Dengue Virus Infection: the pathomechanism

Isra Wahid Dengue Unit, NEHCRI Faculty of Medicine, Hasanuddin University

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Common facts on dengue

A dengerous febril fever due to uncertainty A walking patient for hospitalization can come up with dehospitalisation of a death body, JUST WITHIN HOURS Would attack anyone, including the children of doctors that care dengue patients Do we aware with the following facts?
Isra Wahid July, 2011 NITD-Eijkman-Hasanuddin Clinical Research Initiative

Hundred of million of dengue viruses in patient body: Only 1 % viruses from outside, 99% are made by the patients body themself

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Our immune system is very effective againts DV, and clear the viruses just within days No carrier or chronic dengue

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Patient death not by the viruses, but patients immune responses

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Dengue Virus
Flavirus family RNA virus, withno DNA stage, 4 distinct serotype; DEN-1, DEN-2, DEN-3, DEN-4 Transmitted by Aedes mosquitoes

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

VIRAL STRUCTURES

Surface: 90 dimers E glycoprotein viral entryattachment, membrane fusion, antigenic for neutralizing Ab Membran: Lipid bilayer from host Core: Publish primers acids Capsid protein + Nucleic Lanciotti et al 1989 Genome: +ssRNA, 11.000 nt, no DNA stage

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Viral genome
A (+) sense ssRNA Structural P C prM E direct act as mRNA protein Nonstructural P NS1 NS2a-b NS3 NS4a-b NS5

10.500 nt
Single Open Reading Frame (OPF) one polyprotein 3400 aa
Cleavage by viral and host protease to assembly viral structure, and enveloped by host lipid bilayer when budding from host cytoplasma
Isra Wahid July, 2011 NITD-Eijkman-Hasanuddin Clinical Research Initiative

What acctually happen when dengue viruses enter our body?

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Virus entering blood circulation

Activate innate immune responses:
Cellular mediated immunity (phages, NK) Chemokines: INF, TNF, IL, complement

Triggering development of specific immune responses:

Acute phase: IgM Immune memory :
IgG CT cells
Isra Wahid July, 2011 NITD-Eijkman-Hasanuddin Clinical Research Initiative

Specific antibody responses

Virus has many epitopes to stimulate Ab production Each epitope stimulate one B cell clon to produce mAb Respond to a live virus will be polyclonal Ab response

Isra Wahid July 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

IgG Response to homolog subsequent infection,

Polyclonal Ab response Neutralizing Virus elimination

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

IgG Response to heterolog subsequent infection,

Mono or few clonal Ab responses

cross reactive Ab

Non-Neutralizing Enhancing attachment via Fc-r

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

What happen when dengue viruses enter the cell

HEEEIIII..... Come on and Copy me..... With YOUR OWN EXPENSES

Our bodys machines work for him

We produce the virus that again repeat the order

Isra Wahid July 2011 NITD-Eijkman-Hasanuddin Clinical Research Initiative

How Dengue virus replicate

S protein as mRNA
translasi

Polyprotein NS protein New Virion

+ssRNA

Enzymes Including Rd-RNA polymerase as genome template -ssRNA Progeny RNA

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Non neutralizing Ab-Enhance virus replication

Excessive immune response More severe clinical symptoms

Isra Wahid July 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Viral Load related with clinical severity

10 9 8 7 6 5 4 3 2 1 0 -5 -4 -3 -2 -1 0
DF DHF III DHF I/II 9 DF 8 7 6 10

Viremia Titer (logMID50/ml)

DHF I / II

DHF III / IV
DEN-1 DEN-2

Fever Day
Source: Vaughn et al., 2000 JID, 181:2- 9 Note: Day 0 is the defervescence day

Source: Welcome Trust, Topics in International Health Published May 2005

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Dinamic of IgM and IgG in Dengue Infection, Initial infection (primary) e.g. DV-1
Sudden onset of fever

Recovery

DV-1
Detectable level

Anti DV-1 IgM (90 days) Anti DV-1 IgG

2 NS1 IgM

15 Days

20

Isra Wahid July 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Subsequent infection (secondary) e.g. DV-1 (homotypic infection)

Sudden onset of fever

Recovery

Anti DV-1 IgG DV-1

Detectable level

Anti DV-1 IgM

2 NS1 IgG IgM?

15 Days

20

Isra Wahid July 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Subsequent infection (secondary) e.g. DV-2 (heterotypic infection)

Sudden onset of fever

Recovery

Anti DV-1 IgG DV-2 Anti DV-2 IgM

Detectable level

Anti DV-2 IgG

2 NS1 IgG IgM

15 Days

90

Isra Wahid July 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Severe cases related with secondary infection

Year Serotype DF cases DHF Cases

1977-1979

Den-1

500.000

none

1981

Den-2

344.000

1997

Den-2

5.200

10.300 (98% 2nd) No < 3 yo 205 No < 18 yo

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

A hypothesis for the immunopathology of vascular leakage in DHF IFN inhibitors ADE OAS

Apoptosis Cytokines Mediator release

Fink J, Gu F, Vasudevan SG Published in Rev. Med. Virol. 2006;16:263-275

Increase permeability PLASMA LEAKAGE

Platelet clearance -Ab binding virus -NS1 Ab cross react HEMORRHAGY

Genetic variation affect the extend of immune responses that lead to DHF
Isra Wahid July, 2011 NITD-Eijkman-Hasanuddin Clinical Research Initiative

Pathogenesis of Dengue Infection

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 Increase Permeability Plasma leakage Platelet clearance hemorrhagic: Ab-binding virus reacts with platelet Anti-NS1 Ab cross reacts with platelet Host variation

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

So, Who kill the patient?

Maybe you are killed 90% by your self through: - producing the virus, then - kill the virus with your imun responses - and also, abusely kill your own cells

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Pathogenesis-based diagnosis of dengue infection

Isra Wahid July 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Live virus

Cell Culture

Isra Wahid July 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

VIRAL REPLICATION
S protein as mRNA
translasi

Polyprotein NS protein New Virion

+ssRNA

Enzymes Including Rd-RNA polymerase as genome template -ssRNA Progeny RNA

Sources for diagnosis test

Isra Wahid July, 2011 NITD-Eijkman-Hasanuddin Clinical Research Initiative

Virus Protein Expression

5UTR
5-cap

+ strand RNA genome

Structural Nonstructural

3UTR

One poly-protein cut by enzimes to be fungsional and structural viral proteins NH2
C prM

Translation
COOH

NS1

2A 2B

NS3

4A

4B

NS5
Polymerase Methyltransferase Guanylyl transferase

Furin

pr M

Protease Helicase NTPase 5ter RNAPase

N2B/NS3 protease Signalase

Shi, 2002 Current Opinion in Investigational Drugs 3, 1567

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Isra Wahid July, 2011

Hosts Ab responses

In primary infection, the dominant antibody response is IgM, and characterised by high IgM and low IgG titres. A secondary infection is thus characterised by low IgM, and high and rapidly rising IgG antibody titres, the peak is 2 weeks, decrease slowly over a period of 3 - 6 months. IgM kinetics are much lower to those observed in primary infections

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Principes of Lab. Diagnosis for Viral Infection

Interstitial fluid Cytoplasma Nucleas Blood Cell Culture Ab capture Ag capture PCR, Gene sequencing Other host responses: IL, TNF, INF, Specific cell, etc..
NITD-Eijkman-Hasanuddin Clinical Research Initiative

Isra Wahid July, 2011

Diagnosis timing, dengue infection

Incubation In mosquito Incubation period Febrile period
Sudden onset of fever

Recovery Cytokine storm

IgG (2o)

IgM (90 days)

Viremia ~ -18 -7 0 7 15

IgG
20 Days

DHF/DSS
Modified from: Subhash G. Vasudevan, Dec 4, 2006 (NEHCRI Internal Training)

Isra Wahid July, 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Laboratory diagnosis for dengue

SEROLOGY
Anti Dengue IgM Anti Dengue IgG Dengue NS1

BIOMOLECULAR
One step RT-PCR / q RT-PCR Two step Nested RT-PCR Pr. DG DG specifik Pr. FLv DG specifik Genome sequencing C6/36 Cell BHK Cell Vero Cell PRNT

CYTOKINES
TNF IL 10 IL 1 IL 6 IL 2

CELL CULTURE

Isra Wahid July 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

Isra Wahid July 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative

However... With entomological approaches... we could reduce cases in Makassar for the last 5 years
Monthly cases
1200

1000

800

600

400

200

0 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

Isra Wahid July 2011

NITD-Eijkman-Hasanuddin Clinical Research Initiative