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Detection of JC virus DNA in peripheral lymphocytes from patients with and without progressive multifocal leukoencephalopathy.

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EXPEDITED PUBLICATION
Detection of JC Virus DNA in Peripheral
Lymphocytes from Patients with and without
Progressive Multhocal Ieukoencephalopathy
Carlo Tornatore, MD," Joseph R. Berger, MD,f$ Sidney A. Houff, MD,§ Blanche Curfman, BA,*
Karen Meyers,' David Winfield, MBA,? and Eugene 0. Major, PhD"
Progressive multifocal leukoencephalopathy (PML) results from lytic infection of oligodendrocytes by JC virus UCV).
Although JCV has been identified in mononuclear cells in bone marrow and hematogenous dissemination of thse virus
to the central nervous system has been suspected, JCV has never been clearly demonstrated in the peripheral circulation. Using polymerase chain reaction technology, we examined peripheral lymphocytes of 19 patients with brain
biopsy-proven PML for the JCV genome. Two non-PML control groups, consisting of 26 patients seropositive for
human immunodeficiency virus type 1 (HIV-1) and 30 immunocompetent patients with Parkinson's disease, were also
examined for the presence of the JCV genome in lymphocytes. Cerebrospinal fluid from 10 patients with PML was
examined for the presence of the JCV genome as well. The JCV genome was detected in the lymphocytes of 89'Z (17)
of the patients with PML, 38% (10)of the HIV-1-seropositive patients without PML, and none of the patients with
Parkinson's disease. Sequencing of the JCV regulatory region from the lymphocytes of three patients revealed the
prototype MAD-I strain of JCV in one patient with PML, a MAD-4 strain in a second patient with PML, and a slight1,y
modified MAD-4 strain in an HIV-1-positive patient without PML. Only 3 of 10 patients with PML who had JCV
detected in lymphocytes had the JCV genome in their cerebrospinal fluid. These results demonstrate that the JCV
genome can be found in circulating lymphocytes from patients with PML and suggest that lymphocytes are an
important vector for hematogenous dissemination of JCV to the central nervous system. We also identified the JCV
genome in the lymphocytes of a group of HIV-1-seropositive patients with no clinical evidence of PML, suggesting
that they may be at risk for development of PML and can be identified in a presymptomatic state.
Tornatore C, Berger JR, Houff SA, Curfman B, Meyers K, Winfield D, Major EO. Detection 0.f
JC virus DNA in peripheral lymphocytes from patients with and without progressive
multifocal leukoencephalopathy.Ann Neurol 1992;31:454-462
Progressive multifocal leukoencephalopathy (PML) is
a demyelinating disease resulting from lytic infection of
oligodendrocytes by the papovavirus JC (JCV) { 1-31.
Although the molecular biology of JCV is well characterized, the natural history of the virus is only partially
understood. Serological studies indicate that primary
infection occurs in childhood; antibodies to JCV develop in up to 85% of the population by age nine
14-61. JCV DNA has been detected in the urine of
pregnant women, immunocompetent elderly individuals, and immunocompromised patients who had undergone bone marrow and renal transplant, none of whom
had PML, suggesting that the virus may remain latent
outside the central nervous system (CNS) following
initial infection [7-12). The site and mechanism of
this viral latency is uncertain. As the immune system
becomes compromised, some individuals will experience reactivation of the latent infection, which leads to
infection of the CNS and subsequent leukoencephalopathy. T h e JCV genome has also been found in
lymph nodes, liver, spleen, and lung in a small number
of patients with PML, which is consistent with widespread systemic dissemination of the virus during immunosuppression { 131. The cells harboring the ,virus
in individual organs, however, were not identified in
that study.
In 1788, JCV-infected B cells were detected in the
spleen and bone marrow of two patients with PML
{14], suggesting that B cells or their precursors may
act as a reservoir and a vector for the virion. JCVinfected B cells have subsequently been found in the
CNS of a patient with P N L {lS], suggesting that tdur-
From the "Section of Molecular Virology and Genetics, Laboratory
of Viral and Molecular PathoEenesis, National Institute of Neurolozi d Disorders and Stroke,
MD; the tDepmmencs Of Neurology and $Internal Medicine, University of Miami School of Medicine, Miami, FL, and the $Department of Neurology, Veterans
Administrarion Hospital, Washington, DC.
Received Jan 3 , 1992, and in revised form Jan 27. Accepted for
oublication Ian 28. 1992.
- I
iddress co~respondenceto Dr Tornatore, Section of Molecular Vi.
rology and Genetics,
of
and Molecular Parhogenesis, National Institute of Neurological Disorders and Stroke,
Bethesda, M D 20892.
454 Copyright 0 1992 by the American Neurological Association
ing a period of immunocomprornise t h e infected B-cell
population may further act as a vector for JCV invasion
of t h e CNS. If this observation is correct, then JCV
would also be circulating in peripheral blood lymphocytes (PBLs) during periods of immunocompromise.
We examined t h e circulating peripheral lymphocytes
from patients with brain biopsy-proven PML for t h e
presence of the JCV genome. We also examined lymphocytes from patients without PML w h o were seropositive for human immunodeficiency virus type 1
(HIV-1) and from immunocornpetent but neurologically impaired individuals, represented by a g r o u p of
patients with Parkinson’s disease.
Materials and Methods
Patients
All patients enrolled in this study had the diagnosis of PML
made on the basis of clinical and neuroimaging features and
histopathology of brain biopsy specimens. The diagnosis was
further confirmed by in situ hybridization of the brain biopsy
specimens for JCV. Peripheral lymphocytes, bone marrow
aspirate, and, in some cases, cerebrospinal fluid (CSF) from
19 patients with PML were examined for the presence of the
JCV genome. Seventeen had acquired immunodeficiency syndrome (AIDS)-related immunodeficiency, one had
Wiskott-Aldrich syndrome, and one had no clearly demonstrable immunodeficiency or underlying chronic disease. Two
patients were in a pediatric age group; one had WiskottAldrich syndrome, the other had AIDS. Most patients with
PML (1 1) are part of a prospective study being conducted at
the University of Miami (J.R.B.) examining the efficacy of
alpha-interferon in the treatment of AIDS patients with
PML.
Two other patient populations were used as control
groups. One control group consisted of 26 HIV-l-seropositive patients followed prospectively for the development
of neurological disease, none of whom had PML as demonstrated clinically or by magnetic resonance imaging (MRI).
The other group consisted of 30 immunocompetent patients
with Parkinson’s disease followed periodically for their movement disorder. In these two groups, only peripheral lymphocytes were examined for the presence of JCV DNA.
In Situ Hybridization
In situ hybridization was performed as previously described
[ 16, 171. Briefly, formaiin-fixed, paraffin-embedded brain biopsy sections were placed on gelatin-coated slides, allowed
to air dry, and then incubated at 37°C overnight. The sections
were deparaffinized in xylene, then in alcohol. Endogenous
peroxidase activity was removed by incubation for 30 minutes in 3% hydrogen peroxide in methanol. Bone marrow
aspirates and biopsies were incubated in 3% hydrogen peroxide for one hour. Acid hydrolysis in 0.05 HCI was followed
by a wash in Triton-X, followed by a limited protein digestion with pronase and a wash with glycine buffer. Tissue was
post-fixed in 495 paraformaldehyde and dehydrated in serial
ethanol washes. The slides were then hybridized with 25 to
40 pL of a probe mixture that consisted of 50% formamide,
l o p dextran sulfate, 0.4 mg/mL calf thymus D N A , 2 kg/
mL biotinylated JCV D N A probe ( E N 2 0 Biochem), and
2 x SSC (300 mM sodium chloride and 30 mM sodium citrate; pH, 7). Probe D N A and cellular D N A were denatured
in situ by incubating at 90°C for 7 minutes. Hybridization
was performed at 37°C for 24 to 48 hours. Sections were
then washed at room temperature in 2 x SSC for 2 minutes,
0.1% Triton-X 100 in 0.0067 M phosphate-buffered saline
(1 x PBS) for 2 minutes, and PBS for 3 minutes. Detection
of the biotinylated probe was carried out immediately by
direct-affinity cytochemistry using the streptavidin-biotinhorseradish peroxidase complex kit (Detek I-hrp, E N 2 0
Biochem). A fresh solution of diaminobenzidine tetrahydrochloride (DAB) was used as the chromogen, resulting in a
brown precipitate. Slides were then washed, counterstained
with hematoxylin, dehydrated, and mounted.
Bone marrow aspirates were dried on coverslips, then
fixed in 4% paraformaldehyde for 30 minutes. In situ hybridization was performed as described, starting at the 3% hydrogen peroxide step.
Polymerase Chain Reaction
D N A used as a template for the polymerase chain reaction
(PCR) was extracted from PBLs using the following protocol.
Ten to 15 mL blood were collected in a heparinized tube
and kept at room temperature. The blood was underlayed
with an equal volume of Ficoll-Hypaque and spun at 1,700
X g in an IEC tabletop centrifuge. The PBLs were collected,
washed twice with 1x PBS, and lysed in a final volume of
5 mL 1 x PBS, containing 1% SDS and 100 pg/mL proteinase K. The lysate was incubated at 37°C for 16 hours and
extracted twice in TES-buffered (50 mM Tris, 10 mM
EDTA, 100 mM NaCI; pH, 7.3) phenol, followed by three
extractions with chloroform. Three volumes of 100% ethanol and 1/10 volume of 3 M sodium acetate were added to
the aqueous phase and chilled to - 20°C for 12 to 16 hours.
D N A was collected by centrifugation at 14,000 rpm in a
Sorvall SS-23 rotor, air dried, and brought to final volume
of 200 pL in TE buffer (10 mM Tris, p H 7.4; 1 mM EDTA).
D N A used as a template for PCR was extracted from CSF
as follows. Five hundred pL CSF was aliquoted for each PCR
performed. Fifty pL 10% SDS and 11 pL proteinase K (concentration, 10 pg/pL) were added to each CSF aliquot and
incubated at 65°C for 1 hour, followed by incubation at 37°C
for 5 hours. The CSF was extracted once with equal volumes
of phenol and chloroform, and the D N A was precipitated in
three volumes of 100% ethanol. All the D N A precipitated
from 500 pL CSF was used in one PCR.
Each PCR was run in 100-pL volumes consisting of 10
mM Tris-HCL, p H 8.3; 50 mM KCI; 1.5 mM MgCI; 200
pM of each dNTP; 2.5 U Amplitaq D N A polymerase (Perkin-Elmer Cetus); 1 pg PBL D N A or all the D N A precipitated from 500 kL CSF; and 150 pmol each oligonucleotide
primer pair. Three different sets of primers were used in
three different reactions for each patient (Fig 1).The first set
of primers (T,T’) flanked a conserved portion of the early
region T-antigen gene extending from base pair (bp) numbers
4,481 to 5,000. They were 5‘-AATAGTGGTITACCTTAAAG (T’) on the sense strand and 5’-TGAATAGGGAGGAATCCATG (T) on the antisense strand. The second set of primers (V,V’) flanked a conserved portion of the
late region VP1 gene from bp numbers 2,000 to 2,500. They
Tornatore et al: Detection of JCV D N A in Lymphocytes 455
-
RD
which used the primer pair R and R’ to amplify a 500-hp
segment nested inside the 1,149-bp template.
R’
JCV
GENOME
-+
v’
Fig 1 . Location of .segments of the JC virus genome targeted for
detection using polymerase chain reaction (PCR) technolom. The
JC urus genome comists of double-stranded, super coiled, circular D N A 5.1.30 ba.re pain long (not all genes are shown).
Three .segments targeted for detection are shown. Arrows represent the location and direction of the oligonucleotide primers used
in the PCR to detect a giwn segment. The primers $anking the
regulatoly .sequences were also used in sequencing reactions.
were 5 ‘-AATCTCAAGTCATGAACACA (V’) on the sense
strand and 5’-GTCAACGTATCTCATCATGT (V) on the
antisense strand. The third set of primers (R,R’) flanked the
late region noncoding or regulatory sequences from the origin of replication to bp number 500. They were 5’-GCCTCGGCCTCCTGTATATA (R’) on the sense strand and 5’’ITAC’ITACCTATGTAGCTIT (R) on the antisense
strand. In each reaction, D N A was denatured at 94°C for 1
minute, primers were annealed at 55°C for 2 minutes, and
primers were extended at 72°C for 3 minutes for a total of
40 cycles in a D N A thermal cycler (Perkin-Elmer Cetus).
Three different PCRs were performed as positive and negative controls for each patient tested. The negarive control
consisted of a PCR with all components except a template.
The positive control consisted of a PCR with pMAD-1 as
the template. pMAD-1 is a full-length copy of the JCV genome from the MAD-1 strain cloned into a pBR322 vector.
A third PCR was performed as a control for D N A polymerase integrity, using a lambda-phage D N A template and primers supplied with the PCR kit (GeneAmp; Perkin-Elmer)
Ten pL of each PCR product was run on a 1.59’ agarose
horizontal gel, stained with ethidium bromide, visualized
with ultravioler light, and photographed.
Nested PCR was performed by pairing primer T’ on the
sense strand with primer R on the antisense strand to give an
expected 1,149-bp product. This PCR product was separated
from the residual primers, desalted, and concentrated by spin
dialysis in a Centricon 100 device (Amicon). Five to 10 FL
of the T’/R PCR was used as the template for a second PCR,
456 Annals of Neurology
Vol 31 No 4
April 1992
Southern Transfer a n d Hybridization
To ensure further the specificity of the PCR products, all
reactions underwent Southern blot transfer. The agarose gel
was rinsed once with distilled water, denatured twice in 1 M
NaCl and 0.5 M N a O H for 15 minutes, then neutralized
twice in 0.5 M Tris and 1.5 M NaCl for 15 minutes. The
D N A was then transferred from the gel to a nylon filter by
capillary action according to the method of Southern ClSl.
The D N A was cross-linked to the filter by ultraviolet radiation (Stratalinker, Stratagene) and prehybridized in 50% formamide, 6 x SSPE (0.9 M NaCI, 0.06 M N a H 2 P 0 4 ,0.006
M EDTA-Na,), 5 x Denhardt’s solution, 0.5% SDS, and
100 pg/mL calf thymus D N A at 42°C for one hour. The
filter was hybridized in an identical solution, which also
contained 1 x lo6 dpm/mL nick-translated, ”P-labeled
pMAD-1. The probe was allowed to hybridize to the filter
at 42°C for at least 16 hours. T h e filter was washed twice in
6 x SSPE and 0.1%; SDS for 30 minutes at room temperature, followed by two washes in 1 x SSPE and 0.5% SDS
for 30 minutes at 64°C and a final wash in 0.1 x SSPE and
0.5% SDS for 30 minutes at 64°C. The fiIter was dried and
used for autoradiography.
Sequencing of PCR Prodacts
Two primers were used to amplify the noncoding regulatory
sequences of the late region of the JCV genome: 5‘-CACGCCC’ITACTACTTCTGAG (RD) and 5’-TTAC?TACCT A T G T A G C m (R), which flanked bp numbers 5,090
(across the origin) to bp 500 to give an amplification product
540 bp long (see Fig 1). PCR was performed as described.
The PCRs were run on an agarose gel as described to confirm
the presence of an appropriately sized amplification product.
The remaining PCR was desalted and separated from the
residual primers using a spin dialysis device (Centricon 100;
Amicon). All reactions were dialyzed twice and concentrated
to a final volume of approximately 50 pL. Five to 10 FL of
this reaction were then used as a template in an asymmetrical
PCR to generate single-stranded D N A for the sequencing
reaction. The asymmetrical PCR was performed using the
conditions described for the symmetrical PCR, excepc only
one primer was used in the reaction and the cycling parameters were changed as follows: denaturing of the template at
94°C for one minute, annealing at 55°C for one minute, and
primer extension at 72°C for one minute, for a total of
25 cycles. Primer R’ was used to generate single-stranded
template from the sense strand, and primer R250
(5’-CTCTGGCTCGCAAAACATGT) was used to generate
single-stranded template from the antisense strand in a separate reaction. Asymmetrical products were visualized on a
1.5% agarose gel and again cleaned and concentrated with
the Centricon 100 spin dialysis device. Seven pL of the
concentrated products were subsequently sequenced by the
Sanger dideoxynucleotide termination method (USB Sequenase 11) on an Acugen-automated, 3’P-labeled D N A sequencer (EG + G BioMolecular) using Genquest software.
The nucleotide sequences from all segments were determined from both the sense and the antisense strand.
Table 1 . Detection of the JC Virus Genome by In Situ Hybridization and Polymerase Chain Reaction in Patients with PML
PCR
In Situ Hybridization
Patient
Brain Biopsy
B.A.
W.I.
S.T.
S.C.
B.R.
M.C.
V.E.
M.W.
U.L.
B.Y.
W.I.N.
W.I.L.
M.O.
D.U.
A.C.
H.O.
+
+
+
+
+
+
+
D.O.
K.E.
R.Z.
Bone Marrow
PBLs
CSF’
+
+
NA
3-
+
+
-
+
+
NA
+
+
+
+ (MAD-4)”
+
+ (MAD-1)”
+
+
+
+
+
-
17/19 (89.5%)
16/16 (100%)
-
i-
+
+
+
+
+
+
-
+
+
3.
+
NA
NA
-
NA
NA
NA
NA
NA
NA
NA
-
NA
3/10 (30%)
“Designates similarity of regulatory region to genotype of JCV DNA samples isolated from brain tissue of patients with PML.
PML = progressive multifocal leukoencephalopathy; PCR = polymerase chain reaction; PBL = peripheral blood lymphocyte; CSF
spinal fluid; + = positive; - = negative; NA = not available;JCV = JC virus.
Results
In Situ Hybridization
BRAIN BIOPSY. Brain biopsy material was available for
in situ hybridization from 16 of the 19 patients with
PML. All demonstrated varying amounts of demyelination from oligodendroglial loss, enlargement of the
oligodendroglial nuclei, and occasional bizarre astrocytes. In all I6 patients, the JCV genome was identified
in the biopsy sections by in situ hybridization (Table
1). In three patients, unstained biopsy material was not
available for hybridization; however, clinical and cranial
MRI features were characteristic of PML and brain histopathology was diagnostic in each.
BONE MARROW ASPIRATE.
Of the 19 patients studied,
bone marrow aspirate was available from 16 for in situ
hybridization (see Table I). Five of these patients
(3 1%) demonstrated the JCV genome in mononuclear
cells from bone marrow.
Detection of the JCV Genome in Peripheral
Lymphorytes Sy PCR
Three segments of the JCV genome were targeted for
detection: the large T-antigen gene from the early region, the structural protein VP1 gene from the late
region, and the noncoding regulatory sequence (see
Fig 1). All three segments are critical for viral replica-
=
cerebro-
tion and multiplication and need to be conserved for
the formation of a functional virion.
Sensitivity Assay of PCR Primers
The sensitivity of PCR was determined by successful
amplification of serial dilutions of a positive control.
The positive control was plasmid pMAD-1, a fulllength JCV MAD-1 strain cloned into a pBR322 vector. Serial dilutions starting at 1 ng plasmid and ending
at 10 fg plasmid were used as a template for the PCR
sensitivity assay. As demonstrated in Figure 2, primer
pair V/V’ detected and amplified as little as 10 fg (approximately 1,000 copies) pMAD-1 template. The amplification products were visible on ethidium-stained
gel for all concentrations except the 10 fg template,
which was detected on Southern transfer (see Fig 2).
Similar results were obtained with primer pairs T/T’
and R/R’.
Peripheral Lymphocytes of Patients with PML
Seventeen patients with PML (89.5%)had JCV derectable by PCR in their PBLs (see Table I). The results
from one patient are illustrated in Figure 3. In nine of
the 17 JCV-positive patients, all three segments of the
JCV genome targeted for PCR could be amplified and
detected by ethidium bromide-stained agarose gel or
on autoradiography following Southern hybridization.
Tornatore et al: Detection of JCV D N A in Lymphocytes
457
Fig 2. Sensitivity of polymerase chain reaction IPCR) in detecting thr/C 2:iru.r IJCVi genome. Serial dilutions of
pMAD-1, a plasnzid containing a full-length copy of the JCV
MAD- 1 genome. usere nsed aJ the template with primers VIV'
w
i cl Jer& of PCRs. Lane 2,I ng; Lane 3 , 100 pg: Lane 4,
1 0 pg: Lane 5 , I pg: Lane 6. 100 f g : Lane 7 , 10 fg. Lane 1 is
a Phi S 174lHA.G 111 digest used as a size murker. Upper
panel iJ agarose gel electrophoresis of PCR: lower pariel is re.iulting cliitoradio~qranifollou Yng Southern hybridization.
In eight of the 17 JCV-positive patients, only two of
the three segments of the JCV genome targeted for
PCR could be amplified. The segments that failed to
amplify were those flanked by the VP1 gene primers
in three patients, the large T-antigen primers in three
patients, and the regulatory region primers in two patients.
In some patients, multiple bands were seen when
the PCR products were separated on an agarose gel
458
Annals of Neurology
Vol 31
No 4
April 1992
Fig 3. Detection of the JC z,iriis genome by po(ymera.re &in reaction (PCRI from peripheral fynphocyte DNA. PCRJ shouui
in fanes 2-6 used lyniphocyte DhiA from patient B.R. ui a
template. Primers used in earb PCR are as fo1lou:r: Lane 1 , Phi
X 174lHAE I11 digeJt: Lane 2.V I V primen: Lane 3. RIR'
primers; Lane 4, TIT': Lane 5 , nested PCR ming prirrim
T ' I R in the first reaction. primers R'IU zn the second reac.tion:
Lane 6 , primers RDIR: Lane 7,R'IR primers using PCU
products shown in lane 6 as a templdte: Lane 8. lambda D N A
template and control primen: Lane 9, RIR' primers. n o template; Lane 10. pMAD-1 template. RIR' primers.
(Lane 4, Fig 3); however, on Southern transfer, only
the expected 500-bp product hybridized to the JCV
genome probe. The specificity of Southern hybridization is also illustrated by the failure of probe hybridization to the lambda-positive control PCR and the
negative control lane (no template in this PCR). In
approximately 10% of the PCRs, the expected amplification products were either slightly larger or slightly
smaller than expected, suggesting variability of the amplified template from the prototype MAD-1 strain.
The products from the nested PCR in lane 5 used
primers T' and R in the first reaction and primers R'
and R in the second reaction, as described in the Materials and Methods section. T h e ability of the PCR prod-
Table 2. Summavy of Pobmerase Chain Reaction Analysis for
JC Virus D N A in Peripheral Blood Lymphocytes Derived from
Patients with and Without PM L
With PML"
Without PML
HIV-1-positive
Without AIDS
15/17
10126'
2/2b
0130 (patients with Parkinson's disease)
origin
Mad-1
'Seventeen of 19 (89.5%) patients with clinically and laboratory diagnosed PML have JC virus D N A in their PBLs.
'One patient had Wiskort-Aldrich syndrome, and one patient has no
identifiable immune deficiency [14].
'Thirty-eight percent of patients with AIDS with varying degrees of
immune deficiency have JC virus D N A in their PBLs.
PML = progressive rndtifocal leukoencephalopathy; HIV-1 = human immunodeficiency virus type 1; AIDS = acquired immunodeficiency syndrome; PBL = peripheral blood lymphocyte.
uct from the first reaction (T'/R primers) to act as a
template for the second nested set of primers (R'/R)
further demonstrates the specificity of the PCR. Lanes
6 and 7 represent a second set of nested PCRs. Lane
6 demonstrates the PCR products using primers RD/
R. These PCR products were used as the template in
a second PCR shown in lane 7, which used R/R' as
primers.
Three patients (M.W., B.Y., W.I.) have been tested
several times over the course of their illnesses and have
consistently demonstrated the viral genome in their
lymphocytes. B.R., a prolonged survivor of PML, has
been neurologically stable for four years following diagnosis of PML, yet still has viral genome circulating
in his lymphocytes.
Peripheral Lymphocytes of HlV-1 -positive Patients
Without PML
One of our control groups consisted of 26 HIV-1seropositive men with varying degrees of immunodeficiency who had neither clinical signs and symptoms
nor neuroradiological evidence of PML. All control
patients underwent cranial MRI, which did not reveal
any white matter changes consistent with PML. Only
2 control patients had symptoms referable to the nervous system; both of these were mild peripheral neuropathies. The JCV genome was detectable by PCR in
the peripheral lymphocytes in 10 (38%) (Table 2). In
five of the 10 JCV-positive control patients, all three
segments of the JCV genome targeted for PCR could
be amplified and detected by ethidium bromidestained agarose gels or on autoradiography following
Southern hybridization. In four of the 10JCV-positive
control patients, only two of the three segments of the
JCV genome targeted for PCR could be amplified. The
segments that failed to amplify were those flanked by
the VP1 gene primers (2) and the regulatory region
primers ( 2 ) . In one control patient, only the T-antigen
primers produced an amplification product.
''
98bp
la
11
98bp
-
WI
BY
co
J
.J
F ig 4. Comparison of regulatory region D N A sequence from
three patients. The regulatory sequence of the prototype JC virus
UCV) strain MAD-1 is shown at the top (see text for details).
Horizontal lines represent areas of homology between the
MAD-1 sequence and the regulatory sequences detected from the
three patients. Patient W.I. has the MAD-1 strain of JCV in
his lymphocytes, whereas patients B.Y. and C.O. have a strain
in which the second TATA box has been deleted, known as
MAD-4. Arrows on the M A D 4 sequence from patient C.O.
indicate the two base substitutions described in the text.
Peripheral Lymphocytes of Patients with
Parkinson's Disease
Thirty patients with Parkinson's disease were chosen
as our second control group because they represented
a group of patients with neurological disease occurring
in the absence of immunodeficiency. None had PCRdetectable JCV genome in their peripheral lymphocytes (see Table 2).
Detection of the JCV Genome in the CSF of Patients
with PML
CSF was available in 10 of the 19 patients with PML.
In three, the JCV genome was detected by PCR analysis. All three segments of the JCV genome targeted
for PCR were amplified in two. In the third patient,
two of the three segments of the JCV genome targeted
for PCR were amplified; however, the segment flanked
by the VPl primers failed to amplify.
DNA Seqaencing of the Amplijed
Regulatoy Sequences
In three patients, the amplified regulatory region DNA
from peripheral lymphocytes was sequenced to determine the viral strains of JCV. Two DNA samples were
from the PML-AIDS group; the third was from one of
the JCV-positive, HIV- 1-seropositive patients without PML. The prototype MAD-1 regulatory sequence
consisting of two tandem 98-bp repeats [19} is shown
in Figure 4 . Areas of homology between the MAD-1
strain and the patient sequences are represented by the
horizontal lines. Interruptions of the patient sequences
indicate a deletion when compared with MAD-1. Patient W.I. had a strain of JCV in his peripheral lymphocytes with an identical MAD-1 sequence; no insertions
or deletions were found. Both patient B.Y. and C.O.
Tornatore et al: Detection of JCV DNA in Lymphocytes 459
(who was H1V- 1-seropositive without PML) had a viral strain with a deletion in the second 98-bp repeat,
eliminating the Ti A-rich region also known as the
TATA box. This deletion is found in the MAD-4 strain
ofJCV. The strain detected from the HIV- l-seroposirive patient without PML differed from the original
MAD-4 strain at taw bases (see Fig 4).The position
of the nucleotide changes was identical in both repeats
and consisted of a cytosine-to-guanine substitution.
The identical location of the substitutions in the 98-bp
repeats suggests that the substitution is not an artifact
of the sequencing reaction. N o insertions or deletions
suggestive of an archetype strain (i.e., that had been
previously described in the urine of some individuals
[2o]) were detected in any of the amplified D N A specimens subjected to D N A sequence analysis.
Discussion
Hematogenous spread of JCV to the C N S in PML has
long been suspected because of the multifocal involvement of hemispheric white matter. Demonstration of
JCV D N A in kidney, lymph node, spleen, liver, lung,
and bone marrow has further implicated widespread
extraneural hematogenous distribution of the virus in
patients with PML [12, li}. Our demonstration of the
JCV genome in peripheral lymphocytes supports the
theory of hematogenous spread of JCV and further
suggests that lymphocytes act as a vector for dissemination of the virus to the brain. Additional evidence for
the role of lymphocytes in the pathogenesis of PML
has been provided by results of in vitro experiments,
in which B cells and human glial cells were shown to
share common D N A binding proteins for JCV, a factor
that may make both cells permissive to the virus 1153.
Another member of the papovaviridae family, simian
lymphotrophic virus 12 11, and several other neurotrophic viruses also traffic through peripheral lymphocytes 1221 during their natural history.
It is believed that JCV remains latent in an extraneural location following primary infection. Reactivation
from the site of viral latency, usually in immunocompromised individuals, then allows JCV access to the
CNS. It is not known what triggers either reactivation
of JCV from a latent state or onset of PML; however,
immune surveillance must be a key factor because progression and severity of PML generally correlate with
increasing degrees of immunosuppression. Analysis of
the PCR results from the patients without PML (see
Table 2) suggests that individuals with intact immune
systems are not likely to have JCV present in their
peripheral lymphocytes. None of the patients with Parkinson's disease had detectable JCV genome in their
lymphocytes, whereas 38% of the HIV-1-seropositive
patients without PML did. The fact that the pztients
with Parkinson's disease were negative for JCV D N A
by PCR could mean that either they do not harbor
460 Annals of Neurology
Vol i l
No 4 April 1992
a latent JCV genome or their intact immune system
prevents viral reactivation. Because seroepidemiological data have described JCV infection in up to 85% of
the population worldwide [GI, it is more likely that
immune surveillance protects ,against JCV expression.
This immune-mediated block to infection could occur
several ways: (1) viral infection (either primary or secondary) of lymphocytes or their precursors is directly
prevented, (2) reactivation of persistent lymphocyte infection is prevented, or (3) infected lymphocytes cannot access the vascular tree from either the bone marrow or the lymph nodes.
Detection of JCV in lymphocytes, however, is not
diagnostic for PML in view of the high number of
HIV-1-seropositive patients without PML who were
positive for JCV by PCR. A more appropriate use of
PCR analysis could be in the form of supportive evidence in favor of the diagnosis when used in conjunction with observations of the clinical course of disease
and MRI or computed tomographic findings. This approach may be particularly important in patients for
whom brain biopsy is not feasible. Identification of patients who d o not have PML but have circulating JCV
genome may also be of clinical importance, because
these patients may be at risk for PML. Once effective
antiviral therapy is found for JCV, prophylactic treatment of asymptomatic JCV-positive patients prior to
the development of PML may be feasible. W e are now
following the JCV-positive/HIV- 1-positive group to
determine in whom, if any, PML will develop. In contrast, PCR of the CSF appears to be a relatively insensitive indicator of C N S involvement; only 3 of 10 patients with PML who had JCV detected in lymphocytes
had the JCV genome in CSF. W e cannot comment on
the specificity of this finding because we did not examine the CSF of patients without PML for JCV.
Several strains of JCV have been isolated and partially sequenced from PML-affected brain tissue. Although all these strains have the same basic genomic
configuration as depicted in Figure 1, strain variability
has been classified according to changes within the regulatory sequences {19, 231. The prototype MAD-1
strain has a 98-bp repeat as part of the regulatory sequence (see Fig 4). In contrast, MAD-4 has a deletion
of the second TATA box. Other strains with various
insertions and deletions have been identified. How
these changes affect the behavior of the different viral
strains in humans is not known; however, the MAD-4
strain is known to be highly oncogenic in the CNS of
owl monkeys and hamsters 124-27). Recently, it was
suggested that the different strains all originate from a
common archetypal JCV {20], in which the regulatory
sequence consists of a single 98-bp repeat interrupted
at two points by a 23-base and a 66-base insertion.
Presumably, the archetypal genome undergoes changes
(insertions, deletions, and duplications) of the regula-
tory sequence to give rise to the different strains.
Whether these changes from archetype to a different
strain can occur within the same individual as the virus
passes through different cell types is not known. Although archetypal JCV has been recovered from urine
in patients without PML [ll, 281, it has not been recovered from PML-affected brain tissue E29-3 11. Similarly, the three patients whose viral regulatory region
we characterized from lymphocytes had MAD-4 sequence (2) and M A D 1 sequence (1). No archetypal
insertions were seen. This finding suggests that archetypal JCV was not directly involved in the hematogenous spread of JCV in these patients, but does not
preclude its presence in other organs. We plan to examine the lymphocytes of the other patients with PML
in this study to determine if any have archetypal JCV
in their lymphocytes.
At what point JCV infects peripheral lymphocytes
or their precursors is unknown. In a previous study
1141 and in this one, we were able to detect JCV sequence in mononuclear cells residing in bone marrow,
suggesting that infection occurs prior to hematogenous
dissemination. These cells in the bone marrow could
presumably expand clonally during a period of compromised immune surveillance and then enter the vascular compartment. HIV infection, which has a relatively high association with PML (321, is frequently
associated with B-cell expansion early in its course.
This expansion may promote dissemination of JCVinfected cells and could explain why PML is associated
with all stages of immunosuppression; it is the initial
presentation in some patients with AIDS. Patient
B .R. is a nonimmunocompromised individual with
PML whose only abnormal immune parameter we
were able to detect is B-cell polyclonal expansion, again
suggesting that B-cell proliferation has a role in the
pathogenesis of PML. Also, patient B.R. has been clinically stable for four years after diagnosis of PML, yet
the viral genome is still present in his lymphocytes.
Experiments are in progress to determine in which
lymphocyte subsets the JCV genome resides. In patient W.I. (who has PML), we separated the peripheral
lymphocytes into B-cell and T-cell subsets and found
the JCV genome in the B-cell subpopulation using
PCR technology (preliminary data). We are also comparing immunological data from the JCV-positive/
HIV- I-positive and the JCV-negative1HIV- 1-positive patients without PML to determine which differences might account for the presence or absence of
JCV in these two populations. We also hope to examine the lymphocytes of other immunosuppressed
populations (i.e., in bone marrow and renal transplant
recipients), of pregnant women, and of a cohort of
pediatric AIDS patients without PML to determine the
prevalence of JCV and its strain variation in these
groups.
Supported in part by National Institute of Neurological Disorders
and Stroke Program Project NS25569.
We are indebted to Drs Paul Cimoch, Paul OKeefe, and Richard
Price for enrolling their patients in this study, and to Renee Traub
and Dr Ellen Whitaker for editorial assistance in the preparation of
the manuscript.
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