Figure 1.
Outline of the experimental work presented.
JC virus DNA was detected in urine of 63 out of 254 healthy subjects (HSs) by qPCR targeting the large T antigen (LTAg) gene. For 61 viral shedders the full length VP1 coding sequence and the non-coding control region (NCCR) DNA was obtained via Sanger sequencing. When compared to a reference archetype NCCR (i.e. CY isolate, NCBI acc.nr. AB038249) 44 samples did not contain any rearrangement while 15 samples (24.6%) harbored a deletion (between 1 and 28 bp), 1 sample (1.6%) carried a duplication in the consensus NCCR sequence and 1 sample carried both an insert and a deletion. A subset of 54 urine JCV DNA samples were further analyzed by next generation sequencing (454 pyrosequencing) to specifically look at the presence of JCV quasispecies that were identified in 2 out of 40 archetype samples (5.0%) and 2 out of 12 samples (16.6%) that contained a deletion when analyzed by Sanger sequencing. No quasispecies were detected in both samples carrying a duplication in the consensus NCCR.
Table 1.
Overview of JC virus NCCR rearrangements (i.e. deletions and insertions) identified by Sanger sequencing and 454 sequencing.
Figure 2.
Phylogenetic analysis based on the VP1 coding sequence from healthy subjects.
The full length VP1 coding sequence (1065 bp) was obtained for all healthy subjects for which also the non-coding control region (NCCR) was Sanger sequenced (n = 61). The relatedness of the HS samples to defined JC virus genotypes is illustrated in a phylogenetic tree. For each JCV genotype the following reference VP1 coding sequences were used (NCBI accession number between brackets; reference sequences taken from [26]): genotype 1A (AF015526), genotype 1B (AF015527), genotype 2A (AF015529), genotype 2B (AF015533), genotype 2C (AF015535), genotype 2D (AF015536), genotype 2E (AF281606), genotype 3A (U73500), genotype 3B (U73501), genotype 4 (AF015528), genotype 6 (AF015537), genotype 7 (U61771). Healthy subjects (HS) in which deletions or insertions were identified in the non-coding control region via Sanger sequencing are indicated by * (deletion) or + (insertion). HSs in which JCV quasispecies were identified by 454 sequencing are circled.
Figure 3.
Validation of JC virus quasispecies by nested PCR.
(A) A nested PCR approach was developed to validate the presence of the 28 bps deletion identified in ∼1.5% of the viral DNA sequences in HS11. (B) The nested PCR approach was applied on viral DNA extracted from urine from HS11. Urine aliquots donated at different time points were included: T0, T1, T2 and T3. Viral DNA extracted from HS6 T1 was included as a negative control, together with a no template control (NTC). The upper panel shows successful amplification of the consensus DNA fragment in all samples. In contrast, only in HS11 T1 a PCR fragment could be amplified when using the reverse primer spanning the deletion instead of the consensus reverse primer, confirming the presence of this deletion. L = 25 base pair DNA ladder (Invitrogen). (C) A similar nested PCR approach was used to demonstrate the existence of quasispecies in HS53. DNA fragments were generated when a primer set designed to amplify a consensus sequence from both HS53 and HS26 (lanes HS53 con and HS26 con) were used. When the reverse primer was replaced by a primer specifically targeting the sequence deleted in the quasispecies succesfull amplification was only detected in HS53 (HS53 del), but not in HS26 (HS26 del). NTC: no template control. L = 25 base pair DNA ladder (Invitrogen).