Investigating dengue virus transmission in Malaysia using bioinformatic tools

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Bioinformatics Symposium 2005 HELP University
College
Investigating dengue virus transmission in
Malaysia using bioinformatic tools
M. Y. FONG Faculty of Medicine University of
Malaya
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About dengue

• Dengue is one of the most important mosquito-born
  viral diseases affecting humans.
• Viral life cycle involves humans and the mosquito
  vectors Aedes aegypti and Aedes albopictus.
• The disease is caused by 4 serotypes of the
  dengue virus, a member of the genus Flavivirus
  DEN-1, DEN-2, DEN-3, DEN-4.
• Infection with the dengue virus can result in
  dengue fever (DF), dengue hemorrhagic fever (DHF)
  or dengue shock syndrome (DSS).

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Dengue clinical features

• Dengue fever
• fever, frontal or retro-orbital headache, rash,
  bone pain, nausea
• Dengue haemorrhagic fever
• high fever, internal haemorrhage, enlargement of
  liver, circulatory failure
• Dengue shock syndrome
• severe form of DHF, temperature drops, massive
  circulatory failure, critical state of shock,
  potentially fatal

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Dengue global statistics

• Global prevalence increased in recent decades.
• Endemic in more than 100 countries.
• Africa, the Americas, eastern Mediterranean
  countries.
• Southeast Asia and Western Pacific most seriously
  affected.
• 2500 million people (two fifths of worlds
  population) at risk.
• 50 million cases of infection.

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Dengue distribution
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Dengue in Malaysia

• Dengue fever first reported in 1902.
• Dengue haemorrhagic fever in 1962 in Penang.
• Gazetted as notifiable disease in 1971.
• Four-year cycle outbreaks 1974, 1978, 1982,
  1986, 1990.
• 1991 onwards, no predictable cycle.
• Average 5000 cases annually.
• All four types of dengue virus present.

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Structure of dengue virus
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Molecular biology of dengue virus
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Molecular evolution and distribution of dengue
viruses

• Rebecca Rico-Hesse (1990) determined the
  nucleotide sequence of E/NS1 junction region of
  DEN-1 and DEN-2.
• Junction region is 240 bases in length.
• Bioinformatics programme to align the sequences.
• Programme to draw genetic tree or clustering of
  the viruses.
• Other studies followed
• DEN-2 Deubel et al. (1993)
• DEN-3 Chungue et al. (1993), Chow et al. (1994),
  Lanciotti et al. (1994)

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Multiple sequence alignment
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Clusters of DEN-1(Rico-Hesse, 1990)
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Clusters of DEN-2(Rico-Hesse, 1990)
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Malaysian DEN-2

• DEN-2 viruses isolated from DF and DHF patients.
• 24 samples, over a 25-year period (1968-1993).
• Sequence the 240-nucleotide E/NS1 junction
  region.
• CLUSTAL programme to align the sequences and draw
  the genetic tree.
• Main findings
• Two genotypes (groups) in Malaysia genotypes I,
  and II
• Genotype I consisted of three subgroups
  (1968-1990 1986-1992 1993)
• Genotype II (1982-1991) has no subgroups
• Genotype I related to viruses from Indian Ocean
  region
• Genotype II related to viruses from Western
  Pacific region

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Malaysian DEN-4(AbuBakar et al., 2002)

• DEN-4 predominant in Malaysia in 19671969.
• No isolation reported in 1995-2000.
• Six DEN-4 isolated within 5 months in 2001 from
  patients in University of Malaya Medical Centre.
• Characterization of the envelope (E) gene of five
  of the DEN-4 isolates.
• To trace the potential origin of the virus.
• Evidence of recombination involving Indonesian
  and Malaysia DEN-4

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Malaysian DEN-4 approach

• Compare E gene of these recent DEN-4 with those
  of older (1960s) Malaysian DEN-4 and of other
  regions.
• Multiple alignments of the nucleotide sequences
  were performed using CLUSTAL X.
• Trees were constructed by the neighbour-joining
  method.
• Potential recombinant sequences within the E gene
  were examined using SIMPLOT.
• Recombination was identified when conflicting E
  gene sequence profiles appeared, suggesting
  acquisition of sequences from a different
  parental genotype.

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Malaysian DEN-4 findings

• Phylogenetic tree showed three DEN-4 clusters.
• Genotype I consisting of viruses from Thailand,
  Malaysia (1969), Sri Lanka and Philippines.
• Genotype II comprises mainly isolates from South
  America and the Pacific Islands.
• Sylvatic isolates form a distinctly different
  genotype.
• Recently isolated Malaysian DEN-4 subclustered
  together with Indonesia (1973) into a separate
  subcluster within genogroup II, denoted as
  genotype IIA.

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Malaysian DEN-4 findings

• E gene of Indonesia 73 together with the recent
  Malaysian DEN-4 were mosaics of DENV-4 genotypes
  I and II.
• Evidence of recombination between DENV-4 genotype
  I (MY69) and genotype II (Indonesia 76) was
  obtained from similarity plot analyses.
• Phylogenetic tree, drawn using nucleotides at
  positions 561800 identified from the breakpoint
  analyses.
• Recently isolated Malaysian DENV-4 and Indonesia
  73 grouped into DENV-4 genotype I (note compare
  with tree drawn using whole gene sequence)

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Malaysian DEN-4 conclusion

• Emergence of DEN-4 genotype IIA in Malaysia.
• Different ancestral lineages following
  inter-typic recombination.
• Recombination amongst specific DENV serotypes
  occurs in natural population.
• New genotypes could emerge especially in a
  population where multiple strains of the virus
  are co-circulating.
• Implications in vaccine design and control of
  transmission