Productive infection of cerebellar granule cell neurons by JC virus in an HIV+ individual

BACKGROUND: In the setting of severe immunosuppression, the polyomavirus JC (JCV) can cause a lytic infection of oligodendrocytes. This demyelinating disease of the CNS white matter (WM) is called progressive multifocal leukoencephalopathy (PML). JCV has a very narrow host-cell range and productive infection of neurons has never been demonstrated. Patient, methods, and results: An HIV-1-infected patient presented with signs of pyramidal tract and cerebellar dysfunction. Brain MRI revealed T2 hyperintensities in the WM of both frontal lobes and cerebellar atrophy. His disease progressed despite therapy and he died 6 months later. In addition to classic PML findings in the frontal lobe WM, autopsy revealed scattered foci of tissue destruction in the internal granule cell layer (IGCL) of the cerebellum. In these foci, enlarged granule cell neurons identified by the neuronal markers MAP-2 and NeuN reacted with antibodies specific for the polyomavirus VP1 capsid protein. Electron microscopy showed 40 nm viral particles, consistent with polyomaviruses, in these granule cell neurons. In addition, JCV DNA was detected by PCR after laser capture microdissection of cells from the areas of focal cell loss. Finally, in situ hybridization studies demonstrated that many granule cell neurons were infected with JCV but did not contain viral proteins. Sequence analysis of the JCV regulatory region from cerebellar virions showed a tandem repeat pattern also found in PML lesions of the frontal lobe WM. CONCLUSION: JCV can productively infect granule cell neurons of the IGCL of the cerebellum. This suggests a role for JCV infection of neurons in cerebellar atrophy occurring in HIV-infected individuals.     

Detection and typing of JC virus in autopsy brains and extraneural organs of AIDS patients and non-immunocompromised individuals.

The distribution of JC virus (JCV) variants in the brain, lung, liver, kidney, spleen and lymph nodes collected at autopsy from AIDS patients with (Group A: 10 Ss) and without (Group B: 5 Ss) progressive multifocal leukoencephalopathy (PML) and from HIV-negative patients (Group C: 5 Ss), was examined by amplifying the JCV large T antigen (LT), the regulatory (R) and the VP1 regions. Among the samples from the PML patients, JCV DNA was detected in all of the demyelinating areas, in 60% of the lesion-free brain tissues, in 60% of the lung tissues and in 40% of the spleen and kidney tissues, whereas all liver and lymph node sections were negative. JCV DNA was also found in two of the five brain specimens, in two of the five kidney specimens, in one of the five lung specimens from the HIV-positive patients without PML and in the brain specimens from two of the five HIV-negative subjects. Nucleotide sequence analysis indicated that all of the R region amplified from extraneural tissues had rearrangements similar to those of the Mad-4 strain and that VP1-region amplified products were similar to the Mad-1 strain. In the brain specimens from two PML patients, we found a unique rearranged R region, along with a VP1 region of JCV type 2. In addition, an almost unique variant with multiple rearrangements in the R region and unusual base mutations in the VP1 region was detected in the brain sample from another PML patient. The data indicate that diffuse visceral involvement of JCV is particularly frequent in AIDS patients with PML. Moreover, the presence of rearrangements and mutations, involving different regions of the viral genome, observed in PML-affected brain tissues, could represent a risk factor for the development of PML in immunosuppressed individuals.     

Reappraisal of progressive multifocal leukoencephalopathy due to simian virus 40

Several cases of progressive multifocal leukoencephalopathy (PML) have been associated with simian virus 40 (SV40), rather than with JC virus (JCV), the polyomavirus originally isolated from PML tissue. PML has, therefore, been defined as a demyelinating syndrome with possible multiple viral etiologies. Tissues from three of the cases thought to be associated with SV40 were available for reexamination. Monoclonal antibodies specific for SV40 capsid antigen VP1, virus-specific biotinylated DNA probes for in situ hybridization, and virus-specific primers in the polymerase chain reaction (PCR) were used. Macaque PML brain served as a positive control tissue for SV40 brain infection. Monoclonal antibodies to SV40 VP1 failed to recognize viral antigen in lesions from all three human PML cases. The biotinylated DNA probe, which reacted with SV40 in macaque PML, failed to detect SV40 in human PML. However, JCV could be detected by in situ hybridization with a JCV-specific DNA probe. Moreover, JCV DNA sequences were amplified by PCR from the human PML tissues, whereas SV40 DNA sequences were amplified only from the macaque brain. Thus, we could not confirm the original reports that the demyelinating agent in these three cases of PML was SV40, rather than JCV. We conclude that SV40 infection of the central nervous system need not be ruled out in the differential diagnosis of PML. Received: 1 December 1997 / Revised, accepted: 23 February 1998     

Expression of JC virus agnoprotein in progressive multifocal leukoencephalopathy brain

To examine the function of JC virus (JCV) agnoprotein, we examined the brains of cases of progressive multifocal leukoencephalopathy (PML), which is caused by JCV infection, using a newly generated antibody. The antibody reacted with 8 kDa protein specific for JCV agnoprotein by Western blotting. In vitro analyses showed that JCV capsid protein VP1 and large T antigen (T-Ag) were localized in the nuclei, but that agnoprotein was mainly detected in the cytoplasm of JCV-infected cells with an occasional nuclear staining. In the PML brain, an immunoreactive signal for agnoprotein was distributed in the perinuclear areas and cytoplasmic processes with occasional punctate staining in demyelinating lesions as well as adjacent myelinated areas. Agnoprotein presented mostly in the infected oligodendrocytes and partly in the astrocytes. Using double immunostaining, agnoprotein was seen to be expressed in the cytoplasmic processes of the cells, the nuclei of which were labeled with VP1 and T-Ag, where virus particles existed. Thus, JCV agnoprotein was mostly expressed in the infected oligodendrocytes and mainly localized in the cytoplasmic processes apart from virus particles in the demyelinated lesions.     

An overview: Human polyomavirus JC virus and its associated disorders

JC virus (JCV) is a polyomavirus infecting greater than 80% of the human population early in life. Replication of this virus in oligodendrocytes and astrocytes results in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals, most notably acquired immunodeficiency syndrome (AIDS) patients. Moreover, recent studies have pointed to the association of JCV with a variety of brain tumors, including medulloblastoma. The JCV genome encodes for viral early protein, including large and small T antigens and the newly discovered isoform T′, at the early phase of infection and the structural proteins VP1, VP2, and VP3 at the late stage of the lytic cycle. In addition, the late gene is responsible for the production of a small nonstructural protein, agnoprotein, whose function is not fully understood. Here, we have summarized some aspects of the JCV genome structure and function, and its associated diseases, including PML and brain tumors.     

An overview: Human polyomavirus JC virus and its associated disorders

JC virus (JCV) is a polyomavirus infecting greater than 80% of the human population early in life. Replication of this virus in oligodendrocytes and astrocytes results in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals, most notably acquired immunodeficiency syndrome (AIDS) patients. Moreover, recent studies have pointed to the association of JCV with a variety of brain tumors, including medulloblastoma. The JCV genome encodes for viral early protein, including large and small T antigens and the newly discovered isoform T', at the early phase of infection and the structural proteins VP1, VP2, and VP3 at the late stage of the lytic cycle. In addition, the late gene is responsible for the production of a small nonstructural protein, agnoprotein, whose function is not fully understood. Here, we have summarized some aspects of the JCV genome structure and function, and its associated diseases, including PML and brain tumors.     

Overview of the cellular immunity against JC virus in progressive multifocal leukoencephalopathy

The human polyomavirus JC (JCV) infects most healthy adults without causing any disease. In the setting of severe deficit of cell-mediated immunity, such as in acquired immunodeficiency syndrome (AIDS), malignancies or in organ transplant recipients, JCV can reactivate and cause progressive multifocal leukoencephalopathy (PML), a deadly demyelinating disease of the central nervous system. The humoral immune response, measured by the presence of virus-specific immunoglobulin G (IgG) in the blood or by intrathecal synthesis of IgG in the cerebrospinal fluid (CSF), is unable to contain the progression of PML. CD4+ T lymphocytes recognize extracellular viral proteins that have been degraded into peptides through the exogenous pathway and presented on major histocompatibility complex (MHC) class II molecules at the surface of antigen-presenting cells. Consistent with their underlying immunosuppression, the proliferative response of CD4+ T lymphocytes to mitogens or JCV antigens is reduced in PML patients. CD8+ cytotoxic T lymphocytes recognize intracellularly synthesized viral proteins that have been degraded into peptides through the endogenous pathway, and presented on MHC class I molecules at the surface of virus-infected cells. One of such JCV peptide, the VP1(p100) ILMWEAVTL, has been characterized as a cytotoxic T lymphocyte (CTL) epitope in HLA-A *0201 + PML survivors. Staining with the corresponding A *0201/JCV VP1(p100) tetrameric complex showed that VP1(p100)-stimulated peripheral blood mononuclear cells (PBMCs) of 5/7 (71%) PML survivors had JCV-specific CTL, versus none of 6 PML progressors (P = .02). This cellular immune response may therefore be crucial in the prevention of PML disease progression and the tetramer staining assay may be used as a prognostic marker in the clinical management of these patients.     

JC virus VP1 loop-specific polymorphisms are associated with favorable prognosis for progressive multifocal leukoencephalopathy

JC virus (JCV) is a human polyomavirus that causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease that mainly affects immunocompromised subjects. Since its discovery, PML has been considered a rapidly progressing fatal disease; however, amino acid substitutions in the capsid viral protein have recently been tentatively associated with changes in PML clinical course. In order to provide more insight to PML pathogenesis and identify potential prognostic markers, seven cerebrospinal fluid (CSF) samples and four brain autopsy samples were collected from patients afflicted with PML with different clinical courses (fast- and slow-progressing), and the JCV VP1 coding region was amplified, cloned, and sequenced. In addition, urine samples were collected and analyzed from nine patients with PML or other neurological diseases (ONDs) as a control group. Sequencing analysis of the genomic region encoding the VP1 outer loops revealed polymorphic residues restricted to four positions (74, 75, 117, and 128) in patients with slow PML progression, whereas no significant mutation was found in JCV isolated from urine. Collectively, these data show that JCV VP1 loop mutations are associated with a favorable prognosis for PML. It is therefore possible that slower progression of PML may be related to the emergence of a less virulent JCV strain with a lower replication rate.     

Infection with JC virus and possible dysplastic ganglion-like transformation of the cerebral cortical neurons in a case of progressive multifocal leukoencephalopathy

Infection of the cerebral cortical neurons with JC virus (JCV) with possible dysplastic ganglion-like alteration of the infected neurons found in a case of progressive multifocal leukoencephalopathy (PML) is described. The patient was a 21-year-old man with common variable immunodeficiency who died of PML after a 9-month clinical course. At autopsy, the white matter of the cerebrum, brainstem, cerebellum, and spinal cord exhibited extensive demyelination and necrosis. Numerous inclusion-bearing oligodendrocytes and bizarre astrocytes were found. In the occipital and temporal cortex, thick band-like aggregates of dysplastic ganglion-like cells (DGLCs) were found. These DGLCs showed immunohistochemical properties of neurons, and nuclei of some DGLCs were immunoreactive for large T antigen of SV40/JCV and p53, but not for capsid protein JCV VP1. In situ hybridization for mRNA of JCV large T antigen revealed positive signals in the nuclei of some DGLCs. These results indicate that JCV infected neurons and it is suggested that binding of the large T antigen with cellular proteins could have resulted in the dysplastic, ganglion cell-like change of the infected neurons, although the possibility that the aggregates of DGLCs represent a pre-existent malformative lesion of the cortex cannot be excluded completely.     

High expression of MeCP2 in JC virus‐infected cells of progressive multifocal leukoencephalopathy brains

Mutations of the methyl CpG binding protein 2 (MeCP2) gene are a major cause of Rett syndrome. To investigate whether the expression of this gene was related to JC virus (JCV) infection, we examined brains of four progressive multifocal leukoencephalopathy (PML) patients. JCV infection was confirmed by immunohistochemical labeling with antibodies against JCV VP1, agnoprotein and large T antigen. MeCP2 expression was examined by immunohistochemistry using a specific polyclonal antibody against MeCP2. In normal brains and uninfected cortices of PML brains, MeCP2 expression was observed in the nuclei of neurons, but not observed in glial and endothelial cell nuclei. However, in PML brains intense immunolabeling was observed in abnormally enlarged glial nuclei of JCV‐infected cells. Double immunolabeling using antibodies against large T antigen (visualized as blue) and MeCP2 (visualised as red) revealed dark red JCV‐infected nuclei, which confirmed that the JCV infected nuclei expressed MeCP2. We conclude that MeCP2 is highly expressed in the JCV‐infected nuclei of PML brain and these results may provide a new insight into the mechanism which regulates the MeCP2 expression in glial cells by the infection of JCV.     

Atypical radiological presentation of progressive multifocal leukoencephalopathy following liver transplantation

Progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the brain caused by JC virus (JCV), occurs following transplantation and other conditions associated with immunosuppression. On magnetic resonance imaging (MRI), PML lesions typically appear as hyperintense signal on T2-weighted and FLAIR images located in the subcortical white matter, which are devoid of contrast enhancement or mass effect. The prognosis is poor, but unusual inflammatory forms of PML characterized by contrast enhancement have been associated with a cellular immune response against JCV and a better prognosis. The authors report an atypical presentation of PML with contrast-enhancing lesions and mass effect on the MRI in a liver transplant recipient,who had a progressive course and fatal outcome.     

Development of demyelinating lesions in progressive multifocal leukoencephalopathy (PML): Comparison of magnetic resonance images and neuropathology of post‐mortem brain

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disorder caused by opportunistic infection of JC polyomavirus (JCV). Today, increased attention has been focused on PML development in multiple sclerosis (MS) patients under disease‐modifying therapies (DMT). Although in the acquired immunodeficiency syndrome (AIDS) era, PML was thought to be a rapidly progressive disease with poor prognosis, drug‐associated PML is relatively slow in progress, and a favorable outcome may be expected with early diagnosis. However, early PML diagnosis on magnetic resonance imaging (MRI) is frequently difficult, and JCV DNA copy number in cerebrospinal fluid (CSF) is usually low. To facilitate early PML diagnosis on MRI, the pre‐mortem images were compared with neuropathology of the post‐mortem brain, and underlying pathology corresponding to the MRI findings was evaluated. As a result, PML lesions of the autopsied brain were divided into three parts, based on the disease extension patterns: (A) Progressive white matter lesion in the right frontoparietal lobe including the precentral gyrus. Huge demyelinated lesions were formed with fusions of numerous small lesions. (B) Central lesion including deep gray matters, such as the putamen and thalamus. The left thalamic lesion was contiguous with the pontine tegmentum. (C) Infratentorial lesion of brainstem and cerebellum. Demyelination in the pontine basilar region and in cerebellar white matter was contiguous via middle cerebellar peduncles (MCPs). In addition, (D) satellite lesions were scattered all over the brain. These observations indicate that PML lesions likely evolve with three steps in a tract‐dependent manner: (1) initiation; (2) extension/expansion of demyelinating lesions; and (3) fusion. Understanding of the PML disease evolution patterns would enable confident early diagnosis on MRI, which is essential for favorable prognosis with good functional outcome.     

Cellular and humoral immune response in progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a fatal, demyelinating disease caused by JC virus (JCV) in patients with severe immunosuppression. We studied the JCV-specific cellular and humoral immune response in 7 healthy donors (HD), 6 human immunodeficiency virus-1 (HIV-1)-infected patients without PML (HIV), 4 HIV-1-negative patients with PML (PML), and 8 HIV-1-positive patients with PML (HIV/PML). As antigens, recombinant virus-like particles of the major structural protein VP1 (VP1-VLP) of JCV, tetanus toxoid (TT), or the mitogen phytohemagglutinin (PHA) were used. Proliferation of peripheral blood mononuclear cells (PBMC) after stimulation with the VP1-VLP was significantly suppressed in PML and HIV/PML patients compared to HD. After antigen stimulation the production of interferon-gamma (IFN-gamma) was reduced in PML, in HIV/PML, and in HIV patients. The production of interleukin-10 (IL-10), however, was elevated in HIV/PML patients. Neither proliferation nor cytokine production correlated with the presence of JCV DNA in PBMC. The immunoglobulin G serum antibody titer to the VP1-VLP was slightly elevated in HIV, elevated in PML, and highly elevated in HIV/PML patients compared to HD. The development of PML appears to coincide with a general impairment of the Th1-type T-helper cell function of cell-mediated immunity.     

Spinal cord lesions of progressive multifocal leukoencephalopathy in an acquired immunodeficiency syndrome patient

Progressive multifocal leukoencephalopathy (PML) is a deadly demyelinating disease of the central nervous system, which occurs in immunosuppressed individuals. This disease is caused by a reactivation of the polyomavirus JC (JCV). Clinical presentation can be variable from patient to patient as lesions can occur anywhere in the CNS white matter; however, they appear to spare the optic nerves and the spinal cord. The authors present a case of PML in the setting of acquired immunodeficiency syndrome (AIDS) who developed PML lesions in the spinal cord, discovered during the postmortem examination. This finding is significant because PML has recently been diagnosed in patients with multiple sclerosis (MS) treated with the novel immunomodulatory medication natalizumab. Indeed, spinal cord lesions are frequent in MS. Therefore clinicians should be aware that in addition to the brain, PML may also affect the spinal cord white matter.     

Progressive multifocal leukoencephalopathy

Before the AIDS epidemic, progressive multifocal leukoencephalopathy (PML) was a rare disorder occurring most often in association with leukemia and lymphoma. Current estimates indicate that PML ultimately develops in up to 5% of all patients with AIDS. This demyelinating disease results from infection with JC virus, a papova virus, that most of the world's population is exposed to prior to adulthood. Although PML commonly occurs in the setting of advanced immunosuppression, it may be observed in patients with CD4 lymphocyte counts in excess of 200 cells/mm3. Focal neurological symptoms and signs coupled with hyperintense signals abnormalities of the white matter on T2-weighted cranial magnetic resonance imaging are highly suggestive of the disease. In this setting, a positive CSF polymerase chain reaction for JCV DNA has been felt to be sufficiently diagnostic to eliminate the need for brain biopsy. Survival of AIDS-associated PML is poor with median survivals averaging just 6 months. However, as many as 10% of AIDS patients with PML will have prolonged (>12 months) survival and partial recovery. Highly active antiretroviral therapy (HAART) has been demonstrated to have a salutary effect on survival.     

ISNV Meeting Supplement

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the neurotropic human polyomavirus JC (JCV) lytic infection of oligodendrocytes. PML was first described as a complication of lymphoproliferative disorders more than 50 years ago and emerged as a major complication of human immunodeficiency virus (HIV) infection in the 1980s. Despite the ubiquity of this virus, PML is rare and always seen in association with underlying immunosuppressive condition, such as HIV infection, autoimmune diseases, cancer, and organ transplantation. JCV remains quiescent in the kidneys, where it displays a stable archetypal non-coding control region (NCCR). Conversely, rearranged JCV NCCR, including tandem repeat patterns found in the brain of PML patients, have been associated with neurovirulence. The specific site and mechanism of JCV NCCR transformation is unknown. According to one model, during the course of immunosuppression, JCV departs from its latent state and after entering the brain, productively infects and destroys oligodendrocytes. Although the majority of PML cases occur in severely immunesuppressed individuals, PML has been increasingly diagnosed in patients treated with biological therapies such as monoclonal antibodies (mAbs) that modulate immune system functions: in fact, CD4⁺ and CD8⁺ T lymphopenia, resulting from this immunomodulatory therapy, are the primary risk factor. Furthermore, JCV reactivation in nonpermissive cells after treatment with mAbs, such as intestinal epithelial cells in Crohn’s disease patients, in association with other host tumor-inducing factors, could provide valid information on the role of JCV in several malignancies, such as colorectal cancer.     

Progressive multifocal leukoencephalopathy in a child with hyperimmunoglobulin E recurrent infection syndrome and review of the literature.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease due to infection with polyomavirus JC (JCV). PML occurs almost exclusively in immunocompromised patients, and although it has increased markedly in relation to AIDS, remains exceptional in children. We present the case of an immunocompromised child with hyperimmunoglobulin E recurrent infection syndrome (HIES) and pathologically-proven PML. HIES is a rare congenital immunodeficiency that to our knowledge has never before been reported in association with neurological complications. Following a recurrence of bronchopneumonia, the child's motor and cognitive functions deteriorated progressively in parallel with alterations on cerebral MRI. The neurological onset coincided with lymphocyte subset changes. PCR for JCV DNA did not detect the virus in CSF, and brain biopsy was required to secure the diagnosis. Antiviral treatment with cidofovir produced no benefit. Autopsy revealed the typical neuropathological findings of PML which were associated with inflammatory eosinophilic infiltrate (a marker of HIES). In accordance with the few pediatric PML cases reported and here reviewed, the child died five months after neurological onset.     

An siRNA against JC virus (JCV) agnoprotein inhibits JCV infection in JCV‐producing cells inoculated in nude mice

JC virus (JCV) is the etiological agent of the demyelinating disease progressive multifocal leukoencephalopathy (PML). Because JCV has a very narrow host range, it has been difficult to develop an animal model of JCV infection; as a result, no effective therapy for PML has been established. In this study, we have tried to create an animal model that replaces an in vivo JCV infection. As a result, we have obtained a stable persistence of JCV‐infected human cells in the mouse brain by inoculating the virus‐infected cells into the nude mice brains. In this model, the JCV‐infected cells were well preserved in the nude mouse brains for 2 weeks. We then treated JCV‐injected brains with an siRNA against the JCV agnoprotein that is known to be an effective inhibitor of JCV infection in vitro. A highly purified type I collagen, atelocollagen, was used as a carrier for the siRNA. The siRNA inhibited the expression of JCV protein in inoculated JCV‐infected cells in the mouse brain, compared to the medium containing only atelocollagen used as a placebo. Thus, the combination of siRNA and atelocollagen might be a candidate therapeutic agent for the treatment of JCV infection.     

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

Progressive multifocal leukoencephalopathy (PML) results from lytic infection of oligodendrocytes by JC virus (JCV). Although JCV has been identified in mononuclear cells in bone marrow and hematogenous dissemination of the 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 seopositive 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% (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-1 strain of JCV in one patient with PML, a MAD-4 strain in a second patient with PML, and a slightly 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.(ABSTRACT TRUNCATED AT 250 WORDS)