Virus Reduction Neutralization Test: A Single-Cell Imaging High-Throughput Virus Neutralization Assay for Dengue

Am J Trop Med Hyg. 2018 Dec;99(6):1430-1439. doi: 10.4269/ajtmh.17-0948.


Vaccine immunogenicity and clinical efficacy are often assessed by the measure of serum-neutralizing antibodies. The present gold standard for detecting neutralizing antibodies against many viruses, including dengue, is the plaque/focus reduction neutralization test (P/FRNT). The FRNT is a cell-based assay that inherits high variability, resulting in poor precision and has lengthy turnaround times. The virus reduction neutralization test (VRNT) is a high-throughput alternative to the standard low-throughput and laborious FRNT. The VRNT is similar to FRNT using unaltered wild-type virus and immunostaining, yet uses imaging cytometry to count virus-infected cells 1 day post-infection, reducing assay time and increasing overall throughput 15-fold. In addition, the VRNT has lowered variability relative to FRNT, which may be explained in part by the observation that foci overlap alters foci count and titer over time, in the FRNT. The ability to count one infected cell, rather than waiting for overlapping foci to form, ensures accuracy and contributes to the precision (7-25% coefficient of variation) and sensitivity of the VRNT. Results from 81 clinical samples tested in the VRNT and FRNT show a clear positive relationship. During sample testing, a 96-well plate edge effect was noted and the elimination of this edge effect was achieved by a simple plate seeding technique. The VRNT is an improvement to the current neutralization assays for its shortened assay time, increased precision and throughput, and an alternative to the P/FRNT.

MeSH terms

  • Animals
  • Anthraquinones / chemistry
  • Antibodies, Neutralizing / analysis*
  • Antibodies, Viral / analysis*
  • Chlorocebus aethiops
  • Dengue / immunology
  • Dengue / prevention & control
  • Dengue / virology
  • Dengue Vaccines / analysis
  • Dengue Virus / immunology*
  • Dengue Virus / isolation & purification
  • Fluorescent Dyes / chemistry
  • High-Throughput Screening Assays / standards*
  • Humans
  • Molecular Imaging / methods*
  • Neutralization Tests / standards*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Single-Cell Analysis / methods*
  • Vero Cells
  • Viral Load
  • Viral Plaque Assay


  • 1,5-bis((2-(methylamino)ethyl)amino)-4,8-dihydroxyanthracene-9,10-dione
  • Anthraquinones
  • Antibodies, Neutralizing
  • Antibodies, Viral
  • Dengue Vaccines
  • Fluorescent Dyes