Productive HCMV infection in SGHPL-4 and HFF cells. (A-E) HCMV IE protein expression in human cytotrophoblasts. SGHPL-4 (□) or HFF (■) cells were infected with HCMV strain RVdlMwt-GFP  at a MOI of 2.5 PFU per cell and incubated at 37°C for 1, 4, 8, 12, 24, 48, 72, 96,120 or 144 h. At the indicated times, cells were fixed and stained for HCMV IE 1/2 and DAPI (Molecular Probes) and visualized on a Zeiss Axio Plan II microscope (Thornwood, NY). To determine the number of HCMV-infected cells, three fields of view were considered and the percent of IE-positive cells was calculated as: (average number of IE-stained cells/average number of DAPI-stained cells) × 100. The graph demonstrates an increase in the percentage of SGHPL-4 and HFF cells expressing IE 1/2 over a period of time. Representative images of HCMV IE 1/2 are depicted at 8 h p.i in (B) CTBs and (C) HFFs and at 120 h p.i. in (D) CTBs and (E) HFFs; IE 1/2-red, DAPI-blue, overlaid-purple. (F) Infected CTBs produce and release infectious virions. SGHPL-4 or HFF cells were inoculated with HCMV at a MOI of 0.1 PFU per cell. At the indicated times, cells or culture medium were harvested, freeze-thawed three times, and titers of infectious virus in SGHPL-4 cell lysates (○) and supernatants (△) and HFF cell lysates (●) and supernatants (▲) were determined by a microtiter plaque assay on HFFs . Infectious progeny virus was detected in both cell lysates and culture supernatants of SGHPL-4 and HFF cells. The dashed line represents the lower limit of detection of the plaque assay used to measure viral titers.