Long terminal repeat (LTR) sequences, env, and a region near the 5'' LTR influence the pathogenic potential of recombinants between Rous-associated virus types 0 and 1.

A series of recombinants between Rous-associated virus type 0 (RAV-0), RAV-1, and a replication-competent avian leukosis virus vector (RCAN) have been tested for disease potential in day-old inoculated K28 chicks. RAV-0 is a benign virus, whereas RAV-1 and RCAN induce lymphoma and a low incidence of a variety of other neoplasms. The results of the oncogenicity tests indicate that (i) the long terminal repeat regions of RAV-1 and RCAN play a major role in disease potential, (ii) subgroup A envelope glycoproteins are associated with a two- to fourfold higher incidence of lymphoma than subgroup E glycoproteins, and (iii) certain combinations of 5' viral and env sequences cause osteopetrosis in a highly context-dependent manner. Long terminal repeat and env sequences appeared to influence lymphomogenic potential by determining the extent of bursal infection within the first 2 to 3 weeks of life. This would suggest that bursal but not postbursal stem cells are targets for avian leukosis virus-induced lymphomogenesis. The induction of neutralizing antibody had no obvious influence on the incidence of lymphoma.     

Sequence of the Long Terminal Repeat and Adjacent Segments of the Endogenous Avian Virus Rous-Associated Virus 0

Rous-associated virus 0 (RAV-0), an endogenous chicken virus, does not cause disease when inoculated into susceptible domestic chickens. An infectious unintegrated circular RAV-0 DNA was molecularly cloned, and the sequence of the long terminal repeat (LTR) and adjacent segments was determined. The sequence of the LTR was found to be very similar to that of replication-defective endogenous virus EV-1. Like the EV-1 LTR, the RAV-0 LTR is smaller (278 base pairs instead of 330) than the LTRs of the oncogenic members of the avian sarcoma virus-avian leukosis virus group. There is, however, significant homology. The most striking differences are in the U(3) region of the LTR, and in this region there are a series of small segments present in the oncogenic viruses which are absent in RAV-0. These differences in the U(3) region of the LTR could account for the differences in the oncogenic potential of RAV-0 and the avian leukosis viruses. I also compared the regions adjacent to the RAV-0 LTR with the available avian sarcoma virus sequences. A segment of approximately 200 bases to the right of the LTR (toward gag) is almost identical in RAV-0 and the Prague C strain of Rous sarcoma virus. The segment of RAV-0 which lies between the end of the env gene and U(3) is approximately 190 bases in length. Essentially this entire segment is present between env and src in the Schmidt-Ruppin A strain of Rous sarcoma virus. Most of this segment is also present between env and src in Prague C; however, in Prague C there is an apparent deletion of 40 bases in the region adjacent to env. In Schmidt-Ruppin A, but not in Prague C, about half of this segment is also present between src and the LTR. This arrangement has implications for the mechanism by which src was acquired. The region which encoded the gp37 portion of env appears to be very similar in RAV-0 and the Rous sarcoma viruses. However, differences at the very end of env imply that the carboxy termini of RAV-0, Schmidt-Ruppin A, and Prague C gp37s are significantly different. The implications of these observations are considered.     

Sequence comparison in the crossover region of an oncogenic avian retrovirus recombinant and its nononcogenic parent: genetic regions that control growth rate and oncogenic potential.

NTRE 7 is an avian retrovirus recombinant of the endogenous nononcogenic Rous-associated virus-0 (RAV-0) and the oncogenic, exogenous, transformation-defective (td) Prague strain of Rous sarcoma virus B (td-PrRSV-B). Oligonucleotide mapping had shown that the recombinant virus is indistinguishable from its RAV-0 parent except for the 3'-end sequences, which were derived from td-PrRSV-B. However, the virus exhibits properties which are typical of an exogenous virus: it grows to high titers in tissue culture, and it is oncogenic in vivo. To accurately define the genetic region responsible for these properties, we determined the nucleotide sequences of the recombinant and its RAV-0 parent by using molecular clones of their DNA. These were compared with sequences already available for PrRSV-C, a virus closely related to the exogenous parent td-PrRSV-B. The results suggested that the crossover event which generated NTRE 7 took place in a region -501 to -401 nucleotides from the 3' end of the td-PrRSV parental genome and that sequences to the right of the recombination region were responsible for its growth properties and oncogenic potential. These sequences included a 148-base-pair exogenous-virus-specific region that was absent from the RAV-0 genome and the U3 region of the long terminal repeat. Since the exogenous-virus-specific sequences are expected to be missing from transformation-defective mutants of the Schmidt-Ruppin strain of RSV, which, like other exogenous viruses, grow to high titers in tissue culture and are oncogenic in vivo, we concluded that the growth properties and oncogenic potential of the exogenous viruses are determined by sequences in the U3 region of the long terminal repeat. However, we propose that the exogenous-virus-specific region may play a role in determining the oncogenic spectrum of a given oncogenic virus.     

Induction of neoplasms by subgroup E recombinants of exogenous and endogenous avian retroviruses (Rous-associated virus type 60).

Chickens susceptible to infection with subgroup E viruses were inoculated with four independent isolates of Rous-associated virus type 60 (RAV-60) that are subgroup e recombinants of endogenous and exogenous virus. Neoplasms developed in each inoculated group. Therefore, nontransforming viruses of subgroup E can induce lymphoid leukosis at a moderate rate compared with RAV-0, a subgroup E endogenous virus, suggesting that oncogenicity is not a viral envelope (env)-related characteristic. Since the common (c) regions of the RAV-60s examined were of exogenous origin, we suggest that the c region rather than env is important for a high rate of induction of lymphoid leukosis and related neoplasms.     

Selective shedding and congenital transmission of endogenous avian leukosis viruses.

Shedding and congenital transmission of endogenous avian leukosis viruses were studied in viremic White Leghorn hens exogenously infected with viruses with endogenous long terminal repeats (LTRs) and in four semicongenic lines of hens that naturally express infectious endogenous viruses (EVs). Relatively high titers of infectious virus EV7 (encoded at locus ev7), Rous-associated virus-0 (RAV-0), and recombinant 882/-16 RAV-0 were detected in blood cells and sera from exogenously infected hens, but marked differences were noted in the incidence of congenitally infected progeny. In enzyme immunoassays that detect viral group-specific antigen, little or no p27 was detected in albumens from dams infected with RAV-0. However, hatchmates infected with either EV7 or recombinant 882/-16 RAV-0, which was constructed with an RAV-0 LTR, shed high titers of p27. Similarly, semicongenic hens that expressed RAV-0 (EV2) (encoded at locus ev2) shed little or no p27 into albumens, but hens that harbored ev10, ev11, and ev12 shed high titers of p27. A slower electrophoretic mobility of p27, considered to be characteristic of EVs that are restricted in congenital transmission, was not associated with low levels of shedding or congenital transmission; p27 from other EVs and p27 from an avian leukosis virus field strain, all of which are shed at high levels, had mobilities identical to that of p27 from RAV-0. Although shedding and congenital transmission appear to be controlled by the viral genome, there was no correlation between low efficiency of shedding or congenital transmission and endogenous LTR or p27 sequences.     

Embryonic infection with the endogenous avian leukosis virus Rous-associated virus-0 alters responses to exogenous avian leukosis virus infection.

We inoculated susceptible chicken embryos with the endogenous avian leukosis virus Rous-associated virus-0 (RAV-0) on day 6 of incubation. At 1 week after hatching, RAV-0-infected and control chickens were inoculated with either RAV-1 or RAV-2, exogenous viruses belonging to subgroups A and B, respectively. The chickens injected with RAV-0 as embryos remained viremic with exogenous virus longer and either failed to develop type-specific humoral immunity to exogenous virus or developed it later than the control chickens not inoculated with RAV-0. The RAV-0-injected chickens also developed neoplasms at a much higher frequency than did the control chickens. We suggest that the lower immune responses of the RAV-0-injected chickens were due to an immunological tolerance to envelope group-specific glycoproteins shared among endogenous and exogenous viruses.     

Enhancer activity correlates with the oncogenic potential of avian retroviruses.

Avian retroviruses lacking an oncogene, such as Rous-associated virus 1 (RAV-1), RAV-2, and td mutants of Rous sarcoma virus (RSV), can nevertheless cause leukemias and other neoplastic diseases. During this process, viral DNA integrates near a cellular proto-oncogene, such as c-myc, and thus de-regulates its expression. The virus RAV-0, on the other hand, is known to be non-oncogenic even in long-term in vivo infections of domestic chickens. The major difference between oncogenic and non-oncogenic viruses is found within the U3 region of the long terminal repeat (LTR) which is known to harbor the promoter and enhancer elements. We therefore wanted to see whether viral oncogenicity was correlated with enhancer activity. Using a variety of techniques (including the SV40 'enhancer trap' from which we obtained RSV-SV40 recombinant viruses), we demonstrate that a strong enhancer exists within the LTRs of both RSV and RAV-1. In contrast, no enhancer is present in RAV-0, although RAV-0 has functional promoter elements. Our data therefore strongly support a concept of oncogenesis by enhancer insertion.     

Genetic determinants of neoplastic diseases induced by a subgroup F avian leukosis virus.

Two subgroup F avian leukosis viruses, ring-necked pheasant virus (RPV) and RAV-61, were previously shown to induce a high incidence of a fatal proliferative disorder in the lungs of infected chickens. These lung lesions, termed angiosarcomas, appear rapidly (4 to 5 weeks after infection), show no evidence of proto-oncogene activation by proviral integration, and are not induced by avian leukosis viruses belonging to other subgroups. To identify the viral sequences responsible for induction of these tumors, we constructed recombinant viruses by exchanging genomic segments of molecularly cloned RPV with those of a subgroup A leukosis virus, UR2AV. The ability to induce rapid lung tumors segregated only with the env sequences of RPV; the long terminal repeat of RPV was not required. However, recombinants carrying both env and long terminal repeat sequences of RPV induced lung tumors with a shorter latency. In several cases, recombinant viruses exhibited pathogenic properties differing from those of either parental virus. Recombinants carrying the gag-pol region of RPV and the env gene of UR2AV induced a high incidence of a muscle lesion termed infiltrative intramuscular fibromatosis. One recombinant, EU-8, which carries the gag-pol and LTR sequences of RPV, and the env gene of UR2AV, induced lymphoid leukosis after an unusually short latent period. The median time of death from lymphoid leukosis was 6 to 7 weeks after infection with EU-8 compared with approximately 5 months for UR2AV.     

Common mechanism of retrovirus activation and transduction of c-mil and c-Rmil in chicken neuroretina cells infected with Rous-associated virus type 1.

We previously described the isolation of the IC10 retrovirus which transduced the v-Rmil oncogene, a new member of the mil/raf gene family. This virus was generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina (NR) cells and was selected for its ability to induce proliferation of these nondividing cells. IC10 was isolated after six passages of culture supernatants but was not detected in proliferating NR cells during early virus passages. In this study, we molecularly cloned and sequenced another v-Rmil-containing provirus, designated IC11, from NR cells infected at the third virus passage of the same experiment. Both IC11 and IC10 transduced only the serine/threonine kinase domain of c-Rmil. Comparison of v-Rmil and c-Rmil sequences indicated that amino-terminal truncation is sufficient to activate the mitogenic properties of c-Rmil. IC11 and IC10 have identical 3' ends but differ by their 5' RAV-1-Rmil junctions. The 3' ends of both viruses were generated by recombination between Rmil and env genes, involving partial sequence identity. The 5' RAV-1-Rmil junction of IC11 was formed by a splicing process between the RAV-1 leader and a 37-bp c-Rmil exon located upstream of the kinase domain. NR cells infected with this virus synthesize a unique Rmil protein. IC10 contains most of the gag gene recombined with v-Rmil and encodes a gag-Rmil hybrid protein. Serial passaging of IC11 in NR cells led to the formation of a gag-Rmil-containing retrovirus. These results indicate that IC11 represents an early step in transduction and that this virus further recombined with RAV-1 to generate IC10. They confirm our previously proposed model for the multistep generation of v-mil-transducing retroviruses. Therefore, activation and transduction of c-mil and c-Rmil, in NR cells infected with RAV-1, result from a common mechanism.     

Recombinants between endogenous and exogenous avian tumor viruses: role of the C region and other portions of the genome in the control of replication and transformation.

Endogenous retroviruses of chickens are closely related to exogenous viruses isolated from spontaneous tumors in the same species, yet differ in a number of important characteristics, including the ability to transform cells in culture, ability to cause sarcomas or leukemias, host range, and growth rate in cell culture. To correlate these differences with specific sequence differences between the two viral genomes, the genome RNA of transforming subgroup E recombinants between the Prague strain of Rous sarcoma virus, subgroup B (Pr-RSV-B), and the endogenous Rous-associated virus-0 (RAV-0), Subgroup E, and seven nontransforming subgroup E recombinants between the transformation-defective mutant of Pr-RSV-B and RAV-0 was examined by oligonucleotide fingerprinting. The pattern of inheritance among the recombinant viruses of regions of the genome in which Pr-RSV-B and RAV-0 differ allowed us to draw the following conclusions. (i) Nonselected parts of the genome were, with a few exceptions, inherited by the recombinant virus progeny randomly from either parent, with no obvious linkage between neighboring sequences. (ii) A small region in the Pr-RSV-B genome which maps in the 5' region was found in all transforming but only some of the nontransforming recombinants, suggesting that it plays a role in the control of the expression of transformation. (iii) A region of the Pr-RSV-B genome which maps between env and src was similarly linked to the src gene and may be either part of the structural gene for src or a control sequence regulating the expression of src. (iv) The C region at the extreme 3' end of the virus genome which is closely related in all the exogenous avian retroviruses but distinctly different in the endogenous viruses is the major determinant responsible for the differences in growth rate between RAV-0 and Pr-RSV-B. This latter observation allowed us to redefine the C region as a genetic locus, c, with two alleles cn (in RAV-0) and cx (in exogenous viruses).     

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.     

Component of Strain MC29 Avian Leukosis Virus with the Property of Defectiveness

Three clones of morphologically altered cells (L(-)MC29) of singular properties were isolated from MC29 (subgroup A) leukosis virus-infected chick embryo cells. Supernatant fluids from cultures of the cloned cells produced no transforming or interfering activity on chick embryo cells susceptible to known avian leukosis-sarcoma viruses. No virus associated with the cells was demonstrable by fluorescent-antibody staining or by electron microscopy. All L(-)MC29 clone cells were activated, however, by four strains of Rous-associated viruses (RAV) representative of A, B, C, and D subgroup avian leukosis viruses and by two strains of MC29 virus. Virus L(-)MC29 cells activated by superinfection with RAV-1 and RAV-2 was characterized by helper-dependent and helper-independent properties. These findings suggest that the strain MC29 leukosis virus, or a component thereof, possesses properties of defectiveness similar to those of the Bryan high-titer Rous sarcoma virus.     

Avian acute leukemia virus OK 10: analysis of its myc oncogene by molecular cloning.

Several DNAs representing the genome of the avian acute leukemia virus OK 10 were isolated by molecular cloning from a transformed quail cell line, 9C, which contained at least six OK 10 proviruses. Recombinant lambda phages harboring the OK 10 genome and additional flanking cellular DNA sequences were studied by restriction endonuclease mapping and hybridization to viral cDNA probes. Six of the clones represented complete proviruses with similar, if not identical, viral sequences integrated at different positions in the host DNA. The organization of the OK 10 genome was determined by electron-microscopic analysis of heteroduplexes formed between the cloned OK 10 DNA and DNAs representing the c-myc gene and the genomes of two other avian retroviruses, Rous-associated virus-1 and MC29. The results indicated that the OK 10 proviral DNA is about 7.5 kilobases in size with the following structure: 5'-LTR-gag-delta polmyc-delta env-LTR-3', where LTR indicates a long terminal repeat. The oncogene of OK 10, v-mycOK 10, forms a continuous DNA segment of around 1.7 kilobases between pol and env. It is similar in structure and length to the v-myc gene of MC29, as demonstrated by restriction endonuclease and heteroduplex analyses. Two of the OK 10 proviruses were tested in transfection experiments: both DNAs gave rise to virus with the transforming capacities of OK 10 when Rous-associated virus-1 was used to provide helper virus functions.     

Structural protein markers in the avian oncoviruses.

The proteins of purified avian oncoviruses were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing. Certain members of the avian leukosis-sarcoma viruses (ALSV) had group-specific antigens with altered electrophoretic properties. (i) The p27 protein of Rous-associated virus 0 (RAV-0) had a lower electrophoretic mobility in SDS gels and a lower isoelectric point than the p27 of other ALSV. (ii) The p19 proteins of RAV-1, RAV-2, and the Bryan high-titer strain of Rous sarcoma virus had higher mobilities in SDS gels than did the corresponding protein of other viruses. This altered electrophoretic mobility was correlated with specific differences in the tryptic peptides of radioiodinated p19s. (iii) The p15 protein of RAV-7 had a lower mobility in SDS gels than did the p15 of other ALSV. These markers were used in a study of the structural proteins of subgroup E RAV-60 produced after infection of chicken embryo cells by exogenous ALSV. Although exogenous group-specific protein markers could often be identified in the subgroup E isolates, one RAV-60 had a p27 that comigrated with the p27 of RAV-0. The p19s of two other RAV-60 isolates had electrophoretic properties that were different than those of p19s from either RAV-0 or the exogenous viruses. These results support the hypothesis that RAV-60 is generated by recombination between endogenous and exogenous oncoviruses and indicate that at least the p27 encoded by RAV-0 is closely related to a protein specified by endogenous viral information in chicken cells.     

Molecular and biological properties of c-mil transducing retroviruses generated during passage of Rous-associated virus type 1 in chicken neuroretina cells.

IC1, IC2, and IC3 are novel c-mil transducing retroviruses generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina cells. They were isolated by their ability to induce proliferation of these nondividing cells. IC2 and IC3 were generated during early passages of RAV-1 in neuroretina cells, whereas IC1 was isolated after six consecutive passages of virus supernatants. We sequenced the transduced genes and the mil-RAV-1 junctions of the three viruses. The 5' RAV-1-mil junction of IC2 and IC3 was formed by a splicing process between the RAV-1 leader sequence and exon 8 of the c-mil gene. The 5' end of IC1 resulted from homologous recombination between gag and mil sequences. Reconstitution experiments showed that serial passaging of IC2 in neuroretina cells also led to the formation of a gag-mil-containing retrovirus. Therefore, constitution of a U5-leader-delta c-mil-delta RAV-1-U3 virus represents early steps in c-mil transduction by RAV-1. This virus further recombined with RAV-1 to generate a gag-mil-containing virus. The three IC viruses transduced the serine/threonine kinase domain of the cellular gene. Hence, amino-terminal truncation is sufficient to activate the mitogenic property of c-mil. Comparison of the transforming properties of IC2 and IC1 showed that the transduced mil gene, expressed as a unique protein independent of gag sequences, was weakly transforming in avian cells. Acquisition of gag sequences by IC1 not only increased the rate of virus replication but also enhanced the transforming capacity of the virus.     

Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression of the mouse mammary tumor virus env gene.

To extend our understanding of the organization and expression of the mouse mammary tumor virus genome, we determined the nucleotide sequence of large regions of a cloned mouse mammary tumor virus strain C3H provirus that appears to be a DNA copy of env mRNA. In conjunction with analysis of several additional clones of integrated and unintegrated mouse mammary tumor virus DNAs, we came to the following conclusions: (i) the mRNA for env is generated by splicing mechanisms that recognize conventional eucaryotic signals at donor and acceptor sites with a leader of at least 289 bases in length; (ii) the first of three possible initiation codons for translation of env follows the splice junction by a single nucleotide and produces a signal peptide of 98 amino acids; (iii) the amino terminal sequence of the major virion glycoprotein gp52env is confirmed by nucleotide sequencing and is encoded by a sequence beginning 584 nucleotides from the 5' end of env mRNA; (iv) the final 17 amino acids at the carboxyl terminus of the primary product of env are encoded within the long terminal repeat by the 51 bases at the 5' end of the U3 domain; and (v) bases 2 through 4 at the 5' end of the long terminal repeat constitute an initiation codon that commences an open reading frame capable of directing the synthesis of a 36-kilodalton protein.     

Nucleotide Sequence Relationships of Avian RNA Tumor Viruses: Measurement of the Deletion in a Transformation-Defective Mutant of Rous Sarcoma Virus

Stocks of cloned helper-independent Rous sarcoma virus (RSV) spontaneously segregate transformation-defective (td) mutants that appear to have an RNA genome composed of smaller subunits than those of the patent virus. Differential hybridization and competitive hybridization techniques involving reactions between viral RNA and proviral sequences in host cell DNA (under conditions of initial DNA excess) were used to measure the extent of the deletion in a td mutant of Prague strain (Pr) of RSV (Pr RSV-C). Viral 60 to 70S RNA sequences labeled to 1 to 5 x 10(7) counts per min per mug with (125)I were characterized with respect to their properties in hybridization reactions and used to reinforce data obtained with [(3)H]RNA of lower specific activity. By these techniques, about 13% +/- 3% of the sequences Pr RSV-C that formed hybrids with DNA from virus-induced sarcomas appeared to be deleted from the genome of td Pr RSV-C. Studies comparing hybridization of RNA from Pr RSV-C and td Pr RSV-C with RSV-related sequences in normal cells, and competition experiments with RNA from the endogenous chicken oncornavirus Rous-associated virus type 0 (RAV-0) provided evidence that the majority, if not all, of the RNA sequences of Pr RSV-C deleted from its transformation-defective mutant are not represented in normal chicken DNA. Competition studies with a leukosis virus, RAV-7, indicated this virus also lacks a genome segment of about the same size as the deletion in the td mutant. Finally, the genome of all three "exogenous" viruses was found to lack a small segment (about 12%) of sequences present in the endogenous provirus of RAV-O.     

Nucleotide Sequence of the Long Terminal Repeat and Flanking Cellular Sequences of Avian Endogenous Retrovirus ev-2: Variation in Rous-Associated Virus-0 Expression Cannot Be Explained by Differences in Primary Sequence

A fragment of chicken DNA containing the left long terminal repeat of endogenous retrovirus ev-2 and flanking cellular sequences has been molecularly cloned and analyzed. Comparison with sequence data from the analogous regions of ev-1 and Rous-associated virus-0 viral DNA reveals similarities among flanking regions of the integrated proviruses and among all three long terminal repeats. From the latter finding, we conclude that the difference in level of expression of ev-2 and its progeny Rous-associated virus-0 provirus cannot be due to sequence differences in their upstream long terminal repeats.     

Nucleotide Sequences Derived from Pheasant DNA in the Genome of Recombinant Avian Leukosis Viruses with Subgroup F Specificity

Recombination between viral and cellular genes can give rise to new strains of retroviruses. For example, Rous-associated virus 61 (RAV-61) is a recombinant between the Bryan high-titer strain of Rous sarcoma virus (RSV) and normal pheasant DNA. Nucleic acid hybridization techniques were used to study the genome of RAV-61 and another RAV with subgroup F specificity (RAV-F) obtained by passage of RSV-RAV-0 in cells from a ring-necked pheasant embryo. The nucleotide sequences acquired by these two independent isolates of RAV-F that were not shared with the parental virus comprised 20 to 25% of the RAV-F genomes and were indistinguishable by nucleic acid hybridization. (In addition, RAV-F genomes had another set of nucleotide sequences that were homologous to some pheasant nucleotide sequences and also were present in the parental viruses.) A specific complementary DNA, containing only nucleotide sequences complementary to those acquired by RAV-61 through recombination, was prepared. These nucleotide sequences were pheasant derived and were not present in the genomes of reticuloendotheliosis viruses, pheasant viruses, and avian leukosis-sarcoma viruses of subgroups A, B, C, D, and E. They were partially endogenous, however, to avian DNA other than pheasant. The fraction of these nucleotide sequences present in other avian DNAs generally paralleled the genetic relatedness of these avian species to pheasants. However, there was a high degree of homology between these pheasant nucleotide sequences and related nucleotide sequences in the DNA of normal chickens as indicated by the identical melting profiles of the respective hybrids.