Copy Number Heterogeneity of JC Virus Standards

Abstract

Quantitative PCR is a diagnostic mainstay of clinical virology, and accurate quantitation of viral load among labs requires the use of international standards. However, the use of multiple passages of viral isolates to obtain sufficient material for international standards may result in genomic changes that complicate their use as quantitative standards. We performed next-generation sequencing to obtain single-nucleotide resolution and relative copy number of JC virus (JCV) clinical standards. Strikingly, the WHO international standard and the Exact v1/v2 prototype standards for JCV showed 8-fold and 4-fold variation in genomic coverage between different loci in the viral genome, respectively, due to large deletions in the large T antigen region. Intriguingly, several of the JCV standards sequenced in this study with large T antigen deletions were cultured in cell lines immortalized using simian virus 40 (SV40) T antigen, suggesting the possibility of transcomplementation in cell culture. Using a cutoff 5% allele fraction for junctional reads, 7 different rearrangements were present in the JC virus sequences present in the WHO standard across multiple library preparations and sequencing runs. Neither the copy number differences nor the rearrangements were observed in a clinical sample with a high copy number of JCV or a plasmid control. These results were also confirmed by the quantitative real-time PCR (qPCR), droplet digital PCR (ddPCR), and Sanger sequencing of multiple rearrangements. In summary, targeting different regions of the same international standard can result in up to an 8-fold difference in quantitation. We recommend the use of next-generation sequencing to validate standards in clinical virology.

Keywords: BKV; JC virus; T antigen; clinical standards; deep sequencing; polyomavirus; qPCR; simian virus 40.

FIG 1

Next-generation sequencing of JC virus standards reveals deletions and copy number heterogeneity. (A) Six different JC virus materials were deep sequenced, and five standards were tested by qPCR and ddPCR. Gene organization of each JC virus is shown in green, with regulatory regions depicted in orange. Of note, the 9.4-kb pMAD1 plasmid inserted the backbone at the location of the probe used in the T antigen qPCR and ddPCR assay and cannot be quantitated at that locus. (B) Coverage plot of six different standards of JC virus mapped to the JC virus NCBI reference genome (GenBank accession number NC_001699). The y axis is normalized such that 1 designates the average coverage across the viral genome to highlight relative differences in coverage. Three of the six standards include a large deletion in the T antigen region that constitutes a greater than 4-fold difference in copy number relative to the structural genes. Reductions in coverage in the regulatory repeat region are due both to small deletions and sequence divergence relative to JC virus reference genome. Primers for the Focus PCR analyte-specific reagent targeting the VP2/3 region are shown on the JC virus genome in red, while the pep primers targeting the T antigen region are shown in blue. (C, D) Confirmation of the copy number differences seen by sequencing was performed with qPCR (C) and ddPCR (D) using PCR primers against the VP2/3 gene (red) and T-ag gene (blue). Ten-fold dilutions of each of the standards depicted were quantitated, and the discrepancy in cycle threshold (C_T) and absolute quantitation (D) between the VP2/3 and T-ag assays are depicted (green) for each standard. ΔC_T, change in C_T.

FIG 2

Sanger confirmation of junction reads from next-generation sequencing data. (A) Gel electrophoresis of PCR products amplified with primers between nucleotides 2416 and 4543 based on the JC virus reference genome in NCBI (GenBank accession number NC_001699). NTC, no template control. (B) Expected PCR amplicons in control materials used in this study based on nucleotide distance. The JC virus plasmid pMAD1 contains a 4-kb backbone insert within this PCR amplicon. (C) The PCR amplicon of 340 bp recovered from the WHO standard demonstrates one of the large deletions in the T antigen region that was first identified by next-generation sequencing data. (D) The PCR amplicon of 2,100 bp demonstrates no deletion in the T antigen region in a JC virus from a clinical urine specimen. (E) The PCR amplicon of 700 bp demonstrates a complex rearrangement in the Exact v2 standard and JCV ATCC 1397 strain that was first identified by next-generation sequencing data. (F) Junctional reads with more than 5% allele frequency from the deep sequencing of the WHO JC virus standard are depicted.