Maneth Gravell

Viruses and renal carcinoma of Rana pipiens: IX. The influence of temperature and host cell on replication of frog polyhedral cytoplasmic deoxyribovirus (PCDV)☆

Abstract PCDV replication was studied over the temperature range 8°–33° in cells originating from warm- and cold-blooded vertebrates of three classes: fish, mammals, and birds. These cells were fathead minnow (FHM), baby hamster kidney (BHK), and chicken embryo (CEF). No significant virus replication occurred in any cell type at either 8° or 10°, but at 12° replication occurred in all cell types used. However, yields of PCDV and rate of production at 12° were greater in cells from cold-blooded than from warm-blooded vertebrates (FHM > BHK > CEF). Furthermore, the viral latent period at 12° was shorter in FHM cells (12–24 hours) than in either BHK (2–3 days) or CEF cells (3–4 days). As the temperature was raised from 12 to 30 or 31°, the viral latent period decreased, the rate of multiplication increased, and high titers of infectious virus were obtained over a broad temperature range in all cells. At 30° to 31°, there was little difference in the latent periods (FHM cells, 3–4 hours; BHK cells, 4–6 hours) or in the rates of PCDV replication in cells originating from poikilotherms or homeotherms. Irrespective of cell type, both yield and rate of PCDV replication were reduced at 32°. Infectious progeny virus was not made in any cell type at 33°, but virus-specific macromolecules were formed at this temperature. Data from temperature shift experiments (33.5° → 24°) suggested that a temperature-sensitive event occurred late in the PCDV replication cycle, possibly at assembly of viral components. These results indicate that yields of PCDV and rate of replication are regulated by the host cell, but the permissive temperature zone is controlled by viral genes.

Viruses and renal carcinoma of Rana pipiens: VII. Propagation of a herpes-type frog virus☆

Abstract A frog virus (FV 4) originally isolated by Rafferty (1965) from the urine of a frog bearing a Lucke renal adenocarcinoma has been cultivated and partially characterized. The virus multiplies at 25° with cytopathology in a Rana pipiens embryo cell line and can be plaque-assayed in these cells. Virus multiplication is relatively slow, and late in the growth cycle equivalent amounts of cell-associated and released virus are found. Infectivity is lost by exposure of virus to ether or to pH 3.0. Electron microscopic examination of infected cells reveals a nuclear site of virus synthesis. The virus size (95–100 mμ), morphology (icosahedral), and ultrastructure (162 capsomeres) are indistinguishable from those of the herpes-type virus seen in Lucke tumor cells and are similar to known herpesviruses.

Electron microscopic observations on early events of frog virus 3 replication.

Abstract Attachment, penetration, and uncoating of frog virus 3 (FV 3) has been studied by electron microscopy and electron microscopic radioautography. FV 3 attached to host cell plasma membrane at the vertex of the icosahedral virion. Phagocytosis (viropexis) appeared to be a predominant mode of entry, although evidence that direct penetration may also be operative was obtained. Disruption of FV 3 virions in phagocytic vacuoles was interpreted as virus uncoating.

Mechanisms involved in nongenetic reactivation of frog polyhedral cytoplasmic deoxyribovirus: evidence for an RNA polymerase in the virion.

Abstract Mechanisms involved in nongenetic reactivation of frog polyhedral cytoplasmic deoxyribovirus (PCDV) were studied by plaque assay and the technique of DNA-RNA hybridization. Results of these studies were as follows: (1) Nongenetic reactivation of PCDV occurred predominantly via interaction with single cells of virus aggregates containing protein denatured (heat- or urea-inactivated) and ultraviolet light (UV)-inactivated virions. Little reactivation occurred by coincidence encounters with cells of separate protein denatured and UV-inactivated virions. (2) In untreated and cycloheximide-treated baby hamster kidney and fathead minnow cells, reactivable heat-inactivated PCDV could not initiate synthesis of viral RNA, while PCDV inactivated by brief UV-exposure had limited capacity. Under the same conditions, potentiated amounts of viral RNA were made in cells mixedly infected with a combination of UV- and heat-inactivated PCDV. Additionally, the reactivating capacity of UV-inactivated PCDV was not reduced in direct relationship to the UV-dose it received. From these results, it was concluded that gene expression by UV-inactivated PCDV virions was not essential to obtain reactivation of heat-inactivated PCDV virions. (3) Viral RNA was synthesized in cells blocked in their capacity for protein synthesis by cycloheximide treatment, suggesting that an RNA polymerase is an integral component of the PCDV virion.

Studies on the Viral Etiology of the Renal Adenocarcinoma of Rana pipiens (Lucké Tumor)

The presence of intranuclear acidophilic inclusion bodies and an increased incidence of tumors in frogs receiving cell-free tumor extracts led Lucke (1, 2) to postulate a viral etiology of the renal adenocarcinoma of Rana pipiens (Lucke tumor). Although this conclusion was reached over thirty years ago, propagation of a virus from R. pipiens capable of inducing renal tumors has not been achieved.

Base composition and molecular weight of DNA from a frog polyhedral cytoplasmic deoxyribovirus.

Summary Frog PCDV DNA was estimated from its melting profile to contain 54–55% G + C. The molecular weight of PCDV DNA was confirmed to be about 130 × 106 daltons from its sedimentation behavior in neutral sucrose and by contour length measurements. The mean content of DNA per PCDV virion in two preparations of purified virus was 1.86 × 10-16 g (112 × 106 daltons) and 2.56 × 10-16 g (154 × 106 daltons). Data suggest that PCDV virions contain a single linear double-stranded molecule of DNA.