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 76. 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 CC50 values for EICAR (1.5-8.9 μM) and MPA (0.8-2.5 μM). 6-Azauridine toxicity was 6.1-17.5 μM with NR, VTG, and WST-1, compared to 48-92 μM with ALB-A and ALB-F.(P<0.001). Curiously, the CC50 values for 3-deazaguanine were 83-93 μM with ALB-F versus 2.4-7.0 μM 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 CC50 values, this impacted on the resulting in vitro selectivity indexes (calculated as CC50/EC50 ratio).

In vitro Assessment of Antiviral Effect of Natural Compounds on Porcine Epidemic Diarrhea Coronavirus

“…Our results using an alamarBlue assay support the use of this viability dye to estimate the degree of CPE associated to PEDV infection in Vero cells, allowing the evaluation of the anti-PEDV activity of minimally cytotoxic concentrations of compounds as has already been described for other viruses causing discernible CPE (39,40). The results obtained using this assay confirm the existence of a significant anti-CPE effect of formic acid which could not be previously observed by using an inverted optical microscope.…”

Inactivation of Severe Acute Respiratory Coronavirus Virus 2 (SARS-CoV-2) and Diverse RNA and DNA Viruses on Three-Dimensionally Printed Surgical Mask Materials

“…Cells were maintained in media as previously described. 28,[30][31][32] The VHA supplemental surgical face mask 8 being developed is 3D printed using Multi-Jet Fusion (MJF) technology and a powder-based polyamide-12 (PA12) material (HP 3D HR CB PA 12 -Hewlett-Packard, Palo Alto, CA). The surgical face mask incorporates a removable filter (not tested in this study).…”

Synthesis of Camphecene and Cytisine Conjugates Using Click Chemistry Methodology and Study of Their Antiviral Activity

“…The key characteristic of biological activity for the studied compounds is therapeutic index or selectivity index (SI). Compounds with therapeutic index higher than 8 are considered as showing the studied activity . The analysis of obtained biological data allows us to draw conclusions on the relationship between the structures of the prepared compounds and their activities.…”

“…Compounds with therapeutic index higher than 8 are considered as showing the studied activity. [27] The analysis of obtained biological data allows us to draw conclusions on the relationship between the structures of the prepared compounds and their activities. It should be noted that all the described compounds are nontoxic under studied conditions.…”