2. Four related and antigenically distinct viruses
(DENV 1-4)
Transmitted through mosquito vector (Aedes
sp).
50-100 million people infected with DENV
each year.
DENV infection results in DF, DHF/DSS.
The most significant mosquito-borne viral
diseases in the world today in terms of
illness, death and economic cost.
3. Three Major Hypotheses:
1. Antibodies-dependent enhancement (ADE).
2. Inappropriate T. Cell response (Memory).
3. Viral Virulence.
These three, non-mutually-exclusive processes are
postulated to increase viral load and trigger “cytokine
storm” and activation of the complement system,
resulting in DHF/DSS.
4. Cross-reactive antibodies against a previous
infecting DENV serotype facilitate the Fc-
mediated uptake of a subsequent infecting
heterologous strain by Fc-receptor positive
cells.
Accordingly, This result in a more severe
pathological response to the secondary viral
infection.
Non-neutralizing maternal antibodies is
associated with increased frequency of more
severe cases.
5. Serotype cross-reactive memory T-Cells
(CD8+) contribute to the pathogenesis of
DHF/DSS.
Inappropriate T-cell response is usually
associated with DHF/DSS (original antigenic
sin).
Recent studies contradicted the hypothesis.
6. Viral genotype may play an important role in
the pathogenesis of DENV.
Specific viral genotypes are associated with
more severe disease outbreaks.
Single mutation contributes to viral survival or
replication efficiency, resulting in enhanced
infection in the presence of ADE, a phenomenon
termed increased virus ‘‘fitness’’ in contrast to
‘‘virulence,’’ which is an intrinsic property of the
virus.
7. Fluid replacement with or without blood
factors are currently the standard
management for plasma leakage associated
with DHF/DSS.
Burden of DENV in eight countries range from
587 million to 1.4 billion USD.
Despite low mortality rate, effective treatment
would reduce the morbidity and financial
burden of DENV.
8. Diagnosis:
Low cost, rapid, sensitive, diagnostic test,
predictive of disease severity.
Treatment:
Safe, target multiple serotypes, and
effective even after the onset of severe
clinical disease.
Vaccine:
Safe, effective against all viral serotypes.
9. Mouse Model AG129 for DENV infection.
Lacking both type I and type II interferon (IFN)
receptors.
Infection with DENV leads to splenomegaly,
Viraemia, detectable levels of NS1 protein,
Paralysis and death after 8-10 days.
Recent models showed viral replication in
relevant tissue, tissue damage, increased
vascular permeability without hemorrhage
and death few dpi.
10. The DENV (+)RNA genome and it co-linear polyprotein.
Flavivirus are enveloped viruses having two outer
membrane proteins, the envelope (E) and the
membrane (M) processed from the precursor prM.
The genome is thought to be wrapped/associated
with the capsid protein C.
A single polyprotein is translated from the genome,
and the former is cleaved by a combination of cellular
proteases and a viral serine protease made of NS2B
and NS3(protease N-terminus domain).
11. Proteolysis yields ten proteins, the three
structural proteins (C, prM, and E) and the seven
nonstructural (NS) proteins involved in genome
replication and capping (NS1, NS2A, NS2B, NS3,
NS4A, NS4B, and NS5).
Some of these NS proteins also participate in
pathogenesis and counteract the innate immunity
of the host cell.
Replication of the viral genome does not occur
freely in the cytoplasm. Instead, there is an
extensive intracellular membrane re-
arrangement in the infected cell, with various
observable cell substructures containing most NS
proteins organized along the virus replication
cycle.
12. Pathologic Role.
Excessive consumption of complement proteins
correlated with DHF/DSS.
Complement could enhance DENV infection of
myeloid cells by promoting viral entry through CR3.
DENV nonstructural protein 1 (NS1) could activate
complement, and levels of NS1 and several
complement proteins correlates with disease severity.
Levels of complement factors D, H, and MBL protein
were found to be higher in DHF patients than in DF
patients.
Complement activation is pathogenic in DHF/DSS.
13. Protective Role.
in vitro ADE studies demonstrated that
complement proteins reduce DENV infection.
in vitro studies showed that DENV NS1binds
C4 and C1s or C4BP to antagonize
complement activation.
MBL pathway is critical for neutralization of
DENV.
15. NS3 and NS5 are responsible for all virus-
encoded enzymatic processes during DENV
infection.
Replication of the viral genome does not occur
freely in the cytoplasm. Instead, there is an
extensive intracellular membrane re-
arrangement in the infected cell, with various
observable cell substructures containing most
NS proteins organized along the virus replication
cycle.
16. NS3 (69 kDa) carries two functional domains, a
N-terminal serine protease(~170 aa), and a C
terminus helicase/RNA triphosphatase (~440
aa).
The protease domain is inactive alone, and
needs the presence of 40 aa of NS2b bound to
form a protease active site.
The NS2b/NS3 protease has been the first
dengue protein target actively used in drug
design programs.
17. NS5 is a 900 amino acids protein (~100 kDa)
carrying the enzymatic activities required for RNA
capping and synthesis of the dengue RNA genome.
the full-length NS5 crystal structure is not available.
NS5 may also play a role in pathogenesis, so its
targeting may hit two birds with a single stone.
NS5 has been reported to interact with STAT2,
perturbate interferon signaling, and its traffic within
the nucleus of the infected cell is far from being
understood.
18. The M and E proteins have been considered so
far as drug targets.
The E protein is endowed with a dual function to
recognize cellular receptor, and to fuse the viral
membrane to cellular endosomic membranes.
Five receptors have been found to be involved
in binding to E : DC-SIGN, L-SIGN, the high
affinity laminin receptor, the mannose receptor,
and GRP78.
19. The dengue E protein crystal structure is known.
E belongs to class II fusion proteins.
Upon binding to a receptor and endocytosis under a trimeric
form, the acidic environment of the endosome induces a
structural re-arrangement yielding fusion of cellular and viral
membranes.
Crystal structure study of a E fragment has revealed a pocket
that could be used for antiviral drug-design.
The crystal structure of the prM protein bound to E (prM-E
heterodimer) is known, opening avenues for drug design.
The structure of the capsid protein C has bee elucidated in
solution.
The E protein is the most obvious target for therapeutic
monoclonal antibodies.
20. The dengue genome is a single stranded RNA
molecule of positive polarity.
The replicative form of dengue RNA is not a single
linear molecule but rather, a cyclic or dimerized
genome.
RNA organization proficient for replication carries
many highly ordered secondary and tertiary
structures ensuring proper regulation of dengue
RNA synthesis.
RNA regions contain a significant potential for drug
discovery and design, yet to be addressed and
validated.
21. The host cell is actively involved at many levels
during DENV infection, either at the level of innate
immunity and counteraction thereof, or providing co-
factors and template for replication of the virus.
Many known cellular proteins and pathways that
exert an anti-dengue effect when affected or
inhibited.
Proteases and glucosidases constitute the earliest
discoveries of such host factors, whereas other
candidates (kinases, cholesterol synthesis
enzymes, proteins involved in immune response,…)
are progressively discovered and validated through
siRNA studies.
22. Cholesterol is integral to DENV replication.
Low levels of cholesterol and LDL is a
consequence of severe DENV manifestations
and correlate with severe bleeding, hepatic
dysfunction, and death.
In-vitro, cholesterol pathway inhibitors
inhibited DENV replication.
No in-vitro data is available.
23. DENV blocks the IFN signaling cascade.
TNF- is responsible for vascular leakage in
humans and animal models.
High mobility group box 1 (HGMB1) reduces
DENV titer in infected dendritic cells.
Chemokines and many hemostatic pathways
role are unknown!
24. The use of large-scale siRNA screens has
generated large list (>100 proteins) of host
factors involved in helping DENV to achieve its
replication cycle.
DENV inhibition was achieved through inhibition
of the glycoprotein-processing pathway of the
host cell.
25. No current effective vaccine is available.
T cells, play an important protective role in
controlling dengue virus infection, rather than
creating an aberrant response that can ultimately
worsen the disease as is the prevailing current
belief.
The current thinking in the field is that the goal
of a dengue vaccine should be the induction of
antibodies and not T cells.
Recent studies suggest that both cell types are
needed to produce a strong immune response
against dengue infection.
26. T cell responses in a large group of HLA-
defined Sri Lankan adults naturally infected
by dengue viruses found that T cell immunity
contributed to host protection rather than to
vascular permeability (which occurs in severe
cases).
Suggesting that T cell immunity may be
an important element of the strong cross-
protection that occurs in humans infected
with two or more dengue viruses.
27. Currently, no approved treatment or vaccine is available. The
precise mechanism of DHF/DSS pathogenesis and the
relationships between viral load, cytokine storm, coagulopathy,
and complement activation are uncertain.
why are the vast majority of individuals with either primary or
secondary DENV infections asymptomatic, and why do secondary
infections with DENV result in more severe illness than primary
infections?
Host genetic makeup is unknown.
The number of the studies and centers tripled in the last 5 years.
The prospects for safe, effective treatment, and/or vaccine are
promising (within 5 years).
