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Abstract
Dengue virus and dengue haemorrhagic fever are amongst the most important challenges in tropical
diseases due to their expanding geographical distribution, increasing outbreak frequency,
hyperendemicity and evolution of virulence.
Here, the use of a RT-nested PCR for both the diagnosis and genetic characterization of dengue
infections in clinical samples is described.
Keywords: Dengue, dengue haemorrhagic fever, diagnosis, molecular epidemiology, surveillance, glycoprotein E
gene, NS1 gene.
#
E-mail: cdomingo@isciii.es; Phone: (+34) 918223954, Fax: (+34) 915097966
Inc. Software, Madison, Wisconsin, USA). Scores were calculated to test the significance
The consensus sequence was compared and of each pair-wise alignment by Monte Carlo
aligned to other samples or DNA database simulation on the shuffled sequences.
sequences using the programme CLUSTAL X, Statistical analysis was conducted with the
version 1.83[22]. Programmes from the MEGA SPSS statistical package (SPSS Software,
package[23] were used to produce Chicago, IL).
phylogenetic trees using NJ as the method to
reconstruct the phylogeny and Kimura-2p as
nucleotide substitution calculation method. Results
The statistical significance of a particular tree
topology was evaluated by bootstrap re- Design of the primers
sampling of the sequences 1,000 times. The E/NS1 region of the genome was
Published sequences used in the chosen for the development of a RT-nested
comparisons were obtained from the PCR. The primers selected specifically
GenBank databases. Pair-wise comparisons amplify the four dengue viruses with no
of the dengue virus database were done by cross reactivity to other members of the
global alignment using the Needleman flavivirus family. A mix of degenerate
Wunsch[24] algorithm using the primers representing each serotype was
implementation from EMBOSS, the European used to ensure coverage for the highly
Molecular Biology Open Software Suite[25] . Z- variable dengue serotypes (Table).
Dengue virus RT-n PCR specificity One hundred and sixty-four serum
samples from cases of febrile illness
The specificity of the RT-n PCR was associated with travel were tested with the
determined by analysing serial dilutions of assay. Thirty-seven cases were diagnosed as
RNA from related flavivirus (JEV, MVEV, of classical dengue fever by the WHO
SLEV, TBEV, WNV, YFV) and no criteria[19] . Sixteen of these cases were found
amplification was obtained (data not shown). positive by using our E/NS1 assay.
The amplification was successful with Convalescent sera were available for 13 of
both commercial RNA and serum samples these cases; all were later confirmed to be
for all dengue virus serotypes as shown seroconvert. All serum samples found
(Figure 1), yielding a distinct DNA product positive by RT-n PCR were collected in the
of the expected size (328-pb) in agarose gels. first week after the onset of the symptoms.
366VI03
13VI02
Figure 2. Phylogenetic tree constructed with the E/NS1 fragment which identifies
the four dengue serotypes
[Phylogenetic analysis was performed using the Kimura-two parameter model as a model of nucleotide
substitution and using the neighbor joining method to reconstruct the phylogenetic tree (MEGA version
2.1 software package)]
Den1-Peru9615-00-PERU
Den1-FGA89-FRENCHGUYANA1989
Den1-Peru9581-00-PERU
Den1-BR90-BRASIL1990
Den1-Br01MR-BRASIL2001
Den1-233-BRASIL1997
Den1-111-BRASIL1997
Den1-409-BRASIL1997
Den1-Peru5198-97 DENGUE SEROTYPE 1
Den1-SingaporeS275-90-SINGAPORE1990
Den1-CAMBODIA-CAMBODIA1998
Den1-GZ80-CHINA1980
Den1-DJIBOUTI1998
Den1-MOCHIZUKI-JAPAN1953
Den1-16007-THAYLAND1964
Den1-A88-INDONESIA1988
Den1-clonePDK27
Den1-WestPac-NAURU1974
Den3-80-2-CHINA1980
Den3-H87-PHILIPPINES1956 DENGUE SEROTYPE 3
Den3-11069
Den2-IQT2913-PERU1996
Den2-539-96-PERU1996
Den2-IQT1797-PERU1995
Den2-131-MEXICO1992
Den2-VEN2-VENEZUELA1987
Den2-44-CHINA1989
Den2-NewGuineaC-NEWGUINEA1944
Den2-43-CHINA1987
Den2-04-CHINA1985
Den2-Mara4-VENEZUELA1990 DENGUE SEROTYPE 2
Den2-N.1409-JAMAICA1983
Den2-CookIslands-AUSTRALIA1997
Den2-16681-THAYLAND1964
Den2-ThNH54-93-THAYLAND1993
Den2-ThNHp36-93-THAYLAND1993
Den2-CO371-THAYLAND1995
Den2-CO166-THAYLAND1966
Den2-CO167-THAYLAND1996
Den2-K0008-THAYLAND1994
Den2-K0010-THAYLAND1994
Den4-TRI94-TRINIDAD1994
Den4-HON91-HONDURAS1991
Den4-MON94A-MONTSERRAT1994
Den4-TRI99-TRINIDAD1999
Den4-BAH98A-BAHAMAS1998
Den4-MON94B-MONTSERRAT1994
Den4-SUR94A-SURINAM1994
Den4-BDS93A-BARBADOS1993 DENGUE SEROTYPE 4
Den4-MEX91-MEXICO1991
Den4-JAM83-JAMAICA1983
Den4-TRI82A-TRINIDAD1982
Den4-814669-DOMINICA1981
Den4-SUR82D-SURINAM1982
Den4-JAMAICA1981
Den4-TRI84A-TRINIDAD1984
YellowFever
WestNile
JVE
0.1
Sequences that had no known genotype generate a full taxonomy tree, but did fully
were classified with respect to the group to differentiate the genotypes (data not shown).
which they were most similar. To verify the Even with this simple method, all unknowns
utility of this method, a phylogenetic tree were classified correctly into their genotypic
was built in parallel with the unknown and group (Figure 3 illustrates one example
characterized sequences. Bootstrap values result for each serotype, compared to
in the 220bp region were too low to known sequences).
Figure 3. Pair-wise analysis of four de ngue strains detected by PCR amplification of 328 bp
E/NS1 products from patient sera. Samples are (a) 438VI03, DEN-1 AMERICAN-AFRICAN
genotype, (b) 13VI02, DEN-2 COSMOPOLITAN genotype, (c) 1794F02 DEN-3 INDIAN
genotype, and (d) 366VI03, INDONESIAN DEN-4 genotype
a) Dengue 1 - 438VI03 Nicaragua 2003 d) Dengue 2 - 13VI02 India 2002
1100
1100
1000
1000
NW Score
NW Score
900 900
800 800
700 700
AMAF ASIAN MAL SP THAI COS ASIAN ASIAN AMERICAN ASIAN AFRICAN
GENOTYPE 2 AMERICAN GENOTYPE 1
Genotype
Genotype
1100 1100
1000 1000
NW Score
NW Score
900 900
800 800
700 700
SE Asia/SP THAILAND INDIAN AMERICAS INDONESIA SE ASIA MALAYSIA
SUBCONTINENT
Genotype
Genotype
References
[1] Gubler DJ. Epidemic dengue/dengue [7] Lopez-Velez R, Perez-Casas C, Vorndam AV
haemorrhagic fever: a global public health and Rigau J. Dengue in Spanish travelers
problem in the 21st century. Dengue Bulletin, returning from the tropics. Eur J Clin
1997, 21. Microbiol Infect Dis, 1996, 15: 823-826.
[2] World Health Organization. Dengue and [8] Trofa AF, DeFraites RF, Smoak BL, Kanesa-
dengue haemorrhagic fever. Fact sheet, thasan N, King AD, Burrous JM, MacArthy
2002, No. 117. PO, Rossi C and Hoke CH, Jr. Dengue fever
in US military personnel in Haiti. JAMA,
[3] Clarke T. Dengue virus: break-bone fever.
1997, 277: 1546-1548.
Nature, 2002, 416: 672-674.
[9] Halstead SB. Pathogenesis of dengue:
[4] Lifson AR. Mosquitoes, models, and dengue.
challenges to molecular biology. Science,
Lancet, 1996, 347: 1201-1202. 1988, 239: 476-481.
[5] Jelinek T. Dengue fever in international [10] Lanciotti RS, Lewis JG, Gubler DJ and Trent
travelers. Clin Infect Dis, 2000, 31: 144-147. DW. Molecular evolution and epidemiology
[6] Jelinek T, Dobler G, Holscher M, Loscher T of dengue-3 viruses. J Gen Virol, 1994, 75
and Nothdurft HD. Prevalence of infection (Pt 1): 65-75.
with dengue virus among international [11] Lanciotti RS, Gubler DJ and Trent DW.
travelers. Arch Intern Med, 1997, 157: Molecular evolution and phylogeny of
2367-2370. dengue-4 viruses. J Gen Virol, 1997, 78 (Pt
9): 2279-2284.
[12] Rico-Hesse R. Molecular evolution and [21] Sánchez-Marino JPL-C, G.; Spiteri, I.;
distribution of dengue viruses type 1 and 2 Martín-Sánchez, F. BUSSUB: A virtual
in nature. Virology, 1990, 174: 479-493. amplicon retrieval software. ISMB/ECCB
[13] Chungue E, Deubel V, Cassar O, Laille M 2004. Glasgow, 2004.
and Martin PM. Molecular epidemiology of [22] Thompson JD, Gibson TJ, Plewniak F,
dengue 3 viruses and genetic relatedness Jeanmougin F and Higgins DG. The
among dengue 3 strains isolated from CLUSTAL_X windows interface: flexible
patients with mild or severe form of dengue strategies for multiple sequence alignment
fever in French Polynesia. J Gen Virol, 1993, aided by quality analysis tools. Nucleic Acids
74 (Pt 12): 2765-2770. Res, 1997, 25: 4876-4882.
[14] Leitmeyer KC, Vaughn DW, Watts DM, [23] Kumar S, Tamura K, Jakobsen IB and Nei M.
Salas R, Villalobos I, de C, Ramos C and MEGA2: molecular evolutionary genetics
Rico-Hesse R. Dengue virus structural analysis software. Bioinformatics, 2001, 17:
differences that correlate with pathogenesis. 1244-1245.
J Virol, 1999, 73: 4738-4747. [24] Needleman SB and Wunsch CD. A general
[15] Pandey BD, Morita K, Hasebe F, Parquet method applicable to the search for
MC and Igarashi A. Molecular evolution, similarities in the amino acid sequence of
distribution and genetic relationship among two proteins. J Mol Biol, 1970, 48: 443-453.
the dengue 2 viruses isolated from different [25] Rice P, Longden I and Bleasby A. EMBOSS:
clinical severity. Southeast Asian J Trop Med the European Molecular Biology Open
Public Health, 2000, 31: 266-272. Software Suite. Trends Genet, 2000, 16:
[16] Rico-Hesse R, Harrison LM, Salas RA, Tovar 276-277.
D, Nisalak A, Ramos C, Boshell J, de Mesa [26] Palacios G, Casas I, Tenorio A and Freire C.
MT, Nogueira RM and da Rosa AT. Origins Molecular identification of enterovirus by
of dengue type 2 viruses associated with analyzing a partial VP1 genomic region with
increased pathogenicity in the Americas. different methods. J Clin Microbiol, 2002,
Virology, 1997, 230: 244-251. 40: 182-192.
[17] Rico-Hesse R. Microevolution and virulence [27] Oberste MS, Maher K, Kilpatrick DR,
of dengue viruses. Adv Virus Res, 2003, 59: Flemister MR, Brown BA and Pallansch MA.
315-341. Typing of human enteroviruses by partial
[18] Gubler DJ, Suharyono W, Lubis I, Eram S sequencing of VP1. J Clin Microbiol, 1999,
and Gunarso S. Epidemic dengue 3 in 37: 1288-1293.
central Java, associated with low viremia in [28] Ward CW, McKern NM, Frenkel MJ and
man. Am J Trop Med Hyg, 1981, 30: 1094- Shukla DD. Sequence data as the major
1099. criterion for potyvirus classification. Arch
[19] World Health Organization. Clinical Virol Suppl, 1992, 5: 283-297.
diagnosis. 2 nd edition. Geneva: WHO, 1997.
[20] Twiddy SS, Farrar JJ, Vinh Chau N, Wills B,
Gould EA, Gritsun T, Lloyd G and Holmes
EC. Phylogenetic relationships and
differential selection pressures among
genotypes of dengue-2 virus. Virology, 2002,
298: 63-72.