Journal: Scientific Reports

Article Title: ROCK1/MLC2 inhibition induces decay of viral mRNA in BPXV infected cells

doi: 10.1038/s41598-022-21610-9

Figure Lengend Snippet: ROCK1 inhibition impairs BPXV-induced cell contraction and viral protein synthesis. ( a ) Effect of Thiazovivin on MLC2 phosphorylation (activation). Vero cells were either mock-infected or infected with BPXV at MOI of 5. Thiazovivin or vehicle controls were added at 4 hpi. Cell lysates were prepared at 9 hpi and subjected for detection of the p-MLC2 levels in Western blot analysis (ai). The histogram (aii) shows the band intensity of the protein. The blots were quantified by densitometry (ImageJ) and the data are presented as mean with SD. n = 3 independent experiments. See Supplementary Fig. for full blots. ( b ) Effect of Thiazovivin on BPXV induced cell contraction and levels of viral proteins. HeLa cells were grown in chamber slides and infected with BPXV at an MOI of 5 for 1 h. Thiazovivin was applied at 4 hpi. At 15 hpi, BPXV (FITC) proteins were probed by immunofluorescence assay. Cell morphology and level of viral proteins of Thiazovivin-treated or untreated cells is shown (bi). The histogram shows the relative reduction in cell size (bii) and relative levels of BPXV proteins (biii) in Thiazovivin treated or untreated cells. The area (n = 50 cells) and the intensity of viral proteins (n = 50 cells) were quantified by ImageJ. The data are presented as mean with SD. Pair-wise statistical comparisons were performed using Student's t-test (***P < 0.001; **P < 0.001).

Article Snippet: African green monkey kidney (Vero) and HeLa cells were received from National Centre for Cell Science (NCCS), Pune, India.

Techniques: Inhibition, Phospho-proteomics, Activation Assay, Infection, Western Blot, Immunofluorescence

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Extinction of Zika Virus and Usutu Virus by Lethal Mutagenesis Reveals Different Patterns of Sensitivity to Three Mutagenic Drugs

doi: 10.1128/AAC.00380-18

Figure Lengend Snippet: Favipiravir, ribavirin, and 5-fluorouracil inhibit ZIKV replication. (A and B) ZIKV titers obtained after infection of confluent Vero cell monolayers in the absence (drug concentration of 0 in the abscissa) or presence of each drug at the concentrations indicated. Cells were infected at an MOI of 0.01 and the supernatants collected at 32 h postinfection for titration. (A) ZIKV of Asian lineage (strain PRVABC59); (B) ZIKV African lineage (strain MR 766). Statistically significant differences are highlighted with asterisks (**, P < 0.01; ***, P < 0.001; 2-way ANOVA). Every value represents the average of the results from at least three biological replicas (± standard error of the mean [SEM]). Decitabine (DEC) values are shown as black bars, 5-fluorouracil (FU) as dark gray, ribavirin (RBV) as light gray, and favipiravir (FAV) as white bars. (C) Replication kinetics of ZIKV (Asian lineage, strain PRVABC59) in the presence of different concentrations of decitabine (DEC). Every value represents the average from virus titer determinations of at least three independent biological replicas (± SEM). Each symbol illustrates a different concentration of decitabine used in the assay, as follows: diamond, 200 μM; inverted triangle, 400 μM; black circle, 800 μM. (D to F) Replication kinetics of ZIKV (Asian lineage) in the presence of FU (dark-gray squares), RBV (light-gray triangles), and FAV (white inverted triangles) are compared to those in untreated infected cultures (white circles, dashed lines). Every value is obtained from the analysis of at least three biological replicas (± SEM). Each panel depicts viral replication kinetics in the presence of inhibitors at different concentrations, 200 μM (D), 400 μM (E), or 800 μM (F).

Article Snippet: We used African green monkey kidney epithelial cells (kindly provided by Sylvie Lecollinet, ANSES, France), namely, Vero cells, for ZIKV and USUV propagation, titration, and viral infections in the presence or absence of mutagenic compounds.

Techniques: Infection, Concentration Assay, Titration, Virus

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Extinction of Zika Virus and Usutu Virus by Lethal Mutagenesis Reveals Different Patterns of Sensitivity to Three Mutagenic Drugs

doi: 10.1128/AAC.00380-18

Figure Lengend Snippet: Mutagenic nucleosides inhibit USUV replication in Vero cells. (A) Single-cycle replication kinetics of USUV treated with FU (dark-gray squares), RBV (light-gray triangles), or FAV (white inverted triangles) at 800 μM each, compared to that of untreated virus. Vero cells were inoculated at an MOI of 5 TCID 50 per cell. Cellular supernatants were collected at different time points after infection. Every value in the graph is the average of the results from at least three biological replicas (± SEM). (B and C) USUV titers obtained after multiple rounds of virus replication in Vero cells in the absence (0) or presence of increasing concentrations of each drug. To ensure that the virus titers are the result of several rounds of replication, we employed a low MOI to infect the cells (0.1 or 0.01). Statistically significant differences are represented (**, P < 0.01; ***, P < 0.001; 2-way ANOVA). Each value in the graph is calculated as the average virus titer obtained from at least three independent biological replicates (± SEM). Virus titers obtained in infected cells treated with DEC are represented as black bars, titers in FU-treated cells are in dark gray, RBV cells are in light gray, and FAV cells are in white. (B) Vero cell monolayers were infected at an MOI of 0.1 and supernatants collected for titration at 24 h postinfection. (C) Supernatants of infected cells were collected for virus titer analysis at 48 h postinfection. To ensure that virus titers were obtained during the exponential-growth phase, we used an MOI of 0.01 instead of 0.1.

Article Snippet: We used African green monkey kidney epithelial cells (kindly provided by Sylvie Lecollinet, ANSES, France), namely, Vero cells, for ZIKV and USUV propagation, titration, and viral infections in the presence or absence of mutagenic compounds.

Techniques: Virus, Infection, Titration