BACKGROUND: Characterizing the genomic plasticity of BKV will further our understanding of viral epidemiology, diagnosis, and clinical outcome, and facilitate development of vaccines and drug therapy. Therefore, this study investigated if BKV to virus neutralizing antibodies, cidofovir or leflunomide leads to adaptation evidenced by progressive selection of mutants.

METHODS: BKV Gardner strain was grown in culture in the presence of cidofovir (5-15 ¯o;g/ml), leflunomide(20 ¯o;g/ml) and human intra-venous immunoglobulins (IVIG) containing virus neutralizing antibodies (10 ¯o;g/ml) for up to 4 months, to simulate clinically relevant drug exposure. Genetic adaptation of BKV was assessed by ultra deep pyrosequencing of viral capsid protein-1 (VP-1) and large T antigen (LTA) genes using the Roche 454 platform. Consensus sequences were aligned and reads mapped to the consensus using Bowtie 2.Variant sequences were identified by Samtools 1.18 & VarScan 2.2. All variants identified had a minimum quality value of 15. The minimum coverage was set to 1, minimum supporting reads to 1, and minimum variant frequency at 1e-12, to allow detection of rare variants while correcting for sequencing errors.

RESULTS: The number of sequences obtained varied from 9590 to 29493 per sample with an average read length of 213. Putative mutant populations were identified at very low abundances (average 0.96%). Sequence variations (3-23 substitutions, 1-8 quasispecies) were observed in both IVIG and cidofovir treated samples, but no consistent temporal changes were observed. The number of cidofovir associated LTA mutations increased with time from 3 to 23. DNA derived from sequential urine samples from a patient with BKV nephropathy showed 39-50 variant nucleotides, including synonymous (VP-1 position 45) and non-synonymous (LTA positions 26, 77, 345, 571) changes at putative T-cell epitopes. The number of quasispecies assigned by QuRe v0.995 ranged from 6-8 for VP-1 and 2-3 for LTA.

CONCLUSIONS: The BKV genome does not readily adapt in-vitro to the presence of neutralizing antibodies and drugs affecting viral replication. Serial analyses of clinical urine samples shows viral VP-1 and LTA quasispecies with mutant T-cell epitopes, consistent with selection pressure exerted by cell mediated immunity.

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