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.

References 1. Astrom K-E, Mancall EL, Richardson EP Jr. Progressive multifocal leukoencephalopathy . Brain 1958;81 9 3 - 1 27 2. ZuRhein GM. Association of papovavirions with a human demyelinaring disease (progressive multifocal leukoencephalopathy). Prog Med Virol 1969;11:185-247 3. Major EO, Amemiya K, Tornatore C, e t al. Pathogenesis and molecular biology of progressive multifocal leukoencephalopathy: J C virus induced demyelinating disease of the human brain. Clin Microbiol Rev 1992;3:49-73 4 . Padgett BL, Walker DL. Prevalence of antibodies in human sera against JC virus, an isolate from a case of progressive multifocal leukoencephalopathy. J Infect Dis 1973;127:467-470 5. Padgett BL, Walker DL. Virologic and serologic studies of progressive multifocal leukoencephalopathy. Prog Clin Biol Res 1983;105:107-117 6 . Walker DL, Padgett BL. The epidemiology of human polyomaviruses. In: Sever JL, Madden DL, eds. Polyomaviruses and human neurological diseases. New York: Alan R. Liss, 1983: 99-106 7. Andrews CA, Daniel R, Shah K. Serologic studies of papovavirus infections in pregnant women and renal transplant recipients. In: Sever JL, Madden DL, eds. Human polyomaviruses and human neurologic disease. New York: Alan R. Liss, 1983: 133- 14 I 8 Arthur RA, Dagostin S, Shah K. Detection of BK virus and JC virus in urine and brain tissue by the polymerase chain reaction. J Clin Microbiol 1989;27:1174-1179 9 Arthur RA, Shah KV, Charache P, Saral R. BK and JC virus infections in recipients of bone marrow transplants. J Infect Dis 1988;158:563-569 10. Coleman DV, Wolfendale MR, Daniel RA, e t al. A prospective study of human polyomavirus infection in pregnancy. J Infect Dis 1980;142:1-8 11 Flzgstad T, Sundsfjord A, Arthur RR, e t al. Amplification and sequencing of the control regions of BK and JC virus from human urine by polymerase chain reaction. Virology 1991; 180:55 3-560 12 Myers C , Frisque RJ, Arthur RR. Direct isolation and characterizarion of J C virus from urine samples of renal and bone marrow transplant patients. J Virol 1989;63:4445-4449 13 Grinnel BW, Padgett BL, Walker DL. Distribution of nonintegrated D N A from J C papovavirus in organs of patients with progressive multifocal leukoencephalopathy. J Infect Dis 1983; 147669-67 5 14 Houff SA, Major EO, Katz D, e t al. Involvement of J C virusinfected mononuclear cells from the bone marrow and spleen in the pathogenesis of progressive multifocal leukoencephalopathy. N EngIJ Med 1988;318:301-305 15 Major EO, Amemiya K, Elder G , Houff SA. Glial cells of the human developing brain and B cells of the immune system share a common D N A binding factor for recognition of the regulatory sequences of the human polyomavirus, JCV. J Neurosci Res 1990;27:461-47 1 16 Aksamit AJ, Major EO, Ghatak NR, e t al. Diagnosis of progressive multifocal leukoencephalopathy by brain biopsy with biotin labeled DNA: D N A in situ hybridization. J Neuropathol Exp Neurol 1987:46:556-566

Tornatore et al: Detection of JCV DNA in Lymphocytes 461

17. Aksamit AJ, Mourrain P, Sever JL, Major EO. Progressive multifocal leukoencephalopathy: investigation of three cases using in situ hybridization with JCV virus biotinylated D N A probe. Ann Neurol 1985;18:490-496 18. Southern EM. Detection of specific sequences among D N A fragments separated by gel electrophoresis. J Mol Biol 1975; 98:503-517 19, Frisque R. Regulatory sequences and virus-cell interactions of JC virus. In: Sever JL, Madden DL, eds. Polyomaviruses and human neurological diseases. New York: Alan R. Liss, 1983: 4 1-59 20. Yogo Y , Kitamura T, Sugimoto C, et al. Isolation of a possible archetypal JC virus D N A sequence from nonimmunocompromised individuals. J Virol 1990;64:3139-3143 21. zur Hausen H, Gissmann L. Lymphotropic papovaviruses isolated from African green monkey and human cells. Med Microhiol Immunol 1979;167:137-1 53 22. McChesney MB, Oldstone MBA. Viruses perturb lymphocyte functions: selected principles characterizing virus-induced immunosuppression. Ann Rev Immunol 1987;5:279-304 23. Martin JD, King DM, Slauch JM, Frisque RJ. Differences in regulatory sequences of naturally occurring JC virus variants. J Virol 1985;53:306-311 24. Frisque RJ, Rifkin DB, Walker DL. Transformation of primary hamster brain cells with JC virus and its DNA. J Virol 1980;35:265-269

462 Annals of Neurology Vol 31 No 4 April 1992

25. London WT, Houff SA, Madden DL, et al. Brain tumors in owl monkeys inoculated with a human polyomavirus UC virus). Science 1978;201:1246--1249 26. Major EO, Mourrain P, Cummins C. JC virus induced owl monkey glioblastoma cells in culture: biological properties associated with the viral early gene product. Virology 1984;136:359-367 27. ZuRhein GM. Studies of J C virus-induced nervous system tumors in the Syrian hamster: a review. In: Sever JL, Madden DL, eds. Polyomavirus and human neurological diseases. New York: Alan R. Liss, 1983:205-221 28. Markowitz R, Eaton BA, Kubik MF, et al. BK virus and J C virus shed during pregnancy have predominantly archetypal regulatory regions. J Virol 1991;65:4515-4519 29. Dorries K. Progressive multifocal leukoencephalopathy: analysis of JC virus D N A from brain and kidney tissue. Virus Res 1984;1~25-38 30. Grinnell BW, Padgett BL, Walker DL. Comparison of infectious JC virus DNAs cloned from human brain. J Virol 1983; 45:299-308 31. Rentier-Delrue F, Luhiniecki A, Howley PM. Analysis of JC virus D N A purified directly from human progressive multifocd leukoencephalopathy brains. J Virol I98 1;38:7 6 1-7 69 32. Berger JR, Kaszovitz B, Post MJ, Dickinson G. Progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection. A review of the literature with a report of sixteen cases. Ann Intern Med 1987;107:78-87