Volume 71, Issue 2
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645


Dengue is a serious cause of morbidity and mortality in tropical areas worldwide. We cloned and expressed recombinant polypeptides spanning the entire genome of a Brazilian dengue virus type 2 (DENV-2) strain in contiguous segments to generate antigens for dengue diagnosis and evaluation of the human humoral immune response. When analyzed by Western blot and an enzyme-linked immunosorbent assay (ELISA) using human sera, the most reactive polypeptide (pD2-3(E)) was located in the N-terminal portion of the envelope protein. The sensitivity of an IgG-ELISA using pD2-3(E) versus mouse brain antigen was 100% with convalescent sera and 79% with acute sera, with a specificity of 100%. Sera from patients infected with other DENV serotypes recognized pD2-3(E) equally well, whereas sera positive for yellow fever, rubella, and measles showed little or no reactivity. Using this novel approach, we identified a candidate antigen to facilitate diagnosis of DENV infections and observed a surprising variability in antibody patterns in the clinical response to DENV infections.


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  1. Heinz FX, Collet MS, Purcell RH, Gould EA, Howard CR, Hounghton M, Moormann RJ, Rice CM, Thiel HJ, 2000. Virus Taxonomy. Family Flaviviridae. Van Regenmortel CM, Fauquet DHL, Bishop EB, Cartens MK, Lemon SM, Maniloff MA, Mayo DJ, Pringle CR, Wikner RB, eds. Seventh Report of the International Committee on Taxonomy of Virus. San Diego, CA: Academic Press, 859–878.
  2. Lindenbach BD, Rice CM, 2001. Flaviviridae: the viruses and their replication. Knipe DM, Howley PM, eds. Fields Virology. Philadelphia: Lippincott Williams and Wilkins, 991–1041.
  3. Burke DS, Monath TP, 2001. Flaviviruses. Knipe DM, Howley PM, eds. Fields Virology. Philadelphia: Lippincott Williams and Wilkins, 1043–1126.
  4. Gubler DJ, 2002. Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. Trends Microbiol 10 : 100–103. [Google Scholar]
  5. da Silva JB Jr, Siqueira JB Jr, Coelho GE, Giovanini E, Vilarinhos PTR, Pimenta FG Jr, 2002. Dengue in Brazil: current situation and prevention and control activities. Epidemiol Bull 23 : 3–6. [Google Scholar]
  6. Mandl CW, Guirakhoo F, Holzman H, Heinz FX, Kunz C, 1989. Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-born encephalitis virus as a model. J Virol 63 : 564–571. [Google Scholar]
  7. SeThoe SY, Ng MM, Ling AE, 1999. Retrospective study of Western blot profiles in immune sera of natural dengue virus infections. J Med Virol 57 : 322–330. [Google Scholar]
  8. Valdes K, Alvarez M, Pupo M, Vazquez S, Rodriguez R, Guzman MG, 2000. Human dengue antibodies against structural and nonstructural proteins. Clin Diagn Lab Immunol 7 : 856–857. [Google Scholar]
  9. Gibbons RV, Vaughn DW, 2002. Dengue: an escalating problem. BMJ 324 : 1563–1566. [Google Scholar]
  10. Morens DM, 1994. Antibody-dependent enhancement of infection and the pathogenesis of viral disease. Clin Infect Dis 19 : 500–512. [Google Scholar]
  11. Halstead SB, 1988. Pathogenesis of dengue: challenges to molecular biology. Science 239 : 476–481. [Google Scholar]
  12. Gubler DJ, 1998. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11 : 480–496. [Google Scholar]
  13. WHO, 1997. Dengue Haemorrhagic Dever: Diagnosis, Treatment, Prevention and Control. Geneva: World Health Organization.
  14. Vorndam AV, Kuno G, 1997. Laboratory diagnosis of dengue virus infection. Gubler DJ, Kuno G, eds. Dengue and Dengue Hemorrhagic Fever. New York: CAB International, 313–333.
  15. Kuno G, Cropp CB, Wong-Lee J, Gubler DJ, 1998. Evaluation of an IgM immunoblot kit for dengue diagnosis. Am J Trop Med Hyg 59 : 757–762. [Google Scholar]
  16. Sang CT, Hoon LS, Cuzzubbo A, Devine P, 1998. Clinical evaluation of a rapid immunochromatographic test for the diagnosis of dengue virus infection. Clin Diagn Lab Immunol 5 : 407–409. [Google Scholar]
  17. Palmer CJ, King SD, Cuadrado RR, Perez E, Baum M, Ager AL, 1999. Evaluation of the MRL diagnostics dengue fever virus IgM capture ELISA and the PanBio rapid immunochromatographic test for diagnosis of dengue fever in Jamaica. J Clin Microbiol 37 : 1600–1601. [Google Scholar]
  18. Vaughn DW, Nisalak A, Solomon T, Kalayanarooj S, Nguyen MD, Kneen R, Cuzzubbo A, Devine PL, 1999. Rapid serologic diagnosis of dengue virus infection using a commercial capture ELISA that distinguishes primary and secondary infections. Am J Trop Med Hyg 60 : 693–698. [Google Scholar]
  19. Lam KS, Ew CL, Mitchell JL, Cuzzubbo AJ, Devine PL, 2000. Evaluation of a capture screening enzyme-linked immunosorbent assay for combined determination of immunoglobulin M and G antibodies produced during dengue infection. Clin Diagn Lab Immunol 7 : 850–852. [Google Scholar]
  20. Wu SJ, Paxton H, Hanson B, Kung CG, Chen TB, Rossi C, Vaughn DW, Murphy GS, Hayes CG, 2000. Comparison of two rapid diagnostic assays for detection of immunoglobulin M antibodies to dengue virus. Clin Diagn Lab Immunol 7 : 106–110. [Google Scholar]
  21. Igarashi A, 1978. Isolation of a Singh’s Aedes albopictus cell clone sensitive to dengue and chikungunya viruses. J Gen Virol 40 : 531–544. [Google Scholar]
  22. Gubler DJ, Kuno G, Sather GE, Velez M, Oliver A, 1984. Mosquito cell cultures and specific monoclonal antibodies in surveillance for dengue viruses. Am J Trop Med Hyg 33 : 158–165. [Google Scholar]
  23. dos Santos FB, Miagostovich MP, Nogueira RM, Edgil D, Schatzmayr HG, Riley LW, Harris E, 2002. Complete nucleotide sequence analysis of a Brazilian dengue virus type 2 strain. Mem Inst Oswaldo Cruz 97 : 991–995. [Google Scholar]
  24. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV, 1992. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 30 : 545–551. [Google Scholar]
  25. Nogueira RM, Miagostovich MP, Lampe E, Souza RW, Zagne SM, Schatzmayr HG, 1993. Dengue epidemic in the state of Rio de Janeiro, Brazil, 1990-1: co-circulation of dengue 1 and dengue 2 serotypes. Epidemiol Infect 111 : 163–170. [Google Scholar]
  26. Miagostovich MP, Nogueira RM, dos Santos FB, Schatzmayr HG, Araujo ES, Vorndam V, 1999. Evaluation of an IgG enzyme-linked immunosorbent assay for dengue diagnosis. J Clin Virol 14 : 183–189. [Google Scholar]
  27. Harris E, Roberts TG, Smith L, Selle J, Kramer LD, Valle S, Sandoval E, Balmaseda A, 1998. Typing of dengue viruses in clinical specimens and mosquitoes by singletube multiplex reverse transcriptase PCR. J Clin Microbiol 36 : 2634–2639. [Google Scholar]
  28. Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 : 680–685. [Google Scholar]
  29. Mason PW, Zugel MU, Semproni AR, Fournier MJ, Mason TL, 1990. The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli. J Gen Virol 71 : 2107–2114. [Google Scholar]
  30. Fonseca BAL, Khoshnood K, Shope RE, Mason PW, 1991. Flavivirus, type-specific antigens produced from fusions of a portion of the E protein gene with the Escherichia coli trpE gene. Am J Trop Med Hyg 44 : 500–508. [Google Scholar]
  31. Megret F, Hugnot JP, Falconar A, Gentry MK, Morens DM, Murray JM, Schlesinger JJ, Wright PJ, Young P, van Regenmortel MH, Deubel V, 1992. Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein. Virology 187 : 480–491. [Google Scholar]
  32. Trirawatanapong T, Chandran B, Putnak R, Padmanabhan R, 1992. Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody. Gene 116 : 139–150. [Google Scholar]
  33. Srivastava AK, Putnak JR, Warren RL, Hoke CH Jr, 1995. Mice immunized with a dengue type 2 virus E and NS1 fusion protein made in Escherichia coli are protected against lethal dengue virus infection. Vaccine 13 : 1251–1258. [Google Scholar]
  34. Sugrue RJ, Cui T, Xu Q, Fu J, Chan YC, 1997. The production of recombinant dengue virus E protein using Escherichia coli and Pichia pastoris. J Virol Methods 69 : 159–169. [Google Scholar]
  35. Simmons M, Porter KR, Escamilla J, Graham R, Watts DM, Eckels KH, Hayes CG, 1998. Evaluation of recombinant dengue viral envelope B domain protein antigens for the detection of dengue complex-specific antibodies. Am J Trop Med Hyg 58 : 144–151. [Google Scholar]
  36. Staropoli I, Frenkiel MP, Megret F, Deubel V, 1997. Affinity-purified dengue-2 virus envelope glycoprotein induces neutralizing antibodies and protective immunity in mice. Vaccine 15 : 1946–1954. [Google Scholar]
  37. Kelly EP, Greene JJ, King AD, Innis BL, 2000. Purified dengue 2 virus envelope glycoprotein aggregates produced by baculovirus are immunogenic in mice. Vaccine 18 : 2549–2559. [Google Scholar]
  38. Garcia G, Vaughn DW, DelAngel RM, 1997. Recognition of synthetic oligopeptides from nonstructural proteins NS1 and NS3 of dengue-4 virus by sera from dengue virus-infected children. Am J Trop Med Hyg 56 : 466–470. [Google Scholar]
  39. Cuzzubbo AJ, Endy TP, Nisalak A, Kalayanarooj S, Vaughn DW, Ogata SA, Clements DE, Devine P, 2001. Use of recombinant envelope proteins for serological diagnosis of dengue virus infection in an immunochromatographic assay. Clin Di-agn Lab Immunol 8 : 1150–1155. [Google Scholar]
  40. Wu HC, Huang YL, Chao TT, Jan JT, Huang JL, Chiang HY, King CC, Shaio MF, 2001. Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients. J Clin Microbiol 39 : 977–982. [Google Scholar]
  41. Huang JL, Huang JH, Shyu RH, Teng CW, Lin YL, Kuo MD, Yao CW, Shaio MF, 2001. High-level expression of recombinant dengue viral NS-1 protein and its potential use as a diagnostic antigen. J Med Virol 65 : 553–560. [Google Scholar]
  42. Ludolfs D, Schilling S, Altenschimidt J, Schmitz H, 2002. Serological differentiation of infections with dengue virus serotypes 1 to 4 by using recombinant antigens. J Clin Microbiol 40 : 4317–4320. [Google Scholar]
  43. Makino Y, Tadano M, Saito M, Maneekarn N, Sittisombut N, Sirisanthana V, Poneprasert B, Fukunaga T, 1994. Studies on serological cross-reaction in sequential flavivirus infections. Microbiol Immunol 38 : 951–955. [Google Scholar]
  44. Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, Westaway EG, Brandt WE, 1989. Antigenic relationships between flaviviruses as determined by cross- neutralization tests with polyclonal antisera. J Gen Virol 70 : 37–43. [Google Scholar]

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  • Received : 03 Oct 2003
  • Accepted : 07 Feb 2004

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