Journal: PLoS Pathogens
Article Title: Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation
doi: 10.1371/journal.ppat.1010191
Figure Lengend Snippet: (A) Multi-cycle replication kinetics: Vero cells in six-well plates were infected at an of MOI of 0.01 PFU/cell with wt RSV; Min A; P16 supernatant from Lineages #2, 3, 4, and 5 of the in vitro stress test; or Min A containing the individual indicated P mutations, and incubated at 32°C (left column) or 37°C (right column). For each virus, duplicate wells were harvested daily by scraping cells into the media followed by vortexing for 30 sec to release cell-associated virus. Clarified supernatants were snap-frozen until titrated by immunoplaque assay at the permissive temperature of 32°C. Titers for Min A, Min A-derived mutants, and wt RSV correspond to the mean of two replicate titrations each of two replicate wells at each timepoint. Due to limited sample size, titers of the P16 viruses correspond to the mean of two replicate titrations of one well at each time point. Day 0 titers correspond to the back titration of the inocula. Note that all viruses were evaluated side-by-side in a single experiment but for clarity are shown in four separate graphs for each temperature, with the specific viruses evaluated in each graph identified in the black boxes at the far right-hand side of Part B, with wt RSV (black) and Min A (red) being repeated in each graph. (B) Plaque characterization : Vero cells in six-well plates were incubated with 250 PFU/well of the indicated virus at 32°C. At day seven pi, plates were fixed with 80% methanol and plaques were stained with a cocktail of three anti-RSV F mAbs and a PE-labeled secondary antibodies as described in Materials and Methods. The plaque area (in μm 2 ) and the level of RSV F expression (expressed as median fluorescence intensity, MFI) were evaluated on an average of 2140 (±1167) plaques per virus. Distributions of the virus plaque area and F MFI were compared for statistical significance using the ANOVA test (* = p≤0.05, ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001).
Article Snippet: The primary antibodies were mouse mAbs against RSV N, P, M2-1 and G proteins (1:1,000, Abcam) and a rabbit polyclonal antibody preparation against GAPDH (1:200, Santa Cruz) as a loading control.
Techniques: Infection, In Vitro, Incubation, Virus, Derivative Assay, Titration, Staining, Labeling, Expressing, Fluorescence
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