Figure 3

Functional characterization of recombinant PToV-HE52.7 and PToV-HE52.11 proteins. (A) ANAE in situ staining of rVV plaques produced in BSC40 infected with rVV-HE52.7, rVV-HE52.11 or rVV-HA- (~ 100 PFU per well). After the ANAE assay cell monolayers were stained with crystal violet to visualize all viral plaques. (B) Acetylesterase activity in extracts from BSC40 cells infected with rVV expressing PToV-HEs. Cells infected (MOI 5) with rVV-HE52.7 and rVV-HE52.11 or with the control virus rVV-HA- were harvested at 24 hpi, resuspended in TNE buffer and tested for acetyl esterase activity by incubation with 1 mM p NPA reagent. Background corrected hydrolysis of p NPA (in nmol), measured at 405 nm along a time span (min) is shown in the graph. (C-D) Hemagglutination assays with mouse RBC (0.5%) and 2-fold dilutions of both DFP-treated (+DFP) and untreated (−DFP) rVV-infected cell extracts resuspended in TNE buffer, recorded after incubation at 4 °C (C) and at 37 °C, (D). The negative controls (C-) correspond to wells where RBC were incubated with TNE buffer.