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 sequences (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 (). 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 spectrum of sequences that includes genotypes II, III, and IV at coverage frequencies that are summarized in . 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 mismatches between the viral prototype and PCR primer/probe sequences are depicted in and enumerated in , which shows the relative amplification efficiencies of the different prototype sequences.
GenBank accession numbers of 184 publicly available BKV sequences classified into 14 prototypes
Frequency distribution of BKV genotypes I to IV as represented by prototype sequences 1 to 14a
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.
Comparative amplification efficiencies of prototypes 1 to 14
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/μl (10,000 copies/ml). This is clinically relevant, since plasma BKV loads of this magnitude have been used as a trigger to lower immunosuppression and initiate antiviral therapy. Notably, at lower concentrations, namely, 1 copy/μl, detection of prototype 1 was not possible in 1 of 3 replicates (). Prototype 3, which covered the majority of genotype IV strains, behaved essentially 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 copies/μl and 1E1, i.e., 10 copies/μl. 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/μl or higher. At concentrations of 1E1, i.e., 10 copies/μl, 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/μl, 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 depends principally on two factors: (i) the linearity of the standard curve (prepared using the BKV Dun strain plasmid in our assay) and (ii) the amplification efficiency of the target sequence. Both these factors appear to contribute to the prototype-specific variations seen in our experimental system. The linear part of a real-time PCR standard curve is characterized by a CT difference of approximately 3.3 between serial 10-fold dilutions of the plasmid standard. Using this yardstick, CT measurements were in the linear range for BKV Dun prototype 1 at all concentrations up to 1E2, i.e., 100 copies/μl. All values outside the linear range are indicated in boldface in . 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/μl. 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/μl; 3/13 prototypes at concentrations ≥1E2, i.e., 100 copies/μl; and 0/13 prototypes at concentrations ≤1E1, i.e., 10 copies/μl. Amplification efficiency was also significantly reduced for BKV variants (prototypes 2 to 14) expressed as a percentage of the BKV Dun reference (prototype 1), which was assumed to represent 100% efficiency (). Thus, for prototype 2, the viral-DNA yield was 13.31% of the expected value at 1E7copies/μl and 15.51% of the expected value at 1E6, i.e., 1,000,000 copies/μl. For measurements in the lower nonlinear part of the standard curve (boldface in ), the calculated yields were quite variable and inaccurate, which explains why the mean of several calculated results was >100% of input DNA.
Finally, the experiments conducted illustrate that the amplification 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 sequences is shown in , including the locations of all variant nucleotides in relation to the PCR primers and probe. 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 mismatches. 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 probe mismatch, underestimation of the target copy number was less pronounced and observed deviation was within 5-fold of the expected value.