pmc.ncbi.nlm.nih.gov

JCV infection of human B lymphocytes: A possible mechanism for JCV transmigration across the blood-brain barrier

. Author manuscript; available in PMC: 2011 Jul 15.

Published in final edited form as: J Infect Dis. 2010 Jul 15;202(2):184–191. doi: 10.1086/653823

Abstract

It has been suggested that JCV might traffic to the CNS in infected B-cells. Moreover, recent data suggest presence of JCV in bone marrow plasma cells. However, the evidence for infection and replication of JCV in B cells is unclear. To address this question, we infected EBV-transformed B cells with JCV and found that the viral genome decreased over 1,000-fold from days 0 to 20 after infection, which concurred with the absence of viral early and late mRNA transcripts and proteins. However, immunofluorescent images of B cells infected with FITC-conjugated JCV demonstrated that JCV enters the B cells and DNase protection assay confirmed the presence of intact JCV virions inside the B cells. Moreover, JCV-infected B cells were able to transmit infection to naïve glial cells. These data confirm that JCV non-productively infects B cells and possibly uses them as a vehicle for transmigration across the BBB.

Keywords: Human polyomavirus JC, B lymphocytes, PML, AIDS, BBB, HBMVE cells, human brain microvascular endothelial cells

Introduction

Progressive multifocal leukoencephalopathy (PML), a subacute demyelinating disease of the central nervous system (CNS) [1] results from the lytic infection of oligodendrocytes, the myelin producing cells in the brain, with human polyomavirus JC (JCV) [2-4]. JCV foci in the PML brain are closely related to the blood vessels [5] and JCV is presumably spread by the hematogenous route from the primary site of infection to the secondary sites, such as kidneys, lymphoid tissues and brain, to establish focal areas of infection or persistence [2, 6-13]. However, the precise mechanism(s) of JCV dissemination throughout the body and trafficking across the blood-brain barrier (BBB) remains poorly understood.

Possible role of B cells in JCV transmigration across the BBB has been suggested [8, 12, 14-18]. JCV-infected B cells were first detected in the spleen and bone marrow of two PML patients [15] and subsequently in the CNS of a PML patient [19]. Moreover, JCV DNA was reported to be associated with peripheral blood lymphocytes (PBL) of 89.5% and 38%, PML patients and AIDS patients with varying degree of immunodeficiency, respectively [7, 10]. Demonstration of viral genome in the PBL suggests the possibility of hematogenous spread of JCV. Furthermore, JCV infection of human B cells in vitro was suggested [12, 18, 20] and it was argued that JCV infection of EBV-transformed B cells or B-cell lines resulted in viral DNA replication and infectious virions production [18, 20]. However, evidence for JCV replication in B cells was inconclusive since none of these studies quantitated the viral DNA, and mRNA transcripts were rarely demonstrated [18]. Moreover, infectious JCV virions in the B cells might be the residual virus inoculum used to infect the B cells rather than de novo production of the virus in the B cells.

Possible role of B lymphocytes in JCV transmigration across the BBB came in lime-light again in 2005 after the development of PML in patients with multiple sclerosis and Crohn's disease treated with a monoclonal antibody natalizumab (Tysabri® Biogen Idec and Elan Pharmaceuticals) [21-23]. Natalizumab is a recombinant humanized monoclonal antibody directed against the adhesion molecules α4β1 and α4β7 integrins [24]. Since α4β1 integrin interacts with very late antigen-4 (VLA-4) and this interaction is required for generating T and B cells from bone marrow progenitor cells in adult mice [25], it is argued that α4-integrin blockade mobilizes JCV-infected pre-B cells from bone marrow into the circulation and thus facilitates JCV dissemination and PML development [26, 27]. Recently three patients treated with efalizumab for psoriasis also developed PML [28]. Efalizumab (Raptiva®, Genentech, Inc., South San Francisco, CA) is a humanized monoclonal antibody (IgG1) that binds to the alpha chain (CD11a) of the leukocyte function associated antigen (LFA-1) [29, 30]. LFA-1 is a member of the heterodimeric β2 integrin family and it interacts with intercellular adhesion molecules (ICAM) expressed on antigen presenting cells, and endothelial cells and is necessary for T-cell activation, T-cell helper and B-cell responses, natural killer cell cytotoxicity and antibody-dependent cytotoxicity [30]. Efalizumab can selectively and reversibly block the activation, reactivation and trafficking of T-cells [29, 30]. Natalizumab and efalizumab both could create artificial cell mediated immune deficiency in the CNS by inhibiting the migration of lymphocytes across the BBB and thus may facilitate the development of PML.

To better understand the possible role of B cells in JCV transmigration across the BBB, we studied kinetics of JCV(Mad1) infection of B cells in vitro. Our data confirm that JCV non-productively infects B cells. However, JCV virions persist in B cells and B cells can transmit infectious virions to naïve PHFG cells suggesting that B cells can act as a vehicle for JCV transmigration across the BBB.

Materials and Methods

Virus and cell cultures

JCV (Mad1) was propagated in primary human fetal glial (PHFG) cells, purified and quantitated by the HA assay and real-time PCR [31-34]. EBV-transformed primary B cells were provided by Dr. Allison Imrie, University of Hawaii, and were cultured in RPMI-1640 supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL) and 2 mM L-glutamate at 37°C and 5% CO2 as described previously [35, 36].

JCV infection of EBV-transformed B cells

2.5 × 106 EBV-transformed B cells in suspension were infected with 250, 1,000 or 2,500 HA units of JCV(Mad1) for 2 hr at 37°C, washed with PBS three times and aliquots of 2.5 × 105 cells were harvested at days 0 (2 hr), 5, 10, 15 and 20 after infection for DNA and RNA extraction.

FITC-labeling of JCV

Sucrose purified JCV [31] was labeled with fluorescein isothiocyanate (FITC) using FluoroTag™ FITC Conjugation Kit (Sigma, St. Louis, MO) by modifying the published protocol [37, 38]. Briefly, 850 μL of the sucrose-purified virus containing 100 HAU/μL of JCV was dialyzed overnight with labeling buffer (0.05 M Boric Acid, 0.2 M NaCl, pH 9.2) and the volume was adjusted to 1 mL. The dialyzed virus (1 mL) was then transferred into 2 mL tube containing a small stirrer and 250 μL of FITC solution (1 mg/mL) in 0.1 M carbonate-bicarbonate buffer (pH 9.0) was added slowly drop-by-drop and incubated for 8 hr at room temperature in dark with continuous gentle stirring. The FITC-labeled virus was then dialyzed overnight with PBS (pH 7.2), aliquoted and stored at -80°C. As a control 5 mg BSA in 1 mL of PBS was also conjugated with FITC using the above protocol, aliquoted and stored at -80°C. Protein and FITC in the FITC-conjugated viral- or BSA- suspension were determined according to the company's protocol (Sigma) by measuring the absorbance at 280 nM and 495 nM, respectively. The FITC-conjugated BSA was diluted to get the same absorbance as FITC-conjugated JCV at 495 nM and used as non-specific fluorescence control.

Characterization of EBV-transformed B-cell infection with FITC-conjugated JCV

One million B cells were incubated with 1,000 HAU of FITC-conjugated JCV (FITC-JCV) or FITC-conjugated BSA (FITC-BSA) containing the same amount of fluorescence in 500 μL of RPMI-1640 for 2 hr, washed three times with PBS and the pellet was dissolved in 1 mL of PBS. Forty μL of cell suspension containing approximately 40,000 B cells were transferred into each well of multi-well slide, air dried and fixed with 4% paraformaldehyde for 10 min. Cells were then washed with PBS and permeabilized with 0.4% Triton-X 100 for 5 min. The permeabilized cells were blocked with 5% BSA for 1 hr and incubated with anti-rabbit protein disulphide isomerase (PDI) (1:500), to visualize the plasma membrane, washed three times with PBS and further incubated with anti-rabbit secondary antibody conjugated with Alexa-594 and mounted with Vectashield® mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Fluorescent cells were examined using a Axiocam MRm camera mounted on a Zeiss Axiovert 200 microscope, equipped with appropriate fluorescence filters and objectives as described previously [32].

One million EBV-transformed B cells were incubated with 125, 250, 500 or 1,000 HAU of FITC-JCV for 2 hr and washed with PBS. Cells were incubated with antibody against CD19-conjugated with PE (CD19-PE) and subjected to flow cytometry in Guava EasyCyte™ Plus platform. BSA-labeled with FITC containing the same amount of fluorescence was used as control.

DNA and RNA extraction

Uninfected and JCV-infected B cells in T25 flasks were washed with PBS, counted and an aliquot of 0.25 million cells in duplicate were either frozen for DNA extraction or lysed with 350 μL of RLT plus buffer for RNA extraction (Qiagen) and stored at -80°C. RNA was isolated using previously published protocols [32]. Additionally, 100 μL of DNA was extracted using the Qiagen QIAprep® Spin Miniprep Kit from each 0.25 million cells harvested at different time points, according to the manufacturer's protocol.

Quantitation of viral DNA and mRNA transcripts and RT-PCR

Two μL of template DNA or cDNA were amplified and quantitated in the Bio-Rad's iCycler iQ™ Multicolor Real-Time PCR Detection System using primers, probes and protocols described previously [39]. Copies of JCV TAg or VP-1 genomes or mRNA transcripts in experimental samples were calculated from the standard curve and expressed as copies of viral genome per 35-mm plate or mRNA transcripts per microgram of total RNA [32, 39].

Results

JCV infection of B-lymphocytes is non productive

JCV VP-1 genome copies recovered from infected 2.5 × 105 B cells decreased over 1,000-fold from day 0 to day 20 after infection (figure 1A) and neither early (TAg) nor late (VP-1) mRNA transcripts were detected (data not shown), suggesting that JCV infection of B cells is non-productive. Our qPCR and qRT-PCR assays were sensitive and consistently detected as low as 10 to 100 copies of JCV DNA and mRNA transcripts, respectively [31]. Since, JCV infection of EBV-transformed B cells did not result in viral genome replication or transcripts expression, it is possible that JCV might have remained associated with the B cells by attaching to cell membrane and might not have entered the B cells. To test this hypothesis, 2.5 × 106 B cells were inoculated with 250 HAU of JCV and incubated for 2 hr at 37°C. After incubation, the cells were washed twice with PBS and trypsinized for 10 min with 0.5, 5 or 50 μg/mL of trypsin or with PBS alone followed by two washes of PBS and were cultured for up to 10 days. On days 1, 5 and 10, 2.5 × 105 trypsin-treated and untreated B-cells were harvested and JCV VP-1 DNA was amplified and quantitated by qPCR. The data demonstrate that trypsin treatment had no significant effect in further reducing the JCV genome copies (figure 1B) suggesting that JCV indeed entered into the B cells.

Figure 1.

Figure 1

JCV infection of B cells is non-productive. 2.5 × 106 EBV-transformed B cells were infected with 250, 1,000 or 2,500 HA units of JCV(Mad1) for 2 hr and aliquots of 2.5 × 105 cells were harvested at days 0 (2 hr), 5, 10, 15 and 20 after infection. (A) DNA was extracted from 2.5 × 105 cells and VP-1 DNA was amplified and quantitated by qPCR. VP-1 DNA genome copies decreased exponentially from days 0 to 20 after infection. (B) Trypsin treatment had no effect on further reducing the JCV genome copies at any time point. ID; infection dose - JCV VP-1 genome copies recovered from 25, 100 or 250 HAU of JCV equivalent used to infect 250,000 B lymphocytes was shown for comparison.

Moreover, by infecting EBV-transformed B cells with FITC-labeled JCV and by employing flow cytometry, we confirmed that JCV infects B cells in a dose-dependent manner. When B cells were incubated with 125, 250, 500 or 1,000 HAU of JCV per million B cells approximately 1.5, 1.7, 3.9 or 7.7% of B cells, respectively, were infected with JCV (figure 2A-D).

Figure 2.

Figure 2

JCV infection of B cells is dose-dependent. One million EBV-transformed B cells were incubated with (A) 125, (B) 250, (C) 500 or (D) 1,000 HAU of FITC-JCV or with (A-D) equivalent fluorescence containing BSA similarly labeled with FITC (filled gray histogram) as control for 2 hr and washed with PBS and subjected to flow cytometry.

We further characterized JCV infection of EBV-transformed B cells using FITC-labeled JCV. B cells and human brain microvascular endothelial (HBMVE) cells were either infected with FITC-labeled JCV (figure 3E-L and 3M-P) or inoculated with FITC-conjugated BSA (FITC-BSA) containing the same amount of fluorescence (figure 3A-D) and examined by immunofluorescence microscopy. Interestingly, while all FITC-labeled JCV virions were seen inside the HBMVE cells (figure 3N-O), FITC-labeled JCV virions appeared to be inside the B cells and on the B cells surface (figure 3G-H and 3K-L). These data confirm that JCV indeed infects the B cells. However, in contrast to HBMVE cells where VP-1 and T antigen proteins were observed predominantly in the cell nucleus [32], our repeated attempts to demonstrate T antigen and VP-1 proteins expression in the B cells failed and it is unlikely that JCV infection of B cells is productive. Collectively, our data demonstrates that JCV infects the B cells; however, JCV infection of B cells is non-productive.

Figure 3.

Figure 3

Epifluorescence microscopy demonstrating infection of B cells by FITC-conjugated JCV. One million B cells were inoculated with either (A-D) FITC-labeled BSA as negative control or (E-H and I-L) 1,000 HAU of FITC-labeled JCV. Membranes were labeled with PDI and visualized with Alexa flour 594 (red). Cells were counterstained with DAPI to visualize cell nuclei (blue). Merged images demonstrate FITC-labeled JCV on B cell surface (arrow heads) and inside the B cells (arrow) (H and L). HBMVE cells infected with FITC-labeled JCV (positive control) (M-P). DAPI; 4′,6-diamidino-2-phenylindole; PDI; protein disulphide isomerase; scale bar = 5 μm

Infectious JCV virions persist in the B cells

Since, our data suggest that JCV infects EBV-transformed B cells but does not replicate in these cells, intact virus must survive inside the B cells long enough to be trafficked across the BBB and then have a mechanism to be released to infect susceptible cells, particularly the oligodendrocytes. However, our data suggest that JCV genome copies rapidly decreases in the EBV-transformed B cells after infection and it was unclear whether the decrease in viral genome was the result of degradation of viral DNA inside the B cells or merely resulted from dilution effect of replicating B cells since in vitro EBV-transformed B cells replicate rapidly. To address this question, we infected 2.5 × 106 EBV-transformed B cells with 250 HAU of JCV for 2 hr, washed and 2.5 × 105 cells were seeded in each well of a 24-well plate, cultured and all cells from each well were harvested at each time point. The cells were freeze-thawed for four times to mechanically lyse the cells and triplicate samples at each time point were either untreated or treated with 125 U/mL of DNase for 1 hr at 37°C to digest genomic and free viral DNA. The DNase-treated cells were heat-inactivated at 75°C for 10 min, DNA was extracted from both DNase-treated and -untreated cells, DNA concentration was measured and JCV VP-1 DNA was quantitated by qPCR. Figure 4A demonstrates that the total cellular DNA recovered from the B cells increased approximately 16-fold (10.7±1.5 to 170.9±21.1 μg/mL) from days 0 to 15 after infection suggesting that the B cells were actively replicating. However, JCV VP-1 DNA copies decreased approximately 11-fold (18.0±8.6 × 108 to 16.0±7.5 × 107) from days 0 to 15 after infection further confirming our observation that JCV infection of B cells is non-productive. While DNase treatment was very effective and reduced the total cellular DNA recovered from JCV-infected B cells, up to 96%, (170.9 ± 21.1 μg/mL to 6.9±1.7 μg/mL) on day 15 after infection (figure 4A), DNase treatment had very little effect on the JCV VP-1 DNA copies recovered from the B cells (figure 4B). These results suggest that JCV virions in the B cells remain intact for at least 15 days after infection and are protected from DNase digestion.

Figure 4.

Figure 4

JCV genome in B cells was virion protected. Infected B cells harvested at different time points after infection were mechanically lysed by repeated freeze-thaw, and untreated or treated with DNase. DNA was extracted, concentration was measured and JCV VP-1 DNA was quantitated by qPCR. (A) Total cellular DNA recovered increased from day 0 to day 15 suggesting B cells were actively replicating and DNase effectively reduced DNA concentration. (B) JCV VP-1 DNA copies decreased from days 0 to 15 after infection as expected but DNase treatment had very little effect on the viral DNA suggesting that JCV genome in the B cells was virion protected from DNase digestion.

To further verify that JCV virions inside the B cells were infectious, JCV-infected B cells or lysates after infection of B cells with JCV for 24 hr were further co-cultured for additional 24 hr with naïve PHFG cells. After 24 hr co-culture, the PHFG cells were washed and cells were harvested at days 5, 10 and 15, and analyzed for viral late gene (VP-1) expression. While, no viral transcripts were recovered from PHFG cells co-cultured with uninfected B cells (data not shown), JCV VP-1 mRNA transcripts increased over 100-fold in PHFG cells co-cultured with JCV-infected B cells or JCV-infected B cells lysate clearly indicating that the virions that remained inside the B cell cytoplasm were infectious and replicated efficiently in naïve PHFG cells (figure 5).

Figure 5.

Figure 5

JCV-infected B cells transmit JCV infection to naïve PHFG cells. PHFG cells were co-cultured for 24 hr with JCV-infected B cells or B cells lysate and JCV VP1 mRNA transcripts expression in PHFG cells was quantitated. JCV VP-1 mRNA transcripts increased several fold in PHFG cells co-cultured with JCV-infected B cells or B cells lysate. However, no viral transcripts were detected from PHFG cells co-cultured with uninfected B cells (data not shown).

Discussion

The mechanism of JCV trafficking across the BBB remains poorly understood. PCR analyses have demonstrated that JCV may persist in the brain, tonsils and lymphocytes of individuals with and without PML [2, 6-11, 13, 16, 40, 41] and it was proposed that JCV might employ B-lymphocytes to cross the BBB [8, 12, 14-16], similar to HIV and simian immunodeficiency virus (SIV), which gain entry into the brain via infected monocytes (Trojan horse) [42]. Further studies also suggested presence of JCV DNA in the PBMC of 17.4% of 69 HIV-1-positive immunocompetent patients, in 23.2% of 82 HIV-1-positive immunocompromised patients, and in 60% of AIDS patients with PML [43]. However, there was no expression of JCV early and late mRNA transcripts in the PBMC [43]. Recently, JCV DNA was demonstrated in the PBMC and VP-1 stained CD138+ positive plasma cells were visualized in the bone marrow of a rheumatoid arthritis patient treated with methotrexate, who developed PML and had a rapid fatal outcome, further suggesting that B lymphocytes may play an important role in the JCV latency and dissemination [14, 16]. Similarly, JCV DNA was detected in the bone marrow of HIV-negative and –positive patients with and without PML, and JCV large T antigen but not VP1 was detected by double immunostaining in CD138+ plasma cells in an archival bone marrow specimen from an HIV-positive patient without PML [41]. Moreover, Focosi and colleagues detected JCV DNA in serial bone marrow samples from four hematological patients with histology-confirmed PML. Importantly JCV DNA was first detected in the bone marrow and later in CSF, peripheral blood and brain. Furthermore in one patient who survived, JCV was not detected after resolution of PML suggesting that JCV indeed disseminates by the hematogenous route [44].

It is often argued that JCV establishes low level of productive infection in B-lymphocytes [8, 12, 18, 20]. Atwood and colleagues have demonstrated that 1% of B lymphocytes were JCV positive by in situ DNA hybridization on day 13 after infection when one million B cells were infected with 10,000 HAU of JCV and argued that it was indicative of DNA replication [20]. Whereas Monaco and colleagues claimed that 3-5% of hematopoietic cell line (KG-1a), primary hematopoietic progenitor cells (CD34+) and primary B cells were positive for JCV on day 5 after infection when infected with 300 HAU/mL of JCV (Mad-4) [12]. While TAg and VP-1 transcripts [18] and proteins [8] expression were demonstrated occasionally in JCV-infected B cell line or B lymphocytes in vitro, these studies were often limited to one time point after infection or the transcripts were detected only after second round of PCR (nested-PCR) [18]. Taken together, in spite of several claims of replication of JCV genome and production of infectious virions de novo in the B cells, evidence for JCV replication in B lymphocytes is still lacking.

In an attempt to better understand the mechanism(s) of JCV transmigration across the BBB, we previously demonstrated that JCV productively infects HBMVE cells, principal cells lining the BBB, and proposed that cell-free JCV may cross the BBB by infecting HBMVE cells [32]. Recently VP-1 expressing CD138+ plasma cells were demonstrated in the bone marrow having both rearranged and archetype regulatory regions suggesting that B cells may latently harbor the virus, provide an environment for emergence of rearranged form of JCV and thus may act as a vehicle for JCV dissemination [16]. However, the relative role of B-lymphocytes in JCV transmigration across the BBB remains unclear. To address this issue, we employed EBV-transformed B cells and studied the replication kinetics of JCV. While we did not find any evidence of JCV replication in EBV-transformed B cells in vitro, we observed that JCV non-productively infects EBV-transformed B cells and JCV virions remain intact inside the B cells presumably long enough to employ B cells as a potential vehicle to cross the BBB and transmit infection to the oligodendrocytes in the brain.

Our study does not preclude the possibility that primary B cells, CD34+ hematopetic precursor cells or other cells within this lineage would not support JCV replication. In fact, JCV infection of primary hematopoietic CD34+ cells as well as hematopoietic progenitor cell lines KG-1 and KG-1a was suggested [9, 12]. While, there are no studies comparing JCV replication between primary- and EBV-transformed -B cells, interestingly, our data using EBV-transformed B cells is consistent with the earlier studies that documented low efficiency of JCV infection of primary B cells and hematopoietic precursor cells [9, 12]. Moreover, JCV T antigen and VP-1 expressing primary B cells decreased from 1-3% on day 1 to 0.5% on day 8 after infection [9], suggesting that productive JCV replication is unlikely to occur even in primary B cells and hematopetic precursor cells. These studies used less sensitive immunostaining, HA assay or standard PCR to determine JCV infection. While we always detected JCV TAg and VP-1 DNA, viral mRNA transcripts were never detected in JCV-infected B cells suggesting that JCV transcripts expression was either extremely low or completely shut off in the B cells. Further in vitro and in vivo studies using sensitive state-of-the-art real-time PCR and qRT-PCR assays are warranted to define the role of hematopetic precursor cells, primary B cells and bone marrow-derived plasma cells in JCV replication, latency and dissemination.

graphic file with name nihms201629f6.jpg

On the cover: Epifluoresence images show human polyomavirus JC (JCV) infection of B lymphocytes. One million EBV-transformed B lymphocytes (left) were inoculated with 1000 HAU of FITC-labeled JCV (green). Cell membrane was labeled with anti-protein disulphide isomerase antibody and visualized using Alexa flour 594 (red), and nuclei were counterstained with DAPI (blue). Human brain microvascular endothelial cells (right) were used as a positive control.

Acknowledgments

We thank Dr. Allison Imrie, University of Hawaii, for providing EBV-transformed primary B cells. We also thank Mr. Nelson I. B. Lazaga, Dr. Frederic Mercier, Dr. Pakieli Kaufusi, Ms. Ulziijargal Gurjav, and Ms. Alexandra Gurary for their technical assistance.

Financial support: This work was partly supported by grants from the National Institute of Neurological Disorders and Stroke (R03NS060647), Research Centers in Minority Institutions Program (grants G12RR003061 and P20RR011091) and Centers of Biomedical Research Excellence (P20RR018727), National Center for Research Resources, National Institutes of Health, and Institutional Funds.

Footnotes

Potential conflicts of interest: None

References

  • 1.Astrom KE, Mancall EL, Richardson EP., Jr Progressive multifocal leuko-encephalopathy; a hitherto unrecognized complication of chronic lymphatic leukaemia and Hodgkin's disease. Brain. 1958;81:93–111. doi: 10.1093/brain/81.1.93. [DOI] [PubMed] [Google Scholar]
  • 2.Frisque RJ, White FA., III . The molecular biology of JC virus, causative agent of progressive multifocal leukoencephalopathy. In: R PR, editor. Molecular Neurovirology. Totowa, NJ: Humana Press; 1992. pp. 25–158. [Google Scholar]
  • 3.Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ, Dessel BH. Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy. Lancet. 1971;1:1257–60. doi: 10.1016/s0140-6736(71)91777-6. [DOI] [PubMed] [Google Scholar]
  • 4.Richardson EP., Jr Our evolving understanding of progressive multifocal leukoencephalopathy. Ann N Y Acad Sci. 1974;230:358–64. doi: 10.1111/j.1749-6632.1974.tb14469.x. [DOI] [PubMed] [Google Scholar]
  • 5.Doerries K. Human Polyomavirus JC and BK Persistent Infection. In: Ahsan N, editor. Polyomaviruses and Human Diseases. Vol. 577. Springer Science and Landes Bioscience; 2006. pp. 102–116. [DOI] [PubMed] [Google Scholar]
  • 6.Dorries K, Vogel E, Gunther S, Czub S. Infection of human polyomaviruses JC and BK in peripheral blood leukocytes from immunocompetent individuals. Virology. 1994;198:59–70. doi: 10.1006/viro.1994.1008. [DOI] [PubMed] [Google Scholar]
  • 7.Major EO, Amemiya K, Tornatore CS, Houff SA, Berger JR. Pathogenesis and molecular biology of progressive multifocal leukoencephalopathy, the JC virus-induced demyelinating disease of the human brain. Clin Microbiol Rev. 1992;5:49–73. doi: 10.1128/cmr.5.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Monaco MC, Jensen PN, Hou J, Durham LC, Major EO. Detection of JC virus DNA in human tonsil tissue: evidence for site of initial viral infection. J Virol. 1998;72:9918–23. doi: 10.1128/jvi.72.12.9918-9923.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Monaco MC, Shin J, Major EO. JC virus infection in cells from lymphoid tissue. Dev Biol Stand. 1998;94:115–22. [PubMed] [Google Scholar]
  • 10.Tornatore C, Berger JR, Houff SA, et al. Detection of JC virus DNA in peripheral lymphocytes from patients with and without progressive multifocal leukoencephalopathy. Ann Neurol. 1992;31:454–62. doi: 10.1002/ana.410310426. [DOI] [PubMed] [Google Scholar]
  • 11.White FA, III, Ishaq M, Stoner GL, Frisque RJ. JC virus DNA is present in many human brain samples from patients without progressive multifocal leukoencephalopathy. J Virol. 1992;66:5726–34. doi: 10.1128/jvi.66.10.5726-5734.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Monaco MC, Atwood WJ, Gravell M, Tornatore CS, Major EO. JC virus infection of hematopoietic progenitor cells, primary B lymphocytes, and tonsillar stromal cells: implications for viral latency. J Virol. 1996;70:7004–12. doi: 10.1128/jvi.70.10.7004-7012.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Newman JT, Frisque RJ. Detection of archetype and rearranged variants of JC virus in multiple tissues from a pediatric PML patient. J Med Virol. 1997;52:243–52. doi: 10.1002/(sici)1096-9071(199707)52:3<243::aid-jmv2>3.0.co;2-2. [DOI] [PubMed] [Google Scholar]
  • 14.Houff SA, Berger JR. The bone marrow, B cells, and JC virus. J Neurovirol. 2008;14:341–3. doi: 10.1080/13550280802348222. [DOI] [PubMed] [Google Scholar]
  • 15.Houff SA, Major EO, Katz DA, et al. Involvement of JC virus-infected mononuclear cells from the bone marrow and spleen in the pathogenesis of progressive multifocal leukoencephalopathy. N Engl J Med. 1988;318:301–5. doi: 10.1056/NEJM198802043180507. [DOI] [PubMed] [Google Scholar]
  • 16.Marzocchetti A, Wuthrich C, Tan CS, et al. Rearrangement of the JC virus regulatory region sequence in the bone marrow of a patient with rheumatoid arthritis and progressive multifocal leukoencephalopathy. J Neurovirol. 2008;14:455–8. doi: 10.1080/13550280802356837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Sabath BF, Major EO. Traffic of JC virus from sites of initial infection to the brain: the path to progressive multifocal leukoencephalopathy. J Infect Dis. 2002;186 2:S180–6. doi: 10.1086/344280. [DOI] [PubMed] [Google Scholar]
  • 18.Wei G, Liu CK, Atwood WJ. JC virus binds to primary human glial cells, tonsillar stromal cells, and B-lymphocytes, but not to T lymphocytes. J Neurovirol. 2000;6:127–36. doi: 10.3109/13550280009013156. [DOI] [PubMed] [Google Scholar]
  • 19.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 DNA binding factor for recognition of the regulatory sequences of the human polyomavirus, JCV. J Neurosci Res. 1990;27:461–71. doi: 10.1002/jnr.490270405. [DOI] [PubMed] [Google Scholar]
  • 20.Atwood WJ, Amemiya K, Traub R, Harms J, Major EO. Interaction of the human polyomavirus, JCV, with human B-lymphocytes. Virology. 1992;190:716–23. doi: 10.1016/0042-6822(92)90909-9. [DOI] [PubMed] [Google Scholar]
  • 21.Kleinschmidt-DeMasters BK, Tyler KL. Progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta-1a for multiple sclerosis. N Engl J Med. 2005;353:369–74. doi: 10.1056/NEJMoa051782. [DOI] [PubMed] [Google Scholar]
  • 22.Langer-Gould A, Atlas SW, Green AJ, Bollen AW, Pelletier D. Progressive multifocal leukoencephalopathy in a patient treated with natalizumab. N Engl J Med. 2005;353:375–81. doi: 10.1056/NEJMoa051847. [DOI] [PubMed] [Google Scholar]
  • 23.Van Assche G, Van Ranst M, Sciot R, et al. Progressive multifocal leukoencephalopathy after natalizumab therapy for Crohn's disease. N Engl J Med. 2005;353:362–8. doi: 10.1056/NEJMoa051586. [DOI] [PubMed] [Google Scholar]
  • 24.Rice GP, Hartung HP, Calabresi PA. Anti-alpha4 integrin therapy for multiple sclerosis: mechanisms and rationale. Neurology. 2005;64:1336–42. doi: 10.1212/01.WNL.0000158329.30470.D0. [DOI] [PubMed] [Google Scholar]
  • 25.Arroyo AG, Yang JT, Rayburn H, Hynes RO. Differential requirements for alpha4 integrins during fetal and adult hematopoiesis. Cell. 1996;85:997–1008. doi: 10.1016/s0092-8674(00)81301-x. [DOI] [PubMed] [Google Scholar]
  • 26.Ransohoff RM. Natalizumab and PML. Nat Neurosci. 2005;8:1275. doi: 10.1038/nn1005-1275. [DOI] [PubMed] [Google Scholar]
  • 27.Ransohoff RM. “Thinking without thinking” about natalizumab and PML. J Neurol Sci. 2007;259:50–2. doi: 10.1016/j.jns.2006.04.011. [DOI] [PubMed] [Google Scholar]
  • 28.Genetech. Important Drug Warning regarding Raptiva® (efalizumab) [April 10, 2009]; Available at: http://www.gene.com/gene/products/information/pdf/raptiva_dhcp_0309.pdf.
  • 29.Krueger JG. The immunologic basis for the treatment of psoriasis with new biologic agents. J Am Acad Dermatol. 2002;46:1–23. doi: 10.1067/mjd.2002.120568. quiz 23-6. [DOI] [PubMed] [Google Scholar]
  • 30.Lebwohl M, Tyring SK, Hamilton TK, et al. A novel targeted T-cell modulator, efalizumab, for plaque psoriasis. N Engl J Med. 2003;349:2004–13. doi: 10.1056/NEJMoa030002. [DOI] [PubMed] [Google Scholar]
  • 31.Chapagain ML, Nguyen T, Bui T, Verma S, Nerurkar VR. Comparison of real-time PCR and hemagglutination assay for quantitation of human polyomavirus JC. Virol J. 2006;3:3. doi: 10.1186/1743-422X-3-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Chapagain ML, Verma S, Mercier F, Yanagihara R, Nerurkar VR. Polyomavirus JC infects human brain microvascular endothelial cells independent of serotonin receptor 2A. Virology. 2007;364:55–63. doi: 10.1016/j.virol.2007.02.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Co JK, Verma S, Gurjav U, Sumibcay L, Nerurkar VR. Interferon- alpha and - beta restrict polyomavirus JC replication in primary human fetal glial cells: implications for progressive multifocal leukoencephalopathy therapy. J Infect Dis. 2007;196:712–8. doi: 10.1086/520518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Verma S, Ziegler K, Ananthula P, et al. JC virus induces altered patterns of cellular gene expression: Interferon-inducible genes as major transcriptional targets. Virology. 2006;345:457–67. doi: 10.1016/j.virol.2005.10.012. [DOI] [PubMed] [Google Scholar]
  • 35.Alter A, Duddy M, Hebert S, et al. Determinants of human B cell migration across brain endothelial cells. J Immunol. 2003;170:4497–505. doi: 10.4049/jimmunol.170.9.4497. [DOI] [PubMed] [Google Scholar]
  • 36.Imrie A, Meeks J, Gurary A, et al. Differential functional avidity of dengue virus-specific T-cell clones for variant peptides representing heterologous and previously encountered serotypes. J Virol. 2007;81:10081–91. doi: 10.1128/JVI.00330-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Liu CK, Hope AP, Atwood WJ. The human polyomavirus, JCV, does not share receptor specificity with SV40 on human glial cells. J Neurovirol. 1998;4:49–58. doi: 10.3109/13550289809113481. [DOI] [PubMed] [Google Scholar]
  • 38.Liu CK, Wei G, Atwood WJ. Infection of glial cells by the human polyomavirus JC is mediated by an N-linked glycoprotein containing terminal alpha(2-6)-linked sialic acids. J Virol. 1998;72:4643–9. doi: 10.1128/jvi.72.6.4643-4649.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Chapagain ML, Sumibcay L, Gurjav U, Kaufusi PH, Kast RE, Nerurkar VR. Serotonin receptor 2A blocker (risperidone) has no effect on human polyomavirus JC infection of primary human fetal glial cells. J Neurovirol. 2008;14:448–54. doi: 10.1080/13550280802235916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Newman JT, Frisque RJ. Identification of JC virus variants in multiple tissues of pediatric and adult PML patients. J Med Virol. 1999;58:79–86. [PubMed] [Google Scholar]
  • 41.Tan CS, Dezube BJ, Bhargava P, et al. Detection of JC virus DNA and proteins in the bone marrow of HIV-positive and HIV-negative patients: implications for viral latency and neurotropic transformation. J Infect Dis. 2009;199:881–8. doi: 10.1086/597117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Georgsson G. Neuropathologic aspects of lentiviral infections. Ann N Y Acad Sci. 1994;724:50–67. doi: 10.1111/j.1749-6632.1994.tb38895.x. [DOI] [PubMed] [Google Scholar]
  • 43.Andreoletti L, Dubois V, Lescieux A, et al. Human polyomavirus JC latency and reactivation status in blood of HIV-1-positive immunocompromised patients with and without progressive multifocal leukoencephalopathy. Aids. 1999;13:1469–75. doi: 10.1097/00002030-199908200-00005. [DOI] [PubMed] [Google Scholar]
  • 44.Focosi D, Maggi F, Andreoli E, Lanini L, Ceccherini-Nelli L, Petrini M. The role of bone marrow cells for JCV pathogenicity. J Clin Virol. 2009;45:230–1. doi: 10.1016/j.jcv.2009.05.023. [DOI] [PubMed] [Google Scholar]