterologous DENV serotype has driven efforts to study human DENV-specific antibodies. Most DENV-specific antibodies generated in humans are serotype-cross-reactive, weakly neutralizing, and directed against the immature pre-membrane (prM), envelope (E), and nonstructural 1 (NS1) proteins. To broaden the characterization of human DENV-specific antibodies, we assessed B-cell responses by ELISpot assays and isolated B cells from the peripheral blood of a human subject with previous DENV infection. Forty-eight human IgG monoclonal antibodies (hMAbs) were initially characterized by their potential to bind to an inactivated lysate of DENV-infected cells. Subsequently, most DENV-specific hMAbs were found to bind soluble, recombinant E protein (rE). Two hMAbs were unable to bind rE, despite strongly binding to the DENV-infected cell lysate. Further analyses showed that these two hMAbs bound conformation-dependent, reduction-sensitive epitopes on E protein. These data shed light on the breadth of DENV-specific hMAbs generated within a single immune donor.
doi:10.1089/vim.2012.0010
PMCID: PMC3466914  PMID: 22934599
16.  The Development of Therapeutic Antibodies That Neutralize Homologous and Heterologous Genotypes of Dengue Virus Type 1 
PLoS Pathogens  2010;6(4):e1000823.
Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential.
Author Summary
Dengue virus (DENV) is a mosquito-transmitted virus that infects 25 to 100 million humans annually and can progress to a life-threatening hemorrhagic fever and shock syndrome. Currently, no vaccines or specific therapies are available. Prior studies identified a highly neutralizing monoclonal antibody (MAb) against West Nile virus, a related flavivirus, as a candidate therapy for humans. In this study, we generated 79 new MAbs against the DENV type 1 (DENV-1) serotype, 16 of which strongly inhibited infection in cell culture. Using structural and molecular approaches, the binding sites of these inhibitory MAbs were localized to distinct regions on domain III of the DENV-1 envelope protein. We tested the protective capacity of all of the neutralizing MAbs in mice against infection by a strain of DENV-1 from a distinct genotype. Only two of the MAbs, DENV1-E105 and DENV1-E106, showed efficacy in a post-exposure treatment model, and these antibodies efficiently neutralized all five DENV-1 genotypes. Collectively, our studies define a complex structural binding site on domain III of the envelope protein for MAbs with therapeutic potential against DENV-1.
doi:10.1371/journal.ppat.1000823
PMCID: PMC2848552  PMID: 20369024
17.  Development and Characterization of a Reverse Genetic System for Studying Dengue Virus Serotype 3 Strain Variation and Neutralization 
Dengue viruses (DENV) are enveloped single-stranded positive-sense RNA viruses transmitted by Aedes spp. mosquitoes. There are four genetically distinct serotypes designated DENV-1 through DENV-4, each further subdivided into distinct genotypes. The dengue scientific community has long contended that infection with one serotype confers lifelong protection against subsequent infection with the same serotype, irrespective of virus genotype. However this hypothesis is under increased scrutiny and the role of DENV genotypic variation in protection from repeated infection is less certain. As dengue vaccine trials move increasingly into field-testing, there is an urgent need to develop tools to better define the role of genotypic variation in DENV infection and immunity. To better understand genotypic variation in DENV-3 neutralization and protection, we designed and constructed a panel of isogenic, recombinant DENV-3 infectious clones, each expressing an envelope glycoprotein from a different DENV-3 genotype; Philippines 1982 (genotype I), Thailand 1995 (genotype II), Sri Lanka 1989 and Cuba 2002 (genotype III) and Puerto Rico 1977 (genotype IV). We used the panel to explore how natural envelope variation influences DENV-polyclonal serum interactions. When the recombinant viruses were tested in neutralization assays using immune sera from primary DENV infections, neutralization titers varied by as much as ∼19-fold, depending on the expressed envelope glycoprotein. The observed variability in neutralization titers suggests that relatively few residue changes in the E glycoprotein may have significant effects on DENV specific humoral immunity and influence antibody mediated protection or disease enhancement in the setting of both natural infection and vaccination. These genotypic differences are also likely to be important in temporal and spatial microevolution of DENV-3 in the background of heterotypic neutralization. The recombinant and synthetic tools described here are valuable for testing hypotheses on genetic determinants of DENV-3 immunopathogenesis.
Author Summary
Infectious virus clones are valuable tools for studying how changes in viral genetic codes affect viral biology. Dengue virus is the most important mosquito-borne virus worldwide, yet dengue virus infectious clones have historically been challenging to make and manipulate, making it very difficult to study the variety of genetic changes observed in dengue viruses. Here we describe the construction of a panel of five dengue virus serotype 3 (DENV-3) clones using a novel strategy not previously employed in dengue research. This strategy uses genetic fragments and synthesized genes to introduce genetic changes while minimally affecting the virus. Each of the five recombinant clones was designed to express genetically distinct DENV-3 envelope proteins derived from strains circulating in different regions of the world. We used the recombinant viruses, coupled with DENV-3 sera from geographically defined human cases, to study the impact of E variation on neutralization outcomes. Our data demonstrate that the recombinant viruses varied significantly in their neutralization outcomes, depending on sera. While it has long been presumed that infection, and vaccination, with one serotype confers lifelong protection against all variants of that serotype, our results indicate that this assumption requires a more rigorous assessment by the DENV community.
doi:10.1371/journal.pntd.0001486
PMCID: PMC3289595  PMID: 22389731
18.  Matrix Metalloproteinase 3 Promotes Cellular Anti-Dengue Virus Response via Interaction with Transcription Factor NFκB in Cell Nucleus 
PLoS ONE  2014;9(1):e84748.
Dengue virus (DENV), the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus of immense global health importance. Characterization of cellular factors promoting or inhibiting DENV infection is important for understanding the mechanism of DENV infection. In this report, MMP3 (stromelysin-1), a secretory endopeptidase that degrades extracellular matrices, has been shown promoting cellular antiviral response against DENV infection. Quantitative RT-PCR and Western Blot showed that the expression of MMP3 was upregulated in DENV-infected RAW264.7 cells. The intracellular viral loads were significantly higher in MMP3 silenced cells compared with controls. The expression level of selective anti-viral cytokines were decreased in MMP3 siRNA treated cells, and the transcription factor activity of NFκB was significantly impaired upon MMP3 silencing during DENV infection. Further, we found that MMP3 moved to cell nucleus upon DENV infection and colocalized with NFκB P65 in nucleus. Co-immunoprecipitation analysis suggested that MMP3 directly interacted with NFκB in nucleus during DENV infection and the C-terminal hemopexin-like domain of MMP3 was required for the interaction. This study suggested a novel role of MMP3 in nucleus during viral infection and provided new evidence for MMPs in immunomodulation.
doi:10.1371/journal.pone.0084748
PMCID: PMC3885614  PMID: 24416274
19.  Cross-reactivities between human IgMs and the four serotypes of dengue virus as probed with artificial homodimers of domain-III from the envelope proteins 
BMC Infectious Diseases  2013;13:302.
Background
Dengue fever is the most important vector-borne viral disease. Four serotypes of dengue virus, DENV1 to DENV4, coexist. Infection by one serotype elicits long-lasting immunity to that serotype but not the other three. Subsequent infection by a different serotype is a risk factor for severe dengue. Domain III (ED3) of the viral envelope protein interacts with cell receptors and contains epitopes recognized by neutralizing antibodies. We determined the serotype specificity and cross-reactivity of human IgMs directed against ED3 by using a well-characterized collection of 90 DENV-infected and 89 DENV-uninfected human serums.
Methods
The recognitions between the four serotypes of ED3 and the serums were assayed with an IgM antibody-capture ELISA (MAC-ELISA) and artificial homodimeric antigens. The results were analyzed with Receiving Operator Characteristic (ROC) curves.
Results
The DENV-infected serums contained IgMs that reacted with one or several ED3 serotypes. The discrimination by ED3 between serums infected by the homotypic DENV and uninfected serums varied with the serotype in the decreasing order DENV1 > DENV2 > DENV3 > DENV4. The ED3 domain of DENV1 gave the highest discrimination between DENV-infected and DENV-uninfected serums, whatever the infecting serotype, and thus behaved like a universal ED3 domain for the detection of IgMs against DENV. Some ED3 serotypes discriminated between IgMs directed against the homotypic and heterotypic DENVs. The patterns of cross-reactivities and discriminations varied with the serotype.
Conclusions
The results should help better understand the IgM immune response and protection against DENV since ED3 is widely used as an antigen in diagnostic assays and an immunogen in vaccine candidates.
doi:10.1186/1471-2334-13-302
PMCID: PMC3701519  PMID: 23815496
Cross-reactivity; Dengue Virus; Discrimination; Flavivirus; Human Serums; Immunoglobulin M; MAC-ELISA; ROC Curve; Serotype; Specificity
20.  Detection of Dengue Virus NS1 Antigen in Infected Aedes aegypti Using a Commercially Available Kit 
Epidemic dengue has emerged throughout the tropical world. In the continued absence of a vaccine against dengue virus (DENV), mosquito vector surveillance and control programs are essential to reduce human infections. An effective test to detect DENV in infected mosquitoes would be a valuable addition to the surveillance effort. We investigated DENV detection in infected Aedes aegypti using a commercially available DENV non-structural protein 1 (NS1) ELISA kit (Platelia Dengue NS1 Ag), and by reverse transcription-polymerase chain reaction (RT-PCR) and virus isolation assays. The DENV-infected mosquitoes were subjected to field-relevant conditions and assayed individually and pooled with uninfected mosquitoes. Overall, DENV NS1 antigen was detected in 98% of infected mosquitoes/pools versus 79% for RT-PCR and 29% for virus isolation. Our results indicate that NS1 is an excellent analyte for detection of DENV in Ae. aegypti and that the tested NS1 antigen kit provides a sensitive, rapid, and convenient test for DENV surveillance in mosquitoes.
doi:10.4269/ajtmh.2012.12-0477
PMCID: PMC3583315  PMID: 23185074
21.  Protein Kinase Regulated by dsRNA Downregulates the Interferon Production in Dengue Virus- and dsRNA-Stimulated Human Lung Epithelial Cells 
PLoS ONE  2013;8(1):e55108.
Background
Dengue virus (DENV) is found in the tropical and subtropical regions and affects millions of people annually. Currently, no specific vaccine or antiviral treatment against dengue virus is available. Innate immunity has been shown to be important for host resistance to DENV infection. Although protein kinase regulated by double-stranded RNA (PKR) has been found to promote the innate signaling in response to infection by several viruses, its role in the innate response to DENV infection is still unclear. Our study aimed to investigate the role of PKR in DENV-induced innate immune responses.
Methodology/Principal Findings
By RNAi, silencing of PKR significantly enhanced the expression of interferon (IFN)-β in DENV infected human lung epithelial A549 cells. Western blot and immunofluorescence microscopy data showed that PKR knockdown upregulated the activation of innate signaling cascades including p38 and JNK mitogen-activated protein kinases (MAPKs), interferon regulatory factor-3 and NF-κB, following DENV2 infection. Likewise, a negative regulatory effect of PKR on the IFN production was also observed in poly(IC) challenged cells. Moreover, the PKR knockdown-mediated IFN induction was attenuated by RIG-I or IPS-1 silencing. Finally, overexpression of a catalytically inactive PKR mutant (K296R), but not of a mutant lacking dsRNA binding activity (K64E) or the double mutant (K64EK296R), reversed the IFN induction mediated by PKR knockdown, suggesting that the dsRNA binding activity is required for PKR to downregulate IFN production.
Conclusions/Significance
PKR acts as a negative regulator of IFN induction triggered by DENVs and poly(IC), and this regulation relies on its dsRNA binding activity. These findings reveal a novel regulatory role for PKR in innate immunity, suggesting that PKR might be a promising target for anti-DENV treatments.
doi:10.1371/journal.pone.0055108
PMCID: PMC3555826  PMID: 23372823
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.
doi:10.1128/AAC.00855-10
PMCID: PMC3019636  PMID: 20937790
23.  The Kinase Inhibitor SFV785 Dislocates Dengue Virus Envelope Protein from the Replication Complex and Blocks Virus Assembly 
PLoS ONE  2011;6(8):e23246.
Dengue virus (DENV) is the etiologic agent for dengue fever, for which there is no approved vaccine or specific anti-viral drug. As a remedy for this, we explored the use of compounds that interfere with the action of required host factors and describe here the characterization of a kinase inhibitor (SFV785), which has selective effects on NTRK1 and MAPKAPK5 kinase activity, and anti-viral activity on Hepatitis C, DENV and yellow fever viruses. SFV785 inhibited DENV propagation without inhibiting DENV RNA synthesis or translation. The compound did not cause any changes in the cellular distribution of non-structural 3, a protein critical for DENV RNA synthesis, but altered the distribution of the structural envelope protein from a reticulate network to enlarged discrete vesicles, which altered the co-localization with the DENV replication complex. Ultrastructural electron microscopy analyses of DENV-infected SFV785-treated cells showed the presence of viral particles that were distinctly different from viable enveloped virions within enlarged ER cisternae. These viral particles were devoid of the dense nucleocapsid. The secretion of the viral particles was not inhibited by SFV785, however a reduction in the amount of secreted infectious virions, DENV RNA and capsid were observed. Collectively, these observations suggest that SFV785 inhibited the recruitment and assembly of the nucleocapsid in specific ER compartments during the DENV assembly process and hence the production of infectious DENV. SFV785 and derivative compounds could be useful biochemical probes to explore the DENV lifecycle and could also represent a new class of anti-virals.
doi:10.1371/journal.pone.0023246
PMCID: PMC3157368  PMID: 21858043
24.  Differential Targeting of Viral Components by CD4+ versus CD8+ T Lymphocytes in Dengue Virus Infection 
Journal of Virology  2013;87(5):2693-2706.
Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4+ and CD8+ T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4+ and 21 are CD8+ T cell epitopes. We observe that whereas CD8+ T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4+ epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4+ T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4+ and CD8+ T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.
doi:10.1128/JVI.02675-12
PMCID: PMC3571409  PMID: 23255803
25.  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.
doi:10.1128/JVI.02510-10
PMCID: PMC3126545  PMID: 21507975

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