Fig. 4

Analysis of HCMV progeny from THP-1 latency and reactivation models: pp65 antigen immunofluorescence pattern and viral yield titration. a and d MRC5 fibroblasts were infected with cell culture medium derived from THP-1 monocytes (“pp65-positive MRC5 from THP-1 latency model”) and THP-1 macrophages (“pp65-positive MRC5 from THP-1 reactivation model”) which had been infected with TB40E (a) or Towne (d) HCMV strains at MOI of 0.5 (panels a, a’), 0.25 (panels b, b’) and 0.125 (panels c, c’) for 7 days. At 48 h p.i., MRC5 cells were fixed and labelled with a monoclonal antibody reacting with the viral matrix phosphoprotein pp65. The immunoreaction was revealed by Alexa-Fluor FITC-conjugated goat anti-mouse IgG (panels a, b, c, d: FITC, green nuclei). Cells were counterstained with Evans blue (panels a, b, c, d: red cells) and DAPI (panels a’, b’, c’, d’: blue nuclei). Panels d, d’: uninfected cells. Images were collected using a conventional fluorescence microscopy. Bar: 25 μm. b and e The quantitative evaluation of pp65-positive MRC5 fibroblasts infected with the cell culture medium derived from TB40E (b) or Towne (e) reactivation models. Values were expressed as mean percentages of pp65-positive cells per field (ten randomly selected fields per slide were counted) from two independent experiments; error bars indicate standard deviations. Values were processed using GraphPad Prism 7 software. c and f Virus yields were evaluated by the TCID₅₀ assay from cell culture medium derived from TB40E (c) or Towne (f) reactivation models, as detailed in the “Methods” section. Two independent experiments were performed; error bars in graphs represent standard deviations. Values were processed by the GraphPad Prism 7 software