MINIREVIEW
Genetic polymorphisms of molecules involved in host immune
response to dengue virus infection
Xin Fang1, Zhen Hu1, Weilong Shang1, Junmin Zhu1, Chuanshan Xu2 & Xiancai Rao1
1
IMMUNOLOGY & MEDICAL MICROBIOLOGY
Department of Microbiology, College of Basic Medical Sciences, Third Military Medical University, Key Laboratory of Microbial Engineering under
the Educational Committee in Chongqing, Chongqing, China; and 2School of Chinese Medicine, The Chinese University of Hong Kong, Hong
Kong, China
Correspondence: Xiancai Rao, 30#
Gaotanyan Street, Shapingba District,
Chongqing 400038, China. Tel.: +86 23
6875 2240; fax: +86 23 6875 2240;
e-mail: [email protected]
Received 14 December 2011; revised 29
March 2012; accepted 22 May 2012.
Final version published online 21 June 2012.
DOI: 10.1111/j.1574-695X.2012.00995.x
Editor: Alfredo Garzino-Demo
Keywords
dengue virus; genetics; polymorphisms;
molecules; human immune; pathogenesis.
Abstract
The dengue virus (DENV) belongs to the flavivirus family. Each of the four
distinct serotypes of this virus is capable of causing human disease, especially
in tropical and subtropical areas. The majority of people infected with DENV
manifest asymptomatic or dengue fever with flu-like self-limited symptoms.
However, a small portion of patients emerge with severe manifestations
referred to as dengue hemorrhagic fever, which has a high mortality rate if not
treated promptly. The host immune system, which plays important roles
throughout the whole process of DENV infection, has been confirmed to have
double-edged effects on DENV infection. Recently, much attention has been
paid to the genetic heterogeneity of molecules involved in the host immune
response to DENV infection. This heterogeneity has been proved to be the
determining factor for DENV disease orientation. The present review discusses
the primary functions and single nucleotide polymorphisms of some critical
molecules in the human DENV immunological defense, especially the polymorphism locus associated with the DENV pathogenesis and disease susceptibility.
Introduction
The dengue virus (DENV), a member of the flavivirus
family, has four antigenically distinct yet disease severityassociated serotypes (DENV 1–4; Fried et al., 2010).
Humans are infected by DENV through the bite of female
Aedes aegypti or Aedes albopictus. DENV is the main
pathogen causing dengue fever (DF), dengue hemorrhagic
fever (DHF), and dengue shock syndrome (DSS), which
are still prevalent in many regions of the world (especially
in tropical and subtropical areas; Guzman et al., 2010). It
is estimated that two-fifths of the world population is at
risk of DENV infection (WHO reports, 2009). Infected
people mostly appear asymptomatic or with flu-like DF,
which manifests as a fever, rash or hemorrhage with
systemic pain. Asymptomatic dengue is self-limited and
non-lethal (Endy et al., 2011).
The most important goal of basic medical research is to
determine suitable measures to conquer the diseases that
threaten human health. Aside from symptomatic and
supporting treatments, there is still no known specific
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treatment for DENV infection in spite of extensive
research on this disease, and the importance of maintaining the patient’s circulating fluid volume (Stein et al.,
2011). Vaccination would eventually be a more efficient
and cost-effective approach. However, there are many
obstacles associated with vaccine development (Shang
et al., 2012).
Although most DENV-infected patients manifest
asymptomatic or self-limited DF, epidemiological studies
have revealed that a small portion (9.9%) emerge with
severe, life-threatening DHF/DSS syndromes, such as
thrombocytopenia, massive multiorgan hemorrhage, circulatory failure, and neurological symptoms, after the
primary/secondary infection. DHF/DSS patients will die
in 4–6 h (20–30% fatality rate) if not treated promptly.
DHF remains the leading cause of child deaths in Southeast Asian countries (Guzman et al., 2010).
Many scientists have focused on exploring the mechanisms of the disease severity of DENV-infected people.
The prevailing virus virulence theory (Fried et al., 2010)
and growing evidence have demonstrated that some
FEMS Immunol Med Microbiol 66 (2012) 134–146
135
Genetic polymorphisms for dengue virus pathogenesis
critical molecular events in the host immune process
against DENV may have double-edged effects (pathology
or recovery) on the DENV disease (Kurane, 2007;
Tolfvenstam et al., 2011). Halstead et al. (1970) first
described the “secondary infection enhancement” phenomenon in DENV infection on the basis of an epidemiological study of children with DHF/DSS. They found
that the majority of DHF children (85%) showed higher
heterotypic cross-reactive antibodies, which implied that
pre-existing dengue antibodies (from primary dengue
infection or an infected mother) could result in a serious
pathological reaction rather than neutralization, called
DENV antibody-dependent enhancement (ADE; Halstead
& O’Rourke, 1977; Fig. 1). Many DENV-specific crossreactive non-neutralizing antibodies (usually with limited
protection roles) that mediate dengue pathogenicity have
been found (Dejnirattisai et al., 2010; Schieffelin et al.,
2010; Falconar & Martinez, 2011; Kou et al., 2011;
Table 1). Other plausible immunopathological theories
ascribe the severe development of the DENV disease to
cell apoptosis, cytokine storm and cross-reactive T lymphocyte (Mathew & Rothman, 2008). However, the triggered events such as DENV-specific or cross-reactive
antibodies, T lymphocytes, and a large number of cyto-
B Cells
kines may not result in protection but instead serious
pathological outcomes that may participate in DENV
pathogenesis (Mathew & Rothman, 2008).
As a multifactorial disease, dynamic factors, including
patients’ age, pre-existing chronic diseases and the
infection sequence of different DENV serotypes, partially influence DENV disease outcome. The epidemiological analysis of DENV outbreaks (1997 and 2001) in
a Cuban endemic area showed that people of different
races have notably different morbidities and symptoms
after DENV infection (Kourı́ et al., 1998; De la C
Sierra et al., 2007). These findings eliminate possible
influencing factors of secondary infection and strain
virulence, and provide great inspiration that human
genetic background may be a determining factor of
DENV disease orientation. Furthermore, genetic polymorphisms related to host immunological events may
have a significant effect on DENV disease because of
the importance of host immunity in DENV pathogenesis. We will list in this review the key molecules associated with the host DENV immunologic defense system
and discuss their polymorphism locus, which may have
implications for the susceptibility to severe DENV
disease.
Antigen presenting cells
DENV
Cross-reactive
antibodies
Cytosol
FcγR bearing cells
Mature virions
Immature/partially mature virions
FcγR
Fig. 1. ADE of DENV infection. ADE has been described in several viruses, including DENV. A mixture of immature, partially mature, and mature
virions exist, most likely because of the incomplete processing by furin protease during viral maturation. B cells produce DENV-specific antibodies
after a viral infection. However, the produced cross-reactive antibodies, mostly against prM/M and domain II of the DENV envelope E protein
(Dejnirattisai et al., 2010), can bind with heterologous DENV serotypes to form a virus-antibody complex in a secondary infection. This complex
can enhance the uptake of virions into FccR-bearing cells (primary monocyte and macrophage), mainly through recognition by the FccR on the
surface of these cells. Subsequently, the antiviral immune response is suppressed and an environment beneficial for heterologous DENV entry and
replication is created, which, finally, results in the high growth of progeny DENV virions and serious disease outcome. The following mechanisms
have been proposed to account for this enhancement: one is the suppression of interferon-mediated antiviral responses by upregulating the
negative regulators, and the other is the Th2-type cytokine biasing aroused by the activation of IL-10 (Boonnak et al., 2011).
FEMS Immunol Med Microbiol 66 (2012) 134–146
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X. Fang et al.
136
Table 1. The cross-reactive antibody-mediated damages in DENV diseases
Target
Function
Effect of antibody
Source
prM/M
Constructing DENV virion by
maintaining correct E protein
conformation
Dejnirattisai
et al. (2010)
E
Mediating DENV virion to enter
susceptible cells through receptor
binding and membrane fusion
NS1
A solvable complement binding
protein involved in DENV RNA
replication
A major component of the anti-DENV response or a risk factor of ADE in the
secondary infection by binding to an immature DENV virion (which makes
immature DENV virions highly infectious and increases their efficient binding
and cell entry into FccR-bearing cells)
Accepted as the primary anti-DENV response for antibody-mediated
neutralization (domain III) or mediates the enhancement of secondary infection
and fibrinolysis system imbalance through non-neutralizing cross-reactive
antibodies (domain I/domain II)
NS1 antibody can only be detected in the secondary DENV infection and plays
a protective role in DENV-infected mouse. By cross-reacting with human
platelet surface protein disulfide isomerase, NS1 antibody may account for
serious thrombocytopenia, vascular leakage, and endothelial injury in DENV
patients
Schieffelin
et al. (2010)
Falconar &
Martinez
(2011)
The genome of DENV consists of a single open reading frame (ORF) which can be translated into a polyprotein with a molecular weight of about
380 kDa. The polyprotein is then co- and post-translationally cleaved by cellular and viral proteases into three structural proteins (C, prM/M, and
E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Among these proteins, prM/M, E, NS1, NS3 are both reactogenic and immunogenic, causing their antibodies to mediate contradictory responses (protective and detrimental) (Guzman & Vazquez, 2010).
The NS3 protein (a multifunctional enzyme with serine protease, RNA helicase, and triphosphatase activities) is the major target of DENV-specific
CD4+ and CD8+ T lymphocytes in a secondary infection; however, the role of NS3 antibody is poorly investigated even though anti-NS3 monoclonal antibodies have been developed (Zivna et al., 2002).
Human leukocyte antigen (HLA)
The HLA complex is the most complicated and polygenic
system in humans. The complex is a group of genes
located on chromosome 6p in the human major
histocompatibility complex (MHC). Encoded HLA molecules are widely distributed in various cells and can generally be divided into the following three classes: MHC
classes I (HLA-A, -B, -C, -E, -F and -G), MHC classes II
(HLA-DP, -DQ, -DR, -DO and -DM), and MHC class
III. MHC classes I and II encode structurally similar
membrane surface proteins, whereas MHC class III is
responsible for the production of complementary and
other serum proteins (Stephens, 2010). The polymorphism locus of MHC classes I and II molecules can lead
to the restrictive conjugation of antigens. The association
of HLA molecules with DENV immunopathology may be
directed by HLA-restricted DENV polymorphism antigens
(Appanna et al., 2010). Many case–control studies have
proved that HLA complex polymorphisms are closely
related to DHF/DSS pathogenesis, summarized in the
reviews contributed by Chaturvedi et al. (2006), Coffey
et al. (2009) and Stephens (2010).
Dendritic cell-specific intercellular
adhesion molecule 3 (ICAM-3)- grabbing
non-integrin (DC-SIGN)
Dendritic cells (DCs) have been recognized as major
actors bridging innate and adaptive immune responses.
The number of the circulating DC subsets is reduced after
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DENV infection, although this is not a biomarker of
severe forms of dengue disease (De Carvalho Bittencourt
et al., 2012). DENV-infected DCs can induce initial proliferation of naive CD4+ T cells but they cannot differentiate
into interferon-producing effector T cells, indicating that
inhibition of interferon signaling is conserved among
DENV strains (Chase et al., 2011). DC-SIGN is an important attachment C-type lectin expressed on DCs which
serves as the leading receptor that DENV uses to penetrate
DCs through recognition of the N-glycosylation sites of
DENV E-glycoprotein (Asn153 and Asn67; Yang et al.,
2012). The carbohydrate-binding agents, which have exact
blockade functions on DC-SIGN and DENV interactions,
can protect Raji/DC-SIGN+ cells from the infection of
four DENV serotypes. This behavior may inspire the
development of anti-DENV compounds (Alen et al., 2009,
2011).
The polymorphisms of the DC-SIGN gene has been
considered to affect the outcome of dengue viral disease,
based on the important roles of DC-SIGN in DENV
infection (Selvaraj et al., 2009). DC-SIGN is encoded
by the CD209 gene located on chromosome 19p13.3. A
functional promoter variant of CD209 336 A/G polymorphism associated with DENV infection in Thai individuals was found in 2005 (Desprès et al., 2005). The G
allele can significantly downregulate promoter activity by
affecting the binding of a transcriptional factor Sp1 and
result in downregulation of DC-SIGN level. The G allele
of the variant DC-SIGN-336 is therefore associated with
strong protection against DF (OR = 4.90). However, the
AA genotype is associated with protection against DHF
FEMS Immunol Med Microbiol 66 (2012) 134–146
137
Genetic polymorphisms for dengue virus pathogenesis
(OR = 5.84) by affecting the expression of the DC-SIGN
on the DC surface and the viral replication in DCs from
DENV-infected subjects.
The Chinese population has a significantly lower
frequency of the G allele (3.8%; Wichukchinda et al.,
2007). Wang et al. (2011) demonstrated the existence
of CD209 336 A/G polymorphism in the Taiwanese
population and found a higher G allele frequency in DHF
patients than in healthy people (OR = 4.84) and DF
patients (OR = 2.44). These results are consistent with
the research results from Thai people (Desprès et al.,
2005). However, a retrospective study in the Brazilian
population found that the CD209 336 A/G polymorphism had no association with DHF (Silva et al., 2010).
Hence, the CD209 336 A/G polymorphism may be partially involved in DENV infection. Alternatively, DENV
infection may be caused by population-based genetic heterogeneity. Aside from the 336 A/G polymorphism,
other genetic variants of CD209, such as position 139
and 3′UTR polymorphisms (Wichukchinda et al., 2007),
have been studied for their susceptibility to virus infection (e.g. HIV). However, their influence on the susceptibility to or protection against DENV infection remains
elusive.
ADE effect was abrogated when mature DCs were pretreated with the FccRIIa-specific MAb but not with the
FccRIIb-specific MAb (Boonnak et al., 2008). These findings raise the possibility that increasing the FccRIIa/
FccRIIb ratio would facilitate ADE in DENV-infected
mature DCs as well as downregulate DC-SIGN. The development of a stable BHK-21 cell line, which only expresses
FccRIIa, provides a good model for defining the role of
FccRIIa in the pathogenesis of DENV (Moi et al., 2010).
The encoded gene of FccRII is a locus on chromosome
1q23. Polymorphism of the FccRIIa gene was also
observed to have a significant association with DENV
infection. The alteration of a single amino acid at position
131 His/Arg in the second Ig-like domain of FccRIIa is
critical for human IgG2 binding (Loke et al., 2002). The
homozygosity of the amino acid Arg has been reported to
protect against DHF (OR = 0.09), whereas the wild-type
amino acid His at this position is significantly associated
with serious DENV disease (OR = 10.56) in Vietnamese
children and Cuban adults. The significantly different frequencies of the Arg variant at position 131 around the
world may be partly attributed to the distribution of
DENV infection in different regions and races (Loke et al.,
2002; Garcı́a et al., 2010).
FCcRIIa
Transporter associated with antigen
processing (TAP)
FccR is another important immune-related receptor
expressed on DCs that contributed to ADE pathogenicity
in DENV infection (Green & Rothman, 2006). Since
existing antibodies appear in primary DENV infection,
cross-reactive immunoglobulin G (IgG) can readily be
detected on the first day of a secondary DENV infection.
FCcR is capable of binding IgG, providing an important
link between the humoral and cellular immune systems.
According to the function and affinity of IgG subclasses,
FCcR can be divided generally into three main classes:
FccRI (CD64), FccRII (CD32) and FccRIII (CD16; Bournazos et al., 2009). Several reports have confirmed that
FccRI and FccRII receptor-bearing cells can mediate ADE
of DENV infection (Boonnak et al., 2008, 2011; Nimmerjahn & Ravetch, 2008). FccRII has a broader range of
cellular expression and has been shown to help DENV
enter cells when FccRII binds the Fc portion of nonneutralizing antibodies attached to DENV (Brown et al.,
2006; Ubol & Halstead, 2010).
FccRII has three isoforms: FccRIIa, FccRIIb, and
FccRIIc. Researchers found that mature DCs have a higher
ratio of FccRIIa/FccRIIb compared with immature ones,
which suggests that these changes regulate DC function
and control cellular responses. These two forms of FccRII
play different roles in ADE: activating (FccRIIa) and inhibitory (FccRIIb). Blocking experiments showed that the
FEMS Immunol Med Microbiol 66 (2012) 134–146
CD8+ T lymphocytes as cytotoxic T cells can kill DENVinfected cells (Yauch et al., 2009). However, CD8+
T memory cross-reactive lymphocytes from a primary
infection can alter the production/activity of the cytokines
and host immune response during a secondary heterologous DENV infection. Therefore, these lymphocytes may
contribute to serious clinical symptoms (Green & Rothman, 2006; Beaumier et al., 2008). CD8+ T lymphocytes
perform their role in DENV infection via the interaction
with antigen-loaded MHC class I molecules presented by
DCs through a complex process. First, intracellular DENV
releases its RNA to synthesize and produce new DENV
particles. The virus antigen is transported into the endoplasmic reticulum (ER) lumen by TAP, the only known
ABC transporter in the ER membrane (Lorente et al.,
2012). With the help of some accessory molecules,
including tapasin, calnexin, calreticulin, Bip, ERp57, and
endoplasmic reticulum aminopeptidase (ERAP), the well
processed antigen is then loaded into MHC I molecules,
presented on the surface of DCs, and recognized by CD8+
T lymphocytes (Chang et al., 2005).
Some viruses can escape recognition by CD8+ T lymphocytes by downregulating or blocking the function of
MHC class I molecules. However, flavivirus, including
DENV, can upregulate the expression of MHC I
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X. Fang et al.
138
molecules and increase the virus-specific peptide supply
to the ER post-infection by an interferon (IFN)-independent pathway (Lobigs et al., 1996). The upregulation of
MHC I molecules is beneficial to DENV transmission and
may be a strategy of the NK cell for DENV evasion in the
first few days of infection (Fig. 2). Furthermore, a transient increase in the TAP transport activity (augmented
by up to 50%) during the early phase of flavivirus infection results in an increased supply of peptides for assembly with MHC I molecules, which may account for the
upregulation of MHC I molecule expression (Hershkovitz
et al., 2008). These results reveal that TAP is not only a
key molecule in antigen presentation but may also help
in DENV immune escape and infection.
TAP is composed of two half-transporter subunits,
TAP1 and TAP2. This transporter belongs to the ATPbinding cassette superfamily and needs ATP as an
energy source and allosteric regulator (Sunder et al.,
2011). The TAP gene is located on chromosome 6p21.3.
Association of TAP gene polymorphism with diseases
has been uncovered recently (Soundravally & Hoti,
2007, 2008a; Sunder et al., 2011). Using amplification
refraction mutation system-polymerase chain reaction,
polymorphisms of TAP1 and TAP2 genes were typed in
DF, DHF, DSS patients, and control cases. The results
showed that the frequencies of the AA genotype of
TAP1 gene 333 and the GG genotype of TAP2 gene
379 decreased significantly in DF patients and controls,
compared with DHF patients (OR = 0.611 and 0.605,
respectively; Soundravally & Hoti, 2007, 2008b). By contrast, the AG genotype of TAP1 gene 333 (Ile/Val heterozygote) and the GA genotype of TAP2 gene 379
(Val/Ile heterozygote) increases susceptibility to DHF by
approximately 2.58 and 2.11 times, respectively. These
nVDR
Calcitriol 1,2(OH)2D3
T-cell receptor
T cell
CD28
Th1
CTLA-4
IL-2
IL-3
IFN - γ
TNF -β
CD80/86
DC-SIGN
DENV
MHC I
KAR
IL-4
IL-5
IL-6
IL-10
Th2
CD4+
DC
Endosome
Proteasome
FcγR
KIR
Ribosome
TAP1
MHC II
ADE
Golgi
TAP2
Tapasin
B cell
NK cell
ER
Calreticulin
Type 1 IFN
TNF-α
ERp57
ERAAP
TGF-β1
IFN-γ
Launches
MBL
Neutralization
Viral peptide
presenting
Platelets
damage
Complement system or
Opsono-phagocytosis
Endothelial cells
HPA
Damage
Fig. 2. Overview of the key immune events after DENV infection. DENV binds to DC-SIGN, the viral receptor on the surface of DCs, to mediate
entry. Then, NK cells and MBL, as the early defenders, quickly take measures to kill the infected cells. However, there would be some escaped
infected cells, in which synthetic DENV polyproteins may be processed into peptides and subsequently access the antigen processing and
presentation programs. Afterward, the CD4+ T lymphocytes can be activated and differentiated to promote Th1 and Th2 responses to exert their
adverse complex effects. The migration of Th1/Th2 cells can be directly or indirectly regulated by calcitriol 1,25-(OH)2D3 by binding with nVDR. B
cells can produce diverse antibodies with the help of Th2-related cytokines. The antibodies have three types of effects: ADE, neutralization, and
platelet damage (when binding with HPA). The DENV immune pathogenic mechanism here considers these host immune events to lead to
endothelial cell damage, the increase in vascular permeability, and finally hemorrhagic symptoms. The blue stars represent strategic immune
molecules in which locus polymorphisms have been associated with DENV pathogenesis.
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FEMS Immunol Med Microbiol 66 (2012) 134–146
139
Genetic polymorphisms for dengue virus pathogenesis
findings suggest the potential protective role of TAP1
gene 333 A allele and TAP2 gene 379 G allele against
the development of DHF. In addition, the AA genotype
of TAP1 gene 637 (Asp homozygote) can reduce the
risk of primary DSS by 0.643 times. The AG genotype
of TAP2 gene 665 (Thr/Ala heterozygote) is a risk factor in the development of DHF (Soundravally & Hoti,
2008b).
Vitamin D receptor (VDR)
CD4+ T lymphocytes are another type of important
immunocyte in humans. They can directly engage in
DENV pathogenesis or act as a helper of CD8+ T lymphocyte to participate indirectly in DENV infection (Hatch
et al., 2011). CD4+ T lymphocytes are usually divided into
two antagonistic but cross-reactive subsets: helper T (Th)
1 and 2 cells. The balance of Th1/Th2 cells is relevant to
disease outcome, such as in DENV infection. Clinical
research data demonstrate that patients with DF mostly
manifest the Th1 advantage response, whereas DHF
patients show the Th2 advantage response (Chaturvedi
et al., 2006). Lower expression of the Th1 transcription
factor (T-bet) and higher cytokines levels (interleukin-10,
IL-10) in Southern Taiwan populations are associated with
the pathogenesis of DHF (Chen et al., 2005).
Calcitriol 1,25-(OH)2D3 is the biologically active
metabolite of vitamin D3, a secosteroid hormone that
acts not only as a calcium and phosphorus metabolism
regulator, but also as an immunoregulator via the interaction with the immune adjustment receptor, VDR. There
are two types of VDR, nucleus (nVDR) and membrane
(mVDR). 1,25-(OH)2D3 exerts its immunoregulatory
effects mainly through the nVDR, whereas the mVDR
regulates the effects on immunocytes at the initial phase
by nongenomic effects (Marcinkowska, 2001). After binding with 1,25-(OH)2D3, nVDR changes its conformation
and eventually forms a vitamin D response element to
influence the subsequent mRNA expression and protein
synthesis (Fig. 2). Both DCs and activated T lymphocytes
can express nVDR, which makes them the target of
1,25-(OH)2D3. The 1,25-(OH)2D3-nVDR complex participates in DENV infection by promoting the apoptosis of T
lymphocytes via the Fas/FasL system (Loke et al., 2002).
VDR gene is located on chromosome 12q13.11. Four
types of VDR gene polymorphisms, termed restriction
enzymes and shown as FokI (C/T F, f), BsmI (A/G B, b),
ApaI (T/G A, a), and TaqI (T/C T, t; capital letter signifies
the existence of a restriction enzyme cutting site, whereas
those in lowercase indicate otherwise), have been confirmed to be associated with certain diseases, e. g. low bone
mineral density (Jakubowska-Pietkiewicz et al., 2012). In
case–controlled studies of dengue patients in Vietnam,
FEMS Immunol Med Microbiol 66 (2012) 134–146
Loke et al. (2002) found that the t allele of VDR gene
may play a protective role against severe DHF.
352
Cytotoxic T lymphocyte-associated
antigen-4 (CTLA-4)
Three indispensable signal-stimuli are needed in T lymphocyte activation, proliferation and differentiation. The
first is the combination of antigens with TCR (MHCantigen-TCR-CD3). The second signal is a co-stimulatory
activator (CD28-B7 family). The third is IL-2, which
protects the activated T lymphocytes from apoptosis.
CTLA-4, also known as CD152, is an essential receptor
involved in the negative regulation of T cell activation.
The receptor has nearly 20 times greater affinity to B7
compared with CD28 (Chen et al.,2009). CTLA-4 knockout (KO) mice died approximately 3 weeks after birth
due to lymphoproliferative disorder (Khattri et al., 1999).
Activated CD4+ lymphocytes in 2-week-old CTLA-4 KO
mice tend to produce more Th2 cytokines (preferential
IL-4). However, treatment with murine CTLA-4 antibodies could prolong the survival time of CTLA-4 KO mice
to as much as 45 days, and their Th1 cytokine responses
(IL-2 and IFN-c) were replaced by the Th2 cytokine
skewing response. As an analog of CD28, however, the
role of CTLA-4 in Th cell polarization remains elusive.
The human CTLA-4 gene is a single-copy gene located
on chromosome 2q33. Genetic linkage analysis found two
SNPs (promoter
318 C/T polymorphism and exon
1 + 49 A/G polymorphism) and one (AT)n dinucleotide
repeated polymorphism at exon 3 + 642 to be associated
with host autoimmune and infectious diseases (Jiang
et al., 2007). Exon 1 + 49A/G polymorphism gained particular attention for its notable function in altering the
transcriptional activity of the CTLA-4 gene. This polymorphism could also affect CTLA-4 distribution, IL-2
secretion, and T lymphocyte activation. Exon 1 + 49 A
allele has been reported to increase the risk of DENV
disease development (Fernández-Mestre et al., 2009).
However, an investigation of a DHF/DF case–controlled
study in Taiwan revealed that the exon 1 + 49 A/G polymorphism and TGFb1 gene 509 C/T polymorphism
(TGFb1 is an immunosuppressive cytokine proven to be
associated with DENV pathogenesis) are related to the
DENV-2 clinical manifestation (Chen et al., 2009). Furthermore, different combinations can lead to different
consequences. For example, the CTLA-4 gene exon
1 + 49 G allele combined with the TGFb1 gene 509 CC
genotype is found more frequently in patients with DHF.
However, patients with the CTLA-4 exon 1 + 49 A allele
combined with the TGFb1 gene 509 CC genotype have
a significantly higher virus load, at least in the Taiwanese
population (Chen et al., 2009).
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X. Fang et al.
140
Acute plasma glycoprotein mannose
binding lectin (MBL) and human
platelet-specific antigens (HPA)
MBL, a member of the collectin family, is a kind of liversynthesized, non-enzyme, and non-antibody, early acute
anti-infection plasma glycoprotein (Fuchs et al., 2010),
and can preferentially recognize specific sugar groups on
the surface of various microorganisms with its multiple
carbohydrate-recognition domains (Palaniyar et al., 2004).
MBL performs its function mainly by two mechanisms: (1)
it can act as an activator of the complement system to
form the membrane attack complex to kill pathogens or
infected cells and (2) it can also act as an opsonin by
mediating uptake not only via collectin receptors but also
through the generation of iC3b that coats targets and triggers uptake by CR3 (Ip et al., 2009). Human gene mbl-2,
which encodes the MBL protein, is located on chromosome 10q11.2–24 and comprises four exons. The MBL
structural subunit consists of three identical 32-kDa polypeptide chains. Each chain has a cysteine-rich region
(encoded by exon 1) at its N-terminal, a collagen-like
region (CLR, encoded by exons 1 and 2), a neck region
(encoded by exon 3), and a calcium-dependent carbohydrate recognition domain (encoded by exon 4), successively, at the C-terminal (Turner, 1996). The three
chains are cross-linked through disulfide bonds within the
N-terminal and CLR involvement. Multiple (a maximum
of six) MBL structural subunits further form functional
MBL oligomers through covalent or noncovalent bonds to
exert its function.
Clinical studies have verified that the functions of MBL
are dependent on certain MBL serum levels (Kilpatrick,
2002). The principal factors that influence MBL serum
levels are three genetic polymorphisms at mbl-2, namely,
219 C/T, 226 G/A, and 235 G/A. These mutations
destroy the structure of the CLR to prevent the correct
oligomerization of MBL (Vasconcelos et al., 2011).
Research into DENV infection in the Vietnamese population found that the frequency of mbl-2 226 G/A polymorphism was relatively low, and this SNP was not
significantly associated with DENV clinical outcomes
(Loke et al., 2002). A similar result was obtained from a
Brazilian population (Acioli-Santos et al., 2008), but a
negative relationship between MBL serum levels and
thrombocytopenia (a severe DENV disease manifestation
in DHF patients) was found. This negative relationship
suggests that, although mbl-2 polymorphisms may not be
associated with an augmented or reduced risk of DENV
infection, they have a protective role against development
of thrombocytopenia.
In addition, studies have confirmed that the DENVantibody complex can be detected on platelets of DHF
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
patients, and that DENV that binds to human platelets
can be enhanced by virus-specific antibodies (Wang et al.,
1995). These findings reveal that serious thrombocytopenia in DHF patients may be caused by immune-mediated
damage. The anti-E and anti-NS1 antibodies against
DENV cross-react primarily with HPA, which can, at least
partially, account for the serious hemorrhaging seen in
DENV-infected patients (Saito et al., 2004). HPAs are
membrane glycoproteins on a platelet surface, named by
‘HPA-number’, which are encoded by six genetic fragments located on chromosomes 5, 6, 17, and 22. The
polymorphisms of HPA1a/1b and HPA2a/2b are found
more frequently in DHF patients (Soundravally & Hoti,
2007). SNPs that lead to different single-amino acid substitutions can distinguish HPA1a/1b 176 T/C (aa position 33 Leu for HPA1a and Pro for HPA1b) and
HPA2a/2b 482 C/T (aa position 145 Thr for HPA2a
and Met for HPA2b) polymorphisms. Case–controlled
studies have shown that HPA1a/1a and HPA2a/2b genotypes confer susceptibility to DHF (OR = 3.59 and
OR = 3.71, respectively). The HPA 1a/1a genotype can
increase the risk of DHF (OR = 1.93), and HPA 1b 176
C allele is increased in patients with DSS compared with
controls (OR = 3.59) or DHF (OR = 4.75; Soundravally
& Hoti, 2007). However, whether there is any positive
correlation between two glycoproteins (MBL and HPA)
and DENV pathogenesis remains to be elucidated.
Cytokines
Cytokines are small cell-signaling protein molecules that
are secreted by numerous cells and work actively in
intercellular communication. The biological functions of
cytokines are complicated, extensive, and overlapping
(synergistic or antagonistic). In patients with DF, for
example, the increased circulating IL-1Ra may exert antipyretic actions in an effort to counteract the already
increased concentrations of IL-1b. CXCL10/IP-10
was confirmed as a strong pro-inflammatory marker
(De-Oliveira-Pinto et al., 2012). DF and DHF patients
have significant differences in cytokine levels, and the
involvement of cytokines in DENV pathogenesis has been
identified by many researchers (Mustafa et al., 2001; Gunther et al., 2011). The effects of cytokines on DENV
infection can generally be split into the following, based
on their functions: promotion, restriction, and mixed.
For example, increased serum levels of IL-6 and IL-8 are
positively correlated with DENV infectious dose and have
promotive effects in the beginning of DHF development
(Huang et al., 2000). The dramatically increased levels of
IL-13, IL-18, and transforming growth factor (TGF)-b1 in
DHF patients may result in Th1 to Th2 response transformation and, finally, to an exacerbation of the DENV
FEMS Immunol Med Microbiol 66 (2012) 134–146
FEMS Immunol Med Microbiol 66 (2012) 134–146
Leading receptor of DENV
infection in human
IgG receptor that acts as
linker between humoral and
cellular immune systems
ER particular antigen transporter
Human platelet-specific antigens
Immunoregulator via interaction
with calcitriol 1,25-(OH)2D3
Essential molecule involved in
maintaining T lymphocyte
response homeostasis
DC-SIGN
FCcRIIa
TAP
HPA
VDR
Natural early acute anti-infection
plasma glycoprotein
Regulator of host immune
response and cytokine
responsiveness of target cells
Immune inhibitor
MBL
TGFb1
IL-10
CTLA-4
Function
Molecules
Chromosome 1q31-32
1082 A/G
819 C/T
592 A/C
Chromosome 19q13
promoter 509 C/T
codon 25 G/C and codon 10 T/C
Chromosome 10q11.2-24 codon
52 (CGT-TGT, Arg-Cys)
codon 54 (GGC-GAC, Gly-Asp)
codon 57 (GGA-GAA, Gly-Glu)
Chromosome 6p21.3
TAP1 333 A /G (Ile/ Val)
TAP1 637A/G (Asp/Gly)
TAP2 379 G/A (Val/Ile)
TAP2 665 A/G (Thr/Ala)
Chromosome 17
HPA1a/1b 176 T/C
HPA2a/2b 482 C/T
Chromosome 12q13.11
352 TaqI (T/C T, t)
Chromosome 2q33 exon 1 + 49 A/G
Chromosome 1q23
393 A/G (His/Arg)
Chromosome 19p13
promoter 336 A/G
Locations and SNPs
Polymorphism has no association with DENV
disease orientation, but carriage of 1082/ 819/
592 haplotype ACC/ATA may be a DHF risk factor
+49 A allele is not significantly increased
among DENV patients
+49 G allele, combined with TGFb1 509 CC
genotype, increases the risk of DHF.
+49 A allele, combined with TGFb1 509 CC
genotype, results in higher virus load
No association with DENV disease orientation
mbl-2 polymorphisms have a protective role
against development of thrombocytopenia,
despite not having a significant association
with the augmentation of DENV disease
509 CC genotype can increase the risk of DHF
Codon 10 T/C polymorphism has no association
with DHF, whereas codon 25 GG genotype
plays protective role against DHF
No association with DENV disease orientation
HPA1a/1a and HPA2a/2b genotypes confer
susceptibility to DHF and HPA1b is determined
to be a genetic risk factor for DSS
352 TaqI t allele is protective against severe DHF
336 G allele is protective against DF, but not DHF
336 G allele is more frequent in DHF,
whereas AA genotype is associated with
protection against DHF
No association with DENV disease
orientation found
Arg homozygote is protective against DHF,
His homozygote increases the risk of DHF.
This SNP may contribute to area and race
distribution of DENV infection
TAP1 333 A allele is protective against DHF,
TAP1 637AA genotype reduces the risk of DSS,
TAP2 379 G allele is protective against DHF,
and TAP2-665 AG genotype is a risk factor for DHF
Association with DENV disease
Table 2. Human key molecules in which locus polymorphisms have been associated with DENV pathogenesis and disease susceptibility
Fernández-Mestre
et al. (2004)
Fernández-Mestre
et al. (2004) and
Perez et al. (2010)
Venezuelans
Venezuelans and Cuban
Chen et al. (2009)
Perez et al. (2010)
Loke et al. (2002)
Acioli-Santos
et al. (2008)
Fernández-Mestre
et al. (2009)
Chen et al. (2009)
Loke et al. (2002)
Soundravally &
Hoti (2007)
Soundravally & Hoti
(2007, 2008a, b)
Taiwanese
Cuban
Vietnamese
Brazilian
Taiwanese
Venezuelan
Vietnamese
South Indian
South Indian
Garcı́a et al. (2010)
and Loke et al. (2002)
Silva et al. (2010)
Brazilian
Cuban and Vietnamese
Desprès et al. (2005)
Wang et al. (2011)
Source
Thai
Taiwanese
Population
Genetic polymorphisms for dengue virus pathogenesis
141
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
X. Fang et al.
Vejbaesya et al. (2009)
Loke et al. (2001)
Silva et al. (2010)
Cuban and Venezuelans
Thai
Vietnamese
Brazilian
Key factor involved in
interferon-a/b and-c signal
transduction pathways
JAK1
Chromosome 1p32.3-p31. 3
Intron 3: T/C
Intron 2: C/T
Intron 1: T/G
308A allele is a severity risk factor of DHF
and hemorrhagic manifestation in DF.
308 GG genotype is associated with
protection
238A allele is significantly associated with DHF
No association with DHF
Intron 3 TT genotype and Intron 2 GG genotype
are significantly associated with DHF. Intron 1 TT
genotype is protective against DENV
Garcı́a-Trejo et al.
(2011)
Fernández-Mestre
et al. (2004) and
Perez et al. (2010)
Mexican
238A allele is protective against DHF
Explicit antitumor factor with
serious destructive effects
TNF-a
Chromosome 6p21.3
238 G/A (Ala /Thr)
308 G/A
Function
Molecules
Table 2. Continued
Locations and SNPs
Association with DENV disease
Population
Source
142
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
disease (Mustafa et al., 2001). High levels of human cytotoxic factor (hCF)-autoantibodies are associated with DF
and may be useful as a prognostic indicator of disease
progression (Chaturvedi et al., 2001). The macrophage
migration inhibitory factor induced by DENV infection
may contribute to the increase of vascular permeability
during DHF development (Chuang et al., 2011). The role
of IFN, as a key cytokine in the human DENV immune
response, remains unclear (Shresta et al., 2004). Several
animal studies have shown that mice lacking both IFN-a/
b and IFN-c receptors succumb to primary DENV infection, whereas their wild-type counterparts survive. IFN-c
has been proved to be effective in helping human clearance of DENV by upregulating the expression of HLA
molecules. Furthermore, sustained IFN-c levels during the
acute phase of DENV infection play a protective role
(Gunther et al., 2011). However, IFN-c can not only augment FccR-mediated DENV infection of human monocytic cells (Kontny et al., 1988), but also induce
pathophysiologic events (shock and hemorrhagia) via the
increase of other immune injury cytokines (IL-2, TNF-a)
and anaphylatoxins (C3a, C5a). Scientists have attempted
to use cytokines as a new type of immunotherapeutic target in DENV treatment because of the critical network
role that cytokines play in DENV infection (Bozza et al.,
2008; Feldmann, 2008). Potential clinical benefits of tetracycline and doxycycline in DENV infection treatment act
through the modulation of host cytokines (Castro et al.,
2011).
The variations in the cytokine genes may be correlated
with DENV disease outcome, susceptibility, and process.
TGF-b1 gene 509 CC genotype has been reported potentially to increase the risk of DHF by approximately twofold
(OR = 1.9) in a Taiwanese population (Chen et al., 2009).
However, another TGF-b1 polymorphic locus, the codon
25 GG genotype, played a protective role in a Cuban population (Perez et al., 2010). Furthermore, the polymorphic
locus of pleuripotent vasoactive immunomodulators TNF
is also found. The TNF-a–238 A allele displays a significant protective effect against (in Mexicans) or promotes
an association with (in Thais) DHF (Vejbaesya et al., 2009;
Garcı́a-Trejo et al., 2011). The TNF-a-308 A allele is a
severity risk factor of DHF (in Cubans and Venezuelans).
However, the lymphotoxin-alpha gene +249 A/G polymorphism, +365 G/C polymorphism, and +720 C/A polymorphism, which have gene loci adjacent to TNF genes, have
no independent significance for DENV disease. Although
the IL-10 promoter 1082 A/G, 819 C/T, and 592 C/A
polymorphisms influence the IL-10 production and ADE
in dengue infection (Boonnak et al., 2011), they have no
significant association with DHF in Cubans and Venezuelans. However, the 1082/ 819/ 592 haplotype ACC/
ATA may be a DHF risk factor (OR = 2.54; Perez et al.,
FEMS Immunol Med Microbiol 66 (2012) 134–146
Genetic polymorphisms for dengue virus pathogenesis
2010). Three genotypes at the 5′ end of the Janus kinase 1
(JAK1: involved in the IFN-a/-b and -c signal transduction pathways) gene, namely, intron 3 TT genotype, intron
2 GG genotype, and intron 1 TT genotype, have a significant association with DHF development (Silva et al.,
2010). IFN-c and IL-6 gene polymorphisms have also been
tested but were found to have no significant correlation
with DENV diseases (Fernández-Mestre et al., 2004; Perez
et al., 2010).
Conclusion
Controlling the vector transmission seems to be the only
effective preventive measure to date. However, this method
is expensive and sustaining the universality and substantial
quantity of mosquitoes is difficult. The development of
typing technologies for human genomic SNP analysis, such
as single-stranded conformational polymorphism (Oliveira
et al., 2009) and sequencing-based typing (Garcı́a et al.,
2011) in recent years has provided new and powerful tools
to study human heterogeneity in association with DENV
disease. The present review lists the human key molecules
in which locus polymorphisms have been associated with
DENV pathogenesis and disease susceptibility (Table 2).
The limited findings and paradoxical results encourage further analysis using more populations from diverse DENVendemic areas. The more information is gathered about a
specific disease, the easier will be the development of new
preventive and therapeutic agents against the disease.
Acknowledgements
The present work was supported by grants from
the National Natural Science Foundation of China
(31070811), the New-Drug Development Project of China
(2012ZX09103301-038), the Natural Science Foundation
of Chongqing City (CSTC, 2009BB5015), and the Science
Foundation of Third Military Medical University
(2009XYY04). We would like to sincerely express our
thanks to Mr. Eric Chuck Hey Pun (University of Toronto, Canada) for revising our manuscript.
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