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.     

Role of the environment in the transmission of JC virus

JC virus is etiologically associated with a fatal demyelinating disease known as PML. JCV produces persistent infections in the kidney and is excreted in the urine of healthy individuals and in the urine of PML patients. The characteristics of the JCV excreted in the environment have been studied by analyzing sewage samples from divergent geographical areas. The intergenic region of JCV strains detected in the sewage of Barcelona (Spain), Ume? (Sweden), Nancy (France), Pretoria (South Africa), Patras (Greece), Cairo (Egypt), Washington, D.C. (USA), and diverse areas of Northern India has been sequenced, and the phylogenetic analysis showed their relationships with JCV strains previously described in urine or clinical samples in these geographic areas. The JCV regulatory region of the JCV DNA detected in sewage presented archetypal or archetypal-like regulatory regions with the exception of one of the twenty clones obtained from a sewage sample of the area of Washington, D.C. that presented a tandem repeated structure. Infectivity studies showed that archetypal JCV present in the urine of a pregnant woman productively infected SVG cells. Also JC viral particles showed considerable stability in sewage at 20 degrees C and in front of treatments with acidic pH and trypsin. The high prevalence of JCV in urine and in sewage and the stability of the viral particles observed suggests that contaminated water, food, and fomites could be the vehicles of JCV transmission through the oral route. Virions partially degraded or noninfectious could be a source of JCV DNA and may represent an additional mechanism of entry of viral genes into cells.     

Role of the environment in the transmission of JC virus

JC virus is etiologically associated with a fatal demyelinating disease known as PML. JCV produces persistent infections in the kidney and is excreted in the urine of healthy individuals and in the urine of PML patients. The characteristics of the JCV excreted in the environment have been studied by analyzing sewage samples from divergent geographical areas. The intergenic region of JCV strains detected in the sewage of Barcelona (Spain), Umeå (Sweden), Nancy (France), Pretoria (South Africa), Patras (Greece), Cairo (Egypt), Washington, D.C. (USA), and diverse areas of Northern India has been sequenced, and the phylogenetic analysis showed their relationships with JCV strains previously described in urine or clinical samples in these geographic areas. The JCV regulatory region of the JCV DNA detected in sewage presented archetypal or archetypal-like regulatory regions with the exception of one of the twenty clones obtained from a sewage sample of the area of Washington, D.C. that presented a tandem repeated structure. Infectivity studies showed that archetypal JCV present in the urine of a pregnant woman productively infected SVG cells. Also JC viral particles showed considerable stability in sewage at 20°C and in front of treatments with acidic pH and trypsin. The high prevalence of JCV in urine and in sewage and the stability of the viral particles observed suggests that contaminated water, food, and fomites could be the vehicles of JCV transmission through the oral route. Virions partially degraded or noninfectious could be a source of JCV DNA and may represent an additional mechanism of entry of viral genes into cells.     

Molecular characterization of a JC virus (Sap-1) clone derived from a cerebellar form of progressive multifocal leukoencephalopathy

Summary Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease caused by polyomavirus JC (JCV). In the majority of cases of PML the cerebrum is mainly affected (cerebral PML) but on rare occasions lesions are restricted to the cerebellum and brain stem (cerebellar PML). We report a rare cerebellar PML case which occurred in a Japanese patient undergoing prolonged hemodialysis treatment. To understand the molecular basis of the viral tissue tropism, we molecularly cloned JCV DNA and compared it with those of cerebral PML. Of ten clones analyzed nine showed identical fragment patterns after digestion with various restriction endonucleases, and we designated these clones Sap-1. It could be shown that the basic structures of the regulatory regions are similar between Sap-1 and isolates from cerebral PML. Restriction endonuclease mapping analysis was used to examine the genetic relationship between Sap-1 and urine-derived isolates containing the archetypal regulatory sequence. We found that Sap-1 was genetically related to an archetypal JCV isolate in Japan.Supported by the Suhara Memorial Foundation (Sapporo), and a Special Grant-in-Aid for Promotion of Education and Science in Hokkaido University, provided by the Ministry of Education, Science and Culture. K. Nagashima is supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan, No. 02807036     

Detection of the archetypal regulatory region of JC virus from the tonsil tissue of patients with tonsillitis and tonsilar hypertrophy

The regulatory regions of JC virus (JCV) DNAs in the brain of patients with progressive multifocal leukoencephalopathy (PML) (designated as PML-type regulatory regions) are hypervariable, whereas those in the urine and renal tissue of individuals without PML have the same basic structure, designated as the archetype. It is thought that JCV strains with the archetypal regulatory region circulate in the human population. Nevertheless, Monaco et al (J Virol 70: 7004-7012, 1996) reported that PML-type regulatory regions occur in human tonsil tissue. The purpose of this study is to confirm their findings. Using nested polymerase chain reaction (PCR), the authors detected the regulatory region of JCV DNA in the tonsil tissue from 14 (44%) of 32 donors with tonsillitis and tonsilar hypertrophy. Sequencing of the detected regulatory regions indicated that they were identical with the archetypal regulatory regions detected previously or, in a few cases, slightly deviated from the archetype. This finding suggests not only that tonsil tissue is the potential site of initial JCV infection but also that archetypal JCV strains circulate in the human population.     

Rearrangement of the JC virus regulatory region sequence in the bone marrow of a patient with rheumatoid arthritis and progressive multifocal leukoencephalopathy

The polyomavirus JC (JCV) is the etiologic agent of progressive multifocal leukoencephalopathy (PML). JCV remains quiescent in kidneys, where it displays a stable archetypal regulatory region (RR). Conversely, rearranged JCV RR, including tandem repeat patterns found in the central nervous system (CNS) of PML patients, have been associated with neurovirulence. The precise site and mechanism of JCV RR transformation is unknown. We present herein a patient with rheumatoid arthritis treated with methotrexate, who developed PML and had a rapid fatal outcome. JCV DNA polymerase chain reaction (PCR) was positive in cerebrospinal fluid (CSF), bone marrow, blood, and urine. Double-immunohistochemical staining demonstrated that 9% of bone marrow CD138(+) plasma cells sustained productive infection by JCV, accounting for 94% of JCV-infected cells. JCV RR analysis revealed archetype and rearranged RR forms in bone marrow, whereas RR with tandem repeat was predominant in blood. These results suggest that the bone marrow may be a potential site of JCV pathogenic transformation. Further studies will be needed to determine the prevalence of JCV in bone marrow of immunosuppressed individuals at risk of PML and characterize the RR and phenotype of these JCV isolates.     

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.     

Progressive multifocal leukoencephalopathy: JC virus detection by in situ hybridization compared with immunohistochemistry

In four cases of progressive multifocal leukoencephalopathy (PML), we compared biotin-labeled DNA:DNA in situ hybridization with peroxidase immunohistochemistry for the detection of JC virus (JCV). The localization of JCV DNA and JCV capsid protein was compared in formalin-fixed, paraffin-embedded brain tissues. Infected oligodendrocytes showed both JCV DNA and JCV protein. However, bizarre astrocytes demonstrated JCV capsid protein less often than JCV DNA. In situ hybridization with a biotinylated probe was as sensitive and specific as immunohistochemistry for diagnosis on formalin-fixed tissue. The presence of both JCV DNA and viral capsid protein in bizarre astrocytes suggests that these cells are neither truly transformed nor permissively infected, but are distinctively altered by JCV.     

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.     

Frequent infection of cortical neurons by JC virus in patients with progressive multifocal leukoencephalopathy

The human polyomavirus JC (JCV) infects glial cells and causes progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the brain, in immunosuppressed individuals. The extent of JCV infection of neurons is unclear. We determined the prevalence and pattern of JCV infection in gray matter (GM) by immunostaining in archival brain samples of 49 PML patients and 109 control subjects. Among PML patients, 96% had demyelinating lesions in white matter and at the gray-white junction (GWJ); 57% had them in the GM. Most JCV-infected cells in GWJ and GM were glia, but JCV also infected neurons in PML lesions at the GWJ of 54% and GM of 50% patients and in GM outside areas of demyelination in 11% of patients. The JCV regulatory T antigen (Ag) was expressed more frequently in cortical neurons than the VP1 capsid protein. None of the control subjects without PML had any cells expressing JCV proteins. Thus, the cerebral cortex often harbors demyelinating lesions of PML, and JCV infection of cortical neurons is frequent in PML patients. The predominance of T Ag over VP1 expression suggests a restrictive infection in neurons. These results indicate that JCV infection of cerebral cortical neurons is a previously under appreciated component of PML pathogenesis.     

Association of human polyomavirus JC with peripheral blood of immunoimpaired and healthy individuals

JC virus (JCV) infection is regularly asymptomatic in healthy individuals. In contrast, in immunocompromised individuals, highly activated virus replication may lead to PML. Peripheral blood cells (PBCs) are found to habor JCV DNA in healthy and diseased individuals and it is discussed that they might be responsible for dissemination of the virus to the central nervous system (CNS) during persistence. To better understand the role of JCV DNA in PBCs for persistent infection and pathogenesis, the authors characterized the extent of JCV infection in Ficoll-gradient purified blood cells (peripheral blood mononuclear cells [PBMCs]) of healthy and human immunodeficiency virus type 1 (HIV-1)-infected individuals. Virus activation in PBMCs from healthy JCV-infected individuals was found at a rate of 0% to 38% at low polymerase chain reaction (PCR) sensitivity. In progressive multifocal leukoencephalopathy (PML) patients, a stronger signal was found, indicating increased virus activation. JCV DNA was regularly detected in T and B lymphocytes and in monocytes at low levels. However, granulocytes were shown to be the predominant reservoir of JCV DNA harboring high copy numbers. Although the overall distribution of viral genomes holds true for the population studied, in the individual, a markedly changed pattern of distribution can be found.     

Association of human polyomavirus JC with peripheral blood of immunoimpaired and healthy individuals

JC virus (JCV) infection is regularly asymptomatic in healthy individuals. In contrast, in immunocompromised individuals, highly activated virus replication may lead to PML. Peripheral blood cells (PBCs) are found to harbor JCV DNA in healthy and diseased individuals and it is discussed that they might be responsible for dissemination of the virus to the central nervous system (CNS) during persistence. To better understand the role of JCV DNA in PBCs for persistent infection and pathogenesis, the authors characterized the extent of JCV infection in Ficoll-gradient purified blood cells (peripheral blood mononuclear cells [PBMCs]) of healthy and human immunodeficiency virus type 1 (HIV-1)-infected individuals. Virus activation in PBMCs from healthy JCV-infected individuals was found at a rate of 0% to 38% at low polymerase chain reaction (PCR) sensitivity. In progressive multifocal leukoencephalopathy (PML) patients, a stronger signal was found, indicating increased virus activation. JCV DNA was regularly detected in T and B lymphocytes and in monocytes at low levels. However, granulocytes were shown to be the predominant reservoir of JCV DNA harboring high copy numbers. Although the overall distribution of viral genomes holds true for the population studied, in the individual, a markedly changed pattern of distribution can be found.     

Immune surveillance and response to JC virus infection and PML

The ubiquitous human polyomavirus JC virus (JCV) is the established etiological agent of the debilitating and often fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Most healthy individuals have been infected with JCV and generate an immune response to the virus, yet remain persistently infected at subclinical levels. The onset of PML is rare in the general population, but has become an increasing concern in immunocompromised patients, where reactivation of JCV leads to uncontrolled replication in the CNS. Understanding viral persistence and the normal immune response to JCV provides insight into the circumstances which could lead to viral resurgence. Further, clues on the potential mechanisms of reactivation may be gleaned from the crosstalk among JCV and HIV-1, as well as the impact of monoclonal antibody therapies used for the treatment of autoimmune disorders, including multiple sclerosis, on the development of PML. In this review, we will discuss what is known about viral persistence and the immune response to JCV replication in immunocompromised individuals to elucidate the deficiencies in viral containment that permit viral reactivation and spread.     

Rarity of JC virus DNA sequences and early proteins in human gliomas and medulloblastomas: the controversial role of JC virus in human neurooncogenesis

JC virus (JCV), the agent of progressive multifocal leucoencephalopathy (PML), exerts an oncogenic effect in several laboratory animal models. Moreover, JCV genomic DNA and early viral protein T-antigen have been detected in various types of human central nervous system (CNS) neoplasms. To further explore this association we have studied paraffin-embedded brain biopsy tissue from 60 neoplasms (55 gliomas and five medulloblastomas) and 15 reactive gliosis cases for the presence of JCV DNA sequences and proteins. Four post mortem cases of HIV-associated PML were used as positive controls. Samples were assessed by polymerase chain reaction (PCR) amplification of early (large T antigen) and late (virion protein 3) sequences and immunohistochemistry (IHC) with both PAb 2024 and anti-SV40 large T antigen monoclonal antibodies. Five cases (three neoplasms and two reactive gliosis instances) showed low viral DNA levels when PCR-tested for VP3 or large T, while no case was immunoreactive for any of the two antibodies used. The four PML cases yielded positive results with both PCR and IHC. Additionally, IHC with both antibodies was applied to a tissue micro-array including 109 CNS tumours and 21 reactive gliosis samples. No immunoreactivity was detected in any of these tissue micro-array samples. The rarity of JCV DNA sequences and early proteins in our brain tumours enriches the controversy over the role of JCV in human neurooncogenesis, whose clarification is in need of further molecular and epidemiologic studies.