Impact of Genomic Sequence Variability

on Quantitative PCR Assays for Diagnosis

of Polyomavirus BK Infection

Parmjeet Randhawa, Jeffrey Kant, Ron Shapiro, Henkie
Tan, Antik Basu and Chunming Luo
J . Clin. Microbiol. 2011, 49(12):4072. DOI:
10.1128/JCM.01230-11.
Published Ahead of Print 28 September 2011.




Updated information and services can be found at:
http://jcm.asm.org/content/49/12/4072
















D
o
w
n
l
o
a
d
e
d


REFERENCES


CONTENT ALERTS
These include:

This article cites 17 articles, 7 of which can be accessed free at:
http://jcm.asm.org/content/49/12/4072#ref-list-1

Receive: RSS Feeds, eTOCs, free email alerts (when new
articles cite this article), more

f
r
o
m


h
t
t
p
:
/
/
j
c
m
.
a
s
m
.
o
r
g
/

o
n

A
p
r
i
l

2
8
,

2
0
1
4

b
y

g
u
e
s
t










Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To
subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2011, p. 40724076 Vol. 49, No. 12
0095-1137/11/$12.00 doi:10.1128/JCM.01230-11
Copyright 2011, American Society for Microbiology. All Rights Reserved.

Impact of Genomic Sequence Variability on Quantitative PCR Assays for
Diagnosis of Polyomavirus BK Infection
Parmjeet Randhawa,
1
* Jeffrey Kant,
1,2
Ron Shapiro,
3
Henkie Tan,
3
Antik Basu,
3
and
Chunming Luo
1

Departments of Pathology,
1
Human Genetics,
2
and Surgery,
3
University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

Received 20 June 2011/Returned for modification 6 September 2011/Accepted 22 September 2011

Knowledge of polyomavirus BK (BKV) genomic diversity has greatly expanded. The implications of BKV
DNA sequence variation for the performance of molecular diagnostic assays is not well studied. We analyzed
184 publically available VP-1 sequences encompassing the BKV genomic region targeted by an in-house
quantitative hydrolysis probe-based PCR assay. A perfect match with the PCR primers and probe was seen in
81 sequences. One Dun and 13 variant prototype oligonucleotides were synthesized as artificial targets to
determine how they affected the performance of PCR. The sensitivity of detection of BKV in the PCR assay
was a function of the viral genotype. Prototype 1 (BKV Dun) could be reliably detected at concentrations as
low as 10 copies/ l. However, consistent detection of all BKV variants was possible only at concentrations of
10,000 copies/ l or higher. For BKV prototypes with 2 or more mismatches (representing genotype IV,
genotype II, and genotype 1c strains), the calculated viral loads were 0.57 to 3.26% of the expected values. In
conclusion, variant BKV strains lower the sensitivity of detection and may have a substantial effect on
quantitation of the viral load. Physicians need to be cognizant of these effects when interpreting the results of
quantitative PCR testing in transplant recipients, particularly if there is a discrepancy between the clinical
impression and the measured viral load.



















D
o
w
n
l
o
a
d
e
d

f
r
o
m


h
t
t
p
:
/
/
j
c
m


Polyomavirus BK (BKV) has become an important patho-gen
in kidney transplant patients. Immunosuppression given to prevent
acute rejection triggers BK viruria in 10 to 60%, viremia in 5 to
30%, and biopsy-proven viral nephropathy in 1 to 10% of patients
(8, 10, 11). Initial graft loss rates associated with BKV
nephropathy were very high but have now dropped to 25%. This
success has been attributed to intensive viral monitoring followed
by preemptive reduction in immunosuppression (1, 14). In a
recent survey that had an overall response rate of 55.5%, 173 of
200 (86.5%) kidney transplant centers reported screening for
BKV in blood by quantitative PCR, while 111 of 202 (55.5%)
performed viral screening in urine (2). In the latter category, 90%
of respon-dents preferred PCR screening to urine cytology. While
cytology is a useful modality to screen for viral nephropathy in
low-resource settings, it is less sensitive than quantitative PCR for
detecting viral replication prior to the onset of clinical
nephropathy. In addition, it cannot differentiate BKV infection
from infection by the related polyomavirus JC, which causes
significant graft dysfunction at a substan-tially lower frequency.


Current quantitative PCR assays were developed several years
ago using BKV Dun or similar genotype I strains as reference
sequences for the design of primers and probes. However, our
knowledge of BKV genomic diversity has ex-panded enormously
(6, 7, 12, 16). PCR-based diagnostic and treatment algorithms
must be reevaluated to take into account newly discovered BKV
single-nucleotide polymorphisms. One

* Corresponding author. Mailing address: Division of Transplant
Pathology, University of Pittsburgh, Department of Pathology, E737
UPMC-Montefiore Hospital, 3459 Fifth Ave., Pittsburgh, PA 15213.
Phone: (412) 647-7646. Fax: (412) 647-5237. E-mail: randhawapa
@upmc.edu.
Published ahead of print on 28 September 2011.

approach to define the potential extent of this problem is to assay
the same sample using a panel of different PCR assays
(5). This is a labor-intensive method that is not practical for
routine application by clinical laboratories. We employed an
alternate approach that consists of aligning PCR primer and probe
sequences with large data sets of BKV sequences (3). This
bioinformatic evaluation was followed by experimental
amplification of 14 custom oligonucleotides of extended length
designed to comprehensively represent genetic variability in the
targeted area of the (VP-1) gene. Our results show that variant
BKV strains significantly lower the sensitivity of detect-ing viral
DNA and have a substantial effect on quantitation of the viral
load. We recommend that molecular diagnostic lab-oratories
offering BKV testing regularly reevaluate their cur-rent assays for
the ability to accurately identify and quantitate the majority of
viral strains circulating in the communities they serve. This
recommendation is particularly applicable to geo-graphic
locations with a high incidence of genotypes other than type 1. It
is worth recalling that BKV genotype IV has a reported
prevalence of 54% in Mongolia, while genotype III accounts for
9% of BKV isolates reported from Africa (17).


MATERIALS AND METHODS
Retrieval of public sequences. A total of 184 BKV VP-1 sequences matching
primer and probe sequences of the PCR assay used in our laboratory were retrieved
from GenBank. These sequences were aligned by Clustal X with de-fault
multialignment parameters (13). The alignments were manually adjusted using
BioEdit (T. Hall, Department of Microbiology, North Carolina State University;
available at http://www.mbio.ncsu.edu/BioEdit/BioEdit.html).
Phylogenetic analysis. When not already known, genotype assignment of the
sequences was based on phylogenetic clustering using known reference se-quences,
as previously described (7). Neighbor-joining trees were constructed in Mega 4.1
using Kimuras two-parameter method and the complete deletion option for gaps and
missing data. Trees were viewed using the Tree Explorer program. A bootstrap test
with 1,000 replicates was used to estimate the confi-dence of branching patterns in
the trees.



.
a
s
m
.
o
r
g
/

o
n

A
p
r
i
l

2
8
,

2
0
1
4

b
y

g
u
e
s
t


4072
VOL. 49, 2011 BK VIRUS ASSAYS 4073

TABLE 1. GenBank accession numbers of 184 publicly available BKV sequences classified into 14 prototypes

Prototype (no.) GenBank sequence

1 (81) .............................. Ia_CAF-15; Ia_CAF-5; Ia_CAF-9; Ia_Dunlop; Ia_KEN-1; Ia_KEN-4; Ia_MM; Ia_PittNP4; Ia_PittNP5; Ia_PittVR4;
Ia_PittVR9; Ia_UT; Ia_Z19534; Ia_ZAF-1; Ib1_CAP-h2; Ib1_CAP-h22; Ib1_CAP-h5; Ib1_CAP-h8; Ib1_CAP-
m13; Ib1_CAP-m18; Ib1_CAP-m5; Ib1_CAP-m9; Ib1_Dik; Ib1_GBR-6; Ib1_HI-u5; Ib1_HI-u6; Ib1_HI-u8;
Ib1_J2B-2; Ib1_KEN-3; Ib1_KOM-1; Ib1_KOM-5; Ib1_LAB-18; Ib1_LAB-27; Ib1_MMR-6; Ib1_NER-1;
Ib1_OKN-18; Ib1_PHL-6; Ib1_PHL-7; Ib1_PittNP1; Ib1_PittVM2; Ib1_PittVR8; Ib1_VNM-9; Ib1_WW;
Ib2_ESP-2; Ib2_ETH-4; Ib2_FIN-10; Ib2_FIN-11; Ib2_FIN-13; Ib2_FIN-14; Ib2_FIN-23; Ib2_FNL-12; Ib2_FNL-
22; Ib2_GBR-4; Ib2_GBR-8; Ib2_GBR-9; Ib2_HC-u2; Ib2_HC-u5; Ib2_HC-u9; Ib2_J2B-13; Ib2_J2B-9; Ib2_JL;
Ib2_LAB-14; Ib2_LAB-20; Ib2_LAB-22; Ib2_LAB-25; Ib2_LAB-29; Ib2_LAB-7; Ib2_PittNP2; Ib2_PittVM4;
Ib2_PittVM5; Ib2_PittVR10; Ib2_PittVR2; Ib2_PittVR3; Ib2_PittVR5; Ib2_PittVR6; Ib2_SWE-2; Ib2_TUR-5;
Ic_RYU-2; Ic_TW-8; Ic_TW-8a, II_ETH-3
2 (1) ................................ Ib1_CAP-m2
3 (48) .............................. II_GBR-12; IVa1_MMR-24; IVa1_PHL-8; IVa1_SEC-3; IVa1_VNM-7; IVb1_JPN-32; IVb1_JPN-33; IVb1_JPN-36;
IVb1_THK-8; IVb1_TW-3; IVb1_TW-3a; IVb2_JPN-15; IVb2_JPN-34; IVb2_JPN-35; IVb2_KOM-2;
IVb2_KOM-7; IVb2_MON-8; IVc1_FUJ-18; IVc1_FUJ-32; IVc1_MMR-28; IVc1_MMR-29; IVc1_MON-1;
IVc1_MON-6; IVc1_NEC-14; IVc1_NEC-24; IVc1_NEC-4; IVc1_NWC-14; IVc1_NWC-15; IVc1_NWC-8;
IVc1_SEC-6; IVc1_SWC-1; IVc1_SWC-2; IVc1_SWC-4; IVc1_VNM-1; IVc2_FIN-2; IVc2_FIN-9; IVc2_FNL-17;
IVc2_Fin-4; IVc2_GRC-3; IVc2_GRC-4; IVc2_GRC-5; IVc2_ITA-3; IVc2_LAB-33; IVc2_MON-2; IVc2_MON-
3; IVc2_MON-5; IVc2_NWC-7; IVc2_SWE-4
4 (6) ................................ IVa2_FUJ-13;IVa2_MMR-1;IVa2_MMR-43;IVa2_RYU-3;IVa2_SEC-21;IVa2_VNM-2
5 (5) ................................ III_AS; III_KOM-3; III_Z19536; II_EF376992; II_J2B-11
6 (22) .............................. Ic_J2B-1; Ic_KOM-6; Ic_MT; Ic_NAR-12; Ic_NAR-13; Ic_NEA-25; Ic_NEB-6; Ic_NEC-12; Ic_NEC-8; Ic_NGY-38;
Ic_NGY-5; Ic_OKN-14; Ic_RYU-1; Ic_THK-6; Ic_THK-9; Ic_THK-9a; Ic_TW-1; Ic_TW-1a; Ic_TW-1b;
Ic_TW-4; Ic_TW-5; Ic_TW-7
7 (7) ................................ Ib2_DQ366598; Ib2_LAB-21; Ib2_LAB-8; Ib2_PittNP3; Ib2_PittVM1; Ib2_PittVM3; Ib2_PittVR7
8 (1) ................................ Ib2_V_ITA-5
9 (1) ................................ Ib2_V_PittVR1
10 (4) .............................. Ic_AB181542; Ic_AB181555; Ic_ab268370; Ic_ab276279
11 (3) .............................. II_AB268401;II_AB276173;II_AB276304
12 (2) .............................. Ib2_DQ533639;Ib2_DQ533641
13 (1) .............................. Ia_AB276228
14 (2) .............................. Ic_AB245326;Ia_AB268405




















D
o
w
n
l
o
a
d
e
d

f
r
o
m


h
t
t
p
:
/
/
j
c
m
.
a
s
m
.


Synthesis of oligonucleotides. Fourteen chromatographically purified synthetic
oligonucleotides, each 135 nucleotides in length and representing all known BKV
genetic variation in the VP-1 gene region targeted by the PCR assay, were purchased
for use as artificial targets to compare amplification efficiencies (In-tegrated DNA
Technologies, Coralville, IA). Nucleotides AGGG were incorpo-rated at one end of
these synthetic nucleotides, and AAAT at the other end. Oligonucleotide solutions in
EB buffer (Qiagen) were standardized by measuring the absorbance at 260 nm and
represented target sequence concentrations rang-ing from 1E8 to 1E0 copies/ l.

Real-time PCR. The assay targeted the BKV VP-1 gene as follows: forward
primer, 5 -GCAGCTCCCAAAAAGCCAAA-3 (1600 to 1619; Dun number-ing);
reverse primer, 5 -CTGGGTTTAGGAAGCATTCTA-3 (1726 to 1706; Dun
numbering); probe, 5 -ACCCGTGCAAGTGCCAAAACTACTAATAAA AGG-3
(1623 to 1655; Dun numbering).
The real-time PCR was performed in a total volume of 20 l and contained the
following components: 10 l TaqMan Fast Universal PCR Master Mix (2 ; catalogue
number 4352042), 1.5 l of each primer (prepared at 1 M), 1 l of probe (prepared at 10
M), and 6 l oligonucleotides at specified concentra-tions. The PCR cycling program
consisted of the following steps: 95C for 4 min and then 95C for 10 s, 60C for 30
s, and 72C for 10 s for a total of 40 cycles. Thermal cycling was performed using an
Applied Biosystems 7500 apparatus. Standard precautions were employed to prevent
PCR contamination. Pre- and postamplification steps were done in separate
laboratories. The quantitation of target copy numbers used a standard curve with the
pBKV(34-2) plasmid, which contains the BKV Dun genome (ATCC 45025).

Data analysis. Analysis of the real-time PCR assay was performed using SDS
software (Applied Biosystems). Unknown target concentrations were deter-mined by
linear regression using threshold cycles (C
T
) plotted against the log
10
copy number
of the standard BKV plasmid. Corrections for sample dilution and descriptive
statistics were performed in Microsoft Excel 2007.

RESULTS

The breakdown of genotypes for the 184 sequences retrieved
for the study was as follows: 16 Ia, 30 Ib1, 45 Ib2, 30 Ic, 53 IV,

7 II, and 3 type III. An alignment of these sequences indicated a
perfect match with the PCR primers and probe for 81 se-quences
(13 genotype Ia, 29 genotype Ib1, 35 genotype Ib2, 3 genotype 1c,
and 1 genotype II). All genomic variability in the BKV VP-1
region targeted by our quantitative PCR assay could be
represented by 14 unique prototype sequences (Table 1).
Prototype 1 matches the BKV Dun reference sequence, as well as
several other sequences, all except one of which are genotype 1.
Prototypes 2 thru 14 correspond to a broad spec-trum of
sequences that includes genotypes II, III, and IV at coverage
frequencies that are summarized in Table 2. It is apparent that
prototypes 1, 2, 6, 7, 8, 9, 10, 12, 13, and 14 cover primarily
genotype I strains. Genotype II is represented by prototypes 5 and
11, genotype III by prototype 5, and genotype IV by prototypes 3
and 4. The locations of nucleotide mis-matches between the viral
prototype and PCR primer/probe sequences are depicted in Fig. 1
and enumerated in Table 3, which shows the relative amplification
efficiencies of the dif-ferent prototype sequences.

The results indicate that the sensitivity of detection of BKV is a
function of the viral genotype. Thus, prototype 1, which
represents BKV Dun and similar strains, could be consistently
detected at concentrations as low as 10 copies/ l (10,000 cop-
ies/ml). This is clinically relevant, since plasma BKV loads of this
magnitude have been used as a trigger to lower immuno-
suppression and initiate antiviral therapy. Notably, at lower
concentrations, namely, 1 copy/ l, detection of prototype 1 was
not possible in 1 of 3 replicates (Table 3). Prototype 3, which
covered the majority of genotype IV strains, behaved essen-


o
r
g
/

o
n

A
p
r
i
l

2
8
,

2
0
1
4

b
y

g
u
e
s
t

4074 RANDHAWA ET AL.

TABLE 2. Frequency distribution of BKV genotypes I to IV as
represented by prototype sequences 1 to 14
a

No. of sequences of BKV genotype:

Prototype

I II III IV Total
b
(%)


(n 121) (n 7) (n 3) (n 53)

1 80 1 0 0 81 (44)

2 1 0 0 0 1 (0.5)

3 0 1 0 47 48 (26.1)

4 0 0 0 6 6 (3.3)

5 0 2 3 0 5 (2.7)

6 22 0 0 0 22 (12)

7 7 0 0 0 7 (3.8)

8 1 0 0 0 1 (0.5)

9 1 0 0 0 1 (0.5)

10 4 0 0 0 4 (2.2)

11 0 3 0 0 3 (1.6)

12 2 0 0 0 2 (1.1)

13 1 0 0 0 1 (0.5)

14 2 0 0 0 2 (1.1)


a
Reported frequencies for the major BKV genotypes were 46 to 82% for genotype
I, 0 to 6% for genotype II, 0 to 9% for genotype III, and 12 to 54% for genotype IV.
Genotype IV has the highest reported incidence in China and Mongolia, while
genotype I is the predominant strain in most other parts of the world. Subgroups of
genotype I may also have a predilection for specific geo-graphic regions. Thus,
Zheng et al. report incidence figures of 79% for genotype 1a in Africa, 64% for
genotype 1b in West Asia, and 100% for genotype 1c in northeast Asia (17).


b
Out of 184 sequences.



tially similarly to prototype 1 in terms of the sensitivity of
detection. However, prototype 4, which corresponds to 6/53
(11%) known genotype IV strains, could not be detected in 2 of 3
replicates set up at concentrations of 1E2, i.e., 100 cop-ies/ l and
1E1, i.e., 10 copies/ l. Consistent detection (defined as detection
in 3 of 3 replicates) of all 14 BKV prototypes was possible only at
concentrations of 1E4, i.e., 10,000 copies/ l or higher. At
concentrations of 1E1, i.e., 10 copies/ l, the assay was able to
detect only 6 of 14 prototype sequences in all three replicates,
while lowering of the prototype concentration to 1E0, i.e., 1 copy/
l, reduced the performance of the assay to reproducible detection
of only 3 prototypes.
Another observation of interest is that, compared to the
reference prototype (number 1), the measured viral load is
variably underestimated for all other prototypes (numbers 2 to
14). This effect is most pronounced at low concentrations. In
general, quantitation of the BKV load by real-time PCR de-pends
principally on two factors: (i) the linearity of the stan-dard curve
(prepared using the BKV Dun strain plasmid in our
J. CLIN. MICROBIOL.

assay) and (ii) the amplification efficiency of the target se-quence.
Both these factors appear to contribute to the proto-type-specific
variations seen in our experimental system. The linear part of a
real-time PCR standard curve is characterized by a C
T
difference
of approximately 3.3 between serial 10-fold dilutions of the
plasmid standard. Using this yardstick, C
T
measurements were in
the linear range for BKV Dun proto-type 1 at all concentrations
up to 1E2, i.e., 100 copies/ l. All values outside the linear range
are indicated in boldface in Table 3. For 13 variant BKV strains
(prototypes 2 through 14, taken together), linearity was observed
only at concentrations up to 1E5, i.e., 100,000 copies/ l. The linear
range of the PCR assay for variant strains fell sharply at lower
concentrations: linear measurements were obtained for only 6/13
prototypes at concentrations 1E3, i.e., 1,000 copies/ l; 3/13
prototypes at concentrations 1E2, i.e., 100 copies/ l; and 0/13
prototypes at concentrations 1E1, i.e., 10 copies/ l. Amplification
effi-ciency was also significantly reduced for BKV variants
(proto-types 2 to 14) expressed as a percentage of the BKV Dun
reference (prototype 1), which was assumed to represent 100%
efficiency (Table 3). Thus, for prototype 2, the viral-DNA yield
was 13.31% of the expected value at 1E7copies/ l and 15.51% of
the expected value at 1E6, i.e., 1,000,000 copies/ l. For
measurements in the lower nonlinear part of the standard curve
(boldface in Table 3), the calculated yields were quite variable
and inaccurate, which explains why the mean of sev-eral
calculated results was 100% of input DNA.

Finally, the experiments conducted illustrate that the ampli-
fication efficiency is a function of the number of sequence
mismatches between the viral target and PCR assay primer/ probe
sequences. A sequence alignment of all prototype se-quences is
shown in Fig. 1, including the locations of all variant nucleotides
in relation to the PCR primers and probe. Table 3 enumerates the
mismatches between the target sequence and the forward primer
(F), reverse primer (R), and probe (P) sequences. Prototypes 3, 4,
10, and 11 had 2 or more mis-matches. Prototypes 2, 5, 12, 13,
and 14 had 1 mismatch with the primer sequences, while
prototypes 6, 7, 8, and 9 had 1 mismatch with the probe. For 4/13
BKV variants with 2 or more primer/probe mismatches, the
calculated viral loads were 0.57 to 3.26% of the expected values
(i.e., approximately 100-fold lower).Considering 5/13 BKV
variant prototypes with only 1 primer mismatch, 2/5 and 3/5,
respectively, yielded calculated target copy numbers
approximately 10- and 20-fold lower than the expected values.
Finally, for 4/13 BKV variants with only 1



















D
o
w
n
l
o
a
d
e
d

f
r
o
m


h
t
t
p
:
/
/
j
c
m
.
a
s
m
.
o
r
g
/

o
n

A
p
r
i
l

2
8
,

2
0
1
4

b
y

g
u
e
s
t














FIG. 1. Alignment of 14 prototype BKV sequences (lightface) with PCR primer and probe sequences (boldface and underlined). Identical nucleotides at
the same position are represented by dots.
VOL. 49, 2011 BK VIRUS ASSAYS 4075

TABLE 3. Comparative amplification efficiencies of prototypes 1 to 14



Prototype
Calculated copy no. of prototypes 214
a
No. of mismatches
b



1E7 1E6 1E5 1E4 1E3 1E2 1E1 1E0

F R P


1 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00

2 13.31 15.51 17.07 16.89 39.59 63.4 308.45 120.28 1

3 1.06 0.71 0.63 1.22 12.55 57.61 190.14 48.89 2

4 2.47 1.10 1.00 3.26 7.09 23.07 77.32 136.94 2 1
5 14.74 10.53 9.70 9.36 21.94 47.48 87.32 44.07 1

6 76.66 91.13 59.54 90.81 130.73 116.85 291.55 64.72 1
7 84.51 88.87 83.89 162.56 212.90 139.92 136.48 ND 1
8 54.32 30.68 15.72 24.38 38.89 423.53 342.25 175.00 1
9 18.10 13.74 20.16 39.09 121.29 190.76 270.86 374.07 1
10 0.57 0.68 0.57 2.29 52.9 71.01 453.52 86.11 1 1
11 0.72 1.49 0.84 2.04 16.89 68.95 254.79 48.61 2

12 7.37 4.91 3.69 3.92 26.92 49.54 155.07 72.22 1

13 6.12 3.93 2.96 2.82 17.13 46.22 194.37 90.28 1

14 8.21 5.55 3.53 3.48 21.64 77.73 155.21 46.11 1



a
Each BKV prototype sequence was set up at 8 different concentrations ranging from 1E0 to 1E7 target copies per l. The calculated copy numbers (means of three
independent determinations) for each prototype are expressed as percentages of prototype 1. The values in boldface are outside the linear range of the standard curve. Calculated
values for prototypes set up at these low concentrations were extremely variable, and the mean often appeared to be greater than the expected values ( 100%). Additionally,
detection of the target at these low copy numbers was not reproducible and frequently failed in 1 of 3 replicates (cells shaded in gray) or in 2 of 3 replicates (cells shaded in gray
and underlined). ND, not determined.

b
The number of mismatches between the target sequence and the forward primer (F), reverse primer (R), or probe (P) sequence.




















D
o
w
n
l
o
a
d
e
d

f
r
o
m


probe mismatch, underestimation of the target copy number was
less pronounced and observed deviation was within 5-fold of the
expected value.

DISCUSSION

Quantitative PCR is now widely used to monitor BKV in-
fection after kidney transplantation. Different laboratories em-
ploy different viral targets and primer sequences for amplifying
viral DNA. Assays are typically based on the BKV Dun strain or
similar reference sequences of genotype I. In the assay evaluated
here, primers and probes designed in this manner showed a
perfect match with only 87/184 (47.3%) and one or more
mismatches with 97/184 (52.7%) publicly available se-quences.
Mismatches were seen most often with genotype IV, followed by
genotypes 1c, 1b, II, and III. This rank order is consistent with the
known phylogenetic distances between dif-ferent viral strains.

To study the impact of genetic variability on BKV PCR, 14
prototypesequencesincorporatingrepresentativesingle-nucleo-tide
polymorphisms in 184 viral strains were synthesized. These
prototypes generally corresponded to specific genotypes, but with
occasional exceptions, which may represent more recent
mutational events occurring after the divergence of major ge-
notypes in the course of viral evolution. The ability of the
quantitative real-time PCR assay to accurately detect virus was
maximal for BKV genotypes 1a, 1b1, and 1b2. Sequences rep-
resented by prototype I could be consistently detected at all
concentrations up to 1E1, i.e., 10 copies/ l (i.e., 1E4, or 10,000
copies/ml), which is a threshold that has been frequently used for
reducing immunosuppression in patients with BK viremia or viral
nephropathy (4). For genotype IV, comparable detec-tion was
observed for sequences corresponding to prototype 3, but those
represented by prototype 4 were consistently ampli-fied only at a
concentration that was 2 log units higher. Geno-type II/III
sequences corresponding to prototype 5 could be detected in all
replicates only if the concentration was 1E2, i.e.,

100 copies/ l or higher. For genotype III sequences (prototype
sequence 11), the threshold for consistent detection was 1E4, i.e.,
10,000 copies/ l. The thresholds described are specific for the
assay used and could be altered by modifying the amplifi-cation
conditions. Nonetheless, the comparative data dramat-ically
illustrate the effect of the viral genotype on detection of viral
DNA.
Clinical management of BKV infection depends not only on the
detection of virus, but also on estimation of the viral load in body
fluids. By definition, the amplification efficiency was 100% for
prototype 1, which represents 81 genotype 1a, 1b1, and 1b2
sequences showing a perfect match with PCR primer/ probe
sequences. However, as shown in one prior investigation (5),
quantitation of the viral load was markedly compromised for
variant BKV strains. Thus, at a DNA input of 1E7, or 10,000,000
copies/ l, the calculated viral load for genotype IV sequences was
1.06% of the expected value for sequences represented by
prototype 3 and 2.47% for prototype 4 (Table 3). For genotype II
and III sequences represented by prototype 5, amplification
efficiencies as low as 9.36% were observed. Quantitation of
genotype 1c sequences (prototype 6) was gen-erally not as
severely affected, with the exception of one se-quence from East
Asia (AB245326, prototype 14). Two geno-type 1a sequences,
AB276228 from South Africa (prototype 13) and AB268405 from
Japan (prototype 14), also showed poor amplification. In clinical
practice, changes in the viral load of 10-fold or higher are often
considered to be meaning-ful. Using this criterion, only the
variants corresponding to prototypes 6, 7, 8, and 9 gave
acceptable results, since the calculated copy numbers for other
prototypes were less than 10% of the expected values.
Underestimation of the target copy number was not markedly
dependent on the target con-centration per se, but marked assay
variability was observed for samples tested at the lower end of the
standard curve.
It is notable that in several instances a single-nucleotide primer
mismatch with the target sequence substantially com-promised
detection by PCR. Mismatches at the level of the

h
t
t
p
:
/
/
j
c
m
.
a
s
m
.
o
r
g
/

o
n

A
p
r
i
l

2
8
,

2
0
1
4

b
y

g
u
e
s
t

4076 RANDHAWA ET AL.

probe sequence were less critical. Analogous findings have been
reported in the literature. Thus, single-nucleotide poly-morphisms
have been reported to not always be detrimental to assay
performance (5), while 2 mismatches at the 3 end of a PCR primer
can result in up to 2-log-unit differences in the calculated
cytomegalovirus load (9). Large numbers of mis-matches result in
complete lack of amplification of the viral target sequence (a
false-negative result) (15).
Two previously published studies on the effect of BKV ge-netic
variation on assay performance deserve mention. Hoff-man et al.
used seven different primer/probe sets to perform PCR on urine
specimens and found substantial interassay dis-agreements that
were most striking for genotype III and IV strains (5). Expected
and observed DNA copy numbers varied by as much as 4.2 log
10

templates per reaction. Notably, sig-nificant assay variation was
seen with primers directed at the BKV large T antigen, as well as
VP-1, while the agnogene was not evaluated. Dumoulin and
Hirsch found gene polymor-phisms in the target sequence of their
assay in 32%, 23%, and 82% of sequences corresponding to the
forward primer, re-verse primer, and probe, respectively (3). The
effects of these polymorphisms on amplification of genotype-
specific se-quences were not evaluated. Modification of the PCR
assay using primer or probe sequences with degeneracy at 4
nucle-otide positions was able to correct for the majority of the
observed sequence mismatches. However, no attempt was made to
provide coverage for more divergent variant BKV strains, which
constituted approximately 10% of published se-quences. The
modified PCR assay was found to be comparable to the original
PCR assay, but its performance with respect to detection and
accurate quantitation or uncommon BKV strains other than
genotype I was not evaluated.

In conclusion, our studies indicate that BKV quantitative PCR
assays designed using genotype 1 reference sequences, such as
BKV Dun, do not perform satisfactorily for mutant viral strains.
Detection sensitivity and amplification efficiencies are
particularly compromised for genotypes Ic, II, III, and IV. While
these genotypes are relatively uncommon in the United States,
genotype IV strains occur more frequently in the Far East.
Transplant physicians should suspect variant viral strains when
unexplained graft dysfunction occurs in the setting of low-level
BK viruria and absence of viremia. A high index of suspicion for
the presence of a variant strain should also arise in patients with
rejection-like infiltrates at biopsy that do not respond to steroid
treatment. Sensitive virus detection and accurate quantitation of
the viral load in such patients may be achieved by using alternate
PCR assays targeting a different
J. CLIN. MICROBIOL.

viral gene, employing degenerate primers to account for vari-ant
sequences (3), or by using a multiplex approach that allows
simultaneous amplification of common variant strains (5).

ACKNOWLEDGMENTS
This study was supported by NIH grant RO1 AI 51227 to P.R. The
content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of Health
or the National Institute of Allergy and Infectious Disease.
Jill March provided excellent secretarial assistance.
We have no conflicts of interest.

REFERENCES
1. Brennan, D. C., et al. 2005. Incidence of BK with tacrolimus versus cyclo-
sporine and impact of preemptive immunosuppression reduction. Am. J.
Transplant. 5:582594.
2. Chon, W. J., et al. 2011. A web based survey of U.S. transplant physicians on
BKV surveillance and treatment of BKV nephropathy in renal transplant
recipients. Am. J. Transplant. 11(Suppl. 2):260. (Abstract.)
3. Dumoulin, A., and H. H. Hirsch. 2011. Reevaluating and optimizing polyo-
mavirus BK and JC real-time PCR assays to detect rare sequence polymor-
phisms. J. Clin. Microbiol. 49:13821388.
4. Hirsch, H. H., and P. Randhawa. 2009. BK virus in solid organ transplant
recipients. Am. J. Transplant. 9(Suppl. 4):S136S146.
5. Hoffman, N. G., L. Cook, E. E. Atienza, A. P. Limaye, and K. R. Jerome.
2008. Marked variability of BK virus load measurement using quantitative real-
time PCR among commonly used assays. J. Clin. Microbiol. 46:2671 2680.

6. Krumbholz, A., O. R. Bininda-Emonds, P. Wutzler, and R. Zell. 2009. Phy-
logenetics, evolution, and medical importance of polyomaviruses. Infect. Genet.
Evol. 9:784799.
7. Luo, C., M. Bueno, J. Kant, J. Martinson, and P. Randhawa. 2009. Geno-
typing schemes for polyomavirus BK, using gene-specific phylogenetic trees and
single nucleotide polymorphism analysis. J. Virol. 83:22852297.
8. Nickeleit, V., et al. 2000. Testing for polyomavirus type BK DNA in plasma to
identify renal-allograft recipients with viral nephropathy. N. Engl. J. Med.
342:13091315.
9. Nye, M. B., A. R. Leman, M. E. Meyer, M. A. Menegus, and P. G. Rothberg.
2005. Sequence diversity in glycoprotein B complicates real-time PCR assays
for detection and quantification of cytomegalovirus. J. Clin. Microbiol. 43:
49684971.
10. Ramos, E., et al. 2002. Clinical course of polyoma virus nephropathy in 67 renal
transplant patients. J. Am. Soc. Nephrol. 13:21452151.
11. Randhawa, P. S., et al. 1999. Human polyoma virus-associated interstitial
nephritis in the allograft kidney. Transplantation 67:103109.
12. Sharma, P. M., G. Gupta, A. Vats, R. Shapiro, and P. Randhawa. 2006. A
phylogenetic analysis of polyomavirus BK sequences. J. Virol. 80:88698879.
13. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment through
sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res. 22:46734680.
14. Wadei, H. M., et al. 2006. Kidney transplant function and histological clear-
ance of virus following diagnosis of polyomavirus-associated nephropathy
(PVAN). Am. J. Transplant. 6:10251032.
15. Whiley, D. M., and T. P. Sloots. 2005. Sequence variation in primer targets
affects the accuracy of viral quantitative PCR. J. Clin. Virol. 34:104107.
16. Yogo, Y., C. Sugimoto, S. Zhong, and Y. Homma. 2009. Evolution of the BK
polyomavirus: epidemiological, anthropological and clinical implications. Rev.
Med. Virol. 19:185199.
17. Zheng, H. Y., et al. 2007. Relationships between BK virus lineages and human
populations. Microbes Infect. 9:204213.



















D
o
w
n
l
o
a
d
e
d

f
r
o
m


h
t
t
p
:
/
/
j
c
m
.
a
s
m
.
o
r
g
/

o
n

A
p
r
i
l

2
8
,

2
0
1
4

b
y

g
u
e
s
t