patomekanisme-infeksi-dengue.pdf

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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


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

Isra WahidJuly 2011


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Live virus Cell Culture

Isra WahidJuly 2011


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VIRAL REPLICATION

+ssRNA

as mRNA

asgenometemplate

Polyproteintranslasi

-ssRNA

NS protein

S protein

Progeny

RNA

EnzymesIncludingRd-RNA polymerase

NewVirion


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


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

Isra WahidJuly 2011


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NITD-Eijkman-HasanuddinClinical Research InitiativeIsra WahidJuly 2011


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