Figure 1Identification of dengue virus (DENV) serotypes from (A) the original serum samples and (B) culture supernatants by multiplex RT-PCR. The patient code number is shown at the top. Lane M = 100-bp ladder; Lane C = negative control; Lane 1 = DENV-1 (Hawaii) positive; Lane 2 = DENV-2 (New Guinea C) positive; Lane 3 = DENV-3 (H87) positive; Lane 4 = DENV-4 (H241) positive. The expected sizes of the RT-PCR products were as follows: DENV-1 = 482 bp; DENV-2 = 119 bp; DENV-3 = 290 bp; and DENV-4 = 389 bp.
Figure 2Detection and typing of dengue virus (DENV) isolates using monoclonal antibodies. The culture of C6/36 cells injected with patient serum (patient code. 115/2005) was harvested and stained (A) with monoclonal antibodies reactive with all DENV serotypes (MAB 8705, Chemicon International, USA) and (B) with DENV serotype-1 specific antibodies (D2-1F1-3, Centers for Disease Control and Prevention, USA). (C) is the negative control (non-infected C6/36 cells, reacted with MAB 8705).
Figure 3Phylogenetic tree based on the nucleotide sequences of the envelope gene from 40 dengue type-1 viruses. The tree was constructed by the neighbor-joining method (Tamura-Nei substitution model) and rooted using a reference strain of dengue type-3 virus (GenBank ID: NC_001475). The percentages of bootstrap values are shown at each node (1,000 replicates). Genotypes I, II, IV and V are as defined in previous studies [16,30].
Table 1Summary of the patient information
Patient code/yr |
Travel history |
Serotype by RT-PCRa
|
Virus isolationb
|
IFA |
RT-PCR |
Designation |
32/2004 |
IU |
2 |
– |
– |
|
38/2004 |
IU |
3 |
– |
– |
|
47/2004 |
India, Singapore |
1, 2 |
1 |
1 |
DenKor-01 |
09/2005 |
Indonesia |
1 |
1 |
1 |
DenKor-02 |
10/2005 |
Indonesia |
1 |
1 |
1 |
DenKor-03 |
12/2005 |
Indonesia |
1 |
1 |
1 |
DenKor-04 |
35/2005 |
Thailand |
1 |
1 |
1 |
DenKor-05 |
51/2005 |
Philippines |
4 |
– |
– |
|
108/2005 |
India |
1 |
– |
– |
|
115/2005 |
Thailand |
1 |
1 |
1 |
DenKor-06 |
64/2006 |
Philippines |
1 |
1 |
1 |
DenKor-07 |
Table 2Primers for amplification and sequencing of the envelope (E) gene of dengue type-1 viruses
Name |
Sequence (5′–3′) |
Positiona
|
Use |
D1-820-S |
GAGACACCCAGGATTCACGG |
820–839 |
RT-PCR, Sequencing |
D1-2600-AS |
TGGCTGATCGAATTCCACAC |
2581–2600 |
RT-PCR, Sequencing |
1198-S |
GAACTTTGTGTGYCGACGAAC |
1198–1218 |
Sequencing |
1556-S |
GTCCACAAACAATGGTTTC |
1556–1574 |
Sequencing |
1913-S |
GAAGGAACAGATGCACCATG |
1913–1932 |
Sequencing |
1440-AS |
GGAGCTTGAGGTGTTAT |
1424–1440 |
Sequencing |
1932-AS |
CATGGTGCATCTGTTCCTTC |
1913–1932 |
Sequencing |
Table 3Details of the dengue type-1 viruses used in this study
Strain |
Code |
Location |
Isolation yr |
GenBank IDa
|
RIO H 36589 |
Ang88 |
Angola |
1988 |
AF425610
|
495–1 |
Aru88 |
Aruba |
1985 |
AF425609
|
AUS HATI7 |
Aus83 |
Australia |
1983 |
AF425612
|
BE AR 404147 |
Bra82 |
Brazil |
1982 |
AF425613
|
BR/90 |
Bra90 |
Brazil |
1990 |
S64849
|
BR/01-MR |
Bra01 |
Brazil |
2001 |
AF513110
|
GZ/80 |
Chi80 |
China |
1980 |
AF350498
|
INS 347869 |
Col85 |
Columbia |
1985 |
AF425616
|
D1/H/IMTSSA/98/606 |
Dji98 |
Ethiopia |
1998 |
AF298808
|
FGA/89 |
Fre89 |
French Guiana |
1989 |
AF226687
|
A88 |
Ind88 |
Indonesia |
1988 |
AB074761
|
98901518 DHF DV-1 |
Ind98 |
Indonesia |
1998 |
AB189120
|
02-07-1HuNIID |
Ind02 |
Indonesia |
2002 |
AB111073
|
SC01 |
Ind04 |
Indonesia |
2004 |
AY858983
|
DenKor-02 |
Ind05 |
Indonesia |
2005 |
EF654105
|
PRS 288690 |
Jam77 |
Jamaica |
1977 |
AF425621
|
1298/TVP 951 |
Mex80 |
Mexico |
1980 |
AF425623
|
PRS 228686 |
Mya76 |
Myanmar |
1976 |
AF425615
|
My01D138862 |
Mya01 |
Myanmar |
2001 |
AY618210
|
WestPac 74 |
WestPac 74 |
Nauru |
1974 |
U88535
|
PRS 228682 |
Phi74 |
Philippines |
1974 |
AF425627
|
01St219 |
Phi01 |
Philippines |
2001 |
AY422777
|
02RBD008 |
Phi02 |
Philippines |
2002 |
AY422778
|
DenKor-07 |
DenKor-07 |
Philippines |
2006 |
EF654110
|
Singapore 8114/93 |
Sin93 |
Singapore |
1993 |
AY762084
|
S144/02 |
Sin02 |
Singapore |
2002 |
EU069600
|
T3196/04 |
Sin04 |
Singapore |
2004 |
EU069624
|
DenKor-01 |
DenKor-01 |
India, Singapore |
2004 |
EF654104
|
D1/SG/05K4173DK1/2005 |
Sin05 |
Singapore |
2005 |
EU081262
|
D1/SG/06K2290DK1/2006 |
Sin06 |
Singapore |
2006 |
EU081281
|
TH-SMAN |
Tha54 |
Thailand |
1954 |
D10513
|
2543–63 |
Tha63 |
Thailand |
1963 |
AF425629
|
16007 |
Tha64 |
Thailand |
1964 |
AF180817
|
PUO 359 |
Tha80 |
Thailand |
1980 |
AF425630
|
ThD1_K0229_90 |
Tha90 |
Thailand |
1990 |
AY732466
|
ThD1_K0051_99 |
Tha99 |
Thailand |
1999 |
AY732458
|
ThD1_0075_02 |
Tha02 |
Thailand |
2002 |
AY732398
|
DenKor-05 |
DenKor-05 |
Thailand |
2005 |
EF654108
|
DenKor-06 |
DenKor-06 |
Thailand |
2005 |
EF654109
|
5345 |
Ven95 |
Venezuela |
1995 |
AF425635
|