Flavonoid from Carica papaya inhibits NS2B-NS3 protease and prevents Dengue 2 viral assembly

Dengue virus belongs to the virus family Flaviviridae. Dengue hemorrhagic disease caused by dengue virus is a public health problem worldwide. The viral non structural 2B and 3 (NS2B-NS3) protease complex is crucial for virus replication and hence, it is considered to be a good anti-viral target. Leaf extracts from Carica papaya is generally prescribed for patients with dengue fever, but there are no scientific evidences for its anti-dengue activity; hence we intended to investigate the anti-viral activity of compounds present in the leaves of Carica papaya against dengue 2 virus (DENV-2). We analysed the anti-dengue activities of the extracts from Carica papaya by using bioinformatics tools. Interestingly, we find the flavonoid quercetin with highest binding energy against NS2B-NS3 protease which is evident by the formation of six hydrogen bonds with the amino acid residues at the binding site of the receptor. Our results suggest that the flavonoids from Carica papaya have significant anti-dengue activities.

Abbreviations

ADME - Absorption, distribution, metabolism and excretion, BBB - Blood brain barrier, CYP - Cytochrome P450, DENV - – Dengue virus, DHF - Dengue hemorrhagic fever, DSS - Dengue shock syndrome, GCMS - – Gas chromatography- Mass spectrometry, MOLCAD - Molecular Computer Aided Design, NS - Non structural, PDB - Protein data bank, PMF - Potential Mean Force.     

Galectin-1 Exerts Inhibitory Effects during DENV-1 Infection

Dengue virus (DENV) is an enveloped RNA virus that is mosquito-transmitted and can infect a variety of immune and non-immune cells. Response to infection ranges from asymptomatic disease to a severe disorder known as dengue hemorrhagic fever. Despite efforts to control the disease, there are no effective treatments or vaccines. In our search for new antiviral compounds to combat infection by dengue virus type 1 (DENV-1), we investigated the role of galectin-1, a widely-expressed mammalian lectin with functions in cell-pathogen interactions and immunoregulatory properties. We found that DENV-1 infection of cells in vitro exhibited caused decreased expression of Gal-1 in several different human cell lines, suggesting that loss of Gal-1 is associated with virus production. In test of this hypothesis we found that exogenous addition of human recombinant Gal-1 (hrGal-1) inhibits the virus production in the three different cell types. This inhibitory effect was dependent on hrGal-1 dimerization and required its carbohydrate recognition domain. Importantly, the inhibition was specific for hrGal-1, since no effect was observed using recombinant human galectin-3. Interestingly, we found that hrGal-1 directly binds to dengue virus and acts, at least in part, during the early stages of DENV-1 infection, by inhibiting viral adsorption and its internalization to target cells. To test the in vivo role of Gal-1 in DENV infection, Gal-1-deficient-mice were used to demonstrate that the expression of endogenous Galectin-1 contributes to resistance of macrophages to in vitro-infection with DENV-1 and it is also important to physiological susceptibility of mice to in vivo infection with DENV-1. These results provide novel insights into the functions of Gal-1 in resistance to DENV infection and suggest that Gal-1 should be explored as a potential antiviral compound.     

Immunological and viral determinants of dengue severity in hospitalized adults in Ha Noi, Viet Nam

Background

The relationships between the infecting dengue serotype, primary and secondary infection, viremia and dengue severity remain unclear. This cross-sectional study examined these interactions in adult patients hospitalized with dengue in Ha Noi.

Methods and Findings

158 patients were enrolled between September 16 and November 11, 2008. Quantitative RT-PCR, serology and NS1 detection were used to confirm dengue infection, determine the serotype and plasma viral RNA concentration, and categorize infections as primary or secondary. 130 (82%) were laboratory confirmed. Serology was consistent with primary and secondary infection in 34% and 61%, respectively. The infecting serotype was DENV-1 in 42 (32%), DENV-2 in 39 (30%) and unknown in 49 (38%). Secondary infection was more common in DENV-2 infections (79%) compared to DENV-1 (36%, p<0.001). The proportion that developed dengue haemorrhagic fever (DHF) was 32% for secondary infection compared to 18% for primary infection (p = 0.14), and 26% for DENV-1 compared to 28% for DENV-2. The time until NS1 and plasma viral RNA were undetectable was shorter for DENV-2 compared to DENV-1 (p≤0.001) and plasma viral RNA concentration on day 5 was higher for DENV-1 (p = 0.03). Plasma viral RNA concentration was higher in secondary infection on day 5 of illness (p = 0.046). We didn''t find an association between plasma viral RNA concentration and clinical severity.

Conclusion

Dengue is emerging as a major public health problem in Ha Noi. DENV-1 and DENV-2 were the prevalent serotypes with similar numbers and clinical presentation. Secondary infection may be more common amongst DENV-2 than DENV-1 infections because DENV-2 infections resulted in lower plasma viral RNA concentrations and viral RNA concentrations were higher in secondary infection. The drivers of dengue emergence in northern Viet Nam need to be elucidated and public health measures instituted.     

Synthetic 1,4-Pyran Naphthoquinones Are Potent Inhibitors of Dengue Virus Replication

Dengue virus infection is a serious public health problem in endemic areas of the world where 2.5 billion people live. Clinical manifestations of the Dengue infection range from a mild fever to fatal cases of hemorrhagic fever. Although being the most rapidly spreading mosquito-borne viral infection in the world, until now no strategies are available for effective prevention or control of Dengue infection. In this scenario, the development of compounds that specifically inhibit viral replication with minimal effects to the human hosts will have a substantial effect in minimizing the symptoms of the disease and help to prevent viral transmission in the affected population. The aim of this study was to screen compounds with potential activity against dengue virus from a library of synthetic naphthoquinones. Several 1,2- and 1,4-pyran naphthoquinones were synthesized by a three-component reaction of lawsone, aldehyde (formaldehyde or arylaldehydes) and different dienophiles adequately substituted. These compounds were tested for the ability to inhibit the ATPase activity of the viral NS3 enzyme in in vitro assays and the replication of dengue virus in cultured cells. We have identified two 1,4-pyran naphthoquinones, which inhibited dengue virus replication in mammal cells by 99.0% and three others that reduced the dengue virus ATPase activity of NS3 by two-fold in in vitro assays.     

Phylogenetic and Molecular Clock Analysis of Dengue Serotype 1 and 3 from New Delhi,India

Dengue fever is the most prevalent arboviral disease in the tropical and sub-tropical regions of the world. The present report describes molecular detection and serotyping of dengue viruses in acute phase blood samples collected from New Delhi, India. Phylogenetic and molecular clock analysis of dengue virus serotype 1 and 3 strains were also investigated. Dengue virus infection was detected in 68.87% out of 604 samples tested by RT-PCR between 2011 & 2014. Dengue serotype 1 was detected in 25.48% samples, dengue serotype 2 in 79.56% samples and dengue serotype 3 in 11.29% samples. Dengue serotype 4 was not detected. Co-infection by more than one dengue serotype was detected in 18.26% samples. Envelope gene of 29 DENV-1 and 14 DENV-3 strains were sequenced in the study. All the DENV-1 strains grouped with the American African genotype. All DENV-3 strains were found to belong to Genotype III. Nucleotide substitution rates of dengue 1 and 3 viruses were determined in the study. Time to the most recent common ancestor (TMRCA) of dengue 1 viruses was determined to be 132 years. TMRCA of DENV-3 viruses was estimated to be 149 years. Bayesian skyline plots were constructed for Indian DENV-1 and 3 strains which showed a decrease in population size since 2005 in case of DENV- 1 strains while no change was observed in recent years in case of DENV-3 strains. The study also revealed a change in the dominating serotype in Delhi, India in recent years. The study will be helpful in formulating control strategies for the outbreaks. In addition, it will also assist in tracking the movement and evolution of this emerging virus.     

Inhibition of dengue virus entry and multiplication into monocytes using RNA interference

Background

Dengue infection ranks as one of the most significant viral diseases of the globe. Currently, there is no specific vaccine or antiviral therapy for prevention or treatment. Monocytes/macrophages are the principal target cells for dengue virus and are responsible for disseminating the virus after its transmission. Dengue virus enters target cells via receptor-mediated endocytosis after the viral envelope protein E attaches to the cell surface receptor. This study aimed to investigate the effect of silencing the CD-14 associated molecule and clathrin-mediated endocytosis using siRNA on dengue virus entry into monocytes.

Methodology/Principal Findings

Gene expression analysis showed a significant down-regulation of the target genes (82.7%, 84.9 and 76.3% for CD-14 associated molecule, CLTC and DNM2 respectively) in transfected monocytes. The effect of silencing of target genes on dengue virus entry into monocytes was investigated by infecting silenced and non-silenced monocytes with DENV-2. Results showed a significant reduction of infected cells (85.2%), intracellular viral RNA load (73.0%), and extracellular viral RNA load (63.0%) in silenced monocytes as compared to non-silenced monocytes.

Conclusions/Significance

Silencing the cell surface receptor and clathrin mediated endocytosis using RNA interference resulted in inhibition of the dengue virus entry and subsequently multiplication of the virus in the monocytes. This might serve as a novel promising therapeutic target to attenuate dengue infection and thus reduce transmission as well as progression to severe dengue hemorrhagic fever.     

Specific genetic markers for detecting subtypes of dengue virus serotype-2 in isolates from the states of Oaxaca and Veracruz,Mexico

Background  

Dengue (DEN) is an infectious disease caused by the DEN virus (DENV), which belongs to the Flavivirus genus in the family Flaviviridae. It has a (+) sense RNA genome and is mainly transmitted to humans by the vector mosquito Aedes aegypti. Dengue fever (DF) and dengue hemorrhagic fever (DHF) are caused by one of four closely related virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). Epidemiological and evolutionary studies have indicated that host and viral factors are involved in determining disease outcome and have proved the importance of viral genotype in causing severe epidemics. Host immune status and mosquito vectorial capacity are also important influences on the severity of infection. Therefore, an understanding of the relationship between virus variants with altered amino acids and high pathogenicity will provide more information on the molecular epidemiology of DEN. Accordingly, knowledge of the DENV serotypes and genotypes circulating in the latest DEN outbreaks around the world, including Mexico, will contribute to understanding DEN infections.     

Source and Purity of Dengue-Viral Preparations Impact Requirement for Enhancing Antibody to Induce Elevated IL-1β Secretion: A Primary Human Monocyte Model

Dengue virus is a major global health threat and can lead to life-threatening hemorrhagic complications due to immune activation and cytokine production. Cross-reactive antibodies to an earlier dengue virus infection are a recognized risk factor for severe disease. These antibodies bind heterologous dengue serotypes and enhance infection into Fc-receptor-bearing cells, a process known as antibody-dependent enhancement of infection. One crucial cytokine seen elevated in severe dengue patients is IL-1β, a potent inflammatory cytokine matured by the inflammasome. We used a highly-physiologic system by studying antibody-dependent enhancement of IL-1β in primary human monocytes with anti-dengue human monoclonal antibodies isolated from patients. Antibody-enhancement increased viral replication in primary human monocytes inoculated with supernatant harvested from Vero cells infected with dengue virus serotype 2 (DENV-2) 16681. Surprisingly, IL-1β secretion induced by infectious supernatant harvested from two independent Vero cell lines was not enhanced by antibody. Secretion of multiple other inflammatory cytokines was also independent of antibody signaling. However, IL-1β secretion did require NLRP3 and caspase-1 activity. Immunodepletion of dengue virions from the infectious supernatant confirmed that virus was not the main IL-1β-inducing agent, suggesting that a supernatant component(s) not associated with the virion induced IL-1β production. We excluded RNA, DNA, contaminating LPS, viral NS1 protein, complement, and cytokines. In contrast, purified Vero-derived DENV-2 16681 exhibited antibody-enhancement of both infection and IL-1β induction. Furthermore, C6/36 mosquito cells did not produce such an inflammatory component, as crude supernatant harvested from insect cells infected with DENV-2 16681 induced antibody-dependent IL-1β secretion. This study indicates that Vero cells infected with DENV-2 16681 may produce inflammatory components during dengue virus propagation that mask the virus-specific immune response. Thus, the choice of host cell and viral purity should be carefully considered, while insect-derived virus represents a system that elicits antibody-dependent cytokine responses to dengue virus with fewer confounding issues.     

Concurrent circulation of dengue serotype 1, 2 and 3 among acute febrile patients in Cameroon

Acute febrile patients presenting at hospitals in Douala, Cameroon between July and December 2020, were screened for dengue infections using real time RT-PCR on fragments of the 5’ and 3’ UTR genomic regions. In total, 12.8% (41/320) of cases examined were positive for dengue. Dengue virus 3 (DENV-3) was the most common serotype found (68.3%), followed by DENV-2 (19.5%) and DENV-1 (4.9%). Co-infections of DENV-3 and DENV-2 were found in 3 cases. Jaundice and headache were the most frequent clinical signs associated with infection and 56% (23/41) of the cases were co-infections with malaria. Phylogenetic analysis of the envelope gene identified DENV-1 as belonging to genotype V, DENV-2 to genotype II and DENV-3 to genotype III. The simultaneous occurrence of three serotypes in Douala reveals dengue as a serious public health threat for Cameroon and highlights the need for further epidemiological studies in the major cities of this region.     

IFN-γ production depends on IL-12 and IL-18 combined action and mediates host resistance to dengue virus infection in a nitric oxide-dependent manner

Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1-4). Severe dengue infection in humans is characterized by thrombocytopenia, increased vascular permeability, hemorrhage and shock. However, there is little information about host response to DENV infection. Here, mechanisms accounting for IFN-γ production and effector function during dengue disease were investigated in a murine model of DENV-2 infection. IFN-γ expression was greatly increased after infection of mice and its production was preceded by increase in IL-12 and IL-18 levels. In IFN-γ(-/-) mice, DENV-2-associated lethality, viral loads, thrombocytopenia, hemoconcentration, and liver injury were enhanced, when compared with wild type-infected mice. IL-12p40(-/-) and IL-18(-/-) infected-mice showed decreased IFN-γ production, which was accompanied by increased disease severity, higher viral loads and enhanced lethality. Blockade of IL-18 in infected IL-12p40(-/-) mice resulted in complete inhibition of IFN-γ production, greater DENV-2 replication, and enhanced disease manifestation, resembling the response seen in DENV-2-infected IFN-γ(-/-) mice. Reduced IFN-γ production was associated with diminished Nitric Oxide-synthase 2 (NOS2) expression and NOS2(-/-) mice had elevated lethality, more severe disease evolution and increased viral load after DENV-2 infection. Therefore, IL-12/IL-18-induced IFN-γ production and consequent NOS2 induction are of major importance to host resistance against DENV infection.     

Co-circulation of two genotypes of Dengue virus serotype 3 in Guangzhou, China, 2009

ABSTRACT: 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.     

3-Trifluoromethylpyrazolones derived nucleosides: Synthesis and antiviral evaluation

Dengue (DENV) viral infection is a global public health problem that infrequently develops life threatening diseases such as dengue hemorrhagic fever (DFS) and dengue shock syndrome (DSS). Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human corona virus with 38% fatality rate of infected patients. A series of 4-arylhydrazono-5-trifluoromethyl-pyrazolones, their ribofuranosyl, and 5′-deoxyribofuranosyl nucleosides were synthesized, geometry optimized using Density functional theory (DFT), and evaluated for their antiviral activity. 2-Nitrophenylhydrazonopyra-zolone derivative 5 showed significant activity against MERS-CoV (EC₅₀ = 4.6?μM). The nucleoside analog 8 showed moderate activity against DENV-2 (EC₅₀ = 10?μM), while the activity was abolished with the corresponding 5′-deoxyribonucleoside analogs. The identified hits in this study set this category of compounds for further future optimizations.     

Dengue Virus Activates Membrane TRAIL Relocalization and IFN-α Production by Human Plasmacytoid Dendritic Cells In Vitro and In Vivo

Background

Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro.

Methods & Findings

Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production

Conclusions

This investigation characterizes, during DENV-2 infection, activation of pDCs in vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease.     

Rapid testing requires clinical evaluation for accurate diagnosis of dengue disease: A passive surveillance study in Southern Malaysia

BackgroundDengue fever is the most common mosquito-borne infection worldwide where an expanding surveillance and characterization of this infection are needed to better inform the healthcare system. In this surveillance-based study, we explored the prevalence and distinguishing features of dengue fever amongst febrile patients in a large community-based health facility in southern peninsular Malaysia.MethodsOver six months in 2018, we recruited 368 adults who met the WHO 2009 criteria for probable dengue infection. They underwent the following blood tests: full blood count, dengue virus (DENV) rapid diagnostic test (RDT), ELISA (dengue IgM and IgG), nested RT-PCR for dengue, multiplex qRT-PCR for Zika, Chikungunya and dengue as well as PCR tests for Leptopspira spp., Japanese encephalitis and West Nile virus.ResultsLaboratory-confirmed dengue infections (defined by positive tests in NS1, IgM, high-titre IgG or nested RT-PCR) were found in 167 (45.4%) patients. Of these 167 dengue patients, only 104 (62.3%) were positive on rapid diagnostic testing. Dengue infection was significantly associated with the following features: family or neighbours with dengue in the past week (AOR: 3.59, 95% CI:2.14–6.00, p<0.001), cutaneous rash (AOR: 3.58, 95% CI:1.77–7.23, p<0.001), increased temperature (AOR: 1.33, 95% CI:1.04–1.70, p = 0.021), leucopenia (white cell count < 4,000/μL) (AOR: 3.44, 95% CI:1.72–6.89, p<0.001) and thrombocytopenia (platelet count <150,000/μL)(AOR: 4.63, 95% CI:2.33–9.21, p<0.001). Dengue infection was negatively associated with runny nose (AOR: 0.47, 95% CI:0.29–0.78, p = 0.003) and arthralgia (AOR: 0.42, 95% CI:0.24–0.75, p = 0.004). Serotyping by nested RT-PCR revealed mostly mono-infections with DENV-2 (n = 64), DENV-1 (n = 32) and DENV-3 (n = 17); 14 co-infections occurred with DENV-1/DENV-2 (n = 13) and DENV-1/DENV-4 (n = 1). Besides dengue, none of the pathogens above were found in patients’ serum.ConclusionsAcute undifferentiated febrile infections are a diagnostic challenge for community-based clinicians. Rapid diagnostic tests are increasingly used to diagnose dengue infection but negative tests should be interpreted with caution as they fail to detect a considerable proportion of dengue infection. Certain clinical features and haematological parameters are important in the clinical diagnosis of dengue infection.     

Invasion and maintenance of dengue virus type 2 and type 4 in the Americas

Dengue virus type 4 (DENV-4) was first reported in the Americas in 1981, where it caused epidemics of dengue fever throughout the region. In the same year, the region's first epidemic of dengue hemorrhagic fever was reported, caused by an Asian strain of dengue virus type 2 (DENV-2) that was distinct from the American subtype circulating previously. Despite the importance of these epidemics, little is known about the rates or determinants of viral spread among island and mainland populations or their directions of movement. We employed a Bayesian coalescent approach to investigate the transmission histories of DENV-2 and DENV-4 since their introduction in 1981 and a parsimony method to assess patterns of strain migration. For both viruses there was an initial invasion phase characterized by an exponential increase in the number of DENV lineages, after which levels of genetic diversity remained constant despite reported fluctuations in DENV-2 and DENV-4 activity. Strikingly, viral lineage numbers increased far more rapidly for DENV-4 than DENV-2, indicative of a more rapid rate of exponential population growth in DENV-4 or a higher rate of geographic dispersal, allowing this virus to move more effectively among localities. We propose that these contrasting dynamics may reflect underlying differences in patterns of host immunity. Despite continued gene flow along particular transmission routes, the overall extent of viral traffic was less than expected under panmixis. Hence, DENV in the Americas has a clear geographic structure that maintains viral diversity between outbreaks.     

Role of microparticles in dengue virus infection and its impact on medical intervention strategies

Dengue virus (DV) is one of the most important vector-borne diseases in the world. It causes a disease that manifests as a spectrum of clinical symptoms, including dengue hemorrhagic fever. DV is proficient at diverting the immune system to facilitate transmission through its vector host, Aedes spp. mosquito. Similar to other vector-borne parasites, dengue may also require a second structural form, a virus of alternative morphology (VAM), to complete its life cycle. DV can replicate to high copy numbers in patient plasma, but no classical viral particles can be detected by ultra-structural microscopy analysis. A VAM appearing as a microparticle has been recapitulated with in vitro cell lines Meg01 and K562, close relatives to the cells harboring dengue virus in vivo. VAMs are likely to contribute to the high viremia levels observed in dengue patients. This review discusses the possible existence of a VAM in the DV life cycle.     

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.     

Epidemiological and Molecular Characterization of Dengue Virus Circulating in Bhutan, 2013-2014

Dengue is one of the most significant public health problems in tropical and subtropical countries, and is increasingly being detected in traditionally non-endemic areas. In Bhutan, dengue virus (DENV) has only recently been detected and limited information is available. In this study, we analyzed the epidemiological and molecular characteristics of DENV in two southern districts in Bhutan from 2013–2014. During this period, 379 patients were clinically diagnosed with suspected dengue, of whom 119 (31.4%) were positive for DENV infection by NS1 ELISA and/or nested RT-PCR. DENV serotypes 1, 2 and 3 were detected with DENV-1 being predominant. Phylogenetic analysis of DENV-1 using envelope gene demonstrated genotype V, closely related to strains from northern India.     

Nonstationary influence of El Niño on the synchronous dengue epidemics in Thailand

BackgroundSeveral factors, including environmental and climatic factors, influence the transmission of vector-borne diseases. Nevertheless, the identification and relative importance of climatic factors for vector-borne diseases remain controversial. Dengue is the world''s most important viral vector-borne disease, and the controversy about climatic effects also applies in this case. Here we address the role of climate variability in shaping the interannual pattern of dengue epidemics.ConclusionThe underlying mechanism for the synchronisation of dengue epidemics may resemble that of a pacemaker, in which intrinsic disease dynamics interact with climate variations driven by El Niño to propagate travelling waves of infection. When association with El Niño is strong in the 2–3-y periodic mode, one observes high synchrony of dengue epidemics over Thailand. When this association is absent, the seasonal dynamics become dominant and the synchrony initiated in Bangkok collapses.     

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.