Sequencing and analysis of JC virus DNA from natalizumab-treated PML patients

Abstract

Background: Progressive multifocal leukoencephalopathy (PML) in natalizumab-treated MS patients is linked to JC virus (JCV) infection. JCV sequence variation and rearrangements influence viral pathogenicity and tropism. To better understand PML development, we analyzed viral DNA sequences in blood, CSF and/or urine of natalizumab-treated PML patients.

Methods: Using biofluid samples from 17 natalizumab-treated PML patients, we sequenced multiple isolates of the JCV noncoding control region (NCCR), VP1 capsid coding region, and the entire 5 kb viral genome.

Results: Analysis of JCV from multiple biofluids revealed that individuals were infected with a single genotype. Across our patient cohort, multiple PML-associated NCCR rearrangements and VP1 mutations were present in CSF and blood, but absent from urine-derived virus. NCCR rearrangements occurred in CSF of 100% of our cohort. VP1 mutations were observed in blood or CSF in 81% of patients. Sequencing of complete JCV genomes demonstrated that NCCR rearrangements could occur without VP1 mutations, but VP1 mutations were not observed without NCCR rearrangement.

Conclusions: These data confirm that JCV in natalizumab-PML patients is similar to that observed in other PML patient groups, multiple genotypes are associated with PML, individual patients appear to be infected with a single genotype, and PML-associated mutations arise in patients during PML development.

Figure 1.

Example of NCCRs from different biofluids of 3 natalizumab-treated PML patients. A, Archetype and Mad-1 NCCRs (shown for comparison); B, PML-012; C, PML-006; and D, PML-032. Urine-derived sequences always matched archetype, while sequences from CSF and blood were often identical to each other or contained related patterns of rearrangement. Letter designations and numbering for NCCR segments are described in Table 1 and in Methods section. CSF, cerebrospinal fluid; NCCR, noncoding control region; PML, progressive multifocal leukoencephalopathy.

Figure 2.

The NCCR from CSF or blood of PML patients frequently contained deletion of all or part of region D. A, Schematic of region D from CSF samples. B, schematic of region D from blood samples. Numbering according to Yogo and colleagues and Frisque and colleagues [20, 37]. CSF, cerebrospinal fluid; NCCR, noncoding control region; PML, progressive multifocal leukoencephalopathy.

Figure 3.

PML-associated mutations develop within individual hosts. Comparison of VP1 and NCCR sequences from whole genomes of a natalizumab-treated PML patient (PML-006). Analysis of virus from different biofluids showed that this patient was infected with a single viral genotype and that PML-associated mutations were present in virus found outside of the urine. While multiple VP1 capsid mutations were observed, all mutated viral species contained identical NCCR rearrangements. NCCR, noncoding control region; PML, progressive multifocal leukoencephalopathy; WT, wild type.