Fig 1.
Tissue blocking and experimental scheme.
Fixed postmortem sections corresponding to T2 brain MRI images at 6 weeks post-presentation, ~1.5 weeks prior to death. For indicated brain regions, L, PL, and NL tissue blocks, designated L1–3, PL1–3, and NL1–3, were studied as indicated. T2 - T2-weighted; qPCR—quantitative PCR; MRM/MS–multiple reaction monitoring mass spectrometry; IHC–immunohistochemistry; IF–immunofluorescence; ISH—in-situ hybridization.
Fig 2.
Spectrum of PML histopathology.
H&E (A-C) and IHC (brown, with blue hematoxylin counterstain) for NFP to label neuronal processes (D-F), MBP to label myelin (G-I), GFAP to label astrocytes (black arrows) (J-L), and IBA1 to label microglia/macrophages (blue arrowheads) (M-O) from grossly unaffected, actively infected, and end-stage white matter (blocks NL3, PL3, and L3, respectively). NL3 (left column) had largely normal white matter composed of linear axons/myelin throughout the neuropil, oligodendrocytes with characteristic perinuclear “fried egg” halos (green arrowheads), astrocytes with thin and elongated processes, and microglia with short, thick processes. PL3 (middle column) showed active infection with viral nuclear inclusions (red arrows), hypertrophied astrocytes with thickened processes, swollen myelin, and some enlarged macrophages (this section also had foci of demyelination, not shown here). L3 (right column) had end-stage lesions with rare viral inclusions, reduced number and fragmentation of axons, residual axonal myelin (magenta arrowhead), and variable amounts of MBP within engorged macrophages and massively hypertrophied (“bizarre”) astrocytes. Asterisks in B and C designate thinning of neuropil secondary to axonal loss. Scale bar = 30 μm for all panels.
Table 1.
Qualitative histopathologic features of each tissue block.
Fig 3.
Quantification of JCV DNA and VP1 capsid protein.
Log-log plot of the concentration of JCV genomes vs capsids per microliter tissue for all 9 PML blocks (Table 2). Data are means ± standard deviation based on triplicate measurements. Blue dashed line indicates theoretical relationship for one capsid per viral genome. Linear regression reveals a positive relationship with r2 = 0.96, p<0.0001 (regression line not shown). Asterisk designates the uncertainty of the protein measurement of VP1 in NL3, which was below the assay’s lower limit of quantitation (not shown).
Table 2.
Quantitation of virus components from each tissue block.
Fig 4.
(A) Schematic of circular viral DNA showing sites of transcription initiation and directions of early and late transcription (arrows) adjacent to the NCCR. Regions encoding Early T-Ag (T and t) and late agnoprotein (Agno) and VP1, VP2, and VP3 proteins are indicated by colored boxes. (B) Summary of NCCR variants, with the archetype NCCR shown for reference consisting of an origin of replication (ORI) followed by intact sequence blocks A-F. The percent of sequences showing the schematized rearrangements, duplications, and/or mutant/deleted sequence blocks (indicated by lower case letters) are listed. (C) VP1 variants identified. WT (1B) = wild type, strain 1B. (D) Agnoprotein variants identified; del(51-end) = deletion of C-terminal 21 amino acids. (E) VP2 variants identified; del(283-end) = deletion of C-terminal 60 amino acids. P174S and R207G were identified in the VP1 amplicon, which overlaps the C-terminal region of VP2/3, with asterisks designating percent of VP1 region clones that harbored the VP2 mutation. Representative DNA sequences and alignments are shown in S1 File, and have been deposited at NCBI under accession nos. KX216358-KX216371.
Fig 5.
Histopathology of demyelinated foci.
Adjacent sections of NL1 stained with H&E (A), IHC for VP1 using PAB597 at 0.01 μg/mL (B) or 1.0 μg/mL (C), NFP (D), MBP (E), or IBA1 (F). Thumbnail image in right upper corner of each panel shows low magnification image of stained section. Grey (G) / white (W) matter junction is denoted by a dashed line, and asterisks designate area around the focus of demyelination magnified in each panel. VP1 antibody at 0.01 μg/mL detects individually infected cells (red arrow, B), whereas 1 μg/mL additionally reveals broadly distributed VP1 (red asterisk, C), with no VP1 staining in nearby uninfected white matter (black asterisk) or gray matter. In demyelinated foci, there is no decrease in NFP stain (D) in areas with reduced MBP (E), and increased numbers of IBA+ engorged macrophages (arrowhead, F). Scale bar = 50 μm.
Fig 6.
Spatial correlation of dispersed VP1 and early demyelination.
A) Thumbnail IHC images of each block stained for VP1 (top row, left to right based on ranking from highest to lowest VP1 stain index) and MBP (bottom row). Linear plots of (B) JCV concentration vs. the percentage of section area stained darkly with VP1 at 0.01 μg/ml PAB597 (“VP1 stain index”), (C) JCV DNA concentration vs. the fraction of section area positive for MBP stain (“MBP stain index”) as a measure of residual myelin, and (D) MBP vs. VP1 stain indices. There were positive, statistically significant relationships between VP1 stain index and JCV genome or capsid concentrations (p<0.0001 for both lines in panel B), but not between MBP stain index and JCV concentrations (C), or between VP1 and MBP stain indices (D), with r2 and p values in boxes. E-E”) NL1 stained for VP1 (green; gray scale image of channel in panel E’), MBP (red; gray scale image of channel in panel E”), and DNA (blue) with fluorescence detection. White box on thumbnail image in right upper corner of panel E shows magnified area in E-E”, with gray (G) and white (W) matter regions designated. Demyelinated foci (red asterisks) are spatially coincident with dispersed VP1, whereas numerous VP1-positive cells (green arrowheads) can be seen in areas with intact myelin.
Fig 7.
Distinct VP1 distributions in gray and white matter.
VP1-stained NL1 (A) and L3 (B) with gray (G) and white (W) matter regions labeled, and black arrowheads (and line in A) demarcating the gray/white matter boundary. Red boxes in panels A and B show areas magnified below in panels A' and B', with blue arrows designating VP1-positive late stage (A’) or recently lysed (B’) cells. Note extensive linear VP1 throughout white matter (e.g., blue arrowheads in A') and little to no dispersed VP1 in gray matter. (C, D) JCV DNA distribution in PML white matter. RNAScope ISH for early (C) or Late (D) JCV probes labels individually infected cells with oligodendrocytic morphology (red and green arrows) as well as linear structures consistent with axons (red and green arrowheads). Scale bar = 5 mm in A, B; 100 μm in A', B'; C, D.
Fig 8.
VP1 colocalizes with nearby myelin in recently lysed cells.
Representative 5-μm confocal Z-axis stack images of regions of recent virus-induced cell lysis in otherwise intact white matter (A, B, D, E) or gray matter (C) of human PML, or white matter in an SIV-positive rhesus macaque with SV40 PML-like CNS disease (F), stained for VP1 (green in A-F, A″–F″, and A*-F*) and MBP (red in A-C, F, A′–C′, F′, and A*-C*, F*), GFAP (red in D, D′, D*), or IBA1 (red in E, E′, E*). A*–F* show representative colocalization (coloc) images from a single image from each Z-stack, with magenta pixels designating VP1/MBP colocalization (magenta arrows); Image quantitation of these representative Z-stack images revealed 89% and 86% of VP1 colocalized with MBP in white matter (A* and B*, respectively); 34% colocalized in gray matter (C*); and 13% and 2.9% of VP1 colocalized with GFAP or IBA1 (D* and E*, respectively). In SV40 PML-like disease (F), a subset of VP1 was dispersed in a linear pattern and colocalized with partially demyelinated MBP-positive axons (magenta arrow in F*). Occasional cytoplasmic VP1 viral aggregates were seen in cytoplasm of microglia (green arrow in E) or astrocytes, but nuclei of these cell types did not show VP1 positivity that would be indicative of productive infection. Scale bar in panel A = 10 μm.
Fig 9.
Histopathology and virus protein distributions in SV40 PML-like disease.
Representative section of macaque cerebrum was fixed, stained for H&E (A, D, G), TAg (B, E, H), or VP1 (C, F, I). (A-C) Black asterisks mark cortical area with severe demyelination, and red asterisks mark corpus callosum. Gray (G) and white (W) matter regions are designated, with areas in red and black boxes in A-C magnified in D–F and G–I, respectively. Note foci of infection within white matter (blue arrow) and tracking along gray-white junction (blue arrowheads). (D-F) Magnified area of infected gray-white matter junction, showing actively infected area with TAg-positive cells (red arrow) and VP1-positive infected cells with dispersed VP1 (red arrowhead). (G–I) End-stage lesion showing dispersed TAg and VP1 amid sheets of macrophages (black arrowhead). Inset images in lower right corners of D-I show low magnification view of entire tissue section. Scale bar: 5 mm in A–C, 100 μm in D–I.
Fig 10.
JCV protein distributions during the viral life cycle.
Representative confocal images of JCV-infected white matter oligodendrocytes at various stages of infection, stained for TAg (red), VP1 (green), and DNA (blue), from early to late stages (left to right). Early infection (left two columns) is marked by nuclear foci of TAg (arrow), which increase in number as the nucleus enlarges. Cells transitioning between early and late stages (middle column) show abundant nuclear TAg foci with peripheral nuclear VP1 denoting early viral assembly (arrow). At late stages of infection (right two columns), TAg is absent, and VP1 fills the cytoplasm and proximal extent of some cell processes but does not appear to extend to processes that ensheathe nearby axons (arrow). Cytoplasmic lipofuscin (arrowheads) in some cells was captured with TAg staining with AF594 detection (red channel). Scale bar = 10 μm.
Fig 11.
White matter “wave” of JCV infection.
(A-D) Top of each panel shows low magnification views of sequential sections of block L1, stained for H&E (A) or indicated IHCs (B-D), with higher magnification of region designated by red box shown in bottom of each panel. Black asterisk designates confluent area of demyelination behind wave; blue asterisks designate two foci of MBP pallor ahead of VP1 wave, indicative of recent demyelination. Panel D is a dual IHC, revealing TAg with alkaline phosphatase/fast red detection (red arrows) and VP1 with HRP/DAB detection (green arrow). (E) Schematic of four zones (1–4) relative to wave front of dispersed virus (blue line demarcated by blue arrowheads at the edge of the section) overlying the tissue image in panel D following manual segmentation of TAg-positive cells (red dots) and VP1-positive cells (green dots). Orange arrows indicate putative direction of wave movement through the section. Graphs show cell counts for TAg (red) and VP1 (green) (bottom left), and ratio of early (TAg positive) to late (VP1 positive) infected cells as a function of zone (bottom right). (F) Magnified area of wave front indicated by black box in panels D and E, with TAg-positive cells false-colored red, VP1-positive cells false-colored green, and wave front (gray line) inferred to be progressing in the direction indicated by orange arrow. G–G"–confocal image at wave front stained for TAg (red), VP1 (green), and DNA (blue), with orange arrow showing direction of wave. (Punctate, non-nuclear red stain represents nonspecific autofluorescence due to endogenous lipofuscin.) Scale bar in G" = 2 mm in top panels of A-E, 50 μm in bottom panels of A–D and in G–G" and 70 μm in F.
Fig 12.
Model for PML propagation in white matter from this case.
JCV (green dot) infects myelinating oligodendrocytes (blue, with processes ensheathing axons), which progress from early (red, TAg) to late (green, VP1) infection. Some virus spreads intracellularly within oligodendroglial processes toward the myelin sheath. Infected oligodendrocytes lyse and most viral aggregates associate with nearby myelin, leading to extracellular virus dispersion along white matter tracts (large green arrow), demyelination, and propagation by infection of distant oligodendrocytes (small black arrow). Variable amounts of myelin (blue) and virus are seen in association with astrocytes (magenta), microglia (orange), and macrophages (gray).