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Abstract

There are currently no licensed antivirals available for the treatment of dengue virus (DENV), which causes significant morbidity and mortality throughout tropical areas of the world and is now encroaching on the southern United States. Recent improvements in existing animal models and cell culture systems have been very important in elucidating the mechanisms of DENV pathogenesis in humans, including the identification of potential viral and host proteins that might be targeted for the treatment of DENV infection. The AG129 mouse model is a major advance in the development of antiviral and vaccine candidates for clinical use. It allows for testing of potential therapeutics in a relevant system that exhibits some aspects of disease that are similar to those observed in humans. This review focuses on recent developments in the AG129 mouse model and discusses compounds that have been found to be active in available cell and animal model systems within the past year.

Introduction

Dengue disease is caused by four related and antigenically distinct viruses, which are designated dengue virus (DENV) 1–4. DENV is transmitted from person to person via mosquito vectors (particularly Aedes sp). Approximately 2.5 billion people are at risk of contracting dengue in endemic areas, and as many as 50 million people are infected with DENV each year [1]. DENV infection generally results in dengue fever (DF), a relatively mild and self-limiting disease associated with fever, malaise and other non-specific symptoms. However, in hyperendemic areas with multiple circulating serotypes, the more severe, potentially lethal disease manifestations of dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS) can occur. The hallmark of DHF/DSS is plasma leakage, which is commonly associated with a cytokine storm, thrombocytopaenia and intestinal haemorrhage [2]. Several risk factors, including race, age, sex and host genetic factors (for example, human leukocyte antigen [HLA], dendritic-cell-specific intracellular adhesion molecule-3-grabbing non-integrin [DC-SIGN], tumour necrosis factor [TNF]-α, transforming growth factor [TGF]-β1 and interleukin [IL]-10) appear to be associated with severe disease [3]; however, the single greatest risk factor is the presence of subneutralizing levels of antibodies with specificity to another strain of DENV acquired either in utero from mother to child or from repeat infection by another serotype [4].

Three major hypotheses have been formulated to explain DENV pathogenesis: antibody-dependent enhancement of disease (ADE), aberrant T-cell response and viral virulence factors. According to the ADE hypothesis, cross-reactive antibodies generated against a previous infecting serotype of DENV facilitate the Fc-mediated uptake of a subsequent infecting heterologous strain by Fc-receptor-positive cells. This ultimately results in a more severe pathological response to the secondary virus infection [5]. The involvement of ADE as a cause of severe DENV-associated disease is further supported by the observation of non-neutralizing maternal antibodies to DENV, which could persist for up to 12 months in infants born to immune mothers [6] and might be associated with increased frequency of severe disease in such cases [7]. According to the aberrant T-cell response hypothesis, serotype cross-reactive memory T-cells contribute to the pathogenesis of DHF/DSS. In support of this hypothesis, inappropriate T-cell responses have also been observed with higher frequency in DENV-infected patients with more severe symptoms [8]. Finally, the viral virulence hypothesis postulates that viral genotype might play an important role in DENV pathogenesis, as specific viral genotypes have been shown to be associated with more severe disease outbreaks [9], supporting the contribution of viral determinants in DENV pathogenesis. Recent research has further characterized the host response on a genetic level to give insight into the differences in the development of DSS as compared with less severe disease phenotypes [10].

Currently, fluid replacement, with or without blood factors, is the only course of action for the management of plasma leakage associated with DHF/DSS [11 –14]. Children have the highest risk of developing severe disease manifestations, further underscoring the need for effective care. Most primary infections resolve with a relatively low mortality rate and do not require treatment. In hyperendemic areas, many infected patients must be hospitalized, which puts a tremendous financial burden on these countries. A recent estimate placed the burden of DENV in eight countries between 587 million and 1.8 billion USD annually, which does not include the cost of surveillance and vector control [15]. An effective antiviral could reduce morbidity and thereby ease the financial burden.

Challenges faced in the development of DENV-specific antiviral therapies are numerous and include development of a low-cost rapid diagnostic technique that is predictive of disease severity, and identifying a compound that is safe, targets multiple serotypes and is effective even after the onset of severe clinical disease. These are not easy goals, but recent advances in the understanding of DENV biology and improvements in animal and cell culture models should contribute towards the discovery and advancement of potential anti-DENV agents for clinical use. Here, we will briefly review recent advances in animal models of DENV and discuss, in more detail, the anti-DENV agents that have shown potential for use in the treatment of this disease.

Recent advances in animal models of DENV

The use of a small animal model of disease is crucial to the advancement of antiviral agents toward clinical use, as compounds that possess antiviral properties in vitro might not have the same effect in an in vivo model. For example, the antiviral prodrug ribavirin inhibits DENV replication in vitro [16 –18], but had no effect on viraemia when evaluated in two separate in vivo model systems [19,20]. Optimally, animal models should reflect the clinical pathology of infection observed in humans. Development of an animal model for DENV has been difficult because the virus does not replicate well in species other than its natural hosts, that is, humans and mosquitoes. Some strains have been adapted to cause neurological disease in mice, which might have some utility in the evaluation of antiviral therapies [21,22], but these neuropathological mouse models lack many important and relevant disease features that are seen in human infection cases.

Non-human primate models of DENV infection have been examined (reviewed in [23]). In these models, only transient viraemia is detected and the infected animals do not generally exhibit outward pathology, with the exception of a recent study showing skin rash/haemorrhage in DENV-infected rhesus macaques [24]. Thus, the development of a non-human primate model with relevant disease features is a difficult prospect. At present, the lack of key DHF/DSS manifestations (for example, plasma leakages and cytokine storm), combined with the high cost and ethical considerations associated with conducting studies in higher mammals, has limited the use of primates as model systems for antiviral testing.

A key advance in the field of anti-DENV research is the recent development of a useful mouse model of disease. This allows for the testing of potential antiviral compounds in a small animal model with many disease features that are similar to those observed in humans. Some recent reviews provide a history of the development of mouse models of DENV infection [23,25,26], which will only be addressed briefly here.

129/Sv mice lacking both the type-I and type-II interferon (IFN) receptors (AG129) are highly susceptible to infection with various mouse-adapted and clinical DENV strains, including all four serotypes [20,27,28]. Infection of AG129 mice with sufficient titres of these DENV strains generally results in viraemia and presence of infectious virus in other tissues, splenomegaly, detectable levels of serum NS1 protein and paralysis between 8–12 days post-virus challenge [20,29]. Such models allow the evaluation of potential antiviral agents in the treatment of mild disease and reduction of viraemia, but are complicated by neurological involvement during the final stages of disease, which rarely occurs in humans [30,31].

The development of an AG129 infection model that uses D2S10, a DENV-2 strain alternately passaged through mouse and insect cells has been reported [28]. In this model, animals infected via the intravenous route display an early death phenotype (4–6 days post-infection [dpi]) that does not involve paralysis and includes additional parameters relevant to human infection, such as increased vascular permeability and cytokine responses. A similar disease phenotype is observed with S221, a triple-plaque-purified clone isolated from D2S10. A non-mouse-adapted strain of DENV-2 (D2Y98P) was recently reported to cause similar severe disease manifestations after intraperitoneal administration of 10⁶–10⁷ plaque-forming units of virus, which included cytokine storm, vascular leak, haemorrhage and early death (5–6 dpi) in AG129 mice [32]. Inoculation with lower doses of D2Y98P DENV resulted in viral replication in relevant tissues, tissue damage, increased vascular permeability without haemorrhage and death a few days after virus clearance. This represents a model of less severe disease following infection with a relatively low viral challenge dose. The AG129 mouse model of primary DENV infection has shown utility in antiviral studies evaluating the efficacy of various compounds, including α-glucosidase inhibitors, nucleoside inhibitors and sulfated polysaccharides. Parameters used in these studies included survival, viraemia, levels of inflammatory cytokines and splenomegaly [20,33 –35].

Greater utility of the AG129 mouse model was found after the first demonstration of ADE in vivo using the D2S10 and S221 viruses. Severe DHF/DSS is 40x more frequent during secondary infection as compared with primary infection and is likely to be caused by the presence of enhancing antibodies generated against the heterologous serotype [36]. Therefore, the role of ADE in the context of antiviral therapy of DENV infection is a very important issue to consider. Evaluation of human antibodies generated during natural DENV infection revealed that the majority had specificity to the precursor membrane (prM) protein, with strong enhancing and weak neutralization characteristics [37]. Both Zellweger et al. [29] and Balsitis et al. [38] independently demonstrated that administration of heterologous or subprotective levels of homologous anti-DENV antibodies prior to DENV challenge resulted in an enhancement of disease severity. The S221–AG129 model of ADE-induced disease featured all the hallmarks of severe dengue disease in humans, including increased hematocrit levels, cytokine storm, low platelet count, increased vascular permeability, haemorrhagic manifestations and shock-induced death. Additionally, in this model, only a specific cell subset, liver sinusoidal endothelial cells (LSECs), was found to permit ADE of DENV infection in vivo. On the basis of in vitro studies, macrophages and dendritic cells have been presumed to support ADE, although there is no consensus in the field about cellular tropism of DENV in humans, and no study has defined the cell types that support the ADE model of DENV infection in humans. Hepatitis is more frequently observed in DENV-infected patients that succumb to disease [39], which is consistent with the observation of increased infection of LSECs. Thus, despite the caveat associated with the immunocompromised status of AG129 mice, this finding provides the impetus towards determining the precise cell types that are infected in humans. Importantly, both the S221–AG129 and D2S10–AG129 models of ADE-induced disease are well-suited for evaluating antiviral compounds. Indeed, the D2S10–AG129 model has already been used to demonstrate that a recombinant anti-DENV antibody that does not bind the Fc-γ receptor has prophylactic and therapeutic efficacy against lethal DENV infection [38]. The publication of additional studies evaluating antiviral therapies in these models of ADE-induced disease is anticipated in the near future.

Therapeutic targets

Approximately 2.5 billion people are at risk of contracting DENV; therefore, it is vitally important that antivirals that specifically or broadly inhibit DENV are developed. The development of antiviral treatments for DENV is complicated, as an effective therapy for the treatment of dengue must be safe, inexpensive and active when administered at or soon after the onset of disease. Other factors that reduce the incentive to develop anti-DENV treatments include the short duration of disease, an often mild clinical course and a high survival rate. The National Institute of Allergy and Infectious Disease has supported programmes to encourage the discovery of antiviral therapies for DENV and other important human viral diseases [40].

Intuitively, direct and specific inhibition of the virus would be the most attractive route of therapeutic intervention. Antiviral agents could target structural and non-structural proteins produced during viral infection. Once viral replication is inhibited by host responses, immunopathological events might be more important in later stage disease [41]; hence, reversal or inhibition of immunopathological disease might be a valid therapeutic strategy to pursue. Recent advances in the inhibition of viral and cellular targets, including specific anti-DENV agents, will be discussed below.

Viral protein targets

Similar to other flaviviruses, DENV is composed of a single positive-sense genomic RNA (gRNA) that codes for a polyprotein (Figure 1), which is processed by host and viral proteases into various intermediate and mature structural and non-structural proteins. Recent findings on the cellular and molecular mechanisms of DENV provide insight on how best to inhibit the replicative cycle and pathogenicity through the use of antivirals [42,43]. An in-depth review of specific viral targets and their potential inhibitors was recently published [44]. In the current review, we present a discussion of more recent discoveries in the area of viral targets and newly identified inhibitors (Table 1).

Table 1.

Recently discovered agents with anti-DENV activity

Anti-DENV agent	Target	Active concentration	Model system	Reference
Brequinar	Dihydroorotate dehydrogenase	–	Cell culture	[61]
Anti-DENV monoclonal antibodies	E protein	–	BALB/c mice	[21]
Flavanoid-derived compounds	Unknown	2–25 μg/ml	HepG2 cells	[155]
NITD203 (prodrug of NITD449)	RNA synthesis of DENV	25 mg/kg/day	AG129 mice	[156]
NITD449 (adenosine analogue)	RNA synthesis of DENV	–	AG129 mice	[157]
Lippia essential oils	Virucidal	0.4–33.7 μg/ml	Cell culture	[158]
Glutathione	Reversal of nuclear factor-κB activation	–	HepG2 cells	[145]
Palmatine	Protease inhibitor	26.4 μM	Vero cells	[93]
β-D-2′-ethynyl-7-deaza-adenosine	Polymerase inhibitor	<1 μM	Huh-7 cells	[56]
WSS45	Infection/replication	0.68 μg/ml	BHK cells	[159]
ETAR	IMPDH dehydrogenase	9.5 μM	Vero cells	[72]
IM18	IMPDH dehydrogenase	106.1 μM	Vero cells	[72]
N-sulfonylanthranillic acid derivatives	Polymerase inhibitor	0.7 μM	RdRp assay	[47]
(BIP)(2)B	Helicase	–	Helicase assay	[94]
DC3-9dR-tagged siRNA	Targeted siRNA delivery to DCs	–	DC cultures	[111]
n-Octyl-β-D glucoside competitors	Fusion inhibitor	6.8 μM	Fusion assay	[160]

(BIP)(2)B, 1-N,4-N-bis [4-(1H-benzimidazol-2-yl)phenyl]benzene-1,4-dicarboxamide; DC, dendritic cell; DC3-9dR, dendritic-cell-targeting 12-mer peptide fused to a nona-D-arginine; DENV, dengue virus; ETAR, 1-β-D-ribofuranosyl-3-ethynyl-[1,2,4] triazole; IM18, 1-β-D-ribofuranosyl-4-ethynyl-[1,3] imidazole; IMPDH, inosine monophosphate dehydrogenase; siRNA, small interfering RNA; RdRp, RNA-dependent RNA polymerase.

Figure 1.

A graphical representation of the flavivirus polyprotein

NS5

During DENV infection, all virus-encoded enzymatic processes are accomplished by the NS3 and NS5 proteins, which make these non-structural components attractive targets for antiviral therapy [45]. The flavivirus NS5 protein performs key roles in virus replication and in the modulation of host immune response [46], presenting multiple potential targets because it is a viral polymerase (Figure 2) [46 –48], has methyltransferase function [49,50], binds to various host proteins (forming interactions that are necessary for replication) [51,52] and is involved in inhibition of the antiviral response of the cell [53,54]. Therefore, targeting NS5 will inhibit various processes that the virus uses to establish replication and cause disease.

Figure 2.

DENV envelope, polymerase and protease

Great effort has been undertaken to identify inhibitors of the polymerase function of NS5, including large screening programmes that utilize RNA-dependent RNA polymerase (RdRp) [55], virus-like particle (VLP) [56] and replicon [57] assays. Utilization of these assays in antiviral compound research has resulted in the discovery of several inhibitors of NS5 polymerase function, and will continue to be important in discovering inhibitors in the future.

NITD008, which has broad-spectrum activity against several flaviviruses via the inhibition of NS5, was effective in cell culture as well as in a mouse model of disease in the treatment of DENV-2 [35]. Pyrazine carboxamides, including T-705 and T-1106, might also be useful for the treatment of DENV, having shown efficacy in animal models of yellow fever virus (YFV) [58,59], West Nile virus (WNV) [60] and several other RNA viruses [61 –63]. These compounds target the viral polymerase [61], but might also have other modes of activity. The compound β-D-2′-ethynyl-7-deaza-adenosine triphosphate targets DENV polymerase and causes chain termination during replication in cell culture [64].

NS5-associated methyltransferase activity [50] is another potential target for antiviral development. Methylation of the RNA cap of DENV by this enzyme occurs in two sequential steps and the inhibition of either or both methylation events will attenuate or abolish DENV replication, respectively [49]. Resistance to methyltransferase inhibitors is, however, a concern [44,65], and resistance mutations have been reported [66].

A virtual screening programme identified 263 compounds with favourable NS5 binding capability [67]. Out of these candidates, six were confirmed with 50% inhibitory concentration (IC₅₀) values <100 μM and four with IC₅₀<10 μM in in vitro assays. It will be interesting to see if methyltransferase inhibitors with in vitro activity are effective in animal models of DENV disease.

Depletion of the components necessary for nucleic acid synthesis might also inhibit DENV replication via indirect inhibition of viral polymerase. Treatment with brequinar, an inhibitor of dihydroorotate dehydrogenase, inhibits DENV RNA synthesis in vitro by reducing pyrimidine pools, and might also inhibit assembly or release of the virus [68]. The compound 2′-C-methylcytidine is also active against DENV in cell culture [69] and has been shown to work through a similar mechanism against the related flavivirus, YFV, in a hamster model [70]. Other compounds that inhibit DENV through the reduction of nucleotide pools include mycophenolic acid and ribavirin [16,17,71]. Ribavirin is a broad-spectrum antiviral compound that is active against many different flaviviruses in cell culture [72 –74]. This compound has many modes of action, including inhibition of viral polymerase; however, against flaviviruses, the mode of action is largely caused by the inhibition of inosine monophosphate dehydrogenase with a resulting depletion of GTP pools [71]. Ribavirin has been shown to be active against YFV in a hamster model [75,76]. Also, this compound has been shown to have activity in various primary cells and cell lines infected with DENV [16 –18]. Despite activity in cell culture, however, ribavirin did not show any activity in a mouse model of DENV disease [20] or in a primate model of DENV viraemia [19]. A similar phenomenon, where no activity was observed in animal models after observable activity in cell culture, has also been demonstrated in a hamster model of WNV disease [77]. Ribavirin 2′,3′,5′-triacetate was effective in a mouse model of DENV [78] and further investigation might be warranted. The ribavirin analogues, 1-β-D-ribofuranosyl-3-ethynyl-[1,2,4] triazole (ETAR) and 1-β-D-ribofuranosyl-4-ethynyl-[1,3] imidazole (IM18), have also shown activity in cell culture against several flaviviruses, including DENV [79]. Ribavirin monotherapy has no efficacy against HCV [80], but is quite effective as a part of combination therapy with IFN [81]. Such an approach might be useful in the treatment of DENV.

The NS5 protein is involved in stimulation of immune mediators, including IL-8 [52]. Some aspects of immune stimulation have been elucidated. For example, NS5 normally shuttles between the nucleus and the cytoplasm, which involves exportin proteins, and is essential for replication [51]. Inhibition of the exportin CRM1 through alteration of the nuclear localization sequences results in nuclear accumulation of NS5 and a decrease in IL-8 induction [82]. Improper stimulation of certain inflammatory cytokines has been implicated in severe disease caused by DENV [83 –86]. Specifically, IL-8 has been shown to be significantly increased in cases of severe dengue in humans and might contribute to disease severity [87]. An immature form of NS5 binds to the signal transducer and activator of transcription-2 (STAT-2), causing degradation of this IFN pathway protein [47,88]. Inhibiting the protein–protein interactions of NS5 and key host proteins is therefore also a potential target strategy.

NS3

The NS3 protein, in combination with NS2, functions as a serine protease (Figure 2). This viral-encoded enzyme also functions as a helicase, presenting a second modality that could be targeted. Protease inhibitors have already been developed against other flaviviruses, such as HCV [89], reviewed in [90,91]. Work with in silico docking programmes has led to the identification of potential inhibitors of DENV NS3 protein, two of which were shown to have activity in cell culture models [92,93]. The recent structure determination of the active site of DENV protease will further allow for targeted design of inhibitors of this enzyme [94]. A cyclopeptide derived from the kalata B1 plant protein shows potent inhibition in in vitro assays against DENV protease activity [95].

A fluorescence-based quenching assay, which is sensitive and adaptable to other viral proteins, was recently developed to identify protease inhibitors [96]. Palmatine was shown to inhibit the protease of WNV and had broad-spectrum antiviral activity against several flaviviruses, including DENV, in cell culture [97]. Anti-helicase activity has been observed with the compound 1-N,4-N-bis [4-(1H-benzimidazol-2-yl)phenyl]benzene-1,4-dicarboxamide ((BIP)(4)B), with broad-spectrum activity against DENV, HCV and Japanese encephalitis virus [98].

Structural proteins

DENV structural proteins are important targets (Figure 2). Part of these proteins are found on the surface of virions and might be inhibited by therapeutic antibodies with broad-spectrum efficacy that target common epitopes shared by all four DENV serotypes [99]. A similar approach has been used for the treatment of WNV and a humanized monoclonal antibody has been shown to have potent activity in animal models [100 –102]. Selective and potent antibodies, such as those discovered for WNV [103], might be generated for the treatment of severe DENV infections if the risk of ADE can be avoided [37]. However, neutralizing antibodies are often highly specific and might not be effective even for the treatment of closely-related homologous DENV serotypes, which was demonstrated recently in a mouse model [104].

The majority of antibodies generated during natural DENV infection have specificity for the prM protein and have been shown to have higher disease enhancement and lower neutralization properties [37]. This study also demonstrated that antibodies specific for the E protein generally have a better neutralization capacity, suggesting potential application of anti-E antibodies in the treatment of DENV. Similar conclusions were reached in a study evaluating the functional activity of a large panel of antibodies in cell culture as well as a BALB/c mouse model of intracranial infection. Several antibodies with specificity to various portions of the E protein were effective in the treatment of DENV, even when administered several days post-viral challenge [21,105]. Modification of the Fc portion of an antibody might eliminate the disease enhancement characteristics. Removal or inactivation of the Fc region reduced viraemia and tissue viral burden in an AG129 mouse model, demonstrating the potential for modified antibodies as potential therapeutic agents [38].

Agents with affinity to viral structural proteins can block fusion of the virion with the host cell membrane and several fusion inhibitors have been recently described. A recently described small molecule DENV inhibitor, compound 6 (a pyridinylmethyl-guinazolinylamine with a chlorophenyl-thiophene tail), arrests the virus in vesicles, which results in a downstream inhibition of viral replication [106]. Using high-resolution structures of the E protein and computer-aided design, inhibitory peptides were produced that were effective at μM concentrations [107]. Likewise, soluble receptor protein analogues could potentially block virus entry [108], which might also inhibit pathological modes of action attributed to the E protein domain III [109].

Viral RNA targets

Important structural–functional elements of the viral RNA also provide potential avenues for therapy that, if conserved across different DENV serotypes, could result in broad-spectrum inhibition. Various portions of the 5′- and 3′-ends of the DENV genome are required for replication and might represent one such target [110]. Small interfering RNAs (siRNAs) are effective against DENV and related flaviviruses in various cell culture and animal models [111 –114]. The tagging of siRNA constructs with various peptides allows targeting to specific cell types or tissues, for example, dendritic cells [115]. Hammerhead ribozymes delivered by lentivirus vectors effectively reduce virus production from transduced mosquito cells [116], suggesting potential utility in vector control or vector-delivered therapy. Peptide-conjugated phosphorodiamidate morpholino oligomers have shown promise as anti-DENV agents and were found to inhibit the virus in cell culture [117].

The subgenomic flavivirus RNA (sfRNA) comprises a portion of the 3′ untranslated region and is formed after partial degradation of viral gRNA in host processing bodies [118]. The sfRNA of Kunjin, a related flavivirus, has been shown to contribute to pathogenesis in cell culture as well as in a mouse model of disease [118]. The sfRNA represents another potential target for antiviral therapy. Targeting the 3′ untranslated region could further disrupt cyclization of gRNA, which could further inhibit or impede viral replication [119,120].

Host cell targets

Host proteins play an integral role in many aspects of any viral replication cycle. Viruses, including DENV, have developed specific mechanisms to manipulate their hosts to replicate and evade immune effectors. The difficulty in targeting host proteins or pathways is the potential for toxicity or side effects. However, the potential of such compounds for screening purposes is greater because they are not DENV-specific, many are well-characterized and even compounds that have been approved by the US Food and Drug Administration might be available for testing against the virus. There are recent reviews that discuss options for targeting host cell proteins in detail [121,122]. In this review, we will only cover recent advances in host cell targeting methods for antiviral therapy (Table 1).

Cholesterol biosynthesis

Cholesterol has been shown to be integral to DENV replication. A decrease in cholesterol and very-low-density lipoprotein levels have been reported in individuals with more severe manifestations of DENV disease, which correlated with severe bleeding, hepatic dysfunction and death [123]. DENV replication was inhibited in different cell culture models after treatment with cholesterol pathway inhibitory compounds, including lovastatin, hymeglusin and zaragozic acid A [124]. This has implications in the early diagnosis of the disease and possibly in the treatment of DENV infection, although it is unclear if a similar effect will be seen in animal models.

Cytokines

Many flaviviruses, including DENV, have developed methods of blocking the IFN signalling cascade [47,125,126]. Stimulation of the IFN response or administration of exogenous IFN represents a host response manipulation that could be useful in the treatment of dengue. Recent work identified several IFN-stimulated gene products that are effective in suppressing various processes within the life cycle of DENV and WNV, including IFN-induced transmembrane protein-2 (IFITM2), IFITM3, viperin, IFN-stimulated gene 20 kDa protein (ISG20) and double-stranded RNA-activated protein kinase [127], which were similar to those involved in the response to HCV [128]. The addition of exogenous IFN reversed the effect of DENV inhibition of the cell-mediated host response [125], suggesting potential utility of IFN in the treatment of DENV. However, the AG129 mice used in DENV studies lack IFN-α/β and IFN-γ receptors, presenting an obvious impediment toward the testing of IFN and IFN stimulators against DENV in this model system. Treatment with IFN has been shown to be highly effective against HCV infection in humans [89,129] as well as in various animal models [75,77]. Treatment with pegylated IFN-α (Pegasys®) was effective in a primate model of DENV viraemia [130]. The potential use of IFN for the treatment of vascular disease is further supported by a recent study in DENV-infected human endothelial cells [131].

There is also potential for the use of IFN-γ as a therapeutic agent. IFN-γ treatment administered after virus infection was effective in treating DENV infection in dendritic cells, whereas treatment of these cells with IFN-α was ineffective because of inhibition of IFN type-1 signalling pathways [132]. It has been observed that IFN-γ treatment might increase the number of Fc receptors on promonocytic cells, which augments the uptake of DENV immune complexes in U937 cells [133], but not in peripheral blood mononuclear cells [134]. Overall, this demonstrates the varied effect of IFN-γ depending on the cell type [135].

The inflammatory cytokine, TNF-α, is implicated in triggering vascular leakage in human infection and disease models [28,136,137], suggesting inhibition of TNF stimulation could also be a viable option in the treatment of dengue. Interaction of DENV with C-type lectin domain family 5 member A (CLEC5A) on the surface of myeloid cells results in the phosphorylation of DNAX-activating protein 12 (DAP12), which then stimulates the release of proinflammatory cytokines including TNF-α. The release of TNF-α can be reduced when the CLEC5A–DENV interaction is inhibited [138]. Treatment of DENV-2-infected human umbilical vein endothelial cells with exogenous IFN reduced the production of TNF-α, and prevented virus-induced hyperpermeability [131].

Increased production of the high mobility group box 1 (HMGB1) protein (an important intracellular transcriptional regulator) has been shown to reduce DENV titres in infected dendritic cells [139], potentially suggesting another host target for the inhibition of replication. However, once released extracellularly, HMGB1 acts as a potent proinflammatory protein that might contribute to immunopathogenesis [140].

Other host cell targets

Targeting the glycoprotein-processing pathway of the host has been shown in several models to inhibit DENV. Removal of E protein glycans at residues 153/154 and 67 has been shown to enhance DENV infectivity and decrease efficient release in mammalian cells [141]. A recent review covers much of the salient information on the topic of glycosylation inhibition [122], therefore, only a brief discussion of compounds with activity against DENV is included here. Castanospermine and its derivative, 6-O-butanoyl castanospermine, are derived from the castor bean. These compounds are α-glucosidase inhibitors that disrupt N-glycosylation. They are active in rodent models of dengue disease [20,22]. N-nonyl-deoxynojirimycin, an iminosugar derivative similar to castanospermine, was also effective in reducing disease parameters in an AG129 model [20]. Geneticin is an aminoglycoside that has shown activity against DENV in cell culture, but is inactive against YFV [142]. In the same study, other aminoglycosides, including gentamicin, kanamycin and a guanidinylated geneticin, showed no activity against DENV. Mannose-binding lectin was recently shown to bind the surface of DENV, resulting in neutralization and enhanced clearance of the virus in a mouse model [143]. Host-derived surface components of DENV are useful targets for antiviral therapy.

Cyclophilins regulate cytokine production and have been shown to be involved in the efficient replication of flaviviruses [88] through an interaction with NS5 [144]. Cyclosporine and cyclosporine derivatives and analogues have been shown to be useful in the treatment of flaviviruses, including DENV, in various cell culture [88,145,146] and in the treatment of human HCV infection [147], suggesting potential for the use of such compounds in the treatment of dengue.

Y box-binding protein-1, has been shown to bind to the 3′ stem loop of DENV mediating an antiviral effect in cell culture, which is at least partially caused by the inhibition of virus translation [148].

Infection with DENV reduces intracellular glutathione concentrations, which results in activation of nuclear factor-κB by E protein domain III [149] and increased virus production [150]. The addition of exogenous glutathione reverses this effect and inhibits viral production in cell culture (Table 1).

Many intracellular proteins are used in viral replication and the correct processing of viral proteins. For example, the heat-shock proteins are involved in the life cycles of a diverse array of intracellular pathogen, and inhibition of this group of proteins might result in decreased replication of such pathogens [151]. These proteins are integral in DENV replication [152] and also play an important role in attachment and entry [153,154]. Polypyrimidine tract-binding protein is also involved in DENV replication and knockdown of this protein by siRNA targeting inhibits the production of infectious virus, although this was not observed for YFV [155]. The ubiquitin–proteasome pathway was recently shown to be important in the antiviral response [156], which could possibly be enhanced to improve viral clearance as a means of antiviral therapy.

Other anti-DENV agents that have been recently discovered in cell culture or animal models, and which are not discussed in the text, are included in Table 1. These compounds have diverse mechanisms of action, although many need to be tested in animal models to confirm cell culture activity in vivo. These compounds are included in the table for reference purposes.

Conclusions

Unfortunately, no antivirals are currently approved for the treatment of DENV in human patients. Great efforts are being made in laboratories across the world to discover an effective, safe and readily available therapy to alleviate disease caused by DENV. This effort has been accelerated through the use of various useful small animal models that have been recently developed and improved. With many potential candidates, mouse models of disease and with support of government and private funding, the prospect of an effective antiviral or therapeutic compound for the treatment of dengue is encouraging.

Footnotes

The authors declare no competing interests.

References

Gubler

. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 1998; 11:480–496.

Deen

Harris

Wills

. The WHO dengue classification and case definitions: Time for a reassessment. Lancet 2006; 368:170–173.

Perez

Sierra

Garcia

. Tumour necrosis factor-alpha, transforming growth factor-β1, and interleukin-10 gene polymorphisms: Implication in protection or susceptibility to dengue hemorrhagic fever. Hum Immunol 2010; 71:1135–1140.

Mathew

Rothman

. Understanding the contribution of cellular immunity to dengue disease pathogenesis. Immunol Rev 2008; 225:300–313.

Burke

Kliks

. Antibody-dependent enhancement in dengue virus infections. J Infect Dis 2006; 193:601–603. Author reply 603–604.

Chau

Hieu

Anders

. Dengue virus infections and maternal antibody decay in a prospective birth cohort study of Vietnamese infants. J Infect Dis 2009; 200:1893–1900.

Libraty

Acosta

Tallo

. A prospective nested case-control study of dengue in infants: Rethinking and refining the antibody-dependent enhancement dengue hemorrhagic fever model. PLoS Med 2009; 6:e1000171.

Liu

Huang

Lin

Yeh

Liu

Lei

. Transient CD4/CD8 ratio inversion and aberrant immune activation during dengue virus infection. J Med Virol 2002; 68:241–252.

Rico-Hesse

. Dengue virus virulence and transmission determinants. Curr Top Microbiol Immunol 2010; 338:45–55.

10.

Devignot

Sapet

Duong

. Genome-wide expression profiling deciphers host responses altered during dengue shock syndrome and reveals the role of innate immunity in severe dengue. PLoS ONE 2010; 5:e11671.

11.

Chuansumrit

Teeraratkul

Wanichkul

. Recombinant-activated factor VII for control and prevention of hemorrhage in nonhemophilic pediatric patients. Blood Coagul Fibrinolysis 2010; 21:354–362.

12.

Lee

Yang

Liu

. Comparison of the effects of oral hydration and intravenous fluid replacement in adult patients with non-shock dengue hemorrhagic fever in Taiwan. Trans R Soc Trop Med Hyg 2010; 104:541–545.

13.

Kuo

Chang

Chiu

Chen

Hwang

. Difficulty in diagnosis and treatment of dengue hemorrhagic fever in patients with chronic renal failure: Report of three cases of mortality. Am J Trop Med Hyg 2007; 76:752–756.

14.

Wills

Nguyen

. Comparison of three fluid solutions for resuscitation in dengue shock syndrome. N Engl J Med 2005; 353:877–889.

15.

Suaya

Shepard

Siqueira

. Cost of dengue cases in eight countries in the Americas and Asia: A prospective study. Am J Trop Med Hyg 2009; 80:846–855.

16.

Diamond

Zachariah

Harris

. Mycophenolic acid inhibits dengue virus infection by preventing replication of viral RNA. Virology 2002; 304:211–221.

17.

Takhampunya

Ubol

Houng

Cameron

Padmanabhan

. Inhibition of dengue virus replication by mycophenolic acid and ribavirin. J Gen Virol 2006; 87:1947–1952.

18.

Huang

Lei

Liu

Lin

Liu

Yeh

. Dengue virus infects human endothelial cells and induces IL-6 and IL-8 production. Am J Trop Med Hyg 2000; 63:71–75.

19.

Malinoski

Hasty

Ussery

Dalrymple

. Prophylactic ribavirin treatment of dengue type 1 infection in rhesus monkeys. Antiviral Res 1990; 13:139–149.

20.

Schul

Liu

Flamand

Vasudevan

. A dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs. J Infect Dis 2007; 195:665–674.

21.

Sukupolvi-Petty

Austin

Engle

. Structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2. J Virol 2010; 84:9227–9239.

22.

Whitby

Pierson

Geiss

. Castanospermine, a potent inhibitor of dengue virus infection in vitro and in vivo. J Virol 2005; 79:8698–8706.

23.

Bente

Rico-Hesse

. Models of dengue virus infection. Drug Discov Today Dis Models 2006; 3:97–103.

24.

Onlamoon

Noisakran

Hsiao

. Dengue virus-induced hemorrhage in a nonhuman primate model. Blood 2010; 115:1823–1834.

25.

Williams

Zompi

Beatty

Harris

. A mouse model for studying dengue virus pathogenesis and immune response. Ann N Y Acad Sci 2009; 1171 Suppl 1:E12–E23.

26.

Yauch

Shresta

. Mouse models of dengue virus infection and disease. Antiviral Res 2008; 80:87–93.

27.

Johnson

Roehrig

. New mouse model for dengue virus vaccine testing. J Virol 1999; 73:783–786.

28.

Shresta

Sharar

Prigozhin

Beatty

Harris

. Murine model for dengue virus-induced lethal disease with increased vascular permeability. J Virol 2006; 80:10208–10217.

29.

Zellweger

Prestwood

Shresta

. Enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease. Cell Host Microbe 2010; 7:128–139.

30.

Sundaram

Uppin

Dakshinamurthy

Borgahain

. Acute disseminated encephalomyelitis following dengue hemorrhagic fever. Neurol India 2010; 58:599–601.

31.

Gera

George

. Acute disseminating encephalomyelitis with hemorrhage following dengue. Neurol India 2010; 58:595–596.

32.

Tan

Trasti

Schul

Yip

Alonso

. A non mouse-adapted dengue virus strain as a new model of severe dengue infection in AG129 mice. PLoS Negl Trop Dis 2010; 4:e672.

33.

Lee

Pavy

Young

Freeman

Lobigs

. Antiviral effect of the heparan sulfate mimetic, PI-88, against dengue and encephalitic flaviviruses. Antiviral Res 2006; 69:31–38.

34.

Stein

Huang

Silengo

. Treatment of AG129 mice with antisense morpholino oligomers increases survival time following challenge with dengue 2 virus. J Antimicrob Chemother 2008; 62:555–565.

35.

Yin

Chen

Schul

. An adenosine nucleoside inhibitor of dengue virus. Proc Natl Acad Sci U S A 2009; 106:20435–20439.

36.

Halstead

. Neutralization and antibody-dependent enhancement of dengue viruses. Adv Virus Res 2003; 60:421–467.

37.

Dejnirattisai

Jumnainsong

Onsirisakul

. Cross-reacting antibodies enhance dengue virus infection in humans. Science 2010; 328:745–748.

38.

Balsitis

Williams

Lachica

. Lethal antibody enhancement of dengue disease in mice is prevented by Fc modification. PLoS Pathog 2010; 6:e1000790.

39.

Almas

Parkash

Akhter

. Clinical factors associated with mortality in dengue infection at a tertiary care center. Southeast Asian J Trop Med Public Health 2010; 41:333–340.

40.

Greenstone

Spinelli

Tseng

Peacock

Taylor

Laughlin

. NIAID resources for developing new therapies for severe viral infections. Antiviral Res 2008; 78:51–59.

41.

Clyde

Kyle

Harris

. Recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis. J Virol 2006; 80:11418–11431.

42.

Paranjape

Harris

. Control of dengue virus translation and replication. Curr Top Microbiol Immunol 2010; 338:15–34.

43.

Bollati

Alvarez

Assenberg

. Structure and functionality in flavivirus NS-proteins: Perspectives for drug design. Antiviral Res 2010; 87:125–148.

44.

Noble

Chen

Dong

. Strategies for development of dengue virus inhibitors. Antiviral Res 2010; 85:450–462.

45.

Lescar

Luo

. Towards the design of antiviral inhibitors against flaviviruses: The case for the multifunctional NS3 protein from dengue virus as a target. Antiviral Res 2008; 80:94–101.

46.

Rawlinson

Pryor

Wright

Jans

. Dengue virus RNA polymerase NS5: A potential therapeutic target? Curr Drug Targets 2006; 7:1623–1638.

47.

Ashour

Laurent-Rolle

Shi

Garcia-Sastre

. NS5 of dengue virus mediates STAT2 binding and degradation. J Virol 2009; 83:5408–5418.

48.

Malet

Egloff

Selisko

. Crystal structure of the RNA polymerase domain of the West Nile virus non-structural protein 5. J Biol Chem 2007; 282:10678–10689.

49.

Dong

Chang

Xie

. Biochemical and genetic characterization of dengue virus methyltransferase. Virology 2010; 405:568–578.

50.

Egloff

Decroly

Malet

. Structural and functional analysis of methylation and 5'-RNA sequence requirements of short capped RNAs by the methyltransferase domain of dengue virus NS5. J Mol Biol 2007; 372:723–736.

51.

Pryor

Rawlinson

Butcher

. Nuclear localization of dengue virus nonstructural protein 5 through its importin alpha/beta-recognized nuclear localization sequences is integral to viral infection. Traffic 2007; 8:795–807.

52.

Medin

Fitzgerald

Rothman

. Dengue virus nonstructural protein NS5 induces interleukin-8 transcription and secretion. J Virol 2005; 79:11053–11061.

53.

Mazzon

Jones

Davidson

Chain

Jacobs

. Dengue virus NS5 inhibits interferon-alpha signaling by blocking signal transducer and activator of transcription 2 phosphorylation. J Infect Dis 2009; 200:1261–1270.

54.

Laurent-Rolle

Boer

Lubick

. The NS5 protein of the virulent West Nile virus NY99 strain is a potent antagonist of type I interferon-mediated JAK-STAT signaling. J Virol 2010; 84:3503–3515.

55.

Niyomrattanakit

Chen

Dong

. Inhibition of dengue virus polymerase by blocking of the RNA tunnel. J Virol 2010; 84:5678–5686.

56.

Qing

Liu

Yuan

Shi

. A high-throughput assay using dengue-1 virus-like particles for drug discovery. Antiviral Res 2010; 86:163–171.

57.

Masse

Davidson

Ferron

. Dengue virus replicons: Production of an interserotypic chimera and cell lines from different species, and establishment of a cell-based fluorescent assay to screen inhibitors, validated by the evaluation of ribavirin's activity. Antiviral Res 2010; 86:296–305.

58.

Julander

Furuta

Shafer

Sidwell

. Activity of T-1106 in a hamster model of yellow fever virus infection. Antimicrob Agents Chemother 2007; 51:1962–1966.

59.

Julander

Shafer

Smee

Morrey

Furuta

. Activity of T-705 in a hamster model of yellow fever virus infection in comparison with that of a chemically related compound, T-1106. Antimicrob Agents Chemother 2009; 53:202–209.

60.

Morrey

Taro

Siddharthan

. Efficacy of orally administered T-705 pyrazine analog on lethal West Nile virus infection in rodents. Antiviral Res 2008; 80:377–379.

61.

Furuta

Takahashi

Kuno-Maekawa

. Mechanism of action of T-705 against influenza virus. Antimicrob Agents Chemother 2005; 49:981–986.

62.

Gowen

Wong

Jung

. In vitro and in vivo activities of T-705 against arenavirus and bunyavirus infections. Antimicrob Agents Chemother 2007; 51: 3168–3176.

63.

Sidwell

Barnard

Day

. Efficacy of orally administered T-705 on lethal avian influenza A (H5N1) virus infections in mice. Antimicrob Agents Chemother 2007; 51:845–851.

64.

Latour

Jekle

Javanbakht

. Biochemical characterization of the inhibition of the dengue virus RNA polymerase by beta-d-2′-ethynyl-7-deaza-adenosine triphosphate. Antiviral Res 2010; 87:213–222.

65.

Dong

Zhang

Shi

. Flavivirus methyltransferase: A novel antiviral target. Antiviral Res 2008; 80:1–10.

66.

Zhang

Dong

Zhou

Shi

. Genetic interactions among the West Nile virus methyltransferase, the RNA-dependent RNA polymerase, and the 5' stem-loop of genomic RNA. J Virol 2008; 82:7047–7058.

67.

Podvinec

Lim

Schmidt

. Novel inhibitors of dengue virus methyltransferase: Discovery by in vitro-driven virtual screening on a desktop computer grid. J Med Chem 2010; 53:1483–1495.

68.

Qing

Zou

Wang

. Characterization of dengue virus resistance to brequinar in cell culture. Antimicrob Agents Chemother 2010; 54:3686–3695.

69.

Pierra

Amador

Benzaria

. Synthesis and pharmacokinetics of valopicitabine (NM283), an efficient prodrug of the potent anti-HCV agent 2′-C-methylcytidine. J Med Chem 2006; 49:6614–6620.

70.

Julander

Jha

Choi

. Efficacy of 2′-C-methylcytidine against yellow fever virus in cell culture and in a hamster model. Antiviral Res 2010; 86:261–267.

71.

Leyssen

Balzarini

De Clercq

Neyts

. The predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase. J Virol 2005; 79:1943–1947.

72.

Morrey

Smee

Sidwell

Tseng

. Identification of active antiviral compounds against a New York isolate of West Nile virus. Antiviral Res 2002; 55:107–116.

73.

Anderson

Rahal

. Efficacy of interferon alpha-2b and ribavirin against West Nile virus in vitro. Emerg Infect Dis 2002; 8:107–108.

74.

Crance

Scaramozzino

Jouan

Garin

. Interferon, ribavirin, 6-azauridine and glycyrrhizin: Antiviral compounds active against pathogenic flaviviruses. Antiviral Res 2003; 58:73–79.

75.

Julander

Morrey

Blatt

Shafer

Sidwell

. Comparison of the inhibitory effects of interferon alfacon-1 and ribavirin on yellow fever virus infection in a hamster model. Antiviral Res 2007; 73:140–146.

76.

Sbrana

Xiao

Guzman

Travassos da Rosa

Tesh

. Efficacy of post-exposure treatment of yellow fever with ribavirin in a hamster model of the disease. Am J Trop Med Hyg 2004; 71:306–312.

77.

Morrey

Day

Julander

Blatt

Smee

Sidwell

. Effect of interferon-alpha and interferon-inducers on West Nile virus in mouse and hamster animal models. Antivir Chem Chemother 2004; 15:101–109.

78.

Koff

Pratt

Elm

Jr. Venkateshan

Halstead

. Treatment of intracranial dengue virus infections in mice with a lipophilic derivative of ribavirin. Antimicrob Agents Chemother 1983; 24:134–136.

79.

McDowell

Gonzales

Kumarapperuma

Jeselnik

Arterburn

Hanley

. A novel nucleoside analog, 1-β-D-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR), exhibits efficacy against a broad range of flaviviruses in vitro. Antiviral Res 2010; 87:78–80.

80.

Brok

Gluud

. Ribavirin monotherapy for chronic hepatitis C. Cochrane Database Syst Rev 2005; 4: CD005527.

81.

Feld

Hoofnagle

. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 2005; 436:967–972.

82.

Rawlinson

Pryor

Wright

Jans

. CRM1-mediated nuclear export of dengue virus RNA polymerase NS5 modulates interleukin-8 induction and virus production. J Biol Chem 2009; 284:15589–15597.

83.

Green

Rothman

. Immunopathological mechanisms in dengue and dengue hemorrhagic fever. Curr Opin Infect Dis 2006; 19:429–436.

84.

Lei

Yeh

Liu

Lin

Chen

Liu

. Immunopathogenesis of dengue virus infection. J Biomed Sci 2001; 8:377–388.

85.

Pang

Cardosa

Guzman

. Of cascades and perfect storms: The immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (DHF/DSS). Immunol Cell Biol 2007; 85:43–45.

86.

Rothman

. Immunology and immunopathogenesis of dengue disease. Adv Virus Res 2003; 60:397–419.

87.

Chaturvedi

Agarwal

Elbishbishi

Mustafa

. Cytokine cascade in dengue hemorrhagic fever: Implications for pathogenesis. FEMS Immunol Med Microbiol 2000; 28:183–188.

88.

Qing

Yang

Zhang

. Cyclosporine inhibits flavivirus replication through blocking the interaction between host cyclophilins and viral NS5 protein. Antimicrob Agents Chemother 2009; 53:3226–3235.

89.

Neyts

. Selective inhibitors of hepatitis C virus replication. Antiviral Res 2006; 71:363–371.

90.

Steuber

Hilgenfeld

. Recent advances in targeting viral proteases for the discovery of novel antivirals. Curr Top Med Chem 2010; 10:323–345.

91.

Anderson

Schiffer

Lee

Swanstrom

. Viral protease inhibitors. Handb Exp Pharmacol 2009; 189:85–110.

92.

Frecer

Miertus

. Design, structure-based focusing and in silico screening of combinatorial library of peptidomimetic inhibitors of dengue virus NS2B-NS3 protease. J Comput Aided Mol Des 2010; 24:195–212.

93.

Tomlinson

Malmstrom

Russo

Mueller

Pang

Watowich

. Structure-based discovery of dengue virus protease inhibitors. Antiviral Res 2009; 82:110–114.

94.

Salaemae

Junaid

Angsuthanasombat

Katzenmeier

. Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3. J Biomed Sci 2010; 17: 68.

95.

Gao

Cui

Lam

. Synthesis and disulfide bond connectivity–activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B-NS3 protease. Bioorg Med Chem 2010; 18:1331–1336.

96.

Bodenreider

Beer

Keller

. A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease. Anal Biochem 2009; 395:195–204.

97.

Jia

Zou

Fan

Yuan

. Identification of palmatine as an inhibitor of West Nile virus. Arch Virol 2010; 155:1325–1329.

98.

Belon

High

Lin

Pauwels

Frick

. Mechanism and specificity of a symmetrical benzimidazolephenylcarboxamide helicase inhibitor. Biochemistry 2010; 49:1822–1832.

99.

Rajamanonmani

Nkenfou

Clancy

. On a mouse monoclonal antibody that neutralizes all four dengue virus serotypes. J Gen Virol 2009; 90:799–809.

100.

Morrey

Siddharthan

Olsen

. Humanized monoclonal antibody against West Nile virus envelope protein administered after neuronal infection protects against lethal encephalitis in hamsters. J Infect Dis 2006; 194:1300–1308.

101.

Morrey

Siddharthan

Olsen

. Defining limits of humanized neutralizing monoclonal antibody treatment for West Nile virus neurological infection in a hamster model. Antimicrob Agents Chemother 2007; 51:2396–2402.

102.

Oliphant

Engle

Nybakken

. Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 2005; 11:522–530.

103.

Nybakken

Oliphant

Johnson

Burke

Diamond

Fremont

. Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature 2005; 437:764–769.

104.

Brien

Austin

Sukupolvi-Petty

. Genotypespecific neutralization and protection by antibodies against dengue virus type 3. J Virol 2010; 84:10630–10643.

105.

Shrestha

Brien

Sukupolvi-Petty

. The development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1. PLoS Pathog 2010; 6:e1000823.

106.

Wang

Patel

Vangrevelinghe

. A small-molecule dengue virus entry inhibitor. Antimicrob Agents Chemother 2009; 53:1823–1831.

107.

Costin

Jenwitheesuk

Lok

. Structural optimization and de novo design of dengue virus entry inhibitory peptides. PLoS Negl Trop Dis 2010; 4:e721.

108.

Chen

Maguire

Hileman

. Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nat Med 1997; 3:866–871.

109.

Chen

Yeh

Lin

. The envelope glycoprotein domain III of dengue virus type 2 induced the expression of anticoagulant molecules in endothelial cells. Mol Cell Biochem 2010; 342:215–221.

110.

Friebe

Harris

. Interplay of RNA elements in the dengue virus 5′ and 3′ ends required for viral RNA replication. J Virol 2010; 84:6103–6118.

111.

Bai

Wang

Pal

Bao

Gould

Fikrig

. Use of RNA interference to prevent lethal murine west nile virus infection. J Infect Dis 2005; 191:1148–1154.

112.

Kumar

Lee

Shankar

Manjunath

. A single siRNA suppresses fatal encephalitis induced by two different flaviviruses. PLoS Med 2006; 3:e96.

113.

McCown

Diamond

Pekosz

. The utility of siRNA transcripts produced by RNA polymerase i in down regulating viral gene expression and replication of negative- and positive-strand RNA viruses. Virology 2003; 313:514–524.

114.

Phong

. Construction and characterization of a stable subgenomic dengue virus type 2 replicon system for antiviral compound and siRNA testing. Antiviral Res 2007; 76:222–231.

115.

Subramanya

Kim

Abraham

. Targeted delivery of small interfering RNA to human dendritic cells to suppress dengue virus infection and associated proinflammatory cytokine production. J Virol 2010; 84:2490–2501.

116.

Nawtaisong

Keith

Fraser

. Effective suppression of dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome. Virol J 2009; 6: 73.

117.

Kinney

Huang

Rose

. Inhibition of dengue virus serotypes 1 to 4 in Vero cell cultures with morpholino oligomers. J Virol 2005; 79:5116–5128.

118.

Pijlman

Funk

Kondratieva

. A highly structured, nuclease-resistant, noncoding RNA produced by flaviviruses is required for pathogenicity. Cell Host Microbe 2008; 4:579–591.

119.

Alvarez

Filomatori

Gamarnik

. Functional analysis of dengue virus cyclization sequences located at the 5′ and 3′UTRs. Virology 2008; 375:223–235.

120.

Alvarez

Lodeiro

Luduena

Pietrasanta

Gamarnik

. Long-range RNA–RNA interactions circularize the dengue virus genome. J Virol 2005; 79:6631–6643.

121.

Pastorino

Nougairede

Wurtz

Gould

de Lamballerie

. Role of host cell factors in flavivirus infection: Implications for pathogenesis and development of antiviral drugs. Antiviral Res 2010; 87:281–294.

122.

Sayce

Miller

Zitzmann

. Targeting a host process as an antiviral approach against dengue virus. Trends Microbiol 2010; 18:323–330.

123.

Suvarna

Rane

. Serum lipid profile: A predictor of clinical outcome in dengue infection. Trop Med Int Health 2009; 14:576–585.

124.

Rothwell

Lebreton

Young Ng

. Cholesterol biosynthesis modulation regulates dengue viral replication. Virology 2009; 389:8–19.

125.

Rodriguez-Madoz

Bernal-Rubio

Kaminski

Boyd

Fernandez-Sesma

. Dengue virus inhibits the production of type I interferon in primary human dendritic cells. J Virol 2010; 84:4845–4850.

126.

Munoz-Jordan

. Subversion of interferon by dengue virus. Curr Top Microbiol Immunol 2010; 338:35–44.

127.

Jiang

Weidner

Qing

. Identification of five interferon-induced cellular proteins that inhibit West Nile Virus and dengue virus infection. J Virol 2010; 84:8332–8341.

128.

Jiang

Guo

. Identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis C virus. J Virol 2008; 82:1665–1678.

129.

Melian

Plosker

. Interferon alfacon-1: A review of its pharmacology and therapeutic efficacy in the treatment of chronic hepatitis C. Drugs 2001; 61:1661–1691.

130.

Ajariyakhajorn

Mammen

Jr. Endy

. Randomized, placebo-controlled trial of nonpegylated and pegylated forms of recombinant human alpha interferon 2a for suppression of dengue virus viremia in rhesus monkeys. Antimicrob Agents Chemother 2005; 49:4508–4514.

131.

Liu

Woda

Ennis

Libraty

. Dengue virus infection differentially regulates endothelial barrier function over time through type I interferon effects. J Infect Dis 2009; 200:191–201.

132.

Hung

Weng

. Dengue virus type 2 antagonizes IFN-α but not IFN-γ antiviral effect via down-regulating Tyk2-STAT signaling in the human dendritic cell. J Immunol 2005; 174:8163–8172.

133.

Kontny

Kurane

Ennis

. Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells. J Virol 1988; 62:3928–3933.

134.

Sittisombut

Maneekarn

Kanjanahaluethai

Kasinrerk

Viputtikul

Supawadee

. Lack of augmenting effect of interferon-gamma on dengue virus multiplication in human peripheral blood monocytes. J Med Virol 1995; 45:43–49.

135.

Diamond

Roberts

Edgil

Ernst

Harris

. Modulation of dengue virus infection in human cells by alpha, beta, and gamma interferons. J Virol 2000; 74:4957–4966.

136.

Chen

Hofman

Kung

Lin

Wu-Hsieh

. Both virus and tumor necrosis factor alpha are critical for endothelium damage in a mouse model of dengue virus-induced hemorrhage. J Virol 2007; 81:5518–5526.

137.

Kurane

. Dengue hemorrhagic fever with special emphasis on immunopathogenesis. Comp Immunol Microbiol Infect Dis 2007; 30:329–340.

138.

Chen

Lin

Huang

. CLEC5A is critical for dengue-virus-induced lethal disease. Nature 2008; 453:672–676.

139.

Kamau

Takhampunya

. Dengue virus infection promotes translocation of high mobility group box 1 protein from the nucleus to the cytosol in dendritic cells, upregulates cytokine production and modulates virus replication. J Gen Virol 2009; 90:1827–1835.

140.

Ulloa

Messmer

. High-mobility group box 1 (HMGB1) protein: Friend and foe. Cytokine Growth Factor Rev 2006; 17:189–201.

141.

Lee

Leang

Davidson

Lobigs

. Both E protein glycans adversely affect dengue virus infectivity but are beneficial for virion release. J Virol 2010; 84:5171–5180.

142.

Zhang

Mason

Dubovi

. Antiviral activity of geneticin against dengue virus. Antiviral Res 2009; 83:21–27.

143.

Fuchs

Lin

Beasley

. Direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin. Cell Host Microbe 2010; 8:186–195.

144.

Chatterji

Lim

Bobardt

. HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors. J Hepatol 2010; 53:50–56.

145.

Hopkins

Scorneaux

Huang

. SCY-635, a novel nonimmunosuppressive analog of cyclosporine that exhibits potent inhibition of hepatitis C virus RNA replication in vitro. Antimicrob Agents Chemother 2010; 54:660–672.

146.

Paeshuyse

Kaul

De Clercq

. The nonimmunosuppressive cyclosporin DEBIO-025 is a potent inhibitor of hepatitis C virus replication in vitro. Hepatology 2006; 43:761–770.

147.

Chatterji

Bobardt

Selvarajah

. The isomerase active site of cyclophilin A is critical for hepatitis C virus replication. J Biol Chem 2009; 284:16998–17005.

148.

Paranjape

Harris

. Y box-binding protein-1 binds to the dengue virus 3′-untranslated region and mediates antiviral effects. J Biol Chem 2007; 282:30497–30508.

149.

Leng

Chen

Chang

. A recombinant lipoprotein containing an unsaturated fatty acid activates NF-kappaB through the TLR2 signaling pathway and induces a differential gene profile from a synthetic lipopeptide. Mol Immunol 2010; 47:2015–2021.

150.

Tian

Jiang

Gao

. Inhibitory effects of glutathione on dengue virus production. Biochem Biophys Res Commun 2010; 397:420–424.

151.

Neckers

Tatu

. Molecular chaperones in pathogen virulence: Emerging new targets for therapy. Cell Host Microbe 2008; 4:519–527.

152.

Padwad

Mishra

Jain

Chanda

Karan

Ganju

. RNA interference mediated silencing of Hsp60 gene in human monocytic myeloma cell line U937 revealed decreased dengue virus multiplication. Immunobiology 2009; 214:422–429.

153.

Jindadamrongwech

Thepparit

Smith

. Identification of GRP 78 (BiP) as a liver cell expressed receptor element for dengue virus serotype 2. Arch Virol 2004; 149:915–927.

154.

Reyes-Del Valle

Chavez-Salinas

Medina

Del Angel

. Heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells. J Virol 2005; 79:4557–4567.

155.

Anwar

Leong

Chu

Garcia-Blanco

. The polypyrimidine tract-binding protein is required for efficient dengue virus propagation and associates with the viral replication machinery. J Biol Chem 2009; 284:17021–17029.

156.

Kanlaya

Pattanakitsakul

Sinchaikul

Chen

Thongboonkerd

. Ubiquitin–proteasome pathway is important for dengue virus infection in primary human endothelial cells. J Proteome Res 2010; 9:4960–4971.

157.

Modis

Ogata

Clements

Harrison

. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci U S A 2003; 100:6986–6991.

158.

Yap

Chen

. Crystal structure of the dengue virus RNA-dependent RNA polymerase catalytic domain at 1.85-angstrom resolution. J Virol 2007; 81:4753–4765.

159.

Erbel

Schiering

D'Arcy

. Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus. Nat Struct Mol Biol 2006; 13:372–373.

160.

Poh

Yip

Zhang

. A small molecule fusion inhibitor of dengue virus. Antivir Res 2009; 84:260–266.

Important Advances in the Field of Anti-Dengue Virus Research

Abstract

Introduction

Recent advances in animal models of DENV

Therapeutic targets

Viral protein targets

NS5

NS3

Structural proteins

Viral RNA targets

Host cell targets

Cholesterol biosynthesis

Cytokines

Other host cell targets

Conclusions

Footnotes

References