Journal: Journal of Virology

Article Title: Novel replication-competent reporter-expressing Rift Valley fever viruses for molecular studies

doi: 10.1128/jvi.01782-24

Figure Lengend Snippet: Multicycle growth kinetics. Vero E6 cells (12-well plates, triplicates) were infected (MOI of 0.01) with rRVFV WT (rWT) or with recombinant viruses expressing Nluc ( A and B ) or Venus ( C and D ). At the indicated 24, 48, and 72 h p.i., viral titers in culture supernatants were evaluated by plaque assay (PFU/mL), and the same rWT representation was used in A and D ( A and D ). Comparison of the replication curves of viruses expressing Nluc or Venus versus rWT at different post-infection times revealed statistically significant differences (Dunnett’s method). * P < 0.05; ** P < 0.0021; *** P < 0.0002; **** P < 0.0001. The data represent the means ± SDs of triplicate samples. At the same time, post-infection Nluc activity was evaluated (RLU: relative light units) ( B ), and Venus expression was analyzed using a fluorescence microscope ( C ). Representative images are shown. Scale bars, 100 µm. The dotted line indicates the limit of detection (100 FFU/mL) of the assay.

Article Snippet: The cells were analyzed by flow cytometry by gating on green (Venus) and red fluorescence (mAb anti-N 2B1 Alexa 647 conjugated) ( ) using a CyFlow Cube 8 cytometer (Sysmex).

Techniques: Infection, Recombinant, Expressing, Plaque Assay, Comparison, Activity Assay, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: Novel replication-competent reporter-expressing Rift Valley fever viruses for molecular studies

doi: 10.1128/jvi.01782-24

Figure Lengend Snippet: Plaque assays. Vero E6 cells (10 6 cells/well, 6-well plate format) were infected with rRVFV WT (rWT) or with recombinant viruses expressing Venus (top) or Nluc (bottom) and incubated at 37°C for 3 days. Plaques were evaluated by immunostaining with the mAb anti-N (2B1), the goat pAb anti-GFP (top), or the mAb anti-Nluc (bottom). Immunostaining of rWT was performed with the mAb anti-N (2B1) or a combination of the goat pAb anti-GFP and the mAb anti-Nluc.

Article Snippet: The cells were analyzed by flow cytometry by gating on green (Venus) and red fluorescence (mAb anti-N 2B1 Alexa 647 conjugated) ( ) using a CyFlow Cube 8 cytometer (Sysmex).

Techniques: Infection, Recombinant, Expressing, Incubation, Immunostaining

Journal: Journal of Virology

Article Title: Novel replication-competent reporter-expressing Rift Valley fever viruses for molecular studies

doi: 10.1128/jvi.01782-24

Figure Lengend Snippet: Nluc activity in tissue culture supernatant versus cell extracts and Venus expression by flow cytometry analysis. Vero E6 cells (24-well plates, triplicates) were infected (MOI of 0.01) with recombinant viruses expressing Nluc. At 48 h p.i., viral titers in culture supernatants were evaluated by plaque assay (PFU/mL). The differences between the groups were calculated using one-way ANOVA with the Tukey method. * P < 0.05; *** P < 0.0002; **** P < 0.0001 ( A ). Nluc activity in tissue culture supernatants (TCSs) and cell extracts was measured. RLU: relative light units. The differences between the groups were calculated using two-way ANOVA with the Tukey method. * P < 0.05; *** P < 0.0002; **** P < 0.0001. ( B ). The data represent the means ± SDs of triplicate samples. ( C ) Flow cytometry analysis. Vero E6 cells were infected (MOI of 0.01) with recombinant viruses expressing Venus. At 24 h p.i., the cells were harvested from the culture and stained with a mAb against N conjugated with Alexa 700. The cells were analyzed by flow cytometry by gating on green (Venus) and red fluorescence (mAb anti-N 2B1) and the percentage of N +, Venus+, or Venus+/ N + is indicated.

Article Snippet: The cells were analyzed by flow cytometry by gating on green (Venus) and red fluorescence (mAb anti-N 2B1 Alexa 647 conjugated) ( ) using a CyFlow Cube 8 cytometer (Sysmex).

Techniques: Activity Assay, Expressing, Flow Cytometry, Infection, Recombinant, Plaque Assay, Staining, Fluorescence

Journal: Journal of Virology

Article Title: Novel replication-competent reporter-expressing Rift Valley fever viruses for molecular studies

doi: 10.1128/jvi.01782-24

Figure Lengend Snippet: Multicycle growth kinetics in insect cells. C6/36 mosquito cells (T25 flasks, triplicates) were infected (MOI of 0.01) with rRVFV WT (rWT) or with recombinant viruses expressing Nluc ( A and B ) or Venus ( C and D ). At the indicated days p.i. (3, 5, and 7), viral titers in culture supernatants were evaluated by plaque assay (PFU/mL), and the same rWT representation was used in panels A and C. Dunnett’s method was used to statistically compare viruses expressing Nluc or Venus versus rWT. * P < 0.05; ** P < 0.0021; *** P < 0.0002; **** P < 0.0001 ( A and C ). The data represent the means ± SDs of triplicate samples. At the same time, p.i. Nluc activity (RLU: relative light units) was measured in ( B ), and Venus expression was evaluated using a fluorescence microscope ( D ). Representative images are shown. Scale bars, 100 µm. The dotted line indicates the limit of detection (100 FFU/mL) of the assay.

Article Snippet: The cells were analyzed by flow cytometry by gating on green (Venus) and red fluorescence (mAb anti-N 2B1 Alexa 647 conjugated) ( ) using a CyFlow Cube 8 cytometer (Sysmex).

Techniques: Infection, Recombinant, Expressing, Plaque Assay, Activity Assay, Fluorescence, Microscopy