The CMV MIE transcript was present in the BJAB-B1 and P3HR-1 cells superinfected with CMV at all time points as shown by the real-time PCR data (Figures 1A and 2A, respectively). CMV copy standards were run for calculation of the standard curve. The CMV standards (5 × 10⁵, 5 × 10³, and 5 × 10¹) for the BJAB-B1 cells are shown (Figure 1C) and the standards for the P3HR-1 cells were not shown. Agarose gel analysis was run only with the BJAB-B1 cells (Figure 1D) and the greatest amount of transcript was observed at 24 hours post CMV superinfection with a decline in the latter time points. BJAB-B1 and P3HR-1 cells that were exposed to UV-irradiated CMV had no presence of the CMV MIE transcript, indicating that the virus was inactivated (Figures 1B and 2B, respectively). The uninfected cell lines were negative for the CMV MIE transcript and show that the CMV primers do not cross-react with EBV (data not shown). Table 1 (BJAB-B1) and Table 2 (P3HR-1) show the quantitative analyses calculated from the standard curve. The calculated starting quantity (SQ) values given in both tables suggest that these cell lines, although they are susceptible to CMV, do not show a high amount of infection. Mock RT-PCRs were performed in which PCR grade water was substituted for the RT enzyme. These mock reactions served as a check for DNA contamination and the mock RT-PCRs of the respective experimental groups were negative (data not shown).

Immunofluorescent staining for the p52 antigen was performed to further show the susceptibilities of CMV in the BJAB-B1 and P3HR-1 cell lines. The p52 gene expresses a DNA binding protein that is present in the nuclei of infected cells beginning in the early phase of infection through to the late phase. All time points for both cell lines (BJAB-B1 and P3HR-1) were positive for p52 antigen and a representative slide is shown at 24 hours post CMV superinfection (Figure 3A and 4A, respectively). All BJAB-B1 and P3HR-1 cells exposed to UV-irradiated CMV were consistently negative for the p52 antigen indicating sufficient CMV inactivation (Figures 3B and 4B, respectively). The uninfected cells from both cell lines were also negative showing no cross-reactivities with CMV (data not shown). Flow cytometric analysis was performed only with the BJAB-B1 cells. Data from the flow experiments were able to give a quantitative analysis by analyzing the fluorescence of the individual cells. Positive cells were determined by subtracting the background fluorescence of the uninfected samples from either the superinfected cells or cells exposed to inactivated virus. CMV p52 antigen expression (575 cells) was observed beginning at 72 hours compared to a background staining of 77 cells that were exposed to inactivated CMV. Three hundred seventy nine and 150 p52 positive cells were present at 96 and 168 hours, respectively. The UV-irradiated CMV exposed cells had counts of 34 and 76 cells (96 and 168 hours, respectively). Positive counts in the BJAB-B1 cells exposed to inactivated CMV represents non-specific staining. 10,000 cells were collected and analyzed per experimental group. The flow cytometry data is summarized in Table 3. The amount of CMV positive BJAB-B1 cells, as also observed in the real-time data, show a decrease with respect to time. Both the transcript data as well as the p52 antigen expression data show that the BJAB-B1 cells are susceptible to a CMV superinfection and there was not a high amount of infection. Qualitative analyses of the immunofluorescent staining in BJAB-B1 and P3HR-1 cells also show that a low amount of infection was occurring.

A CMV abortive infection was not determined for the P3HR-1 cells. In order to determine if CMV was undergoing an abortive or replicative infection, separate CMV superinfection experiments were performed. Cells were washed after 5 hours incubation with CMV and fresh media was added. The CMV infections were carried out to 10 days. Supernatants were taken immediately after washing to show absence of unattached CMV. Aliquots were taken at 24 and 48 hours for immunofluorescence analysis to confirm CMV infection (data not shown). After 10 days the cells were vortexed and supernatant was collected and placed into cultures of MRC-5 fibroblasts. MRC-5 infections were carried out to 10 days. No CPE was observed in any of the fibroblast infections, suggesting that an abortive infection was occurring in the BJAB-B1 cells.

This project has shown that the BJAB-B1 and P3HR-1 cells were susceptible to a CMV superinfection by detecting viral transcript and protein. The effect on EBV latency was next examined. The BZLF1 gene product, Zebra (Zta) is an indicator for EBV reactivation. PMA and ionomycin (1 μg/ml) non-specifically reactivates EBV and was used as a positive control for BZLF1 transcript expression in the cell lines. The BJAB-B1 and P3HR-1 cells were incubated with PMA/ionomycin (1 μg/ml) (Figures 5A and 6A, respectively). Presence of the BZLF1 transcript in the superinfected P3HR-1 cells was observed beginning at 24 hours post superinfection, decreased at 48 hours and then was observed to increase after 72 hours (Figure 6B). Detection of BZLF1 in the P3HR-1 cells was performed by real-time PCR only. BZLF1 transcript in the BJAB-B1 cells was first observed at 48 hours post CMV superinfection and was expressed through to 96 hours, as shown in the real-time PCR data (Figure 5B). The amount of BZLF1 transcript expression in the BJAB-B1 cells started to decrease after 48 hours. Agarose gel analysis confirms the real-time PCR data and shows that the PCR was specific (Figure 6D). Due to the relatively low amount of the BZLF1 PCR product in the real-time data and gel analysis, it was expected that the immunofluorescence staining would be minimal. Immunofluorescence analysis was performed only with the BJAB-B1 cells and the experimental group that was superinfected with CMV was observed to have expression of the BZLF1 protein at 168 hours (Figure 7A). BJAB-B1 cells stimulated with PMA/ionomycin were used as a positive IF control (Figure 7B). Uninfected and UV-irradiated CMV treated cells showed no expression of the BZLF1 transcript or protein indicating no EBV reactivation (data not shown). Mock RT-PCRs were not performed for the BZLF1 PCR reactions because the primers recognize mRNA.

Amplification of the housekeeping gene G3PDH served as a control for RNA integrity. The product was analyzed by agarose (2%) gel electrophoresis and stained with ethidium bromide. This transcript was present at all time points among all experimental groups for both cell lines (data not shown).

P3HR-1 (ATCC) and BJAB-B1 cells (generously donated by Dr. George Miller) are EBV latently infected B-cells kept in RPMI 1640 medium (supplemented with 10% FBS and an antibiotic-mycotic solution) (Gibco, Invitrogen Corp., Carlsbad, CA). The AD169 laboratory strain of CMV was from the clinical virology laboratory (Tampa General Hospital, Tampa, FL), and used for the superinfections. CMV was grown and harvested from MRC-5 fibroblast cultures (Biowhittaker, Walkersville, MD) and concentrated by ultra-centrifugation (28000 g for 60 minutes at 4°C). The CMV titer was determined using the shell vial assay. Briefly, this is an immunofluorescence assay in which 1:10 dilutions of the virus were prepared in shell vials and incubated at 37°C overnight. The MRC-5 cells in the shell vials were fixed and permeabilized for 15 minutes in methanol:acetone (1:1) and then washed in PBS (Bartels Inc., Intracel Corp., Frederick, MD, reconstituted in 1 liter of Millipore water, pH 7.0–7.6). The cells were stained with a FITC labelled antibody directed against the major immediate early antigen (MIE) (Chemicon, Temecula, CA) and visualized using a fluorescent microscope. Each green (positive) cell was counted as one viral particle.

500,000 cells were counted using the Cell-DYN 4000 (Abbott Systems, Abbott Park, IL) and placed in a 24-well culture plate with RPMI 1640 medium. The P3HR-1 and BJAB-B1 cells were infected at a multiplicity of infection (MOI) of 20 and were either uninfected, infected, or exposed to UV-irradiated virus. CMV was exposed to UVC light for 2 hours in our biological safety cabinet. The experiments were carried out for 7 days and samples were taken at 24, 48, 72, 96, and 168 hours. Time points for the P3HR-1 cells were the same except for an additional time point at 120 hours. At each time point, cells were analyzed by immunofluorescence and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The P3HR-1 cells were not subjected to flow cytometric analysis. Superinfections were repeated three times and data shown are representative experiments.

Separate superinfection experiments of the BJAB-B1 cells were set up and were incubated with CMV for 5 hours to allow for viral attachment. The cells were then washed to remove any unattached virus and fresh medium was added. The CMV superinfections were carried out to 10 days. The cultures were vortexed, supernatant was collected, and added to cultures of MRC-5 fibroblasts. Supernatant fluid was collected immediately after infection and washing to establish that any unattached CMV was not present. MRC-5 fibroblasts were examined for CPE.

Aliquots of 100 μl (approximately 50,000 cells) from each experimental group were subjected to a cytospin and fixed and permeabilized similarly to the shell vial assay. The anti-p52 and anti-BZLF1 antibodies (Dako Corporation, Carpinteria, CA) were prepared according to package inserts. Evan's Blue was added to the final p52 solution. The p52 antibody was directly labelled with FITC (fluorescein isothiocyanate) and did not require a secondary antibody step. The BZLF1 antibody is unlabelled and required a secondary goat anti-mouse antibody labelled with FITC. The secondary antibody was diluted 1:200 in PBS and Evan's Blue was added. The primary and secondary antibodies were incubated in a humidified chamber at 37°C for 30 minutes. The slides were washed in PBS after each incubation step, prepared with mounting media, and visualized under a fluorescent microscope.

Aliquots of 300 μl (approximately 150,000 cells) were placed in 5-ml Falcon tubes and washed in PBS. The cells were fixed and permeabilized with pre-made solutions from Caltag Laboratories (Burlingame, CA) (Fix & Perm) according to kit instructions. During the permeabilization step, FITC-labelled antibody (p52) was added at a 1:5 dilution. Cells were washed in PBS and then 750 μl of paraformaldehyde was added and read on the FACScalibur flow cytometer (Becton Dickinson, Los Angeles, CA). Fluorescence parameters were set with regards to infected and uninfected samples. CMV infected and uninfected MRC-5 fibroblasts were used as positive controls for viable CMV and antibody staining. 10,000 BJAB-B1 cells were first separated according to size and granularity (forward vs. side scatter) on a dot plot. Viable cells were then selected and gated for further analysis on a histogram for FL1 (FITC, p52 antigen) versus cell counts. The final histogram analyses involved subtracting background of the corresponding mock-infected cells from the BJAB-B1 cells superinfected with CMV or UV-irradiated CMV. Analysis was performed using the CellQuest software (Becton Dickinson, Los Angeles, CA). The P3HR-1 cells were not analysed by flow cytometry.

The nucleic acid isolation was performed on an automated extractor from the Organon Tekinika Corporation (Boxtel, Netherlands) and the extractions were performed according to kit instructions modified from the method by Boom et al. 78. All reagents used for extractions were from the company. Aliquots of 400 μl (approximately 200,000 cells) were used per experimental time point. The final result was approximately 50 μl of nucleic acid in elution buffer. The nucleic acid samples were then subjected to DNase treatment and RNA clean up with Ambion's (Austin, TX) DNA-free kit following their provided protocol.

Using Ribogreen dye and RNA quantitation standards from Molecular Probes (Eugene, OR), the amount of RNA in the experimental samples were calculated. Briefly, an aliquot of the RNA samples were diluted 1:100 in TE buffer. RNA standards were prepared (4000, 2000, 1000, 500, 250, and 125 ng). The Ribogreen dye was diluted 1:200 in TE buffer and 20 μl of the diluted RNA (standard or sample) was mixed with 20 μl of the diluted Ribogreen dye. Samples and standards were placed in a microtiter plate and read on the CytoFluor reader from Applied Biosystems (Foster City, CA) (excitation 485 nm/20 nm and emission 530 nm/25 nm). The standard curve and sample quantitations were determined using an Excel spreadsheet.

RNA for cDNA production was performed using Promega's (Madison, WI) random hexamers, RNAsin, RT buffer, and RT enzyme. For each sample, the RNA (2 μg) was initially incubated with the random hexamers (0.5 μg) and enough PCR grade water to bring the volume up to 20 μl. Incubation of the RNA and random hexamers was performed at 95°C for 5 minutes and then held at 4°C. The RT mix included 5.6 μl of 5X RT buffer, 0.02 mM of dNTPs (with dTTP, not dUTP, Perkin Elmer, Foster City, CA), 14.22 units of AMV RT enzyme, and 20 units of RNAsin per reaction. A total of 9.68 μl of the RT mix was added to the RNA samples (with the random hexamers) and then subjected to RT cycle conditions (42°C for 45 minutes, 95°C for 5 minutes and a 4°C hold). All incubations were done on BioRad's iCycler (Richmond, CA). Mock RTs were set up in which PCR grade water was substituted for the RT enzyme as a check for DNA contamination.

The PCR mix per reaction included 10 μl SYBR Green buffer, 1.5 mM MgCl₂, 1 μM dNTP mix (with dUTP), 0.5 μl CMV primer mix, 2.5 units AmpliTaq gold, 0.05 units of uracil-N-glycosylase (UNG, Epicentre, Madison, WI) that had been diluted 1:10 in UNG buffer, and molecular grade water to bring the volume up to 45 μl with the addition of 5 μl of cDNA or standard. The PCR reagents were obtained from Perkin Elmer. The CMV primers, from Synthetic Genetics, Inc. (San Diego, CA), amplify the MIE gene. The BZLF1 primers recognize only mRNA and the sequences are 5'-TTCCACAGCCTGCACCAGTG-3' and 5'-GGCAGCAGCCACCTCACGGT-3' 79. Primers were generated by Sigma Genosys (The Woodlands, TX). The CMV PCR was quantitative and the BZLF1 PCR was qualitative. The PCR consisted of 2 pre-incubation steps, 37°C/15 minutes and 95°C/10 minutes (for UNG activation and UNG denaturation/Taq activation). The cycles for PCR were 95°C/30 seconds, 55°C/30 seconds, and 72°C/30 seconds for 35 cycles. CMV DNA copy standards (Synthetic Genetics, Inc.) were used to set up a standard curve. Standards were 500000, 5000, and 50 CMV DNA copies. Dilutions were set up using PCR grade water. The software (Bio-Rad, Richmond, CA) calculated the standard curve as well as determined the amount of CMV DNA in the superinfected samples. Agarose (2%) gel electrophoresis was performed in order to determine the specificity of the PCR reactions in the BJAB-B1 cells only. Primers and positive control for the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene were from Clontech (BD Biosciences Clontech, Palo Alto, CA). PCR recipe and conditions were run according to the included protocol. G3PDH transcripts from both cell lines were analyzed on a 2% agarose gel only.

The instrument camera took numerous fluorescence readings during the annealing stages and displayed the average reading for that particular cycle. At the end of the PCR cycling, the base line fluorescence was calculated and all the samples were normalized to zero. The threshold level (C_T) was set at a point when the standards or positive control were in its respective exponential phase of amplification. The C_T value is the cycle number in which a sample's fluorescence crosses the set threshold level, and therefore is called positive. During quantitative analysis, the standard curve was calculated and the sample concentrations were derived from the curve. All calculations were performed with the included software.