The luciferase reporter can be applied to determine the neutralization capacity of HCMV antibodies and sera as well as to comparative analysis of HSV-1 and HCMV.

<p>(A-C) HCMV TB40/E was incubated with serial dilutions of glycoprotein B (gB) antibody (A) or human sera (B-C) prior to infection. Infection efficiency was determined by immunostaining of viral immediate early antigens at 1 d p.i. and luciferase activity was measured in cell lysates harvested at 2 d p.i. (A) The infection efficiency and luciferase signal obtained without antibody was 71% or 5.3e+7 RLU, respectively. (B-C) Serum A, filled circle; serum B, open circle; serum C, filled triangle. The mean values of at least three independent experiments are shown. ND50: 50% neutralization dose. (D) HSV-1 or HCMV or a mixture of both were preincubated with three human sera of known serostatus before infection of MV9Gs. Luciferase activity was measured in cell lysates at 1 d p.i. Error bars: SEM.</p

HCMV IE antigen expression is required and sufficient to activate the heterologous reporter gene.

<p>(A) MV9G cells were transiently transfected with a control plasmid (pmCherry-C1) or an immediate early antigen expression plasmid. The luciferase activity was measured. (B-C) MV9G cells were transfected with non-targeting (NT) siRNA or siRNA targeting the viral immediate early antigens UL122/123 (IE). After 2 days, cells were infected with HCMV TB40/E (B-C) or HSV-1 (C) and luciferase activity was measured after another 2 days (B, left) or at 1 d p.i. (C). In replicate cultures, the efficiency of the IE siRNA was controlled by immunodetection of immediate early antigens at 1 d p.i. (B, right). The data is normalized to the respective control. (B) The graph summarizes data from three independent experiments. (C) One representative experiment is shown (n = 3). Error bars: SEM.</p

MV9G cells are in principle susceptible to epitheliotropic virus strains.

<p>(A) Indicated cells were infected with different laboratory strains of HCMV at a virus dose yielding approximately 60–70% infected cells in HFF. The infection efficiency was visualized by immunostaining of viral immediately early antigens (red) and the nuclei were counterstained with DAPI (blue). (B) The cells were infected with serial virus dilutions and the fractions of infected cells were determined by immunostaining. The relative tropism of each virus strain compared to the reference cell type HFF is given in percent. To calculate the relative tropism, data from three independent experiments was combined.</p

HCMV replicates efficiently in MV9G cells.

<p>(A) The indicated cells were infected with HCMV TB40/E, fixed after 1 or 5 d p.i. and immunostained for viral antigens pUL44 or MCP (red). Nuclei were counterstained with DAPI (blue). (B) Microscopic images of cells showing cytopathic effects (CPE) were taken at 5 d p.i. using phase contrast microscopy. The arrows indicate nuclear inclusions. (C) Supernatants of TB40/E-infected cell cultures were harvested at 1, 5 or 7 d p.i. and analyzed by serial dilution assay on HFF to determine the titers of infectivity. (D-F) Cells and supernatants of TB40/E-infected cell cultures were harvested at the indicated times. The DNA was extracted and the viral genomes were measured by quantitative RT-PCR. (D), copy number of viral DNA in the cell; (F), copy number of viral DNA released into the supernatant. (E) Supernatants of cultures infected for 5 and 7 days with TB40/E were harvested and titrated in HFF. The genome copy number per ml that was determined in the same supernatants by quantitative RT-PCR was divided by the titer (infectious units per ml) to determine the ratio of genome copies per infectious unit. Error bars: SEM (C, D, F); SD (E).</p

The production of infectious HCMV virions in MV9G cells is probably impaired during the nuclear stage of virion morphogenesis.

<p>(A) MV9G cells and HFF were infected with TB40/E and processed for transmission electron microscopy at 5 d p.i. Top row: The representative overviews show the distribution of nuclear capsids within the nucleoplasm of MV9G and HFF cells. Boxed areas are depicted in a higher magnification in the bottom row. Arrows in the left image point at a second patch of nuclear capsids in the MV9G cell nucleus. cy cytoplasm, nu nucleus. RC replication compartment. Scale bars: 1 μm. Bottom row: Higher magnification of the indicated areas. Three different capsid stages (A, B and C capsids) can be distinguished. Scale bars: 200 nm. (B) MV9G cells and HFF were infected with TB40/E and processed for transmission electron microscopy at 5 d p.i. The area of the cytoplasmic viral assembly complex was examined for virions (arrowheads). Representative images are shown. cy, cytoplasm; MTOC, microtubule organizing center; DB, dense bodies. Scale bars: 500 nm.</p

The luciferase gene accurately reports the HCMV infection efficiency.

<p>MV9G cells were infected with serial dilutions of virus strains TB40/E and TB40/F (A-D) or Towne (C-D). The luciferase activity (RLU, relative light units) was measured and plotted against the dilution of the virus preparation (A, C) or against the fraction of infected cells which was determined by immunodetection of viral immediate early antigens in parallel cultures (B, D). The background signal was determined in uninfected MV9G cells. Each graph shows data from one representative experiment out of four (A-B) or two (C-D). Error bars: SEM.</p

MV9G cells allow focal growth of HCMV and are suitable for drug resistance assays.

<p>(A) MV9G or MeWo cells were co-cultured with infected cells, either HFF or homotypic cells as indicated, for 7 days. Infected cells were detected by immunostaining of viral immediate early antigens and the focus expansion (FE) value (left) was determined by counting the number of cells per focus as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169580#pone.0169580.ref042" target="_blank">42</a>]. The graph integrates data from three independent experiments. Statistical significance was assessed by student’s t-test. Example images of immunostained foci are shown (right). (B-C) MV9G cells were incubated with HFF cultures infected with clinical HCMV isolates for two days. The cultures were exposed to serial dilutions of either GCV (left) or FOS (right) and the luciferase activity was measured. The drug resistance assays were performed in quadruplicates. A sigmoidal dose response curve was fitted onto the normalized inhibition values to determine the effective drug concentration yielding 50% inhibition (EC50). Error bars: SEM.</p