Yaba Tumor Research Articles

Analysis of Yaba tumor poxvirus-induced proteins in infected cells by two-dimensional gel electrophoresis

Lack of inhibition of host protein synthesis by Yaba tumor poxvirus and comigration of viral and host proteins did not interfere with high resolution of virus-induced proteins when two-dimensional gel electrophoresis was employed. A total of 84 virus-induced proteins, 44 structural and 40 nonstructural, comprising 62.9% of the coding capacity of Yaba virus DNA, were detected. These proteins may be further categorized in two classes: early, synthesized in the presence of DNA inhibitor, Ara-c; and late, beginning at 6 hr p.i. with the bulk of viral protein synthesis occurring later than 30 hr p.i. The rate of synthesis of viral proteins fell into two patterns: group A, in which the rate of synthesis decreased; and, group B, in which the rate of synthesis increased after the initial appearance of the protein.

A novel cell transformation with DNA-containing cytoplasmic yaba tumor poxvirus

Monkey kidney cells were morphologically transformed in vitro with uv-irradiated Yaba tumor poxvirus. Cell lines established were shown to be virus nonproducers and exhibited biological characteristics typical of transformed cells. These characteristics included increased saturation density, reduced serum requirements for growth, and ability to grow in soft agar. The morphological alterations of transformed cells were similar to Yaba virus-induced tumor cells and were characterized by loss of contact inhibition, multinucleated cells, and cytoplasmic lipid droplets. Southern blot hybridization revealed that sequences homologous to low-molecular-weight viral DNA (5.1, 4.8, 3.9 kbp) were present in the transformed cells. Yaba virus-specific antigens detected by immunofluorescence assays were found to be in the cytoplasm of transformed cells. Four virus-specific proteins, with molecular weights of 160,000, 140,000 107,000, and 74,000 daltons, were detected in transformed cells immunoprecipitated with sera from tumor-bearing monkeys. These results indicate that DNA-containing Yaba tumor virus, which replicates exclusively in the cytoplasm, is capable of inducing cell transformation in vitro.

Surface membrane redistribution and stabilization of concanavalin A-specific receptors following yaba tumor poxvirus infection

Monkey kidney cells productively infected with Yaba tumor poxvirus clearly exhibit plasma membrane alterations when treated with both fluorescein-labeled and unlabeled concanavalin A. The concanavalin A-mediated cytoagglutination reaction for Yaba-infected Jinet and CV-1 cells increased linearly from 12 to 16 h post-infection, reaching a maximum by 24–28 h. Treatment of either Yaba-infected CV-1 or Jinet cells with methyl- D-glucopyranoside before or after addition of concanavalin A completely blocked or reversed the cytoagglutination response. Trypsin treatment of uninfected CV-1 or Jinet cells enhanced concanavalin A-mediated cytoagglutination properties. Conversely, trypsin treatment of Yaba-infected Jinet cells resulted in a reduced cytoagglutination response. Increasing temperature and lectin concentration enhance concanavalin A-mediated cytoagglutination for uninfected, trypsin-treated and Yaba-infected CV-1 cells. Cytosine arabinoside has little or no effect on the Yaba-induced cell cytoagglutination reaction while cycloheximide blocks the cytoagglutination response if added prior to 12 h post-infection. Fluorescein-labeled concanavalin A binding studies have revealed that at 4°C, Yaba-infected CV-1 cells display a predominantly ‘patchy’ pattern of topological fluorescence, while trypsin-treated and uninfected CV-1 cells at 4°C display a uniform pattern of fluorescence binding. Patchy fluorescence, indicative of concanavalin A-susceptible, receptor-site clustering on the surface membrane, was reduced 50% if Yaba-infected CV-1 cells were treated with glutaraldehyde (2.5%) before addition of fluorescein-labeled concanavalin A at 4°C. Similar pre-fixation of trypsin-treated CV-1 cells resulted in uniform, fluorescent labelling patterns at all assay temperatures.

Public health aspects of animal tumor viruses

Some believe animal tumor viruses are frequently involved in our public health, however, over 100 animal tumor viruses have been isolated and only one (Yaba Tumor Virus) has minor public significance: FeLV is highly contagious in cats and some have recommended FeLV infected cats be euthanized because of “possible” public health importance. When all evidence bearing on possible public health relationship is assembled, it is clear that most evidence indicates no likelihood of association between FeLV and any human disease.

Human and primate poxviruses: I. Growth characteristics of cytolytic and tumor variants

The dual potential of poxviruses to be cytolytic and tumorgenic has been extended. Yaba monkey tumor virus formed foci on monkey and human embryonic cells. Cytolytic or plaque-forming virus was isolated from Yaba tumor homogenates by selective sucrose centrifugation and passage through monkey or human embryonic cells. Monkey pox virus (MPV) was cytolytic for monkey cells or human embryonic cells, but upon passage onto young monolayers of human carcinoma cells, HeLa, produced a restricted cytopathic effect on first transfer, foci on second transfer and cytolysis after the fourth transfer. If HeLa monolayers were compact, the rapid growth precluded overt expression of cytolytic MPV. Electron micrographs of cytolytic Yaba indicated that Yaba development was similar to vaccinia and MPV but not totally organized. Growth curves of vaccinia, MPV and cytolytic Yaba were essentially identical in monkey and human embryonic cell lines.

Nucleic acid synthesis in cytoplasm of Yaba monkey tumor virus-infected cells.

Yaba tumor virus progeny appeared in cynomolgus monkey kidney cells at 24 h postinfection and reached a plateau at 72 h in the first cycle of replication. Viral DNA synthesis was first detected at about 3 h and reached a peak in 18 h. Maximum coating of viral DNA in infected cells occurred at 4 days postinfection. Rapidly labeled RNA was synthesized in the cytoplasm of virus-infected cells. At 6 h postinfection 7 to 10S RNA was present; this species was present in greater amount at 12 h; at 24 h a truncated peak indicated the presence of 14 to 15S as well as 7 to 10S RNA. Hybridization data indicated that the largest peak of messenger RNA synthesis occurred at 11 to 13 h postinfection and a second, slightly smaller, peak occurred at 21 to 23 h.

Inhibition of the synthesis of simian virus 40 antigens in cells preinfected with Yaba tumor virus.

The synthesis of tumor and viral antigens after infection of an established line of cynomolgus monkey kidney cells with simian virus 40 (SV40) was compared in cells previously infected with Yaba virus and in cells not preinfected. SV40 failed to induce synthesis of tumor or viral antigens in cells preinfected with Yaba virus. The inhibitory state in preinfected cells was shown to develop sequentially. Increase in the rate of deoxyribonucleic acid synthesis in the nuclei of preinfected cells occurred after infection with SV40. This rate of increase was significantly lower than that which occurred in SV40-infected cells which had not been preinfected. Cytosine arabinoside did not exert significant effect on the development of the inhibitory effect against SV40 in Yaba virus-infected cells.

Biogenesis of poxviruses: Identification of four enzyme activities within purified Yaba tumor virus

The morphogenesis of Yaba monkey tumor virus (YMTV) was investigated in a cynomolgus monkey kidney cell line. YMTV has been shown to grow less rapidly in high-passage cells (i.e., 65 passages) than in low-passage cells (i.e., between 30 and 60 passages). This prolonged growth cycle in high-passage cells allows for a more detailed analysis of the events in morphogenesis. Samples of YMTV-infected cells were prepared for analysis at 2, 3, 4, and 5 days postinfection. At least 12 “stages” of virus morphogenesis can be identified.

Electron microscopy of the tana poxvirus

The fine structure of Tana virus, a poxvirus originally isolated from children in Kenya, has been studied by electron microscopy of infected rhesus embryonic monkey tissue cultures. The intracytoplasmic maturation of the virus has been analyzed and compared with that seen in Yaba-like disease (Y.L.D.), an epidermal pox disease of monkeys. Two types of virus factories have been distinguished, one of which appears to be responsible for formation of nucleoids, the other for virus membranes. Honeycombed crystals, cylindrical tubules, mitochondria in pairs or triplets, and myelin figures were also observed. The nuclei showed areas of decreased electron opacity. The development of Tana virus was identical with that of Y.L.D. virus. These findings are consistent with the hypothesis, based on serologic studies, that Tana poxvirus is identical with Y.L.D. virus but differs from a similar monkey poxvirus, the Yaba tumor virus.

Research Note: Detection of Fowlpox Virus Antigen in Tissue Culture by Fluorescent Antibody Technique

The direct fluorescent antibody (FA) technique has been used to detect vaccinia antigens (6,8,9), small pox virus (2,4,5,7), rabbit pox virus (1), fowlpox virus (9), and Yaba Tumor Pox virus (10). In the present investigation, fowlpox viral antigens were detected in whole chicken embryo or chicken kidney cell cultures by a direct fluorescent antibody technique. Hyperimmune fowlpox virus antiserum was prepared in the chickens by cutaneous and intravenous inoculation of fowlpox virus strain ORT-101. This virus strain had been maintained by serial passages in CAM of developing chicken embryos. On cutaneous inoculation, the virus produced typical lesions of fowlpox in chickens. Specific antibodies against the fowlpox viral antigen were demonstrated by passive hemagglutination test and agar gel precipitation test. The serum was conjugated with fluorescein isothiocynate (General Biochemicals, Ohio) according to methods of Cherry and Goodman (3). The conjugated serum was adsorbed three times with acetone-extracted duck liver powder and stored at -60 C. In preliminary studies, both whole chicken embryo cell culture from nine-day-old embryos and kidney cell cultures from 19-dayold chicken embryos were grown in 10 X 150-mm rubber-stoppered sterile tubes containing 11 X 22-mm glass coverslips. Later, only chicken kidney cells were used. Confluent cell sheets were usually obtained on the coverslips in two to three days. A ten per cent suspension of fowlpox-virus-infected chorioallantoic membranes was made in Eagle's medium (Grand Island

Growth kinetics of Yaba tumor poxvirus after in vitro adaptation to cercopithecus kidney cells.

Yaba tumor poxvirus has been adapted to continuous in vitro cultivation in monolayers of cercopithecus kidney cells. At 35 C, the minimum replicative cycle, after synchronous infection of CV-1 cells with multiplicity of infection of 135 focusforming units per cell, was 35 hr; however, maximum virus yields were not obtained until 75 hr postinfection (PI). Cytoplasmic incorporation of (3)H-thymidine [viral deoxyribonucleic acid (DNA) synthesis] was detected 3 hr PI and was preceded by synthesis of nonstructural associated antigens (YS). Synthesis of YS antigens was not inhibited by the DNA inhibitor, arabinofuranosyl cytosine (ARA-C). Synthesis of at least two virion structural antigens, although not detected by immunofluorescence until 2 hr after the onset of DNA synthesis, occurred in the presence of ARA-C, indicating potential translation of these structural antigens from parental DNA. The first progeny DNA was completed by 20 hr PI but was not detected in infectious form until 35 hr PI. The maximum rate of progeny DNA completion occurred between 20 and 30 hr PI. DNA synthesis continued 45 to 50 hr PI. The adapted virus retained its oncogenicity and, like the wild type, replicated better at 35 C than at 37 C. A synthetic step associated with viral DNA synthesis appears to be temperature-sensitive.

Immunodiffusion analysis of Yaba poxvirus structural and associated antigens.

Yaba poxvirus virions were extracted and purified from Rhesus monkey tumors. A saline-soluble virion fraction (Y-xp), obtained by mechanical fractionation of purified virions with an X-press, contained seven components in acrylamide gel electrophoresis; five of these components were reactive in immunodiffusion with whole virion and Y-xp antisera produced in rabbits and monkeys. The saline-insoluble residue remaining after X-press treatment was hydrolyzed with sodium dodecyl sulfate, urea, and 2-mercaptoethanol (SUM). This fraction, Y-sum, contained five components, four of which were demonstrable by immunodiffusion. There was no evidence of antigenic relationships between Y-xp and Y-sum antigens in immunodiffusion. In acrylamide gel electrophoresis, one Y-xp and one Y-sum component had similar mobilities. Y-xp but not Y-sum antisera contained viral-neutralizing antibodies. Virus-free saline extracts of Yaba tumor prepared with Genetron (YS) were essentially devoid of virion structural antigens. They failed to induce precipitating antibodies for virion antigens, were nonreactive in immunodiffusion with virion antisera, and gave low complement-fixation titers with virion antisera. Yaba virion antigens were recovered from the Genetron tumor sediment by SUM and alkaline hydrolysis. Antisera prepared to YS extracts gave a maximum of 17 precipitin lines in immunodiffusion with YS extracts; none was identified as a virion structural antigen. Saline extracts of tumor prepared without Genetron contained immunogenic amounts of 5 virion antigens and 12 to 14 associated antigens. Animals immunized with infected cell culture extracts (virus-free) formed antibodies to six to seven virion antigens. The implications of using extracts of Yaba poxvirus-infected tissues in complement-fixation tests to measure virion antibodies were discussed.

Yaba tumor virus. II. Studies on inactivation and reactivation.

SummaryA study of the effects of heat, pH, and UV light on Yaba tumor virus indicated that heating at 56° for 1 hr, pH 3 at room temperature for 1 hr, and irradiation for less than 30 sec completely inactivated the virus. Inoculation of a mixture of UV-inactivated Yaba virus and active vaccinia virus into monkeys resulted in the formation of a tumorigenic presumptive hybrid virus which retained, for at least three passages, certain antigenic components of both Yaba and vaccinia viruses. A possible mechanism of the hybridization is discussed.The authors wish to thank Dr. H. A. Wenner for reviewing the manuscript.

Some physiochemical properties of Yaba poxvirus deoxyribonucleic acid.

Yaba tumor poxvirus deoxyribonucleic acid (DNA) has a density of 1.6905 in CsCl and its T(m) value in 0.015 m citrate in saline is 82.3. The guanine plus cytosine content estimated from these properties was taken to be 32.5 +/- 0.5%, a value 2 to 3% less than for DNA from other poxviruses reported to date.

'Spontaneous' occurrence of Yaba tumor in a monkey colony.

Im Laboratorium einer Affenkolonie vonMacaca mulatta, M. irus undM. speciosa, in welchem Experimente mit Yaba-Virus durchgefuhrt wurden, sind 13 Falle von spontan auftretenden Tumoren beobachtet worden. Yaba-Virus ist ein Krankheitserreger, der in empfanglichen Primaten Histiozytome hervorruft. Es besteht kein Anhaltspunkt fur eine Direktubertragung des Virus durch Kontakte. Anzeichen sind aber vorhanden, dass der Virus moglicherweise durch Insekten ubertragen worden sein konnte.

Yaba tumor virus. I. Studies on pathogenesis and immunity.

SummaryTumor development and CF antibody responses of monkeys inoculated s.c. and i.v. with various dilutions of Yaba tumor virus were investigated. Tumors developed at different times and at different sites with CF antibodies appearing soon after the development of tumors and attaining final CF titers comparable in all inoculated animals. No antigenic relationship between Yaba virus and monkey pox viruses was found by in vivo challenge, or between Yaba, monkey pox, and vaccinia virus by cross CF and HI tests. The development of viral antigen(s) in tumor cells removed from infected monkeys at weekly intervals was monitored by the FA technique and CF test. Although the FA technique detected the viral antigen earlier, both procedures were equally effective for detection of antigen in tumors removed 4 or more weeks postinfection.

Yaba tumor poxvirus synthesis in vitro. I. Cytopathological, histochemical, and immunofluorescent studies.

Yohn, David S. (Roswell Park Memorial Institute, Buffalo, N.Y.), Victoria A. Haendiges, and James T. Grace, Jr. Yaba tumor poxvirus synthesis in vitro. I. Cytopathological, histochemical, and immunofluorescent studies. J. Bacteriol. 91:1977-1985. 1966.-Yaba tumor poxvirus synthesis in BSC-1 cell culture was followed sequentially with light microscopy, immunofluorescent microscopy, and various histochemical stains. The first evidence of infection was detected at 24 hr when nucleoli became hypertrophic, reflecting enhanced ribonucleic acid (RNA) synthesis. At 36 hr, deoxyribonucleic acid synthesis was detected in the cytoplasm. This was immediately followed by or associated with antigen synthesis at paranuclear sites and enhanced RNA synthesis in the cytoplasm. Cytoplasmic inclusions were readily apparent at 4 days in initially infected cells. Contiguous spread of virus was judged to have occurred around the third day of infection. The time required to complete the synthetic cycle from time of infection to production of infectious progeny was estimated to be of the order of 60 hr. This cycle is 6 to 10 times longer than for vaccinia virus. By light microscopy, cytopathic effects (CPE) were detectable in 5 days in heavily infected cultures. With 100 units or less of infectious virus, CPE was not readily detected until the 10th to 14th day. At this time, focal areas of infection classified either as microtumors or microplaques were present. Secondary foci appeared during the 4th week of incubation.

Yaba tumor poxvirus synthesis in vitro. II. Adsorption, inactivation, and assay studies.

Yohn, David S. (Roswell Park Memorial Institute, Buffalo, N.Y.), Fanny R. Marmol, Victoria A. Haendiges, and James T. Grace, Jr. Yaba tumor poxvirus synthesis in vitro. II. Adsorption, inactivation, and assay studies. J. Bacteriol. 91:1953-1958. 1966.-Means to increase the efficiency of the Yaba tumor poxvirus assay in BSC-1 cell cultures were sought. A method was devised wherein 0.4 ml of each virus dilution was layered onto BSC-1 cells in 80-mm Leighton tubes and permitted to adsorb at 25 C for 18 hr prior to incubation at 35 C. The diluent found most reliable was medium 199 containing 50% bovine amniotic fluid adjusted to 2.0 mm calcium and 1.0 mm magnesium at pH 7.0. These modifications yielded highly reproducible titrations with a greater than twofold increase in assay sensitivity. Irreversible adsorption of Yaba virus by BSC-1 cells proceeds comparatively slowly at 25 to 33 C. Although the process is more rapid at 35 or 37 C, the increased thermal lability of the virus at these temperatures results in lower titers than with virus adsorbed at 25 C for longer periods of time. Yaba poxvirus appears to be 5 to 10 times more sensitive to thermal inactivation than vaccinia poxvirus.

Yaba tumor poxvirus synthesis in vitro. 3. Growth kinetics.

Yohn, David S. (Roswell Park Memorial Institute, Buffalo, N.Y.), Victoria A. Haendiges, and Etienne de Harven. Yaba tumor poxvirus synthesis in vitro. III. Growth kinetics. J. Bacteriol. 91:1986-1991. 1966.-The infectious synthetic cycle of Yaba poxvirus by BSC-1 cells at 35 C is described and discussed together with related observations obtained by immunofluorescence and electron microscopy. After adsorption of virus at 25 C, at least 6 hr of incubation at 35 C was required before viral eclipse was demonstrable. Infectious progeny appeared after an additional 48 hr of incubation at 35 C. Newly synthesized virus particles were seen at 68 hr but not at 44 hr by electron microscopy. Maximal maturation occurred between 100 and 120 hr. Maximal yields were obtained from the 5th through the 8th day. The differences in kinetics between Yaba and vaccinia poxviruses, as described for the latter in the literature, are discussed. Possible factors accounting for these differences are enumerated.