title: Evaluation of cell viability dyes in antiviral assays with RNA viruses that exhibit different cytopathogenic properties.
abstract: Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue®measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80-90, and 50% maximal cytopathic effect (CPE), respectively, in Vero or Vero 76 cells Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC50) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10-20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC50) assays with dyes in uninfected proliferating cells produced similar CC50values for EICAR (1.5-8.9μM) and MPA (0.8-2.5μM). 6-Azauridine toxicity was 6.1-17.5μM with NR, VTG, and WST-1, compared to 48-92μM with ALB-A and ALB-F (P<0.001). Curiously, the CC50values for 3-deazaguanine were 83-93μM with ALB-F versus 2.4-7.0μM with all other dyes including ALB-A (P<0.001). Overall, ALB minimized the toxicities detected with these two inhibitors. Because the choice of dyes affected CC50values, this impacted on the resulting in vitro selectivity indexes (calculated as CC50/EC50ratio).
authors.affiliation: Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322 USA. Electronic address: don.smee@usu.edu.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Smee
authors.affiliation: Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322 USA.
authors.forename: Brett L
authors.initials: BL
authors.lastname: Hurst
authors.affiliation: Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322 USA.
authors.forename: W Joseph
authors.initials: WJ
authors.lastname: Evans
authors.affiliation: Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322 USA.
authors.forename: Nathan
authors.initials: N
authors.lastname: Clyde
authors.affiliation: Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322 USA.
authors.forename: Sean
authors.initials: S
authors.lastname: Wright
authors.affiliation: Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322 USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Peterson
authors.affiliation: Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322 USA.
authors.forename: Kie-Hoon
authors.initials: KH
authors.lastname: Jung
authors.affiliation: Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322 USA.
authors.forename: Craig W
authors.initials: CW
authors.lastname: Day
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects*; D002646:Chikungunya virus / Q000187:drug effects / Q000472:pathogenicity / Q000502:physiology; D002522:Chlorocebus aethiops; D004396:Coloring Agents / Q000737:chemistry; D003588:Cytopathogenic Effect, Viral; D003716:Dengue Virus / Q000187:drug effects / Q000472:pathogenicity / Q000502:physiology; D018051:Junin virus / Q000187:drug effects / Q000472:pathogenicity / Q000502:physiology; D010078:Oxazines; D012328:RNA Viruses / Q000187:drug effects* / Q000472:pathogenicity / Q000502:physiology; D014709:Vero Cells; D014779:Virus Replication / Q000187:drug effects; D014780:Viruses / Q000187:drug effects*; D014966:Xanthenes
chemical_list: D000998:Antiviral Agents; D004396:Coloring Agents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin
keywords: Antiviral; Chikungunya virus; Dengue virus; Dye assays; Junin virus