Figure 2.

Characterization of the rescued recombinant viruses in vitro. (a) Western blot analysis of cell lysate of Vero cell infected with rNDV-R2BPCVcap-TMCT viruses. (i) Position of NDV specific proteins, namely L, HN, F, NP, P and M (left) and (ii) capsid protein of PCV2 and PCV2-TMCT of approximately 27-kDa (right), along with the marker proteins as indicated. Lane M: Precision plus protein standard dual color marker (Bio-Rad, Hercules, CA, USA). (b) Demonstration of replication of rescued recombinant rNDV-R2B-PCVcap-TMCT virus in Vero cells by fluorescent microscopy (×40) (A) Cells stained with DAPI; (B) Cells stained with anti-NDV antibody and secondary antibody (anti-chicken IgG labelled with Alexa Fluor 568); (C) Cells stained with anti-PCV antibody and secondary antibody (anti-rabbit FITC) (D) Merger of all three frames. (c) Multistep growth kinetics of recombinant viruses in Vero cells. Monolayer of Vero cells were infected at 0.01 MOI for rNDV-R2B, rNDV-R2B-PCVcap-TMCT and rNDV-R2B-PCVcap independently. At every 12-h interval until 72 h, the viral titres were determined by limiting dilution assay and calculated as TCID₅₀ by Reed and Muench method.