Avian Tumor Research Articles

Relationship between A-type and C-type particles in cells infected by Rous sarcoma virus.

Chicken cells infected with avian RNA tumor virus often contain small cytoplasmic A-type particles which commonly exist as clusters of 50--100 particles when viewed in thin sections. These particles were found more consistently in Rous sarcoma virus-infected than Rous-associated virus-infected cultures, but were generally present in only a small fraction of the total infected cells. The results of the survey of cells infected with various strains of leukosis-sarcoma viruses led to the hypothesis that the A particles develop in cells undergoing cytopathological degeneration. The hypothesis explains also the evanescent nature of the appearance of these particles in infected cells. The application of immunoelectron microscopic methods using monospecific antisera against viral internal proteins revealed that the A particles contain components immunologically related to the proteins of C-type virus.

Two active forms of RD-114 virus DNA polymerase in infected cells

Two forms of DNA polymerase are present in RD-114-infected human, dog, and mink cells, but are not detectable in uninfected cells. The two enzymes are indistinguishable catalytically and immunologically, but differ with respect to molecular weight and elution position from (dT) 12–18-cellulose and phosphocellulose. The large enzyme (∼ 95,000 daltons) is found in the infected cells, but not the virions produced by these cells. The virions contain only the smaller enzyme (∼ 70,000 daltons). The larger form may represent a mammalian viral equivalent to the β subunit of avian RNA tumor virus DNA polymerase.

Site on the RNA of an avian sarcoma virus at which primer is bound

In vitro transcription of the avian tumor virus RNA by RNA-directed DNA polymerase is initiated on the unique cellular 4S RNA. Previous studies have shown that on the average there is one such RNA primer hydrogen bonded to each viral 35S RNA. The present study confirms that finding and demonstrates that, at least for the majority of 35S RNA molecules, the primer is bound at a site close to the 5'-terminus.

Isolation of type C virions from a normal human fibroblast strain.

Type C virions were spontaneously released from cultures of a diploid human cell strain. The varions have properties of known type C RNA tumor viruses and share antigenic determinants with the major interspecies-specific antigen (p30) of simian sarcoma virus. Antiserum to reverse transcriptase of gibbon ape leukemia virus inhibits the reverse transcriptase of the putative human virions and that of simian sarcoma virus, but has no effect on the corresponding enzymes of avian or murine RNA tumor viruses.

Genetic Susceptibility of Chicken x Quail Hybrid Embryos to Avian RNA Tumour Viruses

An attempt was made to hybridize the chicken (Gallus domesticus) male with Japanese quail (Coturnix coturnix japonica) female in order to study the genetic susceptibility of hybrid embryos to avian RNA tumour viruses of subgroups, A, B, D and E. In the hybrids the results supported the prevailing concept that susceptibility is dominant over resistance regardless of the dominant trait contributed by either parent. It was also observed that the Ie gene of the chicken was unable to suppress the 'quail-coded' susceptibility to subgroup E virus in the hybrid system, suggesting the lack of penetrance of the Ie gene. Despite the fact that some hybrids were resistant to viruses of subgroups B and D, they were susceptible to subgroup E virus, which was not expected on the basis of the concept that subgroup B-resistant cells cannot be E-susceptible. Also, the hybrids were susceptible to E virus regardless of gs antigen expression and presence of the Ie gene in the genome. This indicates that our earlier suggestion that the Ie gene is another expression of the gs antigen-determining gene is inconsistent.

Specific binding of tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase).

The ability of tryptophan tRNA (tRNATrp) to initiate reverse transcription of the 70S RNA of avian RNA tumor viruses suggested that the reverse transcriptase (RNA-dependent DNA polymerase; deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) might have a specific binding site for the tRNA. A complex of tRNATrp and the avian myeloblastosis virus reverse transcriptase has been demonstrated using chromatography on Sephadex G-100 columns. Of all the chicken tRNAs, only tRNATrp and a tRNA4Met bind to the enzyme with high enough affinity to be selected from a mixture of the chicken cell tRNAs. The ability of tRNATrp to change the sedimentation rate of the enzyme indicates that tRNATrp is not binding to a contaminant in the enzyme preparation. Treatment of the enzyme with monospecific antibody to reverse transcriptase prevented binding of tRNA as well as inhibited the DNA polymerase activity of the enzyme. The ability of reverse transcriptase to utilize tRNATrp aa a primer for DNA synthesis, therefore, appears to involve a highly specific site on the enzyme.

Brief Communication: Oncogenesis by Marek's Disease Herpesvirus in Chickens Lacking Expression of Endogenous (gs, Chick Helper Factor, Rous-Associated Virus-O) and Exogenous Avian RNA Tumor Viruses

Chickens free of exogenous avian leukosis virus (ALV) infection, replicating endogenous ALV (Rous-associated virus-O), gs antigen, and chick helper factor were fully susceptible to induction of Marek's disease (MD) by ALV-free MD viruses. Dual infection with Rous-associated virus-2 and MD virus did not significantly alter the character of the MD lesions. Thus exogenous ALV infection was not requisite for MD virus-induced oncogenesis. Although participation of endogenous RNA tumor virus genes in MD lesion induction could not be excluded, expression of such genes in MD tumors as gs antigen was not established.

Causes of disease in two commercial flocks of laying hens.

Laboratory examination of all birds that were culled or died during an eight-month period in two commerical laying flocks was performed to reveal the causes of disease and their prevalence. The average weekly total of diseased birds was greater in one flock (60-69) than the other (27-37). This resulted mainly from a high incidence in the former flock of leucoses and sarcomas, although losses due to fatty liver syndrome, prolapse and cannibalism and cage layer fatigue were also marginally greater in this flock than the second. Haemangiomas occurred more frequently in the flock with the higher disease level. A total of 273 hens of the 2,000 examined from this flock had single or multiple haemangiomas. Special features of the major causes of disease were outlined and discussed. A conclusion made from this study was that histopathological examination is necessary for accurate diagnosis of avian tumours.

A comparative study of the avian reticuloendotheliosis virus: Relationship to murine leukemia virus and viruses of the avian sarcoma-leukosis complex

Reticuloendotheliosis virus (REV) with several related viruses comprises a group of C-type, avian RNA tumor viruses which is distinct from the avian leukosis-sarcoma virus (ALSV) complex. The emphasis of the present investigation has been on the comparison of the properties of REV with those of a model mammalian RNA tumor virus, murine leukemia virus (MuLV), and with those of the ALSV. The structure and pathway of maturation of these viruses has been examined using electron microscopy. Conclusions derived from this work indicate that while the immature particles of REV can be morphologically distinguished from both MuLV and ALSV, the mature REV particle is very like that of MuLV and quite different from that of ALSV. The properties of the purified DNA-polymerase of these viruses were analyzed with the following significant findings: 1) Unfrozen, purified disrupted REV, but not purified REV-DNA-polymerase is enzymatically active using natural viral RNA as template-primer; 2) the DNA-polymerase copurifies with an RNase-H activity which probably resides on the same polypeptide; 3) the size of the DNA-polymerase-RNase-H complex is indistinguishable from that of the MuLV, a single polypeptide of 84,000 daltons; 4) the divalent cation preference of the REV-DNA polymerase, like that of MuLV, but unlike that of ALSV, is for Mn 2+; and 5) serological cross-reaction between the DNA-polymerase of REV and ALSV could not be demonstrated. Apart from these structural and biochemical analogies, no direct relationship between REV and MuLV has been established. Infectivity of REV in two strains of mouse cells could not be demonstrated. Immunodiffusion tests for reaction of purified, disrupted REV with antisera specific for ALSV structural components and for the interspecies specific reaction characteristic of mammalian RNA tumor virus p 30 protein were uniformly negative. After consideration of all the available data, it seems that the REV must be considered a distinct group of avian RNA tumor viruses with significant structural similarities to mammalian viruses, but nonetheless differing antigenic determinants.

DNA polymerase of reticuloendotheliosis virus: Inability to detect endogenous RNA-directed DNA synthesis

Purified preparations of reticuloendotheliosis virus (REV) appear to lack the endogenous RNase-sensitive DNA polymerase activity present in most, if not all, avian RNA tumor viruses. Although no endogenous RNA-directed DNA polymerase could be detected in REV, we have been able to demonstrate the presence of a virion-associated DNA polymerase by employing exogenous synthetic homopolymers as template · primer. A comparison of the REV-associated DNA polymerase activity with the RNA-directed DNA polymerase of Rous sarcoma virus (RSV) reveals similarities in the preference of the enzymes to utilize certain synthetic template · primer complexes containing ribopolymers as template. For example, both enzymes prefer poly(rA) · oligo(dT) 10 to poly(dA) · oligo(dT) 10 as template · primer. In addition, poly(rC) · oligo(dG) 10 appears to be efficiently utilized by the REV DNA polymerase under conditions whereby a DNA-directed DNA polymerase does not utilize this synthetic homopolymer as template · primer. Although the REV 70 S RNA genome is not transcribed by the DNA polymerase contained within virions of REV, it is as good a template · primer for the purified avian oncornavirus RNA-directed DNA polymerase as RSV 70 S RNA. Furthermore, the virion-associated REV DNA polymerase can transcribe REV 70 S RNA when oligo(dT) 12–18 is present as a source of primer. Therefore, the inability of the REV DNA polymerase to transcribe effectively the REV genome in vitro appears to reflect either some unique property of the REV enzyme or some structural feature of REV 70 S RNA, such as the lack of certain primer molecules required by the REV DNA polymerase for the initiation of DNA synthesis.

Recombination between endogenous and exogenous RNA tumor virus genes as analyzed by nucleic acid hybridization

Certain chicken cells that do not spontaneously release virus particles have been shown to produce a subgroup E avian RNA tumor virus, Rous-associated virus 60 (RAV-60), after infection with viruses of other subgroups. The nucleic acids of RAV-60 were analyzed for sequence homologies with the viral nucleic acids contained in the uninfected cell and with those of RAV-2, the exogenous virus used for the preparation of this particular RAV-60 isolate. In addition, these nucleic acids were compared with those of RAV-0, an endogenous virus spontaneously released from line 100 chicken cells. RAV-60 appears to be intermediate between RAV-0 and RAV-2 in its genetic composition, based on the pattern of hybridization obtained with the nucleic acids of these viruses and on the melting profiles of the various hybrid combinations. Of the three viruses tested, RAV-0 appears to have the greatest sequence homology with the viral nucleic acids of the uninfected cell. Hybridization between RAV-60 3-H-labeled complementary DNA and either DNA or RNA from the uninfected cell indicates that RAV-60 contains some nucleic acid sequences which are not present in the cell. In addition, some RAV-60 sequences which hybridize with the cell nucleic acid contain significant amounts of mismatching, as indicated by the lower thermal stability of these hybrid duplexes. Hybrid formation between these partially homologous sequences was excluded under stringent annealing conditions. The data indicate that RAV-60 is a recombinant between exogenous and endogenous viral genes.

Evidence for a Host Cell Surface Antigen on the Envelope of Avian Tumour Viruses

Avian sarcoma viruses of the A, B, C, D and E subgroups are inactivated about 100-fold by the serum of rabbits immunized against chick embryo (CE) cells, in the presence, but not in the absence, of complement. The inactivation is not due to the action of the antiserum and complement on the CE cell cultures used for virus assay, nor to anti-Forssman antibodies, but it is presumably due to antibodies to some antigen(s) common to the surface of CE cells and to the virus envelope. This host cell surface antigen (HSCA) is also present on the surface of the helper viruses RAV1 and RAV2 of Bryan strain Rous sacroma virus. However, it cannot be said whether it is identical for viruses of all subgroups. A parallel electron microscopical study has revealed a characteristic swelling and loss of opacity to electrons of virus particles treated with the antiserum and complement, which appears to precede virolysis. Avian sarcoma viruses are not inactivated, in the presence or absence of complement, by antiserum to BHK21 hamster cells transformed by RSV and carrying virus-induced surface antigen (VISA). Therefore, the virus particles do not carry any surface antigen common to transformed non-permissive and permissive cells.

Expression of the Major Viral Glycoprotein of Avian Tumor Virus in Cells of chf(+) Chicken Embryos

The expression of gp85, the major viral glycoprotein of avian tumor virus, by certain chicken embryonic cells was studied by the use of sera directed to antigenic determinants of subgroup E viral gp85. As analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immune precipitates prepared with lysates of cells that had been labeled with [(3)H]glucosamine, chf(+) chicken embryo cells synthesize molecules of gp85 which possess type and probably also group antigenic specificities. Under similar conditions of analysis, no gp85 or antigenically related components could be detected in lysates of chf(-) cells.

Virus envelope markers in mammalian tropism of avian RNA tumor viruses

Pseudotypes of vesicular stomatitis virus were prepared with avian sarcoma viruses and avian leukemia viruses representing five different subgroups. These pseudotypes display a host range restricted to that of the avian tumor virus when assayed on avian cells and are neutralized by subgroup-specific antisera. The efficiency of penetration of mammalian cells was assayed by using these vesicular stomatitis virus pseudotypes. Pseudotypes of avian tumor viruses belonging to subgroup D and of B77 virus were able to plate on mammalian cells with a high efficiency, whereas pseudotypes of other strains were not. The efficiency of penetration of the vesicular stomatitis virus pseudotypes was 10-2-to 10-3-fold higher than the efficiency of transformation of the corresponding avian tumor virus strain assayed on mammalian cells, suggesting that there are postpenetration blocks to the expression of transformation in these cells.

Polypeptide and RNA Composition of the Reticuloendotheliosis Viruses

The RNA and polypeptide composition of chick syncytial virus (CSV) and duck infectious anemia virus (DIAV) was investigated and compared to that of reticuloendotheliosis virus (REV) strain T, the prototype of the newly recognized REV group of viruses. CSV and DIAV contain genomic RNA species which cosediment with those of REV in sucrose gradients. Five or six polypeptides, two of which are glycoproteins, were consistently found in CSV and DIAV preparations. The major nonglycosylated polypeptides and glycoproteins of CSV and DIAV comigrated with the corresponding polypeptides of REV strain T. Since the genomic RNA species and the glycoproteins of avian tumor viruses fail to comigrate, this suggests that the REV complex is a more homogeneous group.

Localization of the Major Group-Specific Protein (p27) of Avian Tumor Viruses by Immunofluorescence in Chicken Cells and Tissues

An immunofluorescence technique was developed for the major group-specific (gs) p27 antigen of avian type C viruses. The localization of this antigen virus-infected in chick embryo fibroblasts was perinuclear, intracytoplasmic and at the cell surface in the majority of the cells, while it was at the cell surface only in some of the cells. No antigen was found in the nucleus. When chickens were experimentally infected with RAV-1 or a wild-strain avian leukosis virus (of subgroup A), the viral gs antigen was detected in lymphocytes of bursa of Fabricius and the spleen. Distinct specific staining was seen in the medulla of germinal centers in bursas of Fabricius and in the white pulp of the spleen where B lymphocytes are thought to be located. This is consistent with the possibility that B lymphocytes are the target cells in the infection of chickens with avian leukemia viruses.

Studies on the reverse transcriptase of RNA tumor viruses. Structural relatedness of two subunits of avian RNA tumor viruses.

The structural relationship of the small (alpha) and large (beta) subunits of reverse transcriptase isolated from two avian RNA tumor viruses has been examined by tryptic peptide analysis. Comparison of the tryptic hydrolysates of the isolated subunits by two-dimensional separation on thin-layer cellulose plates indicates that (i) the alpha subunit of reverse transcriptase of avian myeloblastosis virus is structurally related to the beta subunit; (ii) the alpha and beta subunits of the enzyme of Rous sarcoma virus also appear to be related; and (iii) there appears to be an extensive amino-acid sequence homology between reverse transcriptases of avian myeloblastosis virus and Rous sarcoma virus. Evidence is also presented that both alpha and beta subunits can be identified in purified avian myeloblastosis virions.

Avian tumor virus proteins and RNA in uninfected chicken embryo cells.

The content of proteins P19 and P15 (mol wt 19,000 and 15,000, respectively) of avian leukovirus in various types of uninfected chicken embryos has been determined by radioimmunoassay. All chicken embryos examined, including embryos which have thus far been classified as group specific (gs) antigen negative by complement fixation tests, contained these viral proteins as well as P27 as previously reported. The embryos known as "gs antigen-positive" type contained about five times as much of these viral proteins as did the "gs antigen-negative" type. The ratio of the three viral proteins was similar for all types of embryos, suggesting that the genes for these proteins are coordinately controlled. In contrast to the relatively high levels of viral internal proteins in gs antigen-negative cells, the amounts of virus-specific RNA detectable by molecular hybridization were extremely low. The levels of helper activity, which presumably reflect the level of viral envelope glycoprotein, were also generally low or undetectable in these cells. Thus, the expression of the gene for envelope glycoprotein does not appear to be controlled coordinately with the genes for viral internal proteins.

Cell surface antigen induced by avian tumor viruses in hamster cells transformed by a temperature‐sensitive mutant of rous sarcoma virus

AbstractHamster cells (BHK21/13) transformed at the permissive temperature by a temperature‐sensitive mutant (FU 19) of the Schmidt‐Ruppin strain of Rous sarcoma virus exhibited a temperature‐dependent expression of virus‐induced surface antigen (VISA) as assayed by mixed haemadsorption test. This temperature‐dependence of VISA was not observed in cells transformed by the wild‐type virus (SR4), although the number of positive cells was reduced at the restrictive temperature (41° C) when compared to cells incubated at the permissive temperature (37° C). The relations between cell transformation and expression of VISA are discussed.

Evidence for Crossing-Over Between Avian Tumor Viruses Based on Analysis of Viral RNAs

The RNAs of several avian tumor virus recombinants that had inherited their focus-forming ability from a sarcoma virus and their host range marker from a leukosis virus were investigated. Electrophoretic analyses showed that the cloned sarcoma virus recombinants contained only size class a RNA, although they had acquired a marker that resided on class b RNA in the leukosis virus parent. Class a RNA of different recombinant clones, derived from the same pair of parental viruses and selected for the same biological markers, differed slightly in electrophoretic mobility from each other and from the parental sarcoma virus. They were also found to have different fingerprints of RNase T1-resistant oligonucleotides. The average complexity of the 60-70S RNA prepared from Prague Rous sarcoma virus of subgroup B was estimated to be 3.5 x 10(6) daltons from the size of 20 RNase T1-resistant oligonucleotides, which represented 3.9% of the RNA and that of a recombinant to be 3.3 x 10(6) daltons from 23 oligonucleotides, which represented 4.7% of the RNA. This result suggests that the genome of wild-type and of recombinant RNA tumor viruses is polyploid. The sum of these observations led us to propose that recombination among avian tumor viruses occurred by crossing-over between homologous pieces of nucleic acid.