Dengue-Understanding The Virus

Dr Ravindran ThayanVirology Unit, Infectious Diseases Research Centre

Institute for Medical Research, Kuala Lumpur

Dengue and Dengue Hemorrhagic Fever

- caused by dengue virus (DEN-1 to 4)

- members of genus Flaviviridae

- other members include JE, YF, WNF)

-Increasing cases, rapidly spreading to new areas

-Changing demographics, urbanization, environment and global travel

Introduction

Dengue around the world

South

East Asia

52% of

global risk

According to WHO- 2.5 billion people at risk

• 96 million cases reported annually

• 500 000 hospitalized, about 25,0000 deaths

2010

Dengue in Malaysia

Dengue was first reported in Malaysia in 1902

Cases of DHF started in 1950s

1975- Dengue made a notifiable disease

In mid 1990s till now- cases occur throughout

the year

Currently the most important vector borne

disease in Malaysia and important contributor

to mortality and morbidity rates

Year Reported

cases

Fatality cases Percentage of

fatality cases

2009 41486 88 0.21

2010 46171 134 0.29

2011 19884 36 0.18

2012 21900 35 0.2

2013 43346 92 0.21

2014 108,698 215 0.20

2015* 85,488 234 0.27

Dengue situation in Malaysia

* Till 12 September 2015

6CPG Management of Dengue Infection in Adults (3rd Edition)

Dengue Serotypes in Malaysia (1990-2014)

Findings:

(1)Den 1 and Den 2 pre

dominate for the past

13 years

(2) Den 3 pre dominate in

early 1990s

(3) Den 4 –background

7CPG Management of Dengue Infection in Adults (3rd Edition)

0%

10%

20%

30%

40%

50%

60%

70%

DENV1 DENV2 DENV3 DENV4

Dengue Serotypes in Year 2013, 2014 and 2015 (Jan-June)

Findings:

Den 1 and Den 2

co-pre dominate

for the past 2

years

2013 2014 2015

The phenomenon of “double whammy” as both dengue 1

and dengue 2 co- predominate- rather unusual as usually as a

single serotype pre-dominates

Increases risk of secondary dengue infections

Long dengue epidemic- can result in the presence of a

“fitter” virus- able to evade immune response and stay “longer

in the community”

Den 3 (which used to pre dominate in the 1990s ) has not

been a major player since 2001.

Pathogenesis of Dengue

Host factors -Antibody Dependent

Enhancement- Host genetic-Blood group

- Co morbidities

Viral Factors - Dengue

serotype/genotype-mutation

-viral tropism- Fitter virus

Antibody Dependent

Enhancement (ADE)

ADE- refers to role of secondary dengue

infection in dengue pathogenesis

Presence of two types of antibodies- Enhancing

antibodies (“original antigenic sin”) and neutralizing

antibodies (against the current infection)

Virus gets transported elsewhere via Fc∂ receptor cells

and multiply further in target cells- triggers Cytokine

Storm- vascular dysfunction and plasma leak

(Whitehead et al, Nature Reviews Microbiology, 2007)

Are there a New Strain of

Dengue Virus?

-Phylogenetic tree results

Phylogenetic Tree for envelope gene

of Den 1 viruses

Genotype 1

Phylogenetic Tree for envelope

gene of Den 2 viruses

Cosmopolitan

Findings

Both group of patients (severe dengue and dengue

fever) patients share same genotypes

Mutations seen are not significant as yet.

Extend the target for sequencing- to whole virus

Current data do not support any association between

dengue genotypes and severe dengue

What about new strains/ viruses

more virulent?

Not clear yet- based on phylogenetic tree- no new or

novel strains introduced

There are presence of mutations, but not consistent

among different groups of patients, ie DF, severe dengue

or deaths

Even if there are mutations perceived as resulting in

more severe dengue- but was not seen in other people’s

work- lack of clear cut evidence

However there could be viruses that have developed

ability to be “fitter” (evade the immune system and

replicate)

Is there something from the

virus that causes increased

severity?

Is there a fitter virus?

What about prolonged viraemia?

Prolonged viraemia – associated with fitter

virus?

This happens in “extended dengue outbreak”

where virus develops “fitness” against herd

immunity

Results in increased severity?

Identification of Proteomics and

Genomic Markers in Dengue Patients

With Multi Organ Failure

A study started in June 2015, between GICU,

HKL and IMR:-

Preliminary Findings

For each pt, at least 3 samples are collected

serially- Febrile, Critical and Discharge

Out of 45 pts- 29 had dengue virus RNA

detected by Multiplex Real Time RT-PCR

Dengue serotype distribution:-

Den 1 = 16/45

Den 2 = 11/45

Den 3 = 2/45

Den 4 = 0/45

Negative: 16

19 out of 29 samples- virus cleared only just

before discharge- suggestive of prolonged

viraemia.

Viral Load for Den 1

by Real time RT-PCR

Highest load- 7.6 x 1011 at admission and cleared by

Day 7

On average takes about 4-6 days to clear ͠ depends on

initial viral load

Viral Load for Den 2

by Real time RT-PCR

Highest load- 2.19 x 108 at admission and cleared by

Day 4

On average takes about 2-4 days to clear ͠ depends on

initial viral load

Findings

Most patients show high viral load in the febrile stage,

during admission (19 of the 29 patients)

Prolonged viraemia were seen in 15 out of the 19

patients as virus was detected in the critical stage as well

All patients have undetectable levels of virus during

discharge from GICU

37 out of the 45 patients had at least 1 organ

involvement (mostly liver)

Prolonged viraemia could lead to severe dengue

Two papers suggest utility of NS1 as severe dengue

marker

Pt who had detectable NS1 between D5 & D6- 60%

developed severe dengue

Recommended in new Dengue CPG 2015

-To include NS1 as severe dengue marker (Day 5-6)

-To recommend to send liver biopsy in fatal cases

Supports ADE- (presence of enhancing ab vs

neutralizing ab)

NS1 and dengue

pathogenesis

Role of NS1 in dengue pathogenesis

The first of the seven non structural protein and found

secreted in blood. It is found in multiple oligomeric form,

depends on glycosylation status

Important role in pathogenesis by supporting early

viral replication, inhibition of platelet aggregation,

directing complement against endothelial cells

Inducing cell apoptosis and facilitating evasion of DV

particles from complement system-dependent

neutralization and inducing vascular leak with the help of

Toll-line receptor 4 (TLR4) (Thomas, Science Translational Medicine, Sept 2015)

NS1 in Endothelial Integrity

- Dengue virus infected

cells- secreted NS1

- Activate PBMC via TLR4 –

causing dose dependent

increase in mRNAs that

encode pro inflammatory

cytokine (TNF-a, IL6, IFN

b, IF1b, IL12)

- Disrupt endothelial

integrity

- Vascular leak

-There is correlation between the amount of NS1, degree of vascular leak

and production of pro inflammatory cytokines.

-Inducing cell apoptosis and facilitating evasion of DV particles from

complement system-dependent neutralization

-Application of antibody to TLR4 or use of a

TLR4 antagonist prevented the this and

maintain endothelial integrity

Suitable target for vaccine design/ drug target

What’s next?

Antigenic Diversity of

Dengue Viruses

The four genetically divergent dengue virus are

traditionally classified as serotypes

Antigenic and genetic differences among the dengue

serotypes influence disease outcome, vaccine-induced

protection, epidemic magnitude.

But based on antigenic maps constructed from

neutralizing antibody titers- that dengue virus are not

clearly homologous

Implication on vaccination, dynamics of immunity and

the evolution of dengue virus.

Phylogenetic Tree Amino Acid Map

Dengue serotype relationship- Amino acid differences

correspond to amino acid differences.

Antigenic differences as a result of AA changes IS NOT

sufficient. Phenotypic differences are important (Katzelnick

et al, Science, Sept 2015)

Antigenic Map of DENV

panel by antibody

neutralization assay

Dengue serotype-grouped according to current genomic

classification. But clustered more closely- indicating

close antigenic epitopes

Prior immunity to heterologous DENV –strongest risk

factor, but evidence shows that neutralizing responses

to a DENV lineage affects the severity.

Some protects as cross-type neutralizing ab. Important

findings for both natural and vaccine-derived protection

Conclusions

Both Dengue serotype 1 and serotype 2 co circulates

currently- increases risks of secondary dengue

infections

No clear cut evidence on the role of current

circulating dengue genotypes with severity of the

disease.

Prolonged viraemia indicates active viral replication,

may lead to severe dengue

NS1 is good indicator for severe dengue

Dengue serotype classification based on genomic

alone not accurate. Phenotypic changes matters.

Acknowledgement

Some of the studies were funded by MOH

NIH and Tabung Amanah grant