A two-step culture method utilizing secreted luciferase recombinant virus for detection of anti-cytomegalovirus...

Quantification of human cytomegalovirus (HCMV) replication by plaque assay reflects viral infectivity but has several drawbacks. Recombinant virus expressing reporter genes can facilitate quantification of HCMV replication. In this study, a recombinant virus, Towne-BAC(dTT)-MetLuc, was constructed and the secretable Metridia luciferase (MetLuc) gene inserted into it under UL146 promoter. In addition, the UL130 gene was repaired to allow growth of the recombinant virus in both fibroblasts and epithelial cells. As predicted, luciferase activity was secreted into the culture medium and correlated with virus replication in infected fibroblasts and epithelial cells. Furthermore, secreted luciferase activity was correlated with the size of the recombinant virus inoculum with a dynamic range of 3 logs. This recombinant virus was used in a two-step culture protocol for detection of the anti-HCMV activity of compounds; that is, the supernatant of a first-step culture with anti-viral compounds was collected and inoculated into uninfected cells to create a second-step culture. Although secreted luciferase activity leaked in the first-step culture supernatant in the presence of some antiviral compounds, luciferase activity in the second-step culture supernatant reflected the virus yield in the first-step culture. Therefore, comparison of luciferase activity in the first- and second-step cultures indicated the anti-viral activity of the compounds. This two-step culture protocol facilitates screening of anti-viral compounds.