Detection of JC virus DNA sequences in colorectal cancers in Japan

JC virus (JCV), a ubiquitous polyoma virus that commonly infects humans, was first identified as the etiologic agent for the fetal demyelinating disease, progressive multifocal leukoencephalopathy. Recently, a number of reports have documented detection of JCV in samples derived from several types of neural as well as non-neural human tumors. It has been suggested that oncogenicity of JCV depends on a T antigen having a strict structural homology to the T antigen of simian virus 40. To clarify whether JCV might have a potential role with regard to colorectal cancers, we investigated the presence of its genome in a series of cases along with colorectal adenomas and normal colonic mucosa, targeting T antigen, VP and agnoprotein by nested polymerase chain reaction and Southern blotting and T antigen by immunohistochemistry. While VP and agnoprotein were not found in any of the samples examined, T antigen was detected in 6 of 23 colorectal cancers (26.1%) and 1 of 21 adenomas (4.8%), but none of 20 samples of normal colonic mucosa. No clear and diffuse staining with anti-T-antigen antibodies (1:100) could be detected, and there was no correlation with CD20-positive cells, which might have indicated JCV latent infection of B lymphocytes. Presence of T antigen did not influence clinicopathological variables, including survival. In one colonic cancer case positive for T antigen together with lymph node metastasis, DNA extracted from cancer cells in the lymph node revealed no detection of T antigen. Our results are in the intermediate position between the high T antigen rate (81%) in one report and the lack of it (0%) in another focused on colon cancers. It was concluded that T antigen might be integrated in cancer cells in approximately one fourth of Japanese colon cancer cases without clear and diffuse expression of the protein, suggesting a possible role in oncogenesis which might involve a hit-and-run mechanism.     

Detection of herpes simplex virus DNA sequences in human blood and bone marrow cells

Herpes simplex virus type 1 (HSV1) establishes latent infections in neural tissues of humans and experimental animals. Utilizing a sensitive polymerase chain reaction (PCR) assay we detected HSV DNA sequences in blood ceils of healthy prospective bone marrow transplant (BMT) donors and patients. In three healthy individuals studied, HSV DNA sequences were found in all blood cell types and also in bone marrow cells as well as in stem cell progenitor colonies isolated from in vitro cultures. Studies of BMT donor-recipient pairs suggested that HSV reactivation may occur in hematopoietic cells after transplantation, as the PCR signal intensity increased over time simultaneous with an increased antibody liter to HSV. In a mouse model for HSV infection, HSV DNA sequences were found in blood and bone marrow cells at the latent stage of infection, after intravenous (IV) inoculation, but not after ocular inoculation. These studies suggest that bone marrow cells may be an additional site of HSV latency capable of reactivation after BMT. These studies have broad implications for understanding pathogenesis of HSV disease and are of particular significance in situations where allo-geneic bone marrow cells are given therapeutically. © 1994 Wiley-Liss, Inc.     

Detection of JC virus by polymerase chain reaction and colorimetric DNA hybridization assay

A procedure for rapid detection of JC virus (JCV) using the polymerase chain reaction is described. The procedure was tested in eight HIV-1-seropositive patients with progressive multifocal leukoencephalopathy. One-step DNA extraction using a chelating resin was carried out on clinical samples of cerebrospinal fluid (CSF), urine and brain tissue. After amplification, PCR products were detected by a DNA hybridization method. Microplates were coated with a specific probe and hybridized PCR products were revealed by a commercial colorimetric immunoassay. Using this procedure JC virus DNA was detected in all CSF specimens from patients with progressive multifocal leukoencephalopathy. This sensitive and rapid (24 h) procedure could greatly facilitate use of the DNA probe assay for detection of JC virus in clinical laboratories.     

Detection of JC virus DNA in the cerebrospinal fluid of patients with progressive multifocal leukoencephalopathy

Four children with infectious mononucleosis (IM) and one with reactivated Epstein-Barr virus (EBV) infection had concomitant central nervous system disorders. Cerebrospinal fluid (CSF) samples from all five patients contained EBV genomic sequences and EBV-specific antibodies in the neurologic stage, but not during convalescence. Cerebrospinal fluid from two non-neurologic IM patients had neither EBV DNA nor EBV antibodies. The EBV-positive CSF of the five with neurological disorders were aseptic in culture and all negative for other human herpesvirus DNAs and antibodies: herpes simplex virus types 1 and 2, cytomegalovirus, varicella-zoster virus, and human herpesvirus 6. Epstein-Barr virus DNA and EBV antibodies were not detected in the CSF of 17 EBV-seropositive patients with mumps meningitis, rubella encephalitis, unknown febrile convulsion, or partial epilepsy. It is suggested that EBV plays a causal role in neurologic manifestations in patients with acute and reactivated EBV infections, through direct viral invasion and immunopathological reactions. © 1993 Wiley-Liss, Inc.     

Amplification of JC virus regulatory DNA sequences from cerebrospinal fluid: diagnostic value for progressive multifocal leukoencephalopathy

Summary.  Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease in the central nervous system caused by a ubiquitous human polyomavirus designated as JC virus (JCV). PML affects individuals with decreased immune competence and is now one of the common opportunistic infections in patients with AIDS. JCV DNAs in the brain of PML patients contain various PML-type regulatory regions that were generated from the archetypal regulatory region during persistence. Recently, many studies have suggested that detection of JCV DNA from the cerebrospinal fluid (CSF) may offer a tool for diagnosing PML. However, in all of these studies, coding sequences within the T antigen or capsid protein gene have been targeted for amplification. To amplify the JCV regulatory region, we established a nested PCR that could efficiently amplify the regulatory region from most JCV subtypes prevalent in the world. Using this PCR, we amplified JCV regulatory regions from the CSF samples from 4 patients strongly suspected of PML, whereas amplification was negative from 80 CSF samples from patients without PML. Sequencing of the amplified fragments revealed that they had unique deletions and/or duplications. Furthermore, in 3 PML patients, we analyzed the structures of regulatory regions derived from the brain as well as CSF. In each of these cases, the major regulatory sequence of both origins were identical. This finding indicates that JCV DNA in brain lesions is excreted in the CSF. Since the structures of PML-type JCV regulatory regions are unique to individual patients, the current PCR, if the amplified fragments are sequenced, can eliminate false positives that may arise from contaminations. Accepted September 29,1997 Received August 26, 1997     

Detection of pseudorabies virus DNA sequences by the polymerase chain reaction

Summary Genomic sequences of pseudorabies virus, a porcine herpesvirus, were amplified by the polymerase chain reaction from cells of infected cultures, nasal cells and organs from acutely diseased as well as from organs of latently infected pigs.On leave from: Department of Epizootiology, University of Veterinary Sciences, Budapest, Hungary.On leave from: Department of Agricultural Chemical Technology, Technical University, Budapest, Hungary.     

Detection of Epstein-Barr virus DNA sequences in nasopharyngeal carcinoma cells by enzymatic DNA amplification.

The presence of Epstein-Barr virus (EBV) DNA sequences was examined by the polymerase chain reaction in 50 nasopharyngeal carcinoma (NPC) biopsy specimens and in two primary epithelial tumor cell cultures derived from patients with NPC. The detection limit was a single EBV genome equivalent by agarose gel electrophoresis followed by Southern blot analysis of the amplified products. EBV DNA sequences were detected in all 41 undifferentiated NPC cell specimens, in 2 of 4 moderately differentiated NPC cell specimens, and in 3 of 5 keratinized NPC cell specimens. Undifferentiated NPC cells were also found to contain higher copy numbers of EBV than cells of the other two types of NPC. Our data suggest that EBV replication may be closely associated with the differentiation of NPC tumor cells. The results also demonstrated a sensitive and specific method for the detection of EBV DNA sequences in NPC tumor cells.     

Polynucleotide sequences common to the genomes of simian virus 40 and the human papovaviruses JC and BK.

The DNA of the human papovavirus JC (JCV) was examined and compared to the genomes of simian virus 40 (SV40) and human papovavirus BK (BKV). Preparations from a nonplaque purified stock were heterogeneous and the molecular weight of the largest DNA species was calculated to be approximately 90% that of SV40 DNA. Studies of the kinetics of reassociation of radiolabeled JCV DNA in the presence of large excesses of SV40 or BKV DNAs revealed polynucleotide sequence homology of 11% between the genomes of JCV and SV40 and 25% between the genomes of JCV and BKV. Further studies demonstrated that the DNA sequences shared by JCV and SV40 are a subset of the sequences shared by JCV and BKV, and that these DNA sequences are common to the genomes of these three primate papovaviruses.     

Growth of JC virus in adult human brain cell cultures

Summary Adult human brain (AHB) cells infected with JC virus (JCV) developed a cytopathic effect (CPE) beginning 12–14 days after infection. Ultrastructurally, 37–40 nm papova virions were seen in the nuclei of infected cells, and both T and V antigen were demonstrated by indirect immunofluorescence. The hemagglutinating titer of JCV in infected AHB cells was 10–40 times higher than the amount of JCV used to initiate infection. AHB cells are more readily available than primary human fetal brain cells, they can be subcultured 15–25 timesin vitro and they support JCV replication after multiple subcultivations. These properties make the AHB cell line useful for propagating JCV.With 3 Figures     

Detection of adeno-associated virus type 2 sequences in the human genital tract.

Adeno-associated virus (AAV) is a defective parvovirus with unknown pathogenicity. It requires helper functions for its normal replication in human tissue and therefore is not readily isolated from clinical specimens. We have used the PCR method to examine the following clinical samples for the presence of AAV sequences: (i) 15 nasopharyngeal aspirates from symptomatic patients, (ii) 7 swab or fluid specimens from vesicles of patients suspected of having varicella-zoster virus infections, (iii) 21 human papilloma virus-positive genital biopsy specimens, (iv) 61 genital swab specimens from women suspected of having herpes simplex virus (HSV) infection examined either directly or following propagation in tissue culture, (v) 62 samples of first-trimester aborted material, including 38 samples from spontaneous abortions and 24 samples from induced abortions, (vi) 11 samples of chorionic villi taken from women undergoing genetic prenatal diagnosis, and (vii) three lots of cultured human embryonic cells. AAV sequences were detected only in samples taken from the genital tracts of women suspected of having HSV infection and not in any of the other types of samples. Samples from 11 patients were positive for AAV: for 4 patients the original swab sample was positive, for 4 patients the cultured swab sample was positive, and for 3 patients both the original swab samples and the cultures were positive. Five of the 11 patients were infected with HSV. Our study demonstrates the presence of AAV in the female genital tract. However, in contrast to a previous report (E. Tobiasch, M. Rabreau, K. Geletneky, S. Larue-Charlus, F. Severin, N. Becker, and J. R. Schlehofer, J. Med. Virol. 44:215-222, 1994), we did not find solid evidence of its replication in maternal or embryonal tissues from the first trimester of pregnancy. The questions of a potential pathogenic etiology of AAV and the interaction with HSV remain open.     

Detection of latent varicella zoster virus DNA and human gene sequences in human trigeminal ganglia by in situ amplification combined with in situ hybridization

Summary Varicella zoster virus (VZV) establishes latency in sensory ganglia following primary infection (chickenpox) and may reactivate decades later to produce zoster (shingles). The presence of VZV DNA in latently infected ganglia has been demonstrated by Southern blot hybridization as well as by polymerase chain reaction of DNA extracted from latently infected ganglia. Conflicting results have been obtained by in situ hybridization studies to determine the cell type in the ganglia harboring the latent VZV. To address this controversy we have utilized a more sensitive method than the previous studies. We have applied the technique of polymerase chain reaction to sections of ganglia from latently infected individuals and combined this with in situ hybridization to detect the amplified product. Primers specific for VZV were used to amplify VZV DNA in latently infected human trigeminal ganglia and demonstrated the presence of VZV DNA in neurons only. Sections from human kidney and ganglia from neonates as well as monkey ganglia served as controls and did not show amplification of VZV sequences. Amplification using primers for human genes, alpha tubulin and the oncogene Bcl-2, demonstrated the presence of these sequences in nearly all cells in the human tissues while only weak signals were seen in the monkey tissue. This is the first report where in situ amplification has been utilized to detect latent VZV in human ganglia.     

JC [corrected] virus detection in human tissue specimens

AIM: To clarify the advantages and disadvantages of different detection methods for Jamestown Canyon virus (JCV) in human tissue specimens. METHODS: Specimens of lung and gastric carcinomas, and normal lung tissue, gastric mucosa, and tonsil were examined for T-antigen, VP and agnoprotein of JCV by nested PCR, Southern blotting and sequencing. JCV load targeting T-antigen was evaluated by real-time PCR, and JCV existence morphologically by immunohistochemistry, in-situ hybridisation (ISH) and PCR. For these experiments, the JCI cell line (JCV cultured neuroblastoma cell line) was employed as positive control. RESULTS: In lung and gastric carcinomas, T-antigen, VP and agnoprotein of JCV could be detected by nested PCR whose products were confirmed by Southern blots and sequencing. With real-time PCR, frozen samples of gastric carcinomas gave better detection of JCV than their corresponding paraffin-embedded tissues (p<0.05). The positive rate of JCV was high in lung carcinoma, compared with normal lung tissue (p<0.05). It was the same for JCV copies in gastric carcinoma (p<0.05). Only the positive control exhibited JCV in the nucleus by ISH and immunohistochemistry. In-situ PCR showed that JCV genomic DNA was located in the nucleus of the carcinoma cell, some alveolar epithelial cells, and tonsil lymphocytes. In ISH and PCR, NBT/BCIP colouring was stronger than Fuchsin. CONCLUSIONS: Nested PCR whose amplicons should be confirmed by Southern blot and sequencing was a comparatively sensitive approach to detect JCV genomic DNA in human non-neural tissues. Real-time PCR might be employed to quantify copy number of JCV. In-situ PCR was a good method to observe the JCV location in cells, given appropriate modulation of amplification cycles. Combinations of various approaches will be adopted to explore the oncogenic roles of JCV in malignancies.     

Leiomyosarcoma of both kidneys

A rare case of leiomyosarcoma of both kidneys in a female of 45, is described. The origin of tumour from smooth muscles of the arterial walls is demonstrated.     

Identification of integrated hepatitis B virus DNA sequences in human hepatocellular carcinomas in Korea

The rate of HBV DNA integration into the genome of hepatocellular carcinoma (HCC) cells varies greatly among HCC tissues from different geographical districts. Because of the potential etiological significance of identifying HBV DNA integration in HCC tissues from Korea, we investigated 13 aseptically obtained HCC surgical specimens by Southern blot analysis. Among 13 HCCs, eight (61.5%) contained integrated HBV DNA. Of eight HBsAg-positive patients, six (75%) had integrated HBV DNA in their HCCs while two (40%) out of five HBsAg-negative patients had integrated HBV DNA. Those two HBsAg-negative patients with integrated HBV DNA were all anti-HBs-positive. However, it was not demonstrated in the patient without any HBV serological markers. These findings highly suggest a prominent etiologic role of HBV in the development of HCC. On the other hand, it also strongly favors a multifactorial etiology of human HCC in korea.     

Detection of human papillomavirus and Epstein-Barr virus DNA sequences in oral mucosa of HIV-infected patients by the polymerase chain reaction.

The presence of human papillomavirus (HPV) and Epstein-Barr virus (EBV) was analyzed in 21 oral biopsy specimens of HIV-infected patients using the polymerase chain reaction (PCR) method. Biopsies were categorized as hairy leukoplakia (HL) (n = 12), candidiasis (n = 3), oral warts (n = 2), and clinically normal epithelium (n = 4). For HPV detection a modified general primer-mediated PCR method (GP-PCR), which detects a broad spectrum of HPV genotypes at sub-picogram levels, was used. Human papillomavirus DNA was only found in two oral warts and was identified as HPV type 32. Epstein-Barr virus DNA was detected in 16 biopsy specimens, including the 12 HLs, 2 cases of candidiasis, and 2 samples of normal epithelium. Epstein-Barr virus positivity in HL could be confirmed by Southern blot analysis and DNA in situ hybridization using biotinylated DNA probes (bio-DISH). Epstein-Barr virus bio-DISH was also positive in one sample of normal epithelium from a patient with HL. The results indicate that HL is strongly associated with EBV and not with any of the common HPV types that react with general HPV primers in the PCR. However the detection of EBV in normal oral epithelium by PCR and bio-DISH suggests that the presence of this virus is not exclusively related to HL.