Emergence of dengue virus type 4 genotype IIA in Malaysia

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of General Virology (2002), 83, 2437–2442. Printed in Great Britain
Emergence of dengue virus type 4 genotype IIA in Malaysia
Sazaly AbuBakar, Pooi-Fong Wong and Yoke-Fun Chan
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
Phylogenetic analyses of the envelope (E) gene
sequence of five recently isolated dengue virus type
4 (DENV-4) suggested the emergence of a distinct
geographical and temporal DENV-4 subgenotype
IIA in Malaysia. Four of the isolates had direct
ancestral lineage with DENV-4 Indonesia 1973 and
showed evidence of intra-serotypic recombination
with the other recently isolated DENV-4, MY0122713. The E gene of isolate MY01-22713 had
strong evidence of an earlier recombination involving DENV-4 genotype II Indonesia 1976 and
genotype I Malaysia 1969. These results suggest
that intra-serotypic recombination amongst DENV4 from independent ancestral lineages may have
contributed to the emergence of DENV-4 subgenotype IIA in Malaysia.
Dengue is a mosquito-borne disease affecting at least 50
million people around the world annually (WHO, 1998). In
Malaysia, dengue has been endemic since its first description
by Skae in 1902 (Rudnick, 1986). The annual incidence of
dengue in Malaysia is about 367 cases in a ‘ quiet ’ year to about
6628 cases in a ‘ busy ’ year. Similar to other countries within
the region, all four dengue virus serotypes have been
associated with dengue fever (DF) and dengue haemorrhagic
fever in Malaysia. Dengue virus type 4 (DENV-4), the once
predominant (40 to " 64 %) serotype isolated from DF
patients in Malaysia during the period 1967 to 1969 (Rudnick,
1986) has, however, been isolated in less than 5 % of DF cases
for almost a decade with no reported isolation in the last 5
years (WHO, 2000). Nonetheless, against the background of a
very ‘ quiet ’ year in 2001, six DENV-4 were isolated within 5
months from patients attending the University of Malaya
Medical Center, Kuala Lumpur. We report here the characterization of the envelope (E) gene of five of the DENV-4 isolates
in an effort to trace the potential origin of the virus.
Dengue virus was isolated from the serum of DF patients
using C6\36 cells cultured in EMEM supplemented with 10 %
heat-inactivated foetal bovine serum (FBS). After adsorption
Author for correspondence : Sazaly AbuBakar.
Fax j60 3 79675757. e-mail sazaly!ummc.edu.my
0001-8386 # 2002 SGM
for an hour, the infected cell culture was incubated at 28 mC in
EMEM supplemented with 2 % FBS. After cytopathic effects
had been observed in infected C6\36 cell cultures, virus RNA
was extracted from the supernatant. The virus was typed
initially using specific monoclonal antibodies ; this was confirmed by performing multiplex RT–PCR using a forward
primer, DV1, and sets of four serotype-specific reverse primers
DSP1, DSP2, DSP3 and DSP4 to amplify a portion of NS3
region from the different dengue virus serotypes (Seah et al.,
1995). The DENV-2 positive control generated an expected
band size of approximately 362 bp while the DENV-4 control
generated a band of about 426 bp. RT–PCR of all five DENV4 isolates resulted in bands of 426 bp in size indicating that all
five dengue virus isolates used in this study were DENV-4
(data not shown).
The potential phylogenetic relationships of the DENV-4
isolates were examined by determining the complete E gene
sequence following the methods described by Wang et al.
(2000). Reverse-transcription was performed at 42 mC for
1 h ; denaturation at 95 mC for 2 min ; and 35 cycles of
denaturation at 94 mC for 1 min, annealing at 55 mC for 1 min,
extension at 72 mC for 1 min and final elongation at 72 mC for
5 min. The amplified fragments were purified and sequenced
using Applied Biosystems Prism BigDye Terminator Cycle
Sequencing Ready Reaction Kits and Applied Biosystems
model 377 Sequencer (USA). The sequences (accession nos
AJ428556, AJ428557, AJ428558, AJ428559 and AJ428560)
were aligned together with other previously described DENV4 isolates identified by geographical location and year of
isolation (Wang et al., 2000) and the sylvatic DENV-4
(Malaysia 75-P75-215, 73-P73-1120, 75-P75-514) that were
isolated in the 1960s from Aedes niveus group mosquitoes
living in Malaysian forests (Rudnick, 1984). Phylogenetic
analyses were performed using both the distance matrix and
character state methods. For distance matrix analyses, multiple
alignments of the nucleotide sequences and the deduced amino
acids were performed using   version 1.81 (Thompson
et al., 1997) and the resulting alignment was optimized
manually using  version 2.5 (Nicholas & Nicholas,
1997). Phylogenetic trees were constructed by the neighbourjoining method (Saitou & Nei, 1987) using DENV-2 virus
Jamaica strain as the outgroup. The strength of the phylogenetic trees was estimated by bootstrap analyses using 1000
replicates. All trees were displayed using  version
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S. AbuBakar, P.-F. Wong and Y.-F. Chan
Table 1. Summary of amino acid differences within the E gene of the recently isolated Malaysian DENV-4 in comparison
to other known DENV-4
1.6.6 (Page, 1996). Maximum-parsimony and maximumlikelihood analyses performed using  (PAUPSearch,
SeqLab, GCG Wisconsin Package, Accelrys Inc., USA) from
multiple alignments made with  (SeqLab, GCG Wisconsin
Package, Accelrys Inc., USA) yielded results similar to those
obtained from the distance matrix analyses, differing only
within the sylvatic isolates genotype. Hence, only results
obtained from the distance matrix method were presented.
Potential recombinant sequences within the E gene were
examined using  version 3.2 (Lole et al., 1999). Putative
recombinant sequence was queried against two potential
parental sequences after all gaps were stripped with a distant
sequence as the outgroup. A sliding window of 180 nucleotides
was moved in steps of 10 nucleotides at a time and the
resulting similarity values were plotted along the E gene
sequence. Recombination was identified when conflicting E
gene sequence profiles appeared, suggesting acquisition of
sequences from a different parental genotype. Bootscanning
analyses which utilized the bootstrapping procedures of
Salminen et al. (1995) and Worobey & Holmes (1999) were
performed using the maximum-likelihood method with 100
resamplings. Bootstrap values of 70 % were used to indicate
robust support for the topologies.
Pairwise comparisons of the sequences showed that the
recently isolated Malaysian DENV-4 isolates had nucleotide
sequence similarity of at least 92 % to the previously reported
epidemic\endemic strains and 86 % to the sylvatic strains. The
nucleotide changes were distributed throughout the E gene
with most of them located at the third nucleotide of a codon
resulting in no amino acid changes. The amino acid similarity
was " 95 % to the sylvatic strains and ranged from 96 to 98 %
to other epidemic\endemic strains (data not shown). These
findings were comparable to those previously reported for all
other DENV-4 isolates (Lanciotti et al., 1997 ; Wang et al.,
2000). Furthermore, alignment of the deduced amino acid
sequences showed conservation of the 12 cysteine amino acids
involved in disulphide bond formation and the putative Nlinked glycosylation sites at amino acids 67 and 153 among all
the DENV-4 isolates. Only a single amino acid difference
(Phe Val) at amino acid 108, however, was noted within the
glycine-rich putative fusion domain (amino acids 98–111) in
isolate Malaysia 2001-22713 (MY01-22713) in comparison to
the remaining four Malaysian DENV-4 isolates (Table 1). This
single amino acid difference was not surprising, however, since
a number of other DENV-4 isolates had different amino acids
at the same position. Amino acids that were characteristic of
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Emergence of dengue virus type 4 genotype IIA
Fig. 1. Phylogenetic tree depicting the
relationships of DENV-4 genotype IIa with
other known DENV-4. The tree was
constructed by the neighbour-joining
method (Saitou & Nei, 1987) using
nucleic acid sequences of the E gene.
Bootstrap values are shown as
percentages derived from 1000
samplings. The scale reflects the number
of nucleotide substitutions per site along
the branches. DENV-2 Jamaica strain
used as outgroup is not shown.
the sylvatic isolates (amino acids 19, 132, 148, 154, 162, 203,
329, 335, 340, 342, 355, 364, 382, 461 and 478), on the other
hand, were not found in any of the recently isolated Malaysian
DENV-4, suggesting that the isolates could not have evolved
recently from sylvatic origin. Examination of the amino acid
sequences also revealed four distinct amino acids at positions
46, 265, 429 and 494 (Thr, Ala, Phe and Gln) that could be used
to differentiate all the DENV-4 into at least two genogroups
(Table 1). The remaining two amino acids at positions 384 and
455 (Asp and Val) identified by Lanciotti et al. (1997) as
characteristic for DENV-4 genotype I were found also in the
recently isolated Malaysian DENV-4. In addition, only the
recently isolated MY01-23314, -23264, -23096 and -23298
and Indonesia 1973 (ID73) DENV-4 had leucine at position
120 when compared to all other DENV-4 (Table 1), suggesting
that this single amino acid change could be unique to the
recently isolated Malaysian DENV-4 and ID73. A phylogenetic tree drawn using the E gene nucleotide sequences
showed three well-supported DENV-4 clusters (bootstrap
values of 100 %) (Fig. 1). These clusters were similar to that
previously identified as genotype I consisting of viruses from
Thailand (TH), Malaysia 1969 (MY69), Sri Lanka (SE) and
Philippines (PH) ; genotype II comprises mainly isolates from
South America and the Pacific Islands (Lanciotti et al., 1997)
and the sylvatic isolates form a distinctly different genotype
(Wang et al., 2000). The recently isolated Malaysian DENV-4
isolates subclustered together with ID73 into a separate and
well-supported (98 %) subcluster within genogroup II, hence
denoted as genotype IIA in the present study (Fig. 1). A
phylogenetic tree drawn using the deduced amino acid
sequence further supported separation of all but one of the
isolates (MY01-22713) into a different subgenogroup (data not
shown). Except for isolate MY01-22713, all other recently
isolated Malaysian DENV-4 had aspartic acid at position 384,
similar to ID73 virus. MY01-22713, on the other hand, had
asparagine, similar to all other DENV-4 of genotype IIB (Table
1). The presence of aspartic acid has been suggested to be the
reason that DENV-4 ID73 could be effectively neutralized by
DENV-4 genotype I-specific serum and not with genotype IIspecific serum (Lanciotti et al., 1997). This suggested the
possibility that the E gene of DENV-4 ID73 together with the
recent Malaysian isolates were mosaics of DENV-4 genotype
I and II. Evidence of recombination ( 70 % bootstrap support)
between DENV-4 genotype I (MY69) and genotype II (ID76)
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Genotype II
Genotype I
S. AbuBakar, P.-F. Wong and Y.-F. Chan
Fig. 2. For legend see facing page.
was obtained from similarity plot and bootscanning analyses
performed on the recently isolated Malaysian DENV-4
MY01–22713 (Fig. 2a). However, only weak evidence supporting recombination between genotype I and II in DENV-4
ID73 and the remaining recently isolated Malaysian DENV-4
was obtained. This finding was similar to that reported by
Worobey et al. (1999) when the E gene of DENV-4 ID73 was
queried against DENV-4 ID77 (genotype II) and PH73
(genotype I) as possible parental lineages. Despite weak
statistical support, it was argued in that study that ID73 is
indeed a genuine recombinant. However, a phylogenetic tree,
drawn using nucleotides at positions 561–800 identified from
the breakpoint analyses, placed (100 % bootstrap support) the
recently isolated Malaysian DENV-4 and ID73 into DENV-4
genotype I (Fig. 2b), thus lending support to the earlier
assertion that the E gene of DENV-4 ID73 and the recently
isolated Malaysian DENV-4 are mosaics of DENV-4 genotype
I and II.
A phylogenetic tree constructed earlier (Fig. 1) using the
entire E gene suggested that DENV-4 genogroup IIA had
originated from DENV-4 ID73. However, amongst the
recently isolated DENV-4, isolate MY01-22713 varied significantly from the rest by having the strongest evidence of
mosaicism and a possible neutralization site that is similar to
genotype IIB at amino acid 384, unlike the rest of the isolates
which resembled genotype I. Since DENV-4 MY01-22713 was
isolated at least 4 months earlier than the remaining isolates, it
raised the possibility that perhaps the later isolates had
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Emergence of dengue virus type 4 genotype IIA
Fig. 2. Similarity plot analyses for determination of potential recombinant sequences in the E gene of DENV-4 genotype IIA. The
similarity plots were generated using  version 3.2 (Lole et al., 1999). The upper panel shows results of pairwise
comparisons between the query isolates, MY01-22713 (a) and MY01-23298 (c), shown in bold above the plot, and their
potential parents, MY1969 and ID76 and MY01-22713 and ID1973, respectively. The vertical axis is the percent similarity
between the query sequence and each parental sequence plotted at the midpoint of each sliding window of 180 nt at 10 nt
per increment after all gaps were stripped. The horizontal axis is the nucleotide numbers counted from the 5h-terminal of E
gene. The lower panel is the result from bootscan analyses illustrating the likelihood of clustering of the putative recombinants
with respect to the parental isolates.(b) A neighbour-joining tree (Saitou & Nei, 1987) constructed from alignment of the
putative recombinant sequences (nucleotides 561–800) places the recently isolated Malaysian DENV-4 genotype IIA (boxed)
into DENV-4 genotype I. The tree was rooted using the sylvatic DENV-4 E gene sequence (not shown) and the bootstrap value
shown was derived from 1000 replicates.
diverged recently as a result of intra-typic recombination.
Evidence of recombination ( 70 % bootstrap support) within
the E gene of the four isolates, MY01-23096, -23298, -23264
and –23314, was obtained when the sequences were queried
against ID73 and MY01-22713 as the parental isolates (Fig.
2c). These findings suggest that DENV-4 ID73 is potentially a
bona fide ancestor of the recently emerged DENV-4 genotype
IIA, whereas isolate MY01-22713 may have emerged independently from different ancestral lineages, perhaps following an earlier intra-serotypic recombination between
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S. AbuBakar, P.-F. Wong and Y.-F. Chan
DENV-4 genotype I (MY69) and genotype II (ID76) as
indicated above (Fig. 2a).
In summary, evidence supporting the emergence of DENV4 genotype IIA in Malaysia from different ancestral lineages
following inter-typic recombination is presented. Whether
DENV-4 genotype IIA remained localized in Malaysia,
however, remains to be seen. Nonetheless, these findings,
along with others (Worobey et al., 1999 ; Tolou et al.,
2001 ; Uzcategui et al., 2001), strongly suggest that recombination amongst specific DENV serotypes has occurred in a
natural population and new genotypes could emerge especially
in a population where multiple strains of the virus are cocirculating.
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Received 7 February 2002 ; Accepted 7 June 2002
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