Recombinant plasmids containing African swine fever virus (ASFV) DNA f
ragments covering all the virus genome were transfected into infected
cells in order to detect viral origins of DNA replication. Plasmid rep
lication was monitored by sensitivity to Mbol, which cleaves only repl
icated, unmethylated DNA, and resistance to Dpnl, which cleaves only t
he same methylated sequence. All the recombinants replicated to a simi
lar extent, indicating that ASFV does not use a preferred origin for D
NA replication. Circular plasmids without viral inserts were also repl
icated, but linearized plasmids or lambda bacteriophage DNA were not r
eplicated. Replicated plasmid DNA began to accumulate with a time cour
se similar to viral DNA, starting between 6 and 12 hr p.i. and increas
ing steadily for about 18 hr. This apparent dependence on Viral functi
ons was confirmed by the sensitivity of plasmid replication to phospho
noacetic acid and resistance to aphidicolin and by the reduction of re
plication in cells infected with a mutant defective in DNA replication
. Replicated plasmid DNA was present as unit length circles and as lar
ge dimension forms, probably head-to-tail concatemers. The results of
two-dimensional electrophoresis (neutral/alkaline) favor a rolling-cir
cle mechanism for plasmid DNA replication. (C) Press Academic Press, I
nc.