Generation of recombinant vaccinia virus and analysis of virus-induced cell death

Summary Vaccinia virus is a large double-stranded DNA virus that is widely used to express foreign genes from different origins. We generated recombinant vaccinia virus that expresses a viral inhibitor to examine its effect on virus-induced necroptosis. We provide a detailed protocol to describe the generation of recombinant vaccinia virus, validation of protein expression, and determination of necroptosis using live cell imaging. This approach can be adapted to examine the effect of other cell death regulators on virus-induced cell death. For complete details on the use and execution of this protocol, please refer to Liu et al. (2021).
Graphical abstract
Highlights • Construction of targeting vectors for generation of recombinant vaccinia virus • Selection of recombinant vaccinia virus based on plaque size • Amplification of recombinant vaccinia virus • Validation of target protein expression and examine the effect on virus-induced cell death
Vaccinia virus is a large double-stranded DNA virus that is widely used to express foreign genes from different origins. We generated recombinant vaccinia virus that expresses a viral inhibitor to examine its effect on virus-induced necroptosis. We provide a detailed protocol to describe the generation of recombinant vaccinia virus, validation of protein expression, and determination of necroptosis using live cell imaging. This approach can be adapted to examine the effect of other cell death regulators on virus-induced cell death.