Viridot: An automated virus plaque (immunofocus) counter for the measurement of serological neutralizing responses with application to dengue virus

Leah C. Katzelnick, Ana Coello Escoto, Benjamin D. McElvany, Christian Chávez, Henrik Salje, Wensheng Luo, Isabel Rodriguez-Barraquer, Richard Jarman, Anna P Durbin, Sean A. Diehl, Derek J. Smith, Stephen S. Whitehead, Derek A.T. Cummings

Research output: Contribution to journalArticle

Abstract

The gold-standard method for quantifying neutralizing antibody responses to many viruses, including dengue virus (DENV), is the plaque reduction neutralization test (PRNT, also called the immunofocus reduction neutralization test). The PRNT conducted on 96-well plates is high-throughput and requires a smaller volume of antiserum than on 6- or 24-well plates, but manual plaque counting is challenging and existing automated plaque counters are expensive or difficult to optimize. We have developed Viridot (Viridot package), a program for R with a user interface in shiny, that counts viral plaques of a variety of phenotypes, estimates neutralizing antibody titers, and performs other calculations of use to virologists. The Viridot plaque counter includes an automatic parameter identification mode (misses <10 plaques/well for 87% of diverse DENV strains [n = 1521]) and a mode that allows the user to fine-tune the parameters used for counting plaques. We compared standardized manual and Viridot plaque counting methods applied to the same wells by two analyses and found that Viridot plaque counts were as similar to the same analyst's manual count (Lin's concordance correlation coefficient, ρc = 0.99 [95% confidence interval: 0.99-1.00]) as manual counts between analysts (ρc = 0.99 [95% CI: 0.98-0.99]). The average ratio of neutralizing antibody titers based on manual counted plaques to Viridot counted plaques was 1.05 (95% CI: 0.98-1.14), similar to the average ratio of antibody titers based on manual plaque counts by the two analysts (1.06 [95% CI: 0.84-1.34]). Across diverse DENV and ZIKV strains (n = 14), manual and Viridot plaque counts were mostly consistent (range of ρc = 0.74 to 1.00) and the average ratio of antibody titers based on manual and Viridot counted plaques was close to 1 (0.94 [0.86-1.02]). Thus, Viridot can be used for plaque counting and neutralizing antibody titer estimation of diverse DENV strains and potentially other viruses on 96-well plates as well as for formalization of plaque-counting rules for standardization across experiments and analysts.

Original languageEnglish (US)
Pages (from-to)e0006862
JournalPLoS Neglected Tropical Diseases
Volume12
Issue number10
DOIs
StatePublished - Oct 1 2018

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Dengue Virus
Neutralizing Antibodies
Viruses
Neutralization Tests
Antibodies
Gold
Antibody Formation
Immune Sera
Confidence Intervals
Phenotype

ASJC Scopus subject areas

  • Public Health, Environmental and Occupational Health
  • Infectious Diseases

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Viridot : An automated virus plaque (immunofocus) counter for the measurement of serological neutralizing responses with application to dengue virus. / Katzelnick, Leah C.; Coello Escoto, Ana; McElvany, Benjamin D.; Chávez, Christian; Salje, Henrik; Luo, Wensheng; Rodriguez-Barraquer, Isabel; Jarman, Richard; Durbin, Anna P; Diehl, Sean A.; Smith, Derek J.; Whitehead, Stephen S.; Cummings, Derek A.T.

In: PLoS Neglected Tropical Diseases, Vol. 12, No. 10, 01.10.2018, p. e0006862.

Research output: Contribution to journalArticle

Katzelnick, LC, Coello Escoto, A, McElvany, BD, Chávez, C, Salje, H, Luo, W, Rodriguez-Barraquer, I, Jarman, R, Durbin, AP, Diehl, SA, Smith, DJ, Whitehead, SS & Cummings, DAT 2018, 'Viridot: An automated virus plaque (immunofocus) counter for the measurement of serological neutralizing responses with application to dengue virus', PLoS Neglected Tropical Diseases, vol. 12, no. 10, pp. e0006862. https://doi.org/10.1371/journal.pntd.0006862
Katzelnick, Leah C. ; Coello Escoto, Ana ; McElvany, Benjamin D. ; Chávez, Christian ; Salje, Henrik ; Luo, Wensheng ; Rodriguez-Barraquer, Isabel ; Jarman, Richard ; Durbin, Anna P ; Diehl, Sean A. ; Smith, Derek J. ; Whitehead, Stephen S. ; Cummings, Derek A.T. / Viridot : An automated virus plaque (immunofocus) counter for the measurement of serological neutralizing responses with application to dengue virus. In: PLoS Neglected Tropical Diseases. 2018 ; Vol. 12, No. 10. pp. e0006862.
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