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PubMed1999

Archetypal and rearranged sequences of human polyomavirus JC transcription control region in peripheral blood leukocytes and in cerebrospinal fluid.

Two forms of human polyomavirus JC (JCV) genome are known based upon the structure of the transcriptional control region (TCR) of the virus: the archetypal form, which is commonly detected in urine, and the rearranged form, which was first detected in brain tissue from progressive multifocal leukoencephalopathy (PML) patients. The latter actually includes a group of TCR variants that, relative to the former, are characterized by various deletions and/or duplications. The aim of this study was to establish whether or not a correlation exists among the TCR type, the spreading of the virus within the host and its ability to cause PML. JCV TCR sequences from peripheral blood leukocytes (PBL) and cerebrospinal fluid (CSF) obtained from various groups of patients were compared. JCV with archetypal TCR was detected in CSF and PBL specimens from patients without neurological disorders or who eventually received a diagnosis of a non-PML neurological disorder. Rearranged TCR sequences were detected in all the CSF and PBL specimens from PML patients. The high similarity observed between the TCR structure detected in PBL and CSF specimens from individual patients could strengthen the hypothesis that PBL has a role in spreading JCV to the brain. Moreover, heterogeneous TCR patterns have been shown in individual PBL specimens from PML patients. This supports the hypothesis that, in PBL, JCV may replicate and undergo rearrangements of the TCR. The detection of JCV DNA by PCR in CSF independently from PML, although rare, could suggest that this assay is not sufficient for a virological diagnosis of PML. Further studies are required to assess the usefulness of quantitative assays or TCR typing in combination with PCR for diagnostic purposes.

Base Sequence↗

PubMedDate not supplied

Role of cytology in the intraoperative diagnosis of HIV-positive patients undergoing stereotactic brain biopsy.

OBJECTIVE: To determine the accuracy of cytology in the intraoperative diagnosis of central nervous system (CNS) lesions in human immunodeficiency (HIV)-positive patients. STUDY DESIGN: We prospectively studied 75 cases of computed tomography- or magnetic resonance imaging-guided brain biopsies performed with stereotactic instrumentation and a Nashold biopsy cannula over the course of five years. Biopsy samples were sent for both frozen section and immediate cytologic evaluation. Crush preparations (Papanicolaou and Diff-Quik stained) were used for cytologic assessment. There were 23 cases of progressive multifocal leukoencephalopathy (PML), 8 of toxoplasmosis (toxo), 3 of herpes simplex virus, 26 of lymphoma, 3 of HIV encephalitis, 1 of melanoma, 1 of hamartoma and 10 of nonspecific changes (paraffin section). RESULTS: Using permanent hematoxylin and eosin-stained histologic sections as the "gold standard," frozen section had a sensitivity of 78% and a specificity of 90%, while cytology had a sensitivity of 88% and specificity of 90%. Most of the false negative cases in cytology and frozen section were due to the predominance of necrosis and/or gliosis, present in six cases of toxo. Two of the false positive cases in frozen section (diagnosed as lymphoma) showed toxo, while two false positive cases in cytology (diagnosed as PML) showed only gliosis with negative immunoperoxidase staining for PML in the permanent sections. CONCLUSION: First, cytology had higher sensitivity that frozen section. Second, cytology provided faster results in most instances, primarily due to the nature of specimen preparation. Third, most misdiagnoses occurred in infectious diseases, especially toxo; this should therefore be kept in mind when nonspecific changes with an atypical lymphocytic infiltrate are seen. Fourth, cytology can be an alternative to frozen section for the intraoperative diagnosis of CNS lesions in HIV-positive patients. Another advantage of cytology is elimination of the need for cutting potentially infectious fresh tissue.

Biopsy↗

PubMed1998

Human polyomavirus JC control region variants in persistently infected CNS and kidney tissue.

The question of a possible role for JC virus (JCV) genomic rearrangements in the pathogenesis of progressive multifocal leukoencephalopathy (PML) was addressed by analysis of the genomic complexity and the transcriptional control region (TCR) of the JCV DNA population in persistently infected CNS and kidney tissue. After cloning of full-length viral DNA, no extensive changes were detected in the coding regions of the JCV genome by restriction analysis suggesting an intact JCV DNA population. For further analysis of the distribution of JCV subtypes, the non-coding region was amplified by PCR. Molecular analysis revealed homogeneous JCV TCR populations in almost 50% of the individuals. Heterogeneity was found in two CNS samples with three and five different JCV subtypes, respectively, and in four kidney specimens with two TCR subtypes. Altogether, seven TCR subtypes were identified. One in each group represented single promoter element TCRs without duplication of sequences. The TCR of the major variant JCV-W1 was comparable in sequence and structure to that of the PML prototype JCV Mad-1 DNA. The identification of dominant PML-derived JCV TCR subtypes in most persistently infected individuals suggests that rearrangements of the JCV TCR can be associated with the persistent state of infection. However, it appears unlikely that PML-associated JCV subtypes are generated anew in each individual host in the course of persistence. The findings rather suggest that a limited number of stable JCV subtypes circulate in different geographical regions of the world.

Adult↗

PubMed1999

Detection of Toxoplasma gondii, Epstein-Barr virus, and JC virus DNAs in the cerebrospinal fluid in acquired immunodeficiency syndrome patients with focal central nervous system complications.

OBJECT: Toxoplasmic encephalitis (TE), primary central nervous system lymphoma (PCNSL) and progressive multifocal leukoencephalopathy (PML) are major central nervous system (CNS) diseases in patients with acquired immunodeficiency syndrome (AIDS). We assessed the diagnostic value of polymerase chain reaction (PCR) in the detection of DNAs of Toxoplasma gondii (T. gondii), Epstein-Barr virus (EBV) and JC virus (JCV) in the cerebrospinal fluid (CSF). METHODS: We compared the PCR results with those of pathological findings at autopsy. PATIENTS OR MATERIALS: The present study included 23 autopsies representing those in whom CSF samples were obtained before death while the patient was hospitalized or at autopsy. RESULTS: The threshold levels for PCR detection were 4 tachyzoites of T. gondii, 5-15 genomes of EBV and 10 genomes of JCV. We identified T. gondii DNA in 4 out of 5 autopsy-defined cases of TE, EBV DNA in 5 out of 5 cases with PCNSL, and JCV DNA in 2 out of 2 cases with PML. The specificity of PCR was 100% in TE, 78% in PCNSL, and 100% in PML. CONCLUSION: Although the number of cases was relatively small in this study, PCR correctly identified T. gondii DNA in those cases in which PML or PCNSL was the sole clinical diagnosis. Our results indicate that PCR examination of CSF is a clinically useful tool for the diagnosis of focal brain lesions in patients with AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed2005

Investigation on the role of cell transcriptional factor Sp1 and HIV-1 TAT protein in PML onset or development.

JC virus (JCV) causes progressive multifocal leukoencephalopathy (PML), characterized by multiple areas of demyelination and attendant loss of brain function. PML is often associated with immunodepression and it is significantly frequent in AIDS patients. The viral genome is divided into early and late genes, between which lies a non-coding control region (NCCR) that regulates JCV replication and presents a great genetic variability. The NCCR of JCV archetype (CY strain) is divided into six regions: A-F containing binding sites for cell factors involved in viral transcription. Deletions and enhancements of these binding sites characterize JCV variants, which could promote viral gene expression and could be more suitable for the onset or development of PML. Therefore, we evaluated by means of polymerase chain reaction (PCR) the presence of JCV genome in cerebrospinal fluid (CSF) of HIV positive and negative subjects both with PML and after sequencing, we analyzed the viral variants found focusing on Sp1 binding sites (box B and D) and up-TAR sequence (box C). It is known that Sp1 activates JCV early promoter and can contribute in maintaining methylation-free CpG islands in active genes, while up-TAR sequence is important for HIV-1 Tat stimulation of JCV late promoter. Our results showed that in HIV-positive subjects all NCCR structures presented enhancements of up-TAR element, whereas in HIV-negative subjects both Sp1 binding sites were always retained. Therefore, we can support the synergism HIV-1/JCV in CNS and we can hypothesize that both Sp1 binding sites could be important to complete JCV replication cycle in absence of HIV-coinfection.

Adult↗

PubMed1984

Multiple JC virus genomes from one patient.

JC virus was previously isolated from the urine and from diseased brain of a patient with progressive multifocal leukoencephalopathy. The DNAs of these isolates, MAD-7 and MAD-8 respectively, were shown in this study to be mixtures of several different full-length genomes. By differences in their restriction endonuclease cleavage patterns, the genomes were categorized into two types. Type I DNA was defined as that typified by the prototype virus, MAD-1; Type II, that typified by viral DNA molecularly cloned from brain tissue of the MAD-7/8 patient (BrC-8 DNA). MAD-7 DNA was an approximately equal mixture of Type I and Type II; MAD-8 DNA appeared to be homogeneously Type II. These DNAs were molecularly cloned for further studies. Of 17 clones of recombinant MAD-7 (rMAD-7) DNA, nine were Type I and were identical to each other and to MAD-1 DNA. The other eight clones comprised five different species of Type II, none of which was identical to MAD-8 or to BrC-8 DNA. Among 17 recombinant MAD-8 (rMAD-8) clones there were two which contained Type I DNA, which had not been detected in the original viral DNA. The remaining rMAD-8 clones were of Type II. The differences among all these DNAs were primarily in the 'extra' PvuII sites and in the insertions and deletions in the hypervariable regulatory region of the genome (0.67 to 0.72 map unit). Possible explanations for multiple genomes in separate isolates from the same patient are discussed. It is concluded that the observed variation originated in vivo and not during isolation in vitro.

Brain↗

PubMed1985

Neurological manifestations of the acquired immunodeficiency syndrome (AIDS): experience at UCSF and review of the literature.

In this review of the acquired immunodeficiency syndrome (AIDS), the authors have evaluated a total of 352 homosexual patients with AIDS or generalized lymphadenopathy managed at the University of California, San Francisco (UCSF), between 1979 and 1984. Of an initial unselected group of 318 patients, 124 (39%) were neurologically symptomatic, and one-third already had their neurological complaints at the time of presentation. An additional 210 AIDS patients with neurological symptoms have been reported in the literature. Thus, a total of 366 neurologically symptomatic patients with AIDS or lymphadenopathy are reviewed. Central nervous system (CNS) complications, encountered in 315 patients, included the following viral syndromes: subacute encephalitis (54), atypical aseptic meningitis (21), herpes simplex encephalitis (nine), progressive multifocal leukoencephalopathy (six), viral myelitis (three), and varicella-zoster encephalitis (one). Non-viral infections were caused by Toxoplasma gondii (103), Cryptococcus neoformans (41), Candida albicans (six), Mycobacteria (six), Treponema pallidum (two), coccidioidomycosis (one), Mycobacterium tuberculosis (one), Aspergillus fumigatus (one), and Escherichia coli (one). Neoplasms included primary CNS lymphoma (15), systemic lymphoma with CNS involvement (12), and metastatic Kaposi's sarcoma (three). Cerebrovascular complications were seen in four patients with hemorrhage and five with infarction. Five patients in the UCSF series had multiple intracranial pathologies, including two cases of simultaneous Toxoplasma gondii infections and primary CNS lymphoma, two cases of coexistent Toxoplasma gondii and viral infections, and one case of combined Toxoplasma gondii and atypical mycobacterial infection. Cranial or peripheral nerve complications, seen in 51 patients, included cranial nerve syndromes secondary to chronic inflammatory polyneuropathy (five), lymphoma (five), and Bell's palsy (five). Peripheral nerve syndromes included chronic inflammatory polyneuropathy (12), distal symmetrical neuropathy (13), herpes zoster radiculitis (six), persistent myalgias (two), myopathy (two), and polymyositis (one). In light of the protean behavior of AIDS and the problems related to the clinical, radiological, and serological diagnosis of the unusual and varied associated nervous system diseases, patients with AIDS and neurological complaints require a rigorous and detailed evaluation. The authors' experience suggests that biopsy of all CNS space-occupying lesions should be performed for tissue diagnosis prior to the institution of other therapies.

Acquired Immunodeficiency Syndrome↗

PubMed2003

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.

AIDS-Related Opportunistic Infections↗

PubMed1999

Identification of JC virus variants in multiple tissues of pediatric and adult PML patients.

The transcriptional control region (TCR) of JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), undergoes rearrangement during replication of the virus in its human host. The mechanism by which viral promoter/enhancer sequences are deleted and duplicated within the TCR of the archetype form of JCV is not understood, but it is hypothesized that the generation of JCV variants with rearranged TCRs contributes to the virus's pathogenic potential. In a recent study of a pediatric PML patient, we detected extensive rearrangement of the JCV TCR in multiple tissues, and the archetype TCR was amplified from sites other than the kidney. These findings differed from those of previous studies that had examined tissues from adult PML patients. Since exposure to JCV usually occurs early in life, it is likely that some pediatric cases of PML arise as the result of a primary infection, whereas adult cases of PML are thought to result from the reactivation of an infection suffered as an immunocompetent child. To investigate further whether rearrangement of the JCV TCR is affected by the host's age and immune status at the time of exposure, a second pediatric patient and two adult PML patients were examined. As in our first study, multiple tissues were found to contain JCV DNA; however, fewer rearranged variants were detected. In one adult patient, related rearranged variants were detected in the brain, while archetype JCV was found in the other tissues. Based on differences in their VP1 sequences, these two forms represented different JCV genotypes, indicating that this patient had suffered a dual infection. The relevance of these findings to the rearrangement process that alters the JCV TCR is discussed.

Capsid↗

PubMed2006

Analysis of the transcriptional control region of JC polyomavirus in cerebrospinal fluid from HIV-negative patients with progressive multifocal leucoencephalopathy.

The human polyomavirus JC (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system (CNS). JCV has a hyper-variable non-coding transcriptional control region (TCR), which contains the origin of replication and the promoters of viral transcription and replication. The archetype form of TCR-JCV is frequently found in the urine and kidneys of healthy and immunocompromised subjects. However, the rearranged forms, possibly generated by deletion and duplication of segments of the archetype sequence, are found in the peripheral blood, cerebrospinal fluid (CSF), and brain of PML patients. Most experience on this setting has come from the human immunodeficiency virus (HIV) pandemic. Little has been described on the JCV-TCR sequences from PML-HIV-negative patients affected by other immunosuppressive disorders. The aim of this study was to analyze the JCV-TCR detected in CSF samples from 12 HIV-negative immunosuppressed patients suffering from PML and to investigate the possible role of genomic organization in the different incidences of PML in HIV-positive and HIV-negative patients. The results confirm that the JCV-TCR rearrangements play a crucial role in the development of PML, although they do not account for the higher frequency of the disease in HIV infection. These data support the hypothesis that, independently of the rearrangement patterns of JCV-TCR, the direct action of HIV together with other as yet unidentified cellular determinants can be a key to explaining the high rate of PML in HIV infection with respect to other underlying immunosuppressive conditions.

Bone Marrow Transplantation↗

PubMed1996

Genotype profile of human polyomavirus JC excreted in urine of immunocompetent individuals.

The human polyomavirus JC (JCV) causes the central demyelinating disease progressive multifocal leukoencephalopathy in about 5% of AIDS patients. To characterize the type profile of JCV in a control population in the United States, 54 females (10 to 79 years of age; average age, 43.4 years) and 51 males (18 to 94 years of age; average age, 47.9 years) were examined for the excretion of different genotypes of JCV in their urine by PCR followed by direct cycle sequencing. The group consisted of 89 patients of a general medical clinic in addition to 16 healthy volunteers. The overall incidence of JC viruria was 43 of 105 (40.9%) subjects, with a marked increase for those subjects above the age of 30 years. Two men were found to excrete two different types of JCV at the same time, indicating double infections. Of the three different genotypes of JCV identified to date, type 1 strains (European) were the most common in this cohort (64% of total strains) followed by type 2 (East Asian) (18%). No type 3 (East African) strains were detected. Indirect evidence for the existence of JCV type 3 was found in seven individuals (16%) in the form of a type 1/3 recombinant (also called type 4). In addition, a single example of JCV which differs from types 1, 2, and 3 and may represent a phylogenetically older type (type 5) was found in a 59-year-old African-American. Delineation of sequence variations between JCV types is essential for the design of primers for sensitive PCR with clinical samples.

AIDS-Related Opportunistic Infections↗

PubMed2005

Presence of JC virus-specific CTL in the cerebrospinal fluid of PML patients: rationale for immune-based therapeutic strategies.

OBJECTIVES: There is urgent need of a treatment for progressive multifocal leukoencephalopathy (PML), caused by the polyomavirus JC (JCV). To evaluate the rationale for immunotherapy of PML, we explored whether JCV-specific cytotoxic T lymphocytes (CTL) can penetrate the central nervous system (CNS). In addition, we studied the breadth of their T-cell receptor (TCR) repertoire, and sought to establish a reliable method to expand these cells in vitro. DESIGN AND METHODS: We enrolled 18 patients in this study, including 16 with proven or possible PML (15 HIV-positive and one HIV-negative), and two HIV-positive patients with other neurological diseases. Detection of JCV-specific CTL in the blood and the cerebrospinal fluid was performed by Cr release and tetramer staining assays in 15 patients. RESULTS: Of 11 PML patients with analyzable cerebrospinal fluid (CSF), two had no detectable JCV-specific CTL in the blood and CSF and died 3.7 and 7.2 months later. The nine remaining patients had an inactive course of PML and detectable JCV-specific CTL in the blood. In addition, four of them (44%) also had detectable JCV-specific CTL in the CSF. Both HIV-positive patients with OND had detectable JCV-specific CTL in the blood and one in the CSF. Using tetramer technology, we obtained highly enriched JCV-specific CTL lines that were able to kill target cells presenting JCV peptides. The breadth of the TCR repertoire was CTL epitope dependent. CONCLUSIONS: These results indicate that JCV-specific CTL are present in the CNS of PML patients and pave the way for an immune-based therapeutic approach.

Adult↗

PubMed1999

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.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Role of HIV-1 Tat and CC chemokine MIP-1alpha in the pathogenesis of HIV associated central nervous system disorders.

Two syndromes affecting cognitive and motor function in the setting of AIDS have been described as HIV encephalopathy (HIVE) and progressive multifocal leukoencephalopathy (PML). HIVE is characterized by the presence of microglial nodules with accompanying astrocytosis. PML is a fatal demyelinating disease of the white matter induced by the human papovavirus JCV which causes cytolytic destruction of glial cells. In addition to the effect of HIV-1 induced immune suppression, HIV may act directly as a co-factor for stimulation of JCV replication in AIDS patients, in part due to Tat-induced activation of JCV gene transcription. Since Tat has been implicated in CNS pathogenesis, we examined its localization in CNS specimens from HIV infected patients with HIVE and PML as well as controls. Based on the observation of CC chemokine induction in monocytes by Tat, we also examined the cellular localization of the CC chemokine Macrophage Inflammatory Protein-1alpha (MIP-1alpha) and its cognate receptor CCR-5 in these samples. In HIVE, Tat was primarily localized in astrocytes and microglia, within the nodular lesions. In PML, a marked increase in the number of Tat positive astrocytes was observed. In both HIVE and PML, prominent expression of MIP-1alpha and CCR-5 was found within areas containing histopathological lesions. CCR-5 positivity of microglia was localized primarily to nodular lesions in HIVE. In PML, increased numbers of cells with monocyte/microglial morphology were observed relative to HIVE. The increased MIP-1 alpha positivity, and potentially other chemokines, may contribute to the pathogenesis of PML in the setting of HIV infection. Tat may play an important role in the pathogenesis of both HIV associated CNS disease states, acting indirectly through cytokine and chemokine dysregulation.

AIDS Dementia Complex↗

PubMed1996

JCV-DNA and BKV-DNA in the CNS tissue and CSF of AIDS patients and normal subjects. Study of 41 cases and review of the literature.

SUMMARY: We studied the distribution and localization of the human papova-viruses JCV and BKV in the central nervous system (CNS) and cerebrospinal fluid (CSF) of HIV-positive patients with and without progressive multifocal leukoencephalopathy (PML) as compared with HIV-seronegative patients. The presence of JCV-DNA and BKV-DNA was evaluated by nested polymerase chain reaction (PCR) and in situ hybridization (ISH) on CNS autopsy tissues of AIDS patients with (group A, n = 13) and without (group B, n = 16) PML and of HIV-negative patients (group C, n = 12). PCR for JCV-DNA and BKV-DNA was also performed on CSF samples collected 7-420 days before death in all the 29 AIDS patients. Tissue PCR for JCV-DNA was positive in all the cases in group A, in 44 percent of the patients in group B, and in 33 percent of the patients in group C. ISH was positive in all the cases with PML and in three AIDS cases without PML (12 percent), but negative in all the HIV-negative cases. BKV-DNA was detected in two cases from group A and in one case from group B. CSF was PCR-positive for JCV-DNA in 8 of 13 (62 percent) AIDS patients with PML, but in none of the HIV patients without PML, irrespective of the presence of JCV-DNA in CNS tissues. No CSF sample was positive for BKV-DNA. Our data demonstrates that JCV-DNA and, rarely, BKV-DNA can be detected in the CNS of immunocompromised patients with and without PML and also in the CNS of HIV-negative subjects. However, only HIV-positive patients with clinically evident PML and JCV-DNA in the brain have PCR-detectable JCV-DNA in their CSF.

AIDS-Related Opportunistic Infections↗

PubMed2006

Development and utilization of a quantitative polymerase chain reaction assay to evaluate human polyomavirus JC DNA load.

AIM: Quantitative polymerase chain reaction (PCR) analysis to evaluate virus load in comparison with the patient's base-line virus levels would be an optimal diagnostic approach to monitoring human polyomavirus infections and to investigate their possible involvement in the onset of nephropathy in this patient group. Studies on the correlation between viral burden and renal disease have pointed to the incidence of JC virus (JCV) related progressive multifocal leukoencephalopathy (PML) occurring in renal and haematopoietic stem cell transplant recipients. METHODS: We developed a reliable internally-controlled quantitative PCR assay to measure JCV-DNA in fluid samples of urine, serum and cerebrospinal fluid (CSF) by densitometric analysis of the amplification products. The assay was also used to evaluate the JCV load in CFS samples from patients with suspected demyelinating syndrome and in urine and serum samples from healthy subjects and renal transplant recipients. RESULTS: All CSF samples from the 51 patients with suspected demyelinating syndrome tested JCV-DNA negative: none of them had a diagnosed PML. Analysis of the prevalence of JCV-viruria and JCV-viraemia confirmed our previous data. JCV-viruria was detected in 17% of renal transplant recipients and 26.6% of healthy controls; JCV-viraemia was found in 3.4% of transplant patients and 0% in controls. Noteworthy was a lower prevalence of JCV-viraemia in the 116 (3.4%) renal transplant patients than the prevalence previously reported for the 51 (11.8%) patients with suspected demyelinating syndrome. The mean viral load of viruria was much higher in the healthy controls than in the transplant recipients [104020 DNA copies/mL (DS+/-62284) vs 4136 DNA copies/mL (DS+/-77371)]. CONCLUSIONS: The quantitative PCR assay developed in our lab offers in 2 h time a reliable true quantification of viral DNA by densitometric analysis of the amplification product. To check for the possible presence of potential Taq polymerase inhibitors an internal control (the homemade pJCV-C plasmid) is used. The relation between polyomavirus infections and their possible involvement in post-transplant pathologies need further investigation. It would be useful to monitor the JCV-DNA load in urine and serum from more renal transplant recipients, including patients with nephropathy or active graft rejection over a longer period of time.

Base Sequence↗

PubMed1998

Localised 1H-MR spectroscopy for metabolic characterisation of diffuse and focal brain lesions in patients infected with HIV.

OBJECTIVES: To evaluate the role of proton MR spectroscopy (1H-MRS) in detecting metabolic changes in diffuse or focal lesions in the brain of patients infected with HIV. METHODS: Sixty HIV seropositive patients (25 with HIV related encephalopathies, 20 with toxoplasmosis, eight with progressive multifocal leukoencephalopathies (PMLs), and seven with lymphomas) and 22 HIV seronegative neurological controls were examined with a combined MRI and 1H-MRS technique using a Siemens 1.5 Tesla Magnetom. Spectra (Spin Echo sequence, TE 135 ms) were acquired by single voxel, localised on focal lesions in toxoplasmosis, PML, lymphomas, and HIV encephalopathies and on the centrum semiovale of neurological controls. Choline (Cho), creatine (Cr), N-acetyl aspartate (NAA), lactate, and lipids were evaluated in each spectrum and NAA/Cr, NAA/Cho, and Cho/Cr ratios were calculated. RESULTS: A significant decrease in NAA/Cr and NAA/Cho ratios were found in all HIV diagnostic groups in comparison with neurological controls (p<0.003), suggesting neuronal or axonal damage independent of brain lesion aetiology. However, the NAA/Cr ratio was significantly lower in PML and lymphomas than in HIV encephalopathies (p<0.02) and toxoplasmosis (p<0.05). HIV encephalopathies, lymphomas, and toxoplasmosis showed a significant increase in the Cho/Cr ratio in comparison with neurological controls (p<0.03) without between group differences. The presence of a lipid signal was more frequent in lymphomas (71%) than in other HIV groups (Fisher's test, p=0.00003). The presence of mobile lipid resonance together with a high Cho/Cr ratio in lymphomas may be related to an increased membrane synthesis and turnover in tumour cells. A lactate signal (marker of inflammatory reaction), was found in all but one patient with PML lesions (75%), but had a lower incidence in the other HIV diagnostic groups (Fisher's test, p=0.00024). CONCLUSION: 1H-MRS shows a high sensitivity in detecting brain involvement in HIV related diseases, but a poor specificity in differential diagnosis of HIV brain lesions. Nevertheless, the homogeneous metabolic pattern that characterises PML suggests the usefulness of 1H-MRS as an adjunct to MRI in differentiating CNS white matter lesions, such as HIV encephalopathies, from PML.

AIDS Dementia Complex↗

PubMed2003

Propagation of archetype and nonarchetype JC virus variants in human fetal brain cultures: demonstration of interference activity by archetype JC virus.

In immunologically normal individuals, the polyomavirus, JC virus (JCV), produces an asymptomatic primary infection followed by lifelong persistence of the virus in renal tubular epithelial cells. In some immunocompromised patients, however, in particular acquired immunodeficiency syndrome (AIDS) patients, JCV causes an opportunistic central nervous system (CNS) disorder, progressive multifocal leukoencephalopathy (PML). JCV DNA as it persists in kidneys (archetypal JCV) and JCV DNA isolated from PML lesions show differences in their regulatory regions in which transcription and replication are controlled. Archetypal JCV DNA has a single enhancer and no rearrangements or deletions in the regulatory region. In contrast, JCV DNA from PML isolates is characterized by alterations in the regulatory region. Some PML-associated JCVs can be grown in cultures of human fetal brain (HFB) cells. Growth of archetypal JCV in cultured cells has not been reported, however. Here we demonstrate successful propagation of the archetypal JCV, strain GS/K, in HFB cells. Growth occurred more slowly and to lower titers than is seen with the prototypical PML JCV strain Mad-1, with relatively few cells containing viral T antigen (T-Ag) or viral capsid protein, Vp1. Interestingly, GS/K growth could be enhanced, with a large increase in viral DNA and cytopathic effect, by coinfection with GS/B, a nonarchetypal brain-derived JCV variant isolated from the same PML patient as GS/K. The amount of GS/K DNA was also greatly enhanced when it was cotransfected with Mad-1 JCV DNA, the prototypical PML isolate. In contrast to GS/K plus GS/B-cotransfected cells, in GS/K plus Mad-1-infected cells, cytopathic effect was not increased. On subsequent passage of culture lysates to naïve cells, however, the infection produced by either combination of viral DNAs slowed, no cytopathic effect (CPE) was present, and the amount of GS/B or Mad-1 viral DNA was greatly reduced as compared to that of GS/K DNA. These data suggest that GS/K was able to use either GS/B or Mad-1 as a helper and that GS/K was in turn able to interfere with the growth of either helper virus. Archetype JCV can be successfully propagated in HFB cells, although infection develops much more slowly than that caused by the PML JCV variant Mad-1. The ability of archetypal and variant JCVs to enhance or retard each other's replication may have implications in vivo for the maintenance of JCV persistence and the growth of JCV variants.

Brain↗