Construction of an infectious genomic clone of porcine parvovirus: effect of the 5'-end on DNA replication.
The linear single-stranded DNA genome of the porcine parvovirus, an autonomous parvovirus, was cloned in duplex form into the bacterial plasmid pUC18 using a simple and reliable method. These clones were stable during propagation in Escherichia coli JM109. The recombinant clones of porcine parvovirus were infectious when transfected into monolayers of swine testes cells as identified by the development of cytopathic effect, indirect immunofluorescence with specific antiserum, and hemagglutination assays. DNA isolated from progeny virus arising from transfected infectious clones was found to be indistinguishable from wild-type DNA by restriction enzyme analysis. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogeneous, cloned DNA. The presence of the turn of the 5'-end loop seems to be necessary to get stable infectious clones.
Casal
J I
JI
Inmunologia y Genetica Aplicada, S.A. (Ingenasa), Madrid, Spain.
Diaz-Aroca
E
E
Ranz
A I
AI
Manclus
J J
JJ
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004261:DNA Replication; D005814:Genes, Viral; D008969:Molecular Sequence Data; D010321:Parvoviridae / Q000235:genetics* / Q000472:pathogenicity; D015183:Restriction Mapping; D013552:Swine; D014162:Transfection; D014774:Virulence
D004247:DNA