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evelopment of a high-throughput human cytomegalovirusuantitative PCR cell-based assay
onia Tremblay1, Nathalie Dansereau, Scott Balsitis2, Michael Franti3, Louie Lamorte ∗,4
oehringer Ingelheim (Canada) Ltd., Research & Development, Department of Biological Sciences, 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada
rticle history:eceived 6 April 2013eceived in revised form 9 September 2013ccepted 20 September 2013vailable online 4 October 2013
a b s t r a c t
This report describes the development and optimization of a quantitative real-time PCR assay for evalu-ating human cytomegalovirus (CMV) replication in vitro and susceptibility to antiviral drugs. This assaymeasures the level of intracellular CMV DNA in both 96- and 384-well microplate formats. Normalizationof CMV levels using mitochondrial DNA enhanced the robustness of the assay and minimized variability.The assay throughput was further enhanced by eliminating several wash steps and by lysing the cellsdirectly in the presence of cell culture media, both of which had no impact on the assay metrics. The
assay was validated using several known CMV antiviral compounds. The CMV quantitative PCR (qPCR)assay represents a rapid, reliable and reproducible method that can be used with both CMV laboratorystrains and clinical isolates.
Human cytomegalovirus (CMV) represents a clinically seriousathogen for immunocompromised individuals (Jones et al., 2004).vailable treatment options are limited to the antiviral drugs gan-iclovir (GCV), its prodrug valganciclovir (ValGCV), foscarnet andidofovir (Härter and Michel, 2012). Patients infected with CMVften require long-term treatment, which can generate resistanceo currently approved therapies (Li et al., 2007; Martin et al., 2007;mery, 2001) with mutations mapping to UL54 or UL97. Newntiviral agents targeting UL97 or exhibiting novel mechanisms ofction are currently in development and may offer better treatmentptions (Härter and Michel, 2012; Kaul et al., 2011).
Evaluating genotypic drug resistance is the preferred methodf detecting resistance but its liabilities resides in its abilityo only detect known drug-resistance mutations. Although the
laque reduction assay (PRA) is the standard phenotypic screensed to assess drug susceptibility (Landry et al., 2000), it is lowhroughput and labor-intensive. Hence, a rapid, reliable and
reproducible phenotypic assay is needed to evaluate the efficacy ofnew antiviral drugs and to evaluate the impact of novel resistancemutations. This report describes the development and optimiza-tion of a quantitative PCR (qPCR) assay used to monitor CMV viralreplication in vitro.
Different qPCR primers and probes (Integrated DNA Technolo-gies, Coralville, Iowa, USA) from published sources (data not shown)were evaluated and primers specific for CMV US17 (Peres et al.,2010) were selected for all further assay development. US17 ispart of the US12 family, which is highly conserved among clini-cal isolates (Murphy et al., 2003). The 81 bp US17 PCR fragmentwas amplified from AD169 viral DNA and cloned into the pCR4-TAvector (Life Technologies, Burlington, Ontario, Canada). A standardcurve for determination of viral DNA copy number was obtainedby serial dilutions of the pCR4-US17 plasmid (106–102 copies). Thestandard curve was generated by plotting log10 values of the DNAcopy number (x-axis) versus cycle threshold (Ct, y-axis) and pro-duced an r2 > 0.98 (least-squares linear regression, data not shown).
The initial assay development efforts were based on an assayformat described by Schnepf et al. (2009) in which human fibro-blasts were infected in 24-well plates with laboratory or clinicalisolates. The first goal was to improve the throughput of the assayby adapting the assay to a 96-well format. The assay was establishedand optimized for the infection of MRC-5 cells (ATCC, Manassas,
VA, USA) with AD169 cell-free virus (ATCC) in 96-well microplates(Corning Life Sciences, Tewksbury, MA, USA), as depicted in Fig. 1A.Multiplicity of infection (MOI, 0.005–0.1) and assay length (2–5days post-infection) were among the different assay conditions that
68 S. Tremblay et al. / Journal of Virological Methods 195 (2014) 67–71
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Sciences). The following day, the cell culture media was removedand cells were infected with AD169 cell-free virus at an MOI of0.0008 for 3 h at 37 ◦C (in a total volume of 250 �l) with gentle
Table 1GCV EC50 (nM) against AD169 in different assay formats.
qPCR 24-wella qPCR 96-wellb PRAb
Fig. 1. The experimental layout of the different qPCR assays is outlined: 96-well st
ere evaluated. DMEM media (Life Technologies) supplementedith 5% FBS (HyClone, Whitby, Ontario, Canada) and 1% peni-
illin/streptomycin (Life Technologies) was used for all assays withRC-5 cells (except where noted otherwise) and cells were cul-
ured at 37 ◦C in an incubator containing 5% CO2. All chemicals werebtained from Sigma–Aldrich (Oakville, Ontario, Canada), excepthere indicated differently.
Entry inhibitors were added at the time of infection whereasucleoside analogs (GCV, ValGCV) or polymerase inhibitors weredded 3 h post-infection. Compounds were diluted serially inMSO, further diluted with assay media and then added to dupli-ate wells of a 96-well microplate. The DMSO concentration wasaintained constant throughout the assay at 0.64%. All compounds
sed in this study were synthesized at Boehringer IngelheimCanada) Ltd., with the exception of GCV (Sigma–Aldrich). Afterhe first set of washes, 150 �l of assay media was added to all wells,ollowed by 50 �l of diluted antiviral compound (or assay mediaontaining DMSO in lieu of the entry inhibitors). The cytopathicffect (CPE) on day 3 post-infection was weak, with a loss ofiability of 10% or less (data not shown), as measured using theellTiter 96 AQueous MTS Reagent (3-(4,5-dimethylthiazol-2-yl)--(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,romega, Whitby, Ontario, Canada). Cells were lysed with 15 �l of× lysis buffer (as described by Schnepf et al., 2009; 10 mM TrisCl
H 8.0, 50 mM KCl, 2 mM MgCl2, 0.45% Nonidet P-40, 0.45% Tween0, with a final 1:250 dilution of Proteinase K (Qiagen, Toronto,ntario, Canada)) and incubated one hour at 56 ◦C. Cell lysatesere diluted 10-fold with H2O and 10 �l from duplicate wells
d (A), 96-well short (B) and 384-well (C). The indicated volumes are for each well.
were pooled into a new microplate. Proteinase K was inactivatedby incubating the microplates 5 min at 95 ◦C and all samples werefurther diluted 100-fold with H2O (final dilution of 1000-fold).20 �l of master mix containing primers, probe and TaqMan Uni-versal PCR master mix (Applied Biosystems, Burlington, Ontario,Canada) was added to 5 �l of diluted lysate. The final concentrationof primers and probe was 200 nM. qPCR was performed using the7500 real-time PCR system (Applied Biosystems) with an initialdenaturation of 10 min at 95 ◦C, followed by 40 cycles of 15 s at95 ◦C and 60 s at 60 ◦C.
The half maximal effective concentration (EC50) value obtainedfor GCV in the 96-well qPCR assay (Table 1) was comparablewith the value reported by Schnepf et al. (2009) in their 24-wellqPCR assay. A similar activity was obtained for GCV in the PRA(Table 1). The PRA was performed by seeding MRC-5 cells at adensity of 1.4 × 105 cells per well in a 24-well plate (Corning Life
1200 ± 500 1700 ± 800 1800 ± 900
a Value reported by Schnepf et al. (2009).b Value represents the average EC50 (with one standard deviation) from at least 3
independent experiments.
S. Tremblay et al. / Journal of Virologi
Table 2EC50 (nM) of reference compounds in qPCR assay (96-well) and PRA (24-well).
Class Inhibitor qPCR PRA
Entry CFI02 98 ± 40 820 ± 500
Nucleoside analog GCV 1700 ± 800 1800 ± 900Nucleoside analog ValGCV 850 ± 400 n.d.
In the qPCR assay, MRC-5 cells were infected with AD169 at a multiplicity of infection(MOI) of 0.050 and CMV intracellular DNA levels were measured 3 days post-infection. In the PRA, MRC-5 cells were infected with AD169 at an MOI of 0.0008and plaques were scored 7 days post-infection. EC50 values represent the average(n
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(ofltdocdieat1how
infect MRC-5 fibroblasts at an MOI of 0.050, consistent with the
TC
Vn
with one standard deviation) from at least 2 independent experiments..d.: not determined.
ocking. Media was removed and replaced with an equal volumef 0.8% low gelling-temperature agarose (Sigma–Aldrich) and 2×MEM (Life Technologies) supplemented with 10% FBS, 2% peni-illin/streptomycin and either DMSO or serially diluted inhibitors.laques were scored visually 7 days post-infection using an IX-0 microscope equipped with a 10× objective (Olympus Canada,ichmond Hill, Ontario, Canada).
The 96-well qPCR assay was validated further using a panelf 6 reference antiviral compounds: GCV, ValGCV, 2 polymerasenhibitors (Brideau et al., 2002; Knechtel et al., 2002; Hartline et al.,005; Tanis et al., 2010), an entry inhibitor, CFI02 (Jones et al., 2004)nd a terminase inhibitor, AIC246 (Lischka et al., 2010). The activ-ty obtained for CFI02, GCV and ValGCV was in agreement withublished values (Brideau et al., 2002; Jones et al., 2004; Lischkat al., 2010; Tanis et al., 2010) and with results obtained in theRA (Table 2). Polymerase inhibitors exhibited comparable activityn the PRA and qPCR assay (Table 2). The terminase inhibitor wasnactive in the qPCR assay, consistent with its late stage mechanismf action but fully active in the PRA (Table 2). This confirms that thePCR assay is limited to a single cycle of replication.
The 96-well qPCR assay, referred to here-in as the standard assayFig. 1A), was further simplified by reducing the number of steps, asutlined in Fig. 1B. In the qPCR short assay, virus was not removedollowing the 3 h infection, all wash steps were eliminated and 1×ysis buffer was added directly to the cell culture media. In con-rast with the standard assay, the lysates from the short assay wereiluted 40-fold prior to use in the qPCR. To improve the robustnessf the qPCR assay and to reduce the well to well variability, CMVopy numbers were normalized against the levels of mitochon-rial DNA within the samples using primers and probe described
n Butler et al. (2001). Assay reproducibility and robustness wasvaluated using the Z′ value, a statistical parameter which providesn assessment of the variability of the positive and negative con-rols relative to the assay signal to background ratio (Zhang et al.,999). Z′ values between 0.5 and 1 are considered acceptable for
igh-throughput screening. An average Z′ value of 0.61 ± 0.1 wasbtained in the short assay format with normalization, comparedith −0.63 ± 0.3 in the absence of any normalization (Table 3). In
able 3omparison of assay statistics of the 96-well and 384-well CMV qPCR assays.
Assay format CMV DNA-to-Mitochondrial DNA r
Mean Ra
96-well standard version n.d. n.96-well short version 2.5 1.384-well 2.3 1.
alues represent the average of at least 3 independent experiments..d.: not determined.
cal Methods 195 (2014) 67–71 69
addition, all steps of the assay were automated using the BiomekFX liquid handler (Beckman Coulter, Mississauga, Ontario, Canada)equipped with a 96-channel pipetting head. Normalization withmitochondrial DNA reduced the variability of CMV DNA copy num-ber across samples and allowed inhibitors to be tested as singletons,instead of pools of duplicate wells. The 96-well short assay was val-idated using a set of 29 inhibitors, which included the five activereference compounds from Table 2 (GCV, ValGCV, PHA-529311,PNU-183792 and CFI02) and a panel of 24 inhibitors from one ofthe small molecule lead-optimization projects of Boehringer Ingel-heim (Canada) Ltd. (undisclosed target). The correlation betweenthe short and standard forms of the 96-well qPCR assay was evalu-ated by performing Deming regression analysis. Average log10 EC50values (from 2 to 3 independent experiments) from the two differ-ent assay formats were analyzed with Prism 6 (GraphPad Software,La Jolla, CA, USA), using similar uncertainties for both data sets.There was a statistically significant correlation between the twoassay formats, with a p-value < 0.0001 (Fig. 2A).
A 384-well version of the assay was developed to increase fur-ther the throughput of the 96-well short assay. The final conditionsof the 384-well format used 9000 MRC-5 cells per well, infectedwith AD169 at an MOI of 0.015 using the conditions describedin Fig. 1C. Cells were lysed with 40 �l of 1× lysis buffer, sampleswere incubated 1 h at 56 ◦C, diluted 10-fold with H2O and incu-bated at 95 ◦C to inactivate the Proteinase K. All steps of the assaywere performed using liquid handlers equipped with a 384-channelpipetting head (Biomek FX (Beckman Coulter) and MicroLab StarLet(Hamilton Robotics, Reno, Nevada, USA)). 5 �l of master mix con-taining primers, probes and TaqMan Universal PCR master mix wasadded to 3 �l of diluted lysate. The final concentration of primersand probes was 200 nM. qPCR were performed using the ViiA 7real-time PCR system (Applied Biosystems) with the same cyclerparameters as described for the 96-well assays. The Z′ obtained forthe 384-well assay format was 0.55 ± 0.01 (Table 3). The same setof compounds from Fig. 2A was tested in the 384-well assay. Therewas a statistically significant correlation between the 96- and 384-well formats of the qPCR assay, as evaluated by Deming regressionanalysis (p-value < 0.0001, Fig. 2B).
The short form of the 96-well qPCR assay was adapted foruse with several clinical isolates. TR, TB40 (UL32-EGFP-CMV-TB40)and VR1814 isolates were obtained from Jay Nelson and DavidJohnston, ATCC and Giuseppe Gerna, respectively. MRC-5 cellswere infected with cell-free virus at MOI ranging from 0.003 to1 using three different clinical isolates (TR, TB40 and VR1814). Thekinetics of viral replication was monitored by measuring intracel-lular CMV DNA levels at 3, 4 and 5 days post-infection. All threeclinical isolates replicated to high levels by day 4 post-infection,with a reduced increase at day 5 post-infection indicating thatDNA replication plateaued by this time point (data not shown),in agreement with results reported by Schnepf et al. (2009).Based on those results, all three clinical isolates were used to
MOI used in the 96-well AD169 qPCR assay. In addition, a qPCRassay specific for VR1814 and ARPE-19 epithelial cells was alsoestablished using an MOI of 0.050. ARPE-19 cells were cultured
atio Z′ value
nge Mean Range
d. −0.63 −0.23 to −1.037–3.1 0.61 0.50–0.744–2.7 0.55 0.55–0.56
70 S. Tremblay et al. / Journal of Virological Methods 195 (2014) 67–71
Fig. 2. Correlation amongst the different qPCR assay formats. Deming regression analysis was used to analyze the relationship between the 96-well standard and shortformats (A) and 96-well and 384-well formats (B). Average log10 EC50 values from 2 to 3 independent experiments were used for the analysis. The indicated p-values referto the testing of the null hypothesis that the slope is equal to zero. The solid and dotted lines represent the experimental data and absolute identity, respectively.
Table 4EC50 (nM) of GCV and CFI02 against CMV clinical isolates.
Activity of GCV and CFI02 in the 96-well short form qPCR assay against AD169 andthe indicated clinical isolates. Cells were infected with the indicated virus strainand intracellular CMV and mitochondrial DNA levels were measured 4 days post-infection. Values represent the average (with one standard deviation) from at least3n
sing 1:1 DMEM/F12 media (Life Technologies) supplementedith 5% FBS and 1% penicillin/streptomycin. The antiviral activ-
ty of two reference antiviral compounds (GCV and CFI02) againsthe clinical isolates was determined using cells lysed on day 4ost-infection. The potency of GCV was maintained against TB40nd VR1814 but shifted 16-fold against TR (Table 4), a GCV-esistant isolate (Smith et al., 1998). The clinical isolates wereully susceptible to CFI02 (Table 4). CFI02 exhibited a half maxi-
al cytotoxic concentration (CC50) value of 40 �M against ARPE-19ells but no cytotoxicity was observed for all other conditionsmeasured using the CellTiter 96 AQueous MTS Reagent, data nothown).
The improved CMV qPCR assay described in this report repre-ents a rapid, reliable and reproducible method, with a significantlyeduced workflow compared with previously described methodsLandry et al., 2000; Schnepf et al., 2009). The assay conditions areompatible with both laboratory and clinical isolates using differ-nt cell lines. In conclusion, the optimized CMV qPCR assay can besed to evaluate the antiviral activity of inhibitors and to monitorhe replication of clinical isolates.
cknowledgements
The TR CMV strain was kindly provided by Drs. Jay Nelson andavid Johnston, Oregon Health and Science University. The VR1814
train was kindly provided by Dr. Giuseppe Gerna, Servizio diirologia, Fondazione IRCCS Policlinico San Matteo, Italy. We thankr. Annick Gauthier for helpful discussions.
All authors were employees of Boehringer Ingelheim (Canada)Ltd. when this work was performed. This work was supported byBoehringer Ingelheim.
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