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JC [corrected] virus detection in human tissue specimens.

ABSTRACT: 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.

SUBMITTER: Zheng H

PROVIDER: S-EPMC1995782 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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JC [corrected] virus detection in human tissue specimens.

Zheng Huachuan H Murai Yoshihiro Y Hong Mei M Nakanishi Yuko Y Nomoto Kazuhiro K Masuda Shinji S Tsuneyama Koichi K Takano Yasuo Y

Journal of clinical pathology 20070119 7

<h4>Aim</h4>To clarify the advantages and disadvantages of different detection methods for Jamestown Canyon virus (JCV) in human tissue specimens.<h4>Methods</h4>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 ...[more]

PMID: 17237186

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Project description:Virus infections can result in a range of cellular injuries and commonly this involves both the plasma and intracellular membranes, resulting in enhanced permeability. Viroporins are a group of proteins that interact with plasma membranes modifying permeability and can promote the release of viral particles. While these proteins are not essential for virus replication, their activity certainly promotes virus growth. Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease resulting from lytic infection of oligodendrocytes by the polyomavirus JC virus (JCV). The genome of JCV encodes six major proteins including a small auxiliary protein known as agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to viral propagation at various stages in the replication cycle, including transcription, translation, processing of late viral proteins, assembly of virions, and viral propagation. Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV. Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins. Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca(2+); (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity. The viroporin-like properties of agnoprotein result in increased membrane permeability and alterations in intracellular Ca(2+) homeostasis leading to membrane dysfunction and enhancement of virus release.

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JC [corrected] virus detection in human tissue specimens.

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JC [corrected] virus detection in human tissue specimens.

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