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. 2014 Aug 13;9(8):e105081. doi: 10.1371/journal.pone.0105081

Figure 4. Production, protein expression, and replication kinetics of NP-NES3 mutant viruses.

Figure 4

(A) Wild-type and NP-NES3 mutant (Φ1, Φ2, Φ3, and Φ4) viruses were produced by reverse genetics in co-cultures of HEK-293T and MDCK cells. Virus titers in the cell supernatant were calculated in a plaque assay on MDCK cells. Data are expressed as the mean (± SD) PFU/ml from triplicate assays. (B) Comparison of viral protein expression in the producer cells in (A). The cells were collected, lysed, and subjected to Western blotting with an anti-WSN Ab and an anti-actin MAb. The NP, HA, M1, and M2 proteins and molecular weight markers are shown. (C) Replication kinetics were monitored by infecting MDCK cells with equal amounts of each virus (MOI = 0.001). Supernatants containing viruses were collected at the indicated times post-infection and tested in a plaque titration assay. Data are expressed as the mean (± SD) PFU/ml from triplicate titrations.