Volume 81, Issue 5
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645


Dengue virus (DENV) infection is a worsening global health problem. The plaque reduction neutralization test (PRNT) is currently considered to be the “gold standard” to characterize and quantify circulating levels of anti-DENV neutralizing antibody (NAb). Many variations of the PRNT are currently in use and neither the assay nor its performance conditions have been standardized or harmonized between laboratories. We used a well-characterized panel of acute and late convalescent follow-up sera samples from children experiencing primary and secondary DENV infections to evaluate the performance of the dengue PRNT under a variety of testing conditions. Investigators varied cell type, control virus passage, and the use of complement across multiple assay runs of the same sample panel. Our findings indicate wide variation in PRNT titer results in response to varied testing conditions.


Article metrics loading...

The graphs shown below represent data from March 2017
Loading full text...

Full text loading...



  1. Gibbons RV, Vaughn DW, 2002. Dengue: an escalating problem. BMJ 324: 1563–1566. [Google Scholar]
  2. Gubler DJ, 1998. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11: 480–496. [Google Scholar]
  3. 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]
  4. Hotta S, 1952. Experimental studies on dengue. I. Isolation, identification and modification of the virus. J Infect Dis 90: 1–9. [Google Scholar]
  5. Chambers TJ, Hahn CS, Galler R, Rice CM, 1990. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44: 649–688. [Google Scholar]
  6. 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]
  7. Wisseman CL Jr, Sweet BH, 1962. Immunological studies with group B arthropod-borne viruses. III. Response of human subjects to revaccination with 17D strain yellow fever vaccine. Am J Trop Med Hyg 11: 570–575. [Google Scholar]
  8. Henderson JR, Taylor RM, 1959. Arthropod-borne virus plaques in agar overlaid tube cultures. Proc Soc Exp Biol Med 101: 257–259. [Google Scholar]
  9. Hammon W, Sather G, 1964. Problems of typing dengue viruses. Mil Med 129: 130–135. [Google Scholar]
  10. Halstead SB, Sukhavachana P, Nisalak A, 1964. Assay of mouse adapted dengue viruses in mammalian cell cultures by an interference method. Proc Soc Exp Biol Med 115: 1062–1068. [Google Scholar]
  11. Russell PK, Nisalak A, 1967. Dengue virus identification by the plaque reduction neutralization test. J Immunol 99: 291–296. [Google Scholar]
  12. Russell PK, Nisalak A, Sukhavachana P, Vivona S, 1967. A plaque reduction test for dengue virus neutralizing antibodies. J Immunol 99: 285–290. [Google Scholar]
  13. Sukhavachana P, Yuill TM, Russell PK, 1969. Assay of arbovirus neutralizing antibody by micro methods. Trans R Soc Trop Med Hyg 63: 446–455. [Google Scholar]
  14. Fujita N, Tamura M, Hotta S, 1975. Dengue virus plaque formation on microplate cultures and its application to virus neutralization (38564). Proc Soc Exp Biol Med 148: 472–475. [Google Scholar]
  15. Okuno Y, Igarashi A, Fukai K, 1978. Neutralization tests for dengue and Japanese encephalitis viruses by the focus reduction method using peroxidase-anti-peroxidase staining. Biken J 21: 137–147. [Google Scholar]
  16. Morens DM, Halstead SB, Larsen LK, 1985. Comparison of dengue virus plaque reduction neutralization by macro and “semi-micro” methods in LLC-MK2 cells. Microbiol Immunol 29: 1197–1205. [Google Scholar]
  17. Morens DM, Halstead SB, Repik PM, Putvatana R, Raybourne N, 1985. Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization. J Clin Microbiol 22: 250–254. [Google Scholar]
  18. Edelman R, Wasserman SS, Bodison SA, Putnak RJ, Eckels KH, Tang D, Kanesa-Thasan N, Vaughn DW, Innis BL, Sun W, 2003. Phase I trial of 16 formulations of a tetravalent live-attenuated dengue vaccine. Am J Trop Med Hyg 69: 48–60. [Google Scholar]
  19. Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, Sirivichayakul C, Pengsaa K, Pojjaroen-Anant C, Chambonneau L, Saluzzo JF, Bhamarapravati N, 2004. Safety and immunogenicity of a three dose regimen of two tetravalent live-attenuated dengue vaccines in five- to twelve-year-old Thai children. Pediatr Infect Dis J 23: 99–109. [Google Scholar]
  20. Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, Sirivichayakul C, Pengsaa K, Pojjaroen-Anant C, Chokejindachai W, Jagsudee A, Saluzzo JF, Bhamarapravati N, 2002. Safety and immunogenicity of tetravalent live-attenuated dengue vaccines in Thai adult volunteers: role of serotype concentration, ratio, and multiple doses. Am J Trop Med Hyg 66: 264–272. [Google Scholar]
  21. Simasathien S, Thomas SJ, Watanaveeradej V, Nisalak A, Barberousse C, Innis BL, Sun W, Putnak JR, Eckels KH, Hutagalung Y, Gibbons RV, Zhang C, De La Barrera R, Jarman RG, Chawachalasai W, Mammen MP Jr, 2008. Safety and immunogenicity of a tetravalent live-attenuated dengue vaccine in Flavivirus naive children. Am J Trop Med Hyg 78: 426–433. [Google Scholar]
  22. Calisher CH, Nuti M, Lazuick JS, Ferrari JD, Kappus KD, 1981. Dengue in the Seychelles. Bull World Health Organ 59: 619–622. [Google Scholar]
  23. Sangkawibha N, Rojanasuphot S, Ahandrik S, Viriyapongse S, Jatanasen S, Salitul V, Phanthumachinda B, Halstead SB, 1984. Risk factors in dengue shock syndrome: a prospective epidemiologic study in Rayong, Thailand. I. The 1980 outbreak. Am J Epidemiol 120: 653–669. [Google Scholar]
  24. Fukunaga T, Okuno Y, Tadano M, Fukai K, 1983. A retrospective serological study of Japanese who contracted dengue fever in Thailand. Biken J 26: 67–74. [Google Scholar]
  25. McBride WJ, Mullner H, LaBrooy JT, Wronski I, 1998. The 1993 dengue 2 epidemic in Charters Towers, north Queensland: clinical features and public health impact. Epidemiol Infect 121: 151–156. [Google Scholar]
  26. Papaevangelou G, Halstead SB, 1977. Infections with two dengue viruses in Greece in the 20th century. Did dengue hemorrhagic fever occur in the 1928 epidemic? J Trop Med Hyg 80: 46–51. [Google Scholar]
  27. Endy TP, Chunsuttiwat S, Nisalak A, Libraty DH, Green S, Rothman AL, Vaughn DW, Ennis FA, 2002. Epidemiology of inapparent and symptomatic acute dengue virus infection: a prospective study of primary school children in Kamphaeng Phet, Thailand. Am J Epidemiol 156: 40–51. [Google Scholar]
  28. Endy TP, Nisalak A, Chunsuttiwat S, Libraty DH, Green S, Rothman AL, Vaughn DW, Ennis FA, 2002. Spatial and temporal circulation of dengue virus serotypes: a prospective study of primary school children in Kamphaeng Phet, Thailand. Am J Epidemiol 156: 52–59. [Google Scholar]
  29. van Peenen PF, Sunoto, Sumarmo, Sulianti Saroso J, Sinto S, Joseph PL, See R, 1978. Dengue with haemorrhage and shock in Jakarta, Indonesia. Southeast Asian J Trop Med Public Health 9: 25–32. [Google Scholar]
  30. Roehrig JT, Hombach J, Barrett AD, 2008. Guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses. Viral Immunol 21: 123–132. [Google Scholar]
  31. Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, Endy TP, Raengsakulrach B, Rothman AL, Ennis FA, Nisalak A, 2000. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 181: 2–9. [Google Scholar]
  32. Innis BL, Nisalak A, Nimmannitya S, Kusalerdchariya S, Chongswasdi V, Suntayakorn S, Puttisri P, Hoke CH, 1989. An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate. Am J Trop Med Hyg 40: 418–427. [Google Scholar]
  33. Clarke DH, Casals J, 1958. Techniques for hemagglutination and hemagglutination inhibition with arthropod-borne viruses. Am J Trop Med Hyg 7: 561–573. [Google Scholar]
  34. Rosen L, Gubler D, 1974. The use of mosquitoes to detect and propagate dengue viruses. Am J Trop Med Hyg 23: 1153–1160. [Google Scholar]
  35. Kuno G, Gubler DJ, Santiago de Weil NS, 1985. Antigen capture ELISA for the identification of dengue viruses. J Virol Methods 12: 93–103. [Google Scholar]
  36. Kalayanarooj S, Vaughn DW, Nimmannitya S, Green S, Suntayakorn S, Kunentrasai N, Viramitrachai W, Ratanachueke S, Kiatpolpoj S, Innis BL, Rothman AL, Nisalak A, Ennis FA, 1997. Early clinical and laboratory indicators of acute dengue illness. J Infect Dis 176: 313–321. [Google Scholar]
  37. Cutchins E, Warren J, Jones WP, 1960. The antibody response to smallpox vaccination as measured by a tissue culture plaque method. J Immunol 85: 275–283. [Google Scholar]
  38. Edelman R, 2007. Dengue vaccines approach the finish line. Clin Infect Dis 45 (Suppl 1): S56–S60. [Google Scholar]
  39. Henchal EA, Gentry MK, McCown JM, Brandt WE, 1982. Dengue virus-specific and flavivirus group determinants identified with monoclonal antibodies by indirect immunofluorescence. Am J Trop Med Hyg 31: 830–836. [Google Scholar]

Data & Media loading...

  • Received : 01 Dec 2008
  • Accepted : 27 Jul 2009

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error