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Am. J. Trop. Med. Hyg., 74(2), 2006, pp. 263-265
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene

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SHORT REPORT


ORIGIN OF DENGUE TYPE 3 VIRUSES ASSOCIATED WITH THE DENGUE OUTBREAK IN DHAKA, BANGLADESH, IN 2000 AND 2001

GOUTAM PODDER, ROBERT F. BREIMAN, TASNIM AZIM, HLAING MYAT THU, NILUKA VELATHANTHIRI, LE QUYNH MAI, KYM LOWRY, AND JOHN G. AASKOV*
Center for Health and Population Research, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh; Department of Medical Research, Yangon, Myanmar; Department of Microbiology, University of Sri Jayawardenapura, Colombo, Sri Lanka; National Institute of Hygiene and Epidemiology, Hanoi, Vietnam; School of Life Sciences, Queensland University of Technology, Brisbane, Queensland, Australia

 

ABSTRACT

Dengue and dengue hemorrhagic fever re-emerged in Bangladesh in 2000 and 2001 and nearly all viruses isolated were dengue type 3. Phylogenetic analyses of the envelope genes of examples of these viruses indicated that they were most closely related to recently emerged dengue type 3 viruses from neighboring Thailand and Myanmar but distinct from those from India and Sri Lanka. Since this strain of dengue virus type 3 had not been associated with unusual patterns of disease in Thailand or Myanmar, it suggested that the outbreak in Bangladesh was due to local factors after the introduction of viruses from countries to the east rather than to the evolution of an unusually virulent strain of virus in Bangladesh.

Dengue viruses (dengue virus type 3 [DENV-3]) were isolated for the first time from patients in East Pakistan (Bangladesh) in 1964.1 Subsequent reports2–4 suggested that dengue fever may have been occurring sporadically in Bangladesh between 1964 and the outbreak that began in 20005 during which predominantly DENV-3 was recovered from patients.6 The dengue epidemic in Bangladesh in 2000 raised a number of questions. Why had outbreaks not been occurring regularly in Bangladesh as they had in neighboring Myanmar7 and Thailand?8 Was the outbreak in 2000 due to the appearance of a new strain of DENV-3 as a result of evolution of local strains or to the introduction of strains of DENV-3 from outside Bangladesh?

All investigations were reviewed and approved by the relevant Institutional Research/Ethics Committees. Sera obtained from 18 dengue patients at two hospitals in Dhaka, Bangladesh in 2000 and 2001 and from employees with dengue from a recreation club in Dhaka where there had been a focus of intense transmission in 20019 were added to 25-cm2 monolayers of C6-36 mosquito cells and incubated at 30°C for seven days. Cells were recovered and examined by indirect immunofluorescence10 with monoclonal antibodies to flavivirus, dengue virus, and dengue virus serotypes11 for evidence of infection with dengue virus. DENV-3 was recovered from six specimens. Two additional DENV-3 isolates recovered from patients in Bangladesh in 2000 were provided by the U.S. Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand. Contemporary strains of DENV-3 were isolated from patients in Sri Lanka (D3.SriLanka.D39.1999), Myanmar (D3.Myanmar.46881.2002), Cambodia (D3.Cambodia.K0520128.2000), and Vietnam (D3.Vietnam .11598.1998) using similar methodology. Supernatant from the cultures of infected C6-36 cells was used as a source of virus.

Sequencing of envelope (E) protein genes of these viruses was performed as previously described.12,13 Briefly, RNA was extracted from the supernatant of cultures of infected C6-36 cells using a commercial kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. cDNA was generated from the RNA by heating a mixture of random hexamer primers (1 nmol; Boehringer, Mannheim, Germany) and RNA for 10 minutes at 72°C and cooling the mixture on ice. Reverse transcription was carried out at 55°C for 10 minutes followed by 45°C for 60 minutes with Expand reverse transcriptase (Roche, Mannheim, Germany). The cDNA was amplified by a polymerase chain reaction (PCR) using oligonucleotide primers13 composed of nucleotide sequences in the pre-membrane and non-structural protein 1 genes of DENV-3 and Expand DNA polymerase (Roche). After an initial denaturation of the cDNA at 92°C for 2 minutes, 30 cycles at 92°C for 40 seconds, 55°C for 40 seconds, and 68°C for up to 2 minutes were used. cDNA for sequencing was obtained by excising the band of interest after electrophoresis of the PCR product on 1.5% agarose/Trisacetate-EDTA gels and purifying it using a commercial kit (High Pure Gel Extraction Kit; Roche) according to the manufacturer’s instructions.

Approximately 100 ng of cDNA was sequenced using 3.2 pmol of oligonucleotide primer12,13 and the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Wellesley, MA) according to the manufacturer’s instructions. The product was purified using Dye-EX Spin Columns (Qiagen) and analyzed at the Australian Genome Research Facility (Brisbane, Queensland, Australia) on a 3730XL Nucleotide Sequencer (Applied Biosystems, Foster City, CA). Nucleotide sequences were aligned and phylogenetic analyses were performed with EclustalW, Ednapars, Ednadist, Ednakitsch, and Econsense software from the Australian National Genome Information Service (Sydney, New South Wales, Australia). Bootstrap values were derived from 1,000 iterations. Nucleotide sequences of the E genes of additional DENV-3 were obtained from GenBank. The sequences of the Bangladesh viruses have also been deposited in this database (Accession numbers AY656669–AY656674).

The Bangladesh DENV-3 isolates showed little evidence of independent evolution. The consensus nucleotide sequences of the eight DENV-3 isolates from Bangladesh varied at only 18 of 1,479 sites in the E gene. Four of these changes resulted in amino acid changes (E81, Ile->Thr; E134, Asn->Ile; E140, Thr->Ile; and E301, Leu->Ser). The Bangladesh DENV-3 isolates formed a distinct phylogenetic lineage with recent DENV-3 isolates from Thailand (D3.Thailand.HuNII00-27-1.2000) and Myanmar (D3.Myanmar.46881.2002) (Figure 1Go). Two strains of DENV-3 from Thailand (D3.Thailand.313.1996 and D3.Thailand.283.1994) belonged to genotypes IIa and IIb, respectively, of DENV-3 that had replaced an earlier clade of DENV-3 in Thailand in 1992.12 This replacement preceded two consecutive large annual dengue outbreaks in Thailand in 1997 and 19988 that involved predominantly DENV-3. However, strains of DENV-3 from this period were distinct from strains subsequently isolated in Thailand (D3.Thailand.HuNII00-27-1.2000) and Bangladesh. The DENV-3 isolates isolated in Bangladesh in 2000 and 2001 were distinct from viruses from India and Sri Lanka. This was of interest because the spread of genotype III DENV-3 west from the Indian subcontinent to Africa and then to Latin America has been reported.14


Figure 1
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  		    FIGURE 1. Phylogenetic relationships between the nucleotide sequences of the envelope protein genes of dengue virus type 3 (DENV-3) isolated in Bangladesh (shown in bold) and neighboring countries determined by neighbor-joining. Bootstrap values for 1,000 replicates are shown on major nodes of the tree. Representatives of each DENV-3 genotype (I, IIa, IIb, III, IV, and V) are identified. The accession numbers of nucleotide sequences derived from GenBank are shown under the corresponding viruses. The horizontal bar at the lower left shows nucleotide substitutions per site.

 
The close genetic relationship between DENV-3 isolates from Bangladesh, Thailand, and Myanmar and the lack of diversity between the viruses from Bangladesh suggested that the recent dengue outbreak in Bangladesh was associated with the introduction of DENV-3 from countries to its east, rather than to the evolution of a virulent strain of DENV-3 in situ. Although this strain of DENV-3 was associated with cases of dengue fever and dengue hemorrhagic fever in Myanmar and Thailand, it was not the most frequently isolated serotype from patients in these two countries before or during the dengue outbreak in Bangladesh. It is unclear why this strain of virus was associated with an outbreak of dengue in Bangladesh, but not in the countries from which it appeared to originate. It also is notable that at the time of the outbreak of DENV-3 infection in Bangladesh, neighboring Myanmar was having its largest dengue outbreak during which 95% of the viruses recovered from patients were DENV-1.7 It may be useful to compare the competence of mosquitoes from India, Bangladesh, and Myanmar in transmitting genotype III and the Thai/Myanmar/Bangladesh strains of DENV-3.

Received April 18, 2005. Accepted for publication September 9, 2005.

Acknowledgments: We acknowledge the assistance of Dr. Truong Uyen Ninh (National Institute of Hygiene and Epidemiology, Hanoi), Drs. Yukiko Wagatsuma, Mahbubur and Abdul Kashem Siddique (International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka), Drs. Jean-Marc Reynes and Sivuth Ong (Institut Pasteur du Cambodge, Phnom Penh, Cambodia), and Drs. Tim Endy and Ananda Nisalak (Armed Forces Institute for Medical Research, Bangkok) in obtaining material for this study.

Financial support: This study was supported by grants from the Lee Foundation and the Wellcome Trust.

* Address correspondence to John G. Aaskov, School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia. E-mail: j.aaskov{at}qut.edu.au Back

Authors’ addresses: Goutam Podder, Robert F. Breiman, and Tasnim Azim, Center for Health and Population Research, International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh, E-mails: podder{at}icddrb.org, rbreiman{at}cdcnairobi.mimcom.net, and tasnim{at}icddrb.org. Hlaing Myat Thu, Department of Medical Research, Yangon, Myanmar, E-mail: hmthu{at}mptmail.net.mm. Nikula Velathanthiri, Department of Microbiology, University of Sri Jayawardenapura, Colombo, Sri Lanka, E-mail: niluv{at}sjp.ac.lk. Le Quynh Mai, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam, E-mail: lom9{at}hotmail.com. Kym Lowry and John G. Aaskov, School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia, E-mails: lkym{at}hotmail.com and j.aaskov{at}qut.edu.au.

 

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