Replication of bovine parvovirus

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Virginia Polytechnic Institute and State University


Bovine parvovirus (BPV) is a small icosahedral virus containing single stranded DNA and belongs to the group of nondefective parvoviruses. Research on this virus group has revealed that viral replication occurs in cycling cells only, and evidence exists that the required factor provided by cycling cells is produced during early or mid S phase. Due to this S phase dependence, parvoviruses replicate synchronously only in synchronized cells. This study was initiated in order to determine the kinetics of replication of BPV in highly synchronized cells, the effects of viral replication on host macromolecular synthesis, and the properties of the double stranded DNA produced in infected cells.

Bovine fetal spleen (BFS) cells, in which BPV replicates optimally, were synchronized at the G₁/S border by exposure of cells to 2 mM hydroxyurea (HU) for 32 hr. Immediately after release of cells from the HU block by washing, DNA synthesis began. Autoradiographic analysis revealed that within 2 hr, 80 to 85% of the cells were synthesizing DNA.

The latent period for progeny BPV production was 8 hr in HU-synchronized cells infected at the beginning of S phase compared to a 16 hr latent period in asynchronous cells. In addition virus titers and the percentage of cells containing viral specific antigens increased more synchronously in HU-synchronized cells than in asynchronous cultures. Synthesis of BPV DNA always preceded the initial increase in virus titers and appeared to govern the rate of virus maturation. BPV DNA synthesis was always initiated during late S phase in cells infected at the beginning of S. Cellular S phase was not affected by BPV replication, but RNA and protein synthesis declined rapidly after the onset of BPV DNA synthesis in infected cells.

The double stranded BPV DNA produced during infection was isolated in the supernatant after selective precipitation of cellular DNA with NaCl and sodium dodecyl sulfate (Hirt procedure) followed by hydroxylapatite chromatography. Some of the molecules of DNA isolated in this way existed in a covalently closed circular configuration as demonstrated by rapid reannealing rates following thermal denaturation, In addition, agarose gel electrophoresis revealed bands of DNA in this preparation comparable to ØX 174 closed and open circular replicative forms. No double stranded DNA isolated from cells infected with other animal parvoviruses has been shown to contain covalently closed circular forms.

Although BPV is replicated to only a minor extent in stationary BFS cells, BPV was shown to replicate in cells infected 32 hr after the cells were exposed to 2 mM HU but not released. Throughout replication, cells remained in HU and no cellular DNA synthesis was detected. However, BPV DNA and progeny BPV were produced beginning at 16 hr postinfection. This synthesis was not due to viral production in a few cells only, since approximately 75% of the cells were involved in BPV replication as demonstrated by immunofluorescent staining. Therefore, it appears that a factor associated with S phase of the cell cycle and required for optimum BPV replication is produced even in the absence of cellular DNA synthesis per se.