1 s2.0 S016372581200232X Main

Associate Editor: P.
Foster
Dengue virus therapeutic intervention strategies based on viral, vector and host
factors 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
, Weiqiang Chen
a
, Kuo-Ching Sheng
a
,
Adam Taylor
a
, Stefan Wolf
a
, 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, Australia
b
Virus and Inammation Research Group, Faculty of Applied Science, University of Canberra, Bruce ACT 2601, Australia
c
Centre for Medicinal Chemistry, School of Chemistry, University of Wollongong, Wollongong, NSW, 2522, Australia
d
All India Institute of Medical Sciences, New Delhi, India
a b s t r a c t a 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 estimated
to cause 50 million infections annually. Viral factors, such as the genetic composition of the virus strain can
play 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 disease
severity. 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 DV
pathogenesis 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 the
development of vaccines and anti-virals have increased in dramatically. This review summarises the current
understanding 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
2. Dengue disease and pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
3. Dengue viral factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
4. Dengue virus vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
5. Host factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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: +61
7 5552 8098.
E-mail address: s.mahalingam@grifth.edu.au (S. Mahalingam).
1
Authors contributed equally.
270
271
278
278
278
0163-7258/$ see front matter 2012 Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.pharmthera.2012.10.007
Contents lists available at SciVerse ScienceDirect
Pharmacology & Therapeutics
j our nal homepage: www. el sevi er . com/ l ocat e/ phar mt her a
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 humans
by infected Aedes spp. mosquitoes. The World Health Organisation
estimates 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: dengue
fever (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 serious
illness, 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 treatment
is intervention strategies to limit virus spread, coupled with intrave-
nous uid 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 DV
disease. 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 proles
observed in patients. Understanding the role of vector, virus and
host factors in DV pathogenesis will provide valuable insights into
the mechanisms of DV infection and could lead to novel avenues for
therapeutic 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 various
stages 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, arthralgia
and minor hemorrhagic manifestations such as petechiae, epitaxis
and gingival bleeding (Gubler, 1998). In serious DHF and DSS cases
patients present with signs of plasma leakage, haemorrhage and
thrombocytopenia. The onset of micro-vascular plasma leakage and
hemorrhagic 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) have
been 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 develop
an 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 developed
clinical manifestations that have only selective similarity to human
infection (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 to
cause 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, or
for the design of potential vaccines.
3.1. Dengue virus proteins
DV is a positive strand RNA virus with an 11 kb genome coding for
three structural (C, E and prM) and seven non-structural (NS)
proteins and two untranslated regions (UTR) (Fig. 1). The UTRs are
primarily responsible for the regulation of translation and genome
replication. The role of the UTRs in dengue disease remains uncertain
with no denitive correlation between sequence or secondary struc-
ture and disease severity. The roles of the structural and NS proteins
in 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 are
major structural-protein targets of the antibody response in dengue
infection (Rothman, 2011) The E glycoprotein is a major surface pro-
tein of the virus and is responsible for attachment and entry. E protein
is 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 and
DII 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 heterologous
serotype (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 only
relevant to the immature and partially mature virus particle (Fig. 1).
As such, the importance of anti-prM antibodies mounting an immune
response 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 role
in 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 structural
proteins 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 the
development of vascular leakage in severe DV disease. Host-derived
antibodies directed against NS1 have been shown to cross-react
with 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 complement
inhibitory factor clusterin (Clu) (Kurosu et al., 2007). Clu inhibits the
terminal 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 leakage
seen in DHF (Kurosu et al., 2007). Recently it has been demonstrated
that an anti-NS1 antibody can recognise both the epitope on NS1 and
an endothelial cell antigen; human lysine-rich CEACAM1 (LYRIC) (Liu
et al., 2011). This recognition might play a role in the pathogenesis of
DHF/DSS.
267 L.J. Herrero et al. / Pharmacology & Therapeutics 137 (2013) 266282
Further involvement of NS1 in disease pathogenesis can be in-
ferred from the interaction between sNS1 and host antibodies. This
interaction 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 also
been 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 NS2A
and NS4A, as potential IFN antagonists. Expression of these specic
proteins 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). Further
analysis has shown that DV reduces the level of signal transducer
and activator of transcription 2 (STAT2), a key component of the
type I IFN signalling pathway, possibly by inhibiting the Tyk2 protein
kinase (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 addition
it has been shown that NS5 protein alone inhibits IFN- and not
IFN- signaling (Mazzon et al., 2009). Further studies are needed to
fully 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 relationship
between disease severity and DV serotype has been suggested. The
possible link between dengue serotype and disease severity has
been extensively investigated through epidemiological and clinical
studies however a conclusive association remains unsubstantiated.
Thai studies examining DV infected individuals (19992002)
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 severe
clinical disease; plasma leakage, shock, marked thrombocytopenia
and 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 internal
hemorrhage were observed (Balmaseda et al., 2006). Additionally,
plasma viraemia was higher in patients infected with DENV-1 than
that observed in patients infected with either DENV-2 or DENV-3
and 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 primary
DV infection in Thai children (Vaughn et al., 2000) and a prospective
observational 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 critical
for 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 serotype
and/or genotype since different serotypes/genotypes circulate in dif-
ferent regions of the world. Temporal variation in the distribution of
serotypes/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 60
prM-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.
In South America, the endemic American DENV-2 genotype, which
caused a milder, less severe disease (no DHF) in resident populations
was 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 using
chimeric clones showed enhanced viral replication of the Southeast
Asian 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 identied between the level of
viraemia in humans infected with DV, due to enhanced replication of
virus, and the associated disease severity (Cologna & Rico-Hesse, 2003).
Sequence analysis of complete DV genomes has provided insights
into the molecular epidemiology of DVgenotypes globally. For example,
in Vietnam sequence analysis identied a temporal replacement of the
Asian/American DENV-2 genotype by the Asian I DENV-2 genotype
(Vu et al., 2010). When considering the tness of these viruses both ge-
notypes demonstrated similar levels of infectivity in mosquitoes, how-
ever, higher plasma viraemia levels were found in paediatric patients
infected with Asian I genotype compared to those infected with the
Asian/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 the
DV E protein (amino acid 390), nucleotides 68 to 80 of the 5
untranslated region (UTR) and the 300 nucleotides upstream of the 3
UTR 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 correlate
to an increase in disease severity. In a DENV-2 outbreak in Santiago,
Cuba (1997) an intraepidemic increase in disease severity over time
was 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 postulated
that this mutation contributed to increased viral replication and/or
survival efciency, thereby increasing the overall tness of the virus
which may have contributed to the more severe disease observed in
the 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 mutation
H/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 phenotypic
characteristics. Isolates from patients with DSS contained six unique
amino acid changes, had a lower level of replication in mammalian
cells and caused extensive apoptosis in mosquito cells in vitro when
compared to isolates from patients with DF and DHF (Tuiskunen et
al., 2011).
Currently there is limited information on the association between
disease severity and DENV genotype. Changes in the genetic structure
of DENV-2 and the corresponding potential increase in transmission
may impact on the development of vaccines and anti-viral drugs.
Overall, identifying genetic markers within the DV genome that may
be 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 been
comprehensively reviewed (Stevens et al., 2009). It was shown that drug
development 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 human
physiological 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 specic
functions of each of the DV proteins has led to the identication of new
anti-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 being
investigated include viral entry, viral RNApolymerase/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 inhibit
the binding and penetration of dengue virus (Hung et al., 1999).
These ndings led to the development of heparan mimetics, which
were able to inhibit the binding between E protein and immobilized
heparin 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 IC
50
values of up to 46 M. Interestingly, the compounds generate higher
binding 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 a
nonbinding conformation, thus identifying a binding pocket on the
enzyme. 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 inhibitory
characteristics against a variety of DNA and RNA viruses (Sidwell et
al., 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 the
guanosine 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 up
to a 100-fold while conserving their broad anti-viral activity (De
Clercq 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 a
murine 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 or
glycoproteins is highly dependent on the enzyme inosine mono-
phosphate dehydrogenase (IMPDH) (Allison & Eugui, 2000). A clini-
cally used immunosuppressant, mycophenolic acid, is known to
inhibit 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 in
the 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 vitro
has 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
protease an ideal drug target, as inhibition of the enzyme would pre-
vent viral replication (Chanprapaph et al., 2005). Several studies have
investigated 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 the
crystal structure of DV NS3 protease in complex with mung bean
BBI 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 (Hettkamp
et al., 1984). Furthermore they are crucial in protein folding mecha-
nisms (Helenius, 1994). Inhibitors of this enzyme have been able to
inhibit dengue viral budding and viral particle infectivity by
disturbing the folding pathways of prMand 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 signal
transduction 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 kinases
has been hypothesized to have an important role in DV replication
while known Src kinase inhibitors demonstrated inhibition of DV in
vitro (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 been
demonstrated to inhibit the virion assembly of dengue in all four se-
rotypes, hence the protein kinases can be considered as a valuable
target 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 of
aviviruses, which enabled some of these compounds to advance
into clinical trials (Ma et al., 2000; Stein & Shi, 2008). A group of
these 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 the
survival 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 potent
against DV in vitro in low nanomolar concentrations (Gudmundsson,
2006). However, the target is currently unknown. An example of such
a compound is geneticin, which has been demonstrated to inhibit
viral 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 Aedes
spp. mosquitoes, principally by Aedes aegypti, an anthropophilic mos-
quito. A. aegypti can be found over a large geographical area, predomi-
nantly in tropical and subtropical regions between latitudes 35N and
35S. Although A. aegypti is the principle vector for DV, transmission
in outbreaks has also been attributed to other members of the Aedes
spp. including A. albopictus, A. polynesiensis, A. mediovittatus and 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) for
DVand eachof these factors can inuence the role of a particular species
in the transmission and spread of DV disease. Vector competence de-
scribes the ability of a mosquito to acquire, maintain and transmit a
pathogen. Dissemination of virus throughout the mosquito, following
the initial midgut infection, in particular to the salivary glands, is essen-
tial for transmission of arboviral infections.
Studies on vector competence of A. albopictus and A. aegypti for DVin
Central Africa demonstrated that A. aegypti populations in West and
Central Africa were less susceptible to DV than A. aegypti populations
in other parts of the world (Paupy et al., 2010). Additionally, results
froma recent study indicatedthat althoughA. albopictus is more suscep-
tible to DV midgut infection, the virus disseminates poorly within this
species, when compared to A. aegypti, suggesting A. albopictus is not
the major player in the transmission of DV (Lambrechts et al., 2010).
When examining the dissemination of DENV-2 within mosquito
populations it was found that the more virulent Southeast Asian
DENV-2 genotype spreadmore efciently throughA. aegypti populations
compared 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 Asian
DENV-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 from Stevens et al., 2009.
study suggested that differences in mosquito transmission abilities are
probably determined by specic viral tness elements (Cox et al., 2011).
Understanding mosquito vectors and virus/vector interactions is
critical for the establishment of methods for controlling DV outbreaks
by interrupting transmission through vector control. A strategy
targeting elimination or suppression of target mosquito populations
in 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 mosquito
populations 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 human
populations. Control strategies currently employed include commu-
nity based behaviour programs, chemical treatment including the
spraying of larvicides, adulticides and the use of insecticide treated
materials (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 government
and 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 seldom
reaches indoor environments) of spraying and differing habitats of
A. aegypti.
Biological control strategies that are currently used include the use
of larvivorous sh and insects, copepoids (Mesocyclops spp.) and Bacil-
lus thuringiensis (produces toxin with larvicidal activity) in water con-
tainers (Caragata & Walker, 2012). Generally these methods have
been shown to be sustainable and effective as they specically target
mosquito/larval populations, do not adversely affect the environment
and are well accepted in communities (Ballenger-Browning & Elder,
2009)
More recently entomopathogenic fungi such as Beauveria bassiana
and the endosymbiotic bacteria Wolbachia spp. have been exploited
as a means of vector control as these organisms can reduce the
lifespan of vectors to within the extrinsic period for viral replication
and/or confer a pathogen resistance phenotype (Iturbe-Ormaetxe et
al., 2011; Caragata & Walker, 2012). B. bassiana, which can be trans-
mitted through mating, has been shown to be effective against both
adult A. aegypti (B. bassiana conidiospores) and larva (B. bassiana
blastospores) and adversely affect A aegypti competence for dengue
virus (Dong et al., 2012).
Wolbachia spp. is an intracellular bacteria that can be maternally
inherited through successive generations. Strains of Wolbachia have
been shown to reduce the lifespan of transinfected Aedes spp. by 10
50%, greatly reducing the capacity of adult female mosquitoes to trans-
mit dengue and reducing the vector competence of Aedes spp. to den-
gue virus. The release of Wolbachia infected mosquitoes in the eld
has been trailed, however the long-termsuccess of this method of vec-
tor control depends greatly on the persistence of the Wolbachia strain
in 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 wide
range of clinical presentations is generally observed. Host factors,
such as host genotype and the complexity of the immune response
can 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 correlations
between 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 with
increased 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 increased
TNF- levels seen in DHF patients (Fernandez-Mestre et al., 2004).
A study of the genetic polymorphisms present in the TNF and
lymphotoxin-alpha (LTA) genes of DV patients with varying disease
severities found that TNF-238 polymorphism (marking TNF-4.LTA-3
haplotype) occurred with higher incidence in patients with second-
ary DHF compared to patients with secondary DF. Additionally, the
LTA-3 phenotype was associated with in vivo production of TNF and
LTA 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 dengue
disease 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 endoplasmic
reticulum. 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) and
TAP1 637 (ASP/ASP) have been identied as conferring protection
against 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 patients
where 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 genes
are associated with immunity and are specically involved in the func-
tion of T-lymphocytes. Studies have shown that HLA-A*0203 and
HLA-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 DHF
in secondary infections (Stephens et al., 2002; Nguyen et al., 2008). In
a study of DV infected individuals in Cuba, a strong correlation was ob-
served between HLA-B*15 and individuals with DF and between
HLA-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 been
most frequently 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 determinant
of dengue disease susceptibility and severity. Observational studies,
such as those conducted in Haiti, the Caribbean and Africa have
suggested that populations of negroid origin may be less susceptible
to DHF/DSS than Caucasian populations since the incidence of DHF
and DSS was lower in these populations (Kouri et al., 1989;
Halstead, 2001; Sierra et al., 2006, 2007). The genetic reasons behind
this 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
appear to have a greater immunity to severe dengue disease to be elu-
cidated. Differences between ethnic groups, have been reported in
the polymorphic variability of FcRIIa which has been associated
with a higher risk of developing DHF in Caucasian populations
(Marcheco et al., 2010).
By increasing our understanding of genetic factors inuencing
susceptibility 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 of
host genetics on DV severity, further studies are needed as host ge-
netics may inuence the success or failure of both DV vaccine and
anti-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 defence
against pathogen invasion and its components are closely related to
the inammatory response. The cellular armof 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 with
the endosomal membrane results in viral RNA release into the cyto-
plasm for replication and translation (Martn et al., 2009; Nayak et
al., 2009).
It is widely believed that the innate immune response may orches-
trate disease outcome in DV infection. A major component of the innate
immune system is the induction of type I alpha, beta (, ) and type II
gamma () interferons (IFN) by DCs and NK cells respectively (Clyde
et al., 2006; Rodenhuis-Zybert et al., 2010). The production of IFNs in re-
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 also
trigger host adaptive immune response via DC maturation as well as B
and 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 against
DV 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*04
Uninfected 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*52
Milder 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)
This can be driven either by antibody-dependent enhancement (ADE)
of infection or production of T-helper (Th) 1 and Th2 cytokines by
T-lymphocytes (Mathew & Rothman, 2008). Clinically, elevated levels
of pro-inammatory cytokines and chemokines such as tumor necro-
sis factor (TNF)-, Interleukin (IL)-1, IL-2, IL-4, IL-6, IL-7, IL-8, IL10,
IL-13, IL-18, monocyte chemotactic protein (MCP)-1, IFN- and IFN-
have been reported in the sera of DF as well as DHF/DSS patients
(Navarro-Sanchez et al., 2005; Basu & Chaturvedi, 2008).
In response to DV infection, robust production of inammatory me-
diators is essential to initiate rapid viral clearance, hence minimising
further infection and disease progression. However, excessive and
persistent inammation can contribute to the exacerbation of dengue
disease (Garcia et al., 2010; Fagundes et al., 2011). The massive cytokine
secretion (cytokine storm) correlates positively with the hallmark of
dengue disease, including hepatic dysfunction, thrombocytopenia,
hypotension, and haemorrhagic shock (Garcia et al., 2010). From this
perspective, it is crucial to determine potential therapeutic targets
that can inhibit DV replication and assist in viral clearance, while
maintaining a healthy immunostimulatory response.
5.2.2. Cytokines and vascular leakage
Cytokines may also be responsible for the vascular leakage associ-
ated with severe dengue disease. The selective production of IL-8 and
CCL-5, and their accumulation at sinusoidal sites in endothelial cells
infected with DV, has resulted in the suggestion that these soluble
mediators could be responsible for the vascular leakage seen in DSS
(Avirutnan et al., 1998). Lee et al. (2006) observed that high levels
of MCP-1 expression in DV-infected patient sera correlated with seri-
ous disease, with monocytes found to be the primary cell type in-
volved in MCP-1 production. When endothelial cells were exposed
to DV-infected monocyte medium containing MCP-1, endothelial per-
meability was increased and the distribution of zonula occludens-1,
an endothelial-cell tight junction protein, was disrupted on the cell
membrane. Additionally, MCP-1 neutralising antibody reduced the
degree of endothelial permeability by up to 70%, further indicating
that MCP-1 has a role in vascular leakage as a result of dengue infec-
tion (Lee et al., 2006).
A study conducted on an in vitro model of human endothelial cells
reported a decrease the transendothelial electrical resistance with an
increase in TNF- suggesting an increase in vascular membrane per-
meability (Dewi et al., 2004). This was supported by a study on
AG129 mice infected with a specic DV strain, which demonstrated
vascular permeability with increased levels of TNF-. Treatment of
infected mice with anti-TNF was shown to result in a decrease in vas-
cular permeability in the spleen further supporting the role of TNF-
in vascular permeability pathogenesis (Shresta et al., 2006).
A number of other cytokines, including IL-1 and IL-1, IL-2, IL-6,
IL-8 and IFN- have been implemented in the induction of endotheli-
al permeability resulting in vascular leakage. Some of these cytokines;
TNF-, IL-2, IL-6, IL-8 and IFN-, are shown to be elevated during in-
fection or to be released by the dengue infected cells (Fink et al.,
2006)
5.3. Antibody response
Infection with a particular DV serotype results in serotype-specic
life-long immunity and short-term immunity against the other sero-
types. This serotype-specic immunity is believed to be due to either
neutralizing antibodies or DV specic memory T-lymphocytes, or
possibly a combination of both, (reviewed by Clyde et al., 2006). It
is important to note that in an immune host both neutralising and
non-neutralising antibodies are produced against DV-infection.
Neutralising antibodies may prevent infection by means of blocking
the release of viral RNA into the cytoplasm of a susceptible cell,
inhibiting attachment or through complement cascade mediated
lysis of antibody-coated virus. The ability of an antibody to effectively
neutralise the virus is determined by; the level of antibody occupancy
of the E epitopes on the virion, afnity towards the epitope, accessi-
bility of epitopes to antibodies, antibody subclass and the interaction
of antibody with the complement (Murphy & Whitehead, 2011). An-
tibodies against E and PrM protein are shown to be capable of
neutralising DV both in vitro and in vivo (Reviewed by Clyde et al.,
2006). Antibodies and the human antibody response plays a critical
role in the development of therapies against DV-infection and the de-
velopment of a putative vaccine.
5.3.1. Thrombocytopenia
Thrombocytopenia is a well-known symptom of DV infection.
Dengue antigens and anti-dengue antibodies are known to bind to
human platelets (Boonpucknavig et al., 1979) (Fig. 3), but the exact
pathological mechanisms which cause DV-induced thrombocytope-
nia are not fully understood. A study undertaken by Wang et al.
(1995) was the rst to show that DV binds to platelets only in the
presence of virus specic antibody, supporting the hypothesis that
immune-mediated destruction of platelets causes thrombocytopenia
in DHF patients. A more detailed study using dengue patient sera
and human platelets showed that IgM cross-reacted with platelets,
with higher anti-platelet IgM titres in DHF/DSS samples compared
with DF samples (Lin et al., 2001).
An inverse relationship exists between levels of platelet associated
IgG (PAIgG) and patient platelet count (Oishi et al., 2003), with plate-
let count low in the acute phase then rising in the convalescent phase
and vice-versa for PAIgG levels. This suggests that PAIgG may have a
role in platelet destruction related to the thrombocytopenia seen in
dengue infections. This inverse relationship was also seen in a sepa-
rate study, examining both PAIgG and platelet associated IgM
(PAIgM) levels, with higher levels of PAIgG and PAIgM detected in
DHF patients than seen in DF patients (Saito et al., 2004). In addition,
anti-NS1 antibodies have been shown to cross-react with brinogen
and thrombocytes, potentially contributing to thrombocytopenia
seen in severe DV disease. Whilst these studies highlight an impor-
tant role for antibodies in the development of DV-associated throm-
bocytopenia, a recent study observed platelet activation with
increased brinogen-binding qualities, degranulation and an alter-
ation in membrane architecture, suggesting the possibility of a direct
association between DENV-2 and platelets. This direct interaction re-
sults in morphological and characteristic platelet changes which may
also play a role in the development of thrombocytopenia seen in DV
infection (Ghosh et al., 2008) (Fig. 3).
It has clearly been demonstrated that platelets play a critical role
in the development of thrombocytopenia and are therefore a poten-
tial target for pharmaceutical intervention. Anti-D (Rh
0
D IgG) im-
mune globulin (WinRho SDF) has been shown to be highly
effective in producing Fc receptor blockade and in raising the plate-
let count in non-dengue forms of thrombocytopenia. The use of
anti-D as a candidate treatment option against the development of
DV-specic thrombocytopenia has been investigated. Results
reported a higher platelet count after anti-D treatment, thus identify-
ing a potential therapeutic means of enhancing patient platelet count
and preventing the development of severe thrombocytopenia associ-
ated with DHF (de Castro et al., 2007).
5.3.2. Haemorrhage
Apart from thrombocytopenia, antibodies directed against the
dengue NS1 have also been implicated in the inammatory activation
of endothelial cells in vitro and in the adhesion of peripheral blood
mononuclear cells (PBMC) to endothelial cells (Lin et al., 2005). In
the presence of sub-neutralising antibodies, complement is activated
on the surface of DV-infected cells (Avirutnan et al., 1998). This com-
plement activation, specically involving deposition of C3 and C5b-9,
and the subsequent generation of anaphlatoxins C5a abd SC5b-9 is
thought to contribute to the vascular permeability seen in DHF and
DSS in the form of haemorrhage (Avirutnan et al., 2006). Additionally,
direct DV infection of endothelial cells has been shown to cause apo-
ptosis which may further contribute to the increase in vascular per-
meability associated with DSS (Avirutnan et al., 1998) (Fig. 3).
Although there is signicant morbidity associated with DF, it is the
development of DHF and DSS that causes the majority of the mortality
associated with DV infection. Understanding the mechanisms leading
to DV haemorrhage is of signicant importance for the development
of pharmaceuticals. Such treatments may act to curtail the progres-
sion of DF limiting the severe haemorrhagic manifestation, which
may ultimately decrease the current high levels of DV mortality.
5.3.3. Antibody-dependent enhancement of infection
Antibody-dependent enhancement (ADE) describes the immune
phenomenon in which non-neutralising anti-viral antibodies cause
enhanced viral infection of host cells through Fc receptor binding.
Multiple epidemiological studies have shown that DHF almost exclu-
sively occurs in patients who have undergone a secondary heterotyp-
ic dengue infection (Halstead & O'Rourke, 1977; Vaughn et al., 2000).
Early in vitro studies have shown that homotypic and neutralising
anti-dengue antibodies abrogated virus infection, while heterotypic
and non-neutralising antibodies enhanced infection (Halstead &
O'Rourke, 1977; Kliks et al., 1989). In further support of a role for
ADE in dengue infection, infants hospitalised with DHF were born
predominantly to mothers who were seropositive for dengue anti-
bodies. This suggests that maternal antibody transfer can place a
child at risk of DHF in the event of a primary dengue infection
(Kliks et al., 1988). This hypothesis has been supported by epidemio-
logical data from Thailand which demonstrated an increased risk of
developing DHF/DSS in infants less than one year old (Halstead,
2003). Recently an in vivo animal model, analogous to human infants
that develop ADE by transplacental transfer, has been described
(Balsitis et al., 2010). In this model passive administration of anti-
DV antibodies is sufcient to enhance DV infection in mice using
both mouse adapted and clinical DV isolates.
Recent studies have provided some additional insights into the
mechanisms of ADE. In addition to promoting entry into Fc receptor-
bearing cells, preexisting enhancing antibodies have been found to be
involved in IFNevasion by two mechanisms. The rst mechanismis me-
diated by the activation of negative regulators, such as dihydroxyace-
tone kinase (DAK) and autophagy-related 5-autophagy-related 12
(Atg5-Atg12), which result in the disruption of retinoic acid inducible
gene I (RIG-I) and melanoma differentiation associated gene 5
(MDA-5) signalling and type 1 IFN production (Fig. 4A) (Ubol et al.,
2010). The second mechanism is mediated by the induction of IL-10,
which in turn upregulates a suppressor of cytokine signalling 3
(SOCS3), whichin turn is a negative regulator of the JAK-STAT signalling
pathway (Ubol et al., 2010) (Fig. 4B). It has been shown recently that in
ADE infection of human macrophages, down regulation of IFN- is
dependent on IL-6 (Rolph et al., 2011). A detailed study carried out on
infection rates, viral output and cytokine levels in different cell
populations of autologous human myeloid cells undergoing ADE re-
vealed that all cell types (monocytes, macrophages andmature DCs) ex-
cept immature DCs, support ADE (Boonnak et al., 2011). All cells
undergoing ADE express high levels of IL-6 and TNF-, but distinct pat-
terns were obtained for IL-10 and IFN secretion. RFLP studies indicated
that homozygous GCC haplotype was associated with high IL-10 secre-
tion while ACC and ATA haplotypes produced intermediate and low
levels of IL-10. Studies have also identied that antibodies to the struc-
tural (prM) play a major role in the DV ADE response. Anti-DV prM
Fig. 3. Host immune factors involved in the pathogenesis of dengue virus (DV) induced haemorrhagic fever (DHF) and shock syndrome (DSS). DV infect skin dendritic cells (DCs)
following a bite from an infected mosquito. Subsequent activation of the host cell immune response has been shown to involve three possible pathways. Firstly, anti-DV antibodies
(Abs) cause vascular leakage through complement cascade activation. Direct DV infection of vascular endothelial cells causes apoptosis, further contributing to vascular leakage.
Secondly, the infection of PBMCs leads to increased proinammatory cytokine/chemokine production by proliferating T cell populations. Thirdly, thrombocytopenia caused by
DV infection is seen as the result of two pathways: 1) direct infection and activation of platelets by DV. 2) Anti-platelet or anti-DV Ab mediated destruction of platelets. The
three pathways lead to the severe disease symptoms observed in DHF and DSS, including haemorrhage and thrombocytopenia. CXCR3, chemokine (C-X-C motif) receptor 3;
PBMCs, peripheral blood mononuclear cells; IF, infected; IFN-, interferon gamma; TNF-, tumor necrosis factor alpha.
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 of
ADE infection. It has been suggested that the partial cleavage that prM
undergoes on the viral surface during viral maturation may explain
the 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 have
no 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 researchers
striving to develop DV vaccines and therapeutics, however, there has
been some progress achieved so far. An articial antibody variant
(E60-N297Q) that cannot bind to FcR has been genetically engineered
and has been shown to have prophylactic and therapeutic efciency
against ADE induced lethal challenge (Balsitis et al., 2010). In another
recent study using monoclonal antibodies generated from memory B
cells of DV infected individuals, Beltramello et al. (2010) demonstrated
that 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 other
being an incomplete understanding of the immunopathogenesis of den-
gue disease, and possibly the reasons why no dengue vaccine is licensed
as yet, however, tremendous progress has been made in the last few
years. Various live (attenuated, chimeric and live virus vector) and
non-living (inactivated, recombinant subunit and naked DNA) dengue
vaccines have been developed and a number of monovalent and some
tetravalent 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 conducted
in avivirus-nave children (Simasathien et al., 2008) and infants
(Watanaveeradej et al., 2011). In the latter phase I/II trial, after the
second dose, 85.7% of full-dose DV vaccinees developed neutralizing
antibodies to b3 DENV serotypes and 53.6% to all 4 serotypes.
Recombinant live chimeric monovalent DENV vaccine candidate
strains, ChimeriVax DENV1 to DENV4 (CVD14), were developed for
all 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 for
safety 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) pathway
and 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.
(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 replacing
the 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 infectious
cDNA clone as the backbone, and inserting DENV-1, -3 and -4 struc-
tural genes to create a tetravalent vaccine. The DENVax vaccine has
shown immunogenic promise in mice and non-human primates
(Osorio et al., 2011) and is now being prepared for human clinical
trials.
Using an alternative virus as a vector for the expression of DV pro-
teins in vivo has been trialed as a novel vaccine approach. Vectors
trialed include; adenovirus (Raviprakash et al., 2008), Venezuelan
equine 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 have
shown promise in non-human primates, to date no human clinical tri-
als have commenced.
Vaccine development using recombinant subunit approach has
been 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 et
al., 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 and
screening, to express epitopes of the truncated E antigen that are
shared by all four DV serotypes. Recently, the rst phase I trial for a
prototype dengue DNA construct containing DENV-1 prM and E
genes found that T lymphocyte and neutralizing antibody responses
were 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 also
being evaluated by incorporating these in the DNA constructs.
With various vaccine candidates now available, some nearing or
having reached phase III clinical trials, it is increasingly important
for these to be evaluated in endemic areas for safety, immunogenicity
and sustained protection from all the four DV serotypes. It is crucial
that once available, the vaccines should become accessible at a rea-
sonable cost in these high-risk areas, which lie mostly in developing
countries (see Table 2 for a list of vaccines in preclinical or clinical
trials).
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 knownas C-type lectins are believed
to facilitate internalisation of DVentry as well as escaping host immune
responses (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 from Coller and Clements, 2011 current 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).
molecule 3-grabbing nonintegrin (DC-SIGN; CD209), which is mainly
expressed by immature DCs (Tassaneetrithep et al., 2003). It is now
generally accepted that immature DCs are one of the rst 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 et
al., 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 of
serotype, and infection is signicantly reduced in the presence of
anti-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 signalling
can be manipulated by DV, which in turn leads to immune evasion
and viral dissemination to secondary lymphoid organs, suggesting
that they play an instrumental role during DV infection (Alen et al.,
2009). It is also believed that DV target DC-SIGN to shift Th balance
toward 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 in
DC-SIGN results in weakened promoter activity and reduced risk of
severe disease (Sakuntabhai et al., 2005).
One of the most effective strategies to control viral infection is to
prevent 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 by
interacting with the viral glycoproteins (Balzarini et al., 2005;
Bertaux et al., 2007). With respect to DV, an in vitro study has
shown that CBAs can efciently prevent DC-SIGN-mediated infection
in 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-glycoprotein
and DC-SIGN. Together with their low toxicity in animals and high
stability, 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 as
primary target and as a source of immunomodulatory cytokines (Chen
& Wang, 2002; Wong et al., 2012). Recently, Miller et al. (2008) has
shown that mannose receptor (MR; CD206) is a putative receptor for
DV infection in macrophages. MR is a C-type lectin constitutively
internalised 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 is
known to act as an attachment receptor in DV infection, MR is thought
to 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 in
ADAMTS13 promotes microvascular obstruction and results in throm-
bocytopenia (Zhou et al., 2010; Matsumoto et al., 2012). In a recent
study, patients with severe dengue show reduced levels of ADAMTS13
activity 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), also
known 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 and
MR, the author showed that the interaction did not result in viral entry
but 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)-1
and interferon-inducible protein (IP)-10 but not IFN-, which further
support the role of CLEC5Aas 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 in
these 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 in
mice. These ndings suggested that CLEC5Asignalling may play a role in
the 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)-1
decient 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-weight
inhibitors targeting CLEC5A can be difcult to develop. Hence, the use
of humanised antibodies against CLEC5A may possibly prevent DV-
induced manifestations and attenuate excessive pro-inammatory
responses, without affecting viral clearance by the host's adaptive
immune response (Noble et al., 2010).
5.4.4. Macrophage-migration inhibitory factor (MIF)
Macrophage-migration inhibitory factor (MIF) is a pro-inammatory
mediator that plays an important role in the modulation of inammation.
MIF is knownto 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, and
counteracting the anti-inammatory effects of glucocorticoids (Calandra
et al., 1995; Donnelly & Bucala, 1997; Gregory et al., 2006; Bernhagen et
al., 2007).
The serumlevel of MIF indengue patients has beenshown to 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 well
as lethality compared with wild-type (WT) mice. However, the viral
loads 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 (Kimura
et 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 secreted
prior to the amplication of host inammatory responses as observed
in severe dengue. Recently, it has been shown that MIF can enhance
vascular permeability in both DV-infected endothelial cell line and
mice (Chuang et al., 2011). In addition, the author showed that cells
treated with MIF inhibitor ISO-1 or inhibition of MIF receptor CD74
can recover from tight junction protein ZO-1 disarray induced by MIF.
Altogether, these results support MIF as a crucial player in the im-
mune response to DV infection and provide direct evidence for MIF in
DV 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, inammation
and cytokine production. PAF can be produced by numerous cell types,
especially leukocytes (Garcia et al., 2010). The effects of PAF are mediat-
ed through a specic PAF receptor (PAFR) which is distributed on the
plasma membrane or the nucleus surfaces of platelets, leukocytes and
endothelium (Montrucchio et al., 2000; Marrache et al., 2002).
In a previous study, Yang et al. (1995) have shown that macrophages
frompreviously DV-1 infected patients can produce a signicantly higher
amount of PAF compared with healthy controls. When administered sys-
tematically toanimals or humans, PAF canmimic several systemic inam-
matory responses commonly observed in the context of severe dengue
disease, including elevated vascular permeability, thrombocytopenia,
haemoconcentration, hypotension and shock (Ishii & Shimizu, 2000; Xu
et al., 2010). Recently, the role of PAFR in DV pathogenesis has been elu-
cidated in animal models (Souza et al., 2009). The author demonstrated
that DV infection and clinical signs were signicantly less severe in
PAFR
/
mice as compared with WT mice. Notably, treatment with
PAFR antagonist such as UK-74,505 and PCA-4246 was shown to further
inhibit 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 ve
post-infection). In contrast, IFN- was signicantly increased in both
PAFR
/
and PAFR antagonist-treated mice, suggesting that PAFR has
no 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 has
shown 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 et
al., 2012). With a good drug safety prole and blocking efcacy in
humans, 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's
immune response is a signicant factor in dengue disease severity and
induction 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. Recent
development of animal models for DV will help in understanding the
mechanism 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 effective
anti-dengue vaccines and anti-viral pharmaceuticals.
Acknowledgments
SM is grateful for the funding support from the Australian Research
Council (ARC) (Grant #LP0775507). AZ is the recipient of the ARC Post-
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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