PFU virus titration

To evaluate virus replication in vivo, tumor was collected 3 days after virus injection. Viral titers were determined by plaque assays using 100% confluent, either BHK cells (for SFV viruses) or HOS cells (for VVs) in a 6-well plate. Tumors were lysed to extract virus particles, and a 10-fold dilution series of viruses was added to the cell monolayer and incubated for 1 h. Then the monolayer was washed with PBS and overlaid with DMEM containing 0.8% low-melting agarose (Invitrogen). After 72 h incubation, the cells were stained with 1% crystal violet (Sigma, Munich, Germany), and visible plaques were counted. The progeny virus titer from tumor lysate was normalized to tumor weight and represented as PFU/gram of tumor.