Optimized amplification of BK polyomavirus in urine

Abstract

BK polyomavirus (BKPyV) is a ubiquitous pathogen that typically results in asymptomatic infection. However, in immunocompromised individuals, BKPyV viral shedding in the urine can reach 10⁹ copies per mL. These high viral levels within urine provide ideal samples for next-generation sequencing to accurately determine BKPyV genotype and identify mutations associated with pathogenesis. Sequencing data obtained can be further analyzed to better understand and characterize the genetic diversity present in BKPyV. Here, methods are described for the successful extraction of viral DNA from urine and the subsequent amplification methods to prepare a sample for next-generation sequencing.

Keywords: BK polyomavirus (BKPyV); Diversity; Genotype; Rolling circle amplification; Variation.

Fig. 1.

Comparison of RCA methods followed by a 2 h BamHI digestion, visualized on 1% agarose gel. Input DNA was 1 μL in all cases. Subjects 575 and 233 had high viral load samples, while subjects 001 and 028 had low viral loads. Both methods yielded 5 kb bands, although the EquiPhi method had more background. NTC = no template control.

Fig. 2.

Amplification of low viral load patient samples using TempliPhi RCA protocol with varying volumes of input DNA (1, 2, 3 μL). Following BamHI restriction enzyme digest and full-length PCR imaged on a 1% agarose gel. In low viral load subjects, increasing the input DNA to RCA resulted in a visible downstream product in some cases. NTC = no template control.

Fig. 3.

BamHI restriction enzyme digest after TempliPhi RCA. Each digestion contained 1 μL of BamHI, 2.5 μL of buffer, 1 μL of RCA product, and 20.5 μL of water. Reaction conditions were 37 °C for 1, 2, 4, or 24 h, and results were visualized on a 1% agarose gel.

Fig. 4.

Serial dilutions of DNA extracted from a high viral load patient was performed to 1:1b. A) RCA of diluted samples was performed with TempliPhi, followed by a 1 h BamHI digestion. Visible 5 kb products were observed for the first 3 dilutions (1:100). B) The products of A were used as input for full-length PCR, and visible 5 kb products were observed up to the 1:1 m dilution. C) The products of B were used as input for a nested VP1 PCR. Visible 1.6 kb products were observed up to the 1:1 m dilution. NTC = no template control.

Fig. 5.

Subject 717 urine (U) and plasma (P) samples were processed on two separate occasions (labeled 1 or 2). Samples were submitted for next-generation sequencing after RCA and BamHI digest (A), that product run on a 1% agarose gel, the 5 kb band was cut out and DNA extracted (B) or following full-length PCR, 1% agarose separation and extraction (C). Sequences were compared to representative BKPyV sequences (labeled by GenBank accession number and country of origin). All 717 sequences branch from the node circled. Evolutionary distance between these sequences is 0.17 %.