intra-genotypic variation of predominant genotype ii strains of dengue type-3 virus isolated during...
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Intra-genotypic variation of predominant genotype II strainsof dengue type-3 virus isolated during different epidemicsin Thailand from 1973 to 2001
Aimee Zhang • Piyawan Chinnawirotpisan • Yuxin Tang •
Yanfei Zhou • Julia Lynch • Stephen Thomas •
Siripen Kalayanarooj • Robert Putnak • Chunlin Zhang
Received: 10 September 2011 / Accepted: 19 January 2012 / Published online: 13 March 2012
� Springer Science+Business Media, LLC (Outside the USA) 2012
Abstract The prevalence of all four dengue virus
(DENV) serotypes has increased dramatically in recent
years in many tropical and sub-tropical countries accom-
panied by an increase in genetic diversity within each
serotype. This expansion in genetic diversity is expected to
give rise to viruses with altered antigenicity, virulence, and
transmissibility. We previously demonstrated the co-cir-
culation of multiple DENV genotypes in Thailand and
identified a predominant genotype for each serotype. In this
study, we performed a comparative analysis of the com-
plete genomic sequences of 28 DENV-3 predominant
genotype II strains previously collected during different
DENV-3 epidemics in Thailand from 1973 to 2001 with
the goal to define mutations that might correlate with
virulence, transmission frequency, and epidemiological
impact. The results revealed (1) 37 amino acid and six
nucleotide substitutions adopted and fixed in the virus
genome after their initial substitutions over nearly 30-year-
sampling period, (2) the presence of more amino acid and
nucleotide substitutions in recent virus isolates compared
with earlier isolates, (3) six amino acid substitutions in
capsid (C), pre-membrane (prM), envelope (E), and non-
structural (NS) proteins NS4B and NS5, which appeared to
be associated with periods of high DENV-3 epidemic
activity, (4) the highest degree of conservation in C, NS2B
and the 50-untranslated region (UTR), and (5) the highest
percentage of amino acid substitutions in NS2A protein.
Keywords Dengue type-3 virus � Sequence comparison �Intra-genetic variation
Introduction
Dengue (DEN) is one of the most important viral diseases
of the twenty-first century. Its re-emergence is likely due to
increases in the human population, expansion of global
travel networks, and climatic changes altering the distri-
bution of the primary mosquito vector, Aedes aegypti. The
causal agents, the DEN viruses (DENVs), are represented
by 4 serotypes, DENV1-4, belonging to the family Flavi-
viridae, which co-circulate widely in the tropics and sub-
tropics. Infection with any one of the four DENV serotypes
can lead to disease ranging from relatively mild dengue
fever (DF) characterized by fever, rash, malaise, body
aches, and pains, to much more severe dengue hemorrhagic
fever (DHF) associated with capillary leakage and hem-
orrhage sometimes progressing to dengue shock syndrome
(DSS). It is not clear why some individuals experience only
uncomplicated DF while others progress to DHF or DSS.
Electronic supplementary material The online version of thisarticle (doi:10.1007/s11262-012-0720-2) contains supplementarymaterial, which is available to authorized users.
A. Zhang
Science and Engineering Apprentice Program at the Walter Reed
Army Institute of Research, Silver Spring, MD, USA
P. Chinnawirotpisan � C. Zhang
Department of Virology, U.S. Army Medical Component-Armed
Forces Research Institute of Medical Sciences, Bangkok,
Thailand
Y. Tang � Y. Zhou � J. Lynch � S. Thomas � R. Putnak �C. Zhang (&)
Division of Viral Diseases, Walter Reed Army Institute
of Research, 503 Robert Grant Avenue, Silver Spring,
MD 20910, USA
e-mail: [email protected]
S. Kalayanarooj
Queen Sirikit National Institute of Child Health,
Bangkok, Thailand
123
Virus Genes (2013) 46:203–218
DOI 10.1007/s11262-012-0720-2
However, it is thought due to both the inherent virulence of
the infecting virus, i.e., viral genetics [1–3] and predis-
posing host factors including humoral and cell-mediated
immunity from previous DENV infection [1, 4–7].
The DENV is a single-stranded, positive-sense RNA
virus with a genome of approximately 11 kb, which
contains one open reading frame flanked by 50- and
30-untranslated regions (50-UTR/30-UTR). The virus gen-
ome encodes three structural proteins, capsid (C),
premembrane/membrane (PrM/M), and envelope (E) and
seven non-structural (NS) proteins, NS1 though NS5, in the
following order: 50-UTR–C–PrM/M–E–NS1–NS2A–NS2B
–NS3–NS4A–NS4B–NS5–30-UTR. The DENV genome is
expected to rapidly accumulate mutations due to the error-
prone nature of viral RNA polymerase, resulting in genetic
changes in DENVs as they spread worldwide. Such genetic
changes may have significant implications for the emer-
gence of new genotype(s) and/or viruses with altered
antigenicity, virulence, or tissue tropism, and also may
influence disease patterns and transmission [8]. DENVs
have evolved rapidly, and genotypes associated with
increased virulence have expanded from South and
Southeast Asia into the Pacific Rim and the Americas. Co-
circulation of multiple genotypes in the same community
has been commonly reported in many countries. Five
DENV-3 genotypes, genotype I–V, have been identified [9,
10]. Genotype I strains, which circulate mainly in Indo-
nesia, Malaysia, and Philippines, have recently been iso-
lated in the South Pacific islands. Genotype II strains are
present in Thailand, Vietnam, and Malaysia. Genotype III
strains are found mainly in Venezuela, Central America,
Sri Lanka, India, and Samoa. Genotype IV strains have
been isolated from outbreaks in China, Philippines, and
Malaysia. Genotype V strains have been isolated from
outbreaks in Puerto Rico.
DENV infection is endemic in Thailand with cases
reported every year. Since the first large DEN epidemic in
1958, the country has experienced two major epidemics in
1987 and 1998 [11, 12]. Until 2003, Thailand reported the
greatest number of DEN cases among the South East Asia
Region (SEAR) countries. In 2006, 23% of the reported
DEN cases in the SEAR were from Thailand. Bangkok is
the epicenter of DEN in Thailand and the place where DHF
was first described [6]. The records of DEN positive cases
at Children’s Hospital, Queen Sirikit National Institute of
Children Health (QSNICH) show that DENV-3 is the most
prevalent serotype followed by DENV-2, DENV-1, and
DENV-4 [12–14]. DENV-3 and DENV-1 appear to cause
more severe DEN diseases after primary infection than the
other two serotypes [4, 12, 15, 16]. Among the co-circu-
lating DEN serotypes, DENV-3 was the leading cause of
the major disease outbreaks in 1987 and 1998 in Thailand
[12]. Although the DENV-3 genotypes I–III are reported in
Thailand, our previous study demonstrated that genotype II
is predominant and has been circulating in the country for
over three decades [17, 18]. Due to the lack of in vivo and
in vitro correlates of virulence, the precise mechanism by
which DENVs cause severe disease is still not well known.
However, to successfully control DEN it is essential to
fully understand its etiology. In the absence of good
experimental disease models, a thorough comparative
analysis of complete viral genomic sequences of clinical
isolates sampled sequentially over time from the same
community may be an effective alternative approach for
correlating viral genetic changes with transmission, epi-
demic behavior, and possibly with pathogenesis. This
approach is especially powerful when the sequence anal-
yses can be cross referenced and correlated with accurate
clinical records detailing disease severity and other factors
associated with each viral isolate. In this way, potentially
important mutations can be identified for the study of
disease mechanism, and the information can be used to
develop more effective virus and vector control measures.
This study, a comparative analysis of the complete
genomic sequences of DENV-3 predominant genotype
clinical isolates associated with different epidemics in
Thailand from 1973 to 2001, is an extension of our pre-
vious studies [18]. The goal of this study was to better
define potentially important genetic changes in the DENV-
3 genome that might correlate with increased incidence,
transmission frequency, and virulence and hopefully pave
the way for more comprehensive phenotypic analyses in
future studies.
Materials and methods
Study samples and pertinent background information
The DENV-3 isolates (N = 28) analyzed in this study
represent predominant genotype II strains circulating in
Thailand for the past three decades. Of 28 DENV-3 iso-
lates, 18 were sampled in 2001 during a period of inter-
mediate DENV-3 epidemic activity (Supplement Figure 1).
Ten additional DENV-3 isolates from the children’s hos-
pital, QSNICH, Bangkok repository were collected in 1973
(N = 2), a period of relatively low DEN incidence, in 1987
(N = 2), during the first major DEN epidemic in Thailand
in recent times where DENV-3 was the most prevalent
serotype followed by DENV-2 and DENV-1, in 1993
(N = 2) and 1994 (N = 2) during a period intermediate
DENV-3 epidemic activity, and in 1998 (N = 2) during a
second major DEN epidemic in Thailand where DENV-3
was the most prevalent serotype followed by DENV-1,
DENV-2, and DENV-4 [12]. Six of the 28 DENV-3 iso-
lates were sequenced for this study in the Department of
204 Virus Genes (2013) 46:203–218
123
Virology, AFRIMS, Bangkok, and the sequences of other
22 isolates were retrieved from GenBank representing all
available Thai DENV-3 complete genomic sequences
currently deposited in GenBank. The viruses sampled from
QSNICH were serotyped by both ELISA and nested
RT-PCR assay. All 28 DENV-3 strains were genotyped by
phylogenetic analysis of the virus E gene sequences
(Supplement Figure 2). The background data pertaining to
virus isolation, serological, molecular, and epidemiological
characterization, are described elsewhere [12, 19, 20] for
the viruses sampled from the QSNICH.
Viral RNA extraction, RT-PCR, and genomic
sequencing
Virus RNA was extracted either from infected cell culture
supernatants (for viruses sampled before 2000) or directly
from patient’s sera (for viruses sampled after 2000) using
Trizol (Invitrogen) LS reagent according to manufacturer’s
instructions. The extracted viral RNA was used for RT-PCR
amplification. The detailed methodologies for RT-PCR
amplification and genomic sequencing are described else-
where [18]. The accession numbers of all 28 DENV-3
complete sequences are presented in Table 1.
Sequence alignment analysis
The complete viral genomic sequences and deduced amino
acid (aa) sequences of the 28 Thai DENV-3 strains were
aligned using the Clustal W (1.81) software program
available online at http://www.genome.jp/tools/clustalw/.
The strain, CH53489-1973 sampled in 1973, the earliest
isolate of the 28 strains analyzed in this study, was used as
the reference strain, and all the other strains, the later
isolates, were compared with this reference strain gene/
region by gene/region at the level of each individual aa and
nucleotide (nt) in the sequences. The terms high, interme-
diate, and low are used here to refer to the relative DEN
epidemic activity (i.e., case rates) during the time when the
individual virus isolates were collected, and are based on
the DEN case rates of the QSNICH.
RNA secondary structure prediction
The MFOLD software program [21] available online
(www.bioinfo.rpi.edu/applications/mfold/cgi-bin/rna-forml.
cgi) [22] was used to perform RNA folding to predict the
secondary structures of nt sequences comprising the
50-UTR (96 nt), 30-UTR (451 nt), and the cyclized 50–30
termini for each strain. For 50–30end cyclization (291 nt),
the first 143 nt, including the entire 50-UTR (96 nt) plus the
first 47 nt of the C gene (containing the AUG start codon),
were juxtaposed with the last 106 nt of the 30-UTR. A
poly-A sequence (N = 42) was inserted between the 50-end
and the 30-end to represent the rest of the viral genome.
Results
Genetic variation within each gene
Comparative analyses of the complete viral genomic
sequences, gene by gene, for the 28 DENV-3 isolates
revealed that the most conserved proteins were NS2B and
C. Except for single sporadic aa substitutions that occurred
at certain sites in the C protein for four isolates (ThD3-
0104-93, ThD3-0055-93, CO331-94, and ThD3-1687-98),
and in the NS2B protein for isolates, CO331-94 and ThD3-
1687-98 [aa labeled with () in Table 3], there were no other
aa substitutions in these proteins. However, these sporadic
aa substitutions were not present in the viral genomes of
succeeding generations, which suggests that they might
have been deleterious to virus survival and were eventually
eliminated by purifying selection acting on the DENV-3
genome. The least conserved protein, NS2A, had 10 aa
substitutions, which was the highest percentage (4.59%) of
aa substitutions, followed by M [ PrM [ NS1 [ NS4A [30-UTR [ NS5 [ E = NS3 = NS4B (Table 2).
Genetic variation among viruses collected
from different sampling times (early vs. late isolates)
The 28 isolates analyzed in this study were divided into six
groups according to the sampling years. The sampling time
spanned 28 years from the group-1 isolates collected in 1973
to the group-6 isolates collected in 2001. Groups 1–5 each
contained two isolates sampled in the same year, but Group-6
contained 18 isolates sampled in 2001. Comparative analysis
of their complete viral genomic sequences revealed that virus
isolates sampled more recently generally had more aa/nt
substitutions than those sampled earlier (Table 2). For
instance, there were 11 aa and nt substitutions in the 1973
isolate (BID-V3360-73), 37 to 38 in the 1987 isolates, 45 to 46
in the 1993 isolates, 56 in the 1994 isolates, 55 to 58 in the
1998 isolates. After 1994, the number of aa substitutions
tended to decrease, but only slightly, with 46 to 48 in the 1998
isolates, and 42 to 44 in the 2001 isolates (Table 2). This
trend toward increasing numbers of aa/nt substitutions over
time suggests that the viruses currently circulating in Thailand
have been evolving. It is notable that 37 aa substitutions (one
in PrM protein, two in M protein, four in E protein, five in NS1
protein, nine in NS2A protein, four in NS3 protein, one in
NS4A and two in NS4B proteins, and nine in NS5 protein) and
six nt substitutions (one in the 5’-UTR and five in the 3’-UTR)
were adopted and fixed in virus genome after their initial
substitutions (Table 3, aa/nt denoted by italics). These results
Virus Genes (2013) 46:203–218 205
123
suggest that a process of microevolution was acting on the
viral genome over time. The mutations at these sites did not
appear to be deleterious or adversely affect virus fitness, but
were perhaps even favorable for virus survival, which might
explain why they were adopted and fixed in virus genome.
Genetic variation among viruses sampled from different
epidemic periods
According to the clinical case rates of DENV-3 infection
from records maintained by QSNICH, Bangkok (Supple-
ment Figure 1), two isolates (CH53489-73 and BID-
V3360-73) were associated with a period of relatively low
DENV-3 epidemic activity in 1973; four isolates (ThD3-
0104-93, ThD3-0055-93, CO360-94, and CO331-94) were
associated with a period of intermediate DENV-3 epidemic
activity during 1993–1994; two isolates (ThD3-0010-87
and ThD3-0007-87) were sampled from the first large DEN
outbreak in the country which occurred in 1987; two iso-
lates (ThD3-1283-98 and ThD3-1687-98) were sampled
during the second large DEN outbreak in the country in
1998; and the remaining 18 isolates were sampled during a
period of intermediate DEN epidemic activity in 2001
(Table 3). Comparison of the complete viral genomic
sequences of these isolates revealed that viruses circulating
in different DENV-3 epidemics were genetically different,
and that the aa/nt differences were distributed in each gene
except for the 50-UTR (Table 3). For instance, comparing
the complete genomic sequences of virus isolates sampled
in the 1987 and the 1998 DEN epidemics, eight different aa
substitutions and one nt substitution were observed. Of the
eight aa substitutions in the 1998 isolates, four occurred in
residues E-132, E-172, NS4B-274, and NS5-389 while the
same substitutions were not observed in the 1987 isolates.
Table 1 Sample information of 28 DENV-3 isolates sampled from Thailand
Sample name Genotype Dengue
epidemics
Disease
severity
Dengue
infection
Isolation
(Years)
GenBank
accession #
DENV-3/TH/BID-V3360-1973 II Low –a – 1973 GQ868593
CH53489-1973 II Low – – 1973 DQ863638
ThD3-0007-87 II High DF Primary 1987 AY676353
ThD3-0010-87 II High DHF Secondary 1987 AY676352
ThD3-0104-93 II Interb DF Secondary 1993 AY676350
ThD3-0055-93 II Inter DHF Primary 1993 AY676351
CO360-94 II Inter DF – 1994 AY923865
CO331-94 II Inter DHF – 1994 AY876794
ThD3-1283-98 II High DF Primary 1998 AY676349
ThD3-1687-98 II High DHF Secondary 1998 AY676348
DENV-3/TH/BID-V2312-01 II Inter – – 2001 FJ744726
DENV-3/TH/BID-V2313-01 II Inter – – 2001 FJ744727
DENV-3/TH/BID-V2314-01 II Inter – – 2001 FJ744728
DENV-3/TH/BID-V2315-01 II Inter – – 2001 FJ744729
DENV-3/TH/BID-V2316-01 II Inter – – 2001 FJ744730
DENV-3/TH/BID-V2317-01 II Inter – – 2001 FJ744731
DENV-3/TH/BID-V2318-01 II Inter – – 2001 FJ687448
DENV-3/TH/BID-V2319-01 II Inter – – 2001 FJ810413
DENV-3/TH/BID-V2320-01 II Inter – – 2001 FJ744732
DENV-3/TH/BID-V2321-01 II Inter – – 2001 FJ744733
DENV-3/TH/BID-V2322-01 II Inter – – 2001 FJ810414
DENV-3/TH/BID-V2323-01 II Inter – – 2001 FJ744734
DENV-3/TH/BID-V2324-01 II Inter – – 2001 FJ744735
DENV-3/TH/BID-V2325-01 II Inter – – 2001 FJ744736
DENV-3/TH/BID-V2326-01 II Inter – – 2001 FJ744737
DENV-3/TH/BID-V2327-01 II Inter – – 2001 FJ744738
DENV-3/TH/BID-V2328-01 II Inter – – 2001 FJ744739
DENV-3/TH/BID-V2329-01 II Inter – – 2001 FJ744740
a Information is unavailableb Intermediate dengue epidemic
206 Virus Genes (2013) 46:203–218
123
Ta
ble
2S
um
mar
yo
fto
tal
nu
mb
eran
dp
erce
nta
ge
of
amin
oac
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a)/n
ucl
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de
(nt)
sub
stit
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on
sin
each
gen
ean
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Rs
of
28
Th
aiD
EN
V-3
stra
ins
Item
sT
ota
l#
of
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tal
%
of
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tal
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mb
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erce
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ge
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oac
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bst
itu
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ns
inea
chg
ene
To
tal
#an
d%
of
nt
dif
.
at50 -
UT
R/30 -
UT
R3
C (11
4aa
)
Pre
M
(91
aa)
M (75
aa)
E (49
3aa
)
NS
1
(35
2aa
)
NS
2A
(21
8aa
)
NS
2B
(13
0aa
)
NS
3
(61
9aa
)
NS
4A
(15
0aa
)
NS
4B
(24
8aa
)
NS
5
(90
0aa
)
50 -
UT
R
(96
nt)
30 -
UT
R
(45
1n
t)
Vir
use
s#
%#
%#
%#
%#
%#
%#
%#
%#
%#
%#
%#
%#
%
CH
53
48
9-7
30
00
00
00
00
00
00
00
00
00
00
00
00
00
0
BID
-V3
36
0-7
31
00
.29
00
00
00
00
20
.57
41
.83
00
10
.16
00
10
.40
20
.22
00
1*
0.2
2
Th
D3
-00
10
-87
29
0.8
60
01
1.1
02
2.6
71
0.2
05
1.4
25
2.2
90
03
0.4
80
02
0.8
11
01
.11
11
.04
81
.77
Th
D3
-00
07
-87
34
1.0
00
01
1.1
02
2.6
73
0.6
15
1.4
28
3.6
70
03
0.4
80
02
0.8
11
01
.11
11
40
.89
Th
D3
-01
04
-93
39
1.1
51
0.8
82
2.2
02
2.6
74
0.8
15
1.4
29
4.1
30
04
0.6
51
0.6
72
0.8
19
1.0
01
1.0
45
0.1
1
Th
D3
-00
55
-93
40
1.1
81
0.8
82
2.2
02
2.6
74
0.8
15
1.4
21
04
.59
00
40
.65
10
.67
20
.81
91
.00
11
.04
50
.11
CO
33
1-9
45
01
.47
10
.88
22
.20
34
.00
71
.42
61
.70
10
4.5
91
0.7
74
0.6
51
0.6
72
0.8
11
31
.44
11
.04
50
.11
CO
36
0-9
44
81
.42
00
11
.10
22
.67
11
2.2
35
1.4
29
4.1
30
05
0.8
11
0.6
74
1.6
11
01
.11
11
.04
71
.55
Th
D3
-12
83
-98
48
1.4
20
01
1.1
04
5.3
36
1.2
25
1.4
29
4.1
30
04
0.6
53
2.0
03
1.2
11
31
.44
11
.04
61
.33
Th
D3
-16
87
-98
46
1.3
61
0.8
81
1.1
02
2.6
77
1.4
25
1.4
29
4.1
32
1.5
45
0.8
11
0.6
73
1.2
11
01
.11
11
.04
12
2.6
6
BID
-V2
32
8-0
14
31
.27
00
22
.20
22
.67
61
.22
51
.42
94
.13
00
50
.81
21
.33
20
.81
10
1.1
11
1.0
48
*1
.77
BID
-V2
32
9-0
14
31
.27
00
22
.20
22
.67
61
.22
51
.42
94
.13
00
50
.81
21
.33
20
.81
10
1.1
11
1.0
47
*1
.55
BID
-V2
31
2-0
14
31
.27
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
06
0.9
72
1.3
32
0.8
11
01
.11
11
.04
8*
1.7
7
BID
-V2
31
3-0
14
31
.27
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
06
0.9
72
1.3
32
0.8
11
01
.11
11
.04
10
*2
.22
BID
-V2
31
4-0
14
31
.27
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
06
0.9
72
1.3
32
0.8
11
01
.11
11
.04
8*
1.7
7
BID
-V2
31
5-0
14
41
.30
00
22
.20
22
.67
51
.01
51
.42
10
4.5
90
05
0.8
12
1.3
33
1.2
11
01
.11
11
.04
6*
1.3
3
BID
-V2
31
6-0
14
21
.24
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
05
0.8
12
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
BID
-V2
31
7-0
14
21
.24
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
05
0.8
12
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
BID
-V2
31
8-0
14
21
.24
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
05
0.8
12
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
BID
-V2
31
9-0
14
21
.24
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
05
0.8
12
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
BID
-V2
32
0-0
14
31
.27
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
06
0.9
72
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
BID
-V2
32
1-0
14
21
.24
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
05
0.8
12
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
BID
-V2
32
2-0
14
31
.27
00
22
.20
22
.67
40
.81
61
.42
10
4.5
90
05
0.8
12
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
BID
-V2
32
3-0
14
31
.27
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
06
0.9
72
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
BID
-V2
32
4-0
14
21
.24
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
05
0.8
12
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
BID
-V2
32
5-0
14
21
.24
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
05
0.8
12
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
Virus Genes (2013) 46:203–218 207
123
However, four other aa substitutions at residues E-479,
NS2A-215, NS3-399, and NS4A-90, in addition to one nt
substitution at the 30UTR-13, were present only in the 1987
isolates (Table 3).
It was notable that aa substitutions at certain sites
seemed to vary with the degree of DEN-3 epidemic
activity. For example, the viruses sampled in 1993–1994, a
period of intermediate DENV-3 epidemic activity, had
threonine at residue C-27, but it was replaced by serine for
the viruses sampled during low and high DENV-3 epi-
demic periods (Table 3). Viruses sampled during the
intermediate DENV-3 epidemic period also exhibited a
similar pattern of aa substitution at PrM-15 (aa labeled with
[ ] in Table 3). The alanine at residue PrM-15 was present
only in those viruses sampled during the period of inter-
mediate DENV-3 epidemic activity. In contrast, those
viruses sampled before and after 1993–1994, during peri-
ods of lower or higher DENV-3 epidemic activity, had
different associated substitutions suggesting that there
might be a link between genetic changes in the viruses and
DENV-3 epidemic activity. A similar pattern of aa sub-
stitutions associated with the second large DENV-3 out-
break was observed at residues E-132, E-172, NS4B-247,
and NS5-389 in virus strains, ThD3-1283-98 and ThD3-
1687-98 sampled in 1998 (aa labeled with { } in the
Table 3). Likewise, the viruses sampled from the 1998
DEN outbreak had a tyrosine residue at E-132, valine at
E-172, arginine at NS4B-247, and lysine at NS5-389,
whereas, isolates sampled from other time periods had a
histidine substitution at E-132 resulting in an aa charge
change, isoleucine at E-172, lysine at NS4B-247, and
arginine at NS5-389. Although the apparent correlation of
genetic substitutions at certain sites with DENV-3 epi-
demic activity must be confirmed by the analysis of a larger
number of clinical isolates collected from the same com-
munity, the results of this study strongly suggest that viral
genetic factors might play a key role in the disease impact
in DENV-3 epidemics.
Genetic variation in the 50-UTR and 30-UTR
The 50-UTR of all strains was highly conserved with only
one nt substitution at 50-UTR-90. This substitution was
adopted and fixed in viral genome after the initial substi-
tution before or during 1987, suggesting that strict con-
servation of the 50-UTR is essential for virus survival.
In contrast, nucleotides in the 30-UTR of all viral
genomes analyzed varied significantly (see Table 3). It is
notable that there were two insertions that occurred at
30-UTR-28 and 30-UTR-29 in viral isolates, ThD3-0010-87
sampled in 1987, and BID-V2313-01 sampled in 2001, as
well as one insertion at 3’UTR-29 for BID-V2312-01,
BID-V2314-01 and BID-V2315-01 sampled in 2001.Ta
ble
2co
nti
nu
ed
Item
sT
ota
l#
of
aad
if1
To
tal
%
of
aad
if2
To
tal
nu
mb
eran
dp
erce
nta
ge
of
amin
oac
idsu
bst
itu
tio
ns
inea
chg
ene
To
tal
#an
d%
of
nt
dif
.
at50 -
UT
R/30 -
UT
R3
C (11
4aa
)
Pre
M
(91
aa)
M (75
aa)
E (49
3aa
)
NS
1
(35
2aa
)
NS
2A
(21
8aa
)
NS
2B
(13
0aa
)
NS
3
(61
9aa
)
NS
4A
(15
0aa
)
NS
4B
(24
8aa
)
NS
5
(90
0aa
)
50 -
UT
R
(96
nt)
30 -
UT
R
(45
1n
t)
Vir
use
s#
%#
%#
%#
%#
%#
%#
%#
%#
%#
%#
%#
%#
%
BID
-V2
32
6-0
14
21
.24
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
05
0.8
12
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
BID
-V2
32
7-0
14
21
.24
00
22
.20
22
.67
40
.81
51
.42
10
4.5
90
05
0.8
12
1.3
32
0.8
11
01
.11
11
.04
6*
1.3
3
1T
ota
ln
um
ber
(#)
of
aasu
bst
itu
tio
ns
or
dif
fere
nce
sin
the
cod
ing
reg
ion
2P
erce
nta
ge
(%)
of
tota
laa
sub
stit
uti
on
so
rd
iffe
ren
ces
inth
eco
din
gre
gio
n3
To
tal
nu
mb
er(#
)an
dp
erce
nta
ge
(%)
of
nt
sub
stit
uti
on
sin
the
50
UT
Ran
d30
UT
R*
Nu
cleo
tid
eco
mp
aris
on
wit
hin
on
lyth
efi
rst
40
1n
tat
the
30 -
UT
Rd
ue
tou
nav
aila
ble
com
ple
te30 -
UT
Rse
qu
ence
sin
Gen
Ban
k
208 Virus Genes (2013) 46:203–218
123
Ta
ble
3T
ota
lam
ino
acid
/nu
cleo
tid
esu
bst
itu
tio
ns
inea
chg
ene
of
28
Th
aiD
EN
V-3
pre
do
min
ant
gen
oty
pe
IIst
rain
s
Vir
use
sC
H5
34
89
V3
36
00
01
00
00
70
10
40
05
5C
O3
31
CO
36
01
28
31
68
7V
23
28
V2
32
9V
23
12
V2
31
3aIS
O.y
ears
19
73
19
73
19
87
19
87
19
93
19
93
19
94
19
94
19
98
19
98
20
01
20
01
20
01
20
01
bE
pid
emic
sL
ow
Lo
wH
igh
Hig
hIn
ter
Inte
rIn
ter
Inte
rH
igh
Hig
hIn
ter
Inte
rIn
ter
Inte
r
C-2
7S
SS
S[T
][T
][T
]S
SS
SS
SS
C-8
6K
KK
KK
KK
KK
(R)
KK
KK
PrM
-15
GG
GG
[A]
[A]
[A]
GG
G[A
][A
][A
][A
]
PrM
-55
HH
L*
LL
LL
LL
LL
LL
L
M-2
8Q
Q(P
)Q
M-5
8T
TT
TT
TT
T(I
)T
TT
TT
M-6
5I
II
II
II
I(V
)I
II
II
M-6
8I
IM
MM
MM
MM
MM
MM
M
M-7
5A
AT
TT
TT
TT
TT
TT
T
E-3
4M
MM
MM
MM
MM
M(I
)(I
)M
M
E-6
8I
II
II
I(V
)I
II
II
II
E-7
1D
DD
DD
DD
(G)
DD
DD
DD
E-8
1I
II
II
II
II
(T)
II
II
E-1
23
EE
EE
EE
EE
EE
EE
EE
E-1
24
SS
SP
PP
PP
PP
PP
PP
E-1
32
HH
HH
HH
H{Y
}*{Y
}*{Y
}*H
HH
H
E-1
49
HH
HH
HH
H(P
)H
HH
HH
H
E-1
54
EE
DD
DD
DD
DD
DD
DD
E-1
60
AA
AV
VV
VV
VV
VV
VV
E-1
69
VV
VV
VV
(A)
VV
VV
VV
V
E-1
72
II
II
II
I{V
}{V
}{V
}I
II
I
E-1
74
PP
PP
PP
P(L
)P
PP
PP
P
E-1
80
GG
GG
GG
(V)
GG
GG
GG
G
E-1
82
EE
EE
EE
E(K
)E
EE
EE
E
E-2
01
NN
NN
NN
N(T
)N
NN
NN
N
E-3
80
II
II
II
II
II
(V)
(V)
II
E-4
79
AA
AA
VV
VV
VV
VV
VV
NS
1-1
39
HN
*N
NN
NN
NN
NN
NN
N
NS
1-1
74
EV
*V
VV
VV
VV
VV
VV
V
NS
1-1
88
VV
II
II
II
II
II
II
NS
1-2
17
LL
LF
FF
FF
FF
FF
FF
NS
1-2
61
HH
HH
HH
(Y)
HH
HH
HH
H
NS
1-2
67
PP
(T)
PP
PP
PP
PP
PP
P
NS
1-3
46
VV
VV
VV
VV
VV
VV
VV
Virus Genes (2013) 46:203–218 209
123
Ta
ble
3co
nti
nu
ed
Vir
use
sC
H5
34
89
V3
36
00
01
00
00
70
10
40
05
5C
O3
31
CO
36
01
28
31
68
7V
23
28
V2
32
9V
23
12
V2
31
3aIS
O.y
ears
19
73
19
73
19
87
19
87
19
93
19
93
19
94
19
94
19
98
19
98
20
01
20
01
20
01
20
01
bE
pid
emic
sL
ow
Lo
wH
igh
Hig
hIn
ter
Inte
rIn
ter
Inte
rH
igh
Hig
hIn
ter
Inte
rIn
ter
Inte
r
NS
1-3
50
AA
VV
VV
VV
VV
VV
VV
NS
2A
-5V
VV
VV
VV
VV
VV
VM
M
NS
2A
-12
FV
VV
VV
VV
VV
II
II
NS
2A
-37
FL
LL
LL
LL
LL
LL
LL
NS
2A
-38
LL
FF
FF
FF
FF
FF
FF
NS
2A
-41
LV
VV
VV
VV
VV
VV
VV
NS
2A
-68
MM
MM
MM
(T)
MM
MM
MM
M
NS
2A
-10
2F
LL
LL
LL
LL
LL
LL
L
NS
2A
-13
3A
AA
TT
TT
TT
TT
TT
T
NS
2A
-18
0V
VV
LL
LL
LL
LL
LL
L
NS
2A
-19
5A
AA
TT
TT
TT
TT
TT
T
NS
2A
-21
5L
LL
LP
PP
PP
PP
PP
P
NS
2A
-21
7R
RR
RR
(K)
RR
RR
RR
RR
NS
2B
-58
DD
DD
DD
DD
D(N
)D
DD
D
NS
2B
-88
DD
DD
DD
DD
D(N
)D
DD
D
NS
2B
-95
LL
LL
LL
(P)
LL
LL
LL
L
NS
3-1
20
TT
TT
TT
TT
TT
TT
TT
NS
3-2
48
TT
TT
TT
TT
TT
II
II
NS
3-3
24
DD
EE
EE
EE
EE
EE
EE
NS
3-3
38
RR
RR
RR
RR
RR
RR
(K)
(K)
NS
3-3
56
VV
VV
VV
VV
VV
VV
VV
NS
3-3
58
KK
KK
KK
KK
K(R
)K
KK
K
NS
3-3
99
KK
KK
RR
RR
RR
RR
RR
NS
3-4
06
VV
VV
VV
V(L
)V
VV
VV
V
NS
3-4
76
RT
TT
TT
TT
TT
TT
TT
NS
3-5
89
KK
RR
RR
RR
RR
RR
RR
NS
4A
-58
LL
LL
LL
LL
(M)
LL
LL
L
NS
4A
-90
AA
AA
VV
VV
VV
VV
VV
NS
4A
-10
0V
VV
VV
VV
V(I
)V
VV
VV
NS
4A
-14
9A
AA
AA
AA
AA
AT
TT
T
NS
4B
-11
5A
AV
VV
VV
VV
VV
VV
V
NS
4B
-14
2T
MM
MM
MM
MM
MM
MM
M
NS
4B
-23
3A
AA
AA
AA
(T)
AA
AA
AA
NS
4B
-24
1K
KK
KK
KK
KK
KK
KK
K
210 Virus Genes (2013) 46:203–218
123
Ta
ble
3co
nti
nu
ed
Vir
use
sC
H5
34
89
V3
36
00
01
00
00
70
10
40
05
5C
O3
31
CO
36
01
28
31
68
7V
23
28
V2
32
9V
23
12
V2
31
3aIS
O.y
ears
19
73
19
73
19
87
19
87
19
93
19
93
19
94
19
94
19
98
19
98
20
01
20
01
20
01
20
01
bE
pid
emic
sL
ow
Lo
wH
igh
Hig
hIn
ter
Inte
rIn
ter
Inte
rH
igh
Hig
hIn
ter
Inte
rIn
ter
Inte
r
NS
4B
-24
7K
KK
KK
KK
{R}
{R}
{R}
KK
KK
NS
5-5
0I
IT
TT
TT
TT
TT
TT
T
NS
5-5
2R
RH
HH
HH
HH
HH
HH
H
NS
5-1
99
KK
K(R
)K
KK
KK
KK
KK
K
NS
5-2
00
HH
YY
YY
YY
YY
HH
HH
NS
5-2
88
NN
SS
SS
SS
SS
SS
SS
NS
5-3
38
II
TT
TT
TT
TT
TT
TT
NS
5-3
89
RR
RR
RR
R{K
}{K
}{K
}R
RR
R
NS
5-4
19
DD
DD
DD
DD
(N)
DD
DD
D
NS
5-4
32
KK
KK
KK
(N)
KK
KK
KK
K
NS
5-4
34
VV
VV
VV
(W)
VV
VV
VV
V
NS
5-4
82
YY
YY
YY
YY
(F)
YY
YY
Y
NS
5-4
91
FL
LL
LL
LL
LL
LL
LL
NS
5-6
31
AA
AA
AA
AA
AA
VV
VV
NS
5-6
49
TT
TT
TT
(P)
TT
TT
TT
T
NS
5-6
56
KK
KK
KK
KK
KK
RR
RR
NS
5-7
42
RQ
NS
5-8
31
TT
(A)
TT
TT
TT
TT
TT
T
NS
5-8
57
AA
AA
AA
(S)
AA
AA
AA
A
NS
5-8
76
NN
D*
DD
DD
DD
DD
DD
D
NS
5-8
90
KK
RR
RR
RR
RR
RR
RR
NS
5-8
95
SS
SS
SS
SS
(T)
SS
SS
S
50
UT
R-9
0C
CT
TT
TT
TT
TT
TT
T
30
UT
R-1
2G
GA
AA
AA
AA
AA
AA
A
30 U
TR
-13
GG
GG
AA
AA
AA
AA
AA
30 U
TR
-28
––
\A[
––
––
––
––
––
\A[
30 U
TR
-29
––
\G[
––
––
––
––
–\
G[
–
30 U
TR
-32
GG
GG
GG
G(A
)G
GG
GG
G
30 U
TR
-35
CC
AA
AA
AA
AA
AA
AA
30 U
TR
-50
TT
TT
TT
TT
(C)
TT
TT
T
30 U
TR
-68
GG
GG
GG
GG
G(C
)G
GG
G
30 U
TR
-96
AA
AA
AA
AA
AA
GA
GG
30 U
TR
-10
3T
TT
TT
TT
TT
TT
(C)
TT
30 U
TR
-12
1C
CC
CC
CC
CC
C(T
)C
CC
Virus Genes (2013) 46:203–218 211
123
Ta
ble
3co
nti
nu
ed
Vir
use
sC
H5
34
89
V3
36
00
01
00
00
70
10
40
05
5C
O3
31
CO
36
01
28
31
68
7V
23
28
V2
32
9V
23
12
V2
31
3aIS
O.y
ears
19
73
19
73
19
87
19
87
19
93
19
93
19
94
19
94
19
98
19
98
20
01
20
01
20
01
20
01
bE
pid
emic
sL
ow
Lo
wH
igh
Hig
hIn
ter
Inte
rIn
ter
Inte
rH
igh
Hig
hIn
ter
Inte
rIn
ter
Inte
r
30 U
TR
-13
5T
TT
TT
TT
(C)
TT
TT
TT
30 U
TR
-15
1G
GG
GG
GG
GG
G(A
)G
GG
30 U
TR
-15
9G
(T)
GG
GG
GG
GG
GG
GG
30 U
TR
-19
8T
TC
CC
CC
CC
CC
CC
C
30 U
TR
-26
8G
GG
GG
GG
GG
(A)
GG
GG
30 U
TR
-34
0C
CT
TT
TT
TT
TT
TT
T
Vir
use
sV
23
14
V2
31
5V
23
16
V2
31
7V
23
18
V2
31
9V
23
20
V2
32
1V
23
22
V2
32
3V
23
24
V2
32
5V
23
26
V2
32
7aE
pid
emic
s2
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
1
Gen
esIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
r
C-2
7S
SS
SS
SS
SS
SS
SS
S
C-8
6K
KK
KK
KK
KK
KK
KK
K
PrM
-15
[A]
[A]
[A]
[A]
[A]
[A]
[A]
[A]
[A]
[A]
[A]
[A]
[A]
[A]
PrM
-55
LL
LL
LL
LL
LL
LL
LL
M-2
8Q
Q
M-5
8T
TT
TT
TT
TT
TT
TT
T
M-6
5I
II
II
II
II
II
II
I
M-6
8M
MM
MM
MM
MM
MM
MM
M
M-7
5T
TT
TT
TT
TT
TT
TT
T
E-3
4M
MM
MM
MM
MM
MM
MM
M
E-6
8I
II
II
II
II
II
II
I
E-7
1D
DD
DD
DD
DD
DD
DD
D
E-8
1I
II
II
II
II
II
II
I
E-1
23
E(K
)E
EE
EE
EE
EE
EE
E
E-1
24
PP
PP
PP
PP
PP
PP
PP
E-1
32
HH
HH
HH
HH
HH
HH
HH
E-1
49
HH
HH
HH
HH
HH
HH
HH
E-1
54
DD
DD
DD
DD
DD
DD
DD
E-1
60
VV
VV
VV
VV
VV
VV
VV
E-1
69
VV
VV
VV
VV
VV
VV
VV
E-1
72
II
II
II
II
II
II
II
E-1
74
PP
PP
PP
PP
PP
PP
PP
E-1
80
GG
GG
GG
GG
GG
GG
GG
E-1
82
EE
EE
EE
EE
EE
EE
EE
E-2
01
NN
NN
NN
NN
NN
NN
NN
212 Virus Genes (2013) 46:203–218
123
Ta
ble
3co
nti
nu
ed
Vir
use
sV
23
14
V2
31
5V
23
16
V2
31
7V
23
18
V2
31
9V
23
20
V2
32
1V
23
22
V2
32
3V
23
24
V2
32
5V
23
26
V2
32
7aE
pid
emic
s2
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
1
Gen
esIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
r
E-3
80
II
II
II
II
II
II
II
E-4
79
VV
VV
VV
VV
VV
VV
VV
NS
1-1
39
NN
NN
NN
NN
NN
NN
NN
NS
1-1
74
VV
VV
VV
VV
VV
VV
VV
NS
1-1
88
II
II
II
II
II
II
II
NS
1-2
17
FF
FF
FF
FF
FF
FF
FF
NS
1-2
61
HH
HH
HH
HH
HH
HH
HH
NS
1-2
67
PP
PP
PP
PP
PP
PP
PP
NS
1-3
46
VV
VV
VV
VV
(A)
VV
VV
V
NS
1-3
50
VV
VV
VV
VV
VV
VV
VV
NS
2A
-5M
MM
MM
MM
MM
MM
MM
M
NS
2A
-12
II
II
II
II
II
II
II
NS
2A
-37
LL
LL
LL
LL
LL
LL
LL
NS
2A
-38
FF
FF
FF
FF
FF
FF
FF
NS
2A
-41
VV
VV
VV
VV
VV
VV
VV
NS
2A
-68
MM
MM
MM
MM
MM
MM
MM
NS
2A
-10
2L
LL
LL
LL
LL
LL
LL
L
NS
2A
-13
3T
TT
TT
TT
TT
TT
TT
T
NS
2A
-18
0L
LL
LL
LL
LL
LL
LL
L
NS
2A
-19
5T
TT
TT
TT
TT
TT
TT
T
NS
2A
-21
5P
PP
PP
PP
PP
PP
PP
P
NS
2A
-21
7R
RR
RR
RR
RR
RR
RR
R
NS
2B
-58
DD
DD
DD
DD
DD
DD
DD
NS
2B
-88
DD
DD
DD
DD
DD
DD
DD
NS
2B
-95
LL
LL
LL
LL
LL
LL
LL
NS
3-1
20
TT
TT
TT
TT
T(M
)T
TT
T
NS
3-2
48
II
II
II
II
II
II
II
NS
3-3
24
EE
EE
EE
EE
EE
EE
EE
NS
3-3
38
(K)
RR
RR
RR
RR
RR
RR
R
NS
3-3
56
VV
VV
VV
(A)
VV
VV
VV
V
NS
3-3
58
KK
KK
KK
KK
KK
KK
KK
NS
3-3
99
RR
RR
RR
RR
RR
RR
RR
NS
3-4
06
VV
VV
VV
VV
VV
VV
VV
NS
3-4
76
TT
TT
TT
TT
TT
TT
TT
Virus Genes (2013) 46:203–218 213
123
Ta
ble
3co
nti
nu
ed
Vir
use
sV
23
14
V2
31
5V
23
16
V2
31
7V
23
18
V2
31
9V
23
20
V2
32
1V
23
22
V2
32
3V
23
24
V2
32
5V
23
26
V2
32
7aE
pid
emic
s2
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
12
00
1
Gen
esIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
rIn
ter
Inte
r
NS
3-5
89
RR
RR
RR
RR
RR
RR
RR
NS
4A
-58
LL
LL
LL
LL
LL
LL
LL
NS
4A
-90
VV
VV
VV
VV
VV
VV
VV
NS
4A
-10
0V
VV
VV
VV
VV
VV
VV
V
NS
4A
-14
9T
TT
TT
TT
TT
TT
TT
T
NS
4B
-11
5V
VV
VV
VV
VV
VV
VV
V
NS
4B
-14
2M
MM
MM
MM
MM
MM
MM
M
NS
4B
-23
3A
AA
AA
AA
AA
AA
AA
A
NS
4B
-24
1K
(R)
KK
KK
KK
KK
KK
KK
NS
4B
-24
7K
KK
KK
KK
KK
KK
KK
K
NS
5-5
0T
TT
TT
TT
TT
TT
TT
T
NS
5-5
2H
HH
HH
HH
HH
HH
HH
H
NS
5-1
99
KK
KK
KK
KK
KK
KK
KK
NS
5-2
00
HH
HH
HH
HH
HH
HH
HH
NS
5-2
88
SS
SS
SS
SS
SS
SS
SS
NS
5-3
38
TT
TT
TT
TT
TT
TT
TT
NS
5-3
89
RR
RR
RR
RR
RR
RR
RR
NS
5-4
19
DD
DD
DD
DD
DD
DD
DD
NS
5-4
32
KK
KK
KK
KK
KK
KK
KK
NS
5-4
34
VV
VV
VV
VV
VV
VV
VV
NS
5-4
82
YY
YY
YY
YY
YY
YY
YY
NS
5-4
91
LL
LL
LL
LL
LL
LL
LL
NS
5-6
31
VV
VV
VV
VV
VV
VV
VV
NS
5-6
49
TT
TT
TT
TT
TT
TT
TT
NS
5-6
56
RR
RR
RR
RR
RR
RR
RR
NS
5-7
42
NS
5-8
31
TT
TT
TT
TT
TT
TT
TT
NS
5-8
57
AA
AA
AA
AA
AA
AA
AA
NS
5-8
76
DD
DD
DD
DD
DD
DD
DD
NS
5-8
90
RR
RR
RR
RR
RR
RR
RR
NS
5-8
95
SS
SS
SS
SS
SS
SS
SS
50
UT
R-9
0T
TT
TT
TT
TT
TT
TT
T
30
UT
R-1
2A
AA
AA
AA
AA
AA
AA
A
30 U
TR
-13
AA
AA
AA
AA
AA
AA
AA
214 Virus Genes (2013) 46:203–218
123
Ta
ble
3co
nti
nu
ed
Vir
use
sV
23
14
V2
31
5V
23
16
V2
31
7V
23
18
V2
31
9V
23
20
V2
32
1V
23
22
V2
32
3V
23
24
V2
32
5V
23
26
V2
32
7aE
pid
emic
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00
12
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12
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ter
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Inte
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30 U
TR
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––
––
––
––
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30 U
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––
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GG
GG
GG
GG
GG
GG
GG
30 U
TR
-35
AA
AA
AA
AA
AA
AA
AA
30 U
TR
-50
TT
TT
TT
TT
TT
TT
TT
30 U
TR
-68
GG
GG
GG
GG
GG
GG
GG
30 U
TR
-96
GG
GG
GG
GG
GG
GG
GG
30 U
TR
-10
3T
TT
TT
TT
TT
TT
TT
T
30 U
TR
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1C
CC
CC
CC
CC
CC
CC
C
30 U
TR
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TT
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1G
GG
GG
GG
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9G
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CC
CC
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CC
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GG
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Virus Genes (2013) 46:203–218 215
123
In addition, five substitutions at 30-UTR-12, 30-UTR-13,
30-UTR-35, 30-UTR-198, and 30-UTR-340 were fixed in the
viral genome after their initial substitutions (Table 3).
RNA secondary structure of the cyclized 50–30 termini
We limited our analysis of the 30-UTR RNA secondary
structure to 10 of the 28 DENV-3 isolates because these
were the only isolates with complete 50-UTR and 30-UTR
sequences. All secondary structures of 30-UTRs analyzed
by MFOLD exhibited a similar topology and none of the
observed nt substitutions were predicted to disrupt or sig-
nificantly alter the RNA secondary structures (data not
shown). As the replication of DENVs requires circular-
ization of the 50–30termini involving the entire 50-UTR, part
of the N-terminus of the C protein and the terminus of the
30-UTR (we included the first 43 nt of the C protein and the
last 106 nt of the 30-UTR.), we performed secondary
structure analysis for the circularized RNA to determine
whether mutations in these regions disrupted the predicted
viral RNA secondary structure. The results showed that the
50–30 cyclized termini for all genotype II virus strains
displayed similar image patterns (data not shown); there-
fore, it is unlikely that these mutations significantly
affected viral RNA secondary structure.
Discussion
A process of microevolution acting on the DENV genome
over time resulted in some aa/nt substitutions that were
adopted and became fixed in virus genome after their initial
substitution. It seems reasonable to suggest that these
adopted and fixed mutations are advantageous to the virus,
leading to strains with higher epidemic potential, resulting
in increased DEN incidence, transmission frequency, and
epidemiological impact in Thailand. The observation of
multiple, co-circulating DENV genotypes with one domi-
nant genotype for each DENV serotype in Thailand [18–
20] tends to support a hypothesis that more virulent, highly
transmissible genotypes are now displacing those with
lower epidemiological impact [23]. The DENV-3 genotype
II has retained its predominance in Thailand for over
30 years, and along with other co-circulating serotypes is
responsible for the large 1987 and 1998 DEN epidemics in
the country [12], suggesting that it is highly transmissible
and has potentially more epidemiological impact than other
co-circulating non-predominant genotype I and III viruses.
Previously, it has been demonstrated that a strong neg-
ative selection pressure is acting on the DENV genome
during virus evolution [18–20]. This negative selection
pressure quickly eliminates deleterious aa/nt substitutions
that are continually generated by mutation in new DENV
lineages [18]. Although our previous results [18–20] do not
support a process of positive selection acting on the DENV
genome during virus evolution, the finding of adopted and
fixed aa/nt substitutions suggests that a process of positive
selection is probably acting at certain sites in the viral
genome even though this could not be detected by current
methods. The process of positive selection allows the virus
to adapt quickly to environmental changes and survive in
the host. Although the genetic mutations observed in our
study are limited to certain sites, if they occur at key
positions in the viral genome they would be expected to
have significant effects on virus phenotype.
Two potentially important aa substitutions at residues
C-27 and PrM-15 appeared to be associated with an
intermediate period of DENV-3 epidemic activity during
the early 1990s, while four aa substitutions at residues
E-132, E-172, NS4B-247, and NS5-389 appeared to be
associated with the large 1998 DEN epidemic in Thailand.
We also observed that two aa substitutions at C-102 and
PrM-16 appeared to be associated with periods of inter-
mediate DENV-2 epidemic activity in Thailand. This
observation raises the question: Do these aa changes play
any role in modulating DENV-3 epidemics? Results from
previous studies suggest that in addition to serving as a
structural protein to encapsidate the virion RNA, the
DENV C protein is also implicated in the regulation of
cellular gene expression and proliferation. It interacts with
the transcription factor heterogeneous nuclear ribonucleo-
protein K (hnRNP K) in the nucleus and enhances c-Myc
transcription to promote apoptosis [24]. Three nuclear
localization signals (NLS) have been identified in DENV C
protein [25]. Apoptosis due to DENV infection has been
observed in the liver of some infected patients [26], as well
as in virus-infected primary endothelial and hepatic cells in
culture [27–29]. Apoptosis could be a host-induced defense
mechanism designed to limit the ability of viruses to rep-
licate. It can be speculated that the mutation at C-27 (being
in close proximity to the NLS1) of the C protein is able to
affect apoptosis and thereby contribute to a reduction in
viral replication during periods of intermediate DENV
epidemic activity. Similarly, the mutation at PrM-15 might
alter E protein-mediated fusion because the PrM protein
serves as a chaperone for E protein in virion maturation and
a precursor to M protein on the virion surface. Cryoelectron
microscopy has revealed that the PrM protein, in its
chaperone role, covers various fusion peptides in E protein
thereby inhibiting virus-cell fusion until specific structural
changes have taken place [30, 31]. As fusion with the host
cell membrane is a critical early step in infection, viruses
with mutations in PrM might be expected to exhibit alter-
ations in fitness which can be correlated with periods of
greater or lesser epidemic activity. Although requiring
corroboration from the analysis of a larger number of
216 Virus Genes (2013) 46:203–218
123
DENV-3 isolates collected from different epidemics in
Thailand, the mutations identified in C protein and at PrM-
15 may have given rise to viruses with reduced virulence.
These mutations may have had a direct impact on disease
frequency and severity during the period of intermediate
DENV-3 epidemic activity in the early 1990s. It can be
speculated that four aa substitutions at E-132, E-172,
NS4B-247, and NS5-389, associated with the 1998 DEN
outbreak in Thailand, might co-act to modulate the virus
life cycle in a number of ways, e.g., (1) by increasing virus
fusion activity, (2) enhancing E protein dimerization and
fusion with the host membrane, (3) inhibiting host inter-
feron (IFN) responses allowing the virus to better evade the
host immune response, and (4) increasing the activity of
the viral RNA dependant RNA polymerase, thus increasing
the viral replication rate during periods of high DEN epi-
demic activity.
Of all the DENV-3 proteins, the 218 aa long NS2A
showed the highest rate (percentage) of aa substitutions.
However, mutations in NS2A do not appear to be as lethal
to the virus as those in NS2B and C proteins. Although the
functions of NS2A, NS4A, and NS4B proteins have not yet
been fully elucidated, previous research has demonstrated
that these proteins might play a role in the inhibition of the
host IFN response [32–34]. In addition, the NS2A serves as
a cis-acting protease that directly cleaves itself from NS1
or provide sequences for recognition by a specific cellular
protease that cleaves at the NS1–NS2A junction [35], and
an intact NS2A sequence is required for this cleavage
reaction [36]. It is possible that certain mutations in the
NS2A protein alter the rate of viral protein cleavage and
affect the ability of virus to escape the host IFN response.
The precise cause of the increased rate of aa substitutions
in the NS2A protein is unknown and merits further
investigation.
In summary, this study revealed that a genetic link might
exist between some specific aa substitutions in the viral
genome and the rate of DENV transmission in Thailand.
Genetic variation in the DENV-3 genome might correlate
with changes in the incidence of DENV-3 infections. The
visual timeline of DENV-3 genome evolution in a specific
community provided by this study will hopefully lead to
more comprehensive studies of the effects of mutations at
particular DENV genomic sites on virus phenotype, and
ultimately, to novel strategies for disease control.
Acknowledgments We thank our field unit, Department of Virol-
ogy, AFRIMS, Bangkok, Thailand for sample characterization and
storage as well as information record; and the doctors and nurses of
QSNICH/AFRIMS for sample collection and clinical grading. This
research was supported by the US Military Infectious Disease
Research Program at Fort Detrick, Maryland, USA and the Science
and Engineering Apprentice Program (SEAP) sponsored by George
Washington University/Walter Reed Army Institute of Research.
Disclaimer The opinions and assertions contained herein are the
private views of the authors and are not to be construed as reflecting
the official views of the U.S. Army or the Department of Defense.
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