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Journal of Virology, May 2004, p. 4993-4998, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.4993-4998.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

Annabel Rector,1 Ruth Tachezy,2 and Marc Van Ranst1*

Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven, 3000 Leuven, Belgium,1 Department of Experimental Virology, Institute of Hematology and Blood Transfusion, 128 22 Prague, Czech Republic2

Received 30 October 2003/ Accepted 13 January 2004

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with {phi}29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 x 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.

* Corresponding author. Mailing address: Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, BE-3000 Leuven, Belgium. Phone: 32-16-347908. Fax: 32-16-347900. E-mail: marc.vanranst{at}uz.kuleuven.ac.be.

Journal of Virology, May 2004, p. 4993-4998, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.4993-4998.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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