Molluscum contagiosum virus (MCV), a poxvirus pathogenic for humans, replicates 
well in human skin in vivo, but not in vitro in standard monolayer cell 
cultures. In order to determine the nature of the replication deficiency in 
vitro, the MCV infection process in standard culture has to be studied step by 
step. The method described in this chapter uses luciferase and GFP reporter 
constructs to measure poxviral mRNA transcription activity in cells in standard 
culture infected with known quantities of MCV or vaccinia virus. Briefly, MCV 
isolated from human tissue specimen is quantitated by PCR and used to infect 
human HEK293 cells, selected for ease of transfection. The cells are 
subsequently transfected with a reporter plasmid encoding firefly luciferase 
gene under the control of a synthetic early/late poxviral promoter and a control 
plasmid encoding a renilla luciferase reporter under the control of a eukaryotic 
promoter. After 16 h, cells are harvested and tested for expression of 
luciferase. MCV genome units are quantitated by PCR targeting a genome area 
conserved between MCV and vaccinia virus. Using a GFP reporter plasmid, this 
method can be further used to infect a series of epithelial and fibroblast-type 
cell lines of human and animal origin to microscopically visualize MCV-infected 
cells, to assess late promoter activation, and, using these parameters, to 
optimize MCV infectivity and gene expression in more complex eukaryotic cell 
culture models.