- Volume 13, Issue 2, 1971

In vitro transformation of rat fibroblasts by the schmidt–ruppin strain of Rous sarcoma virus is described. Primary rat embryo fibroblasts were transformed in 2 to 6 weeks after infection with the virus. The transformed cells were large, refractile and polygonal in appearance and produced multilayer colonies. Injection of the transformed cells to young and weanling Sprague–Dawley rats induced fibrosarcomas. The continuous cell line of rat fibroblasts was transformed by Rous sarcoma virus (schmidt–ruppin) after 14 to 25 weeks. The transformation developed gradually from 1 or 2 islands of transformed cells per culture to a solid sheet of transformed cells. Cell cultures initiated tumours in young and weanling rats at the site of injection. Tumours grew slowly in weanling rats, while younger animals died from large tumours. These differences between young and old rats may reflect the immunological status of the host and/or the decrease in susceptibility of host cells to transformation by inoculated cell cultures.

In foetal cell cultures and leucocytes, Sendai virus induced considerably higher levels of interferon than rubella, whereas in placental cultures this was reversed. A low passage strain of rubella virus, ko-i, which was originally isolated in Japan and is non-teratogenic in rabbits, produced particularly high levels of interferon in placental cultures, this being most marked at an m.o.i. of 1 and 15. Although the number of experiments conducted with brain, spleen, heart and amnion cultures was far fewer than with lung cultures, it appeared that comparable levels of interferon were produced by different cell cultures derived from the same foetus. Foetal cultures, leucocytes and placental cultures, derived from foetuses varying in gestational age from 10 to 23 weeks, were capable of producing interferon when infected by Sendai or rubella virus, but levels were unrelated to gestational age.

Feline leukaemia virus and the feline leukaemia virus pseudotype of murine sarcoma (MSV(FeLV)) readily propagate in cat embryo cells. Feline leukaemia virus will rapidly attain high titres even if minimal virus inocula are used initially, and undiminished virus production will be maintained in the chronically infected cells. Feline leukaemia virus, which was previously quantitated as helper activity for defective MSV (FeLV), has now been related to the amount of autonomously replicating feline leukaemia virus. Rapid growth of MSV (FeLV) to high titre is obtained only if each sarcoma infected cell also becomes the recipient of a replicating unit of feline leukaemia virus. Conditions approximating one-step growth curves for both feline leukaemia virus alone and MSV (FeLV)–feline leukaemia virus complex can be achieved and show that first cycles of growth are completed within about 40 and 30 hr, respectively. The rate of growth of the sarcoma-leukaemia virus complex is somewhat faster than that of leukaemia virus by itself, which can result in ratios of replicating leukaemia to sarcoma viruses of less than unity. Inhibition of DNA synthesis during the first 12 hr after infection with either virus prevents virus replication. The requirement for DNA synthesis is not seen beyond the first 12 hr after infection. No stimulation of DNA synthesis is discernible after either feline leukaemia virus or MSV (FeLV) inoculation at any time after infection.

The filamentous inclusions associated with the development of vesicular stomatitis virus were investigated by the combined use of ferritin-conjugated antibody and high-resolution radioautography. The specific attachment of ferritin-antibody complexes to the filaments as well as their consistent labelling with [3H]-uridine indicated that the inclusions were the sites of accumulation of virus specific ribonucleoprotein.

Potato aucuba mosaic virus and potato virus C were transmitted by the aphid Myzus persicae, not only from plants also infected with a helper virus, but also from plants infected with them alone, provided the aphids fed first on plants infected with the helper virus. Several different viruses acted as helpers but all are in the potato virus Y group. Helper viruses differed in the efficiency with which they aided potato aucuba mosaic virus and potato virus C, and some potato aucuba mosaic virus isolates were transmitted more frequently than others. With potato virus Y as helper, up to 30% of the aphids transmitted potato aucuba mosaic virus. Aphids were usually fed for brief periods on plants infected with the helper virus but aphids fed for 2 days also transmitted potato aucuba mosaic virus readily. Starving the aphids for 1 to 2 hr between their acquisition feeds on plants infected with helper and aided virus decreased but did not eliminate transmission.

The helper virus need not be infective; potato aucuba mosaic virus and potato virus C were transmitted as frequently when transmission of the helper virus was prevented by exposing the source leaf to u.v. radiation as when it was not. Virus was not transmitted by aphids fed through artificial membranes on extracts of leaves infected with potato virus Y, potato aucuba mosaic virus or a mixture of the two. However, potato aucuba mosaic virus was transmitted from extracts by aphids fed through membranes when the aphids had previously fed on a potato virus Y-infected leaf. Similarly, potato virus Y was transmitted from leaf extracts by aphids fed through membranes when the aphids had previously fed on a potato virus Y-infected leaf that had been irradiated with u.v. to prevent transmission of potato virus Y from this source.

Possible mechanisms for the transmission of the helper and aided viruses are discussed.

Samples of Madin-Darby bovine kidney cells were taken for electron microscopy at various times after infection with bluetongue virus. A quantitative electron microscopic cell sampling technique was used in observing ultrathin sections of the cell population, and various morphological features were tabulated. The appearance of virus within phagocytic vesicles and lysosomes, and the distension of the rough endoplasmic reticulum, were early ultrastructural changes resulting from the virus infection. Granular inclusions which were usually juxtanuclear and tubular structures were other features observed later in cells infected with bluetongue virus. Progeny virus particles were seen within granular inclusions, amidst tubular structures, and within cytoplasmic vesicles. There was agreement between the infectivity data from plaque counts of cell-associated virus and direct electron microscopic counts of cells containing intracellular virus, granular inclusions, and cytoplasmic tubular structures. The large tubules associated with bluetongue infection had a mean outer diameter of 47.2 nm., more than twice the size of classic cellular microtubules. The mean diameter of bluetongue virus measured in ultrathin sections and negatively stained preparations was 63 nm. Even at late stages of infection, small numbers of intracellular virus particles were observed with the electron microscope. This agreed with the low titres obtained from plaque counts of cell-associated virus. Bluetongue virus did not, therefore, accumulate in the cell and undergo a burst-like release. Instead, it appeared to be extruded from the cell as it was made. There was no evidence for virus release by budding from the plasma membrane, nor for the presence of an envelope around complete particles.

Kirsten sarcoma virus induces foci of morphologically altered cells in rat, mouse, and human cells in tissue culture. The kirsten sarcoma virus stock contains two viruses; one forming foci and the other plaques on XC cells. The sarcoma virus particle transforms cells in the absence of murine leukaemia virus but requires murine leukaemia virus for its replication. The high susceptibility of human cells to transformation by kirsten sarcoma virus is related to a function provided to the sarcoma virus by murine leukaemia virus. A large number of kirsten sarcoma virus-transformed mouse and rat cell foci has been isolated, and from these many non-producer clonal lines have been derived and characterized.

Cells from crossbred embryos from the inbred Reaseheath R and W lines, which were segregating at the tva and tvb loci, and at a possible third locus, tvc, were examined for responses to avian sarcoma viruses of subgroups A, B and C. Responses to subgroup A were independent of those to subgroup B, as were those for subgroups B and C. Genetic analysis of the results suggested absence of linkage between the tvb locus and the tva or tvc loci.

Responses to subgroup A and C viruses were strongly associated and were consistent with linkage between the tva and tvc loci, or with the presence of a single locus which controlled responses to both subgroups.

Several arthropod-borne viruses were grouped on the basis of relative stability to lipid solvents and sodium deoxycholate, lability at pH 3.0, and lack of antigenic relationship to major arbovirus serologic groups A, B, and Bunyamwera. These viruses, previously ungrouped or in minor serogroups, include bluetongue, epizootic haemorrhagic disease of deer, Eubenangee, IbAr 22619, B1327, Colorado tick fever, African horse sickness, Irituia, Changuinola, BeAr 35646, BeAr 41067, Kemerovo, Chenuda, Tribec, Wad Medani, Mono Lake, Huacho, Lebombo, Palyam, D’Aguilar, G8886, G15534, Corriparta, Acado, MP359, CH9935, and MRM 10434. In contrast to other arboviruses, the reduction in infectivity of these viruses caused by lipid solvents or sodium deoxycholate was less than 101.5 suckling mouse intracerebral LD50. This degree of sensitivity was reproducible and was significantly different from the absolute resistance of reoviruses and picorna-viruses. After exposure of each of these viruses to pH 3.0 for 3 hr, no residual infectivity was recovered. By complement-fixation testing no serological relationship to members of other virus taxonomic groups was found. The viruses themselves had no common group antigen but were placed into ten serologically distinct subgroups on the basis of individual cross-reactions. Although probably containing a double-stranded RNA genome, the relatively solvent resistant arboviruses were distinguished from reoviruses by acid lability, slight solvent sensitivity, and serology.

The morphology and mode of maturation of a number of relatively solvent resistant arboviruses were examined by thin-section and negative-stain electron microscopy of infected mouse brain and cell culture specimens. These viruses, which have physicochemical properties distinct from other arboviruses, included Colorado tick fever, Tribec, Wad Medani, Chenuda, Irituia, Palyam, Lebombo, epizootic haemorrhagic disease of deer and bluetongue. They were 65 to 80 nm. in diameter and matured in the cytoplasm as unenveloped particles with an electron-dense core. Virus development occurred in association with a cytoplasmic granular matrix and was accompanied by formation of regularly substructured filaments and tubules. Surface architecture was compatible with icosahedral symmetry with T = 3 (32 capsomeres). The combination of taxonomic parameters, morphologic and morphogenetic as well as physicochemical, was distinct from that of any presently recognized virus group. The independent classification of these viruses with bluetongue as the type virus is thus proposed.

Non-transforming helper viruses were isolated from pools of two hamster-specific sarcoma viruses in which isolations by terminal dilution procedures had previously been unsuccessful. This presumably resulted from approximately equivalent levels of sarcoma and helper viruses in the original pools. Helper virus was isolated from morphologically normal cells selected from tissue culture plates showing relatively few transformed colonies, and by end-point dilution from cloned transformed cells. Focus-formation by the hamster-specific sarcoma viruses was independent of the spread of virus and non-producer cell lines carrying the sarcoma virus genome were isolated; thus these viruses were apparently unable to replicate independently of helper virus. Hamster-specific virus antigens were not detected in hamster cells infected productively or non-productively with a murine sarcoma virus; thus, evidence for virus activation in vitro by the sarcoma virus genome was not obtained.

Four major species of single-stranded RNA (38s, 30s, 19s and 10 to 16s) and one double-stranded species of RNA (13s) were found in BHK cells infected with vesicular stomatitis virus. A portion of the 38s and 19s was associated with ribonucleoprotein particles sedimenting at 140s and 80s respectively. Some of the 38s RNA and the 10 to 16s RNA could be isolated from the polyribosome region of fractionated cells. The latter RNA could be fractionated further into at least five (possibly eight) peaks by electrophoresis on polyacrylamide gels. The molecular weight of the RNAs in these peaks ranged from 0.24 to 1.0 × 106. Hybridization studies revealed that the 10 to 16s RNA was complementary to the RNA extracted from purified virion, suggesting that the different sizes represent monocistronic (negative) messengers. A high molecular weight RNA complex was also isolated from virus-infected cells. Denaturation of the complex revealed the constituent strands to consist of 38s, 19s and 10 to 16s RNA. This may be the active transcription complex.

Molluscum contagiosum virus was purified by two methods involving differential centrifugation; treatment with ribonuclease and sucrose density gradient centrifugation. When the final gradient centrifugation was preceded by two consecutive centrifugations through 36% sucrose, the percentage of DNA in the purified virus particles was reduced. The purified virus was similar to purified vaccinia virus in DNA content, in the absence of detectable RNA and in sedimentation characteristics. Treatment of secondary mouse embryo monolayers with purified molluscum virus caused a reduction in the number of viable cells and in the amount of DNA/culture but had little effect on the amounts of RNA and protein/culture. The depression in the rate of DNA synthesis was maximal at about 24 to 36 hr after infection.