Electron microscopy of bovine Parvovirus DNA replication intermediates

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Virginia Polytechnic Institute and State University


Several low molecular weight DNA species have been isolated from autonomous parvovirus-infected cells that could·not be demonstrated in mock-infected cells. The DNA species were termed replicative forms and intermediates. The purpose of this study was to isolate and characterize the replicative forms from bovine parvovirus (BPV)-infected cells.

Low molecular weight DNA was isolated from BPV-infected cells by the guanidine hydrochloride method and the Hirt procedure. Heterogeneity was present in DNA samples isolated by both procedures; however the Hirt procedure seemed better suited for studies with BPV-infected cells since some of the heterogeneity could be eliminated by further purification by hydroxyapatite chromatography and sedimentation through neutral sucrose gradients.

Characterization of the purified Hirt BPV RF DNA by electron microscopy showed that the RF DNA was linear and had a mean length of 1.69 µm or a mode length of 1.66 µm. Several branches molecules were also observed, however length measurements were not performed due to the small sample size. The molecular weight of the RF DNA based upon the mean length was 3.7 x 10⁶ d and 3.6 x 10⁶ d when based upon the mode length. Molecules approaching dimer and trimer length were also observed.

Sedimentaton analysis of the BPV RF DNA showed that the DNA had a bouyant density of 1.689 g/cm³ in neutral CaCl and an estimated sedimentation coefficient of 16S in neutral sucrose gradients. The molecular weight of the RF DNA based upon the sedimentation coefficient was 3.4 x 10⁶ d.

Hybridization studies showed that the RF DNA was of viral origin, but contained some cellular DNA contamination. The cellular DNA contamination could be the source of the heterogeneity observed.