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[Cancer Research 61, 4287-4293, May 15, 2001]
© 2001 American Association for Cancer Research

Virology

Detection of JC Virus DNA Sequences and Expression of the Viral Regulatory Protein T-Antigen in Tumors of the Central Nervous System1

Luis Del Valle, Jennifer Gordon, Martha Assimakopoulou, Sahnila Enam, Jennian F. Geddes, John N. Varakis, Christos D. Katsetos, Sidney Croul and Kamel Khalili2

Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122 [L. D. V., J. G., S. E., S. C., K. K.]; Department of Anatomy, University of Patras School of Medicine, GR-26500 Patras, Greece [M. A., J. N. V.]; Department of Histopathology and Morbid Anatomy, Queen Mary, University of London, London E1 4NS, United Kingdom [J. F. G.]; Department of Pediatrics, Medical College of Pennsylvania Hahnemann University and St. Christopher’s Hospital for Children, Philadelphia, Pennsylvania 19134 [C. D. K.]; and Department of Pathology and Laboratory Medicine, Medical College of Pennsylvania Hahnemann University, Philadelphia, Pennsylvania 19102 [S. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
JC virus (JCV) is a neurotropic polyomavirus infecting greater than 70% of the human population worldwide during early childhood. Replication of JCV in brains of individuals with impaired immune systems results in the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Furthermore, JCV possesses an oncogenic potential and induces development of various neuroectodermal origin tumors including medulloblastomas and glioblastomas in experimental animals. The oncogenecity of JCV is attributed to the viral early gene product, T-antigen, which has the ability to associate with and functionally inactivate well-studied tumor suppressor proteins including p53 and pRb. The observations from laboratory animal experiments have provided a rationale for examining the presence of the JCV DNA sequence and expression of the viral oncogenic protein in human brain tumors. We have examined 85 clinical specimens from the United Kingdom, Greece, and the United States, representing various human brain tumors including oligodendroglioma, astrocytoma, pilocytic astrocytoma, oligoastrocytoma, anaplastic astrocytoma, anaplastic oligodendroglioma, glioblastoma multiforme, gliomatosis cerebri, gliosarcoma, ependymoma, and subependymoma, for their possible association with JCV. We performed gene amplification techniques using a pair of primers that recognize the JCV DNA sequence, and we demonstrated the presence of the viral early sequence in 49 (69%) of 71 samples. More importantly, our results from immunohistochemistry analysis revealed expression of JCV T-antigen in the nuclei of tumor cells in 28 (32.9%) of 85 tested samples. These observations, along with earlier in vitro and in vivo data on the transforming ability of this human neurotropic virus invite additional studies to re-evaluate the role of JCV in the pathogenesis of human brain tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Brain tumors comprise a group of neoplasms originating from various tissues including neuroectoderm, mesenchymal structures, retained embryonal structures, or metastases from other organs (1) . Neuroectodermal tumors, which are predominantly of glial origin, constitute more than 60% of brain tumors. Tumors derived from astrocytes include pilocytic astrocytomas, diffuse astrocytomas, anaplastic astrocytomas, and glioblastoma multiforme; those from oligodendrocytes include oligodendrogliomas and anaplastic oligodendrogliomas; and those from ependymal cells include ependymomas (2) .

With the exception of a few inherited neoplastic syndromes, the etiology of brain tumors remains largely unknown. Mutational inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations in human astrocytic brain tumors (3) . p53 mutations are mainly located in the highly conserved region of the gene, with clusters at codons 175, 248, and 273 (4) . These codons are among the six hot spots found in a variety of human tumors. Other genetic alterations include allelic deletions on chromosome 19q in more than 60% of cases of oligodendrogliomas (5) , trisomy 7, monosomy 21 and 22, and deletion of p22 in ependymomas (6) , which appear to have the largest number of chromosome abnormalities.

Several lines of recent studies have suggested the association of oncogenic viruses including polyomaviruses with various types of human cancer (7, 8, 9, 10, 11, 12) . Specifically, investigation of ventricular tumors revealed frequent association of polyomavirus DNA such as SV40 with a large number of choroid plexus papillomas and ependymomas (7 , 11) . In more recent studies, an association of DNA with the human neurotropic polyomavirus, JCV,3 has been detected in several primitive neuroectodermal tumors including medulloblastoma (13 , 14) . These observations are of concern in light of epidemiological studies showing that by the age of 15, >75% of the human population worldwide are infected with JCV (15 , 16) .

Although evidence for the role of JCV in human CNS neoplasms is mounting, the oncogenic potential of this human virus has been well established in several experimental animal models (17 , 18) . For example, intracerebral inoculation of JCV into owl and squirrel monkeys results in the development of astrocytomas (19 , 20) . Intracerebral inoculation of newborn Golden Syrian hamsters with JCV has induced a broad range of tumors including medulloblastoma, astrocytoma, glioblastoma, primitive neuroectodermal tumors and peripheral neuroblastomas in more than 85% of inoculated animals (21 , 22) . Whereas no evidence for the lytic infection of the tumor cells with JCV was observed, expression of the viral oncogenic protein, T-antigen, was prominent in the tumor cells. Injection of JCV into the brains of newborn rats caused undifferentiated neuroectodermal origin tumors in the brain of 75% of the animals (23 , 24) . Interestingly, in all instances, neoplasia induced by JCV were of neuroectodermal origin presumably because of unique neurotropism of the viral early gene promoter, which makes expression of the JCV genome more active in cells of neural origin (25) .

Transgenic mice containing the entire gene for JCV T-antigen under the control of its own promoter develop adrenal neuroblastomas (17 , 26) and primitive neuroectodermal tumors (18) . T-antigen was expressed only in the tumor tissues of transgenic mice. Altogether, these observations have established that, in the absence of viral replication, the product of the JCV early gene can induce tumors of neuroectodermal origin in various laboratory animals. With this notion we sought to evaluate a collection of various well-characterized tumors of CNS origin obtained from hospitals in the United States, United Kingdom, and Greece for the presence of the JCV DNA sequence and the expression of its early protein, T-antigen. Because the oncogenic potential of JCV T-antigen may be attributed, at least in part, to its ability to associate with and functionally inactivate tumor suppressors, including p53, we examined the presence of p53 in the tumor cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Clinical Samples.
A total of 85 samples of different varieties of brain tumors were collected from the pathology archives of Queen Mary, University of London, London, United Kingdom; University of Patras, Patras, Greece; and MCP Hahnemann University, Philadelphia, Pennsylvania, USA.

Histological and Immunohistochemical Analysis.
Formalin-fixed, paraffin-embedded samples were sectioned at 4-µm thickness and were stained with H&E for routine histological examination and classification. Immunohistochemistry was performed by using the avidin-biotin-peroxidase complex system, according to the manufacturer’s instructions (Vectastain Elite ABC Peroxidase Kit; Vector Laboratories). Briefly, sections were deparaffinized in xylene and rehydrated through alcohols up to water. For non-enzymatic antigen retrieval, sections were heated in 0.01 M sodium citrate buffer (pH 6.0) to 95°C under vacuum for 40 min and allowed to cool for 30 min at room temperature. Then the slides were rinsed with PBS and incubated in methanol/3% H2O2 for 20 min to quench endogenous peroxidase. Sections were then washed with PBS and blocked in 0.1% BSA/PBS containing 5% normal horse serum for 2 h at room temperature. Incubation with primary antibodies took place overnight at room temperature in a humidifier chamber with antibodies specific for viral proteins and cellular markers. Antibodies that were used in this study included pAb416, for the detection of SV40 T-antigen, which cross-reacts with JCV T-antigen (1:100 dilution, Oncogene Science), and p53 antibody (1:100 dilution, Dako clone D0–7). Paraffin-embedded tumor tissue from Syrian hamsters intracerebrally inoculated with JC virus was used as a positive control for detection of T-antigen. Cellular markers were analyzed using monoclonal antibodies specific for GFAP (1:100 dilution, clone 6F2; Dako); Synaptophysin (1:500 dilution, clone SY38; Boehringer-Mannheim); Pan-axonal neurofilament (1:1000 dilution, clone SMI-312; Sternberger Monoclonals Inc.) and rabbit anticow antibody specific for S-100 (1:250 dilution; Dako). Next, biotinylated horse antimouse or antirabbit IgGs were incubated for 1 h at room temperature and avidin-biotin peroxidase complex steps were performed according to the manufacturer’s instructions (Vector Laboratories). Finally, the sections were developed with a diaminobenzidine substrate, counterstained with hematoxylin, and coverslipped with Permount.

DNA Extraction and PCR Amplification.
DNA was prepared from 10 sections of 10 µm each of paraffin-embedded tissue by using the QIAamp Tissue Kit (Qiagen) according to the manufacturer’s instructions. To avoid cross-contamination of the samples, several precautions were taken as described previously (14) .

PCR amplification was performed on extracted DNA using the following primers: Pep1 (nucleotides 4255–4274), 5'-AGTCTTTAGGGTCTTCTACC-3'; and Pep2 (nucleotides 4408–4427), 5'-GGTGCCAACCTATGGAACAG-3'. Amplification was performed on 500 ng of template in a total volume of 50 µl with AmpliTaq DNA Polymerase (Perkin-Elmer) in the presence of 2.5 µM MgCl2 and 0.5 µM each primer (Oligos, Etc.) using the Perkin-Elmer Gene Amp 9600 PCR System dedicated for amplification of human tissue samples. The samples were amplified by denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. A final extension at 72°C for 7 min was performed for termination. Negative controls consisted of samples amplified in the absence of template DNA and the plasmid pBJC as template, containing the JCV genome, served as a positive control. Southern blot analysis was performed on 10 µl of PCR product separated by electrophoresis on 2% agarose gels. The gels were then treated for 15 min each with 0.2 M HCl for depurination, 1.5 M NaCl/0.5 molar NaOH for denaturation, and 1.5 M NaCl/0.5 M Tris-HCl (pH 7.4) for neutralization, followed by transfer of the amplified fragments from the gel to nylon membranes (Hybond-N, Amersham). The membranes were then prehybridized for 1 h in Ultrahyb solution (Amersham), followed by hybridization in the same solution containing 5 x 106 cpm/ml radiolabeled oligonucleotide specific for JCV (JC probe: 5'-GTTGGGATCCTGTGTTTTCATC-3'). Membranes were hybridized overnight, washed, and autoradiographed as described previously (14) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The brain tumor samples used in this study were classified according to the criteria from the latest classification of tumors of the CNS from WHO (27) . Of the 85 samples, 11 were classified as oligodendrogliomas, 16 as fibrillary astrocytomas, 6 as pilocytic astrocytomas, 8 as mixed oligoastrocytomas, 4 as anaplastic astrocytomas, 3 as anaplastic oligodendrogliomas, 3 as anaplastic oligoastrocytomas, 26 as glioblastoma multiforme, and 5 as ependymomas. In addition, one case of gliosarcoma, one gliomatosis cerebri, and one case of subependymoma were included in this study.

Fig. 1Citation illustrates a representative histological assessment of the tumors and production of GFAP in some of these cells. In general, the oligodendrogliomas were characterized by groups of cells with a clear halo around the nuclei, a distinct capillary network, and contained calcifications. Those cells were GFAP negative (Fig. 1A)Citation . Pilocytic astrocytomas were characterized by spindle-shaped cells, which formed interdigitating fasicles, in a dense fibrillary matrix containing Rosenthal fibers. Those cells were GFAP positive (Fig. 1B)Citation . Fibrillary astrocytomas had a low cell density and formed a prominent fibrillary network with numerous microcysts. These neoplastic cells and their fibrillary network were GFAP positive (Fig. 1C)Citation . Gemistocytic astrocytomas were characterized by numerous cells containing abundant eosinophilic cytoplasm-producing GFAP (Fig. 1D)Citation . Anaplastic oligodendrogliomas had similar features as the oligodendrogliomas (shown in Fig. 1ACitation ) with the exception that they contained atypical pleomorphic nuclei and several areas of necrosis (Fig. 1E)Citation . Anaplastic astrocytomas were characterized by numerous pleomorphic cells with atypical nuclei, mitotic activity and multinucleated giant cells. These neoplastic cells were GFAP positive (Fig. 1F)Citation . The cases of glioblastoma multiforme exhibited numerous giant multinucleated cells, nuclear pleomorphism, mitotic figures, necrosis, pseudopalisading, and endothelial cell proliferation. As anticipated, these tumors were GFAP positive (Fig. 1G)Citation . Ependymomas showed moderate cellularity with the characteristic formation of several rosettes containing blepharoplasts (Fig. 1H)Citation .



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  		Fig. 1. Histological and immunohistochemical characterization of human brain tumors. Morphological characteristics of oligodendrogliomas (A, x400) include numerous cells with a clear halo around the nucleus, a capillary network, and calcifications. A pilocytic astrocytoma (B, x400) shows fasicles of spindle cells. A diffuse fibrillary astrocytoma (C, x400) exhibits a prominent fibrillary background. A diffuse gemistocytic astrocytoma contains numerous cells with abundant eosinophilic cytoplasm (D, x400). An anaplastic oligodendroglioma (E, x400) demonstrates numerous atypical cells with mitotic figures. Anaplastic astrocytomas (F, x400) show numerous atypical cells with pleomorphic nuclei. Glioblastoma multiforme shows numerous pleomorphic cells, some of them giant and multinucleated. Mitotic figures and neovascularization are shown (G, x400). Ependymoma (H, x1000) shows the characteristic rosette formation. GFAP immunostaining to confirm the glial nature of the tumors is positive in the cytoplasm of the astrocytic cells and fibrillary background (B, C, D, F, and G, insets, x400) and negative in the oligodendroglial cells (A and E, x400).

 
To examine the association of JCV with the tumor samples, first we selected several well-preserved paraffin-embedded tissues from each group of tumors and extracted total DNA for PCR analysis. In the initial studies, a pair of primers that amplified the NH2 terminus of JCV T-antigen between nucleotides 4255 to 4427 were used. In some instances, other primers that amplify other regions of the viral genome, including Agno and the viral sequences corresponding to the capsid protein (VP1), were also used. Fig. 2Citation illustrates representative data depicting results from PCR that were obtained by Southern blot analysis of the amplified DNA using the T-antigen NH2-terminal primers and hybridization with oligonucleotide probe that is specific for JC virus.



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  		Fig. 2. Schematic representation of the JCV genome and detection of the JCV DNA sequence in various human brain tumors. A, outside the circle, the position of the oligonucleotide primers used for PCR are shown. The numbers in the inner circle represent the map positions. Arrows, the coding regions for the early protein, T-antigen (solid), and the late capsid proteins (shaded). The location of the amplified DNA fragment corresponding to the NH2-terminal region of JCV T-antigen is shown outside the circle and depicts the size of the amplified DNA. B, DNA from various brain tumors was analyzed for the presence of the JCV sequence by PCR using primers derived from the NH2-terminal of T-antigen as described in "Materials and Methods." PCR products were analyzed by Southern blot analysis using DNA probe specific for JCV as described in "Materials and Methods." Negative controls represent amplification reaction in the absence of template DNA, whereas the positive samples contain reaction with the JCV genome. The numbers under the blots, the case numbers in Table 1Citation .

 
Results from multiple experiments revealed that 4 (57.1%) of 7 oligodendrogliomas, 10 (76.9%) of 13 astrocytomas, 4 (80%) of 5 pilocytic astrocytomas, 5 (62.5%) of 8 oligoastrocytomas, 2 (66.6%) of 3 anaplastic oligodendrogliomas, 5 (83.3%) of 6 anaplastic astrocytic tumors, 12 (57.1%) of 21 glioblastoma multiformes, and 5 (83.3%) of 6 ependymomas contained the JCV early gene sequence. Also, the only cases of gliomatosis cerebri, gliosarcoma, and subependymoma that were available for this study were positive for the JCV DNA sequence and the expression of T-Antigen. Table 1Citation Citation summarizes the characterization of the tumor samples based on pathological diagnosis, location of tumors, immunohistochemical evaluation of the marker proteins and the results from PCR of the selected tumor samples. Several controls were included in the PCR experiments. Examination of normal human and mouse brain showed no evidence of the presence of JCV sequences, whereas DNA from a JCV-induced tumor in a hamster brain showed detection of the JC viral genome (data not shown).


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  		Table 1 Clinical and immunohistochemical analysis of brain tumors

 

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  		Continued

 
Detection of the JCV early gene sequence in the various human brain tumors prompted us to examine production of the early gene product T-antigen by an immunohistochemical technique. Conditions for T-antigen immunostaining were previously optimized in our laboratory with various sections of paraffin-embedded tumors derived from JCV in experimental animals as well as in several well-established clinical samples (14 , 18) . As illustrated in Fig. 3Citation , T-antigen-positive nuclei were found by immunohistochemical analysis of oligodendroglioma (Fig. 3A)Citation , anaplastic oligodendroglioma (Fig. 3B)Citation , pilocytic astrocytoma (Fig. 3C)Citation , astrocytoma (Fig. 3D)Citation , glioblastoma multiforme (Fig. 3E)Citation , and ependymoma (Fig. 3F)Citation . Immunohistochemistry against VP1 demonstrated no evidence of expression of the viral late proteins in any of the samples (data not shown). Table 1Citation illustrates the summary of immunostaining of the tumor samples with anti-T-antigen antibody. It is evident that with the exception of anaplastic astrocytoma, every type of tumor was positive for T-antigen expression. We also noticed a discrepancy in the number of JCV positive samples as determined by PCR amplification to those obtained by immunohistochemistry. For instance, the results from PCR showed that whereas four of five pilocytic astrocytomas contained the JCV DNA sequence, only one of six samples was positive for T-antigen expression, which suggested that, perhaps, mutations in the viral genome abrogated expression of T-antigen in tumor cells. As mentioned earlier, JCV T-antigen has the ability to interact with and functionally inactivate the p53 tumor suppressor protein. This association increases the steady-state level of p53 and allows its detection in cancer cells. It should be noted that the stability of p53 can also be increased because of mutation, which results in inactivation of this protein (28) . To examine the level of p53 in tumor cells exhibiting positive staining for T-antigen, we performed immunohistochemistry using an anti-p53 antibody that recognizes both wild-type and mutant human p53. As shown in Fig. 3Citation (insets) the various T-antigen-positive brain tumor cells showed nuclear immunoreactivity for p53. The summary of the results from these studies is shown in Table 1Citation . With the exception of the oligoastrocytomas, glioblastoma multiforme, and ependymoma, the number of tumor samples that were positive for T-antigen closely paralleled those that showed p53 immunoreactivity in the nuclei. It is likely that in oligoastrocytoma, anaplastic astrocytoma, and glioblastoma multiforme, mutations in the p53 gene stabilized this protein and permitted its detection in T-antigen negative cells. Double-labeling immunostaining for T-antigen and p53 was performed in selected cases and demonstrated the presence of both proteins in the nuclei of the same neoplastic cells (data not shown). All together, results from gene amplification and immunohistochemistry of the various human brain tumor samples point to the possible association of JCV with a wide variety of CNS neoplasias.



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  		Fig. 3. Immunohistochemical analysis of human glial tumors for JCV T-antigen and p53. Detection of T-antigen demonstrates intense nuclear reactivity in neoplastic cells of an oligodendroglioma (A), anaplastic oligodendroglioma (B), pilocytic astrocytoma (C), fibrillary astrocytoma (D), glioblastoma multiforme (E), and ependymoma (F). Immunohistochemistry for p53 shows the same pattern of nuclear positivity (insets). All panels and insets, x1000.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Unlike other members of the polyomavirus family, including the well-studied SV40, JCV has a narrow tropism attributable to the viral regulatory region that restricts efficient expression of the early protein, T-antigen, to neural origin cells (25) . Similar to other members of this family, the T-antigen of JCV has multiple regulatory functions, which are required for the successful progression of the virus through the lytic cycle. This latter feature of JCV causes destruction of oligodendrocytes, the myelin-producing cells of the CNS, and the appearance of phenotypically abnormal astrocytes in brains of patients with PML (15 , 29) . Activation of the viral early promoter under circumstances that are not optimal for efficient lytic infection cycle can result in the accumulation of T-antigen in the brain. This, in turn, may trigger a cascade of events that results in uncontrolled proliferation of CNS cells. In support of this notion, previous studies have demonstrated that intracellular injection of JCV into hamsters, in which the virus is unable to initiate DNA replication, perhaps because of the incompatibility of the {alpha}-primase subunit of DNA polymerase (30 , 31) , induces CNS neoplasia with no sign of viral replication or demyelination.

Earlier observations on the development of primitive neuroectodermal tumors in transgenic mice expressing only JCV T-antigen (18) renewed interest in studying the association of JCV with brain tumors, in particular, medulloblastoma. Results from more sensitive assays such as PCR in concert with immunohistological techniques have allowed detection of the JCV sequence and expression of T-antigen in as many as 25% of medulloblastomas (13 , 14 , 32) .

Detection of the JCV DNA sequence and expression of T-antigen in various human brain tumors is an important observation because it suggests that this ubiquitous human virus may be associated with the development of some, if not all, of the various types of brain tumors. Indeed, a large scale multi-institutional study is required to establish the association of JCV with CNS tumors. The T-antigen of JCV, by interacting with the tumor suppressor proteins such as p53 and pRb, can deregulate the cell cycle pathway and cause uncontrolled proliferation of cells (33 , 34) . The cell cycle pathway is controlled by well-balanced expression and activity of a series of positive factors such as cyclins and their partner cyclin-dependent kinases, and negative factors such as tumor suppressors and p15, p16, and p21. Accordingly, deregulation of both positive and negative factors has been repeatedly observed in various brain tumors including malignant astrocytoma. Indeed, functional inactivation of these regulatory proteins can be due to genetic variations, which may occur in the locus corresponding to genes encoding these proteins.

The number of tumors showing the presence of the JCV DNA sequence in the absence of T-antigen expression is interesting because it suggests that an alternative pathway independent of T-antigen-mediated inactivation of the tumor suppressors may be operative. Therefore, one may also envision a hit-and-run mechanism for JCV-induced uncontrolled proliferation of tumor cells. In support of this notion, results from previous studies on JCV-induced medulloblastoma in a murine model suggest that T-antigen, by associating with p53, may cause genomic instability and promote the occurrence of mutations in various important genes including p53 itself (10) . Under these conditions, the uncontrolled proliferation of cells becomes independent of T-antigen, and that may lead to the extinction of T-antigen in the cells.

Our findings presented in this report are significant in light of earlier observations that indicated that: (a) JCV is mutagenic and has the ability to transform human cells in culture (35) ; (b) JCV induces a variety of CNS tumors in laboratory animals and primates (19, 20, 21, 22, 23, 24) ; (c) the tumors induced in animals closely resemble those seen in pediatric patients (36) ; (d) reactivation of JCV in PML brain induces giant bizarre astrocytes with pleomorphic hyperchromatic nuclei resembling malignant astrocytes of pleomorphic glioblastomas (29) ; and, finally, (e) JCV is widespread in the human population inasmuch as >75% of adults are exposed to JCV and the virus may establish a persistent infection (37 , 38) .

These observations, along with several case reports on the association of JCV and human neoplasia in immunosuppressed and non-immunosuppressed patients (32 , 39, 40, 41, 42) , suggest a potential etiological role for JCV in multiple types of human brain tumors.


   ACKNOWLEDGMENTS
 
We thank past and present members of the Center for Neurovirology and Cancer Biology for their insightful discussion, sharing of ideas, and sharing of reagents, and C. Schriver for editorial assistance and preparation of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant PO1 NS36466 (to K. K). Back

2 To whom requests for reprints should be addressed, at Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, 1900 North 12th Street, 015-96, Room 203, Philadelphia, PA 19122. Phone: (215) 204-0678; Fax: (215) 204-0679; E-mail: kkhalili{at}astro.temple.edu Back

3 The abbreviations used are: JCV, JC virus; CNS, central nervous system; GFAP, glial fibrillary acidic protein; PML, progressive multifocal leukoencephalopathy. Back

Received 7/28/00. Accepted 3/15/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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