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Associate Editor: P. Foster

Dengue virus therapeutic intervention strategies based on viral, vector and hostfactors involved in disease pathogenesis

Lara J. Herrero a,b,1, Andrew Zakhary b,1, Michelle E. Gahan b, Michelle A.Nelson b, Belinda L.Herring a,Andrew J. Hapel b, Paul A. Keller c, Maheshi Obeysekera a, WeiqiangChen a, Kuo-Ching Sheng a,Adam Taylor a, Stefan Wolfa, Jayaram Bettadapura a,b, Shobha Broor d, Lalit Dar d, Suresh Mahalingam a,b,a Emerging Viruses and Inammation Research Group, Institute for Glycomics, Grifth University, Gold Coast, QLD, 4222, Australiab Virus and Inammation Research Group, Faculty of Applied Science, University of Canberra, Bruce ACT 2601, Australiac Centre for Medicinal Chemistry, School of Chemistry, University of Wollongong, Wollongong, NSW, 2522, Australiad All India Institute of Medical Sciences, New Delhi, India

a b s t r a c ta r t i c l e i n f o

Keywords:

Dengue virus

Immunity

Pathogenesis

Arbovirus

Flavivirus

Disease severity

Dengue virus (DV) is the most widespread arbovirus, being endemic in over 100 countries, and is estimatedto cause 50 million infections annually. Viral factors, such as the genetic composition of the virus strain canplay a role in determining the virus virulence and subsequent clinical disease severity. Virus vector compe-

tence plays an integral role in virus transmission and is a critical factor in determining the severity and im-pact of DV outbreaks. Host genetic variations in immune-related genes, including the human leukocyte

antigen, have also been shown to correlate with clinical disease and thus may play a role in regulating diseaseseverity. The host's immune system, however, appears to be the primary factor in DV pathogenesis with the

delicate interplay of innate and acquired immunity playing a crucial role. Although current research of DVpathogenesis has been limited by the lack of an appropriate animal model, the development of DV therapeu-

tics has been a primary focus of research groups around the world. In the past decade advances in both thedevelopment of vaccines and anti-virals have increased in dramatically. This review summarises the currentunderstanding of viral, vector and host factors which contribute to dengue virus pathogenesis and how this

knowledge is critically important in the development of pharmaceutical interventions. 2012 Published by Elsevier Inc.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2672. Dengue disease and pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

3. Dengue viral factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2674. Dengue virus vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

5. Host factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2676. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

Pharmacology & Therapeutics 137 (2013) 266282

Abbreviations: (A), Aedes spp.; (ADE), Antibody dependent enhancement; (Clu), clusterin; (DC), dendritic cell; (DC-SIGN), dendritic cell-specic intracellular adhesion

molecule-3-grabbing non-integrin; (DENV-1-4), Dengue virus serotype 14; (DF), Dengue fever; (DHF), Dengue haemorrhagic fever; (DSS), Dengue shock syndrome; (DV), Dengue

virus; (E), Envelope; (HLA), human leukocyte antigen; (IFN), Interferon; (IL), Interleukin; (LTA), lymphotoxin-alpha; (MBL), mannose binding lectin; (MCP), Macrophage

chemoattractant protein; (MHC), major histocompatibility complex; (NK), Natural killer cell; (NS), non-structural protein; (PAIgG), platelet associated IgG; (PBMC), peripheral

blood mononuclear cells; (PrM), Pre-membrane; (STAT2), signal transducer and activator of transcription 2; (TAP), transporters associated with antigen processing; (TNF),

tumor necrosis factor; (UTR), untranslated region.

Corresponding author at: Emerging Viruses and Inammation Research Group, Institute of Glycomics, Grifth University, 4222, QLD, Australia. Tel.: +61 7 5552 7178; fax: +617 5552 8098.

E-mail address:[email protected](S. Mahalingam).1 Authors contributed equally.

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0163-7258/$ see front matter 2012 Published by Elsevier Inc.

http://dx.doi.org/10.1016/j.pharmthera.2012.10.007

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1. Introduction

Dengue virus (DV), a member of the genus Flavivirus, is a vector-transmitted virus with four circulating serotypes (DENV-1, DENV-2,

DENV-3 and DENV-4), each of which can be transmitted to humansby infected Aedes spp. mosquitoes. The World Health Organisationestimates that DV is endemic in over 100 countries with 50 million

cases occurring annually worldwide (WHO, 2009).

DV can cause three distinct forms of disease in humans: denguefever (DF), dengue haemorrhagic fever (DHF) and dengue shock syn-drome (DSS). DF is a self-limiting febrile illness that is rarely fatal but

produces a variety of non-specic symptoms. DHF is a more seriousillness, with symptoms including severe fever, liver enlargement,plasma leakage, haemorrhage and thrombocytopenia. DHF can fur-ther progress to the potentially lethal DSS if plasma leakage is pro-found. There are no vaccines or anti-viral treatments currently

available for DV infections. To date, the best method of DV treatmentis intervention strategies to limit virus spread, coupled with intrave-nousuid replacement therapy in cases of DSS.

Decades of research have implicated various elements of both the

innate and adaptive host immune response in the pathogenesis of DVdisease. More recently, other host factors, such as genetic polymor-phisms in infected populations, as well as viral factors, such as geno-type, have also been correlated with the different disease prolesobserved in patients. Understanding the role of vector, virus and

host factors in DV pathogenesis will provide valuable insights intothe mechanisms of DV infection and could lead to novel avenues fortherapeutic intervention. This review discusses the current under-standing of DV pathogenesis and will explore how this knowledge

can be used in the development of therapeutics to target the variousstages of DV disease.

2. Dengue disease and pathogenesis

DV can affect people of all ages including infants, young children,adults and the elderly causing a spectrum of illnesses ranging from

DF to the most severe forms of DHF and DSS. Signs and symptoms in-

clude fever, retro-orbital pain, severe headache, myalgia, arthralgiaand minor hemorrhagic manifestations such as petechiae, epitaxisand gingival bleeding (Gubler, 1998). In serious DHF and DSS cases

patients present with signs of plasma leakage, haemorrhage andthrombocytopenia. The onset of micro-vascular plasma leakage andhemorrhagic manifestations is one of the life-threatening complica-tions in DV-infection, however, the pathogenic mechanisms are not

well understood.As humans are the only natural host of DV infection, recapitulating

DV disease in an in-vivo model has been a challenge and therefore in-sights into the pathogenesis of DV disease has been limited (Simmons

et al., 2012). Numerous models including both small (e.g. mice, rats,rabbits) and large animal models (e.g. dogs, pigs, monkeys) havebeen used as potential dengue models, each with their limitations

(Zompi & Harris, 2012). Even with the genetic proximity of non-human primates to humans researchers have been unable to developan overt infection as found in humans (Lavinder & Francis, 1914;Rosen, 1958; Yauch & Shresta, 2008). Currently mouse models show

the most promise and both immunodecient and immunocompro-mised mice have been studied but have been shown to developedclinical manifestations that have only selective similarity to humaninfection (An et al., 1999; Bente et al., 2005; Shresta et al., 2006;

Balsitis et al., 2009; Williams et al., 2009 ).

3. Dengue viral factors

The primary element that determines the potential for a virus tocause disease comes from a combination of viral factors including

the viral proteins, virion structure, virus genotype and virus serotype.

Elucidating the complexity of DV biology is essential for the develop-ment of anti-virals, which may target one of the many viral factors, orfor the design of potential vaccines.

3.1. Dengue virus proteins

DV is a positive strand RNA virus with an 11 kb genome coding forthree structural (C, E and prM) and seven non-structural (NS)

proteins and two untranslated regions (UTR) (Fig. 1). The UTRs areprimarily responsible for the regulation of translation and genome

replication. The role of the UTRs in dengue disease remains uncertainwith no denitive correlation between sequence or secondary struc-ture and disease severity. The roles of the structural and NS proteinsin dengue disease have been extensively studied and will be

discussed further below.

3.1.1. Structural proteins

The envelope (E) and precursor membrane (prM) proteins aremajor structural-protein targets of the antibody response in dengueinfection (Rothman, 2011) The E glycoprotein is a major surface pro-

tein of the virus and is responsible for attachment and entry. E proteinis made up of three domains, DI, DII and DIII. DI contains the central

region, DII is the site of dimerisation and is involved in membrane fu-sion and DIII has an immunoglobulin domain most likely important

for receptor binding. In the host, antibody responses are directed pri-marily against the E protein. While antibodies directed against DI andDII of the E protein are cross-reactive among DV serotypes and other

aviviruses (Crill & Chang, 2004; Lisova et al., 2007), antibodies di-

rected against DIII are neutralising and serotype specic (Crill &Chang, 2004). Cross reactivity of non-neutralizing antibodies contrib-ute to higher DHF/DSS upon secondary infection with a heterologousserotype (Lai et al., 2008).

Antibodies against the prM protein are also highly serotype specif-ic; however in the mature virion the prM protein is completely inac-cessible to antibodies, suggesting that anti-prM antibodies are onlyrelevant to the immature and partially mature virus particle (Fig. 1).

As such, the importance of anti-prM antibodies mounting an immuneresponse against DV infection has not been fully evaluated. Recently,it has been shown that both E protein-specic and prM protein-specic antibodies can mediate antibody dependent enhancement

(ADE), indicating that anti-prM antibodies may play a greater rolein DV disease than rst hypothesized (Rothman, 2011). The ADE phe-nomenon will be discussed in detail in Section 5.4.3. The identica-tion of immuno-dominant DV proteins is of particular relevance in

vaccine studies and several studies have focused on using structuralproteins as targets by combining protective epitopes of all four sero-types in a chimeric envelope (Guy & Almond, 2008; Webster et al.,

2009).

3.1.2. Non-structural proteins

DV non-structural protein 1 (NS1) is thought to be involved in thedevelopment of vascular leakage in severe DV disease. Host-derivedantibodies directed against NS1 have been shown to cross-reactwith endothelial cells and induce inammatory cellular activation

resulting in endothelial cell damage (Lin et al., 2005). Additionally,soluble NS1 (sNS1) has been shown to interact with the complementinhibitory factor clusterin (Clu) (Kurosu et al., 2007). Clu inhibits theterminal pathway of the complement system and the interaction of

Clu with sNS1 is thought to decrease Clu levels and ultimately con-tribute to complement activation, resulting in the vascular leakageseen in DHF (Kurosu et al., 2007). Recently it has been demonstratedthat an anti-NS1 antibody can recognise both the epitope on NS1 and

an endothelial cell antigen; human lysine-rich CEACAM1 (LYRIC) (Liuet al., 2011). This recognition might play a role in the pathogenesis of

DHF/DSS.

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Further involvement of NS1 in disease pathogenesis can be in-ferred from the interaction between sNS1 and host antibodies. Thisinteraction has been observed to result in activation of the SC5b-9

complement complex, which is hypothesised to contribute to vascu-lar leakage (Avirutnan et al., 2006). Soluble levels of NS1 have alsobeen identied as an indicator of severe DV disease, with DHF pa-tients exhibiting higher plasma levels of NS1 than DF patients

(Libraty et al., 2002).Pathogenic strains of DV are resistant to the anti-viral effects of

type I interferons (IFNs) (Munoz-Jordan et al., 2003). Research into

the non-structural proteins has identied NS4B, and possibly NS2Aand NS4A, as potential IFN antagonists. Expression of these specicproteins was shown to down-regulate IFN--stimulated gene expres-sion with NS4B strongly blocking the IFN-induced signal-transduction

cascade by interfering with signal transducer and activator of tran-scription 1 (STAT1) function (Munoz-Jordan et al., 2003). Furtheranalysis has shown that DV reduces the level of signal transducerand activator of transcription 2 (STAT2), a key component of the

type I IFN signalling pathway, possibly by inhibiting the Tyk2 proteinkinase (Jones et al., 2005). NS5 protein was shown to specically in-duce interleukin-8 (IL-8) expression and secretion, suggesting that

virus-induced chemokine production may contribute to the inam-matory response in dengue disease (Medin et al., 2005). In additionit has been shown that NS5 protein alone inhibits IFN- and notIFN-signaling (Mazzon et al., 2009). Further studies are needed tofully characterise the role that DV non-structural proteins play in vas-

cular leakage during severe DV disease.

3.2. Dengue virus serotype

All four dengue serotypes circulate globally and a relationshipbetween disease severity and DV serotype has been suggested. Thepossible link between dengue serotype and disease severity has

been extensively investigated through epidemiological and clinicalstudies however a conclusive association remains unsubstantiated.

Thai studies examining DV infected individuals (1999

2002)

demonstrated that DENV-1 and DENV-3 caused DHF in primary infec-tions, whilst DENV-2 and 4 were only associated with DHF in second-ary infection, conrming the results of (Nisalak et al., 2003;

Anantapreecha et al., 2005). A study where four features of severeclinical disease; plasma leakage, shock, marked thrombocytopeniaand internal haemorrhage were evaluated with respect to the circu-lating dengue serotype of either DENV-1 or 2, found that when

DENV-1 was the dominant serotype more plasma leakage was ob-served whereas when DENV-2 predominated shock and internalhemorrhage were observed (Balmaseda et al., 2006). Additionally,

plasma viraemia was higher in patients infected with DENV-1 thanthat observed in patients infected with either DENV-2 or DENV-3and that the viremic period lasted longer (Tricou et al., 2011).

In contrast to these studies, DENV-2 has been identied as con-

tributing to more severe dengue clinical presentations in primaryDV infection in Thai children (Vaughn et al., 2000) and a prospectiveobservational study reported signs of severe clinical disease, includ-ing DHF, ascites and larger pleural effusions associated with DENV-2

serotype (Fried et al., 2010). It should be noted though that more se-vere clinical manifestations such as DHF occur more frequently in pa-tients undergoing a subsequent secondary DV infection with a

different serotype (Vaughn et al., 2000).Further research on the different DV serotypes and their associa-

tion with both clinical manifestations and disease outcome is criticalfor the design of therapeutic interventions.

3.3. Dengue virus genotype

Within each of the four antigenic groups there is genetic variation,

which delineates a small number of genotypes within each of the se-rotypes. Variation in the severity of DV disease has raised the possibil-ity that disease severity may be linked to a particular viral serotypeand/or genotype since different serotypes/genotypes circulate in dif-

ferent regions of the world. Temporal variation in the distribution ofserotypes/genotypes may also play a role in the differing pattern of

disease severity.

Fig. 1.The structural and genomic features of dengue virus (DV). The dengue virion contains a positive single-stranded genomic RNA, which is encapsulated within the nucleocap-

sid and enveloped in a glycoprotein-embedded lipid bilayer. The genomic RNA comprises of a single open reading frame encoding 10 viral proteins: three structural proteins

(C, prM and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Immature dengue virions have a spiky glycoprotein shell with 60prM-Envelope arranged in icosahedral protrusions. Matured dengue virions have a smooth glycoprotein shell with 90 Envelope protein dimers arranged in a herringbone pattern.

C, capsid; E, envelope; prM, pre membrane; NS, non-structural; ss-RNA, single stranded ribonucleic acid.

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In South America, the endemic American DENV-2 genotype, whichcaused a milder, less severe disease (no DHF) in resident populationswas displaced by the introduction of the Southeast Asian DENV-2 geno-type resulting in the emergence of DHF in the region (Rico-Hesse et al.,

1997; Watts et al., 1999). Analysis of these two DENV-2 variants usingchimeric clones showed enhanced viral replication of the SoutheastAsian genotype in target human monocyte and dendritic cells during

dengue infection (Rico-Hesse et al., 1997; Cologna & Rico-Hesse, 2003).

Furthermore, a correlation has been identi

ed between the level ofviraemia in humans infected with DV, due to enhanced replication ofvirus, and the associated disease severity (Cologna & Rico-Hesse, 2003).

Sequence analysis of complete DV genomes has provided insightsinto the molecular epidemiology of DV genotypes globally. For example,in Vietnam sequence analysis identied a temporal replacement of theAsian/American DENV-2 genotype by the Asian I DENV-2 genotype(Vuet al., 2010). When considering thetness of these viruses both ge-

notypes demonstrated similar levels of infectivity in mosquitoes, how-ever, higher plasma viraemia levels were found in paediatric patientsinfected with Asian I genotype compared to those infected with theAsian/American genotype (Vu et al., 2010).

Broad genome comparisons of some DENV-2 genotypes have identi-

ed variations that may relate to disease severity (Leitmeyer et al.,1999). Sequence polymorphisms identied in the gene encoding theDV E protein (amino acid 390), nucleotides 68 to 80 of the 5untranslated region (UTR) and the 300 nucleotides upstream of the 3UTR have been implicated in disease severity (Leitmeyer et al., 1999).Molecular analysis of DENV-2 isolates from Thailand showed no segre-gation between genotypes causing DF and those causing DHF(Leitmeyer et al., 1999).

Other studies have also reported sequence changes that correlateto an increase in disease severity. In a DENV-2 outbreak in Santiago,Cuba (1997) an intraepidemic increase in disease severity over timewas reported. It was observed that individuals who acquired disease

late in the epidemic were infected with a variant that contained a sin-gle amino acid change (T164S) in the NS1 protein. It was postulatedthat this mutation contributed to increased viral replication and/orsurvival efciency, thereby increasing the overall tness of the virus

which may have contributed to the more severe disease observed inthe latter part of the epidemic (Rodriguez-Roche et al., 2011). Phylo-genetic analysis of DENV-2 strains circulating in Bangkok in 2010 also

identied two different DENV-2 virus clusters with a single mutationH/Y346 delineating each cluster (Puiprom et al., 2011).

A preliminary study on DV from patients with varying disease se-verities (DF, DHF and DSS) in Cambodia (2007) identied a correla-

tion between disease severity and virus genotype and phenotypiccharacteristics. Isolates from patients with DSS contained six uniqueamino acid changes, had a lower level of replication in mammaliancells and caused extensive apoptosis in mosquito cells in vitro when

compared to isolates from patients with DF and DHF (Tuiskunen etal., 2011).

Currently there is limited information on the association between

disease severity and DENV genotype. Changes in the genetic structureof DENV-2 and the corresponding potential increase in transmissionmay impact on the development of vaccines and anti-viral drugs.Overall, identifying genetic markers within the DV genome that maybe responsible for increased virulence is critical for designing viable

therapeutic interventions.

3.4. Dengue virus and anti-viral developments

The medicinal chemistry of therapeutics against DV has recently beencomprehensively reviewed (Stevens et al., 2009). It was shown that drugdevelopment is still in its infancy and this can be seen as a result of inad-

equate rodent models, which are not capable of mimicking the humanphysiological response to dengue infection (Schul et al., 2007) and its

molecular biology. However, recent elucidation of the molecular

mechanisms behind the dengue replication cycle including the specicfunctions of each of the DV proteins has led to the identication of newanti-viral targets. In addition, with the help of in silico screening tech-niques and ligand-based design strategies, new leads and an increasing

number of novel compounds for the treatment of dengue are emerging(Tomlinson et al., 2009). Different proteins for potential therapeutic in-tervention are beginning to emerge (Fig. 2) and targets currently beinginvestigated include viral entry, viral RNA polymerase/methyltransferase,

nucleotide synthesis, viral serine protease, -glucosidases and kinases.

3.4.1. Inhibition of viral entry

The inhibition of the virus entering the host cells is the rst point

of intervention. It has been shown, that in the early stage of infection,the viral E protein binds to a highly sulfated glycosaminoglycan(GAG), heparan sulfate, on the host cell membrane ( Chen et al.,1997). Monoclonal antibodies and heparin have been able to inhibitthe binding and penetration of dengue virus (Hung et al., 1999).

These ndings led to the development of heparan mimetics, whichwere able to inhibit the binding between E protein and immobilizedheparin in vitro (Marks et al., 2001).

Cinnamic acid derivatives are one of the more studied classes of

compounds and show modest to high binding properties with IC50values of up to 46 M. Interestingly, the compounds generate higherbinding between the virus and the host cell, which disrupts the viral

entry and leads to inhibition of the virus (Rees et al., 2008).Another attempt to inhibit viral entry is to target a small binding

pocket on the E protein, which locks this fusion peptide into anonbinding conformation, thus identifying a binding pocket on theenzyme. The National Cancer Institute (NCI) screened 142,000 com-

pounds in silico into this pocket; with hits showing activity in the mi-cromolar range (Zhou et al., 2008).

3.4.2. Inhibition of viral RNA polymerase/methyltransferase

The broad anti-viral agent Ribavirin is known for its inhibitorycharacteristics against a variety of DNA and RNA viruses (Sidwell etal., 1972). However, only weak activity against DV has been reported

(Gabrielsen et al., 1992). Nevertheless it has been shown, that Ribavi-

rin inhibits the methyltransferase activity of NS5 and binds to theguanosine 5-triphosphate (GTP)/RNA binding cap (Benarroch et al.,2004). These ndings led to the rational drug design of new com-

pounds for binding with these residues, improving the potency upto a 100-fold while conserving their broad anti-viral activity (DeClercq et al., 1991). Another nucleoside analogue with inhibitory ac-tivity against DV has also been shown to impede NS5B, which is re-

sponsible for RNA-dependent RNA polymerase activity (Olsen et al.,2004). Decreased viraemia and cytokine production was shown in amurine dengue model for this compound (Schul et al., 2007).

3.4.3. Inhibition of nucleotide synthesis

De novo guanosine production for the synthesis of DNA, RNA orglycoproteins is highly dependent on the enzyme inosine mono-

phosphate dehydrogenase (IMPDH) (Allison & Eugui, 2000). A clini-cally used immunosuppressant, mycophenolic acid, is known toinhibit IMPDH and was capable of lowering cellular GTP levels(Leyssen et al., 2005). This compound has shown potent anti-viral

activity in sub-immunosuppressive doses (Diamond et al., 2002).Another target of nucleotide synthesis is the enzyme orotidine

monophosphate decarboxylase (ODCase), which plays a key role inthe pyrimidine biosynthesis (Miller et al., 1998). Inhibition of ODCase

causes a depletion of intracellular pyrimidine, which results in the in-hibition of viral replication. Potent inhibitory activity on DV in vitrohas been demonstrated for ODCase inhibiters (Crance et al., 2003).

3.4.4. Inhibition of serine protease

The serine protease of NS3 is responsible for the proteolytic pro-

cessing of the polyprotein after translation. This makes the serine


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protease an ideal drug target, as inhibition of the enzyme would pre-vent viral replication (Chanprapaph et al., 2005). Several studies haveinvestigated different peptidomimetics to inhibit the viral serine pro-tease, out of which one has been patented (Chanprapaph et al., 2005;Yin et al., 2006a, 2006b).

The BowmannBirk inhibitors (BBIs) are natural protease inhibi-tors that are part of defense mechanisms in plants. Analyses of thecrystal structure of DV NS3 protease in complex with mung beanBBI demonstrated the efcacy of BBIs and offered a starting point

for further drug design investigations (Murthy et al., 2000).

3.4.5. Inhibition of-glucosidase I

-Glucosidase enzymes are responsible for the correct biosynthe-

sis and processing of asparagine-linked oligosaccharides (Hettkampet al., 1984). Furthermore they are crucial in protein folding mecha-nisms (Helenius, 1994). Inhibitors of this enzyme have been able toinhibit dengue viral budding and viral particle infectivity by

disturbing the folding pathways of prM and E proteins. The natural al-kaloid Castanospermine, derived from the black bean chestnut tree(Castanospermum australae), is an inhibitor of various murine disac-charidases (Pan et al., 1993). It showed potent activity against all

four serotypes of DV both in vivo and in vitro and increased the

time of survival in lethal DV challenge (Whitby et al., 2005).

3.4.6. Kinase inhibitors

Phosphorylation by protein kinases is signicant in the signaltransduction responsible for improving cell survival during viral in-fection (Lee et al., 2005; Chang et al., 2006), immune evasion(Munoz-Jordan et al., 2003; Ho et al., 2005) and regulation of endocy-

tosis in some viruses (Pelkmans et al., 2005). In addition, phosphory-lation regulates the location of the NS5 and most likely its activity(Kapoor et al., 1995; Forwood et al., 1999). The Src family of kinaseshas been hypothesized to have an important role in DV replication

while known Src kinase inhibitors demonstrated inhibition of DV invitro (Chu & Yang, 2007). The c-Scr subfamily of Src kinases was iden-tied to be crucial in the budding of DV from the ER lumen ( Chu &

Yang, 2007). Two Src inhibitors, dasatinib and AZD0530, have beendemonstrated to inhibit the virion assembly of dengue in all four se-rotypes, hence the protein kinases can be considered as a valuabletarget for Dengue anti-virals (Chu & Yang, 2007).

3.4.7. Antisense oligonucleotides

The dissection of DV via RNA interference has enabled the identi-

cation of putative anti-viral targets. Approaches with antisense oli-

gonucleotide compounds have been able to alter gene expression ofaviviruses, which enabled some of these compounds to advanceinto clinical trials (Ma et al., 2000; Stein & Shi, 2008). A group ofthese antisense compounds are the phosphorodiamidate morpholino

oligomers (PMOs), which have been shown to suppress the magni-tude and duration of replication of all four types of DV in vitro. Fur-

thermore peptide conjugated PMOs have been capable of anti-viral

DENV-2 activity in an AG129 mouse model, where it prolonged thesurvival times of DENV-2 infected mice (Stein et al., 2008).

3.4.8. Compounds with an unknown target

There are examples of compounds, which are extremely potentagainst DV in vitro in low nanomolar concentrations (Gudmundsson,

2006). However, the target is currently unknown. An example of sucha compound is geneticin, which has been demonstrated to inhibitviral replication of DV in vitro, protecting against the cytopathic effect

of DV, reducing the viral yield and blocking DV RNAand protein synthe-sis (Zhang et al., 2009).

4. Dengue virus vectors

All four DV serotypes are transmitted to humans by infected Aedesspp. mosquitoes, principally by Aedes aegypti, an anthropophilic mos-quito.A. aegyptican be found over a large geographical area, predomi-

nantly in tropical and subtropical regions between latitudes 35N and35S. AlthoughA. aegypti is the principle vector for DV, transmissionin outbreaks has also been attributed to other members of the Aedesspp. includingA. albopictus,A. polynesiensis,A. mediovittatusand mem-bers of the A. scutellaris complex. In recent decades A. albopictus has

spread from Asia to Africa, the Americas and Europe, thereby theoreti-cally expanding the geographical range for future DV epidemics. How-

ever, each mosquito species has a particular ecology, behaviour,geographical distribution and competence (vector competence) forDV and each of thesefactors can inuence the role of a particular speciesin the transmission and spread of DV disease. Vector competence de-

scribes the ability of a mosquito to acquire, maintain and transmit apathogen. Dissemination of virus throughout the mosquito, followingthe initial midgut infection, in particular to the salivary glands, is essen-tial for transmission of arboviral infections.

Studies on vector competence ofA. albopictus andA. aegypti for DVinCentral Africa demonstrated that A. aegypti populations in West andCentral Africa were less susceptible to DV thanA. aegypti populationsin other parts of the world (Paupy et al., 2010). Additionally, results

froma recent study indicated that althoughA. albopictus is moresuscep-tible to DV midgut infection, the virus disseminates poorly within thisspecies, when compared to A. aegypti, suggesting A. albopictus is notthe major player in the transmission of DV (Lambrechts et al., 2010).

When examining the dissemination of DENV-2 within mosquitopopulations it was found that the more virulent Southeast AsianDENV-2genotypespreadmore efciently throughA. aegypti populationscompared to the American DENV-2 genotype (Cologna et al., 2005). This

data suggests that the Southeast Asian genotype has a replicative advan-tage in being able to infect a larger proportion of the mosquito popula-tion, enabling more extensive dissemination of the Southeast AsianDENV-2 virus compared to the American genotype (Cologna et al.,

2005). This may also explain the replacement of less virulent DV geno-types with the more virulent genotypes as seen in South America

(Rico-Hesse et al., 1997; Watts et al., 1999). Additionally, a recent

Fig. 2.Different proteins for potential therapeutic intervention and targets currently being investigated. Adapted with modication fromStevens et al., 2009.

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study suggested that differences in mosquito transmission abilities areprobably determined by specic viral tness elements (Cox et al., 2011).

Understanding mosquito vectors and virus/vector interactions iscritical for the establishment of methods for controlling DV outbreaks

by interrupting transmission through vector control. A strategytargeting elimination or suppression of target mosquito populationsin areas with endemic DV is commonly used as a means of controlling

the spread of dengue disease in many countries. Genetic elimination

of the vector by introducing dominant lethal mutations into mosquitopopulations and control of breeding by the release of transgenic ster-ile males are promising strategies being investigated for vector con-

trol (Harris et al., 2011; Wise de Valdez et al., 2011).Vector control for the prevention of transmission of dengue stands

as the only option for reducing the impact of dengue on humanpopulations. Control strategies currently employed include commu-nity based behaviour programs, chemical treatment including the

spraying of larvicides, adulticides and the use of insecticide treatedmaterials (e.g. curtains and bed nets), the release of sterile and trans-genic mosquitoes and biological methods.

Vector control using education and/or behavioural programs re-

quires the involvement of individuals, communities and governmentand therefore its widespread success has been limited. Similarly, al-though the use of insecticides/larvicides has been relatively success-ful, there are a number of drawbacks associated with insecticides/larvicides as a means of vector control, namely; insecticide resistance,

the transient nature and limited dissemination (spraying seldomreaches indoor environments) of spraying and differing habitats of

A. aegypti.Biological control strategies that are currently used include the use

of larvivoroussh and insects, copepoids (Mesocyclopsspp.) andBacil-lus thuringiensis(produces toxin with larvicidal activity) in water con-tainers (Caragata & Walker, 2012). Generally these methods havebeen shown to be sustainable and effective as they specically target

mosquito/larval populations, do not adversely affect the environmentand are well accepted in communities (Ballenger-Browning & Elder,2009)

More recently entomopathogenic fungi such asBeauveria bassiana

and the endosymbiotic bacteria Wolbachiaspp. have been exploitedas a means of vector control as these organisms can reduce thelifespan of vectors to within the extrinsic period for viral replication

and/or confer a pathogen resistance phenotype (Iturbe-Ormaetxe etal., 2011; Caragata & Walker, 2012). B. bassiana,which can be trans-mitted through mating, has been shown to be effective against bothadult A. aegypti (B. bassiana conidiospores) and larva (B. bassianablastospores) and adversely affect A aegypti competence for denguevirus (Dong et al., 2012).

Wolbachiaspp. is an intracellular bacteria that can be maternallyinherited through successive generations. Strains of Wolbachia have

been shown to reduce the lifespan of transinfected Aedesspp. by 1050%, greatly reducing the capacity of adult female mosquitoes to trans-mit dengue and reducing the vector competence ofAedesspp. to den-

gue virus. The release of Wolbachia infected mosquitoes in the eldhasbeen trailed, howeverthe long-term successof this method of vec-tor control depends greatly on the persistence of the Wolbachia strainin the mosquito population and the pathogen interference phenotype(Iturbe-Ormaetxe et al., 2011; Caragata & Walker, 2012).

5. Host factors

In an outbreak of any pathogen, including DV infection, a widerange of clinical presentations is generally observed. Host factors,such as host genotype and the complexity of the immune responsecan all contribute to disease presentation and severity. Correlating

disease outcomes with host factors has major implications for both

the development of safe anti-virals and efcacious vaccines.

5.1. Host genotype

Recently, a growing body of research has investigated correlationsbetween certain host genetic factors and the clinical prole of DV in-

duced disease (Table 1). The presence of the wild-type mannose-binding lectin (MBL) gene, MBL2, has been strongly correlated withincreased susceptibility to thrombocytopenia, a common feature of

DV infection. Thrombocytopenia is thought to be due to the comple-

ment and binding roles of MBL which promote activation of the com-plement cascade and ultimately, platelet lysis (Acioli-Santos et al.,2008). Individuals that possess the tumor necrosis factor alpha(TNF-) TNF--308A gene also have an increased chance of develop-

ing DHF compared to DF, a fact that may contribute to the increasedTNF-levels seen in DHF patients (Fernandez-Mestre et al., 2004).

A study of the genetic polymorphisms present in the TNF andlymphotoxin-alpha (LTA) genes of DV patients with varying disease

severities found that TNF-238 polymorphism (marking TNF-4.LTA-3haplotype) occurred with higher incidence in patients with second-ary DHF compared to patients with secondary DF. Additionally, theLTA-3 phenotype was associated with in vivo production of TNF andLTA during the acute vireamic phase of infection (Vejbaesya et al.,

2009).Gene polymorphisms in transporters associated with antigen pro-

cessing (TAP) have also been correlated with susceptibility to denguedisease and disease severity. TAP, a member of the ATP-binding cas-

sette transporter family, delivers cytosolic peptides to major histo-compatibility complex (MHC) class 1 molecules in the endoplasmicreticulum. A heterozygous pattern at TAP1 333 (ILE/VAL), TAP2 379(ILE/VAL) and TAP2 665 (THR/ALA) have all been associated with sus-

ceptibility to DHF (Soundravally & Hoti, 2007, 2008a). Conversely, ho-mozygous alleles at TAP1 333 (ILE/ILE), TAP2 379 (VAL/VAL) andTAP1 637 (ASP/ASP) have been identied as conferring protectionagainst DHF and DSS (Soundravally & Hoti, 2008b).

The dendritic cell-specic intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) promoter variant, DC-SIGN1-336,in the CD209 gene has been associated with an increased risk of de-veloping DHF. Genotypes GG and GA in DC-SIGN1-336 are strongly

associated with the development of DHF, as opposed to DF patientswhere these genotypes were found to be rare (Sakuntabhai et al.,2005). It was hypothesized that the promoter variant was responsible

for determining the disease prole of dengue infection, which sug-gests the pathogenic mechanisms of DF and DHF may differ substan-tially (Sakuntabhai et al., 2005).

A strong correlation has been identied between human leukocyte

antigen (HLA) alleles and severity of DV induced disease. HLA genesare associated with immunity and are specically involved in the func-tion of T-lymphocytes. Studies have shown that HLA-A*0203 andHLA-B*52 are associated with the less severe DF, while HLA-A*0207,

HLA-B*51 and HLA-A*24 are associated with the development of DHFin secondary infections (Stephens et al., 2002; Nguyen et al., 2008). Ina study of DV infected individuals in Cuba, a strong correlation was ob-

served between HLA-B*15 and individuals with DF and betweenHLA-A*31 and individuals with DHF (Sierra et al., 2007a, b). Additional-ly, the HLA class II allotypes HLA-DRB1*07 and HLA-DRB1*04 have beenmostfrequently detected in uninfected individuals, alluding to a protec-tive role in the development of dengue disease (Sierra et al., 2007a, b).

Host phenotype has also been explored as a possible determinantof dengue disease susceptibility and severity. Observational studies,such as those conducted in Haiti, the Caribbean and Africa havesuggested that populations of negroid origin may be less susceptible

to DHF/DSS than Caucasian populations since the incidence of DHFand DSS was lower in these populations (Kouri et al., 1989;Halstead, 2001; Sierra et al., 2006, 2007). The genetic reasons behindthis observation are the focus of ongoing research.

Studies into differences in immune responses to DV between eth-

nic groups may enable the mechanisms by which certain ethnicities


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appear to have a greater immunity to severe dengue disease to be elu-

cidated. Differences between ethnic groups, have been reported inthe polymorphic variability of FcRIIa which has been associatedwith a higher risk of developing DHF in Caucasian populations(Marcheco et al., 2010).

By increasing our understanding of genetic factors inuencingsusceptibility to DV disease mechanisms could be established to per-mit targeted use of therapeutic or prophylactic pharmaceuticals. Al-

though considerable research has been focused on the inuence ofhost genetics on DV severity, further studies are needed as host ge-netics may inuence the success or failure of both DV vaccine andanti-viral development.

5.2. Immune response to dengue infection

5.2.1. Cross-talk between innate and adaptive immune response

The host innate immune response is the front line of defenceagainst pathogen invasion and its components are closely related tothe inammatory response. The cellular arm of the innate immune re-sponse is comprised of leukocytes such as natural killer cells (NK),

mast cells and phagocytic cells, including monocytes, neutrophils,macrophages and dendritic cells (DC) (Navarro-Sanchez et al.,

2005). Infectious DV is thought to enter target cells such as DCs and

macrophages either via non-classical or classical endocytosis,

depending on the host's cell types and DV strains (Acosta et al.,2009). In the case of the classical endocytic pathway, receptor-bound DV enters cells via clathrin-coated vesicles and fusion withthe endosomal membrane results in viral RNA release into the cyto-

plasm for replication and translation (Martn et al., 2009; Nayak etal., 2009).

It is widely believed that the innate immune response may orches-

trate disease outcome in DV infection.A major component of theinnateimmune system is the induction of type I alpha, beta (,) and type IIgamma () interferons (IFN) by DCs and NK cells respectively (Clydeet al.,2006; Rodenhuis-Zybert et al., 2010). Theproduction of IFNs inre-sponse to DV infection represents an early host defence that limits viral

replication and enhances clearance (Navarro-Sanchez et al., 2005). Be-sides, the production of IFNs as an innate anti-viral response can alsotrigger host adaptive immune response via DC maturation as well as Band T-lymphocytes activation (Erickson & Gale, 2008; Rodenhuis-

Zybert et al., 2010).The host adaptive immunity plays a pivotal role in the immune

response to DV by providing antibody-mediated protection againstDV E-protein as well as acute viral clearance by T-lymphocytes

(Murphy & Whitehead, 2011). Ironically, the host adaptive immune

response can, at times contribute to more severe form of the disease.

Table 1

Genes and alleles that correlate with dengue disease severity.

Gene/Allele Disease correlation Reference

Allelic phenotypes

HLA-DRB1*07 Uninfected individuals (Sierra, et al., 2007a)

HLA-DRB1*04Uninfected individuals,

less severe disease

(Sierra, et al., 2007a;

Soundravally & Hoti,

2007, 2008a, 2008b)

HLA-B*15 Infection with DV (Sierra, et al., 2007)

HLA-A*0203

HLA-B*52Milder disease (DF) (Nguyen, et al., 2008)

HLA-A*0207

HLA-B*51

HLA-A*24

HLA-A*31

More severe disease (DHF)

(Nguyen, et al., 2008;

Sierra, et al., 2007a;

Stephens, et al., 2002)

Gene polymorphisms

TAP1 333 (ILE/ILE)

TAP2 379 (VAL/VAL)

TAP1 637 (ASP/ASP)

Protection against severe disease



2007, 2008a, 2008b)

MBL2 Increased susceptibility to thrombocytopenia (Acioli-Santos, et al., 2008)

TNF--308A Increased chance of developing DHF

(Fernandez-Mestre,

Gendzekhadze, Rivas-

Vetencourt, & Layrisse,

2004)

TAP1 333 (ILE/VAL)

TAP2 379 (ILE/VAL)

TAP2 665 (THR/ALA)

Susceptibility to DHF



2007, 2008a, 2008b)

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antibodies show high cross-reactivity between serotypes as well as in-complete neutralization of DV-infection, even at high concentrations.

Instead these antibodies have been shown to be potent promoters ofADE infection. It has been suggested that the partial cleavage that prMundergoes on the viral surface during viral maturation may explainthe inability of anti-prM antibodies to neutralize the virus, thus

resulting in viral enhancement. This has major implications for the de-velopment of safe vaccines, which must be rigorously tested and haveno potential to cause ADE. Overall, the disease enhancement is an im-portant factor in the dengue pathogenesis mosaic, providing a basis

for host immune mechanisms to propagate severe disease symptoms,and may explain why secondary heterotypic dengue infections are as-sociated with the development of DHF and DSS.

The existence of ADE poses additional difculties for researchersstriving to develop DV vaccines and therapeutics, however, there hasbeen some progress achieved so far. An articial antibody variant(E60-N297Q) that cannot bind to FcR has been genetically engineeredand has been shown to have prophylactic and therapeutic efciency

against ADE induced lethal challenge (Balsitis et al., 2010). In anotherrecent study using monoclonal antibodies generated from memory Bcells of DV infected individuals,Beltramello et al. (2010)demonstratedthat antibodies engineered to prevent FcR binding neutralized DV in-

fection in vitro and in vivo in a mouse model of lethal DV infection(Beltramello et al., 2010).

5.3.4. Host antibody response and vaccine development

An ideal vaccine against dengue should produce a life-long protective

immune response with neutralizing antibodies that are equally effective

against all the four serotypes of DV, without development of sub-neutralizing antibodies that may induce ADE. This is one of the major

challenges in the development of an effective dengue vaccine, the otherbeing an incomplete understanding of the immunopathogenesis of den-gue disease, and possibly the reasons why no dengue vaccine is licensedas yet, however, tremendous progress has been made in the last few

years. Various live (attenuated, chimeric and live virus vector) andnon-living (inactivated, recombinant subunit and naked DNA) denguevaccines have been developed and a number of monovalent and sometetravalent dengue vaccines have even completed phase I and II clinical

trials.A live attenuated tetravalent DV vaccine, has undergone phase I

and II clinical trials in populations in the United States (Edelman et

al., 2003; Sun et al., 2009). In Thailand, trials have been conductedin avivirus-nave children (Simasathien et al., 2008) and infants(Watanaveeradej et al., 2011). In the latter phase I/II trial, after thesecond dose, 85.7% of full-dose DV vaccinees developed neutralizingantibodies to b3 DENV serotypes and 53.6% to all 4 serotypes.

Recombinant live chimeric monovalent DENV vaccine candidatestrains, ChimeriVax DENV1 to DENV4 (CVD14), were developed forall dengue serotypes by Acambis (now acquired by Sano Pasteur).In these, the prM and E genes of the well established yellow fever

live attenuated vaccine strain virus 17D (YFV 17D) have been re-placed with the corresponding genes from the respective DV sero-types. Initially, monovalent chimeras were tested individually forsafety and immunogenicity (Kanesa-thasan et al., 2001). Various

tetravalent formulations were then evaluated in multiple doses,

with results showing even the lowest dose causing fever and rash

Fig. 4.Mechanisms of immune evasion by dengue virus (DV) and pre-existing antibody complex. Two mechanisms of DV-antibody (Ab) dependent enhancement involving an in-

hibition of the interferon (IFN)-mediated anti-viral pathway by DV-Ab complexes interacting with Fc receptors (FcR) have been proposed.A: DV-Ab binds to FcR and activates DAK

and Atg5-Atg12, which act as negative regulators, disrupting the RIG-I/MDA-5 cascade and consequently results in the suppression of type I IFN induction as well as the

interferon-mediated anti-viral pathway.B: The interaction of DV-Ab with FcR induces the expression of IL-10, possibly through the Sp1 transcription factor, which at high concen-

trations is responsible for the activation of SOCS3 gene. The expression of SOCS3 inactivates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathwayand inhibits the IFN-mediated anti-viral pathway. Atg5-Atg12, autophagy-related 5-autophagy-related 12; DAK, dihydroxyacetone kinase; IFNAR1, interferon alpha/beta receptor

1; IFNAR2, interferon alpha/beta receptor 2; IL-10, interleukin 10; IRF, interferon regulatory factors; ISGF3, interferon-stimulated gene factor 3; ISGs, interferon-stimulated genes;

ISRE, interferon-stimulated response element; MDA-5; melanoma differentiation associated gene 5; RIG-I, retinoic acid inducible gene I; SOCS3, suppressor of cytokine signalling 3;Sp-1, specicity protein 1; TYK2, tyrosine kinase 2.

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(Sabchareon et al., 2002). Subsequent improved tetravalent formula-tions of this vaccine have led to the requisite low-level viraemia and

induced seroconversion to all four serotypes, without any serious ad-verse effects in avivirus naive populations (Kanesa-thasan et al.,

2001; Morrison et al., 2010; Poo et al., 2011; Sabchareon et al.,2012). Based on the results of phase I and II clinical trials, a phase III

trial of ChimeraVax tetravalent dengue vaccine was initiated in Octo-ber 2010 and is ongoing (Guy et al., 2011).

Another chimeric vaccine has been developed by partly replacingthe 3 stem and loop structure of the DENV-2 PDK-53 strain with a

part of the West Nile virus genome. The Center for Disease Control(CDC), along with their commercial partners (e.g. Inviragen) has pro-duced this chimeric vaccine candidate, DENVax, using an infectiouscDNA clone as the backbone, and inserting DENV-1, -3 and -4 struc-

tural genes to create a tetravalent vaccine. The DENVax vaccine hasshown immunogenic promise in mice and non-human primates(Osorio et al., 2011) and is now being prepared for human clinicaltrials.

Using an alternative virus as a vector for the expression of DV pro-teins in vivo has been trialed as a novel vaccine approach. Vectorstrialed include; adenovirus (Raviprakash et al., 2008), Venezuelanequine encephalitis virus (VEE) (Chen et al., 2007; White et al.,

2007) and live-attenuated measles virus Schwarz vaccine strain(Brandler et al., 2007). While some of these vaccine strains haveshown promise in non-human primates, to date no human clinical tri-als have commenced.

Vaccine development using recombinant subunit approach hasbeen trialed by several groups with ranging success (Simmons et al.,2001; Konishi & Fujii, 2002; Robert Putnak et al., 2005; Imoto &Konishi, 2007; Chiang et al., 2011; Coller et al., 2011 ). Recently, low

doses of the DENV-2 component of the truncated 80% E (r80E)subunit tetravalent vaccine (Hawaii Biotech, Inc., USA), has been

shown to induce complete protection in mice and non-human

primates and is now undergoing human phase I trials (Clements etal., 2010).

DNA vaccines have the advantage of in vivo antigen expression,proper protein folding and post-translation modications, as well as

the potential to induce good cytotoxic immune responses. Early stud-ies assessed the safety and immunogenicity of monovalent DV DNA

vaccine plasmid constructs in mice (Kochel et al., 1997). Subsequent-ly, chimeric DNA vaccines have been prepared by DNA shufing andscreening, to express epitopes of the truncated E antigen that areshared by all four DV serotypes. Recently, the rst phase I trial for a

prototype dengue DNA construct containing DENV-1 prM and Egenes found that T lymphocyte and neutralizing antibody responseswere mounted for a proportion of subjects in a avivirus-nave popu-lation (Beckett et al., 2011). Since DNA vaccines usually induce low

levels of neutralizing antibodies, various intrinsic adjuvants are alsobeing evaluated by incorporating these in the DNA constructs.

With various vaccine candidates now available, some nearing orhaving reached phase III clinical trials, it is increasingly important

for these to be evaluated in endemic areas for safety, immunogenicityand sustained protection from all the four DV serotypes. It is crucialthat once available, the vaccines should become accessible at a rea-sonable cost in these high-risk areas, which lie mostly in developing

countries (seeTable 2for a list of vaccines in preclinical or clinicaltrials).

5.4. Host factors as potential therapeutic targets in dengue infection

5.4.1. DC-specic intracellular adhesion

molecule 3-grabbing nonintegrin (DC-SIGN)

Calciumdependent lectins,also known as C-type lectins are believed

to facilitate internalisation of DV entryas well as escapinghost immuneresponses (van Kooyk & Geijtenbeek, 2003; Lozach et al., 2005). A

well-studied C-type lectin is the DC-specic intracellular adhesion

Table 2

Dengue vaccines currently in clinical trials.

Vaccine type Candidate

designation

Developer/Commercial partner Clinical trial

phase

Reference

Live

chimeric

ChimeriVax Sanofi Pasteur III (Guy, Almond,

& Lang, 2011)

DENVax Inviragen/CDC II (Osorio, et al.,

2011)

Live

attenuated

LAV GlaxoSmithKline/Walter Reed Army

Institute of Research

II Edelman, 2007;

Edelman, et al.,

2003; Sun, et

al., 2003)

TV vaccine

formulations

NIH/NIAID/LID,

Butantan, Biological E, Panacea

Biotec and Vabiotech

I/II (Durbin, et al.,

2011)

Live

recombinant,DNA &

subunit

D1ME-VR-P Naval Medical Research Centre I (Beckett, et al.,

2011)

DEN-80E Merck (Hawaii Biotech) I (Clements, et

al., 2010)

Table modied fromColler and Clements, 2011current opinion in immunology (Coller and Clements, 2011) and Murrell et al., 2011 Biotechnology advances (Murrell et al., 2011)

(Durbin et al., 2011; Edelman, 2007; Sun et al., 2003 ).

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molecule 3-grabbing nonintegrin (DC-SIGN; CD209), which is mainlyexpressed by immature DCs (Tassaneetrithep et al., 2003). It is nowgenerally accepted that immature DCs are one of therst cells that in-teract with DV at sites of infection (Wu et al., 2000; Alen et al., 2011).

Emerging evidence suggests that DC-SIGN can bind to various patho-gens such as human immunodeciency virus (HIV) (Cunningham etal., 2007), hepatitis C virus (HCV) (Ludwig et al., 2004), bacteria as

well as yeasts (Appelmelk et al., 2003). In vitro, DC-SIGN has been

shown to bind to the highly glycosylated envelope of DV, regardless ofserotype, and infection is signicantly reduced in the presence ofanti-DC-SIGN antibodies (Navarro-Sanchez et al., 2003; Alen et al.,

2011; Hidari & Suzuki, 2011).The unique contribution of DC-SIGN in DV infection as a viral

binding receptor is further highlighted by the ability to enhance in-fection and viral transmission in permissive cells. DC-SIGN signallingcan be manipulated by DV, which in turn leads to immune evasion

and viral dissemination to secondary lymphoid organs, suggestingthat they play an instrumental role during DV infection (Alen et al.,2009). It is also believed that DV target DC-SIGN to shift Th balancetoward Th2 or T-regulatory responses, which are favourable for viral

persistence in the host (Pulendran, 2004; Mabalirajan et al., 2005).Furthermore, it is believed that genetic variation in DC-SIGN can dic-tate dengue disease severity, where 336A/G polymorphism inDC-SIGN results in weakened promoter activity and reduced risk ofsevere disease (Sakuntabhai et al., 2005).

One of the most effective strategies to control viral infection is toprevent attachment during the early stages of infection. Indeed,carbohydrate-binding agents (CBAs) have been shown to prevent vi-ruses such as HIV and HCV from attaching to DC-SIGN+ cells byinteracting with the viral glycoproteins (Balzarini et al., 2005;Bertaux et al., 2007). With respect to DV, an in vitro study hasshown that CBAs can efciently prevent DC-SIGN-mediated infectionin Raji/DC-SIGN+ cells (Alen et al., 2009). Recently,Alen et al. (2011)

also reported that primary human monocyte-derived DC (MDDC)treated with plant-derived CBAs such as Hippeastrum hybrid (HHA)can exert signicantly higher anti-DV activity than in Raji/DC-SIGN+

cells. This suggests that CBAs can block the early stages of DV infec-

tion by interfering with the interaction between DV E-glycoproteinand DC-SIGN. Together with their low toxicity in animals and highstability, CBAs can be used as potential therapeutic targets in DV in-

fection (Balzarini et al., 2004).

5.4.2. Mannose receptors (MR)

Besides DCs, macrophages are important in dengue disease both asprimary target and as a source of immunomodulatory cytokines (Chen& Wang, 2002; Wong et al., 2012). Recently, Miller et al. (2008) hasshown that mannose receptor (MR; CD206) is a putative receptor for

DV infection in macrophages. MR is a C-type lectin constitutivelyinternalised into early endosomes and recycled back to cell surface. Al-though both MR and DC-SIGN are C-type lectins, they recognise differ-

ent mannose moieties (Engering et al., 2002). While DC-SIGN isknown to act as an attachment receptor in DV infection, MR is thoughtto function as an internalisation receptor (Navarro-Sanchez et al., 2003;Tassaneetrithep et al., 2003; Miller et al., 2008).

Interestingly, MR has been shown to play a role in DV pathogenesis.

Von Willebrand factor (VWF), a large glycoprotein responsible for me-diating platelet adhesion can be cleaved by a plasma metalloproteinase,ADAMTS13 (Zheng et al., 2001; Zhou et al., 2010). A deciency inADAMTS13 promotes microvascular obstruction and results in throm-

bocytopenia (Zhou et al., 2010; Matsumoto et al., 2012). In a recentstudy, patients with severe dengue show reduced levels of ADAMTS13activity and high circulating levels of VWF (Rossi et al., 2010;Djamiatun et al., 2012). Furthermore,Sorvillo et al. (2012) demonstrat-

ed that MR can facilitate the endocytosis of ADAMTS13 and subsequent

antigen processing by DCs. This suggests that MR can contribute to

severe complications of dengue and may be a potential therapeutic tar-get in dengue infection.

5.4.3. C-type lectin-like domain family 5, member A (CLEC5A)

The C-Type Lectin-Like Domain Family 5, Member A (CLEC5A), alsoknown as myeloid DAP12-associating lectin (MDL-1) is a type II trans-membrane protein expressed by both monocytes and macrophages(Bakker et al., 1999; Ingersoll et al., 2010; Watson et al., 2011). Recently,

Chen et al. (2008)demonstrated that CLEC5A can interact with DV di-rectly and leads to DAP12 phosphorylation. In contrast to DC-SIGN andMR, the author showed that the interaction did not result in viral entrybut stimulated the release of pro-inammatory cytokines in macro-phages. The blockade of CLEC5A and DV interaction suppressed the re-

lease of TNF-, IL-6, IL8, macrophage inammatory protein (MIP)-1and interferon-inducible protein (IP)-10 but not IFN-, which furthersupport the role of CLEC5A as a pro-inammatory cytokine signalling re-ceptor (Chen et al., 2008). In a more recent study,Cheung et al. (2011)

observed the inltration of CLEC5A+ immature myeloid cells after con-canavalin (ConA)-induced liver injury in mice. CLEC5A activation inthese inltrates using DV leads to an increase in the levels of shock me-

diators such as nitric oxide (NO) and TNF-, followed by lethal shock inmice. These ndings suggested that CLEC5A signalling may play a role inthe development of severe shock, where the progression to severe den-gue disease is closely associated with liver damage.

Notably, signal transducers and activators of transcription (STAT)-1decient mice infected with DV showed amelioration of vascular leak-age, reduced levels of inammatory mediators and lethality after treat-ment with anti-CLEC5A monoclonal antibodies, compared with non-

infected controls (Chen et al., 2008). Small low-molecular-weightinhibitors targeting CLEC5A can be difcult to develop. Hence, the useof humanised antibodies against CLEC5A may possibly prevent DV-induced manifestations and attenuate excessive pro-inammatory

responses, without affecting viral clearance by the host's adaptiveimmune response (Noble et al., 2010).

5.4.4. Macrophage-migration inhibitory factor (MIF)

Macrophage-migration inhibitory factor (MIF) is a pro-inammatory

mediator that playsan important role in the modulation of inammation.MIFis known to be constitutively expressedby many different cells and is

released in response to a variety of stimuli including cytokines, patho-gens, immune complexes and glucocorticoids (Calandra et al., 1995;Calandra & Roger, 2003; Paiva et al., 2009). Once released, MIF can stim-ulate monocytes to secrete other pro-inammatory factors such as

TNF-, acting as a potent monocyte/macrophage chemoattractant, andcounteracting the anti-inammatory effects of glucocorticoids (Calandraet al., 1995; Donnelly & Bucala, 1997; Gregory et al., 2006; Bernhagen etal., 2007).

The serum level of MIFin dengue patients has been shownto be pos-itively correlated with disease severity and fatality (Chen et al., 2006;Assuno-Miranda et al., 2010). Assuno-Miranda et al. (2010)showed that DV-infected MIF knockout (Mif/) mice exhibit signi-

cantly reduced viral load, thrombocytopenia and inammation as wellas lethality compared with wild-type (WT) mice. However, the viralloads were similar to that of WT mice at later time points. Interestingly,the blockade of MIF production has been shown to have no effect on

hepatitis B virus (HBV) replication but can reduce liver injury (Kimuraet al., 2006). Similarly, MIF blockade can reduce pro-inammatory me-diators such as TNF- and IL-6 without affecting viral replication(Assuno-Miranda et al., 2010). This indicates that MIF can regulate

pro-inammatory cytokines at a transcriptional level and is secretedprior to the amplication of host inammatory responses as observedin severe dengue. Recently, it has been shown that MIF can enhancevascular permeability in both DV-infected endothelial cell line and

mice (Chuang et al., 2011). In addition, the author showed that cellstreated with MIF inhibitor ISO-1 or inhibition of MIF receptor CD74

can recover from tight junction protein ZO-1 disarray induced by MIF.


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Altogether, these results support MIF as a crucial player in the im-mune response to DV infection and provide direct evidence for MIF inDV pathogenesis. Therefore, through the blockade of MIF using its in-hibitors or neutralising antibodies, it may open up new avenues for

therapeutic interventions to DV infection.

5.4.5. Platelet-activating factor receptor (PAFR)

The platelet-activating factor (PAF) is a potent phospholipid media-

tor well-known for the induction of platelet aggregation, in

ammationand cytokine production. PAF can be produced by numerous cell types,especially leukocytes (Garcia et al., 2010). Theeffects of PAFare mediat-

ed through a specic PAF receptor (PAFR) which is distributed on theplasma membrane or the nucleus surfaces of platelets, leukocytes andendothelium (Montrucchio et al., 2000; Marrache et al., 2002).

In a previous study,Yang et al. (1995)have shown that macrophagesfrom previously DV-1 infected patients can produce a signicantly higher

amount of PAF compared with healthy controls. When administered sys-tematicallyto animalsor humans,PAF can mimic several systemicinam-matory responses commonly observed in the context of severe denguedisease, including elevated vascular permeability, thrombocytopenia,

haemoconcentration, hypotension and shock (Ishii & Shimizu, 2000; Xuet al., 2010). Recently, the role of PAFR in DV pathogenesis has been elu-cidated in animal models (Souza et al., 2009). The author demonstratedthat DV infection and clinical signs were signicantly less severe inPAFR/ mice as compared with WT mice. Notably, treatment withPAFR antagonist such as UK-74,505 and PCA-4246 was shown to furtherinhibit dengue manifestations including vascular permeability changes,hypotension and decreased TNF- secretion as well as lethality. The

drug treatment was effective even at peak clinical signs (day vepost-infection). In contrast, IFN- was signicantly increased in bothPAFR/ and PAFR antagonist-treated mice, suggesting that PAFR hasno effect on host ability to control DV infection (Souza et al., 2009).

In vivo, these studies have provided strong evidence for the in-volvement of PAFR in DV pathogenesis and blocking PAFR hasshown an amelioration of dengue disease. Similarly, patients com-monly seek medical advice from pain between day three and ve

post-DV infection and treatment with PAFR antagonists may prevent

development to severe dengue disease (Binh et al., 2009; Biswas etal., 2012). With a good drug safety prole and blocking efcacy inhumans, the use of PAFR antagonists in human as a therapeutic inter-

vention for dengue may be a feasible option (Kuitert et al., 1995;Garcia et al., 2010; Fagundes et al., 2011).

6. Conclusion

The research presented in this review suggests that the host'simmune response is a signicant factor in dengue disease severity andinduction of symptoms. Viral factors also play a role, particularly in

explaining differences in virulence and geographic distribution. An un-derstanding of vector competence will help devise strategies for con-trolling dengue outbreaks. The exact mechanisms by which DV causes

the wide range of observed symptoms are not fully understood. Recentdevelopment of animal models for DV will help in understanding themechanism of disease pathogenesis in a complete immunological sys-tem. A greater understanding of the precise mechanisms of DV patho-

genesis and disease could accelerate the development of effectiveanti-dengue vaccines and anti-viral pharmaceuticals.

Acknowledgments

SM is grateful for the funding support from the Australian ResearchCouncil (ARC)(Grant # LP0775507). AZ is the recipientof the ARCPost-graduate Award Industry (APAI) scholarship. SM is the recipient of the

ARC Future Fellowship (# FT0991272).Conict of interest

The authors declare that there are no conicts of interest.

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