Vitamin D modulates inflammatory response of DENV-2-infected macrophages by inhibiting the expression of inflammatory-liked miRNAs.

Dengue disease caused by dengue virus (DENV) infection is the most common vector-borne viral disease worldwide. Currently, no treatment is available to fight dengue symptoms. We and others have demonstrated the antiviral and immunomodulatory properties of VitD3 as a possible therapy for DENV infection. MicroRNAs (miRNAs) are small non-coding RNAs responsible for the regulation of cell processes including antiviral defense. Previous transcriptomic analysis showed that VitD3 regulates the expression of genes involved in stress and immune response by inducing specific miRNAs. Here, we focus on the effects of VitD3 supplementation in the regulation of the expression of inflammatory-liked miR-182-5p, miR-130a-3p, miR125b-5p, miR146a-5p, and miR-155-5p during DENV-2 infection of monocyte-derived macrophages (MDMs). Further, we evaluated the effects of inhibition of these miRNAs in the innate immune response. Our results showed that supplementation with VitD3 differentially regulated the expression of these inflammatory miRNAs. We also observed that inhibition of miR-182-5p, miR-130a-3p, miR-125b-5p, and miR-155-5p, led to decreased production of TNF-α and TLR9 expression, while increased the expression of SOCS-1, IFN-ß, and OAS1, without affecting DENV replication. By contrast, over-expression of miR-182-5p, miR-130a-3p, miR-125b-5p, and miR-155-5p significantly decreased DENV-2 infection rates and also DENV-2 replication in MDMs. Our results suggest that VitD3 immunomodulatory effects involve regulation of inflammation-linked miRNAs expression, which might play a key role in the inflammatory response during DENV infection.

Effect of high doses of vitamin D supplementation on dengue virus replication, Toll-like receptor expression, and cytokine profiles on dendritic cells.

Dengue, caused by dengue virus (DENV) infection, is a public health problem worldwide. Although DENV pathogenesis has not yet been fully elucidated, the inflammatory response is a hallmark feature in severe DENV infection. Although vitamin D (vitD) can promote the innate immune response against virus infection, no studies have evaluated the effects of vitD on DENV infection, dendritic cells (DCs), and inflammatory response regulation. This study aimed to assess the impact of oral vitD supplementation on DENV-2 infection, Toll-like receptor (TLR) expression, and both pro- and anti-inflammatory cytokine production in monocyte-derived DCs (MDDCs). To accomplish this, 20 healthy donors were randomly divided into two groups and received either 1000 or 4000 international units (IU)/day of vitD for 10 days. During pre- and post-vitD supplementation, peripheral blood samples were taken to obtain MDDCs, which were challenged with DENV-2. We found that MDDCs from donors who received 4000 IU/day of vitD were less susceptible to DENV-2 infection than MDDCs from donors who received 1000 IU/day of vitD. Moreover, these cells showed decreased mRNA expression of TLR3, 7, and 9; downregulation of IL-12/IL-8 production; and increased IL-10 secretion in response to DENV-2 infection. In conclusion, the administration of 4000 IU/day of vitD decreased DENV-2 infection. Our findings support a possible role of vitD in improving the innate immune response against DENV. However, further studies are necessary to determine the role of vitD on DENV replication and its innate immune response modulation in MDDCs.

A Multi-Perspective Review on Dengue Research.

Dengue fever is a disease which is caused by a family of viruses named Flaviviridae which are transmitted by female Aedes mosquitoes. Today, this is endemic in more than 100 nations in the World Health Organization's African, Americas, Eastern Mediterranean, South-East Asia and Western Pacific locales. The treatment of typical dengue is focused on relieving the symptoms and signs. Carica papaya is a very common plant whose leaf extract is used in the treatment of this disease. Despite extensive research on Dengue, not a single vaccine or anti-viral drug was available until 2016 (a partially effective Chimeric Yellow fever virus treated by DENV-Tetravalent Dengue Vaccine for dengue fever made by Sanofi Pasteur). This review highlights dengue fever's current situation and explains the importance of Natural chemical moieties like methionine-proline anilides, tetrapeptide aldehyde uncovered via Structure Activity Relationship studies. Also, we have reviewed the drug candidates currently in the clinical trials that have the potential to solve these issues. Important patents in the past 20 years have been outlined in this review. An in depth Protein Data Bank analysis of the different possible target proteins that can potentially have a major role in curing Dengue fever has been conducted.

Effect of freeze-dried Carica papaya leaf juice on inflammatory cytokines production during dengue virus infection in AG129 mice.

BACKGROUND: Carica papaya leaves have been used for traditional treatment of dengue fever and have been reported to exhibit an immunomodulatory activity by affecting the level of cytokine production in vitro and in vivo. Due to the lack of adequate in vivo evidence in dengue disease model, the present study was initiated to screen and identify the cytokines affected by freeze-dried C. papaya leaf juice (FCPLJ) treatment in AG129 mice infected with DEN-2 dengue virus. METHODS: The AG129 mice were fed orally with FCPLJ for 3 consecutive days after 24 h of dengue virus inoculation. Plasma cytokines were screened by using ProcartaPlex immunoassay. The gene expression in the liver was analyzed by using RT2 Profiler PCR Array. RESULTS: The results showed that FCPLJ treatment has increased the plasma CCL2/MCP-1 level during peak of viremia. Gene expression study has identified 8 inflammatory cytokine genes which were downregulated in the liver of infected AG129 mice treated with FCPLJ. The downregulated inflammatory cytokine genes were CCL6/MRP-1, CCL8/MCP-2, CCL12/MCP-5, CCL17/TARC, IL1R1, IL1RN/IL1Ra, NAMPT/PBEF1 and PF4/CXCL4. CONCLUSION: The findings indicated the possible immunomodulatory role of FCPLJ during dengue virus infection in AG129 mice.

Could Homeopathy Become An Alternative Therapy In Dengue Fever? An example Of 10 Case Studies.

Background:Dengue fever is one of the most rampant epidemics in India of late and any therapy that may help limit the sickness and hospital admissions is worth considering. In India complementary and alternative medicine physicians are medically trained and hence have a role to play in delivery of public health. Case Series:We present a retrospective case series of 10 Indian patients who were diagnosed with dengue fever and treated exclusively with homeopathic remedies at Bangalore, India. This case series demonstrates with evidence of laboratory reports that even when the platelets dropped considerably there was good result without resorting to any other means. Conclusions:A need for further, larger studies is indicated by this evidence, to precisely define the role of homeopathy in treating dengue fever. This study also emphasises the importance of individualised treatment during an epidemic for favourable results with homeopathy. Abbreviations:DF: dengue fever, NS1: non-structural protein 1 antigen, IgG: immunoglobulin G, IgM: immunoglobulin M, +ve: positive, -ve: negative, WBC: white blood cells, RBC: red blood cells, ESR: erythrocyte sedimentation rate.

Dengue virus causes changes of MicroRNA-genes regulatory network revealing potential targets for antiviral drugs.

BACKGROUND: Dengue virus (DENV) is an increasing global health threat and associated with induction of both a long-lived protective immune response and immune-suppression. So far, the potency of treatment of DENV via antiviral drugs is still under investigation. Recently, increasing evidences suggest the potential role of microRNAs (miRNAs) in regulating DENV. The present study focused on the function of miRNAs in innate insusceptible reactions and organization of various types of immune cells and inflammatory responses for DENV. Three drugs were tested including antiviral herbal medicine ReDuNing (RDN), Loratadine (LRD) and Acetaminophen. RESULTS: By the microarray expression of miRNAs in 165 Patients. Results showed that 89 active miRNAs interacted with 499 potential target genes, during antiviral treatment throughout the critical stage of DENV. Interestingly, reduction of the illness threats using RDN combined with LRD treatment showed better results than Acetaminophen alone. The inhibitions of DENV was confirmed by decrease concentrations of cytokines and interleukin parameters; like TNF-α, IFN-γ, TGF-ß1, IL-4, IL-6, IL-12, and IL-17; after treatment and some coagulants factors increased. CONCLUSIONS: This study showed a preliminary support to suggest that the herbal medicine RDN combined with LRD can reduce both susceptibility and the severity of DENV.

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7.

BACKGROUND: Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features. METHODS: Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. FINDINGS: The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAIN. CONCLUSIONS: The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

A recombinant live attenuated tetravalent vaccine for the prevention of dengue.

INTRODUCTION: Dengue is an important and still growing public health problem associated with substantial morbidity, as well as significant social and economic impact. The present review describes the main features and development of the first dengue vaccine (CYD-TDV, Dengvaxia®), which has been licensed by several dengue-endemic countries in Asia and Latin America for use in populations above 9 years of age. Areas covered: The review focuses on the large clinical development of CYD-TDV, which includes in particular two pivotal phase III efficacy trials conducted in Asia and Latin America and supported vaccine licensure. Based on these clinical data, the WHO Strategic Advisory Group of Experts (SAGE) on Immunization recommended considering introduction of the vaccine in geographic settings (national or subnational) with high burden of disease. Long-term safety follow-up studies of the efficacy trials are currently ongoing, and post-licensure studies will evaluate the vaccine effectiveness and safety in 'real-life' following vaccine introduction. Expert commentary: During vaccine development, a number of complexities were tackled, innovation pursued, and risk managed. These aspects, as well as the potential impact of CYD-TDV on public health are also discussed.

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7

ABSTRACT BACKGROUND Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features. METHODS Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. FINDINGS The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAIN CONCLUSIONS The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

Antibody Epitopes Identified in Critical Regions of Dengue Virus Nonstructural 1 Protein in Mouse Vaccination and Natural Human Infections.

Dengue is a global public health problem and is caused by four dengue virus (DENV) serotypes (DENV1-4). A major challenge in dengue vaccine development is that cross-reactive anti-DENV Abs can be protective or potentially increase disease via Ab-dependent enhancement. DENV nonstructural protein 1 (NS1) has long been considered a vaccine candidate as it avoids Ab-dependent enhancement. In this study, we evaluated survival to challenge in a lethal DENV vascular leak model in mice immunized with NS1 combined with aluminum and magnesium hydroxide, monophosphoryl lipid A + AddaVax, or Sigma adjuvant system+CpG DNA, compared with mice infected with a sublethal dose of DENV2 and mice immunized with OVA (negative control). We characterized Ab responses to DENV1, 2, and 3 NS1 using an Ag microarray tiled with 20-mer peptides overlapping by 15 aa and identified five regions of DENV NS1 with significant levels of Ab reactivity in the NS1 + monophosphoryl lipid A + AddaVax group. Additionally, we profiled the Ab responses to NS1 of humans naturally infected with DENV2 or DENV3 in serum samples from Nicaragua collected at acute, convalescent, and 12-mo timepoints. One region in the wing domain of NS1 was immunodominant in both mouse vaccination and human infection studies, and two regions were identified only in NS1-immunized mice; thus, vaccination can generate Abs to regions that are not targeted in natural infection and could provide additional protection against lethal DENV infection. Overall, we identified a small number of immunodominant regions, which were in functionally important locations on the DENV NS1 protein and are potential correlates of protection.

Low sensitivity of the tourniquet test for differential diagnosis of dengue: an analysis of 28,000 trials in patients.

BACKGROUND: The aim of this study was to evaluate the utility of the tourniquet test (TT) for dengue diagnosing. To our knowledge, no previous study with such a large sample, of this duration, with as many laboratory methods referenced, or relating the results of the TT to the 2009 WHO classification of severity has been conducted thus far. METHODS: In this study, we analyzed the records of 119,589 suspected dengue cases in a Brazilian city, with 30,670 confirmed cases. The Cohen's Kappa test was applied to evaluate the degree of agreement between the tests, and the sensitivity and specificity was calculated for the TT. RESULTS: Twenty-eight thousand six hundred thirty-five TT were performed. No association between the outcome of the TT and greater severity of infection, according to the 2009 guideline, was observed (P = 0.28); furthermore, relevant agreement with the final diagnosis (κ = 0.01; 95 % CI = 0.00 to 0.02) or individually with the IgM enzyme-linked immunoassay was not observed (κ = 0.05; 95 % CI = 0.04 to 0.06), and was even lower with PCR (κ = 0.27; 95 % CI = 0.06 to 0.49). Most importance of the TT was shown in relation to specificity (88.9 %; 95 % CI = 0.88 to 0.89) and negative predictive value (70.3 %; CI 95 % = 0.70 to 0.71). CONCLUSIONS: TT was more effective in detecting cases that were truly negative than positive. These results suggest that the TT should not be used as diagnosis of dengue.

Mouse models of dengue virus infection for vaccine testing.

Dengue is a mosquito-borne disease caused by four serologically and genetically related viruses termed DENV-1 to DENV-4. With an annual global burden of approximately 390 million infections occurring in the tropics and subtropics worldwide, an effective vaccine to combat dengue is urgently needed. Historically, a major impediment to dengue research has been development of a suitable small animal infection model that mimics the features of human illness in the absence of neurologic disease that was the hallmark of earlier mouse models. Recent advances in immunocompromised murine infection models have resulted in development of lethal DENV-2, DENV-3 and DENV-4 models in AG129 mice that are deficient in both the interferon-α/ß receptor (IFN-α/ß R) and the interferon-γ receptor (IFN-γR). These models mimic many hallmark features of dengue disease in humans, such as viremia, thrombocytopenia, vascular leakage, and cytokine storm. Importantly AG129 mice develop lethal, acute, disseminated infection with systemic viral loads, which is characteristic of typical dengue illness. Infected AG129 mice generate an antibody response to DENV, and antibody-dependent enhancement (ADE) models have been established by both passive and maternal transfer of DENV-immune sera. Several steps have been taken to refine DENV mouse models. Viruses generated by peripheral in vivo passages incur substitutions that provide a virulent phenotype using smaller inocula. Because IFN signaling has a major role in immunity to DENV, mice that generate a cellular immune response are desired, but striking the balance between susceptibility to DENV and intact immunity is complicated. Great strides have been made using single-deficient IFN-α/ßR mice for DENV-2 infection, and conditional knockdowns may offer additional approaches to provide a panoramic view that includes viral virulence and host immunity. Ultimately, the DENV AG129 mouse models result in reproducible lethality and offer multiple disease parameters to evaluate protection by candidate vaccines.

Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection.

UNLABELLED: With over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre(+) Ifnar(flox/flox) [denoted as Ifnar(f/f) herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre(+) Ifnar(f/f) mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre(+) Ifnar(f/f) mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents. IMPORTANCE: Although dengue virus (DENV) infects hundreds of millions of people annually and results in morbidity and mortality on a global scale, there are no approved antiviral treatments or vaccines. Part of the difficulty in evaluating therapeutic candidates is the lack of small animal models that are permissive to DENV and recapitulate the clinical features of severe human disease. Using animals lacking the type I interferon receptor only on myeloid cell subsets, we developed a more immunocompetent mouse model of severe DENV infection with characteristics of the human disease, including vascular leakage, hemoconcentration, thrombocytopenia, and liver injury. Using this model, we demonstrate that pathogenesis by two different DENV serotypes is inhibited by therapeutic administration of a genetically modified antibody or a RIG-I receptor agonist that stimulates innate immunity.

Vitamin D: a new anti-infective agent?

Before the antibiotic era, treatment of tuberculosis patients was restricted to sun exposure in sanatoria. Years later, it was found that 1,25-dihydroxyvitamin D3 stimulates production of cathelicidins, a family of polypeptides found in lysosomes of macrophages and polymorphonuclear leukocytes. Cathelicidins serve a critical role in innate immune defense, which plays an important role in the suppression of Mycobacterium infections and other pathogens. It is believed that the increased incidence of the common cold and pneumonia during winter is related, in part, to decreased exposure to sunlight, resulting in a decreased synthesis of 1,25-dihydroxyvitamin D3. An association has been established between low levels of vitamin D and upper respiratory and enteric infections, pneumonia, otitis media, Clostridium infections, vaginosis, urinary tract infections, sepsis, influenza, dengue, hepatitis B, hepatitis C, and HIV infections. Accumulating evidence suggests that 1,25-dihydroxyvitamin D3 exerts protective effects during infections by upregulating the expression of cathelicidin and ß-defensin 2 in phagocytes and epithelial cells. Vitamin D may be acting as a panaceal antibiotic agent and thus may be useful as an adjuvant therapy in diverse infections.

Rhodiola inhibits dengue virus multiplication by inducing innate immune response genes RIG-I, MDA5 and ISG in human monocytes.

Recognition of virus infection by retinoic acid-inducible gene (RIG) I and melanoma differentiation-associated protein (MDA) 5, which are RNA helicases, and interferon-stimulated gene (ISG) 15 activates cascades of signal transduction pathways leading to production of type I interferons and proinflammatory cytokines that orchestrate the elimination of the viruses. However, it has been demonstrated that RNA-helicase-mediated innate immunity plays an essential role in defending the host from infection. In our efforts to identify plant-derived antivirals that selectively enhance ISG- and RNA-helicase-mediated antiviral immune responses, we identified a plant, rhodiola, that significantly promoted ISG, RIG-I and MDA 5 gene expression and an antiviral immune response against dengue virus (DENV) infection. Rhodiola induced interferon (IFN) ß and other cytokines, including IL-1ß, TNF-α, IL-6 and IL-8, in infected cells. It was also found that rhodiola upregulated phosphorylated eIF-2α, PKR and NF-kB in infected cells. In addition, the number of NK cells was also increased by rhodiola treatment in dengue-virus-infected human PBMCs. Treatment with a crude extract of rhodiola (RAE) resulted in effects in the 20 % range, which is similar to the magnitude of the same effects observed in DENV infections. Taken together, our results imply that rhodiola induces pharmacological modulation of RIG-I, MDA 5 and ISG signal transduction pathways in favor of the induction of a beneficial antiviral immune response against dengue virus, which can be a novel therapeutic strategy for management of infection.

Utility of humanized BLT mice for analysis of dengue virus infection and antiviral drug testing.

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. IMPORTANCE Infection with dengue virus remains a major medical problem. Progress in our understanding of the disease and development of therapeutics has been hampered by the scarcity of small animal models. Here, we show that humanized mice, i.e., animals engrafted with components of a human immune system, that were infected with a patient-derived dengue virus strain developed clinical symptoms of the disease and mounted virus-specific immune responses. We further show that this mouse model can be used to test preclinically the efficacy of antiviral drugs.

Identifying protective dengue vaccines: guide to mastering an empirical process.

A recent clinical trial of a live-attenuated tetravalent chimeric yellow fever-dengue vaccine afforded no protection against disease caused by dengue 2 (DENV-2). This outcome was unexpected as two or more doses of this vaccine had raised broad neutralizing antibody responses. Data from pre-clinical subhuman primate studies revealed that vaccination with the monotypic DENV-2 component failed to meet established criteria for solid protection to homotypic live virus challenge. Accordingly, it is suggested that preclinical testing adopt more rigorous criteria for protection and that Phase I testing be extended to require evidence of solid monotypic protective immunity for each component of a dengue vaccine by direct challenge with live-attenuated DENV. Because live-attenuated tetravalent DENV vaccines exhibit evidence of immunological interference phenomena, during Phase II, volunteers given mixtures of DENV 1-4 vaccines should be separately challenged with monotypic live-attenuated DENV. Immune responses to live-attenuated challenge viruses and vaccine strains should be studied in an attempt to develop useful in vitro correlates of in vivo protection. Finally, it will be important to learn if DENV non-structural protein 1 (NS1) contributes to pathogenesis of the vascular permeability syndrome in humans. If so, immunity to dengue 1-4 NS1 may be crucial to prevent severe disease.

Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection.

Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection.

Animal models of dengue virus infection.

The development of animal models of dengue virus (DENV) infection and disease has been challenging, as epidemic DENV does not naturally infect non-human species. Non-human primates (NHPs) can sustain viral replication in relevant cell types and develop a robust immune response, but they do not develop overt disease. In contrast, certain immunodeficient mouse models infected with mouse-adapted DENV strains show signs of severe disease similar to the 'vascular-leak' syndrome seen in severe dengue in humans. Humanized mouse models can sustain DENV replication and show some signs of disease, but further development is needed to validate the immune response. Classically, immunocompetent mice infected with DENV do not manifest disease or else develop paralysis when inoculated intracranially; however, a new model using high doses of DENV has recently been shown to develop hemorrhagic signs after infection. Overall, each model has its advantages and disadvantages and is differentially suited for studies of dengue pathogenesis and immunopathogenesis and/or pre-clinical testing of antiviral drugs and vaccines.

A dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs.

Dengue fever is an emerging arboviral disease for which no vaccine or antiviral treatment exists and that causes thousands of fatalities each year. To develop an in vivo test system for antidengue drugs, AG129 mice, which are deficient for the interferon- alpha / beta and - gamma receptors, were injected with unadapted dengue virus, resulting in a dose-dependent transient viremia lasting several days and peaking on day 3 after infection. Additionally, nonstructural protein 1, increased levels of proinflammatory cytokines, and neutralizing IgM and IgG antibodies were found, and mice had splenomegaly. Oral administration of the antiviral compounds 7-deaza-2'-C-methyl-adenosine, N-nonyl-deoxynojirimycin, or 6-O-butanoyl castanospermine significantly reduced viremia in a dose-dependent manner, even after delayed treatment, leading to a reduction of splenomegaly and proinflammatory cytokine levels. The results validate this dengue viremia mouse model as a suitable system for testing antidengue drugs and indicate that antiviral treatment during the acute phase of dengue fever can reduce the severity of the disease.