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PREFACE

First of all, I would like to thank God for the blessing and health so I could finish this

paper on time. I also want to thank my supervisor, Prof. Dr. Widyasari K, Sp. MK. for his

guidance and help on this paper.

Thanks to my family that has supported me during this writing. And thanks to my

friends for their helps. Without their helps and supports, I wouldnt be able to finish this paper.

This paper is far from perfect. There are a lot of mistakes in the writings, whether the

grammar or the theory. I hope after reading this paper, readers could give me some advice and

critics. Hopefully, with the critics and advice, I will be able to develop myself. I apologize for all

the mistakes Ive made in this paper.

I hope this paper could be useful to all the readers. Thank you.

Jakarta, January 2011

Aji Patriajati

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CONTENTS

Preface .. 1

Contents .. 2

Abstract .. 3

CHAPTER I Introduction ...... 4

I. 1 Background ...... 4

I. 2 Problems ...... 4

I. 3 Limitation of the Problems.. 5

I. 4 Objectives ...... 5

I. 5 Methods of Writing ...... 5

I. 6 Frame of Writing ...... 6

CHAPTER II Dengue Virus ...... 7

II. 1 Morphology ...... 7

II. 2 Genome Organization .................. 7

II. 3

II. 4

Replication cycle

Antibody Responce

......................................................

..

8

10

CHAPTER III Comparison NS1 with IgM ELISA Capture ...... 13

CHAPTER IV Conclusion ......... 16

BIBLIOGRAFI ..... 18

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Abstract

Dengue is a viral infection of humans that is transmitted by mosquitoes. Dengue is a very

important public health problem in many developing countries. Recently, new tests to help

diagnose patients with dengue have been developed. Evaluating these tests to see how well they

perform in different countries and in different health care settings is an important process that

helps to guide health care policy on whether these assays are likely to be useful in making a

diagnosis, and if so, when best to use them. Our hospital-based results, using two different types

of NS1 tests for diagnosing dengue, indicates that these tests are most sensitive when used during

the first 3 days of illness and are most likely to be positive if the patient has primary dengue. Our

results also show that a positive NS1 test result is a reflection of the amount of virus in the blood,

so that patients with high amounts of virus in the blood are more likely to be NS1 positive.

Collectively, the results indicate these NS1 tests deserve inclusion in the diagnostic approach to

dengue.

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CHAPTER I

Introduction

I. 1. Background

Dengue is a major public health problem in many parts of the tropical developing world

[1,2]. Dengue is caused by infection with one of four serotypes of dengue virus (DENV1-4),

which are arboviruses belonging to the Flaviviridae family. Although most DENV infections are

asymptomatic, a proportion result in clinically apparent disease that varies in severity from mild

undifferentiated fever through to more severe syndromes, primarily dengue haemorrhagic fever

(DHF) and dengue shock syndrome (DSS). DHF is a vasculopathy characterized by capillary

leakage and haematological dysregulation; in severe case hypovolaemic shock (DSS) may

develop. There are no licensed vaccines or specific antiviral therapies for dengue, and patient

management relies on good supportive care. Nowadays scientist develop Detection of the dengue

NS1 antigen during the symptomatic phase of illness represents an important advance in thediagnosis of dengue fever.

I.2. Problems

Dengue hemorrhagic fever, is an increasing cause of morbidity and mortality throughout the

tropical world. There are an estimated 50 to 100 million cases of dengue infection each year,

including about half a million cases of dengue hemorrhagic fever. The number of cases of bothdengue fever and dengue hemorrhagic fever has increased dramatically for the past few decades,

and the geographic range has extended to involve most tropical countries.

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I. 3. Limitation of the Problems

1. Early and accuratediagnosis can assist in patient management by directing clinicalattention to the appearance of major warning signs of severe or even life threatening

complications

2. Accurate dengue diagnosis prevents unnecessary and possibly expensive antibiotic usage

3. Prompt diagnosis of index cases can facilitate vector control activities in the community

so as to mitigate further transmission

4. Expanded use of accurate dengue diagnostics provides important data on the

epidemiology and health burden of dengue

I. 4. Objectives

The objectives of writing this paper are to describe :

Comparison Non-Structural Protein NS1 sero-types specific Ig-G with IgM dengue Blot

I. 5. Methods of Writing.

The writing of paper is carried out by a library research and also via internet.

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I. 6. Frame of Writing

Preface .. 1

Contents .. 2

Abstract .. 3

CHAPTER I Introduction ...... 4

I. 1 Background ...... 4

I. 2 Problems ...... 4

I. 3 Limitation of the Problems.. 5

I. 4 Objectives ...... 5

I. 5 Methods of Writing ...... 5

I. 6 Frame of Writing ...... 5

CHAPTER II Dengue Virus ...... 6

II. 1 Morphology ...... 6

II. 2 Genome Organization .................. 7

II. 3

II. 4

Replication cycle

Antibody Responce

......................................................

..

7

7

CHAPTER III Comparison NS1 with IgM dengue blot .... 9

CHAPTER IV Conclusion .......... 14

BIBLIOGRAFI .. 16

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CHAPTER II

Dengue Virus

Dengue is caused by one of four closely related virus serotypes of the genus Flavivirus, family

Flaviviridae, each serotype is sufficiently different that there is no cross-protection and

epidemics caused by multiple serotypes (hyperendemicity) can occur. The Dengue virus is a

member of the virus family Flaviviridae and is transmitted to people through the bite of the

mosquitoesAedes aegypti andAedesalbopictus.

II. 1 Morphology of the Virus

Electron micrographs showed that dengue virions are characterized by a relatively smooth

surface, with a diameter of approximately 500 , and an electron-dense core surrounded by a

lipid bilayer. In addition to the plus-sense RNA genome of 10,700 nucleotides, there are three

structural proteins that occur in stoichiometric amounts in the particle: core (C, 100 amino acids),

membrane (M, 75 amino acids), and envelope (E, 495 amino acids).

II.2 Genome Organization of Dengue Virus

Dengue Virus (DV) belongs to the family Flaviviridae. The four serotypes of dengue virus

(designated DEN-1, DEN-2, etc) can be distinguished by serological methods. Infection in

humans by one serotype produces life-long immunity against reinfection by that same serotype,

but only temporary and partial protection against the other serotypes. Dengue viruses share many

characteristics with other flaviviruses, having a single-stranded RNA genome surrounded by an

icosahedral nucleocapsid and covered by a lipid envelope. The virion is approximately 50nm in

diameter. The flavivirus genome is approximately 11kb (kilobases) in length, and the complete

genome sequence is known for isolates of all four serotypes of dengue virus. The genome is

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composed of three structural protein genes, encoding the nucleocapsid or core protein C, a

membrane-associated protein (M), an envelope protein (E) and seven non-structural (NS) protein

genes. The domains responsible for neutralization, fusion and interactions with virus receptors

are associated with the envelope protein. The order of proteins encoded is 5-C-prM(M)-E-NS1-

NS2ANS2B- NS3-NS4A-NS4B-NS5-3. NS1, a glycoprotein is detected in high titers in patients

with secondary dengue infections but its function is unknown. NS2 region, is known to code for

2 proteins (NS2A and NS2B), which are assumed to play a role in polyprotein processing. NS3,

the viral proteinase functions in the cytosol. NS4 region codes for two small hydrophobic

proteins involved in the membrane bound RNA replication complex establishment. NS5 codes

for a protein with a molecular weight of 105,000 and is the most conserved flavivirus protein.

This protein is assumed to be the virus encoded RNA dependent RNA polymerase. NS6 and NS7

function yet to be found.

A. Schematic of the single stranded RNA genome with highly structured RNA elements in

the 5' and 3' NTRs.

B. DV genomic organization and functions of viral proteins. For some proteins their

function in the viral life cycle is not yet established; they are marked with a ?

C. Putative membrane topology of DV proteins and proteinases involved in polyprotein

cleavage.

II.3 Dengue virus Replication cycle

Dengue virus (DV) particles bind to cells via interactions between the surface glycoprotein and

one or several poorly defined cellular receptor(s). In addition, particles may enter cells via Fc-

receptor upon opsonization. Virions are internalized by receptor-mediated endocytosis resulting

in release of the viral genome from the nucleocapsid in a low pH dependent manner. Soon after

infection, viral proteins induce rearrangments of intracellular membranes forming distinct

structures that have designated vesicle packets and convoluted membranes. It appears that

vesicle packets are sites of RNA replication that is probably catalyzed by a multi-protein

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complex composed of viral proteins, cellular membranes and presumably also cellular proteins.

DV RNA is replicated via a negative strand intermediate that serves as a template for the

production of excess amounts of positive strand progeny. Virus particles are thought to assemble

by budding into the ER and are transported through the host secretory pathway.

Within the mosquito, the virus replicates during an extrinsic incubation period of eight to twelve

days. The mosquito then bites a susceptible person and transmits the virus to him or her, as well

as to every other susceptible person the mosquito bites for the rest of its lifetime. The virus then

replicates in the second person and produces symptoms. The symptoms begin to appear in an

average of four to seven days after the mosquito bite this is the intrinsic incubation period, within

humans. While the intrinsic incubation period averages from four to seven days, it can range

from three to 14 days also. The viremia begins slightly before the onset of symptoms. Symptoms

caused by dengue infection may last for three to 10 days, with an average of five days, after theonset of symptoms. So the illness persists several days after the viremia has ended. Flaviviruses

vary widely in their pathogenic potential and mechanisms for producing human disease.

However, it is useful to consider them in three major categories: those associated primarily with

the encephalitis syndrome (prototype: St. Louis encephalitis), with fever-arthralgia-rash

(prototype: dengue fever), or with hemorrhagic fever (prototype: yellow fever). Human infection

with both mosquitoborne flaviviruses is initiated by deposition of virus through the skin via the

saliva of an infected arthropod. Virus replicates locally and in regional lymph nodes and results

in viremia.

Dengue viruses of all four serotypes cause three distinct syndromes: classic dengue fever, dengue

hemorrhagic fever, and dengue shock syndrome. Although caused by the same viruses, dengue

and dengue hemorrhagic fever are pathogenetically, clinically, and epidemiologically distinct.

Dengue viruses appear to replicate in macrophages at the site of the mosquito bite, in regional

lymph nodes, and then throughout the reticuloendothelial system. Viremia is concurrent with

clinical illness. Virus is present in the serum and in association with circulating monocytes.

Severe leukopenia is often present.

The mechanism by which flaviviruses enter the cells probably involves an interaction between

the E protein and cellular receptors, followed by a postattachment fusion event that occurs in

acidic intracytoplasmic vacuoles. Naked genomic RNA is infectious if introduced into the

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cytoplasm. The genomic RNA is capped but not polyadenylated; it serves as mRNA for all

proteins. Structural proteins are encoded at the 5' end of the genome, and

nonstructural proteins (e.g., NS-1 and RNAdependent RNA polymerase) are encoded in the

3'two-thirds. Complementary (negative-sense) RNA, made from genomic RNA, serves as a

template to generate genomic RNA. Replication occurs in the cytoplasm.

Virions are formed in perinuclear regions of the cytoplasm in association with Golgi or smooth

membranes. Virions appear within cytoplasmic vacuoles and appear to exit the cell as vacuoles

fuse with the plasma membrane. Unlike alpha viruses, no evidence of budding has been seen in

flavivirus infected cells, and the mechanisms of virion assembly and release remain obscure.

The 4 subtypes of dengue virus have 60-80% homology between each other. The major

difference for humans lies in subtle differences in the surface proteins of the different dengue

subtypes. After a person is infected with dengue, they develop an immune response to thatdengue subtype. The immune response produces specific antibodies to that subtypes surface

proteins that prevents the virus from binding to macrophage cells (the target cell that dengue

viruses infect) and gaining entry. However, another type of dengue virus infects the individual,

the virus will activate the immune system to attack the first subtype. The immune system is

tricked because the 4 subtypes have very similar surface antigens. The antibodies bind to the

surface proteins but do not inactivate the virus. The immune response attracts numerous

macrophages, which the virus proceeds to infect because it has not been inactivated. This

situation is referred to as Antibody-Dependent Enhancement (ADE) of a viral infection. This

makes the viral infection much more acute. The body releases cytokines that cause the

endothelial tissue to become permeable which results in hemorrhagic fever and fluid loss from

the blood vessels.

II.4 Antibody Responce

Dengue infection will result in lifelong immunity to that serotype, but only temporary immunity

to other serotypes.

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Primary Infection

1. IgM antibodies appear approximately 5 days after onset of symptoms and rise for the next

1-3 weeks.

2. IgM antibodies detectable for up to 6 months.

3. IgG are detectable at approximately 14 days after onset of symptoms and are maintained

for life.

Secondary Infection

Approximately 5% patients do not produce detectable levels of specific IgM.

1. IgM titer can be slower to rise in secondary infection.2. IgG appears approximately 2 days after symptoms appear.

3. IgG titre significantly higher in secondary infection.

The World Health Organization estimates that there may be 50 to 100 million cases of dengue

virus (DENV) infections worldwide every year, resulting in 250,000 to 500,000 cases of dengue

hemorrhagic fever (DHF) and approximately 25 000 deaths annually (Guzman and Kouri, 2002;

Halstead, 2007; Kyle and Harris, 2008). Four serotypes of DENV serotypes cause the disease in

humans (DENV-1 to DENV-4), producing a broad spectrum of illnesses, which ranges from

asymptomatic infection, undifferentiated fever, and classic dengue fever (DF) to the more severe

and sometimes fatal forms (DHF) and dengue shock syndrome (Deen et al., 2006). In addition,

other nonclassic clinical forms has been described, such as encephalitis and hepatitis (Deen et al.,

2006). One of the most challenging problems associated with management of the infected patient

is to achieve a rapid and specific diagnosis of DENV infection during the acute phase,

particularly in those countries where dengue coexists with other acute tropical febrile illnesses,

presenting with similar symptoms and signs. Thus, a specific and early diagnosis is determinant

to provide an adequate supportive and timely clinical treatment. Currently virologic diagnostic

methodsare based on virus isolation or detection of viral RNA in acute serum; however, both

methodologies are time consuming, expensive, and mainly restricted to reference laboratories.

Serologic tests, which rely on the detection of DENV-specific immunoglobulin M (IgM) and

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immunoglobulin G (IgG) antibodies by enzyme-linked immunosorbent assay (ELISA), are more

commonly used for dengue diagnosis. During the acute phase, the presence of IgM antibodies

alone suggests primary infection, whereas detection of both IgM and IgG antibodies is

suggestive of secondary or later infection. Nevertheless, detectable levels of IgM antibodies

appear approximately 4 to 6 days after the fever onset and remains in serum for 90 days

afterward. This late and persistent IgM response, together with the Flavivirus cross-reactivity,

restricts the efficacy of ELISA tests for the diagnosis of dengue infections (WHO, 2000, 2007).

Several studies have shown that the DENV nonstructural 1 (NS1) antigen, a highly conserved

glycoprotein, produced in both membrane-associated and secreted forms, is abundant in the

serum of patients in the early stages of DENV infection. Because of this, NS1 antigen constitutes

a suitable DENV biomarker, which can be detected before seroconversion and, therefore,

represents a new approach for the diagnosis of acute dengue infection (Alcon et al., 2002;Dussart et al., 2006; Libraty et al., 2002; Xu et al., 2006; Young et al., 2000)

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positive detection within the rst four days of illness. However, the NS1 antigen-capture ELISA

has the added advantage of continuing to give good detection rates up to seven days of the

illness. In this evaluation, the NS1 antigen-capture ELISA gave a signi cantly higher detection

rate in acute primary dengue than in acute secondary dengue. Despite the lower detection rate for

serum samples from patients with acute secondary dengue, the Platelia NS1 antigen-capture

ELISA still gave a higher detection rate than the other dengue diagnostic methods used in this

laboratory. Fig. 1 shows the positive detection rate of Platelia NS1 antigen-capture ELISA,

which on the whole, gave a higher detection rate than the other test methods at the various

sample ages. The sensitivity rate of IgM assay for early diagnosis of dengue was poor in the rst

three days of the illness, notwithstanding the presence of dengue speci c IgM was merely

indicative of recent dengue infection, and not con rmative of acute dengue infection. The

nding of this evaluation shows that no dengue speci c IgM was detected within the rst twodays of the fever and only 50% of patients had detectable dengue IgM in their sera, even at the

fth post-fever day. Thus, the Platelia NS1 antigen-capture ELISA should be considered as the

test of choice for patients suspected of acute dengue illness, especially those with fever lasting

ve days or less. For those patients with a history of fever for more than six days and are

suspected to have acute dengue infection, the test could also be considered concurrently with an

assay of dengue specic IgM.

Fig. 1 Positive detection rate of each dengue test method with respect to the sample age.

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The Platelia NS1 antigen-capture ELISA test has the prospect of wide usage for early

diagnosis of acute dengue virus infection in dengue endemic countries, since it uses the same

instruments as that of the dengue IgM-capture ELISA (MAC ELISA) test, which is normally

carried out in the hospital diagnostic laboratories. This study was limited by the lack of negative

controls to evaluate the speci city of the test kit. Further work is ongoing to evaluate the speci

city of the Platelia NS1 antigen-capture ELISA kit and the possibility of cross-reactivity with

NS1 antigens of other aviviruses. The possibility of a correlation between a high level of

circulating dengue NS1 antigen with the occurrence of dengue haemorrhagic fever, as

demonstrated by other studies, is also included in the ongoing evaluation work.(18-20)

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Chapter IV

Conclusion

Early laboratory diagnosis of acute dengue virus infection still remains a major problem

in many parts of the world especially in regions where dengue is hyper-endemic but resources

are limited. Three basic methods used commonly by most laboratories in resource rich countries

for the diagnosis of acute dengue virus infection are viral isolation and identication, molecular

detection of viral genomic sequence by a nucleic acid amplication assay, and detection of

dengue virus-specic IgM antibodies by IgM-capture enzyme-linked immunosorbent assay

(MAC-ELISA) and/or rapid dengue immunochromatography test device for detection of dengue

specic IgM. Although virus isolation and characterization are considered the gold standard

for laboratory diagnosis of acute dengue virus infection, it is expensive and at least 610 days are

required for the virus to replicate in tissue culture cells or laboratory mosquitoes (adult or

larvae). Reverse transcriptase-polymerase chain reaction (RT-PCR) is also an expensive method

and is not available widely, especially in hospital diagnostic laboratories in developing countries.

The usefulness of anti-dengue specic IgM assays depends on the time taken for the immuneresponse to produce IgM antibodies against dengue virus antigens. Thus both rapid dengue

immunochromatography test device for detection of dengue specic IgM (often considered as the

rapid test for the diagnosis of acute dengue infection) and MAC-ELISA do not provide early

diagnosis of acute dengue infection, as in most cases the rst detectable IgM only appears on

days 45 of the illness. A single serological detection of IgM is merely indicative of a recent

exposure to dengue virus and should not be interpreted as a diagnosis of acute infection without a

paired second serum sample for conrmation.

Recently, a highly sensitive and specic commercial dengue NS1 antigen-capture ELISA

kit has been evaluated and found to be better in comparison to virus isolation and RT-PCR for

early laboratory conrmation of acute dengue virus infection based on a single serum sample

(Kumarasamy et al., 2007). This NS1 antigen-capture ELISA has the prospect of wide usage for

early diagnosis of acute dengue virus infection since it uses the same instruments as for the

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dengue IgM-capture ELISA (MAC-ELISA). However, it is still limited by the need for

sophisticated instrumentation and higher technical skill which is normally only available in large

hospital diagnostic laboratories. A simple yet highly sensitive and specic rapid dengue test that

does not require instrumentation will be highly desirable

for wide application to conrm acute dengue even in an outpatient clinic setting or for

application in the eld. The nding of this study shows that the rapid dengue NS1 antigen

immunochromatography test device meets the intended purpose. With its high specicity

(99.5%) and positive predictive value (99.6%), this rapid immunochromatography test device is

highly recommended for use in a dened population group with clinical features suggestive of

acute dengue virus infection, but not as a routine screening test for an asymptomatic population.

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Bibliografi

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