VIRUS DIVERSITY AND THE EMERGENCE OF DENGUE

Hlaing Myat Thu

M.B.,B.S, M.Med.Sc.

Centre for Molecular Biotechnology, School of Life Sciences,

Queensland University of Technology, Brisbane, Australia.

A thesis submitted for the degree of Doctor of Philosophy of the Queensland University of Technology, 2004

DEDICATION

To my parents, my husband Win Maw and my daughter Thawdar (Mee Mee).

ABSTRACT

The aims of this study were to investigate the role of the diversity of dengue

virus populations in changing patterns of virus transmission and disease. Prior to the

commencement of this study, dengue 2 virus (DENV-2) had been associated most

frequently with severe disease, so the study commenced with this serotype. Because it

was not possible to quantitate diversity in the entire 11 kb of the viral genome, the

study focussed on the envelope (E) gene, because the E protein is the major protein on

the surface of the virion and thus might be under strong selective pressure from the

host immune system and from the requirement to engage specific receptors on host

cells. This study was the first direct quantification of the diversity of dengue virus

populations in individual hosts. The nucleotide sequences of more than 70 per cent of

the E genes in each virus population differed from the consensus nucleotide sequence

for the population. In the course of quantitating genetic diversity in DENV-2 virus

populations in patients and in mosquitoes, recombinant DENV-2 and both parental

virus populations were detected in a single mosquito. This was the first such report.

In 2001, just after the commencement of this study, Myanmar had the largest

outbreak of dengue on record. Unlike previous outbreaks, 95 per cent of dengue

viruses isolated from patients were of a single serotype, DENV-1. Despite the large

number of cases of dengue, the proportion of patients with severe dengue was low. In

the light of these observations, the direction of this study changed to focus on DENV-

1. Phylogenetic analysis of the E genes of DENV-1 collected before and after the

2001 dengue outbreak suggested that some time before 1998, an early lineage of

DENV-1 had become extinct and had been replaced by two new lineages. There was

no evidence that these changes were due to selection or to recombination within the E

protein genes of the old clade of viruses and the newly introduced viruses.

A more detailed analysis was undertaken, of the entire genome of 11 human

DENV-1 isolates and of 4 from mosquitoes recovered in Yangon between 1971 and

2002, to determine whether the extinction of the pre-1998 lineage of DENV-1 (clade

A) and the appearance of the two new lineages (clades B and C) could have been due

to selective pressures acting on genes other than E. Evidence of only weak selection

was found in the NS5 gene (at amino acids 127,135 and 669) but the resultant amino

acid changes did not distinguish all recent viruses from viruses belonging to the

extinct clade. The phylogenetic relationships between individual genes from these

viruses and between the open reading frames were similar. No evidence was found of

recombination that might have given rise to two new clades of virus with enhanced

fitness. Collectively, these data suggested that the extinction of clade A viruses and

their replacement by the two new clades, between 1998 and 2000 was a stochastic

event in an inter-epidemic period when rates of virus transmission were low. This was

the first report of such an extinction of a lineage of DENV-1 and its replacement by

new lineages.

At about the same time as the 2001 outbreak of DENV-1 infection in

Myanmar, an outbreak of DENV-1 began in the Pacific. A comparison of the

nucleotide sequences of the E genes of viruses from the Pacific with those of viruses

from throughout south-east Asia suggested that the outbreak in the Pacific was due to

the introduction of multiple genotypes of DENV-1 from Asia and that some of these

DENV-1 could have originated in Myanmar.

The principal observations from this study are: -

(a) Dengue virus populations in individual hosts are extremely heterogenous and may

contain a significant proportion of non-infectious genomes.

(b) Intra-serotypic recombination between dengue viruses may be far more common

than the literature suggests but it may not be detected because of the almost universal

use of consensus nucleotide sequences.

(c) Significant changes in dengue virus genotypes that occur at single localities may

be due to genetic bottlenecks rather than to selection or to recombination.

(d) Dengue viruses can be transported more than 10,000 km to cause outbreaks in

non-endemic areas.

Key words: Dengue viruses, diversity, recombination, selection, genetic bottleneck

LIST OF PUBLICATIONS

The following references are to publications and manuscripts prepared in conjunction

with this thesis.

Craig, S., Thu, H.M., Lowry, K., Wang, X.F., Holmes, E.C., and Aaskov, J (2003).

Diverse dengue type 2 virus populations contain recombinant and both parental

viruses in a single mosquito host. Journal of Virology, 77, 4463-4467. (Joint first

author)

Thu, H.M., Lowry, K., Myint, T.T., Shwe, T.N., Han, A.M., Khin, K.K., Thant, K.Z.,

Thein, S. and Aaskov, J.G (2004). Myanmar dengue outbreak associated with

displacement of serotypes 2, 3 and 4 by dengue 1. Emerging Infectious Diseases 10,

593-597.

A-Nuegoonpipat, A., Berlioz-Arthaud, A., Chow, V., Endy, T., Lowry, K., Mai, L.Q.,

Ninh, T.U., Pyke, A., Reid, M., Reynes, J.M., Su Yun, S-.T., Thu, H.M., Wong, S-S,

Holmes, E.C and Aaskov, J.G (2004). Sustained transmission of dengue virus type 1

in the Pacific due to repeated introduction of different Asian strains. Virology 329,

505-512.

Thu, H.M., Lowry, K., Jiang, L., Hlaing, T., Holmes, E.C. and Aaskov, J.G (2004).

Lineage extinction and replacement in dengue type 1 virus populations due to

stochastic events rather than to natural selection. Virology (accepted for publication).

TABLE OF CONTENTS

PAGE

TITLE PAGE 1 CERTIFICATE RECOMMENDING ACCEPTANCE 2 DEDICATION 3 ABSTRACT AND KEYWORDS 4 LIST OF PUBLICATIONS AND MANUSCRIPTS 7 TABLE OF CONTENTS 8 DECLARATION 9 ACKNOWLEDGEMENTS 10 CHAPTER 1. INTRODUCTION 1.1 A DESCRIPTION OF THE SCIENTIFIC PROBLEM INVESTIGATED 11 1.2 THE OVERALL OBJECTIVES OF THE STUDY 13 1.3 THE SPECIFIC AIMS OF THE STUDY 13 1.4 AN ACCOUNT OF SCIENTIFIC PROGRESS LINKING PAPERS 13 CHAPTER 2. LITERATURE REVIEW 18 CHAPTER 3. DIVERSE DENGUE TYPE 2 VIRUS POPULATIONS CONTAIN 59 RECOMBINANT AND BOTH PARENTAL VIRUSES IN A SINGLE MOSQUITO HOST CHAPTER 4. MYANMAR DENGUE OUTBREAK ASSOCIATED WITH DISPLACEMENT OF SEROTYPES 2,3 AND 4 BY DENGUE 1 66

CHAPTER 5. LINEAGE EXTINCTION AND REPLACEMENT IN DENGUE TYPE 1 VIRUS POPULATIONS DUE TO STOCHASTIC EVENTS RATHER THAN TO NATURAL SELECTION 73 CHAPTER 6. SUSTAINED TRANSMISSION OF DENGUE VIRUS TYPE 1 IN THE PACIFIC DUE TO REPEATED INTRODUCTION OF DIFFERENT ASIAN STRAINS 91 CHAPTER 7. GENERAL DISCUSSION 112

DECLARATION

The work presented in this thesis has not been submitted previously for a

degree at this or any other university to the best of my knowledge and belief, this

thesis contains no material previously published or written by another person except

where due reference is made.

Signed ..................................... Hlaing Myat Thu Date ........................................

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my supervisors Dr John

Aaskov and Dr Soe Thein for their invaluable advice and encouragement.

My thanks to the World Health Organization and the Wellcome Trust for

allowing me to undertake this project and providing the scholarship and research

funding.

I would also like to thank Dr Edward C. Holmes, from the Department of

Zoology, University of Oxford, Oxford, U.K. for helping me with analysis of my data.

My sincere thanks to the academic and administrative staff of the School of

Life Sciences, Queensland University of Technology for all their help and support.

To my friends and colleagues from the Arbovirology group (past and present),

CMB and CRC, all your help and support have made my hardships away from home

bearable.

My sincere thanks to Dr Kyaw Moe, deputy director and head of the Virology

Research Division and all the staff of the Virology Research Division and the Medical

Entomology Research division, Department of Medical Research, Yangon, Myanmar

for their support and help during sample collections.

Finally, to my parents, my husband Win Maw and my daughter Mee Mee, for

their love and continuous encouragement throughout my PhD.

CHAPTER 1

INTRODUCTION

1.1 A DESCRIPTION OF THE SCIENTIFIC PROBLEM INVESTIGATED

Dengue fever / Dengue haemorrhagic fever (DF/DHF) is caused by a

flavivirus, of the same name from the family Flaviviridae. DHF can progress from a

mild, non-specific, viral disease to irreversible shock and death within a few hours

and is the major cause of hospitalization and death in children in Southeast Asian

countries (Gubler, 1997; Gubler and Meltzer, 1999). The case fatality rate can be as

high as 44% (Rigau-Perez et al., 1998). In the last few decades, both the incidence of

dengue and the areas affected have increased dramatically (Monath, 1994). Currently,

more than 100 countries have endemic dengue (Gubler, 1998). The global burden of

DHF is estimated to be 750,000 disability adjusted life years (DALYs) per year per

million population (Meltzer et al., 1998).The burden of DHF in Myanmar was

estimated to be 83.8 DALYs per year per million population (Naing, 2000).

There are four antigenically distinct dengue virus serotypes (DENV 1, 2, 3 and

4) and multiple genotypes have been identified in each virus serotype (Holmes, 1998,

Lanciotti et al., 1994, Lanciotti et al., 1997, Lewis et al., 1993 and Rico-Hesse, 1990).

The increased epidemic activity and the co-circulation of multiple serotypes increases

the possibility of genetic changes and diversity in virus populations. Recombination

(Tolou et al., 2001, Uzcategui et al., 2001, Worobey et al., 1999), natural selection

(Twiddy et al, 2002, Bennett et al., 2003) and genetic bottlenecks (Sittisombut et al.,

1997, Wittke et al., 2002) have been implicated as factors contributing to diversity in

virus populations and to the emergence of new strains.

The vector responsible for transmitting dengue is the mosquito of the Aedes

species. Despite the huge effort put into mosquito control programs, today this species

is more abundant and widespread than at any time in human history (Halstead, 2000).

The World Health Organization designated the development of a safe and

effective tetravalent vaccine the most important approach for dengue prevention. For

a dengue vaccine to be effective, it must give protection against all dengue viruses in

all geographical locations (Wittke et al., 2002). The development of a live tetravalent

vaccine began in Bangkok, Thailand in the late 1970s (Bhamarapravati and Yoksan,

1997). Other approaches such as recombinant vaccines have been explored (Trent et

al., 1997). Despite a major effort put into vaccine development, with various

candidate vaccines being evaluated, a safe and effective dengue vaccine is not yet

available.

Myanmar is a typical dengue endemic country in Southeast Asia that has

experienced dengue outbreaks since 1970 (Ming et al., 1974). Myanmar is bordered

in the northeast by China, in the northwest by India, in the west by Bangladesh and in

the southeast by Thailand. The capital city, Yangon, has a population of three and a

half million. The Yangon Children's Hospital (YCH) is a large teaching hospital in the

central part of the city and is a major referral centre for most dengue patients. Because

of high rates of dengue transmission essentially in the paediatric population (Thein et

al., 1997), the YCH provides an ideal opportunity to study this health problem.

1.2 THE OVERALL OBJECTIVE OF THE STUDY

The objective of this study was to investigate the contribution of dengue virus

genetics to the changing patterns of dengue virus transmission and to dengue disease.

1.3 THE AIMS OF THE STUDY

1) To determine the genetic diversity within, and between, dengue virus populations

in sera from dengue patients and in mosquitoes.

2) To correlate the nature and magnitude of nucleotide and amino acid changes with

the frequency of transmission of virus from mosquitoes to humans.

3) To determine the causes of genetic changes and recombination events that give

rise to the emergence of new strains of virus.

1.4 AN ACCOUNT OF PROGRESS LINKING THE SCIENTIFIC PAPERS

The first publication describes the diversity of dengue virus type 2 (DENV-

2) populations collected from dengue patients attending the Yangon Children's

Hospital and in mosquitoes collected from the homes of patients. It also describes the

first example of recombinant and both parental dengue viruses in a single mosquito

host.

In 2001, the largest outbreak of dengue on record occurred in Myanmar. There

were 15,361 cases of dengue and 95% of the viruses isolated from patients were

DENV-1.The second publication in this thesis is a description of this 2001 outbreak

and an analysis of the viruses involved. A phylogenetic analysis of the E protein gene

of these and other DENV-1 isolates revealed that a lineage of DENV-1 had become

extinct some time between 1994 and 1998, before the outbreak, and that 2 new

lineages had emerged and were associated with this unusual epidemic in 2001.

Because of reports of selection acting on genes other than the E gene in other

dengue serotypes (Bennett et al., 2003), and in an attempt to reveal the cause/s

contributing to the extinction of the ancestral strain and emergence of new strains, the

complete genome of 15 DENV-1 representing ancestral (pre-1998) viruses and the

two post 1998 lineages were sequenced and analysed (Virology; accepted for

publication).

At about the same time as the dengue outbreak due to DENV-1 occurred in

Myanmar, an outbreak due to DENV-1 began 10,000 km away in the Pacific. This

provided an opportunity to investigate the relative contribution of local evolution and

introduction of new virus genotypes to outbreaks of disease. The outbreak in the

Pacific was due to multiple introductions of DENV-1 to the region, at least one of

which may have been from Myanmar (Third publication).

A study of the diversity of DENV-1 populations within humans and mosquito

hosts in Myanmar was also undertaken by myself and other scientists in the

Arbovirology group, QUT and will be published later. The data has not been included

in this thesis.

REFERENCES

Bennet, S.N., Holmes, E.C., Chirivella, M., Rodriguez, D.M., Beltran, M.,

Vordam, V., Gubler, D.J. and McMillan, W.O. (2003). Selection-driven evolution

of emergent dengue virus. Mol Biol Evol 20,1650-1658.

Bhamarapravati, N. and Yoksan, S. (1997). Live attenuated tetravalent dengue

vaccine. In Dengue and dengue haemorrhagic fever, pp. 367-377. Edited by Gubler,

D.J. and Kuno, G. CAB International, Oxford.

Gubler, D.J. (1997). Dengue and dengue haemorrhagic fever: its history and

resurgance as a global public health problem. In Dengue and dengue haemorrhagic

fever, pp. 1-22. Edited by Gubler, D.J. and Kuno, G. CAB International, Oxford.

Gubler, D.J. (1998). Dengue and dengue haemorrhagic fever. Clin Microbiol Rev 11,

480-496.

Gubler, D.J. and Meltzer, M. (1999). Impact of dengue/ dengue haemorrhagic fever

on the developing world. Adv Virus Res 53, 35-70.

Halstead, S.B. (2000). Global perspectives on dengue research. Deng Bull 24, 71-76.

Holmes, E.C. (1998). Molecular epidemiology and evolution of emerging infectious

diseases. British Medical Bulletin 54, 533-543.

Lanciotti, R.S., Lewis, J.G., Gubler, D.J. and Trent, D.W. (1994). Molecular

evolution and epidemiology of dengue-3 viruses. J Gen Virol 75, 65-75.

Lanciotti, R.S., Gubler, D.J. and Trent, D.W. (1997). Molecular evolution and

phylogeny of dengue 4 viruses. J Gen Virol 78, 2279-2286.

Lewis, J.A., Chang, G., Lanciotti, R.S., Kinney, R.M., Mayer, L.W. and Trent,

D.W. (1993). Phylogenetic relationships of dengue 2 viruses. Virology 197, 216-224.

Meltzer, M.I., Rigau-Perez, J.G., Clark, G.G., Reiter, P. and Gubler, D.J. (1998).

Using disability-adjusted life years to assess the economic impact of dengue in

Puerto-Rico: 1984-1994. Am J Trop Med Hyg 59, 265-271.

Ming, C.K., Thein, S., Thaung, U., Tin, U., Myint, K.S., Swe, T., Halstead, S.B.

and Diwan, A.R. (1974). Clinical and laboratory studies on haemorrhagic fever in

Burma, 1970-72. Bull.World Health Organ 51, 227-235.

Monath, T.P. (1994). Dengue: the risk to developed and developing countries. Proc

Natl Acad Sci U S A 91, 2395- 2400.

Naing, C.M. (2000). Assessment of Dengue hemorrhagic fever in Myanmar. South

East As J Trop Med and Pub Health 31, 636-641.

Rico-Hesse, R. (1990). Molecular evolution and distribution of dengue type 1 and 2

in nature. Virology 174, 479-493.

Rigau-Perez, J.G., Clark, G.G., Gubler, D.J., Reiter, P., Sanders, E.J., Vorndam,

A.V. (1998). Dengue and dengue haemorrhagic fever. Lancet 352, 971-977.

Sittisombut, N., Sistayanarain, A., Cardose, M.J., Salminen M.,

Damrongdachakul, S., Kalayanrooj, S., Rojanasupot, S., Supawadee, J. and

Maneekarn, N. (1997). Possible occurrence of a genetic bottleneck in dengue

serotype 2 viruses between the 1980 and 1987 epidemic seasons in Bangkok,

Thailand. Am J Trop Med Hyg 57, 100-108.

Thein, S., Aung, M.M., Shwe, T.N., Aye, M., Zaw, A., Aye, K., Aye, K.M. and

Aaskov, J. (1997). Risk factors in dengue shock syndrome. Am J Trop Med Hyg 56,

566-572.

Tolou, H.J.G, Couissinier-Paris, Durand, J-P., Mercier, V., de Pina, J-J., de

Micco, P., Billoir, F., Charrel, R.N. and de Lamballerie, X. (2001). Evidence for

recombination in natural populations of dengue virus type 1 based on the analysis of

complete genome sequences. J Gen Virol 82, 1283-1290.

Trent, D.W., Kinney, R.M. and Huang, CY.-H. (1997). Recombinant DNA

vaccines. In Dengue and dengue haemorrhagic fever, pp. 379-403. Edited by Gubler,

D.J. and Kuno, G. CAB International, Oxford.

Twiddy, S.S., Woelk, C.H. and Holmes, E.H. (2002). Phylogenetic evidence for

adaptive evolution of dengue viruses in nature. J Gen Virol 83, 1679-1689.

Uzcategui, N.Y., Camacho, D., Comach, G., Cuello de Uzcategui, R., Holmes,

E.C. and Gould, E.A. (2001). Molecular epidemiology of dengue type 2 virus in

Venezuela: evidence for in situ virus evolution and recombination. J Gen Virol 82,

2945- 2953.

Wittke, V., Robb, T.E., Thu, H.M., Nisalak, A., Nimmannitya, S., Kalayanrooj,

S., Vaughn, D.W., Endy, T.P., Holmes, E.C. and Aaskov, J.G. (2002). Extinction

and rapid emergence of strains of dengue 3 virus during an interepidemic period.

Virology 301, 148-156.

Worobey, M., Rambaut, A. and Holmes, E.C. (1999). Widespread intra-serotype

recombination in natural populations of dengue virus. Proc Natl Acad of Sci U S A 96,

7352-7357.

CHAPTER 2

LITERATURE REVIEW

EMERGING INFECTIOUS DISEASES

Sixty three per cent of all childhood deaths and 48 per cent of premature adult

deaths are due to infectious diseases (World Health Organization, 1998). Of the

seventeen million deaths, annually, from infectious diseases almost 41 per cent

occurred in the Southeast Asia region. In addition to the deaths, there is extensive

morbidity associated with infectious diseases. The World Health Organization and the

World Bank estimated the burden of disease due to infectious and parasitic diseases to

be 1830,000,000 disability adjusted life years (DALYs) for females and 1888,800,000

DALYs for males annually (World Development Report, 1993). Of this disability, an

estimated 750,000 DALYs resulted from dengue haemorrhagic fever each year

(Gubler and Meltzer, 1999). Newly emerging and re-emerging infections will add to

the already heavy global burden of infectious diseases (World Health Organization,

1998).

THE EMERGENCE OF DENGUE

Dengue/ dengue haemorrhagic fever (DHF) is a global health problem

and one of the most important emerging tropical viral diseases of humans (Monath,

1994). About 2.5 billion people are at risk of infection with dengue viruses and there

is more morbidity and mortality associated with dengue than any other arboviral

disease (Gubler, 1997). Prior to 1970 only nine countries had experienced epidemics.

By 1995, the number had increased more than four fold (Gubler, 1998). In 1998, 1.2

million cases of dengue and DHF including 3442 deaths were reported to the World

Health Organization (Halstead, 2000).

In the last few decades, both the incidence of dengue and the areas affected

have increased dramatically and hyperendemic transmission, that is the co-circulation

of multiple serotypes has been established in many countries (Monath, 1994).

Currently, more than 100 countries have endemic dengue and DHF has been reported

from over 60 countries (Gubler, 1998). It has been estimated that there are 50-100

million cases of dengue fever and 500,000 cases of DHF every year (Gubler and

Meltzer, 1999).

The factors responsible for this global emergence and spread include rapid and

uncontrolled population growth, unplanned urbanization with substandard housing,

crowding, deterioration in water supplies leading to the need for water storage in

containers and insufficient waste disposal management systems. These factors have

created ideal conditions for increased transmission of mosquito-borne diseases. The

increase in international travel and growing trade and tourism also provide ideal

mechanisms for the transport of viraemic hosts between population centres of the

world (Gubler, 1997).

Events contributing to changes in the virus itself resulting in the formation of

new, more virulent viral strains, may also be a contributing factor to the increase in

outbreaks of dengue (Leitmeyer et al., 1999). The dengue outbreaks occurring in

Cuba before 1981 were associated only with dengue fever, but after 1981, with the

introduction of a Southeast Asian strain of virus, outbreaks occurred which were

associated with very high morbidity and mortality. In the 1981 outbreak, a total of

344,203 cases were reported among which 10,312 cases were severely ill (Kouri et

al., 1989, Watts et al., 1999).

THE ECONOMIC IMPACT OF DENGUE

Children suffering from DHF/DSS, have an average hospital stay of 5-10

days. Intensive care including intravenous fluids, blood or plasma transfusion and

medicines such as antipyretics and oral rehydration salts are required for severely ill

patients. Adults may need to miss work in order to care for their children during

illness. The cost of the 1994 DHF outbreak in Thailand was estimated to be between

US$19.3 million to US$ 51.49 million. The 1994 dengue outbreak in Nicaragua

which had an estimated 60,916 cases was thought to cost US$2.7 million (Meltzer et

al., 1998). The estimated cost of the 1997 epidemic in Puerto Rico ranged from US$

6.1 to $ 15.6 million. In 1996 and 1997, twenty-three countries in the South American

region invested US$ 331 million and US $671 million respectively, on control

programs (Guzman and Kouri, 2003). The total cost of the 1981 Cuban DHF

epidemic alone was estimated to be US$103 million. There was a large scale

environmental sanitation campaign all of which cost US$ 43 million plus US$41

million for medical services (Kouri et al., 1989, World Health Organization, 1997).

The global burden of DHF is estimated to be 750,000 disability adjusted life

years (DALYs) per year per million population (Meltzer et al., 1998). The burden of

DHF in Myanmar from 1970-1997 was estimated to be 83.8 DALYs per year per

million population (Naing, 2000).

DENGUE VIRUS AND THE VECTOR MOSQUITO

Dengue is caused by a virus of the same name which has four antigenically

distinct serotypes (DENV-1, 2, 3 and 4). Dengue belongs to the genus Flavivirus of

the family Flaviviridae, a family of viruses including about 70 different viruses.

Dengue virus infections may be asymptomatic or result in a disease spectrum ranging

from a mild febrile flu-like illness known as dengue fever (DF) characterized by

fever, joint pain and retro-orbital pain to fever with haemorrhage known as dengue

haemorrhagic fever (DHF). According to the classification by the World Health

Organization, there are four grades of DHF. Grade I with skin haemorrhage or a

positive tourniquet test, Grade II with bleeding from orifices, Grade III with

impending shock and Grade IV with circulatory collapse and cardiac failure. Grade III

and IV are designated as dengue shock syndrome, DSS (World Health Organization,

1997).

The mosquito vector Aedes is responsible for the transmission of dengue,

although other routes of transmission such as mucocutaneous transmission through

contact with patients’ blood has been reported (Chen and Wilson, 2004). The

primitive enzootic transmission cycle of dengue viruses occurs in the rain forests of

Asia and Africa and involves Aedes mosquitoes and lower primates (Gubler, 1998).

The Aedes mosquitoes involved in the transmission of dengue include Ae. aegypti

usually in an urban environment , Ae. albopictus usually in rural settings and Ae.

polynesiensis in the Pacific. The urban endemic/ epidemic cycle which involves. Ae.

aegypti - human- Ae. aegypti is the most important transmission cycle from a public

health point of view and occurs in most large urban centres and is the major cause of

huge epidemic outbreaks in the tropics (Gubler and Meltzer, 1999). This mosquito

breeds mostly in artificial water containers around human habitation. The female

mosquito acquires the virus by feeding on a viremic human and once infected,

probably is able to transmit virus for her life, the average life span being 8 to 15 days

and the flight range for females is about 30 to 50 metres per day (Rodhain and Rosen,

1997). The reason Ae. aegypti is such an efficient epidemic vector is because of its

multiple feeding habit. The infected Ae. aegypti females will often feed on several

persons during a single blood meal and thus may transmit dengue virus to multiple

persons in a short period of time. It is not uncommon to see several members of the

same household contract dengue infection within 24 to 36 hours (Gubler, 1997).

Three to 14 days after being bitten by an infective mosquito, the person will undergo

an acute febrile viremic stage for an average of 5 days during which the virus

circulates in the peripheral blood. If an uninfected mosquito bites this person during

the viremic stage, it may become infected and transmit the virus to other uninfected

persons. When compared to yellow fever which is also transmitted by Ae. aegypti ,

dengue viruses are better suited to the urban environment and no longer seem to

require a sylvatic host for their reservoir. All four serotypes of dengue virus can give

rise to epidemic outbreaks throughout the tropical region wherever Ae. aegypti is

present in high densities (Gould et al, 2001).For infection and transmission to be

sustained in the vector mosquito, virus titers in the blood of human hosts must exceed

105 –107 virus particles per ml (Monath, 1994). The vector itself is thought to function

as an important biological filter for maintaining virus titers at a high level, since only

virus strains that replicate efficiently in humans and produce high viremias have the

potential to be transmitted to mosquitoes (Monath, 1994) and high viraemia has been

a factor suggested in generating severe dengue disease (Vaughn et al., 2000 ). In

periods of low virus transmission, virus may survive through vertical transmission

from parent to progeny (Khin and Than, 1983). Although Ae.aegypti is the dominant

peri-domestic vector of dengue viruses, evolutionary studies suggested that Ae.

albopictus may be the original vector and that dengue viruses from sylvatic

progenitors were transmitted from canopy-dwelling Aedes mosquitoes to Ae.

Albopictus and then later to Ae.aegypti (Wang et al., 2000).

For decades, mosquito control has relied on the application of insecticides to

larval habitats, destroying unwanted water containers and on health education. These

measures have been complemented by insecticide spraying targeting adult mosquitoes

during epidemics (World Health Organization, 2000).

HISTORICAL BACKGROUND

The earliest records of dengue-like illness come from the Chin Dynasty in AD

265 to 420. In a Chinese encyclopedia of disease symptoms and remedies, the disease

was called water poison. Outbreaks which could have been dengue also were reported

from the French West Indies in 1635 and in Panama in 1699. Some epidemics of

dengue-like illness also were reported in 1748-88 in Spain, 1818 in Peru, 1836 in

India, 1837 in Bermuda, 1850-51 in Texas to Florida, 1889-1890 in Taiwan, 1896-99

in Charters Towers and Brisbane, Australia (Gubler, 1997).

The origin of the name dengue is unclear and this illness has been called many

names over the years. In 1801, the Queen of Spain, Maria Luisa was reported to have

a febrile disease called "dengue". However, the most likely origin of the word is from

Swahili. In the epidemics occurring in 1823 and 1870, in Zanzibar on the East African

coast, the disease was called Ki-Dinga pepo, which meant a disease, caused by an evil

spirit and characterized by a sudden cramp-like seizure (Gubler, 1997).

EPIDEMIOLOGY OF DENGUE / DHF

The first DHF outbreak was reported in Charters Towers in 1896. In the

Southeast Asia region, the first DHF outbreak occurred in Manila, the Philippines, in

1953-54. After the first epidemic, a pattern of epidemic activity was established every

3-5 years in most Southeast Asian countries (Gubler and Meltzer, 1999). DHF later

spread west to India, Pakistan, Sri Lanka and east to China. The first evidence of DHF

in the South Pacific was a small outbreak of DEN-3 in Tahiti in 1965. After that

outbreak epidemic DHF was not seen in the South Pacific for 25 years.

In the 1950s and 1960s, most countries in Central and South America were

certified to have eradicated Ae. aegypti through the efforts of a coordinated mosquito

control programme (Rigau-Perez et al., 1998). However, in the late 1970s, there was

reinfestation of Ae. aegypti. In 1977, dengue 1 was reported for the first time causing

a widespread DF epidemic which spread from Jamaica to Cuba, Puerto Rico and

Venezuela (Guzman and Kouri, 2003). It then spread to the rest of the Caribbean

countries, Mexico, Central America and northern South America (Gubler, 1998).

With the introduction of a Southeast Asian strain of DEN 2 in 1981, Cuba

experienced one of its largest outbreaks of DHF with 344,203 cases (Kouri et al.,

1989). There also were reports of the spread of DENV-3 from the Indian sub-

continent into Africa in the 1980s and then to Latin America in the 1990s (Messer et

al., 2003). In 2002, more than 1,000,000 cases of DF were reported from more than

30 countries in Latin America. 17,000 cases of DHF, including 225 fatal cases, were

reported from 20 of these countries (Guzman and Kouri, 2003).

CURRENT GLOBAL STATUS OF DENGUE

Fig 1. Global distribution of DEN/DHF and the principal mosquito vector,

Aedes aegypti (Centres for Disease Control, 2000)

DENGUE IN MYANMAR (BURMA)

The first DHF epidemic occurred in 1970 in Yangon, the capital city

(Ming et al., 1974). It spread to other states and divisions (Myanmar is divided into 14

states and divisions) from the beginning of 1975 (Hlaing and Thein, 1983). A total of

83381 cases and 3242 deaths were recorded from 1970 to 1995. The burden of DHF

in Myanmar from 1970-1997 was estimated to be 83.8 DALYS per year per million

population (Naing, 2000). DHF is endemic in Myanmar with a 3-5 year epidemic

cycle. The incidence has been increasing over the past 20 years with an upward trend

(Mon et al, 1998). In 2001, Myanmar experienced its largest dengue 1 outbreak on

record (Thu et al., 2004).

Table1. DF and DHF cases in Myanmar from 1991- 2003 Year No. of cases No. of deaths Case fatality rate % Remarks

1991 6772 282 4.16 1992 1685 37 2.19 1993 2279 67 2.94 1994 11,647 444 3.81 Epidemic 1995 2477 53 2.14 1996 1854 18 0.97 1997 4005 82 2.05 1998 12,668 192 1.51 Epidemic 1999 5828 88 1.51 2000 1884 15 0.74 2001 15,361 193 1.32 Epidemic 2002 16,047 170 1.05 2003 7,907 78 0.98 (Data from Vector Borne Diseases Control, Ministry of Health, 2003).

PATHOGENESIS OF DENGUE / DHF

There are two distinct hypotheses to explain severe disease caused by

infection with dengue viruses antibody-mediated enhancement and viral virulence.

However, the most plausible explanation now seems to be a combination of both.

Studies in Thailand revealed that heterotypic antibodies from a primary dengue

infection contributed to a higher risk of developing severe disease (DHF/DSS)

following a second infection with dengue virus (Burke et al., 1998). The mechanism

for this phenomena could be dengue virus infection being augmented in Fc receptor

positive cells through non-neutralizing cross reactive antibodies, a process called

antibody mediated enhancement (Halstead, 1970). The DHF epidemic associated with

dengue 2 infections in Cuba in 1981 following a dengue 1 epidemic in 1977,

supported the concept that secondary infection is an important risk factor for severe

disease. Children born after the epidemic in 1977 did not develop DSS when infected

with dengue 2 in 1981 (Guzman and Kouri, 2003). However, this mechanism could

not account for primary infections in patients without antibody to dengue virus and

also the ability of dengue virus to infect cell types that do not express Fc receptors

(Klicks et al., 1989, Morens and Halstead, 1987). It also is possible that dengue virus

specific memory T lymphocytes recognize epitopes on viral proteins after secondary

infections and these cross reactive T lymphocytes release cytokines and other

mediators which contribute to severe disease by promoting plasma leakage and

abnormalities in homeostasis (Rothman and Ennis, 1999).

Specific dengue virus serotypes and genotypes have been associated with

severe disease. A study by Thein et al. (1997) determined that DENV 2 virus had

been associated with severe disease in Myanmar more often than the other three

dengue virus serotypes. Eighty nine per cent of dengue shock syndrome (DSS) cases

were associated with DEN 2. This has also been seen in prospective epidemiological

studies in Thailand (Vaughn et al., 2000). The sequence of virus serotypes have

important associations with severe disease. Although all four serotypes of dengue

viruses have been associated with severe disease, DEN 2 and less frequently DEN 3

have been most commonly detected in secondary infections leading to severe disease.

Some of the sequences of infection seen in association with outbreaks are DEN 1/

DEN 2 in the 1981 Cuban outbreak, DEN 3/DEN 2 in the 2000 El Salvador outbreak,

DEN 1/DEN 3 in Cuba 2001-2002 and DEN 2/DEN 3 in Brazil 2001 (Guzman and

Kouri, 2003). Transmission of dengue viruses in Cuba and some other Carribean

islands may have ceased because of the elimination of a source of susceptible hosts.

Specific genotypes have been associated with severe disease especially in the

Americas (Rico-Hesse et al., 1997). The American DENV 2 and DENV 3 genotypes

had not been associated with haemorrhagic fever. DHF caused by DENV 2 was only

experienced in Cuba when a South East Asian strain of DENV 2 was introduced in

1981. The dengue experience in Peru also demonstrated the importance of genotypes.

It was seen that the sera from dengue 1 immune individuals neutralized the American

genotype of DENV 2 more effectively than the Asian genotype. On sequencing the

full genome of eleven dengue 2 viruses, there were sequence differences which were

associated with the potential to cause severe disease. There were six amino acid

changes found in the prM, E, NS4B and NS5 genes and also some nucleotide changes

seen in both 5' and 3' untranslated regions which were associated with RNA

secondary structure changes (Leitmeyer et al., 1999). Cologna and Rico-Hesse,

(2003) substituted the American DEN 2 genotype E390 (previously thought to be the

sole virulence marker) together with 5' and 3' UTRs for those of the Southeast Asian

genotype sequence and found that viral replication only increased when all three

Southeast Asian genotype structures were present. Similarly, the American DEN 3

genotype in the 1960s was not associated with severe disease. The DEN 3 genotype

which is similar to the DEN 3 strains from Sri Lanka and India had been associated

with DHF epidemics in the Americas (Gubler, 1997).

STRUCTURE AND REPLICATION OF DENGUE

C prM E ns1 ns2a ns2b ns3 ns4a ns4b ns5

3’ UTR5’ UTR

Immature virion Mature virion

E dimer

M

C

Lipid bilayer

E

prM

Fig 2. Dengue virus genome organisation and virion structure.

Dengue virus has a genome of approximately 11Kb and the complete

nucleotide sequence is known for the genome of isolates of all four serotypes. The

genome codes for three structural proteins [the capsid protein (C), a membrane

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

proteins which are flanked by the two untranslated regions (5' and 3' UTR). The order

of proteins encoded in a single open reading frame are 5'-C-prM (M)-E-NS1-NS2A-

NS2B-NS3-NS4A-NS4B-NS5-3' (Rice et al., 1986).

Viral polyprotein processing involves both host and viral proteases. The C-

terminal hydrophobic membrane anchor sequence in C, prM and E proteins is

followed by charged amino acids (Chang, 1997). These C-terminal anchor domains

apart from being membrane anchor domains, take part in the transfer of the

polypeptide into the lumen of the rough endoplasmic reticulum (ER) by acting as

internal signal sequences. The charged amino acids serve as a signalase cleavage

recognition site by the host cell signal peptidase in the ER lumen. The host signalase

in the lumen of the ER mediates the initial processing events separating the structural

protein precursors and N-terminus of NS1 (Markoff, 1989). Penetration of host cells

and uncoating of the virion occur by endocytosis. When the virus enters the cell, the

nucleocapsid is uncoated by acid dependant fusion of viral and endosomal membrane

and once uncoating is complete, replication proceeds with immediate translation of

the uncoated viral genome (Chang, 1997).

The capsid protein lies at the 5' end of the genome. It is cleaved by the

NS2B/NS3 protease to generate the mature capsid protein and this is involved in

packaging of genomic RNA. Studies on DEN 4 show that the 21 amino acid residues

(45 to 65) which comprise the internal hydrophobic segment serve as a membrane

anchor domain and are also thought to play a role in viral assembly (Markoff et al.,

1997). The hydrophobic residues (100 to 113) which precede the N-terminus of the

prM act as a candidate signal sequence for translocation and cleavage of both prM and

E (Markoff et al., 1989). The membrane protein has 2 forms: prM which is found in

intracellular immature virions and M found in extracellular mature virions. The prM

is processed to M by a cellular protease. Only the mature virions containing M are

able to mediate the pH- dependent membrane fusion (Guirakhoo et al., 1993). The

hydrophobic residues, 246 to 279 and 735 to 773 in the C-termini of the prM and E

proteins, serve as candidate transmembrane signals for the translocation of the E and

NS1 proteins, respectively. The E protein is the major protein component on the

surface of the virion and comprises 494 to 501 amino acids. It is capable of inducing a

protective immune response and haemagglutination of erythrocytes (Mandl et al,

1989). The part of the domains which are exposed on the outer surface would be more

likely to have an effect on the immunological response of the host to the virus than the

other functions of the protein (Seligman and Bucher, 2003). The E protein is also

responsible for mediation of virus specific membrane fusion in acid pH endosomes

(Guirakhoo et al., 1993) and interaction with virus receptors and virus assembly. Two

potential glycosylation sites, Asn-67 and Asn-153, are present in the E protein of all

four dengue serotypes (Chang, 1997). The structural and functional organization of

the E protein is of central interest for the understanding of the biology of flaviviruses

including dengue (Mandl et al., 1989).

Studies of dengue virus binding to target cells have shown that a site occurring

between amino acids 281 and 423 of the envelope protein was responsible for the

binding of virus to cells which did not have Fc receptors (Chen, 1996). There are

three distinct regions in the E protein namely (1) the distal face of domain III where

mutations are likely to affect cell attachment (2) the contact between the domain I /

domain III interphase and (3) the base of domain II which is likely to influence

virulence by affecting the low-pH conformational transition (Rey et al., 1995).

The NS1 protein contains 353-354 amino acids. It has at least one conserved

N-linked glycosylation site at position 208 or 209 of all flaviviruses and also contains

12 conserved cysteine residues. The N- linked glycosylation is believed to be

important in the processing and secretion of NS1. It has also been suggested that NS1

plays a role in virion maturation and the synthesis of the negative strand in the RNA

replication cycle (Lindenbach and Rice, 1997).

NS2A is a small hydrophobic protein which is suggested to have a role in

viral assembly (Liu et al., 2003). The NS2B protein forms a complex with NS3 to

become the viral serine protease. There are 40 hydrophilic amino acid residues in

NS2B essential for protease activity. This NS2B-NS3 complex cleaves NS2A/NS2B,

NS2B/NS3, NS3/NS4A and NS4B/NS5. These viral protease sensitive sites are

characteristically found after a pair of basic amino acids or before a small side chain

amino acid ( Falgout et al., 1993).

The nonstructural protein NS3 is a multifunctional protein which has

protease, NTPase and helicase activities. The protease activity is found within the 167

N-terminal residues and the C-terminal region contains functional motifs which have

both NTPase and helicase activity (Li et al, 1999). A proteolytic cleavage of NS3 in

the helicase domain may have a role in regulation of RNA replication (Arias et al.,

1993).

NS4A is also a small hydrophobic protein thought to have an interaction with

NS1 and plays a significant role in RNA replication (Lindenbach and Rice, 1999).

NS4B is suggested to maintain the balance between efficient replication of the virus

in the two hosts, humans and mosquitoes. It was seen that a single mutation at

nucleotide 7129 of the NS4B in dengue virus type 4 decreased replication in mosquito

cells (C6/36 cells) whereas increased replication was observed in simian Vero cells

(Hanley et al., 2003).

The NS5 protein has two distinct functions, the N-terminal region has the

methyltransferase domain (Koonin, 1993) and the C-terminal domain has eight

conserved motifs which have RNA- dependent RNA polymerase activity which is

essential for virus replication (Koonin, 1991, Tan et al., 1996).

The untranslated regions of positive stranded RNA viruses are folded in

stable secondary stem loop structures that interact with viral or cellular proteins. At

the 5' untranslated region (5' UTR), ribosomes bind to the 5' terminus of the positive

strand to initiate translation. Mutations that modify the structures in the untranslated

regions were found to affect virulence or cause attenuation (Cahour et al., 1995,

Durbin et al., 2001). A study on dengue 4 viruses revealed that the RNA transcripts

with deletions altering the local base-pairing in the 5'UTR did not yield infectious

virus (Cahour et al., 1995). A single nucleotide mutation in nucleotide position 57 of

the 5'UTR of a Southeast Asian strain derived virus resulted in a lowered efficiency to

replicate in mosquito cells (Butrapet et al., 2000). In polio virus, a single nucleotide

mutation in the 5' UTR was found to be associated with neurovirulence (Evans et al.,

1985) and attenuation resulted when the secondary structure was disrupted

(MacAdam et al., 1992). In Sindbis virus, an Alphavirus, 51 nucleotides of the

5’UTR are thought to be important for viral replication in mosquito cells. Mutations

in this region had no apparent effect on virus replication in cells of vertebrate origin

but had deleterious effects on replication of virus in mosquito cells (Fayzulin and

Frolov, 2004).

The 3' untranslated region (UTR) in flaviviruses contains a highly

conserved secondary structure which is thought to play an important role in viral

replication and transcription (Proutski et al., 1999). A hypervariable region was

identified in the sequence immediately following the NS5 stop codon ( Shurtleff et al,

2001). Leitmeyer et al., (1999) suggested that the 3'UTR had a role in determining

virus virulence since a particular secondary structure was correlated with isolates

from dengue haemorrhagic fever (DHF) patients. It was seen that a live attenuated

dengue virus type 1 vaccine candidate with a 30-nucleotide deletion in the 3'UTR was

attenuated in rhesus monkeys and highly protective against the wild type dengue 1

strain (Whitehead et al., 2003).In the 5' and 3' untranslated regions of Flavivirus

RNA, two conserved sequences, CS1 and CS2 have been described for all flaviviruses

which are proposed to initiate viral transcription (Hahn et al., 1987). The base pairing

between the complementary 5' and 3' sequences are essential for replication in

mosquito-borne and tick-borne Flaviviruses. An experiment determined the base

pairing interactions between the covalently linked 5' genomic region (first 160

nucleotides) and the 3'UTR (last 115 nucleotides) for a range of mosquito-borne

Flaviviruses. The RNAs with mutations only in the 5' or 3' cyclization sequences were

not able to replicate after transfection into BHK cells but those with compensatory

mutations in both 5' and 3' cyclization sequences were able to replicate (Khromykh et

al., 2001).

GENETIC DIVERSITY IN VIRUS POPULATIONS

Dengue viruses, being RNA viruses, have high mutation frequencies with

mutation rates in the range of 10-3 – 10-5 per nucleotide per round of replication

(Drake, 1993). For a 10Kb genome, there is an average of 0.1 to 10 mutations in the

genome of each progeny (Domingo and Holland, 1997). The rate of mutation in RNA

genomes is 104 to 106 fold greater than the mutation rates of DNA genomes (Holland

et al., 1982). Also, the absence of proof reading-repair activities in RNA replicases

and transcriptases contributes to the high mutation rate and RNA virus diversity

(Steinhauer et al., 1992). In DNA based organisms, DNA polymerases have a 3'- 5'

exonuclease activity as well as polymerase activity. The exonuclease acts

preferentially on incorrectly paired bases. If by chance, the wrong base becomes

added to the 3' end of a growing chain, it is removed, thus allowing the DNA

polymerase to insert the correct base (Steinhauer et al., 1992, Watson et al., 1976).

Genetic diversity among arboviruses is thought to be significantly

contrained by their requirement to replicate in alternating host cycles between

vertebrate and invertebrate hosts (Gould et al., 2001). To test this hypothesis, eastern

equine encephalomyelitis virus was passaged in both BHK (baby hamster kidney)

cells and mosquito cells individually, as well as alternative passages in both cell

culture systems. It was observed that single host-cell passages increased fitness no

more than alternating cell passages. Interestingly, however, single host passages gave

rise to more mutations than alternating passages, since it was observed that mosquito

cell adaptation alone gave rise to a replacement of the stop codon in nsP3 with

arginine or cysteine. Also, BHK cell adaptation resulted in a 238- nucleotide deletion

in the 3' untranslated region. It was therefore suggested that alternating host

transmission cycles in arboviruses placed a constraint on the evolution rate but had no

effect on their fitness for either host alone (Weaver et al., 1999).

The evolution of RNA viruses is affected by the population size of the

virus as well as the mode of transmission, whether it is vertical or horizontal. With

large population sizes, the factor which affected evolution is selection and the result

of competition between genomes carrying different beneficial mutations assured the

best possible candidate became fixed (Elena et al., 2001). Novel lineages of virus

strains could emerge and proliferate replacing ancestral strains through selection,

multiple introductions or genetic bottlenecks. There has been little evidence of

positive selection of dengue viruses in nature. A study based on the envelope (E)

protein gene in DEN 2 viruses showed that the E protein of most DEN 2 genotypes

was subjected to relatively strong selective constraints (Twiddy et al., 2002a).

However, some positive selection was seen in the E protein gene of two DEN 2

genotypes and in DEN 3 and DEN 4 but none in DEN 1 (Twiddy et al., 2002b). A

study on DEN 4 viruses in Puerto Rico showed some evidence of positive selection in

the NS2A gene (Bennett et al., 2003). New virus lineages could also emerge by

introduction/s from external sources such as seen in DEN-1 viruses from Argentina

from the 1999-2000 outbreak (Barrero and Mistchenko, 2004). Phylogenetic analysis

of these viruses revealed two distinct clades had emerged and clade I was linked to

Brazilian samples and clade II linked to samples from Paraguay and Northeastern

Argentina. The authors explained the phenomena as two different introductions from

evolution in countries with different ethnic backgrounds, resulting in the appearance

of two different clades. Also, in the 2001 DHF outbreak which occurred in the

Pacific, the authors determined the cause of the outbreak to be due to multiple

introductions of Asian strains rather than transmission of local strains (A-

Nuegoonpipat et al, 2004; personal communication).

A situation called a genetic bottleneck occurs when a virus population is

periodically reduced to one or a few virions (Duarte et al., 1993). Attenuation caused

by genetic bottlenecks in vertically transmitted viruses is caused by two different

forces acting on virus populations. The first one is when mutation rates are high, and

deleterious mutations accumulate, the resulting Muller's ratchet effect causes

irreversible loss of fitness (Duarte et al., 1992). The second is, in strict vertical

transmission in viruses, different viral genotypes are separated into distinct

evolutionary lineages reducing the level of competition and thus reducing the force of

natural selection to increase viral fitness. Also, vertical transmitted strains are strictly

dependent on the survival and successful reproduction of their hosts and it is in their

interest in keeping virulence low (Bergstrom et al., 1999). Vertically transmitted

pathogens never move among lineages and competition occurs at the intrahost level.

In horizontal transmission, different genotypes compete in hosts and the "fittest" ones

expected to become fixed (Duarte et al., 1992). The replicative capacity of RNA

viruses in a given environment is defined as "fitness" (Novella, 1999). Horizontal

transmitted pathogens can spread to every member of the host population in a short

period of time and competition could occur at the interhost level (Bergstrom et al.,

1999). A genetic bottleneck was the mechanism explained in the transmission of

dengue 2 viruses between epidemics in Thailand in the years 1980 and 1987. There

were nucleotide and amino acid substitutions which were only seen among subtypes

isolated throughout 1987 from various localities, but absent from the corresponding

subtypes from 1980. This indicated a genetic bottleneck occurring during the seven

year period which allowed only one or a few of the co-circulating viruses from 1980

to be transmitted after seven years (Sittisombut et al., 1997). A genetic bottleneck was

also the most possible cause of extinction of dengue 3 virus populations circulating

prior to 1992 in Thailand and the replacement by 2 new lineages. Although there was

implication of natural selection, the ratio of nonsynonymous to synonymous

nucleotide changes did not indicate evidence of positive selection. Also, 1992 was an

inter-epidemic year which would favour the occurrence of a genetic bottleneck rather

than selection (Wittke et al., 2002).

Gene reassortment also contributes to genetic diversity in RNA viruses

with segmented genomes. Two viruses with segmented genomes can produce new

offspring by reassorting their gene segments in a variety of combinations (Strauss et

al., 1996). This may result in a progeny virus with completely new potential and this

phenomenon was thought to be the cause of global pandemics of influenza by major

antigenic shifts. The sudden emergence of the influenza virus strain H2N2 in 1956

and strain H3N2 in 1968 are examples of the effect on virus transmission from gene

re-assortment. A human influenza virus can be reassorted with one from an animal

resulting in a new strain which has the ability to replicate in humans but has totally

new surface antigens which the human population does not recognize. The 1997

outbreak of influenza in Hong Kong is evidence of direct infection of humans by

Avian influenza viruses. These viruses showed genetic reassortment, genetic

polymorphism and antigenic variation among strains and were associated with high

pathogenicity in the lungs as well as neurovirulence of mice (Hiromoto et al., 2000).

Recombination is another source of genetic diversity in viruses. It has been

known to occur for more than two decades but has only recently been shown to occur

in RNA viruses (Lai et al., 1992). The first RNA virus in which recombination was

seen was the Poliovirus (Cooper et al., 1974). Highly variant forms of RNA genomes

may be produced by recombination events and these may provide greater evolutionary

jumps than those mediated by point mutations (Domingo et al., 2001). A new RNA

pathogen emerged in the form of western equine encephalomyelitis virus (WEE)

which resulted from a recombination between two other alphaviruses, Sindbis-like

virus (SIN) and eastern equine encephalomyelitis virus, EEE (Hahn et al, 1988).

Recombination has also been detected in human immunodefiency virus (Rhodes et al,

2003). Evidence of recombination in flaviviruses was first demonstrated in 1999

(Monath, 1994). Among all mosquito-borne flaviviruses, dengue viruses were

estimated to have the highest frequency of recombination. The Aedes aegypti

mosquito, the major vector of dengue, has multiple feeding habits allowing co-

infection with more than one virus strain to occur in the vector, fulfilling a condition

required for recombination to occur. Also the global distribution of dengue virus and

the extensive travel of the human host species to areas where these viruses circulate,

provides more chance for co-circulation of different virus genotypes contributing to

the relatively high level of recombination in dengue viruses, unlike yellow fever (YF)

virus in which recombination has not been detected. Most transmission of YF virus

occurs in forests in Africa, Central and South America and most human cases would

be rural workers or inhabitants of villages in Africa who are unlikely to engage in

extensive travel between areas where African and the new world strains of YF virus

circulate (Twiddy and Holmes, 2003). The widely accepted mechanism for

recombination is the copy-choice mechanism where the RNA- dependent RNA

polymerase jumps from one strand, the donor, to the other, the acceptor strand (Flint

et al., 2004). The RNA secondary structure may also influence template switching in

recombination. A study on bovine viral diarrhoea virus (BVDV) revealed that sites of

dissociation in recombination of this virus were frequently associated with regions

having bulges in their predicted secondary structure and sites of re-initiation

frequently associated with loop structures or in the junction of multiple stem

structures (Desport et al., 1998). Widespread intra serotype recombination has been

detected among all four serotypes (Craig et al., 2003, Tolou et al., 2001, Uzcategui et

al., 2001, Worobey et al., 1999). A study done to determine the time scale and evolution of dengue viruses

revealed the rates of nucleotide substitution in dengue viruses ranged from 4.55 x10-4

substitutions/site/year in dengue 1 viruses to 11.58x10-4 substitutions/site/year in

genotype 3 of dengue 3 viruses. This analysis also revealed that all 4 serotypes

considered separately had a molecular clocklike or near-clocklike behavior (Twiddy

et al., 2003). Zanotto et al. (1996) estimated the rate of non-synonymous substitutions

in the envelope gene of dengue viruses as 7.5 x10-5 substitutions/site/year. An

estimate of DEN 1 virus evolution according to substitutions in third codon position

sites alone showed the evolution rate to be 16.2 +/- 1.5 x 10-4 substitutions/ third

position codon site/ year (Goncalvez et al., 2002). Comparatively, the evolutionary

rates for alphaviruses ranged from 2.8 x10-4 to 5 x10-4 substitutions/site/year for

eastern equine encephalitis virus (EEE), western equine encephalitis (WEE) and

Venezuelan equine encephalitis (VEE) viruses in the tropics (Weaver et al., 1997).

Phylogenetic analyses of dengue viruses have identified genetic subtypes of viruses in

each of the four serotypes which have up to 12% difference in the nucleotide

sequence of the envelope E protein gene. There are five known subtypes in dengue 1,

six in dengue 2, four in dengue 3 and two in dengue 4. (Holmes, 1998, Lanciotti et al.,

1994, Lanciotti et al., 1997, Lewis et al., 1993 and Rico-Hesse, 1990).

In a study done by Holmes, (2003) to determine the patterns of intra

(within) and interhost (between) virus variation, the genetic variation was compared

for different virus populations, namely, (a) virus populations within individual hosts

(intrahost variation), (b) virus populations which are epidemiologically linked, that is

from the same country within a limited time span and (c) virus populations over a full

range of diversity comprising all genotypes in each virus serotype. It was seen that

the frequency of non-synonymous mutations as greatest in intrahost populations and

as greatly reduced in comparisons made from more distantly related viruses, interhost

populations. The ratio of non-synonymous to synonymous mutations in interhost

populations was less than 10 % of its mean value within hosts indicating that over

90% of synonymous mutations found within hosts were deleterious which indicated a

strong purifying selection in dengue virus populations. When mutation rates are high

and a significant proportion of the mutations are deleterious, there will be a gradual

decrease in the mean fitness of the population in an irreversible manner, known as the

Muller's ratchet (Duarte et al., 1992).

A study on genetic diversity in a 393 nt region of the E gene of DEN 3

virus populations revealed that 5.8% of clones were defective (Wang et al., 2002).

Also, in DEN 1 virus strains from Argentina, the minor subpopulation showed a

defective variant with deletions in the NS4A region of the genomes. (Aviles et al.,

2003).These particles have a defective nature as they are unable to reproduce because

of loss of protein coding sequences and are known as defective interfering particles

(Turner et al., 1999). These defective genomes may arrest the disease process by

competing with the wild type for structural proteins and replication enzymes and may

hitch-hike their way into host cells. Once in the host cell, without any access to

functional proteins, they will be unable to replicate and move into other cells (Huang

and Baltimore, 1970).

Because of genetic variation, there exists a population of viruses which co-

infects an individual often referred to as "viral quasispecies". This is defined as a

population of viruses that share a common origin but which have distinct genome

sequences as a result of mutation, a heterogenous mixture of viral genomes that

circulate within an infected individual and mutate with time (Eigen, 1993).

A viral quasispecies has a master sequence and a mutant spectrum. The master

sequence is the dominant nucleotide sequence in the genome distribution which may

or may not coincide with the consensus or average sequence of the distribution.

Experiments on a Qβ phage population revealed a closely related mutant distribution,

the hallmark of quasispecies (Eigen, 1993). Not only was the consensus sequence

stable over multiple passages, but mutants showed close relation in that they only

differed from the consensus (average sequence) by an average of only one or two

nucleotides. In studying viral quasispecies, there is a region called a "sequence space"

in which the variant nucleotide sequences are mapped. In this space, mutations arise

from the centre and those which survive longer and have a better chance of producing

more offspring, have protrusions which reach out further than the others (Eigen,

1993). It is estimated that for any viral genome, the number of possible sequences

which can be present in a sequence space is 4V, v being the number of nucleotides in

the genome (Eigen, 1993).

The continuous production of mutants favours adaptability of viruses in the

process of environmental changes (Domingo and Holland, 1997). Relative fitness

assays done with vesicular stomatitis virus by mixing two virus populations with

initial equal fitness showed that after many passages in vitro one population

eventually dominated over the other. However, there were absolute gains in fitness of

both the winners and losers, this being called the Red Queen's Hypothesis (Clarke et

al., 1994).

The accumulation of mutations is a continuing process, which, together with

the possibility of intramolecular recombination due to dual infection with different

dengue virus serotypes, could lead to the emergence of a fifth dengue virus serotype

differing at one or more critical neutralizing epitopes (Monath, 1994). The diversity in

dengue virus populations may pose obstacles in the design of a safe and effective

dengue vaccine. For a dengue vaccine to be effective, it must give protection against

all dengue viruses in all geographical locations. Diversity may limit the effectiveness

of engineered vaccines which have a narrow spectrum of neutralizing epitopes and

give rise to the emergence of neutralization-escape mutants with new phenotypic

properties. Problems have been encountered with live vaccines (Seligman and Gould,

2004). There has been evidence in live attenuated poliovirus vaccines of loss of the

attenuated phenotype from intraserotypic recombination (Worobey et al, 1999). The

17D yellow fever vaccine, considered to be one of the safest, was found to be

associated with fatal side effects (Chan et al, 2001 Martin et al, 2001). There has been

intensive efforts made to develop a safe and effective dengue vaccine supported by

the World Health Organization since the late 1970s. Candidate vaccines currently on

trial are live attenuated vaccines from the Mahidol University, Bangkok and from the

Walter Reed Army Institute, USA. Chimeric vaccines using a yellow fever virus

backbone or constructs using different dengue virus serotypes themselves are

approaches currently on trial (Monath et al, 2002). Despite all efforts, a safe and

effective dengue vaccine has not yet been developed.

A greater knowledge of genetic variation in dengue populations by comparing

gene sequences from viruses with different biological properties such as

transmissibility, virulence and tropism, may reveal the exact nucleotides which

control these factors and thus lead to heightened knowledge crucial for effective

control of dengue and may provide more answers for why dengue is an emerging

disease (Holmes, 1998).

REFERENCES

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CHAPTER 3

DIVERSE DENGUE TYPE 2 VIRUS POPULATIONS CONTAIN RECOMBINANT AND BOTH PARENTAL VIRUSES IN A SINGLE

MOSQUITO HOST

Scott Craig, 1 Hlaing Myat Thu, 1 Kym Lowry, 1 Xiao-fang Wang, 2 Edward C. Holmes, 3 and John Aaskov1

1. Centre for Molecular Biotechnology, School of Life Sciences,

Queensland University of Technology, Brisbane, Australia.

2. Institute of Virology,

Beijing, China.

3. Department of Zoology, University of Oxford,

Oxford, United Kingdom.

Journal of Virology (2003), 77:4463-4467

CHAPTER 4

MYANMAR DENGUE OUTBREAK ASSOCIATED WITH DISPLACEMENT OF SEROTYPES 2,3 AND 4 BY DENGUE 1

Hlaing Myat Thu,1 Kym Lowry,1 Thein Thein Myint,2 Than Nu Shwe,2 Aye Maung Han,2

Kyu Kyu Khin,3 Kyaw Zin Thant,4 Soe Thein4 and John Aaskov1

1.Centre for Molecular Biotechnology, School of Life Sciences,

Queensland University of Technology, Brisbane, Australia.

2.Yangon Children's Hospital,

Yangon, Myanmar.

3. Mawlamyaing General Hospital, Mawlamyaing, Myanmar.

4. Department of Medical Research (Lower Myanmar), Yangon, Myanmar

Emerging Infectious Diseases (2004), 10: 593-597

CHAPTER 5

LINEAGE EXTINCTION AND REPLACEMENT IN DENGUE TYPE 1

VIRUS POPULATIONS DUE TO STOCHASTIC EVENTS RATHER THAN TO NATURAL SELECTION

Hlaing Myat Thu,1 Kym Lowry,1 Limin Jiang,1 Thaung Hlaing,2 Edward C. Holmes3

and John Aaskov1

1. Centre for Molecular Biotechnology, School of Life Sciences,

Queensland University of Technology, Brisbane, Australia.

2. Department of Medical Research (Lower Myanmar),

Yangon, Myanmar.

3. Department of Zoology, University of Oxford,

Oxford, United Kingdom

Virology (accepted for publication).

CHAPTER 6

Sustained transmission of dengue virus type 1 in the

Pacific due to repeated introductions of different Asian strains

Atchareeya A-Nuegoonpipat1, Alain Berlioz-Arthaud2, Vincent Chow3, Tim

Endy4, Kym Lowry5, Le Quynh Mai6, Truong Uyen Ninh6, Alyssa Pyke7, Mark

Reid8, Jean-Marc Reynes9, Se-Thoe Su Yun10, Hlaing Myat Thu11, Sook-San

Wong12, Edward C. Holmes13, John Aaskov5.

CHAPTER 7

GENERAL DISCUSSION

7.1. DIVERSITY IN DENGUE VIRUS POPULATIONS

Dengue virus populations in individual hosts are diverse and minority sub-

populations could be a source of "new" viruses. The first study reported in this thesis

(Chapter 3) described the diversity of DENV-2 populations in humans and in

mosquitoes from Yangon, Myanmar. There were seventeen variable sites at which the

nucleotide sequences of three or more envelope (E) protein genes differed from the

consensus nucleotide sequence for that population. The mean diversity (number of

substitutions/total length of sequence x 100) in DENV-2 populations from humans

and mosquitoes ranged from 0.12 to 0.32 per cent. These values were comparable to

those reported by Wang et al. (2002) for a 393 nt segment of the envelope protein in

DENV-3.

A number of the nucleotide changes in these DENV-2 population resulted in

stop codons. The genomes of several Alphaviruses (e.g. Sindbis virus, eastern equine

encephalomyelitis virus, western equine encephalomyelitis virus, Venezuelan equine

encephalomyelitis virus and Ross River virus) contain an OPAL stop codon in the

nsP4 gene. Production of non-structural protein nsP4 requires "read through" of the

stop codon (Strauss and Strauss, 1994). It remains to be determined whether similar

read through of the stop codon in dengue viruses occurs or whether the stop codons

lead to defective virus particles (Huang and Baltimore, 1970).

Another significant finding was that some polymorphisms were detected in

virus populations recovered from patients over a two-year period (1998 to 2000). This

meant, that if Aedes aegypti mosquitoes survived for approximately a week after

taking a blood meal and the average duration of viremia in a dengue patient was a

week, these polymorphisms had survived, perhaps, fifty cycles of human-mosquito

transmission.

Recombination contributes to changes in virus populations by producing

highly variant genomes (Hahn et al., 1988, Domingo et al., 2001). In the first study

(Chapter 3), phylogenetic analyses identified recombinant and both parental viruses in

a single mosquito. Intra-serotypic recombination between dengue viruses may be far

more common than the literature suggests but it may not be detected because of the

almost universal use of consensus nucleotide sequences. Although others had

reported recombination in dengue viruses previously (Tolou et al., 2001, Uzcategui et

al., 2001,Worobey et al., 1999), this study was the first to identify recombinant and

both parental virus strains in a single host.

7.2. CHANGES IN VIRUS POPULATIONS AND THEIR SIGNIFICANCE IN

VIRUS TRANSMISSION AND PATHOGENESIS

The outbreak of DENV-1 in Myanmar in 2001 (Chapter 4) was the first

example of one dengue virus serotype displacing the other three serotypes almost

completely. A significant observation in this outbreak was the proportion of dengue

patients experiencing primary infections. Previously, Thein et al. (1997) had observed

that 15 per cent of dengue patients who were admitted to the Yangon Children's

Hospital had primary infections but in 2001 the proportion was 46 per cent. This

confirmed a previous report by Vaughn et al. (2000) that primary infections with

DENV-1 resulted in clinical disease more frequently than primary infections with

DENV-2 or with DENV-4. The relatively small number of dengue shock syndrome

(DSS) cases in 2000 and in 2001, compared to years prior to 1988, confirmed

previous observations that DSS occurred most commonly when the host had a

secondary infection with DENV-2 following a prior infection with one of the other

three DENV serotypes (Halstead et al., 1970, Thein et al., 1997). Phylogenetic

analyses of the E genes of DENV-1 collected before and after the 2001 dengue

outbreak suggested an early lineage of DENV-1 had become extinct some time

between 1998 and 2000 and had been replaced by two new lineages of DENV-1.

There was no evidence that selection or recombination in the E gene of DENV-1 that

may have contributed to this change.

The third manuscript (Chapter 5) described an attempt to identify the cause (s)

of the extinction of the ancestral strain of DENV-1 and the emergence of the two new

lineages, and to determine if genes other than the E gene were under selective

pressure (Bennett et al, 2003). An analysis of DENV-1 strains from Myanmar, from

the earliest recovered, in 1971, to 2002 failed to find evidence of strong selective

pressures having contributed to the extinction of pre-1998 strains of virus or to the

appearance of the two new clades of viruses. However, sites found to be under weak

selective pressure in the NS5 protein (NS5-127, 135 and 669) were close to two

critical functional determinants in that protein. Amino acids NS5-127 and NS5-135

are in motif III of the RNA cap (nucleoside-2’-O-) – methyltransferase portion of NS5

and straddle amino acids NS5 -131 and 132 which interact with S-adenosyl-L-

methionine to aid capping of the viral RNA (Egloff et al., 2002). The second site

found to be subjected to selective pressure (NS5-669) is adjacent to a potential

nucleoside triphosphate binding motif 661GDD663 in motif VI (Koonin, 1991) of the

polymerase component at the C-terminal end of NS5. However, amino acid changes

at these sites were not common to all members of the extinct clade of viruses or to

either of the two clades which replaced it suggesting that selection, at an amino acid

level, was not responsible for the extinction of the pre-1996 strains of DENV-1 in

Myanmar.

Dengue viruses are thought to have the highest frequency of recombination

among all mosquito borne viruses and widespread intraserotype recombination has

been detected in all four serotypes (Tolou et al., 2001, Twiddy and Holmes, 2003,

Uzcategui et al., 2001, Worobey et al., 1999). No evidence was found that the two

new lineages of DENV-1 had arisen by recombination between the ancestral

Myanmar DENV-1 and other DENV-1. Collectively these data suggested that the

extinction of the ancestral clade of viruses was due to some stochastic event which

occurred in the 199-2000 inter-epidemic interval. Genetic bottlenecks are believed to

have contributed to strain extinction and to the emergence of new strains of DEN 2

and 3 viruses in Thailand (Sittisombut et al., 1997, Wittke et al., 2002). This study

(Chapter 5) demonstrated the importance of the role of stochastic events in changing

patterns of disease.

The 2001 DENV-1 outbreak in Myanmar also raised the issue of the

importance of virus surveillance at a national level in disease outbreak prediction. The

emergence of two new strains of dengue 1 occurred a few years before the 2001

epidemic and the increase in the number of DENV-1 isolates in the previous year

2000, could have been an indicator that DENV-1 had become the dominant serotype

and an epidemic outbreak could follow. Constant virological surveillance and

detection of new strains as they emerge could lead to timely measures for the

prevention of an epidemic.

An outbreak of dengue due to DENV-1 began in the Pacific (Chapter 6) at

approximately the same time as the dengue outbreak in Myanmar in 2001. The Pacific

outbreak was believed to have spread from an index outbreak in Palau in 2000

Phylogenetic analyses of the E genes of DENV-1 from the Asia-Pacific region

suggested that the dengue outbreak in the Pacific was caused by multiple

introductions of DENV-1 from several Asian countries rather than to local spread.

DENV-1 isolated in New Caledonia was closely related to strains recovered in

Myanmar in 2001. Another significant observation was that only DENV-1 had

become established in the Pacific despite all 4 serotypes circulating in the countries

where the viruses originated. This suggested that there may be a level of protection

arising from infections with other dengue serotypes in the Pacific in the previous

decade (Kiedrzynski et al, 1998).

7.3. CONCLUSIONS

This study found dengue virus populations in individual hosts to be diverse.

The difference between the diversity within dengue virus populations and between

virus populations and the difference in their dN/dS ratios, suggested that dengue virus

populations are subjected to extensive purifying selection. The use of consensus

nucleotide sequences to characterise dengue, and other RNA virus populations leads

to a significant under-estimate of this diversity because it may fail to reveal within-

population variation. The detection of large numbers of stop codons in the genomes of

dengue viruses raised the question of what role these " defective particles" might play

in regulating virus infectivity and hence disease severity. This warrants further

investigation.

These diverse viral populations are a potential source of "new" viruses, which

could appear as a result of selection or a genetic bottleneck. Major changes in

genotype, such as that which preceded the dengue outbreak in Myanmar in 2001,

appeared to be due to extinction of one virus lineage as a result of a genetic

bottleneck(s) that occurred in an inter-epidemic period of low virus transmission.

In contrast to the situation in Myanmar where two co-circulating clades of

viruses replaced a third, the outbreak of dengue due to DENV-1 in the Pacific

appeared to be due to the introduction of "new" strains of virus to the region. Some of

the viruses introduced into New Caledonia may have come from Myanmar. This

study has highlighted the important role for comprehensive virological surveillance at

an international level in interpreting and understanding different patterns of dengue

virus transmission and disease.

7.4. FUTURE WORK

(a). Further investigation of what role "defective interfering particles" might play in

regulating virus infectivity and hence disease severity.

(b). To undertake comprehensive virological surveillance in order to identify

significant genotypic and phenotypic changes in populations of dengue viruses and to

determine the mechanism(s) of these changes.

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