JOURNAL OF VIROLOGY, June 2008, p. 5494–5500
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 11
Microevolution of Dengue Viruses Circulating among Primary School
Children in Kamphaeng Phet, Thailand?
Richard G. Jarman,1* Edward C. Holmes,2,3Prinyada Rodpradit,1Chonticha Klungthong,1
Robert V. Gibbons,1Ananda Nisalak,1Alan L. Rothman,4Daniel H. Libraty,4
Francis A. Ennis,4Mammen P. Mammen, Jr.,1and Timothy P. Endy5
Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand 104001; Center for Infectious
Disease Dynamics, Department of Biology, Mueller Laboratory, The Pennsylvania State University, University Park,
Pennsylvania 168022; Fogarty International Center, National Institutes of Health, Bethesda, Maryland 208923;
Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester,
Massachusetts 016554; and State University of New York Upstate Medical University, Syracuse, New York 132105
Received 22 December 2007/Accepted 28 February 2008
To determine the extent and structure of genetic variation in dengue viruses (DENV) on a restricted
spatial and temporal scale, we sequenced the E (envelope) genes of DENV-1, -2, and -3 isolates collected
in 2001 from children enrolled in a prospective school-based study in Kamphaeng Phet, Thailand, and
diagnosed with dengue disease. Our analysis revealed substantial viral genetic variation in both time and
space, with multiple viral lineages circulating within individual schools, suggesting the frequent gene flow
of DENV into this microenvironment. More-detailed analyses of DENV-2 samples revealed strong clus-
tering of viral isolates within individual schools and evidence of more-frequent viral gene flow among
schools closely related in space. Conversely, we observed little evolutionary change in those viral isolates
sampled over multiple time points within individual schools, indicating a low rate of mutation fixation.
These results suggest that frequent viral migration into Kamphaeng Phet, coupled with population
(school) subdivision, shapes the genetic diversity of DENV on a local scale, more so than in situ evolution
within school catchment areas.
Dengue is the most common mosquito-borne viral disease
in tropical and subtropical regions of the world, and hence,
dengue virus (DENV) is an emerging human pathogen of
major importance (10). The numbers of cases of dengue
fever (DF) and the more severe conditions dengue hemor-
rhagic fever (DHF) and dengue shock syndrome, as well as
the number of countries affected by dengue, have increased
dramatically. Current estimates place over 2 billion people
in areas of dengue endemicity, with more than 50 million
DENV infections and over 20,000 deaths each year (8, 9).
The majority of dengue cases are characterized by a self-
limited febrile illness (DF) associated with viremia, tran-
sient mild laboratory abnormalities, and sometimes mild
bleeding. In a small percentage of infections, overt plasma
leakage occurs, with hemoconcentration, hypoalbuminemia,
and extravasation of fluid (DHF). Dengue shock syndrome
can result when plasma leakage is severe and is responsible
for most of the severe morbidity and mortality associated
with DENV. The causative RNA virus (family Flaviviridae,
genus Flavivirus) has a single-strand, positive-sense genome
approximately 11 kb in length and consists of four antigeni-
cally distinct serotypes (DENV-1 to DENV-4), which now
cocirculate in many populations.
Over the last 20 years, many studies have documented the
extent and structure of genetic variation in all four DENV
serotypes (reviewed in reference 11). In general, these studies
have revealed that each of the four serotypes contains a num-
ber of phylogenetically distinct “subtypes” (or genotypes), the
genetic structures of which clearly reflect a complex pattern of
viral gene flow (migration among locations) coupled with pop-
ulation subdivision. Most notably, some DENV genotypes ap-
pear to be restricted to specific localities, commonly South
East Asia, while others have more-cosmopolitan distributions
across the tropical and subtropical world. Whether these dif-
ferences in geographic distribution reflect underlying differ-
ences in viral fitness, manifest as differences in transmissibility
and/or virulence, is still a subject of debate.
Despite the growing database of partial and complete
DENV genome sequences (17) and the increasing number of
studies addressing various aspects of the molecular epidemiol-
ogy of DENV infection, none to date have considered the
nature of viral genetic diversity on a scale of populations sep-
arated by only a few kilometers and sampled over a time period
of months. However, such studies of DENV “microevolution”
are essential to obtain a full understanding of the mechanisms
shaping DENV evolution. Further, given forthcoming phase
III clinical trials of DENV vaccines, which may be imperfect in
their protection against all four serotypes and hence shape
viral evolution (7), it is critical to document the extent and
structure of DENV genetic variation at vaccine trial sites and
the pace of viral evolution within a limited time frame. To this
end, we performed a detailed genetic analysis of the envelope
(E) genes of DENV serotype 1, 2, and 3 isolates collected in
Kamphaeng Phet, Thailand, during 2001 as part of a school
absence-based prospective study.
* Corresponding author. Mailing address: Department of Virology,
USAMC-AFRIMS, APO, AP 96546, 315/6 Rajvithi Road, Bangkok,
Thailand 10400. Phone: (662) 644-5644. Fax: (662) 644-4760. E-mail:
?Published ahead of print on 26 March 2008.
MATERIALS AND METHODS
Prospective study with primary school children in Kamphaeng Phet. Twelve
primary schools from the Kamphaeng Phet Province in northern Thailand were
selected to participate in a prospective dengue study (Fig. 1). Details of this study
have been previously published (5). Children were recruited during January 1998
from school grades 1 through 5 and were eligible to remain in the study until
graduation from sixth grade. New first-grade students were enrolled in the cohort
each January. The enrollment criteria were attendance at a study school, enroll-
ment in a grade between first and sixth, and parental informed consent. Blood
samples for dengue serology were obtained from the entire population four times
each year (January or February, 1 June, 15 August, and 15 November). Active
acute-illness-case surveillance occurred during the peak DENV transmission
season, from 1 June to 15 November. Absent students were visited by village
health workers and evaluated with a symptom questionnaire, and oral temper-
atures were obtained. Samples collected outside this surveillance period were
based on subject visits to the hospital during which a study nurse identified the
subjects as enrolled in this study. Acute-phase and 14-day convalescent-phase
blood samples were obtained from absent students with histories of fever within
7 days of school absence or oral temperatures of ?38°C. Laboratory assays,
including an immunoglobulin M (IgM)/IgG enzyme-linked immunosorbent assay
(ELISA) and PCR, were used to diagnose dengue as previously described (5, 6).
Classification of dengue disease. The criteria for DF were school absence
associated with a febrile illness and laboratory confirmation of acute DENV
infection without evidence of DHF by World Health Organization (WHO)
criteria (2). DF was further classified as symptomatic nonhospitalized or symp-
tomatic hospitalized DF. The criteria for DHF were school absence associated
with a febrile illness and laboratory confirmation of acute DENV infection with
evidence of DHF by WHO criteria (2).
Viral isolation and sequencing. For all suspected acute dengue cases, DENV
serotype-specific PCR was performed on the first serum sample obtained for
acute-illness evaluation. Reverse transcription-PCR (RT-PCR) was performed
according to the protocol of Lanciotti et al. (14), with the following modifica-
tions. Avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI)
was used in the first-round RT-PCR. The concentrations of the primers used in
the RT-PCR and nested reactions were reduced from 50 pmol to 12.5 pmol per
reaction, and the number of nested PCR amplification cycles was increased to 25.
For DENV PCR-positive samples, virus isolation was performed with C6/36
cells and/or Toxorhynchites splendens mosquitoes as previously described (13, 16).
DENV serotype identification was performed using a dengue serotype-specific
enzyme immunoassay as previously described (18, 20). Viral RNA for sequencing
was extracted from patient samples first amplified in T. splendens mosquitoes,
followed by one passage in C6/36 cells by use of a QIAamp viral RNA mini kit
(Qiagen, Germany) according to the manufacturer’s instructions. RT was per-
formed using random hexamer oligonucleotides with the SuperScript first-strand
synthesis system (Invitrogen) according to the manufacturer’s instructions. The
DNA fragments of the envelope gene regions of 9 DENV-1, 40 DENV-2, and 18
DENV-3 isolates were amplified by PCR using 5 ?l of cDNA in a 50-?l reaction
mixture containing 0.3 mM deoxynucleoside triphosphates, 2.5 U AmpliTaq
DNA polymerase (Applied Biosystems), 1? PCR buffer, 1.5 mM MgCl2, and 15
pmol of each forward and reverse primer. The PCR mixtures of DENV-1 and -3
were subjected to 1 cycle of 95°C for 5 min; 35 cycles of 94°C for 30 s, 50°C for
1 min, and 72°C for 2 min; and 1 cycle of 72°C for 15 min. The PCR mixtures of
DENV-2 were subjected to the same thermal conditions as those of DENV-1 and
-3, except for the annealing temperature, which was changed to 55°C. The
PCR-amplified DNA fragments were purified using QIAquick PCR purification
kits (Qiagen) according to the manufacturer’s instructions. Purified DNA
fragments were used for sequencing.
Sequencing reactions were performed by using a DYEnamic ET dye terminator
sequencing kit (GE Healthcare Bio-Sciences) according to the manufacturer’s in-
structions. The sequencing primers are available upon request. The sequencing
products were cleaned by standard precipitation before sequencing with a Mega-
BACE 500 automated DNA sequencer (GE Healthcare Bio-Sciences). Overlap-
ping nucleic acid sequences were combined for analysis and edited by using
SEQUENCHER software (Gene Code Corporation).
Unfortunately, the number of viruses available for sequencing was a relatively
small percentage of the viruses circulating in our cohort during the 2001 dengue
season. In all, 244 infections, including inapparent infections, were detected in
our cohort. Of the overt infections, only 86 had virus detectable by PCR, while
FIG. 1. Map of study area in subdistrict of Muang, Kamphaeng Phet, Thailand, showing the locations of participating schools.
VOL. 82, 2008MICROEVOLUTION OF DENGUE VIRUS5495
the remaining 38 were detected based on a serological response consistent with
acute dengue. Hence, we were able to sequence 78% of the PCR-positive sam-
ples but only 67 of 244 sequences (27%).
Phylogenetic analysis. The following sets of DENV E-gene sequences from
Kamphaeng Phet were compiled for evolutionary analysis: for DENV-1, 9 se-
quences (genotype I) from three schools; for DENV-2, 40 sequences (genotype
Asian I) from nine schools; and for DENV-3, 18 sequences (genotype II) from
three schools. To place these isolates within the wider context of DENV evolu-
tion, particularly that from South East Asia, we also compiled, from GenBank,
phylogenetically representative “background” data sets of sequences from each
of the respective genotypes, many of which also came from Thailand. This
resulted in final data sets of the following sizes in the phylogenetic analyses: for
DENV-1, 116 sequences (1,485 nucleotides [nt]); for DENV-2, 168 sequences
(1,485 nt); and for DENV-3, 124 sequences (1,479 nt).
Phylogenetic trees for all three data sets were inferred using the maximum
likelihood (ML) method available in PAUP* (19), with the best-fit model of
nucleotide substitution (usually GTR?I??4) determined using the MODELTEST
program (15) and employing tree bisection-reconnection branch swapping. Boot-
strap resampling (1,000-replicate neighbor-joining trees under the ML substitu-
tion model) was used to determine the phylogenetic support for individual nodes.
All parameter values are available from the authors on request.
Analysis of spatial and temporal structure. In the case of DENV-2, the larger
number of E-gene sequences allowed us to determine whether there were dis-
tinct spatial and temporal components to the evolutionary patterns observed.
This was achieved by using a parsimony approach which had previously proven
highly informative in a study of DENV in the Caribbean (4). In the case of spatial
structure, we tested whether there was significant clustering according to the
school from which the virus was isolated. In the case of temporal structure, we
tested whether viruses were clustered according to their months of isolation. In
each of these analyses, DENV-2 sequences were assigned a character state
dependent on the school (spatial) or month (temporal) of origin. Given the ML
phylogeny for these sequences (determined as described above) and the above-
defined isolate states, the minimum number of changes in character state needed
to give rise to the observed distribution of states was then estimated using
parsimony (with all ambiguous changes excluded). To determine the expected
number of changes under the null hypothesis of complete mixing among states by
space or time, the states of all isolates were randomized 500 times, and for each
randomization, the number of changes in state was calculated in the manner
described above. The difference between the mean numbers of observed and
expected changes for each pair of states indicates the level of geographic or
temporal isolation, with statistical significance calculated by comparing the total
number of observed state changes to the number expected under random mixing.
All these analyses were performed using PAUP*.
Analysis of selection pressures. Overall and site-specific selection pressures in
the E genes of all three serotypes circulating in Kamphaeng Phet (excluding
other Asian sequences) were measured as ratios of nonsynonymous (dN) to
synonymous (dS) substitutions per site (dN/dS), estimated using the single-like-
lihood-ancestor-counting and random-effect-likelihood methods. Both methods
utilized the GTR substitution model with input phylogenetic trees inferred using
the neighbor-joining method available at the Datamonkey Web facility (12).
Nucleotide sequence accession numbers. All DENV gene sequences have
been submitted to GenBank and assigned accession numbers EU117304 to
EU117312, EU117313 to EU117352, and EU117353 to EU117370 for DENV-1,
-2, and -3, respectively.
RESULTS AND DISCUSSION
DENV prevalence and serotypes in Kamphaeng Phet. From
1998 to 2002, a prospective study of DENV infection was
conducted with ?2,000 primary school age children in Kam-
phaeng Phet, Thailand. Overt illness was detected using school
absences to identify sick children, and DENV infections were
confirmed using IgM/IgG ELISA, hemagglutination inhibition,
and RT-PCR (5). Inapparent infections were identified based
on a fourfold rise in hemagglutination inhibition between se-
rial serum samples. Over the 5-year study period, 312 inapparent
detected in children attending every school in the study, and all
four serotypes were identified by RT-PCR (1, 5, 6).
The highest incidence of disease occurred in 2001, with 120
inapparent infections and 124 cases of overt disease (Table 1).
Because of the high dengue incidence during 2001, samples
were collected throughout the year, with the majority identi-
fied during the peak dengue season of June to November as
part of the school absence study and the remainder identified
from hospitalization of study participants (Table 2). Through
ELISA typing of clinical isolates or RT-PCR of serum samples,
we were able to identify the infecting DENV serotype in 86
(69%) symptomatic cases; the remaining 38 (31%) were iden-
tified as acute DENV infection by serology only. DENV-2 was
the predominant serotype, identified in 52 of the 86 (60%)
cases, followed by DENV-3 (25 cases; 29%) and DENV-1 (9
cases; 10%), collected in 10 of the 12 schools.
Microevolution of DENV in Kamphaeng Phet. To determine
the extent and structure of genetic diversity during the 2001
dengue season, we sequenced the E genes of all available
TABLE 1. Numbers of DENV infections in school absence-based
cohort during 2001
No. of subjects
Total 12080 13 31
TABLE 2. Numbers of viral isolates sequenced according to school
and month of sampling
(no. of isolates)
Mo of sampling
July, August, November
June, July, August, November
June, July, August, September
June, August, September
May, June, July, August,
April, May, June, July, August
June, July, August, September,
5496JARMAN ET AL.J. VIROL.
viruses collected during this year (Table 2). In total, all nine
DENV-1 virus samples, collected from students in schools 8,
11, and 12 from July to November 2001, were available. All
were assigned to genotype I, which is common in South East
Asia, especially in Thailand. Similarly, 40/52 DENV-2 viruses,
from 9 of the 12 schools, were available for sequencing, all of
which represented the Asian I genotype, which is common in
Thailand. The period of collection of these viruses was diverse,
ranging from February to November and hence covering peri-
ods of both low and high DENV transmission. A total of 18/25
DENV-3 viruses, obtained from three schools in 2001 and
largely from school 11 between June and November, were
available for sequencing. All DENV-3 viruses were assigned to
genotype II, which is again common in South East Asia. Vi-
ruses that were unavailable for sequencing were unavailable
because of a lack of clinical material, a failure to grow a viral
stock during the study, or insufficient genetic material for se-
ML phylogenetic trees for the DENV-1, DENV-2, and
DENV-3 E-gene sequences from Kamphaeng Phet, combined
with the background isolates, are presented in Fig. 2, 3, and 4,
respectively. In the case of DENV-1, viral isolates fall into two
distinct clades, separated by other Asian viruses. That the
DENV-1 isolates from school 11 fall in both clades provides
compelling evidence for the independent entry of genetically
distinct viruses into this spatially restricted region. A similar
pattern was observed in DENV-2. Here, those viruses sampled
from schools 1, 5, 8, 9, and 10 formed school-specific clusters
(although only those from school 1 were clearly phylogeneti-
cally distinct from those from the other schools), while multi-
ple genetic variants were seen in schools 2, 4, 11, and 12 (with
one highly divergent variant observed in school 11). Finally, in
the case of DENV-3, single clades were observed in schools 4
and 10, while two clades were again observed in school 11,
although with weak bootstrap support. Notably, children from
school 11 come from a densely populated urban environment
at the center of the study area, close to the major transporta-
tion throughway and a public health clinic, which may have
contributed to the appearance of multiple viral variants in this
FIG. 2. ML tree of selected isolates of genotype I of DENV-1. Different schools from within Kamphaeng Phet are designated by different
colors. All horizontal branch lengths are drawn to scale according to the numbers of nucleotide substitutions per site, with bootstrap support values
shown next to relevant nodes. The tree is midpoint rooted for purposes of clarity only.
VOL. 82, 2008MICROEVOLUTION OF DENGUE VIRUS5497
Overall, these phylogenetic results reveal a marked cluster-
ing by school (reflected in the grouping of colors in Fig. 2 to 4),
indicating that multiple genetic variants of DENV circulate
within a spatially restricted area during a single dengue season
but that individual schools represent distinct evolutionary en-
tities that have experienced clear population subdivision. In
only a relatively small number of cases do multiple variants of
individual serotypes cocirculate, most notably in some cases
involving school 11. Further, the observation that these distinct
clusters are often separated by viruses isolated from outside
Kamphaeng Phet (and in the cases of DENV-1 and DENV-2
outside Thailand) indicates that there were multiple introduc-
tions of DENV into this region during 2001. We believe that
this is the first report of evolution on a localized scale for
DENV, and this report indicates that even viruses sampled
from spatially restricted regions may harbor extensive genetic
To determine the extent of spatial clustering more rigor-
ously, we examined DENV-2 in more detail as this represented
our best-sampled serotype. A parsimony-based analysis con-
firmed that there is a highly significant clustering of viral iso-
lates by school (P ? 0.005). However, despite this strong ge-
netic segregation, there is also clear evidence (P ? 0.005) of
viral migration between schools 8 and 9. Notably, these two
schools are geographically the closest in the study population,
separated by only 5 km, and the furthest from the city, at
distances of 36 and 33 km from the field site, respectively (Fig.
1). Further, the villages that feed these schools have low pop-
ulation densities, are highly rural, and possess poor road in-
frastructure, making access to this area more difficult than
access to other regions of Kamphaeng Phet. As such, our data
suggest that patterns of viral gene flow are determined by local
geographic and economic variables. The villages that make up
the Kamphaeng Phet region are diverse, with the most devel-
FIG. 3. ML tree of selected isolates of the Asian I genotype of DENV-2. Different schools from within Kamphaeng Phet are designated by
different colors. All horizontal branch lengths are drawn to scale according to the numbers of nucleotide substitutions per site, with bootstrap
support values shown next to relevant nodes. The tree is midpoint rooted for purposes of clarity only.
5498JARMAN ET AL.J. VIROL.
oped and densely populated villages lying close to the city
center, where frequent travel into and out of the city is com-
mon. In contrast, villages become less developed and densely
populated further from the city center, in turn restricting travel
and resulting in the genetic isolation of viruses from schools 8
In contrast, these DENV-2 sequence data exhibited no sig-
nificant clustering by month of sampling, indicating that there
was little replacement of viral lineages at this level of spatial
and temporal resolution. Similarly, at the level of the E-gene
sequences analyzed, there was little evidence for in situ evolu-
tion, such that viruses sampled from the same school over time
periods ranging from days to months exhibited few mutational
differences (in some cases only one mutation over a 6-month
sampling period). Such relative intraschool stability sits in
marked contrast to the often strong genetic differentiation
among schools. This observation, coupled with the strong pop-
ulation subdivision exhibited by each school, indicates that the
importation of DENV into the study population, rather than in
situ evolution within school catchment areas, was the most
important factor shaping viral genetic diversity on this spatial
FIG. 4. ML tree of selected isolates of genotype II of DENV-3. Different schools from within Kamphaeng Phet are designated by different
colors. All horizontal branch lengths are drawn to scale according to the numbers of nucleotide substitutions per site, with bootstrap support values
shown next to relevant nodes. The tree is midpoint rooted for purposes of clarity only.
VOL. 82, 2008MICROEVOLUTION OF DENGUE VIRUS5499
To determine the selection pressures acting on the DENV Download full-text
E-gene sequences sampled from Kamphaeng Phet, we com-
pared the relative numbers of nonsynonymous (dN) and syn-
onymous (dS) substitutions per site. This analysis provided no
evidence for positive selection acting on any nucleotide site,
although all dN/dSmethods are inherently conservative. In-
deed, the overall picture obtained was that of strong purifying
selection, with mean dN/dSvalues of 0.084, 0.065, and 0.008 for
DENV-1, DENV-2 and DENV-3, respectively. Such strong
purifying selection has previously been observed for these se-
rotypes in Thailand (21, 22), highlighting the predominance of
genetic drift over natural selection in shaping DENV substitu-
tion dynamics in the short term. However, the reasons for the
greater selective constraints in DENV-3, which is character-
ized by very low dN/dSvalues, are unclear. Further, as random
genetic drift is the most common process determining substi-
tution dynamics, allele frequencies are not expected to change
greatly over the period of sampling, even given the high mu-
tation rate of DENV (3).
Overall, these results have mixed implications for how
DENV populations might respond to imperfect vaccination in
the near future. DENV sampled on a short spatial and tem-
poral scale evidently exhibits remarkably high levels of genetic
diversity, thereby providing the raw material for adaptive evo-
lution. However, this variation is more often generated by
migration than by mutation accumulation, with purifying se-
lection the dominant evolutionary force.
We acknowledge the invaluable contributions of the clinical, labo-
ratory, and entomological personnel of AFRIMS and Kamphaeng
Phet AFRIMS Research Unit (KAVRU). We are indebted to the staff
of the Kamphaeng Phet AFRIMS Research Unit (KPP) Governor and
Chief, the KPP Provincial Medical Office and Chief, and the KPP
Provincial School Office as well as participating village leaders. Finally,
we are grateful to the children of the KPP and their parents for their
lasting enthusiasm and cooperation.
Funding was partially provided by the U.S. Military Infectious Dis-
ease Research Program, Ft. Detrick, MD, and the National Institutes
of Health (NIH-P01-AI34533).
The opinions and assertions contained herein are the personal views
of the authors and are not to be construed as official or reflecting the
views of the Armed Forces Research Institute of Medical Sciences,
the U.S. Department of the Army, the U.S. Department of Defense, or
the National Institutes of Health.
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