Molecular typing of dengue virus type 2 in Brazil

Tipificação molecular do vírus da dengue tipo 2 no Brasil

Molecular typing of dengue virus type 2 in Brazil

Tipificação molecular do vírus da dengue tipo 2 no Brasil

Marize P. Miagostovich^I; Patrícia C. Sequeira^I; Flávia B. dos Santos^I; Amanda Maia^I; Rita Maria R.Nogueira^I; Hermann G. Schatzmayr^I; Eva Harris^II; Lee W. Riley^II

^ILaboratório de Flavivirus, Departmento de Virologia, Instituto Oswaldo Cruz/FIOCRUZ, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil

^IIDivision of Infectious Diseases, School of Public Health, University of California, Berkeley, USA

^{Address to correspondence}

SUMMARY

Strain typing is a critical tool for molecular epidemiological analysis and can provide important information about the spread of dengue viruses. Here, we performed a molecular characterization of DEN-2 viruses isolated in Brazil during 1990-2000 from geographically and temporally distinct areas in order to investigate the genetic distribution of this serotype circulating in the country. Restriction site-specific polymerase chain reaction (RSS)-PCR presented the same pattern for all 52 Brazilian samples, showing the circulation of just one DEN-2 variant. Phylogenetic analysis using progressive pairwise alignments from 240-nucleotide sequences of the E/NS1 junction in 15 isolates showed that they belong to genotype III (Jamaica genotype).

Keywords: Dengue virus type 2; Molecular epidemiology; Brazil.

RESUMO

A tipagem de cepas é uma ferramenta fundamental para a análise epidemiológica molecular e pode fornecer informações importantes sobre a dispersão dos vírus dengue. Neste trabalho, foi realizada a caracterização molecular de amostras de vírus DEN-2 isoladas no Brasil entre 1990-2000, de áreas geograficamente e temporalmente distintas, com o objetivo de investigar a distribuição genética deste sorotipo circulante no país. A reação em cadeia pela polimerase baseada em sítios de restrição específicos (RSS-PCR) apresentou o mesmo padrão para as 52 amostras Brasileiras, mostrando a circulação de apenas uma variante de vírus DEN-2. A análise filogenética utilizando alinhamento progressivo de sequências de 240 nucleotídeos da junção E/NS1 de 15 cepas mostrou que estas pertencem ao genotipo III (genotipo Jamaica).

INTRODUCTION

Dengue (DEN) viruses are grouped as four antigenically distinct serotypes designated DEN-1, 2, 3, and 4 (SABIN, 1952; HAMMON et al., 1960). Evidence that intratypic strain variation occurs among DEN viruses has been demonstrated since the 1970s (McCLOUD et al., 1971; RUSSEL & McCOWN, 1972), although only with the advances in molecular technologies it has been possible to determine the genetic variability of each serotype.

Sequencing replaced the original fingerprinting assays, since it allowed a greater discrimination of genetic relations among samples. Partial sequencing of DEN viruses characterized five genomic groups for DEN-1 and DEN-2, four for DEN-3 and two for DEN-4 viruses (RICO-HESSE, 1990, LANCIOTTI et al., 1994, 1997).

For DEN-2 viruses the five genotypes are represented by samples from Caribbean and South Pacific (genotype I); Taiwan, Philippines, the New Guinea prototype virus and Thailand 1964 strain (genotype II); Vietnam, Jamaica and Thailand strains (genotype III); Indonesia, Seychelles, Burkina Faso and Sri Lanka (genotype IV) and rural Africa (genotype V) (RICO-HESSE, 1990).

Genome sequencing provides accurate characterization of strain differences but requires labor-intensive procedures. Recently, a new PCR-based approach has been used to rapidly subtype dengue viruses. The method consists of a single reverse transcriptase PCR (RT-PCR) amplification using four primers that target regions spanning polymorphic endonuclease restriction site-specific (RSS-PCR) within the envelope (E) gene (HARRIS et al., 1999; MIAGOSTOVICH et al., 2000). It was validated against well characterized panels of dengue viruses and was shown to correlate with previous classifications determined by sequence analysis, thus functioning as an alternative subtyping method.

A variety of methods have been used to monitor the distribution and movement of distinct dengue virus genotypes within each serotype, and such genotypic analysis has provided important information about dengue viral spread, transmission and severity of disease with certain genotypes (RICO-HESSE et al., 1997).

In this study, we analyzed DEN-2 viruses isolated in Brazil between 1990 and 2000 from geographically and temporally distinct areas to investigate the genetic distribution and diversity of this serotype circulating in the country.

MATERIALS AND METHODS

Viruses - The DEN-2 viruses analyzed in this study were obtained from the collection of the Flavivirus Laboratory, Department of Virology, IOC - FIOCRUZ. The number of each strain, in addition to the location and year of isolation, are listed in Table 1. Viruses were isolated from sera by inoculation into Aedes albopictus cell line C6/36 (IGARASHI, 1978) and were identified by immunofluorescence using type-specific monoclonal antibodies (GUBLERet al., 1984). Viral seeds were propagated once in C6/36 grown in Leibovitz-15 medium containing 10% fetal bovine serum.

RNA extraction - Viral RNA was extracted from the supernatant of infected cells using QIAamp Viral RNA Mini Kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer's instructions.

(RSS)-PCR. - The reaction mixture and electrophoresis conditions were performed as described previously, except that 25 µl reaction volumes were used (HARRIS et al., 1999). Briefly, 2.5 µl of viral RNA was added to 22.5 µl of an RT-PCR mixture consisting of 50 mM potassium chloride, 10 mM Tris (pH 8.5), 0.01% gelatin, 200 µM of each of the four deoxynucleotide triphosphates, 1.5 mM magnesium chloride, 30 mM tetramethylammonium chloride, 0.5 M betaine, 5 mM dithiotreitol, 0.5 µM of each of four specific RSS-PCR primers for DEN-2 (RSS1-4), 0.025 U/µl of reverse transcriptase RAV-2 (Amersham Corp., Arlington Heights, IL) and 0.025 U/µl of Taq DNA polymerase (AmpliTaq^®, Perkin Elmer Corp., Foster City, CA). Reverse transcription was conducted at 42 °C for 60 min, followed directly by 30 amplification cycles of 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 2 min, with a final extension at 72 °C for 5 min. Amplification was conducted in 0.5 ml tubes (USA Scientific, Ocala, FL) in a PTC-200 thermocycler (MJ Research, Inc., Watertown, MA). Ten microliters of the product were analyzed by electrophoresis in a 1.5% agarose gel in 1X TBE (89 mM Tris borate, 2 mM EDTA, pH 8.3) containing 4 ng/ml ethidium bromide. A 100-basepair ladder was used as a size standard (Gibco BRL). The products predict by pairs of primers are as follows: RSS3-RSS4 = 848bp; RSS2-RSS3 = 754bp; RSS1-RSS4 = 676bp; RSS1-RSS2 = 582bp.

RT-PCR amplification - Nucleotides from positions 2311 to 2550 coding for the fragment of E/NS1 gene junction were amplified using RT-PCR. The primers, reaction mixture and electrophoresis conditions were performed as previously described (RICO-HESSE et al., 1997). Briefly, 5 µl of viral RNA were added to Superscript II RT buffer (Gibco BRL), 100 mM DTT and 50 pmoles antisense primer D2/2578. The solution was heated at 90 °C for 90 sec and cooled down on ice. Each of the four deoxynucleotides was added (1mM) along with 40 U RNasin (Promega) and 200 U reverse transcriptase Superscript II RT (Gibco BRL). The mixture was incubated for 1 h at 42 °C and cooled on ice. Both PCR primers (150 pM), PCR buffer consisting of 100 mM Tris pH 8.5, 500 mM KCl, 15mM MgCl₂ and 0.1% gelatin, and 200 µM of each deoxynucleotide were added to the mix, and the volume was brought up to 50 µl with water. Taq polymerase (5U/Gibco BRL) was added last, and the solution was overlayed with mineral oil. The samples were placed in a thermal cycler (Perkin Elmer CENS) for 30 cycles of 94 °C for 60 sec, 55 °C for 2 min, and 72 °C for 3 min.

Sequencing - The RT-PCR product was precipitated with 3M sodium acetate, pH 5.2 and ethanol, electrophoresed in a 1% agarose gel and stained with 1 mg/ml ethidium bromide. Bands (408 bp) were excised and purified using the QIAquick Gel extraction Kit (Gibco BRL). Purified DNA was then sequenced using Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystem, Inc, USA). Cycle sequencing parameters used were as described by the manufacturer's protocol. Sequencing was performed using an ABI Prism 310 (Applied Biosystem). Dendrogram were generated by progressive pairwise alignment (Genetics Computer Group, INC. Wisconsin Package version 9.1).

RESULTS

The analysis of RSS-PCR products from 52 isolates of Brazilian DEN-2 viruses showed that all of them presented the same pattern, except in a few cases, where extra bands were observed (Fig. 1A, lanes 12-16; Fig. 1B). Although Brazilian RSS-PCR pattern was consistent, it did not match any of the DEN-2 RSS-PCR patterns previously described by Harris et al., 1999 (Fig. 1A, lanes 2-8). The Brazilian pattern presented consistent and reproducible bands of 582bp and 100bp and, occasionally extra bands of 676 bp or 150 bp.

Fig. 2 shows a dendogram generated by pairwise alignment of the nucleotide sequence by using progressive pairwise alignments of 15 DEN-2 Brazilian isolates and 24 DEN-2 isolates obtained from GenBank, representing the 5 genotypes described by Rico-Hesse, 1990. According to the dendogram generated all Brazilian DEN-2 viruses are placed together in the same group of Jamaica 81 (GenBank Access # M32950) strain showing that Brazilian strain was more closely related to Asian-like DEN-2 American strain.

The comparison of the 240-base pair sequence spanning the gene junction between the E structural protein and the NS1 nonstructural protein among 15 Brazilian DEN-2 viruses showed homology ranging from 90% to 100% and 94% to 100% for nucleotide sequence and deduced amino acid sequence, respectively. All the nucleotides differences were single base substitutions; no deletions or insertions were observed. In relation to DEN-2 virus isolated from Jamaica 1981, the homology of Brazilian isolates ranged from 95% to 99% for nucleotide and deduced amino acid sequences (Fig. 3).

DISCUSSION

DEN-2 virus was first isolated in the American continent in 1953 in Trinidad (ANDERSON et al., 1956) and was responsible for epidemics of classic dengue fever in the 1960s and 70s (GUBLER, 1992). This genotype, called "Puerto Rico", is genetically related to other viruses from the Pacific Region and India (TRENT et al., 1983; RICO-HESSE, 1990) and was the only DEN-2 genotype circulating in the Americas until the 80's, when a new virus related to isolates from Southeast Asia (RICO-HESSI, 1990; LEWIS et al., 1993) was introduced during an extensive epidemics in the Caribbean islands in 1981. This genotype, known as "Jamaica", is associated with severe disease and its introduction into the continent resulted in an increase in both numbers of severe cases and epidemics in which there was a significant number of dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS) cases (RICO-HESSE et al., 1997). For instance, circulation of this variant in 1994-96, especially in Venezuela, resulted in a large number of DHF/DSS cases and high cases fatality (SALAS et al., 1998). The "Jamaica" genotype is closely related to the Southeast Asian genotype; in fact recent analysis have merged the two genotypes into one (RICO HESSE et al., 1998).

In Brazil, DEN-2 virus was first isolated in 1990 in the State of Rio de Janeiro during an effective dengue surveillance program that included the ongoing systematic collection and sample analysis of suspected cases (NOGUEIRA et al., 1990). The introduction of DEN-2 virus increased the number of severe disease cases with the appearance of several cases of DHF/DSS (NOGUEIRA et al., 1993; ZAGNE et al., 1994). Beginning with its introduction in 1990 until 2000, a total of 928 DHF/DSS cases were reported with 48 deaths (MINISTRY OF HEALTH, BRAZIL).

The DEN-2 virus genetic analysis performed in the present study confirms previously data obtained by using different protocols that identified the "Jamaica" genotype as the only one circulating in the country (RICO-HESSE, 1990; DEUBEL et al., 1993; VORNDAM et al., 1994; MIAGOSTOVICH et al., 1998).

By using a low cost subtyping method (RSS-PCR) we were able to analyze larger number of viruses, performing a retrospective study and characterizing recent isolates during an epidemic period. Although we could not match the RSS-PCR products obtained with the others previously described by HARRIS et al., 1999, we established a consistent and reproducible pattern for DEN-2 Brazilian isolates. The RSS-PCR method was developed with DEN-2 isolates obtained from 1964-1986 and the ongoing evolution of dengue virus over the last 15 years could explain the genetic diversity observed in DEN-2 viruses analyzed in this study, since they were isolated between 1990 and 2000.

The nucleotide changes observed by E/NS1 sequencing of these isolates showed the variability produced during ten years of DEN-2 transmission. Despite this variability, all samples belong to the "Jamaica" genotype. Since sequencing of the entire envelope (E) gene in DEN-2 virus (LEWIS et al., 1993) established essentially the same genotypic groups as those suggested by E/NS1 sequencing (RICO-HESSE, 1990), we recommend sequencing of this small region in programs of virological surveillance performed in countries with high dengue viruses activity.

For RSS-PCR results our data suggested that further studies should be performed using different geographic and temporally samples for a better representation of those genotypes since the method showed to be reproducible and can be used as an important tool for assessing the genetic variability of larger numbers of samples.

ACKNOWLEDGMENTS

To José da Costa Farias Filho and José de Carvalho Filho for dengue virus isolation and identification and to the Directors of Central Laboratories from Bahia, Ceará, Espírito Santo and Rio Grande do Norte for supplying serum samples.

Received: 24 July 2002

Accepted: 22 October 2002

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq- 40.0164/98-1), Oswaldo Cruz Foundation, and the Fogarty International Center of the National Institutes of Health (TW-00905).

• 1. ANDERSON, C.R.; DOWNS, W.G. & HILL, A.E. - Isolation of dengue virus from a human being in Trinidad. Science, 124: 224-225, 1956.
• 2. DEUBEL, V.; NOGUEIRA, R.M.R.; DROUET, M.T. et al - Direct sequencing of genomic cDNA fragment amplified by the polymerase chain reaction for molecular epidemiology of dengue-2 viruses. Arch. Virol., 129: 197-210, 1993.
• 3. GUBLER, D.J. - Dengue/dengue hemorrhagic fever in the Americas: prospects for the year 2000. In: HALSTEAD, S.B. & GOMEZ-DANTES, H. Dengue, a worldwide problem, a common strategy. Proceedings of the International Conference on Dengue and Aedes aegypti Community-based Control. México, Ministry of Health, 1992. p. 19-27.
• 4. GUBLER, D.J.; KUNO, G.; SATHER, G.; VELEZ, M. & OLIVER, A. - Mosquito cell cultures and specific monoclonal antibodies in surveillance for dengue viruses. Amer. J. trop. Med. Hyg., 33: 158-165, 1984.
• 5. HAMMON, W.M.C.D.; RUDNICK, A.; SATHER, G.E. - Viruses associated with epidemic hemorrhagic fever of the Philippines and Thailand. Science, 31: 1102-1103, 1960.
• 6. HARRIS, E.; SANDOVAL, E.; JOHNSON, M.; XET-MULL, A.M. & RILEY, L.W. - Rapid subtyping of dengue viruses by restriction site-specific (RSS)-PCR. Virology, 253: 86-95, 1999.
• 7. IGARASHI, A. - Isolation of a Singh's Aedes albopictus cell clone sensitive to dengue and chikungunya viruses. J. gen. Virol., 40: 531-544, 1978.
• 8. KOURI, G.; GUZMAN, M.G. & BRAVO, J. - Why dengue haemorrhagic fever in Cuba? II. An integral analysis. Trans. roy. Soc. trop Med. Hyg., 81: 821-823, 1987.
• 9. LANCIOTTI, R.S.; GUBLER, D.J. & TRENT, D.W. - Molecular evolution and phylogeny of dengue-4 viruses. J. gen. Virol., 78: 2279-2286, 1997.
• 10. LANCIOTTI, R.S.; LEWIS, J.G.; GUBLER, D.J. & TRENT, D.W. - Molecular evolution and epidemiology of dengue-3 viruses. J. gen. Virol., 75: 65-75, 1994.
• 11. LEWIS, J.A.; CHANG, G.J.; LANCIOTTI, R.S. et al - Phylogenetic relationship of dengue-2 viruses. Virology, 197: 216-224, 1993.
• 12. McCLOUD, T.G.; CARDIFF, R.D.; BRANDT, W.E.; CHEIWSILP, D. & RUSSEL, P.K. - Separation of dengue strains on the basis of a nonstructural antigen. Amer. J. trop. Med. Hyg., 20: 964-968, 1971.
• 13. MIAGOSTOVICH, M.P.; NOGUEIRA, R.M.R.; SCHATZMAYR, H.G. & LANCIOTTI, R.S. - Molecular epidemiology of DEN-2 virus in Brazil. Mem. Inst. Oswaldo Cruz, 93: 625-626, 1998.
• 14. MIAGOSTOVICH, M.P.; SANTOS, F.B.; GUTIERREZ, C.M.; RILEY, L.W. & HARRIS, E. - Rapid subtyping of dengue viruses serotypes 1 and 4 by restriction site-specific-pcr. J. clin. Microbiol., 38: 1286-1289, 2000.
• 15. NOGUEIRA, R.M.R.; MIAGOSTOVICH, M.P.; LAMPE, E. & SCHATZMAYR, H.G. - Isolation of dengue virus type 2 in Rio de Janeiro. Mem. Inst. Oswaldo Cruz, 85: 253, 1990.
• 16. NOGUEIRA, R.M.R.; MIAGOSTOVICH, M.P.; LAMPE, E. et al - Dengue epidemic in the state of Rio de Janeiro, Brazil, 1990-1991: co-circulation of dengue 1 and dengue 2. Epidem. Infect., 111: 163-170, 1993.
• 17. RICO-HESSE, R. - Molecular evolution and distribution of dengue viruses type 1 and 2 in nature.Virology, 174: 479-493, 1990.
• 18. RICO-HESSE, R.; HARRISON, L.M.; SALAS, R.A. et al. - Origins of dengue type 2 viruses associated with increased pathogenicity in the America.Virology, 230: 244-251, 1997.
• 19. RICO-HESSE, R.; HARRISON, L.M.; NISALAK, A. et al - Molecular evolution of dengue type 2 virus in Thailand. Amer. J. trop. Med. Hyg., 58: 96-101, 1998.
• 20. RUSSELL, P.K. & MCCOWN, J.M. - Comparison of dengue 2 and dengue 3 virus strains by neutralization tests and identification of a serotype of dengue 3. Amer. J. trop. Med. Hyg., 21: 97-99, 1972.
• 21. SABIN, A.B. - Research on dengue during World War II. Amer. J. trop. Med Hyg., 1: 30-50, 1952.
• 22. SALAS, R.A.; TOVAR, D.; BARRETO, A. et al - Serotipos y genotipos de virus dengue circulantes en Venezuela, 1990-1997. Acta cient. venez., 49(suppl.1): 33-37, 1998.
• 23. TRENT, D.W.; GRANT, J.A.; ROSEN, L. & MONATH, T.P. - Genetic variation among dengue 2 viruses of different geographic origin. Virology, 128: 271-284, 1983.
• 24.VORNDAM, V.; NOGUEIRA, R.M.R. & TRENT, D.W.- Restriction enzyme analysis of American region dengue viruses. Arch. Virol., 136: 191-196, 1994.
• 25. ZAGNE, S.M.O.; ALVES, V.G.F.; NOGUEIRA, R.M.R. et al - Dengue haemorrhagic fever in the state of Rio de Janeiro, Brazil: a study of 56 confirmed cases. Trans. roy. Soc. trop. Med. Hyg., 88: 677-679, 1994.

Publication Dates

• Publication in this collection
  03 Apr 2003
• Date of issue
  Jan 2003

History

• Received
  24 July 2002
• Accepted
  22 Oct 2002