Detection of truncated circular DNA species in Escherichia coli with a PCV2-containing plasmid with a single PCV2 origin of replication.
OBJECTIVE
Porcine circovirus type 2 (PCV2) is a small circular single-stranded DNA virus that causes postweaning multisystemic wasting syndrome in pigs. Cloning of the PCV2 genome in a plasmid allows the construction of infectious clones. Our objective was to clone single PCV2 genomes from an isolate containing a mixture of strains, in a plasmid in order to obtain pure PCV2 strains.


METHODS
PCR amplification of PCV2 genomes and cloning followed by restriction enzyme analysis and sequencing. Transfection and PCV2 titration on PK-15 cells.


RESULTS
Single-copy PCV2 genomes from three Belgian PCV2 strains were cloned. Unexpectedly, agarose gel analysis revealed that additional circular DNA species were generated in Escherichia coli. Restriction enzyme analysis and sequencing showed that the circular DNA species were truncated and derived from the plasmid containing the PCV2 genome. Mutagenesis of the PCV2 replicase gene abolished the formation of these DNA species. The infectious clones were transfected in PK-15 cells and pure PCV2 viral strains were obtained.


CONCLUSIONS
Infectious clones were obtained that can be used for antigenic mapping and mutagenesis. In addition, our findings suggest that the replicase protein was expressed in E. coli and involved in the generation of the truncated DNA species.
Van Doorsselaere
J
J
Department of Health Care and Biotechnology, KATHO Catholic University College of South-West Flanders, Roeselare, Belgium. jan.vandoorsselaere @ katho.be
Lefebvre
D J
DJ
Nauwynck
H J
HJ
Delputte
P L
PL
D017925:Circovirus / Q000235:genetics*; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics; D004270:DNA, Circular / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D009154:Mutation; D010957:Plasmids; D018741:Replication Origin; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
D004270:DNA, Circular; D015534:Trans-Activators; D014764:Viral Proteins; C045264:replication initiator protein; D004265:DNA Helicases