Single-Molecule Sequencing Revealing the Presence of Distinct JC Polyomavirus Populations in Patients With Progressive Multifocal Leukoencephalopathy

Seppälä, Hanna; Virtanen, Elina; Saarela, Mika; Laine, Pia; Paulín, Lars; Mannonen, Laura; Auvinen, Petri; Auvinen, Eeva

doi:10.1093/infdis/jiw399

Cited by 13 publications

(17 citation statements)

References 43 publications

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“…NCCR structure organization is important because of the association of the rearranged variants with specific human diseases, such as PML for JCPyV and nephropathy for BKPyV [52–55]. In light of that, we investigated the sequence variability of the HPyVs NCCRs in the gastrointestinal tract of hematological patients, in effort of advancing our understanding of HPyVs biology.…”

Section: Discussionmentioning

confidence: 99%

Polyomaviruses shedding in stool of patients with hematological disorders: detection analysis and study of the non-coding control region’s genetic variability

Prezioso

Ciotti

Obregón

et al. 2019

Med Microbiol Immunol

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Fragmented data are available on the human polyomaviruses (HPyVs) prevalence in the gastrointestinal tract. Rearrangements in the non-coding control region (NCCR) of JCPyV and BKPyV have been extensively studied and correlated to clinical outcome; instead, little information is available for KIPyV, WUPyV and MCPyV NCCRs. To get insights into the role of HPyVs in the gastrointestinal tract, we investigated JCPyV, BKPyV, KIPyV, WUPyV and MCPyV distribution among hematological patients in concomitance with gastrointestinal symptoms. In addition, NCCRs and VP1 sequences were examined to characterize the strains circulating among the enrolled patients. DNA was extracted from 62 stool samples and qPCR was carried out to detect and quantify JCPyV, BKPyV, KIPyV, WUPyV and MCPyV genomes. Positive samples were subsequently amplified and sequenced for NCCR and VP1 regions. A phylogenetic tree was constructed aligning the obtained VP1 sequences to a set of reference sequences. qPCR revealed low viral loads for all HPyVs searched. Mono and co-infections were detected. A significant correlation was found between gastrointestinal complications and KIPyV infection. Archetype-like NCCRs were found for JCPyV and BKPyV, and a high degree of NCCRs stability was observed for KIPyV, WUPyV and MCPyV. Analysis of the VP1 sequences revealed a 99% identity with the VP1 reference sequences. The study adds important information on HPyVs prevalence and persistence in the gastrointestinal tract. Gastrointestinal signs were correlated with the presence of KIPyV, although definitive conclusions cannot be drawn. HPyVs NCCRs showed a high degree of sequence stability, suggesting that sequence rearrangements are rare in this anatomical site.

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Section: Discussionmentioning

confidence: 99%

Polyomaviruses shedding in stool of patients with hematological disorders: detection analysis and study of the non-coding control region’s genetic variability

Prezioso

Ciotti

Obregón

et al. 2019

Med Microbiol Immunol

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“…This study adds to the many uses of next-generation sequencing in the clinical virology laboratory ( 30 , 31 ). The main limitation of our study is the use of short-read sequencing to sequence the strain, as we are thus unable to link rearrangements across the multiple viruses present in each standard ( 32 ). Deep sequencing provides single-nucleotide resolution of the sequences present and the relative copy numbers of loci across the genome.…”

Section: Discussionmentioning

confidence: 99%

Copy Number Heterogeneity of JC Virus Standards

et al. 2017

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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.

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“…Although the f section was initially excluded from the classification of NCCR variants for purposes of simplification [ 25 ], these results suggest that its role in the evolutionary outcome of JCV infection may be underestimated. Indeed, the deletion of the f section has been previously described in several patients, mostly combined with duplications [ 15 , 24 , 36 , 55 , 56 ], and rarely as a single rearrangement [ 32 , 34 , 37 , 38 , 41 , 52 , 57 ]. Since NCCRs from urine and CSF samples from Patient 1 were very similar except for the ef Δ66 deletion, they represent a good model to investigate the importance of the f region in the context of a naturally occurring polymorphism.…”

Section: Resultsmentioning

confidence: 99%

Exploring the role of NCCR variation on JC polyomavirus expression from dual reporter minicircles

L'Honneur¹,

Leh²,

Laurent-Tchenio³

et al. 2018

PLoS ONE

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JC virus (JCV), a ubiquitous human polyomavirus, can cause fatal progressive multifocal leukoencephalopathy (PML) in immune compromised patients. The viral genome is composed of two conserved coding regions separated by a highly variable non-coding control region (NCCR). We analyzed the NCCR sequence from 10 PML JCV strains and found new mutations. Remarkably, the NCCR f section was mutated in most cases. We therefore explored the importance of this section in JCV expression in renal (HEK293H) and glioblastoma (U-87MG) cell lines, by adapting the emerging technology of DNA minicircles. Using bidirectional fluorescent reporters, we revealed that impaired NCCR-driven late expression in glioblastoma cells was restored by a short deletion overlapping e and f sections. This study evidenced a relevant link between JCV NCCR polymorphism and cell-type dependent expression. The use of DNA minicircles opens new insights for monitoring the impact of NCCR variation.

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Single-Molecule Sequencing Revealing the Presence of Distinct JC Polyomavirus Populations in Patients With Progressive Multifocal Leukoencephalopathy

Abstract: We report here, for the first time, full characterization of individual neurotropic JCPyV strains in the cerebrospinal fluid of patients with PML. It remains to be established whether PML pathogenesis is driven by one or several neurotropic strains in an individual.

Cited by 13 publications

References 43 publications

Polyomaviruses shedding in stool of patients with hematological disorders: detection analysis and study of the non-coding control region’s genetic variability

Polyomaviruses shedding in stool of patients with hematological disorders: detection analysis and study of the non-coding control region’s genetic variability

Copy Number Heterogeneity of JC Virus Standards

Exploring the role of NCCR variation on JC polyomavirus expression from dual reporter minicircles

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