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Dengue and Chikungunya virus
van Duijl-Richter, Mareike
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Chapter
2
Dengue Virus Life Cycle
and Pathogenesis
Chapter 2
2
18
DENV Life Cycle and Pathogenesis
Dengue Virus Life Cycle and Pathogenesis
Introduction
Dengue virus (DENV) – mainly transmitted by the Aedes aegypti mosquito
– is the most common mosquito-borne viral infection worldwide. DENV causes an
estimated 390 million infections per year, of which about 100 million are symptomatic
[1]. Each of the four serotypes of DENV (designated DENV 1-4) can cause disease
with symptoms including fever, rash, headache, and musculoskeletal pain [2].
Approximately 500,000 to 1 million individuals however develop more severe disease
including abdominal pain, bleedings, organ impairment, hematological aberrations,
and plasma leakage. Severe dengue can be fatal if not properly treated, and each year
about 25,000 patients die from dengue infection [3,4].
Severe dengue is almost exclusively seen during secondary infection with a
heterotypic DENV serotype and during primary infection of infants with declining
levels of maternal antibodies [5-7]. This observation points towards a pathogenic role
of the immune system in severe disease development. In contrast, upon homotypic
re-infection, the newly infecting DENV is effectively neutralized and individuals do
not develop disease [8]. Also, third and tertiary infections usually do not lead to
disease symptoms [9]. In this chapter, the viral structure and life cycle of DENV
will be described, followed by a review of the host cell tropism and the mechanisms
controlling disease outcome.
2
Viral structure & life cycle
Dengue virus (DENV) belongs to the flavivirus genus within the Flaviviridae
family. Other flaviviruses are for example West Nile virus (WNV), Yellow Fever virus
(YFV), Japanese encephalitis virus (JEV), Tick-borne encephalitis virus (TBEV), and
the emerging Zika virus (ZIKV) [10,11]. DENV virions are small spherical particles
with a diameter of approximately 50 nm. Each virion contains a single-stranded,
positive-sensed RNA genome of 10.8kB in length, which is packaged by the capsid
(C) protein to form the nucleocapsid. The nucleocapsid is surrounded by a hostcell derived lipid bilayer in which 180 copies of the membrane protein (M) and
envelope protein (E) are anchored. The E ectodomain consists of three structural
domains (DI, DII and DIII). The E proteins are arranged as homodimers in a headto-tail orientation. The homodimers are lying tangential to the viral membrane in a
herringbone-like pattern, thereby giving the particle a “smooth” surface (figure 1
a,b) [12-15].
The first step in the viral life cycle (figure 2) involves binding of the E
glycoprotein to a receptor or attachment factor on the host target cell. The putative
19
Chapter 2
2
Figure 1 DENV envelope proteins on the mature virion
The E protein consists of three functional domains (DI, DII & DIII). Receptor-binding is presumably
mediated by DIII (blue). The fusion loop (green) is located at the tip of DII (yellow). DI is depicted in
red. On the mature virions, E homodimers lie flat on the membrane in a head-to-tail orientation, giving the particle a smooth appearance. Adapted from [39] and reprinted with permission from Elsevier
receptor binding domain has been allocated to E DIII [16-19]. Upon binding, DENV
particles enter the host cell via clathrin-mediated endocytosis. The internalized
virions are delivered to Rab5-positive early endosomes, which further mature into
Rab7-positive late endosomes [20,21]. Upon exposure of the virus to the acidic lumen
of the endosome, the E protein homodimers dissociate, which results in the exposure
of the fusion loop situated on DII (figure 1, green). The hydrophobic fusion peptide
subsequently inserts into the endosomal membrane and E homotrimers are formed.
Then, DIII folds back towards the fusion loop, which forces the opposing outer
leaflets of the membranes together. The outer membranes subsequently merge, which
is referred to as hemifusion. This short-lived intermediate is followed by complete
fusion and expansion of the fusion pore. Finally, the nucleocapsid is released to the
cytosol [12,22-24]. It has been suggested that fusion mainly occurs from within
Rab7-positive late endosomes [20], although contrasting reports have been published
considering the location of membrane fusion [25]. In addition to low pH, negatively
charged lipids were shown to support membrane fusion of DENV. This observation
strongly points towards fusion from within late endosomal compartments as
negatively charged lipids are abundantly present within these compartments [26].
Upon the release of the nucleocapsid to the cytosol, the capsid proteins
dissociate from the viral genome. The viral genome is translated at the rough
endoplasmic reticulum (ER) into a single polyprotein and processed into 3 structural
proteins (prM, E and C) and 7 non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B,
and NS5) proteins. The ectodomains of prM and E, NS1 and parts of NS4A and NS4B
are facing the ER lumen whereas the other proteins are present at the cytosolic side of
the ER. NS4A, presumably together with other viral and host proteins, subsequently
triggers membrane rearrangements and vesicle formation by ER membrane
20
DENV Life Cycle and Pathogenesis
invagination. These vesicles were found to contain NS proteins, viral RNA, and host
cell factors and serve as sites for viral RNA replication. Newly synthesized viral RNA
is released to the cytosol and associates with C proteins to form the nucleocapsid.
The nucleocapsid subsequently buds into the ER lumen, thereby acquiring a lipid
membrane in which prM and E proteins are anchored. The newly assembled virus
particles have a “spiky” appearance with prM and E heterodimers arranged as 60
trimeric spikes on the viral surface [27-29] (figure 3).
2
The virions are then transported via the Golgi and Trans Golgi Network
(TGN) to the plasma membrane of the cell. During exocytosis the virus particles
further mature. The acidic lumen of the TGN triggers a global conformational
re-arrangement of the viral spike proteins leading to dissociation of the prM-E
heterodimers and formation of E homodimers which are capped by prM. This
Figure 2 DENV life cycle
DENV enters the cell via receptor-mediated endocytosis followed by fusion from within acidic endosomes and release of the nucleocapsid. Viral replication takes place in endoplasmic reticulum (ER)
membrane invaginations. Virus assembly occurs by budding of a newly formed nucleocapsid into the
ER lumen. The newly assembled immature particles are transported through the Golgi and Trans Golgi
Network (TGN) to the surface of the cell. Within the TGN, enzymatic cleavage of the prM protein occurs by the host protease furin. The pr peptide remains associated with the virion till its release to the
extracellular milieu. Reprinted from [39] with permission from Elsevier.
21
Chapter 2
rearrangement allows proteolytic cleavage of prM by the host protease furin, which
separates the pr peptide from the M protein (figure 2). The pr peptide functions
to stabilize the E protein while transiting acid compartments during virus egress. It
stays associated with M at low pH, and is released after secretion of the particle in the
pH-neutral extracellular space [30-33]. DENV-infected cells have been described to
secrete virions with varying maturation degree [34]. Indeed, electron micrographs
revealed the presence of virions with a spiky surface (immature), smooth surface
(mature), and surfaces with both spiky and smooth characteristics in varying
distributions (partially immature). At least 40% of all particles are partially immature
and 3% is fully immature when produced in mosquito cells [35,36]. Comparable
levels of prM were seen in viruses produced in mammalian endothelial and human
embryonic kidney cells, whereas dendritic cells appear to produce more mature virus
[37]. Fully immature DENV virions were found to be essentially non-infectious, as
the pr peptide is capping the E protein thereby preventing virus-cell binding and
membrane fusion [32,38]. It is not known to which degree prM cleavage must have
taken place to enable viral infectivity, yet it is clear that not all prM proteins have to
be cleaved [39].
2
Figure 3 DENV envelope proteins on the immature virion
DENV virions are assembled as immature virions (left side). On these particles, the fusion loop of the
E protein is covered by the prM protein (purple). Color coding of EDI, DII, DIII is same as in figure 1.
Three E-prM heterodimers form one spike on the immature particles, with the prM protein at the tip of
the spike (right side). Adapted from [39] and reprinted with permission from Elsevier.
Primary dengue virus infection
Dengue is transmitted to humans via mosquito bites. The mosquito usually
probes the skin several times before a blood vessel is reached. Most DENV particles
are inoculated in the skin and a minor fraction of particles is directly delivered to the
blood. DENV particles can infect cells resident in the skin, including keratinoctyes,
Langerhans cells and dendritic cells (DCs) [40-43]. Infected Langerhans cells and
DCs migrate to the lymph nodes, where other DENV target cells are infected,
22
DENV Life Cycle and Pathogenesis
including monocytes (which can also be directly infected by virus injected in the
blood vessels) and macrophages [40,44,45]. Moreover, monocytes are recruited to
the dermis were they differentiate into DCs that can be infected as well [46]. The
virus then spreads to other parts of the body and becomes a systemic infection [2].
Although cells derived from the mononuclear lineage are the main target cells of
DENV, also a variety of other cell types is permissive to infection, like for example
cells of hepatic and endothelial origin or fibroblasts [47,48]. Such a wide cell tropism
suggests that DENV uses several receptors or attachment factors. Indeed, molecules
that were described to interact with DENV are (amongst others) heat shock protein
70, heat shock protein 90, GRP78/BiP, a 37/67kDa high-affinity laminin receptor,
heparin sulphate, CD14, and C-type lectin receptors [45,49-58]. C-type lectin
receptors are expressed on cells of the myeloid lineage. For example, Dendritic Cell
Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN/
CD209) is abundantly expressed on immature dendritic cells (imDC) and on certain
types of macrophages [59]; the mannose receptor is mainly expressed on imDCs
and macrophages, but also on dermal fibroblasts and keratinocytes [60]; and C-type
lectin domain family 5, member A (CLEC5A/MDL-1) is expressed on monocytes
and macrophages [58]. The infected cells produce large amounts of progeny virus,
and approximately three days after the mosquito bite, viremia can be detected in
the blood of infected patients. Peak titers are usually measured three days after the
appearance of fever. Viral load can reach 106-108 viral copies/ml, though in patients
with severe disease, higher titers are observed as well. DENV viremia is cleared on
average 5 days after onset of fever [61,62].
Early in DENV infection the innate immune response is activated, which
is important for restricting the infection and viral clearance [63,64]. The expression
of IFNs and other pro-inflammatory cytokines is triggered by activation of pattern
recognition receptors, including Toll-like receptor (TLR) 3, TLR 7 and TLR 8; but
also the C-type lectins DC-SIGN, CLEC-5, and MR [58,65-68]. Subsequent binding
of IFNs to infected and neighboring cells leads to activation of the JAK/STAT pathway
and induces an antiviral state of the cell. Moreover, activation of natural killer (NK)
cells leads to secretion of type-II IFNγ, which is likewise important in the antiviral
defense [63,69]. DENV can however counteract the antiviral effects of IFNα/β
binding by downregulating the JAK/STAT pathway [70]. Therefore, activation of the
adaptive immune system is essential for complete clearance of infection.
IFN production not only induces an antiviral state, it also stimulates the
adaptive immune system by inducing imDC maturation and activation of lymphocytes
[71]. Approximately 6 days after infection, DENV specific B and T cells appear.
The majority of antibodies against DENV are generated against E and prM, which
suggests that the secreted virus particles indeed consist of a mixture of virions with
varying maturation state [72-74]. Serotype-specific, strongly neutralizing antibodies
are often targeting the E protein and can efficiently prevent infection [75]. NS1
antibodies are produced as the NS1 protein is expressed on the surface of infected
2
23
Chapter 2
cells and secreted in the extracellular space [76,77]. NS1 antibodies were shown
to mediate complement-mediated lysis of DENV infected cells [78,79]. Activated
CD4+ and CD8+ T cells also induce lysis of DENV-infected target cells and produce
a range of antiviral cytokines. Thus, infection is cleared through natural killer cells
and macrophages, assisted by the presence of neutralizing antibodies and activated T
cells [8,80].
2
Re-infection: Neutralization versus enhancement of infection
Upon re-infection with DENV, the adaptive immune response is rapidly
activated and anti-DENV antibodies are secreted. In case of a homologous reinfection, the pre-existing and newly produced antibodies efficiently prevent
infection and together with a rapid activation of memory T-cells, the individual is
protected from disease [81]. If heterotypic re-infection occurs shortly after primary
infection (estimations range from six months up three years), cross-reactive
antibodies are present in high titers thereby protecting the individual from disease
[82,83]. However, if re-infection occurs after a longer time-span, the pre-existing
cross-reactive antibodies are no longer protective against heterologous infection
and may even enhance the severity of disease. Indeed, severe disease development
is strongly associated with a heterologous secondary infection [62,84]. In contrast,
third and tertiary DENV infections only rarely lead to disease as cross-immunity is
then sufficient to prevent infection [9].
Role of antibodies in homologous re-infection
During homologous re-infection, antibodies play an important role in
neutralization. NS1 antibodies have been described to induce complement-dependent
lysis of infected cells [76] and lysis of antibody-opsonized viruses [85]. Antibodies
directed against the viral spike proteins can directly neutralize viral infectivity. The
dogma is that antibody-bound virions are taken up by Fc-receptor expressing cells and
are delivered to the endocytic pathway for degradation. Importantly, Fc-receptors are
expressed on myeloid cells, which are the main natural target cells of DENV. Therefore,
in case of dengue, neutralization has to occur at a post-attachment step [86,87].
Indeed, multiple high-affinity antibodies were identified that neutralized infection
by prevention of membrane fusion [88-90]. Most neutralizing antibodies were found
to target the E protein. In murine studies, antibodies binding to the DIII domain were
most potent, whereas in humans, antibodies targeting quaternary structures and
cross-linking multiple E proteins have been described as strongly neutralizing. These
antibodies likely act by preventing the essential conformational changes required for
membrane fusion [37,91-95]. Neutralization of DENV can be described as a “multiple
hit” phenomenon, in which a certain amount of available epitopes must have bound to
prevent infection. For strongly neutralizing antibodies, only a fraction of epitopes has
24
DENV Life Cycle and Pathogenesis
to be covered to efficiently prevent infection, whereas weakly neutralizing antibodies
have to bind more available sites to mediate neutralization [96]. Thus, in homotypic
re-infection, type-specific antibodies displaying high affinity to the virus are present
in high titers and prevent infection.
Enhanced disease during heterologous secondary infection
2
Approximately 2.5% of the individuals experiencing a secondary heterologous
re-infection develop severe disease [2,3]. It is generally believed that antibodies play
an important role in the development of severe disease as infants with declining
levels of maternal antibodies were found to have an increased risk for severe disease
during primary infection [7,84,97]. During a secondary heterologous re-infection,
pre-existing cross-reactive antibodies can recognize newly infecting virus serotype.
Functional studies revealed that cross-reactive antibodies generally have weakly
neutralizing properties [72,73,98,99]. The presence of weakly-neutralizing crossreactive antibodies is further increased through original antigenic sin, a phenomenon
in which cross-reactive memory B-cells are expanded, but the number of immune
cells against the currently remaining serotype remains low [8,73,98,100].
The cross-reactive antibodies bind to the virions, which are then internalized
by Fc-receptor bearing DENV target cells. Intriguingly, these particles escape from
degradation and fuse from within acidic endosomes [87]. This phenomenon is
called antibody-dependent enhancement of infection (ADE). In vitro, all antibodies
that can neutralize flavivirus infectivity also show enhancement of infection when
antibody concentrations are low enough. Important factors in ADE are: antibody
affinity, epitope accessibility, and antibody concentration. High affinity antibodies
that bind to accessible epitopes are more prone to neutralization than low affinity
antibodies that bind to cryptic sites. ADE occurs when the occupancy of viral particle
by a neutralizing antibody is below the threshold of neutralization [39,74,96,101].
At ADE conditions, antibody-opsonized virions not only escape from
degradation, these particles also have a higher chance to induce membrane fusion
and productive infection [102]. This might be related to the alternative entry route by
which antibody-opsonized DENV is taken up into the cell [103]. In addition, we and
others showed that virtually non-infectious fully immature particles are rendered
infectious by antibodies [104-106]. Upon entry of antibody-opsonized immature
particles in Fc-receptor bearing target cells, furin cleavage and maturation occurs
within the endosome, followed by infection of the cell [105,107]. ADE of infection of
both mature and immature DENV particles leads to an enhanced infected cell mass
and viral load early in infection. Epidemiological studies showed that an increased
viral load is associated with a higher risk of developing severe dengue [61].
Furthermore, not only memory B cells but also memory T cells specific for
the primary infecting serotype are preferentially expanded. Original antigenic sin of
T cells leads to the generation of low avidity T cells that are less efficient in clearing
25
Chapter 2
the infection. This results in the secretion of a vast amount of pro-inflammatory
cytokine and modulators which can cause capillary leakage as seen in severe dengue
[2,8,73,98,108,109]. Though these and other aspects like virulence of the infecting
strain and host genetic factors are clearly associated with severe disease, the complete
picture is not understood given the small percentage of individuals with a heterologous
secondary infection that develop severe disease [2,3].
2
Concluding remarks
DENV has a tremendous clinical impact, yet no antiviral treatment or vaccine
has been approved. The development of an efficient and – most importantly – safe
vaccine is challenging, as it is of upmost importance to prevent vaccine enhanced
disease through ADE and original antigenic sin [110]. Therefore, an optimal vaccine
has to neutralize all four DENV serotypes and elicit a long-term antibody response.
The most promising candidate vaccine so far is from Sanofi Pasteur. Their tetravalent
chimeric DENV/Yellow Fever (YF) vaccine (prM and E proteins of DENV and the
replicative backbone of the YF vaccine 17D) reduced hospitalization by 67%-80%
and dengue hemorrhagic fever by 80%-90% [111,112]. The efficacy to protect against
natural DENV infection was surprisingly low (30%-60%, depending on the study
population) [111-114]. The low protection against disease was rather unexpected,
as antibody titers towards all DENV serotypes were found using the in vitro plaque
reduction neutralization test (PRNT) [114,115].
The “golden standard” PRNT involves cells of endothelial origin [116].
Infection of these cells by DENV can be prevented by neutralizing antibodies.
However, as endothelial cells do not express Fc-receptors, the enhancing properties
of antibodies by Fc-receptor mediated entry cannot be assessed. Therefore,
neutralization in DENV target cells might require higher titers than the ones found
in standard PRNTs. Indeed, several groups have shown that Fc-receptor bearing
target cells often require higher antibody titers for neutralization, especially when
cross-reactive antibodies are tested [117-119]. For this, assays employing Fc-receptor
bearing cells might represent a better predicting neutralization test for DENV, yet
this approach is challenging in an industrial setting. Alternatively, the definition of
the correlate of protection should be re-defined in retro perspective based on the
results of the recent clinical trials, since the current correlate of protection of DENV
is based on early vaccine-related studies on the flaviviruses JEV, YFV, and TBEV; but
does not take the genetic variability between and within the different serotypes of
DENV into account [110].
In summary, dengue is a complex disease and multiple factors are important
in controlling disease outcome. More fundamental research into the virus life cycle
and host immune response is imperative to fully understand disease pathogenesis
and should guide the rational design of antiviral therapies and vaccines.
26
DENV Life Cycle and Pathogenesis
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