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NITD-Eijkman-HasanuddinClinical Research Initiative
Isra WahidDengue Unit, NEHCRI
Faculty of Medicine, Hasanuddin University
Dengue Virus Infection:
the pathomechanism
Isra WahidJuly, 2011
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Common facts on dengue A dengerous febril fever due to uncertainty
A walking patient for hospitalization cancome up with dehospitalisation of a deathbody, JUST WITHIN HOURS
Would attack anyone, including thechildren of doctors that care denguepatients
Do we aware with the following facts?
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Hundred of million of dengue viruses
in patient body:
Only 1 % viruses from outside,99% are made by the patients body
themself
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Our immune system is very effective againtsDV, and clear the viruses just within days
No carrier or chronic dengue
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Patient death not by the viruses, but
patients immune responses
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Dengue Virus Flavirus family
RNA virus, withno DNAstage,
4 distinct serotype; DEN-1,
DEN-2, DEN-3, DEN-4
Transmitted by Aedesmosquitoes
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VIRAL STRUCTURES
Publish primersLanciotti et al 1989
Surface:
90 dimers E glycoprotein viral
entryattachment, membrane fusion,antigenic for neutralizing Ab
Membran:
Lipid bilayer from host
Core:
Capsid protein + Nucleic acids Genome:
+ssRNA, 11.000 nt, no DNA stage
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Viral genome
C prM E NS1 NS2a-b NS3 NS4a-b NS5
Structural P Nonstructural P
10.500 nt
one polyprotein
3400 aa
Single Open Reading Frame (OPF)
Cleavage by viral and host protease to assembly viral structure, and enveloped
by host lipid bilayer when budding from host cytoplasma
A (+) sense ssRNA direct act as mRNAprotein
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What acctually happen when dengueviruses enter our body?
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Virus entering blood circulation Activate innate immune responses:
Cellular mediated immunity (phages, NK)
Chemokines: INF, TNF, IL, complement
Triggering development of specificimmune responses:
Acute phase: IgM
Immune memory : IgG
CT cells
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Virus has many epitopes
to stimulate Abproduction
Each epitope stimulateone B cell clon to
produce mAb
Respond to a live viruswill be polyclonal Ab
response
Isra WahidJuly 2011
Specific antibody responses
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IgGResponse to homolog subsequent infection,
Polyclonal Abresponse
Neutralizing
Virus elimination
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IgGResponse to heterolog subsequent infection,
Mono or few clonal
Ab responses
Non-Neutralizing
Enhancing
attachment via Fc-r
Isra WahidJuly, 2011
cross reactive Ab
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How Dengue virus replicate
+ssRNA
as mRNA
asgenometemplate
Polyproteintranslasi
-ssRNA
NS protein
S protein
Progeny
RNA
EnzymesIncludingRd-RNA polymerase
NewVirion
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Non neutralizing Ab-Enhance virus replication
Isra WahidJuly 2011
Excessive immune responseMore severe clinical symptoms
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Viral Load related with clinical severity10
9
-3-4-5
8
7
6
5
4
3
21
0
0-1-2
ViremiaTiter(logMID50
/m
l)
Fever Day
DHF III
DHF I/II
DF
Source: Vaughn et al., 2000 JID, 181:2- 9
Note: Day 0 is the defervescence day
6
10
7
8
9
DF DHF
I / II
DHF
III / IV
Source: Welcome Trust,
Topics in International Health
Published May 2005
DEN-1
DEN-2
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Dinamic of IgM and IgG in Dengue Infection,
Initial infection (primary) e.g. DV-1
Isra WahidJuly 2011
0 15 20
Anti DV-1 IgM (90 days)
Recovery
7
Days
Anti DV-1 IgG
Suddenonset of
fever
2
Detectablelevel
DV-1
NS1IgM
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Subsequent infection (secondary)
e.g. DV-1 (homotypic infection)
Isra WahidJuly 2011
0 15 20
Anti DV-1 IgG
Recovery
7
Days
Suddenonset of
fever
2
Detectablelevel
DV-1
Anti DV-1 IgM
NS1IgGIgM?
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Subsequent infection (secondary)
e.g. DV-2 (heterotypic infection)
Isra WahidJuly 2011
0 15 90
Anti DV-1 IgG
Recovery
7
Days
Suddenonset of
fever
2
Detectablelevel
DV-2Anti DV-2 IgM
Anti DV-2 IgG
NS1IgGIgM
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Severe cases related with secondary infection
Year Serotype DF cases DHF Cases
1977-1979 Den-1 500.000 none
1981 Den-2 344.000 10.300(98% 2nd)
No < 3 yo
1997 Den-2 5.200 205No < 18 yo
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A hypothesis for the immunopathology of vascular leakage in DHF
ADE
IFN inhibitors
OAS
Apoptosis
Cytokines
Mediator
release Platelet clearance-Ab binding virus-NS1 Ab cross react
Increase
permeability
Genetic variation affect the extend of immune responses that lead to DHF
Fink J, Gu F, Vasudevan SG
Published in Rev. Med. Virol.2006;16:263-275
HEMORRHAGY
PLASMA LEAKAGE
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Viral factors
Viral Load
Viral virulence(Replication rate, IFN inh)
Infection status:
primer / sekunder
Homotypic /heterotypic infection
Host Responses
Non-neutralising Ab
Antibody-Dependent Enhancement(ADE)
Cytokines release IncreasePermeability Plasma leakage
Platelet clearance hemorrhagic:
Ab-binding virus reacts with platelet
Anti-NS1 Ab cross reacts withplatelet
Host variation
Pathogenesis of Dengue Infection
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So, Who kill the patient?
Maybe you are killed 90% by your selfthrough:
- producing the virus, then
- kill the virus with your imun responses
- and also, abusely kill your own cells
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Pathogenesis-based diagnosis ofdengue infection
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Live virus Cell Culture
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VIRAL REPLICATION
+ssRNA
as mRNA
asgenometemplate
Polyproteintranslasi
-ssRNA
NS protein
S protein
Progeny
RNA
EnzymesIncludingRd-RNA polymerase
NewVirion
Isra WahidJuly, 2011
Sources for diagnosis test
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5UTR
Translation
Structural Nonstructural
+ strand RNA genome3UTR
5-cap
NH2 COOH
E 4A NS54BNS32B2ANS1prMC
pr M
Furin Protease
Helicase
NTPase 5ter RNAPase
Polymerase
Methyltransferase
Guanylyl transferase
N2B/NS3 protease
Signalase
Shi, 2002 Current Opinion in
Investigational Drugs 3, 1567
Virus Protein Expression
Isra WahidJuly, 2011
One poly-protein cut byenzimes to be fungsional
and structural viral proteins
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Hosts Ab responses
In primary infection, the dominant antibodyresponse is IgM, and characterised by high IgMand low IgG titres.
A secondary infection is thus characterised bylow IgM, and high and rapidly rising IgGantibody titres, the peak is 2 weeks, decreaseslowly over a period of 3 - 6 months.
IgM kinetics are much lower to those observedin primary infections
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Principes of Lab. Diagnosis for Viral Infection
BloodInterstitial fluid
Cytoplasma
Nucleas
CellCulture
Ab capture
Ag capture
PCR, Genesequencing
Other hostresponses:
IL, TNF, INF,Specific cell,etc..
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Diagnosis timing, dengue infection
Modified from: Subhash G. Vasudevan, Dec 4,2006 (NEHCRI Internal Training)
~ -18 0 15 20
IgM (90 days)
Incubationperiod
Febrileperiod
Recovery
7
Viremia
-7
IgG (2o)
Days
IgG
DHF/DSS
Suddenonset of
fever Cytokine storm
IncubationIn mosquito
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Laboratory diagnosis for dengue
SEROLOGY
Anti Dengue IgM
Anti Dengue IgG
Dengue NS1
CYTOKINES
TNF IL 10
IL 1
IL 6
IL 2
BIOMOLECULAR
One step RT-PCR / q RT-PCR
Two step Nested RT-PCR
Pr. DG DG specifik
Pr. FLv DG specifik
Genome sequencing
CELL CULTURE C6/36 Cell
BHK Cell
Vero Cell
PRNT
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