JC [corrected] virus detection in human tissue specimens.
OBJECTIVE
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.
Zheng
Huachuan
H
Division of Pathology, The Second Affiliated Hospital of China Medical University, Shenyang, China.
Murai
Yoshihiro
Y
Hong
Mei
M
Nakanishi
Yuko
Y
Nomoto
Kazuhiro
K
Masuda
Shinji
S
Tsuneyama
Koichi
K
Takano
Yasuo
Y
D000957:Antigens, Viral, Tumor / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D036022:Capsid Proteins / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis; D002141:Encephalitis Virus, California / Q000235:genetics / Q000302:isolation & purification*; D005753:Gastric Mucosa / Q000821:virology; D006801:Humans; D017403:In Situ Hybridization; D008168:Lung / Q000821:virology; D008175:Lung Neoplasms / Q000821:virology; D008969:Molecular Sequence Data; D014066:Palatine Tonsil / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods; D013274:Stomach Neoplasms / Q000821:virology; D014764:Viral Proteins / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins; D000086082:Viroporin Proteins
D000957:Antigens, Viral, Tumor; D036022:Capsid Proteins; D004279:DNA, Viral; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C042559:agnoprotein, polyomavirus; D000086082:Viroporin Proteins