The Viridot package is now available on GitHub at https://github

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The Viridot package is now available on GitHub at https://github.com/leahkatzelnick/Viridot.. plaque from background and size (in pixels) of the window for applying the thresholding algorithm to the image. Step 6: dilate plaques to ensure they are counted as single objects. Step 7: cut overlapping plaques so they are counted separately. Step 8: define the minimum and maximum pixel size to count as a plaque. Details are provided in the section of the Viridot manual (S1 Supporting File).(PDF) pntd.0006862.s001.pdf (2.5M) GUID:?8A4DE3A8-01D7-4F63-B959-2A1581492C95 S2 Fig: Comparison of manual plaque counts among analysts. Manual plaque counting by four separate analysts for (A) a well of uniform, evenly colored plaques or (B) a well of plaques with non-uniform size and intensity.(PDF) pntd.0006862.s002.pdf (7.4M) GUID:?0E1A40C5-C9FA-4DD1-9211-7364FCDEE8AB S1 Table: Plate, AZD6244 (Selumetinib) camera, and color settings used for taking images of plaques using a CTL Immunospot Analyzer, corresponding to the Costar 96 well template built into the software. (DOCX) pntd.0006862.s003.docx (54K) GUID:?FC0C7BDA-48C0-4CD5-9259-54F353DECC3E S2 Table: Manual and Viridot plaque count comparison as shown in Figs ?Figs55 and ?and66. (DOCX) pntd.0006862.s004.docx (86K) GUID:?13539345-B072-41E7-B3C5-BBC0FF606C4C S3 Table: Neutralizing antibody titers (PRNT50) estimated with manual and Viridot plaque counting (Data correspond to Fig 6). (DOCX) pntd.0006862.s005.docx (51K) GUID:?877F2361-758B-4017-9DC4-27BFCCDF2EA1 S1 Supporting File: The Viridot manual. (PDF) pntd.0006862.s006.pdf (579K) GUID:?3E243E79-6573-42DC-8BA7-15CDA659E8E2 S2 Supporting File: PRNT and image acquisition protocols. (PDF) pntd.0006862.s007.pdf (85K) GUID:?D13D2F9E-C1B2-4CB9-BF6F-5F2D92CE7A5F S1 Supporting Data: A .zip file that includes plaque count data used for Figs ?Figs55 and ?and66. (ZIP) pntd.0006862.s008.zip (4.2K) GUID:?FB2F9B17-5331-4C1E-8923-02F83558D566 Data Availability StatementRelevant data are within the paper and its Supporting Information files. The Viridot package is now available on GitHub at https://github.com/leahkatzelnick/Viridot. Abstract The gold-standard method for quantifying neutralizing antibody AZD6244 (Selumetinib) 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 IKK-gamma antibody 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, AZD6244 (Selumetinib) 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 (Lins concordance correlation coefficient, c = 0.99 [95% confidence interval: 0.99C1.00]) as manual counts between analysts (c AZD6244 (Selumetinib) = 0.99 [95% CI: 0.98C0.99]). The average ratio of neutralizing antibody titers based on manual counted plaques to Viridot counted plaques was 1.05 (95% CI: 0.98C1.14), similar to the average ratio of antibody titers based on manual plaque counts by the two analysts (1.06 [95% CI: 0.84C1.34]). Across diverse DENV and ZIKV strains (n = 14), manual and Viridot plaque counts were mostly consistent (range of c = 0.74 to 1 1.00) and the average ratio of antibody titers based on manual and Viridot counted plaques was close to 1 (0.94 [0.86C1.02]). 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. Author summary Although the plaque reduction neutralization test (PRNT) is an important assay for measuring neutralizing antibody responses against many viruses, no free, open-source programs specifically designed for virus plaque counting and neutralizing antibody titer estimation are available. We have developed Viridot, a package for R with a user-interface in shiny, which is designed for use by laboratory-based virologists and researchers with minimal coding experience. The program includes: automatic and user-specification of.