SCREENING HUMAN TUMOR SAMPLES WITH A BROAD-SPECTRUM POLYMERASE CHAIN-REACTION METHOD FOR THE DETECTION OF POLYOMAVIRUSES

Polyomaviruses induce tumors of different histological types when inoc ulated into experimental animals. An etiological role for this virus g roup in the development of malignant tumors in humans remains question able, despite several reports demonstrating the presence of SV40, JCV, and BKV DNA in human cancers. Only two human polyomavirus types are k nown to date: JCV, causing progressive multifocal leukoencephalopathy (PML) under severe immunosuppression, and BKV, first isolated from the urine of a renal transplant recipient and associated with hemorrhagic cystitis. We developed a degenerate polymerase chain reaction assay i n an attempt to identify additional, presently unknown human polyomavi rus types that may be involved in the malignant transformation of huma n tissues. A large part of the gene coding for the viral capsid protei n VP1 is highly conserved in nine polyomavirus types (and their strain s) and was therefore selected as most suitable for the primer design. Degenerate oligonucleotide primers were deduced from four different co nserved amino acid motifs in this region. Three different sets of prim ers were included in each test to obtain the highest sensitivity in co mbination with primers with the lowest degeneracy numbers. The sensiti vity obtained ranged from 1 copy/cell for bovine polyomavirus to 100 c opies/cell for LPV after ethidium bromide staining and was increased a t least 10-fold after hybridization with a radiolabeled probe. A subse quent seminested amplification allowed for the detection of 1 copy/cel l for LPV. These degenerate primers were applied to analyze bladder ca rcinomas, Hodgkin lymphomas, meningiomas, Kaposi tumors, and Kaposi-de rived cell lines. No polyomavirus DNA sequences could be detected. (C) 1997 Academic Press.