High sensitivity detection of JC-virus DNA in postmortem brain tissue by in situ PCR

Opportunistic infection of the central nervous system by human polyomavirus JC can cause a devastating disease, progressive multifocal leukoencephalopathy (PML). To gain new neuropathological insights into JC-virus (JCV) infection patterns in PML at the light microscopic level, the highly sensitive indirect in situ polymerase chain reaction (in situ PCR) was employed in up to 15-year old formalin-fixed and paraffin-embedded postmortem brain tissue derived from nine AIDS patients with PML. In situ PCR, in which target DNA is amplified intracellularly and detected by a specific labelled probe in morphologically intact tissue, was compared with conventional in situ hybridization (ISH). Validity was ensured by the inclusion of 13 controls. JCV detection with in situ PCR proved to be highly sensitive since in all nine brain samples the number of positive cells exceeded the ISH results by 2-3-fold. Whereas by routine staining the brain tissue of each individual patient showed regions with severe, mild or no involvement by PML, improved detection of JCV DNA by in situ PCR allowed a regrading into five different degrees of JCV infection. Significant myelin staining was observed, suggesting that cell-to-cell contact may not be the only means of virus spread but that new cells could also be infected by virus released after cell lysis. Furthermore, using in situ PCR hitherto unreported intracellular distribution patterns of JCV DNA in oligodendro- and astrocytes were observed by light microscopy.     

AIDS-associated progressive multifocal leukoencephalopathy (PML): comparison to non-AIDS PML with in situ hybridization and immunohistochemistry

We studied brain sections from 10 patients with the acquired immunodeficiency syndrome (AIDS) and progressive multifocal leukoencephalopathy (PML) by in situ hybridization with a biotin-labeled JC virus (JCV) DNA probe and by immunohistochemistry using antibody against the JCV capsid antigen. We compared the results with brain sections studied in the same fashion from 10 PML patients without AIDS. The pathology of JCV infection in AIDS was similar to non-AIDS PML except for minor differences in degree. AIDS-associated pathologic material showed a greater tendency toward necrosis and a higher density of JCV-infected cells. Replication of JCV was restricted to glial cells in all tissue studied. Bizarre astrocytes were less frequent in the AIDS patients, and perivascular inflammatory cells were more frequent. We could not demonstrate JCV in macrophages or microglial cells known to harbor HIV infection. In situ hybridization with nonradioactive probes serves as a useful technique for the confirmation of PML in AIDS.     

Early events in the life cycle of JC virus as potential therapeutic targets for the treatment of progressive multifocal leukoencephalopathy

The human polyomavirus, JC virus (JCV), is the etiological agent of progressive multifocal leukoencephalopathy (PML). PML occurs almost exclusively in the setting of severe and prolonged immunosuppression and it remains an important and life-threatening complication in the acquired immunodeficiency syndrome (AIDS) population. Several drugs that target DNA replication have shown efficacy at inhibiting JCV replication in vitro but none to date have shown in vivo efficacy. The authors' laboratory has been studying early events that contribute to infection of susceptible cells by JCV. They previously demonstrated that infection of glial cells by JCV requires clathrin-dependent endocytosis and that this early step in the viral life cycle can be blocked by the antipsychotic drug, chlorpromazine. As chlorpromazine is associated with the development of extrapyramidal symptoms that may be heightened in AIDS patients, the authors sought to test the atypical antipsychotic, clozapine, for antiviral activity against JCV. In this report, the authors show that clozapine is as effective as chlorpromazine at inhibiting infection. They further demonstrate that low-dose combinations of both drugs synergistically inhibit infection.     

Early events in the life cycle of JC virus as potential therapeutic targets for the treatment of progressive multifocal leukoencephalopathy

The human polyomavirus, JC virus (JCV), is the etiological agent of progressive multifocal leukoencephalopathy (PML). PML occurs almost exclusively in the setting of severe and prolonged immunosuppression and it remains an important and life-threatening complication in the acquired immunodeficiency syndrome (AIDS) population. Several drugs that target DNA replication have shown efficacy at inhibiting JCV replication in vitro but none to date have shown in vivo efficacy. The authors’ laboratory has been studying early events that contribute to infection of susceptible cells by JCV. They previously demonstrated that infection of glial cells by JCV requires clathrin-dependent endocytosis and that this early step in the viral life cycle can be blocked by the antipsychotic drug, chlorpromazine. As chlorpromazine is associated with the development of extrapyramidal symptoms that may be heightened in AIDS patients, the authors sought to test the atypical antipsychotic, clozapine, for antiviral activity against JCV. In this report, the authors show that clozapine is as effective as chlorpromazine at inhibiting infection. They further demonstrate that low-dose combinations of both drugs synergistically inhibit infection.     

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.     

Genotypes of JC virus,DNA of cytomegalovirus,and proviral DNA of human immunodeficiency virus in eyes of acquired immunodeficiency syndrome patients

JC virus (JCV) is a human polyomavirus that exists in at least eight different genotypes as a result of coevolution with different human populations all over the world. Well adapted to its host, it usually persists in the kidneys and possibly the brain. If the host becomes immunodeficient, JCV can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). There is increasing evidence that JCV is transactivated by cytomegalovirus (CMV) and the human immunodeficiency virus (HIV). Both CMV and HIV can infect the retina of acquired immunodeficiency syndrome (AIDS) patients, causing severe necrosis in the case of CMV retinitis or a mild HIV-associated vasculopathy, with bleeding and cotton wool spots. The authors therefore investigated by polymerase chain reaction (PCR) whether DNA of these three viruses was detectable in paraffin-embedded eyes of AIDS patients with a clinical history of CMV retinitis. From a total of 65 eyes, JCV was detected in 21 (32%). Thirty-six (55%) were positive for CMV and 6 (9%) for proviral DNA of HIV. JCV and CMV were found in 13 eyes, JCV and HIV in 3 eyes, CMV and HIV in 1 eye, and DNA from all three viruses in 1 eye. The JCV genotypes were types 1A, 2A, 2E, 3, and 4. In 21 eyes of patients without AIDS, only one sample was JCV positive. In conclusion, JCV DNA can be detected in ocular tissue of AIDS patients at a significantly higher level than in eyes of nonimmunosuppressed patients. Further investigations will help to decide if JCV contributes to the retinopathy caused by CMV and HIV.     

Progressive multifocal leukoencephalopathy (PML) in AIDS and in the pre-AIDS era

Summary Twenty-five brains with definite, and three brains with possible, progressive multifocal leukoencephalopathy (PML), including six brains of AIDS patients, were studied with special regard to the detection of papovaviruses. Formalin-fixed serial paraffin sections were immunostained with monospecific anti-JC virus (JCV) and genus-specific anti-simian virus (SV) 40 antisera, and hybridized in situ with DNA probes for JCV and SV 40, respectively. Immunocytochemistry (ICC) and in situ hybridization (ISH) were similarly sensitive in detecting virus in classical PML lesions. In all but one definite PML cases at least one method detected virus (96%). Possible PML tissue was never labeled. Labeling patterns were generally similar in ICC and ISH: mainly oligodendroglia and, less frequently, astroglia harbored virus, whereas labeling of neurons and endothelia was absent. Bizarre giant astrocytes were occasionally labeled by ICC and ISH. Burnt-out lesions harbored JCV DNA but not virus antigens. SV 40 DNA was never detectable. PML morphology in AIDS cases did not usually differ from the disease process seen in the pre-AIDS era. However, two AIDS brains presented extremely extended and, in one case, unusually necrotizing PML damage; in the latter case, PML lesions contained large amounts not only of JCV, but also of human immunodeficiency virus (HIV) antigens. We conclude that ICC and ISH are methods of comparable sensitivity for detection of papovavirus in flourishing PML lesions. In burnt-out PML lesions only ISH may detect virus. The possibility of an exceptional non-JCV (e.g., SV 40) etiology of PML could be neither confirmed for disproved. In AIDS, massive coinfection by HIV of PML lesions may increase damage to tissue, resulting in unusually extended and necrotizing PML.Supported by the Lord Mayor's Medical-Scientific Fund Vienna. Major parts of this study were presented at the Scientific Winter Meeting in Kitzbühel, Austria, March 15–18, 1989     

Progressive multifocal leukoencephalopathy in AIDS: a clinicopathologic study and review of the literature

We reviewed the clinical, radiographic, and pathologic features of 15 patients with the acquired immune deficiency syndrome (AIDS) and progressive multifocal leukoencephalopathy (PML). Brain tissue from 10 autopsy and 6 biopsy specimens was studied using: in situ hybridization (ISH) for JC virus (JCV), immunohistochemistry for human immunodeficiency virus (HIV) p24 antigen, and electron microscopy. Thirteen patients presented with focal neurologic deficits, while 2 presented with a rapid decline in mental status. PML was commonly the initial opportunistic infection of AIDS and produced hemiparesis, dementia, dysarthria, cerebellar abnormalities; and seizures. Magnetic resonance imaging was more sensitive than computed tomography in detecting lesions, and often showed multifocal areas of PML. CD4+ T-cell counts were uniformly low (mean 84/mm³), except in 1 patient who improved on 3-azido-3-deoxythymidine (AZT). PML involved the cerebral hemispheres, brain stem, cerebellum, and cervical spinal cord. The distribution of brain involvement was consistent with hematogenous dissemination of the virus. In 2 brain specimens, multiple HIV-type giant cells were present within the regions involved by PML. When co-infection by HIV and papovavirus was present, PML dominated the pathological picture. ISH for JCV showed virus in the nuclei of oligodendrocytes and astrocytes. Occasionally there was staining for JCV in the cytoplasm of glial cells and in the neuropil, the latter possibly a correlate of papovavirus spread between myelin sheaths, as seen by electron microscopy. ISH demonstrated more extensive foci of PML than did routine light microscopy.     

Relation of JC virus DNA in the cerebrospinal fluid to survival in acquired immunodeficiency syndrome patients with biopsy-proven progressive multifocal leukoencephalopathy.

The detection and semiquantitation of JC virus (JCV) DNA in cerebrospinal fluid (CSF) is prognostic of survival and is a marker of the course of progressive multifocal leukoencephalopathy (PML). CSF samples from 15 acquired immunodeficiency syndrome (AIDS) patients with biopsy-proven PML were analyzed by semiquantitative polymerase chain reaction (PCR). A low JCV burden was predictive of longer survival compared with a high JCV burden (median survival from entry, 24 [2-63] vs 7.6 [4-17] weeks). Further analyses indicated a possible threshold of 50 to 100 copies/microl separating high- and moderate-risk cases. Patients with a JCV load below this level survived longer than those with a JCV load above it.     

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     

Detection of JC virus by anti-VP1 immunohistochemistry in brains with progressive multifocal leukoencephalopathy

We have assessed the diagnostic efficacy of a novel polyclonal rabbit antiserum directed to the recombinant major capsid protein VP1 of JC virus (JCV). Immunohistochemistry for VP1 was compared to non-radioactive JCV DNA in situ hybridization (ISH) in ten cases of progressive multifocal leukoencephalopathy (PML). Tissue sections from postmortem brains were studied from PML patients suffering from immunodeficient conditions of various causes: immunodeficiency syndrome (AIDS, n = 7), severe combined immune deficiency due to adenosine deaminase deficiency (n = 1), sarcoidosis (n = 1) and leukemia (n = 1). VP1 immunohistochemistry demonstrated the presence of JCV in lesional oligodendrocytes of all PML patients, whereas ISH was able to detect JCV in nine out of ten cases. We conclude that VP1 immunohistochemistry is a specific, sensitive and rapid method for confirming the diagnosis of PML. Received: 11 September 1996 / Revised, accepted: 12 March 1997     

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.     

Analysis of cerebrospinal fluid and cerebrospinal fluid cells from patients with multiple sclerosis for detection of JC virus DNA

OBJECTIVE: 1) To determine whether JC virus (JCV) DNA was present in the cerebrospinal fluid (CSF) and blood from patients with multiple sclerosis (MS) in comparison with controls and 2) to find out if our clinical material, based on presence of JCV DNA, included any patient at risk for progressive multifocal leukoencephalopathy (PML). METHODS: The prevalence of JCV DNA was analyzed in CSF and plasma from 217 patients with MS, 86 patients with clinically isolated syndrome (CIS), and 212 patients with other neurological diseases (OND). In addition, we analyzed CSF cells, the first report of JCV DNA in CSF cells in a single sample, and peripheral blood cells in a subgroup of MS (n = 49), CIS (n = 14) and OND (n = 53). RESULTS: A low copy number of JCV DNA was detected in one MS cell free CSF sample and in one MS CSF cell samples. None of these had any signs of PML or developed this disease during follow-up. In addition, two OND plasma samples were JCV DNA positive, whereas all the other samples had no detectable virus. CONCLUSION: A low copy number of JCV DNA may occasionally be observed both in MS and other diseases and may occur as part of the normal biology of JC virus in humans. This study does not support the hypothesis that patients with MS would be at increased risk to develop PML, and consequently screening of CSF as a measurable risk for PML is not useful.     

Diagnosis of progressive multifocal leukoencephalopathy by brain biopsy with biotin labeled DNA:DNA in situ hybridization

DNA:DNA in situ hybridization using a cloned JC virus (JCV) DNA probe labeled with biotin confirmed the presence of JCV DNA in formalin-fixed, paraffin-embedded brain biopsies from four cases of progressive multifocal leukoencephalopathy (PML). Only small pieces of tissue were available in each case. Detection of the JC DNA:DNA hybrids was carried out by affinity cytochemistry. JCV DNA was identified predominantly in the nuclei of interfascicular oligodendrocytes in demyelinated areas of the biopsies. JC virus was isolated from one case, and the diagnosis of PML was substantiated in all cases by electron microscopic identification or immunocytochemical labeling of JC viral antigen. In situ hybridization using a biotin labeled JCV DNA probe is a specific, sensitive and convenient method for confirming the diagnosis of PML in suspected cases evaluated by brain biopsy.     

Cellular changes in the cerebellar granular layer in AIDS-associated PML*

Six cases of AIDS-associated progressive multifocal leukoencephalopathy (PML) exhibited peculiar cellular changes in the cerebellar granular layer. These cells without discernible cytoplasm showed hypochromatic nuclei about twice as large as those of normal granule cells. They were restricted exclusively to the granular layer and always surrounded PML foci. An astrocytic, leukocytic or macrophage/microglial nature was largely excluded by immunocytochemistry. Human immunodeficiency virus (HIV) antigen p 24 could not be found in these cells and there was no unequivocal detection of JC virus (JCV) DNA and no ultrastructural evidence of papovavirus particles in them. They possibly represent altered cerebellar granule cells abortively or latently infected with JCV.     

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.     

Rearrangement patterns of JC virus noncoding control region from different biological samples

The JC virus (JCV) is generally considered the etiological agent of progressive multifocal leukoencephalopathy (PML), a demyelinating brain illness, often associated with immunosuppression and significantly frequent in acquired immunodeficiency syndrome (AIDS) patients. The primary infection by JCV is usually asymptomatic and the virus can remain in a latent status in the kidney. As a consequence of immunological alterations of the host, the virus can show a genetic variability in the noncoding control region (NCCR) due to deletions, duplications, and insertions as compared with the archetype. The NCCR of the archetype strain can be divided into six regions, named boxes A to F. In this study, the authors evaluated the presence of the JCV genome in different biological samples, such as urine, peripheral blood mononuclear cells (PBMCs) and cerebral spinal fluid (CSF) by means of polymerase chain reaction (PCR). After sequencing of the PCR fragments, the NCCR structure of isolated JCV strains was analyzed in order to verify the presence of different viral variants. An analysis of the homology and of the multiple alignment of the obtained sequences in comparison with the archetype strain has been carried out. The results indicated the presence of different rearrangements among the analyzed samples. Whereas in the urine, the NCCR structure always appeared very similar to that of the archetype, in the PBMCs and CSF, the NCCR sequences showed specific and characteristic rearrangements as compared to the archetype. These different rearrangements could be correlated with the emerging of an NCCR organization more suitable for the development of PML.