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Progressive multifocal leukoencephalopathy Investigation of three cases using in situ hybridization with JC virus biotinylated DNA probe.

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Progressive Multhocal Leukoencephalopathy:
Investigation of Three Cases Using In Situ
Hybridization with JC Virus Biotinylated
DNA Probe
Allen J. Aksamit, MD, Pascale Mourrain, BA, John L. Sever, MD, PhD, and Eugene 0. Major, PhD
Using the technique of in situ DNA-to-DNA hybridization, a JC virus biotinylated DNA probe was developed and
applied to formalin-fixed, paraffin-embedded, or fixed, frozen sections of brain tissue from three subjects with progressive multifocal leukoencephalopathy (PML). Light microscopy was carried out to correlate the presence of JC virus
DNA with the selective infection of oligodendrocytes and astrocytes in PML. Oligodendrocytes (lytically infected)
showed the greatest evidence of viral DNA. More astrocytes showing bizarre morphological changes had evidence of
viral DNA than did astrocytes that were simply reactive. Viral DNA was not evident in vascular endothelial cells using
this technique. Viral DNA replication may be an important initial step which produces the bizarre “transformed”
astrocytes of PML. Findings in this study do not support the hypothesis that vascular endothelial replication is
important in the pathogenesis of JC virus-induced PML. In situ hybridization with biotinylated JC virus probe may be
useful in the diagnosis of PML on brain biopsy specimens.
Aksamit AJ, Mourrain P, Sever JL, Major EO: Progressive multifocal leukoencephalopathy:
investigation of three cases using in situ hybridization with JC virus biotinylated DNA probe.
Ann Neurol 18:490-496, 1985
Progressive multifocal leukoencephalopathy (PML) is a
subacute multifocal demyelinating disease that has
been recognized for more that 25 years 11). Though
rare, the disease usually occurs in patients who have
some form of immunosuppressive illness. A human
papovavirus, designated JC Y ~ Y U S has
been implicated
as the etiological agent in all but two published cases
15, 20, 22, 28). In the two exceptions, a related virus,
simian virus 40 (SV40),was found 127).
Neuropathologically, the disease has been called an
“oligodendrocytopathy” because of the characteristic
changes in the infected oligocytes 124). Also, some
astrocytes undergo a characteristic bizarre morphological change similar to that seen in astrocytomas. This
finding has resulted in speculation that these cells are
“transformed,” a phenotype that can be induced by
oncogenic viruses 11, 10, 28). Virions, however, have
not been visualized by electron microscopy in the
“transformed” cells of PML 116, 25, 28). JC virus has
been shown to induce central nervous system neoplasms without virion production when injected into
experimental animals 112, 291.
Materials and Methods
The clinical and electron microscopic findings for the three
study cases are summarized in Table 1. Patient 1 was 51
years old when diagnosed as having Hodgkin’s disease, lymphocyte-depleted type, in 1977 at the National Institutes of
Health, Bethesda, MD. He received radiation therapy to the
involved vertebrae and six courses of chemotherapy, with
remission for one year. Lymphoma recurred and was treated
with a splenectomy and another course of chemotherapy.
From the National Institute of Neurological and Communicative
Disorders and Stroke, National Institutes of Health, Bethesda, MD
Address reprint requests to D r Aksamit, Unit on Molecular Biology
and Genetics, Building 36, Room 5D-06, NINCDS, Bethesda, MD
Received Jan 21, 1985, and in revised form Mar 4. Accepted for
publication Mar 5, 1985.
In situ hybridization is a method of annealing
labeled known nucleic acid sequences (probes) to complementary strands in tissue sections or tissue culture
cells. The present study was conducted to apply these
new techniques using biotinylated probes to study the
presence of JC virus DNA in various cell types in the
brains of patients with PML. These findings were correlated with histopathological changes in the same tissue. In situ hybridization was carried out using a
biotinylated D N A probe made from cloned JC virus
Table 1. Summary of Clinical and Electron Microscopic Findings of Three Cases of Progvessiue Multifocal Leukoencephalopathy
Patient No.,
Age (yr)
Underlying Disease
1, 51
Hodgkin’s disease
2, 34
3, 61
Duration of
Disease (mo)
Electron Microscopy
Cyclophosphamide, vincristine, procarbazine,
prednisone, splenectomy
Renal transplantation Prednisone, azathioprine,
35-45 nm virions,
filamentous forms
.New case
33-35 nm particles,
Rheumatoid arthritis
18-24 nm diameter
filamentous forms
Immunoelectron microscopy positive with
monospecific JC virus
{20 (Case 7)l
Glucocorticoids, gold therapy, Plaquenil (hydroxychloroquine sulfate)
Two years after the initial diagnosis, the patient developed
night sweats, a low-grade fever, mild right hemiparesis, and
word-finding difficulty over the course of two weeks. The
patient was pancytopenic, with a prolonged prothrombin
time. Lumbar puncture results were normal, but a computed
tomographic scan of his head showed an area of subcortical
decreased attenuation in the left frontoparietal area that did
not become enhanced with contrast material. The patient’s
condition deteriorated, with increasing right hemiparesis that
progressed to hemiplegia and increasing global aphasia over
the following month. Left hemiparesis developed subacutely,
and the patient became comatose and died approximately
two months after the onset of neurological symptoms. A
general postmortem examination at the National Institutes of
Health showed residual Hodgkin’s disease in the pelvis, the
para-aortic nodes, and several vertebrae. The brain grossly
displayed multiple 1-to 5-mm lesions of the white matter
bilaterally in the cerebral hemispheres, the basal ganglia regions, and the midbrain. Microscopic evaluation demonstrated the typical histological changes of PML, including
oligodendrocytes with enlarged nuclei at the borders, enlarged reactive astrocytes, occasional bizarre astrocytes, and
macrophages [23,24,29]. Electron microscopy revealed 35to 45-nm icosahedral virions and filamentous forms in the
involved oligodendrocytes.
The cases of Patients 2 and 3 have been reported previously C17, 20 (Case 7)J
Pathological Material and Controls
Formalin-fixed, paraffin-embedded brain sections were available from autopsies of Patients 1 and 2. Unfixed, frozen
brain tissue was examined from Patient 3. The period of
autolysis for these cases was unknown. Formalin-fixed brain
tissue from patients with herpes simplex encephalitis and
from routine autopsy subjects without neurological disease
served as negative controls when the JC virus DNA probe
was used. Normal frozen brain tissue was used as the control
for Patient 3. JC virus-infected primary human feral glial
(PHFG) cells grown on glass coverslips served as positive
controls. These positive control preparations were also compared with uninfected PHFG cell cultures.
Preparation of the JC Virus DNA Probe
JC virus genomic D N A was prepared from the cloned plasmid vector pJC in Escherichia colz K-12 (strain HB101) bacteria as previously described [ 181. Biotinylation of the purified
JC virus genome was carried out by nick translation as described in the method of Brigati and colleagues 141. Biotinylated d-UTP and other reagents used in the nick translation
procedure were purchased from E N 2 0 Biochem, New
York, NY. The prepared probe was separated from unicorporated nucleotide triphosphates by the “spin column” technique and stored at 4°C until use 1141. Probe size was determined to be 80 to 300 bp by sizing on a 1.4% agarose gel
Preparation of Tissue Culture Cwersiips
PHFG cells cultured to serve as controls and to be infected
with JC virus were isolated as previously reported [13].
PHFG cells from the first passage were prepared on glass
coverslips and allowed to grow to semiconfluence. PHFG
cells were then infected with the Mad-1 strain of human JC
papovavirus or the HEK-adapted strain of JC virus 1197.
Virus titers were measured by hemagglutination (HA) of
human type 0 erythrocytes C21). Each coverslip of PHFG
cells was inoculated with 100 H A units. Coverslips were
fixed in 4% paraformaldehyde at the time of the earliest
cytopathogenic effect. The coverslips were then mounted on
clear microscope slides with fast-drying epoxy.
Mounting of Brain Tissue Sections
Glass slides for mounting tissue sections were pretreated
with a 0. l-mdml solution of poly-D-lysine to prevent loss of
tissue adherence during the pretreatment and hybridization
steps. Formalin-fixed, paraffin-embedded sections of brain
tissue were cut into 6-p-thick sections and placed on precoated slides. Frozen brain sections were cut at 10 p. on a
cryostat and fixed with 5% acetic acid in absolute ethanol.
This was followed by dehydration in 95% ethanol and 100%
Aksamit et al: In situ Hybridization on PML
Pretreatment of Coyerslips and Tissue Sections
Paraffin-embedded sections were dewaxed in xylene, then in
ethanol, and endogenous peroxidase activity was removed by
incubation for 30 minutes in 3% hydrogen peroxide in
methanol. Pretreatment washes using mild acid and detergent, followed by limited protein digestion and post fixation
in 4% paraformaldehyde were similar to those previously
described 14). All pretreatment steps were performed at
room temperature. Digestion was carried out with 0.1- to
0.3-mg of pronase (Boehringer Manhein Biochemicals, Indianapolis, IN) per milliliter of 50 mM Tris (pH, 7.6) and 5
mM EDTA buffer, for 5 minutes. The concentration of pronase used was titrated depending on the tissue. After dehydration, slides were ready for application of the probe mixture.
Application of Probes
Histologically acute and chronic lesions, as previously
defined [ l l , 281, were studied with JC virus biotinylated
DNA probes. Herpes simplex virus type 1 (HSV-1) biotinylated DNA probe purchased from E N 2 0 Biochem served
as a control probe on all tissues.
In Situ Hybridization
In situ hybridization was carried out under stringent conditions as described by Brigati and co-workers {4].The final
probe mixture contained 50% deionized formamide, 10%
dextran sulfate, 0.4 mdml of sheared herring sperm DNA
(Boeringer Mannheim), 2 pdml of biotinylated probe, and
2 X SSC (300 mM sodium chloride and 30 m~ sodium citrate; pH, 7.0). Brain sections were encircled using a wax
pencil. The volume of probe mixture applied per slide was
determined by the coverslip size required to cover the section or tissue culture. Siliconized glass coverslips were then
placed over the probe mixture, and the contents were sealed
along the edges with rubber cement.
Probe DNA and cellular DNA were denatured in situ by
incubating for 7 minutes at 80°C. Hybridization was allowed
to proceed at 37°C for 22 to 26 hours. After careful removal
of the siliconized coverslips, washing was carried out at room
temperature in 2 x SSC for 2 minutes, 0.1% Triton X-100
in phosphate buffered saline (PBS) for 2 minutes, and PBS
Affinity Cytocbemicai Detection
Detection of the biotinylated probe was carried out immediately by direct-affinity cytochemistry using the streptavidin-biotin-horseradish peroxidase complex kit (Detek
I-hrp) purchased from E N 2 0 Biochem and was done
according to the method suggested by the manufacturer. A
fresh solution of diaminobenzidine tetrahydrochloride
(DAB) served as the indicator. The reaction product was a
golden brown precipitate. The slides were then washed,
counterstained with hematoxylin, dehydrated, and mounted.
Some serial tissue sections were stained using the antiglial fibrillary acidic protein (anti-GFAP) peroxidase-antiperoxidase kit from DakopattdAccurate Chemical, Westbury, NY, for comparison with hybridized sections.
492 Annals of Neurology Vol 18 N o 4 October 1985
Histological Findings a f t r In Situ
Hybridization of PML Tissues
In the formalin-fixed, paraffin-embedded tissues from
Patients 1 and 2, oligodendrocytes showed the most
staining by in situ hybridization for JC virus DNA.
The greatest staining was seen in oligodendrocytes exhibiting the typical pathological changes of productive
infection 123, 24, 281. At low-power magnification,
the cells that stained the darkest were most numerous
in early lesions or at the advancing edges of a demyelinated area (Fig 1A). At higher power, the histological characteristics of these cells were typical, with enlarged, often hyperchromatic, nuclei, occasionally with
inclusions. The brown stain from the DAB reaction
product was essentially confined within the nuclear
membrane of the affected cells, except where the cells
showed obvious evidence of cytolysis (Fig 1B). Other
microscopic foci of altered oligodendrocytes were seen
scattered throughout the otherwise normal white matter. These foci were stained with the JC virus probe.
The altered oligodendrocytes were not necessarily
perivascular in location. Some oligodendrocytes, usually pathologically altered, showed staining in the cortex, particularly in ateas adjacent to subcortical
Some astrocytes from the formalin-fixed, paraffinembedded sections stained for JC virus DNA. Although reactive gemistocytic astrocytes were a prominent part of advanced lesions, only very rarely could
staining by in situ hybridization be found in these nuclei. In contrast, a significant number of bizarre, malignant-appearing astrocytes did show staining with the
JC virus DNA probe (Fig 2). These cells were characterized by morphological changes such as enlarged
nuclei (25 to 60 p), lobulated nuclei, multiple nuclei,
nuclear hyperchromasia with a coarse chromatin pattern, and occasional frank mitosis, as have been previously described [l, 28). Proportionally more bizarre
astrocytes than reactive astrocytes were stained with
the JC virus D N A probe (Table 2 ) . The staining was
exclusively intranuclear in the bizarre astrocytes that
were positive for JC virus DNA.
Other cells identified in the brain sections from Patients l and 2 did not stain with the biotinylated
JC virus DNA in situ hybridization technique. Specifically, macrophages seen in more chronic and severe lesions did not stain. Mononuclear cells, mainly
seen in perivascular locations near more severe lesions,
showed no evidence of staining. Plasma cells were occasionally encountered in tissue from Patient 2 but
demonstrated no JC virus DNA by this technique.
Neurons in normal areas, as well as those adjacent to
subcortical areas involved by demyelination, did not
stain with the JC virus probe. N o endothelial cells
were stained, even in areas where a small, early lesion
Fig 3. Patient 3. In situ hybridization with biotinylatedJC
virus probe on frozen tissue. Enlarged oligodendrocytenuclei are
stained brown, indicating the presence of JC virus DNA.
(Hematoxylincounterstain; x 500 befire 30% reduction.)
Fig 1. Patient 2. (A)Progressive multifocal leukoencephalopathy
brain tissue subjected to in situ hybridization withJC virus
biotinylated D N A probe. Cells stained with the brown probe
reaction product indicateJC virus D N A in the area of lesser
demyelination (long arrow) but not in the most severeh demyelinated area (arrowhead). (Hematoxylincounterstain; x 100
before 30% reduction.) (B) Higher mugnijication of A. Typiralh altered oligolindrocytes show nuclear staining, except where
there is cytolysis. (Hematoxylincounterstain; x 500 before 30%
F i g 2. Patient 1. In situ hybridization with JC virus probe is
seen in a severely limyelinated area of white matter. Nuclear
staining, indicating the presence of JC virus DNA, is seen in a
bizarre astrocyte. (Hematoxylan counterstain; x 500 before 30%
Fig 4. Patient 2. In situ hybridization with herpes simplex virus type 1 biotinylated D N A probe. There is no hybridization
in typicalJC virus-infected oligodendmytes. (Hemutoxylin
counterstain; x 500 bofre 30% reduction.)
Fig 5. Patient 2. Neighboringsection of brain tissue to that
depicted in Figure 4, hylwidized with JC virus biotinyhted
D N A probe. Infected oligodendwytesare stained with the
brownJC virus D N A probe reactionproduct. (Hemutoxylin
counterstain; x 500 before 30% reduction.)
Aksamit et al: In situ Hybridization on PML 493
Tabie 2. Comparison of In Situ Hybridization
in Reactive and Bizawe Astrocytes in Progressive
Multifocal Leukoencephalopathy
Astrocytes (%O)a
Astrocytes (%)b
“Determined by counting labeled astrocytes from a representative
lesion after in situ hybridization and comparing with adjacent serial
section stained with anti-dial fibrillary acidic protein antibody.
hCharacterized morphologically by enlarged nuclei, lobulated nuclei,
multiple nuclei, nuclear hyperchromasia, and occasional frank
surrounded a small blood vessel or where lymphocytic
infiltrates were prominent. Ependymal cells, even adjacent to an area of demyelination, did not stain.
Sections from Patient 3 were also evaluated with the
biotinylated JC virus D N A probe. These sections
were frozen, and because of resulting artifacts, cell
identification based on morphological appearance was
very difficult. Those cells that stained and could be
identified were oligodendrocytes (Fig 3). Labeling was
again confined to the nucleus unless there was disruption of the cell. No other cell types, either pathologically altered or histologically normal, hybridized
with the JC virus probe. Bizarre astrocytes were not
seen histologically in these sections.
Control Hybridization Findings
Hybridization to JC virus biotinylated D N A probe
stained the nuclei of PHFG cells infected with the JC
virus or JC-HEK-adapted strain { 191. N o hybridization signal was detected in uninfected PHFG cells.
Sections of normal human brain tissue and of brain
tissue from herpes simplex encephJtis patients produced no reaction product after probing with the JC
virus D N A probe. HSV-1 biotinylated D N A probe
did not stain tissue from PML subjects (Figs 4 , 5).
HSV-1 probe did hybridize to herpes simplex encephalitis sections. Pretreatment with pancreatic
RNAase (100 pglml) and T1 ribonuclease (5 pglml)
for 1 hour did not markedly reduce the staining of
positive tissues.
Rationale for Use of In Situ Hybridization
as an Investigational Tool for PML
The continued investigation for PML is important for
several reasons. First, it remains the only human demyelinating disease that has been shown conclusively to
have a viral cause. Second, the JC virus is genetically
relatively simple, and the regulation of its gene expression may be understood in the context of current
494 Annals of Neurology Vol 18 No 4 October 1985
molecular biology technology {9]. Third, by understanding the molecular events involved in the selective
tropism of this virus for oligodendrocytes, new light
may be shed on mechanisms that play a role in other
human, possibly virus-related, demyelinating disease.
Fourth, JC virus causes central nervous system neoplasia in experimental animals and is related to other oncogenic viruses [lo, 12, 291. Finally, understanding the
pathogenesis of the JC virus infection associated with
immunosuppression may provide information on the
mechanisms of viral latency.
In situ hybridization offers many advantages as a
useful technique in the investigation of PML. It can be
applied to tissue fixed and processed for standard histopathologic examination, and it can be performed in
conjunction with standard histological counterstains
and immunostains. In addition, lytic (permissive) infection and transforming (nonpermissive) infection of different cell populations are postulated to occur simultaneously in PML. This selective effect on different cell
types by the same virus is determined by molecular
interactions between the JC virus genome and the host
cells. In situ hybridization provides a means of investigating these events.
Spec6city ofthe JC Virus DNA Probe
We have shown that highly specific hybridization with
a biotin-labeled D N A probe made from cloned JC
virus D N A could be carried out in situ using previously published methodology {4]. There was no crosshybridization with either normal or herpes simplex encephalitis tissue. The HSV-1 probe likewise did not
cross-react with PML tissue. In situ hybridization with
the JC virus D N A biotinylated probe provides a
specific means for the study of viral DNA replication
in formalin-fixed or frozen tissue sections from PML
subjects and allows correlation with standard histopathological findings.
The pattern of intranuclear staining after in situ hybridization is one that would be expected, as DNA
replication of the JC virus occurs in the nucleus {lo,
2 11. Immunohistochemical and electron microscopic
data have shown that viral antigen and intact virion are
present in the nuclei of infected cells 16, 11, 15, 28).
This finding is consistent with the model in which JC
virus enters the nucleus of a susceptible central nervous system cell, undergoes DNA replication, and is
packaged into infectious virions at that site.
The labeled JC virus D N A probe produced for this
hybridization was free from contaminating sequences.
By using cloned JC virus D N A and restriction endonuclease digestions, we were able to remove
chromosomal contaminants and plasmid vector sequences 118). The nick translation procedure could
then be carried out on purified JC virus D N A to make
a specifically labeled D N A probe.
Preservation of Viral D N A Sequences in Tissue
As has been previously noted in other publications
using in situ hybridization on tissue sections 13, 4, 261,
methods of fixation are important. Fixation affects
both the quality of the hybridization signal and the
morphological preservation of the tissue, often in opposing directions. Our goal in undertaking these studies was to adopt a sensitive and specific technique that
could be applied to tissue routinely processed for standard histopathological examination. As can be seen
from the results, the methodology seems to fulfill
these criteria. Our experience in working with tissue
cultures (data not shown) indicates that the time of
fixation is important in preservation of DNA for hybridization. However, the duration of storage did not
seem to be a factor, as tissue specimens from Patients 1
and 2 were stored in paraffin blocks for 5 and 9 years,
respectively, and tissue from Patient 3 was stored frozen for 12 years.
Sensitivity of the Biotinylated D N A Probe
The sensitivity of this assay is difficult to quantitate.
Estimation of the sensitivity for DNA-to-DNA hybrids using the biotinylated adenovirus DNA probe
on A549 cells infected with adenovirus type 2 suggests
detection limits of 750 to 1500 genomes per cell [4].
Another study using the biotinylated D N A probe 1261
found that, on the average, 1000 copies per cell of
actin messenger RNA were required in order to be
detected in cultures of myoblasts [26).
Quantitation studies using the JC virus biotinylated
DNA probe on PHFG cells are under way. However,
it appears that current biotinylated probe systems used
in situ are still significantly less sensitive than radiolabeled probes 12-41.
Comparison to Previous Studies on PML Tissue
In one previous study of PML by in situ hybridization
using tritium-labeled complementary RNA made from
DNA purified directly from PML brain virions [ S ] ,
affected oligodendrocytes were also found to be the
most heavily labeled, as seen by autoradiography.
Some of the bizarre astrocytes were labeled as well,
although they were believed to be a minority of the
total. As opposed to our study, however, nuclei that
were believed to represent vascular endothelium were
labeled. N o such cells were labeled when evaluated
with the biotinylated probe. Whether this finding represents a difference in sensitivity between biotinylated
and radiolabeled probes can only be answered by direct comparison of the two techniques.
The present study is in agreement with other previous immunohistochemical viral antigen-labeling studies, showing mainly oligodendrocyte labeling 16, 11).
As in one study Ell], there was no relationship between stained single oligodendrocytes or small clusters
(representing early lesions) and blood vessels. Another
study 167 also found no staining of endothelial cells or
neurons but did find staining of a small proportion of
the bizarre astrocytes.
Significance of Bizawe Astroqte Staining
The importance of JC virus DNA replication in producing bizarre astrocytes is unclear. The current study
suggests that JC virus DNA replication does occur in a
large number of these cells. This replication may represent a superinfection of “transformed” cells with limited viral replication, as intact virions have not been
seen in bizarre astrocytes Cl6, 25, 28). Alternatively,
and more attractively, limited viral DNA replication
may take place in astrocytes until the cellular mechanisms in the infected astrocytes respond to earlyregion viral gene product and develop “abortively”
the malignant, bizarre morphological characteristics.
“Abortive” is the most appropriate term because of the
infrequent occurrence of frank astrocytoma with PML
17, 251. The infrequent staining of reactive astrocytes
is compatible with previous immunohistochemical and
electron microscopic data suggesting limited infection in some “nontransformed” astrocytes [l 1, 161.
Whether these astrocytes represent the early stage of
astrocyte infection before undergoing bizarre morphological alteration is speculative.
Usejdness of In Situ Hybridization
in the Diagnosis of PML
Finally, in situ hybridization with the biotinylated
probe may be a particularly useful diagnostic, as well as
investigative, tool in the evaluation of PML. Theoretically, an unlimited amount of probe material is available because the JC virus genome has been cloned and
the biotinylated probe is stable as a reagent. Futher
evaluation will be necessary to see if the technique can
be made to be as rapid as immunohistochemical labeling for viral antigen of brain biopsy specimens. Preliminary information suggests that it is as sensitive as
the peroxidase-antiperoxidase technique for antigen
detection in formalin-fixed, paraffin-embedded tissue.
The authors thank Drs David Katz, Sidney Houff, William McCormick, and David Asher for providing clinical material. Mrs Renee
Traub provided excellent technical assistance.We are also grateful to
Ms Lin Aspinall for assistance with preparation of the manuscript.
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