QPCR and RT-QPCR assays allow highly sensitive and specific detection of DNA and RNA sequences as well as offering the ability to quantify the target sequence. Specific instruments (ABI Sequence Detection Systems (SDS) 7700/7900) are able to detect fluorescence during QPCR, allowing the ‘real- time’ detection of PCR product accumulation via the hydrolysis of probes labeled with both a fluorescent reporter and a quencher. Quantitative data is derived from a determination of the cycle at which the amplification product signal crosses a pre-set detection threshold (threshold cycle, CT value), which is proportional to the amount of starting material.
3.2 Assay Validation
It is necessary that analytical procedures are appropriately validated to demonstrate that they are suitable for the intended use. The intention of the QPCR (and RT-QPCR) assays described in this thesis is to measure the amount of viral targets present in cell and supernatant harvests. In light of this information, the following validation characteristics have been evaluated for all QPCR and RT-QPCR assays utilised during this project. Assay validation was carried out in accordance with the ICH Topic Q2A and Q2B.
3.2.1 Accuracy and Precision
The accuracy of the assay was assessed by spiking known amounts of BPyV synthetic control into various substances such as water, MDBK DNA, and contaminants expected to be present in the testing products (trypsin and protein). The precision (and repeatability) of the QPCR’s was assessed by carrying out multiple runs with different operators and machines.
3.2.2 Specificity
Specificity of the assay was assessed by testing each QPCR assay in the presence of components most likely to be present. For these QPCR’s DNA from various species was considered the most appropriate. Bovine (MDBK), Human (Human Placental DNA), and Rodent (Chinese Hamster Ovary) DNA were all used to asses specificity. Other polyoma viruses were unavailable during validation to determine specificity.
3.2.3 Quantitation Limit, Linearity and Range
The quantitation limit was assessed by analysing each positive control in replicates of 8, and determining the lowest concentration to show precise and accurate results. Linearity and range was assessed by analysing each standard in triplicate and determining the level that had a suitable level of precision, accuracy and linearity. The r2 data generated refers to the squared coefficient of correlation and should be above 0.98 (recommended by the manufacturers of the TaqMan system). For the slope, an efficiency of 100% would relate to a value of -3.3. An acceptable reaction should have an efficiency between 90% and 110%, which corresponds to a slope between - 3.6 and -3.0.
3.2.4 Primer/Probe Optimisation
The primer and probe concentrations were optimised prior to validation to ensure optimal reaction concentrations. This is achieved by varying the concentrations of firstly the primers, and then the probe.
CHAPTER 4 - Results
What levels of large T mRNA expression are observed with BPyV in comparison to other polyomaviruses such as SV-40, and does infection of rapidly growing cells with BPyV result in eventual disappearance of the virus from the culture system?
What are the levels of large T mRNA prior to the onset of viral replication, and could a more efficient infectivity assay be developed by inoculating material onto cells expressing high levels of the large T antigen? The ability of other polyomavirus to move the infected cell into S-phase has been determined, but it is the same for cells infected with BPyV?
4.1 Validation Results
Primer and probe optimisation studies demonstrated the optimal primer probe combination to be 900:900:225µM (Forward Primer: Reverse Primer: Probe) for all assays. All assays were shown to be precise, specific, accurate and robust (assay sensitivity or linearity consistent using different batches of reagent on different days). The range was determined to be 1x101 to 1x106 copies (or genome equivalents) per reaction for both QPCR’s, and 1x102 to 1x106 copies/reaction for the RT-QPCR. The limit of quantitation for both QPCR’s was determined to be 1x101 copies per reaction, the lowest level at where precision and accuracy where observed. The limit of quantitation of the RT-QPCR’s was determined to be 1x102 copies per reaction. Quantities above 1x106 will still be reported for all assays but it should be noted that these results may not be accurate.
Figure 8 - Example of amplification plots (using large T mRNA primers and probes). Amplification plots for serial dilutions of large T RNA positive controls (tested in triplicate) are shown in green (marked with arrows). The highest concentration (1x108) is shown at the left of the plot, and the lowest concentration (1x102) is shown at the right of the plot. The red line is referred to as the ‘threshold’ and is set as close to the exponential phase of amplification as possible. The red arrows are referred to as the ‘baseline’ and are set to minimize background signals. The point at which each dilution crosses the threshold is plotted in a standard curve (see figure 9) and quantities of unknowns calculated (Source – MSc experimental data).
Figure 9 - Example of a Standard Curve. The data from the amplification plot detailed in figure 8 was plotted in this standard curve. The CT numbers refers to the threshold cycle, which is the cycle that the amplification plot passes through the red threshold line (see figure 8). The CT values of unknown samples are plotted against the curve (as shown with the red x’s) and quantities calculated. The statistical analysis of the slope indicates how linear and therefore quantitative the assay has been. The optimal slope is -3.3, Y-Inter 40 (as this assay was run over 40 cycles) and R2 1.00. Slope values of -3.0 and -3.6, and R2 values above 0.98 are indicative of a linear assay.
4.2 Are there differences between BPyV cultured on cells supplemented with 1 and 10% FCS?
Nairn et al documented in 2003 that the mode of replication for BPyV was unusual when compared to other polyomaviruses. It was observed that viral replication was only detected after 5-8 weeks of continuous culture (with no passaging) on cells doubling at a low rate in media supplemented in 1% Foetal Calf Serum (FCS) (Personal Reference, Nairn et al., 2003). What is
the mechanism of replication, and is the inoculation and subsequent growth of BPyV on cells supplemented with 10% FCS different?
To assess this an 8-week infectivity assay was initiated as detailed in section 2.4 of the Methods and Materials (Eukaryotic Cell Culture). Supernatant and cells were harvested weekly from MDBK cells inoculated with BPyV and cultured in DMEM supplemented with 1% and 10% FCS. A heat inactivated (HI) control (which was inactivated by incubation at 95ºC for 24 hours) was also included in order to show that positive signals in cells were due to infectious virus particles, and not from residual inoculum. During preliminary studies (Personal Reference, Nairn et al., 2003), amplification signals were detected in cells not able to support BPyV viral replication. Inoculation of these cultures with HI inactivated BPyV generated similar results, proving that signals detected were due to residual inoculum, and not true amplification. Supernatants were harvested from flasks and nucleic acid extracted using a Qiagen DNA Mini kit (Section 2.2.2 of Materials and Methods) and tested by Q-PCR with primers and probes (BPyV LargeT-945F, 1016R and 971T) designed to the large T region of the BPyV viral genome. Extracted material was tested along with a synthetic DNA oligonucleotide diluted to represent 101-106 genome equivalents in order to quantitate any viral genomes present. During the first 3 weeks of the infectivity assay BPyV viral genomes were detected in cells supplemented with both 1% and 10% FCS, although signals detected in 1% FCS samples were several logs higher following week 1. From week #’s 4 to 8 significant increases in BPyV viral genomes was only detected in cells grown in 1% FCS, with no viral sequences detected following week 3 in cells grown in 10% FCS (results illustrated in figure 10).
In summary, infection of rapidly growing cells with BPyV (being passaged and fed with media supplemented with 10% FCS) resulted in the eventual disappearance of the virus from the culture system. In contrast it takes 4-5 weeks of continuous culture in cells at a very low doubling rate, only being fed
weekly with media supplemented with 1% FCS and not passaged, before exponential virus replication is detected. This is uncharacteristic among polyomaviruses in which the complete replication cycle takes 48-72 hours. Polyomaviruses only have 4-5 functional genes so rely heavily on cellular mechanism for replication. Therefore BPyV inoculated onto cells that are constantly multiplying (as they would be if grown in media supplemented in 10% FCS and passaged regularly) should be replicating. In this period no viral replication was observed in the negative or HI controls (figure 11).
1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 1.00E+10 Genome equivalent BPyV 1% BPyV 10%
BPyV Infectivity Results (s/n)
wk.1 wk.2 wk.3 wk.4 wk.5 wk.6 wk.7 wk.8
Figure 10 - Comparison of BPyV viral genomes in infected MDBK cells grown in DMEM supplemented with 1% and 10% FCS. Exponential amplification can be observed from wk 4 in MDBK cells infected with BPyV and grown in media supplemented with 1% FCS. No amplification is observed from week # 4 in the 10% cultures. Data shown is from duplicate flasks tested and mean quantities calculated.
1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 1.00E+10 Genome equivalent
Negative Control HI Control BPyV Infectivity Results (s/n)
wk.1 wk.2 wk.3 wk.4 wk.5 wk.6 wk.7 wk.8
Figure 11 - Assessment of BPyV viral genomes (DNA) in negative and heat inactivated (HI) flasks. No amplification is observed in any of the controls. Data shown is from duplicate flasks tested.
4.3 At what stage is large T mRNA expressed?
During the same 8 week period that supernatant was harvested as detailed in section 4.2, cells were also harvested and total RNA extracted (as detailed in section 2.2.3) in an attempt to quantitate BPyV large T mRNA.
Of the early phase antigens that are expressed during BPyV replication, the most critical is the large T antigen, which is thought to be directly responsible in the initiation of viral replication. Extracting RNA from cells harvested each week would indicate when large T mRNA was expressed but also show if there was any correlation between large T mRNA expression and BPyV viral genome replication. Extracted RNA was tested by RT-QPCR with primers and probes (SpliceLargeT-60F, 137R and 86T) designed to the large T region (intron-exon region in an attempt to only detect mRNA). RT-QPCR’s were analysed using a synthetic RNA oligonucleotide in the range of 101 – 106 genome equivalents in order to quantitate mRNA targets. Samples were tested with and without RT (which reverse transcribes RNA to DNA) in order to distinguish between viral mRNA and viral genomic material. Primers and
probe are designed in an intron-exon region, which should only amplify reverse transcribed RNA, but there is always a possibility that viral genomic material will also be amplified. Analyzing samples plus and minus RT allows determination of mRNA levels by subtracting the levels of minus RT reactions from plus RT reactions.
Large T mRNA was not detected until weeks 4-5 of the culture period, when exponential replication of BPyV was also detected in the supernatant. The expression of mRNA from both flasks is similar, although mRNA expression was only detected week 4 in one of the duplicate flasks (results illustrated in figure 12).
Virus inoculated onto cells grown in media supplemented with 10% FCS showed no production of large T mRNA, although detection of replicating virus in these cells during wk #’s 1-3 indicates that large T mRNA must be present, but potentially at a level lower than the limit of quantitation for the RT-QPCR assay (which is 1x102 copies per reaction) (results illustrated in figure 13). This may also be the case for cells grown in media supplemented with 1% FCS (figure 10). No mRNA expression was detected in the negative and heat inactivated cells (figure 14).
It is possible that large T mRNA is expressed in the first 3-4 weeks of culture, but errors during transcription is resulting in large T mRNA being unable to be detected by the current primer/probe design. To determine if large T antigen was present in these initial harvests, cells were prepared for protein analysis by SDS-PAGE as detailed in section 2.7 (figure 15).
Gels were set up with kD protein markers (lane 1 and 12), week 1 and 4 1% FCS MDBK negative controls from the 8-week infectivity assay (lane 2 and 3) and wks 1 - 8 1% FCS A MDBK samples from the 8-week infectivity assay (lanes 4-11). There is no protein band present in the samples that can correlated to the large T antigen (approx 65kD). It may be noted that the production of the large T antigen is heavily regulated, and therefore may not
be at a level that would allow visualization. There is also the potential that some cellular proteins are masking the appearance of the large T antigen. Can these large T antigens be detected by western blot analysis with antibodies specific to large T?
Western blots were prepared (section 2.7 of the methods and materials) using a large T specific primary antibody in an attempt to detect the large T antigen. Western blots were set up with a kD protein marker (lane 1), week 1, 4 and 8 1% FCS MDBK negative controls from the 8-week infectivity assay (lane 2 - 4), wks 2 - 8 1% FCS A MDBK samples from the 8-week infectivity assay (lanes 5-11), and a positive control (SV-40 infected Vero cells - lane 12). No bands were observed in lanes 2 (week 1, 1% negative control) and 6 (wk 4 1% FCS A sample). No bands of the excepted size (approx. 65kD) were present in the remaining lanes. Faint bands are present above the 60kD marker for week 4 and 8 1% FCS negative control, and weeks 2, 3 and 5 – 8 1% FCS A sample, however these must be non-specific due to observations in the negative controls (lanes 3 and 4) (figure 16). No cells were available for assessment due to poor cell growth for week 1 (1% FCS A or B samples) inoculated flasks. The only commercial large T antibody that was available was produced to the large T antigen of SV-40, and it was unclear if this antibody would also be specific to the large T antigen of BPyV. The SV-40 large T antigen is approx 79kD. Confirmation of large T mRNA expression was shown by RT-QPCR, but subsequent experiments proved inconclusive, although several factors (low levels of expression, unspecific primary antibodies) may have been responsible for this.
1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 1.00E+10 genome equivalent 1 2 1= 1%A, 2= 1%B LargeT mRNA data
wk.1 wk.2 wk.3 wk.4 wk.5 wk.6 wk.7 wk.8
Figure 12 - Data from duplicate flasks of MDBK cells grown in media supplemented with 1% FCS and inoculated with BPyV. Expression of large T mRNA can be observed wk 4 for the 1%A harvests, and wk 5 for the 1%B harvests.
1.00 10.00 100.00 1000.00 10000.00 100000.00 1000000.00 10000000.00 100000000.00 1000000000.00 10000000000.00 genome equivalent 1 2 1= 10%A, 2= 10%B LargeT mRNA data
wk.1 wk.2 wk.3 wk.4 wk.5 wk.6 wk.7 wk.8
Figure 13 - Data from duplicate flask of MDBK cells grown in media supplemented with 10% FCS and inoculated with BPyV. Expression of large T mRNA was not detected in any of the harvests, although the detection of viral genomes at the start of the infectivity assay indicates that large T mRNA may have been present in the first 3 weeks.
1.00 10.00 100.00 1000.00 10000.00 100000.00 1000000.00 10000000.00 100000000.00 1000000000.00 10000000000.00 genome equivalent 1 2
1= Negative Control, 2= HI Control LargeT mRNA data
wk.1 wk.2 wk.3 wk.4 wk.5 wk.6 wk.7 wk.8
Figure 14 - Data from negative and heat inactivated (HI) control flasks. No amplification is observed in any of the controls. Data shown is from duplicate flasks tested.
Figure 15 – SDS-PAGE using 8-week infectivity cell cultures (stained with GelCode Blue Stain). Approximately 6µg of protein was assessed. No bands are present that would indicate the presence of the large T antigen (65kD). Gels were run with kD protein markers (lane 1 and 12), infectivity negative controls (MDBK) cultured in 1% FCS and harvested week 1 and 4 (lane 2 and 3) and infectivity samples (MDBK) inoculated with BPyV, cultured with 1% FCS, and harvested week 1 to week 8 (lanes 4-11). Large T mRNA was detected by RT-QPCR in infectivity samples inoculated with BPyV and harvested week 4 to week 8 (lanes 7-11) (figure 12).
Figure 16 – Western Blot analysis of 8-week infectivity cell cultures using SV40 large T primary antibody and Anti-Mouse IgG Peroxidase secondary antibody. Membranes were developed using the DAB Substrate system. Approximately 6µg of protein was assessed. No large T antigen bands are present (approx. 65kD). Faint bands are present just above the 60kD marker in lanes 3-6 and 8-11, however must be non-specific due to observations in the negative controls (lanes 3 and 4). The large T antibody was raised against SV-40 large T antigen – it is unclear if this antibody will also be specific to BPyV. Western Blots were set up with a kD protein marker (lane 1), infectivity negative controls (MDBK) cultured in 1% FCS and harvested week’s 1, 4 and 8 (lane 2 - 4), infectivity samples (MDBK) inoculated with BPyV, cultured with 1% FCS, and harvested week 2 to week 8 (lanes 5-11), and a positive control (SV-40 infected Vero cells - lane 12).
4.4 Are infected cells in S phase?
During the same 8-week infectivity period that BPyV and large T mRNA sequences were assessed (sections 4.2 and 4.3), cells concentrations, viability and cyclin A expression were also assessed.
If polyomaviruses require cellular mechanisms in order to replicate, and one of the functions of the large T antigen is to move cells into S phase to support viral replication, then performing cell counts at each harvest could potentially
explain why we are only detecting high titres of virus in cells grown in media supplemented with 1% FCS. Assessing viability of cells during this 8-week infectivity period will also be of interest, to determine if viral infection is causing any cell death. Of particular interest are the cells grown in media supplemented with 1% FCS as they are not passaged for the entire 8 week experiment, and only fed with fresh media weekly.
When exponential amplification is observed (4-5 weeks), all cell counts decreased gradually, with the exception of the cells grown in media supplemented with 1% FCS, which are the cells in which BPyV amplification is being detected (figure 17). Counts for cells grown in the 10% FCS B flask increased after this period, but from week 6 the cell counts gradually decline, unlike the cells grown in media supplemented in 1% FCS which remain constant (figure 18). There were no differences in cell viability which remained between 95% and 100% throughout the experiment (figure 19). Does the large T antigen induce S-phase allowing cellular and viral replication, as reported for other polyomaviruses?
If BPyV does have the ability to move cells into S-Phase, which has been indicated by counting viable cells, then would it be possible to detect any S- Phase promoting factors such as cyclin A or the absence of proteins such as cyclin E, which are destroyed during this phase? In order to determine if the cells were indeed moving into S-Phase towards the end of the infectivity period, samples that were previously analysed (section 4.2 and 4.3) for the presence of BPyV and large T mRNA were also assessed by western blot analysis (as detailed in section 2.7) with an antibody specific to cyclin A (figure 20).
Western blots were set up with a kD marker (lane 1), week 1, 4 and 8 1% FCS negative MDBK control samples from the 8-week infectivity assay (lane 2 - 4), wks 2 - 8 1% FCS A MDBK samples from the 8-week infectivity assay (lanes 5-11), and a positive control (HeLa cells - lane 12). Lanes 2 (week 1, 1% negative control) and 7 (wk 4 1% FCS A sample) did not show any bands.
However, in lanes 9 – 11 (wks 6, 7 & 8 1% FCS A samples), bands can be observed that are the size expected from cyclin A (approx. 65kD). These bands do not appear to be present in earlier harvests or negatives. Closer examination of the gels does show bands in other samples, but this is to be expected as there will be some growth or turnover of cells even in fully confluent cultures. These results confirm the theory that BPyV is able to move the cells into S-Phase to necessitate replication. FACS (fluorescence- activated cell sorter) analysis would have been a useful tool is confirming these observations, but unfortunately this technology was not available during this experiment.
1% FCS cell count 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 2000000 1 2 3 4 5 6 7 8 Week #'s ce ll s/ m l 1% -ve 1% A 1% B
Figure 17 - Cell counts for MBK cells inoculated with BPyV and maintained with media supplemented with 1% FCS. With the exception of the –ve cells, counts from the A and B flasks remained constant from wk’s 4-8, at the same time that exponential amplification was observed. 10% FCS cell count 0 500000 1000000 1500000 2000000 2500000 1 2 3 4 5 6 7 8 Week #'s cell s/m l 10% -ve 10% A 10%B
Figure 18 - Cell counts for MDBK cells inoculated with BPyV and maintained with media supplemented with 10% FCS. With the exception of counts carried out during week 6 of the 10% B cells, counts gradually decreases from week’s 3-4. No viral amplification is detected from this time.
% Viabilty 70.00 75.00 80.00 85.00 90.00 95.00 100.00 105.00 1 2 3 4 5 6 7 8 Week #'s % Vi a b il it y 1% -ve 1% A 1% B 10% -ve 10% A 10%B
Figure 19 - Percentage viability for MDBK cells inoculated with BPyV and maintained with media supplemented with 1% and 10% FCS. % viabilities vary little over the 8 week infectivity assay.