Large-plaque Virus Research Articles

Characterization of rabies viruses recovered from persistently infected BHK cells

Persistent rabies virus infection with a cyclic rising and falling pattern was easily initiated in BHK cells. In striking contrast with the same chronic infection established by T. J. Wiktor and H. F. Clark ((1972). Infect. Immun. 6, 988–995), this virus and cell interaction was not mediated by interferon, and the infected cells were not resistant to a heterologous virus challenge. The persistently infected cells produced small-plaque viruses (SPV) and, after a number of subcultures, these SPV replaced entirely the wild large-plaque viruses (LPV). Due to the marked cytopathic effects (CPE) on BHK cells, SPV was less efficient than LPV for the establishment of viral persistence. A fraction containing defective interfering (DI) rabies virus particles was obtained by density gradient centrifugation of virus stocks from the persistently infected cells. The DI particles could be quantitatively assayed to the degree that they suppressed the CPE by the standard rabies virus. Cyclic production of DI particles in the persistent infection was demonstrated by this method. This evidence plus the fact that the homologous virus resisted superinfection indicate that the DI particles of rabies virus play a major role in the establishment and maintenance of a persistent state of infection in vitro.

Comparative studies of the viruses rescued from polyoma tumor cell line.

SummaryBiological studies were made on the viruses rescued from two polyoma tumor cell lines induced by the parental large plaque virus, St-Lp. The virus rescued from one of the cell lines, KIT-5/2, consisted of two different plaque formations, a large plaque, R5/2-Lp and a small plaque, R5/2-Sp, and the one rescued from the other cell line, KIT-20W, entirely of small plaques, R20-Sp.The antigenic differences between the small plaque and the large plaque viruses were disclosed by HI test. Regardless of plaque size, all of the rescued viruses were shown to be less tumorigenic while the parental virus was highly tumorigenic. This situation held true also in the in vitro transformation study.

The replication of polyoma DNA.

SUMMARY Measurement of replicating molecules of polyoma virus DNA after digestion with endonuclease R1 shows that DNA replication is bidirectional, starting predominantly at a specific site. In both large plaque and small plaque polyoma virus DNA this site is 29 ± 2% of the total length of the DNA from the cleavage site of the endonuclease R1.

Infectious SV40 and SV40 DNA: rapid fluorescent focus assay.

SummaryA method utilizing the indirect immunofluorescent technique is described that permits a more rapid assay for the infectivity of the oncogenic virus, SV40. With this method both large and small plaque viruses as well as the DNA extracted from them give titer estimates that are comparable to those obtained in the standard plaque method.

Factors influencing virulence and plaque properties of attenuated Venezuelan equine encephalomyelitis virus populations.

A minority of stable large-plaque virus increased proportionally in stored unstable attenuated (9t) Venezuelan equine encephalomyelitis virus populations. L-cell-grown progeny (9t2) of stored 9t showed large amounts of large-plaque virus and increased virulence. Small-plaque virus inhibited large-plaque virus but not the reverse. Serial passage of small-plaque virus from 9t2 yielded a strain (20t) that was more attenuated than 9t.

"Rescued" SV40: increased transforming efficiency in mouse and human cells.

Plaque-size mutants of simian virus 40 (SV40) have been characterized with respect to their ability to transform mouse and human cells in vitro. Large plaque mutants of SV40 were extremely inefficient at transforming mouse or human cells, requiring approximately 50 times more infectious particles per transformation event than did the small plaque mutants. The minute plaque SV40 was the most efficient, requiring less than 10(3) plaque-forming units per transformation in mouse Balb/3T3 cells and only 1.5 x 10(4) plaque-forming units per transformation unit in the "susceptible" human strains. Both small and minute plaque virus were unable to help the large plaque virus to transform. Virus "rescued" from transformed 3T3 cells by cocultivation with monkey kidney cells was more efficient at transformation than the parental type. The increased efficiency could be shown for each plaque type and in human as well as in mouse cells. The property of "high efficiency transformation" is stable through at least two passages in monkey kidney cells.

Markers of Rubella Virus Strains in RK 13 Cell Culture

When tested on RK(13) cell cultures, strains of rubella virus could be differentiated by their ability to form small or large plaques. Large plaques were produced by the HPV-77 and Cendehill strains, and also by a laboratory stock strain (West Point), after only 14 passages in RK(13) culture. Five wild-type rubella viruses, isolated and passaged only a few times in African green monkey kidney tissue culture, grew well in RK(13) cell culture, but they were sensitive to agar inhibitors and, therefore, formed small plaques. On the other hand, RA27/3, an attenuated strain grown in WI-38 human fibroblast cells, developed low titers in RK(13) cells and also produced small plaques. We concluded that the morphological differences between small-plaque and large-plaque viruses depended on their sensitivity to agar inhibitors and on the pH of the medium during plaque formation.

Unusually Large-Plaque Mutant of Sindbis Virus

SummaryWhen Sindbis virus plaques were formed in chick embryo fibroblasts (CEF) at 40°C, plaques varying in size were observed. Plaques measuring less than 2 mm (sp) were subjected to cloning. Out of 51 sp plaques so examined, three yielded predominantly, on passage through CEF, unusually large-plaque (u) mutants. This mutant as well as the parent large-plaque (lp) and sp viruses were purified by consecutive clonings. Mixed infection of CEF with the sp and u viruses tended to exclude the sp virus. Contrarily, mixed infection with the lp virus and the u mutant tended to exclude the latter. Thus for maintenance of the u mutant, frequent clonings had to be done, because otherwise an incidental appearance of the parent type virus by back mutation seemed to result in exlusion of the u mutant virus. The u mutant as well as the sp virus was more sensitive to interferon than the lp virus. The reason why the interferon-sensitive u mutant can grow more rapidly and yield a higher amount of virus than the parent lp v...

Properties of Venezuelan equine encephalomyelitis virus accompanying attenuation in vitro.

Virus obtained during serial plaque passage of the virulent parent egg seed (PES) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus produced only large plaques during either 3 serial plaque passages in chick fibroblasts or 10 plaque passages in L cells, and was lethal for mice by the intraperitoneal route. Virus showing these characteristics was designated the stable large-plaque (Ls) type. In contrast, virus obtained during serial plaque passage of the attenuated 9t strain in chick fibroblasts formed only very small plaques and was not lethal for mice by the intraperitoneal route. Virus showing these properties was designated the stable small-plaque (Ss) type. Under other passage conditions, however, large-plaque virus that yielded about 90% large and 10% small plaques was obtained; this virus was designated the unstable large or Lu type because it differed from the Ls type, which yielded only large plaques. The Lu type continued to yield the same ratio of large to small plaques for several plaque-to-plaque passages. In addition, small-plaque virus that yielded both large and small plaques and that showed a reduced capability to infect mice was also recovered. This virus was designated the unstable small or Su type because it differed from the Ss type in its higher level of virulence and in its plaque-forming properties. Thus, based upon the properties of virulence for mice and plaque size, four viral types could be discerned. The evidence suggests that serial passage in cell culture imposed environmental pressures that sequentially selected the following viral types: Ls, Lu, Su, and Ss.

Transplant immunity to polyoma virus-induced tumor cells. IV. A polyoma strain defective in transplant antigen induction.

Abstract Differences in the capacity of polyoma virus strains to stimulate transplant resistance in hamsters as well as induce measurable quantities of the polyoma-specific transplant antigen in tumor cells were demonstrated. The large (Ip-D) and small plaque (sp-D) virus strains isolated by Dulbecco from the LID-1 polyoma virus isolated at the National Institute of Allergy and Infectious Diseases, were found to be defective in their capacity to stimulate transplant immunity against tumor cell lines known to contain the polyoma transplant antigen which had been induced by several other strains. Furthermore, both strains were defective in the quantity of transplant antigen induced in tumor cells. There was no correlation between the lack of transplant antigen activity and the neoplastic characteristics of the tumor cells induced by these virus strains; this finding suggests that the cell membrane alteration responsible for the transplant antigen function does not play a direct, mediating role in the neonlastic event.

A cause of variation in clonal morphology of polyoma transformed hamster cells

When a mixed cell population of hamster embryo cells is transformed in vitro by polyoma virus (PV), a broad spectrum of morphologically different transformed cell clones can be observed at 7–9 days after virus infection. To test the cause of this morphological variation, two series of experiments were carried out. Series A: 21 homogeneous cell populations, each originating from a single cell, were transformed by PV. In all cases, all the transformed clones in any one such homogeneous population showed the same morphology. In addition, 2 homogeneous populations showed the same clone morphology when transformed either by large-plaque virus or a small-plaque mutant. Series B: Cell cultures made from different embryonic organs were transformed by PV. In each case there was, in comparison to a mixed population from whole embryos, a significant reduction in the spectrum of normal and transformed clone morphologies. The hereditability of clonal morphology in transformed clones was established by recloning 15 clones. Cultures of transformed clones with similar morphologies were examined under several physiological conditions. Morphologically similar clones isolated from a mixed population of embryo cells showed physiological differences, whereas morphologically similar clones isolated from a homogeneous cell population were similar under these conditions. The results indicate that when examined at 7–9 days after virus infection, the normal cell type that is transformed is a major factor controlling the morphology of the transformed clone. The uniformity of transformed clones derived from homogeneous normal cell populations indicates that there is a common mechanism for PV-induced cell transformation.

Electrophoretic properties and purification of large and small plaque-forming strains of polyoma virus

Radioactive preparations of polyoma virus purified by treatment with ribonuclease and deoxyribonuclease, fluorocarbon extraction, and banding in rubidium chloride density gradients were found to be electrophoretically heterogeneous when examined by the sucrose-gradient zone electrophoresis method. Infectivity and hemagglutinin activity moved with the same negative mobility, constant in different preparations; other distinctly resolved radioactive components, probably consisting of DNA bound to cellular and viral material, also were generally found. Large-plaque virus migrated at about 1.5 times the speed of small plaque-virus at pH 8.5 and pH 7.2. “Empty” particle fractions from rubidium chloride gradients contained hemagglutinating components of similar mobility to those of the “full” particle fractions indicating that the capsid alone determines the electrophoretic properties of the virus particle. Attempts at further purification of “full” band preparations were made. Treatment with trypsin released components which sedimented more slowly than the virus and gave virus preparations of increased electrophoretic homogeneity. Electrophoretic analysis of trypsin-treated preparations showed that transforming activity migrated at the same rate as plaque-forming activity.

Cell transformation in vitro and tumor induction in vivo by large- and small-plaque polyoma virus

Large-plaque and small-plaque polyoma virus suspensions transformed the same percentage of hamster kidney cells in vitro when similar exposure multiplicities were used. However, measurements on the adsorbed multiplicities showed that more small-plaque than large-plaque virus actually adsorbed to the cells and, therefore, that more transformed colonies per adsorbed hemagglutinin or plaque-forming unit were produced by large-plaque virus suspensions than by small-plaque. Large-plaque virus suspensions induced a high proportion of kidney tumors and few peritoneal tumors when inoculated intraperitoneally into newborn hamsters. With small-plaque virus suspensions, the incidence of tumors in the two anatomical sites was reversed. It is suggested that the difference in tumor sites is related to the difference in the adsorption characteristics of the two plaque types.

Some characteristics of large-plaque and small-plaque lines of polyoma virus

Suspensions of small-plaque and large-plaque lines of polyoma virus showed marked differences in the adsorption of virus particles to cells and to the hemagglutinin inhibitor released from cells. Small-plaque virus was more readily adsorbed than large-plaque virus, and this difference could be used as an adjunct to plaque size distribution in determining the predominant plaque type of virus suspensions. The suspensions of large- and small-plaque virus contained similar proportions of “full” particles of buoyant density 1.32 and “empty” particles of buoyant density 1.29.

Studies on the lytic interaction and cell transformation with a large- and a small-plaque mutant of polyoma virus

Quantitative studies were undertaken, with the small-plaque polyoma mutant SP2 and the large-plaque virus BP5, on the lytic interaction in mouse and hamster cells and on transformation of hamster cells under various conditions. In the study on the lytic interaction, one-step growth curves of the two viruses showed a higher total virus yield per culture for SP2 than for BP5. The higher total virus yield with SP2 was explained by a higher percentage of virus-yielding cells. The percentage of such cells was 3.8 to 7.0 times higher than with BP5 after inoculation of mouse cultures, and 4.2 to 20.0 times higher after inoculation of hamster cultures. There was a similar average virus yield per virus-yielding cell [2 to 4 × 10 3 plaque-forming units (PFU)] with both viruses. No effect was found on the plaque size of SP2 on mouse embryo monolayers after addition of DEAE-dextran or protamine to the agar nutrient overlay. In the study on cell transformation, the percentage of transformed clones (PTC) was determined by cloning cultures of hamster cells after exposure to either virus. After exposure of cultures grown at 37° at a virus: cell ratio of 20 PFU per cell, the PTC was 0.1%–0.4% for BP5, and 0.8%–2.0% for SP2. This showed a 4.0–14.0 times higher PTC for SP2. The increased efficiency of SP2 with respect to PTC was thus of about the same order of magnitude as the increased efficiency of SP2 in the lytic interaction. Similar differences in the PTC of the two viruses, as compared with those obtained after a single exposure at 20 PFU per cell, were found after a single exposure at a virus: cell ratio of 1000 PFU per cell, and after three successive exposures at 10 PFU per cell at 24-hour intervals. Exposure at 1000 PFU did not appreciably increase the PTC, and even after the three successive exposures, the highest PTC was still only 3.0% for SP2 and 0.4% for BP5. The PTC could, however, be increased to 10.0% for SP2 and to 3.0% for BP5, by maintaining cultures at 24° before cloning, with virus exposure either at the beginning or at the end of the period at 24°. The results thus indicate that the use of a virus mutant, and differences in the culture conditions, can change the rate of polyoma-induced cell transformation.

A comparison of some properties of polyoma virus and pneumonia virus of mice.

Polyoma virus (PY) and pneumonia virus of mice (PVM) are not related to each other serologically (1), but because of their similarity in size and their semi-ubiquitous presence in a latent form in mice, the possibility was considered that they belong to the same or related groups of viruses. Since sensitivity to ether serves to distinguish groups of viruses (2, 3), and since it is known that PY is ether resistant (4), the reaction of PVM towards ether was investigated. Ability of the 2 viruses to grow in tissue culture, and their in vivo effects on mouse lung were also compared.Materials and Methods. Three strains of PY were used in these experiments. Strain LID-1 (5) was kindly provided by Dr. R. J. Huebner. Strain Lp or large plaque virus was derived by Dr. R. Dulbecco by single plaque isolation from a stock originally provided by Dr. W.P. Rowe(6,7). Sp or small plaque virus was isolated from transformed hamster embryo cells after exposure to Lp virus(7). Both Lp and Sp viruses were kindly provided by D...