Search

Avian leukosis virus subgroup J has a high tropism for myeloid lineage cells and frequently induces neoplastic transformation of myelocytes. The impact of congenital avian leukosis virus subgroup J infection on the function of circulating heterophils and susceptibility to staphylococcal infection was investigated. Six-week-old broiler chickens negative for exogenous avian leukosis viruses or congenitally infected with avian leukosis virus subgroup J were inoculated intravenously with 10(6) colony-forming units of Staphylococcus aureus, and pre- and postinoculation heterophil function was assessed. All chickens developed a leukocytosis with heterophilia after inoculation, but total leukocyte and heterophil counts were significantly higher in leukosis-negative chickens than in virus-infected chickens. Tenosynovitis was more severe in leukosis-negative chickens, and 2/10 (20%) of the virus-infected chickens had no histologic evidence of tenosynovitis. Osteomyelitis in the tibiotarsus or tarsometatarsus developed in 5/10 (50%) of the chickens in each group. S. aureus was recovered from the hock joint of 6/10 (60%) of the chickens in each group. Heterophils from all chickens exhibited similar phagocytic ability pre- and postinoculation. Heterophils from virus-infected chickens exhibited less bactericidal ability preinoculation than did heterophils from leukosis-negative chickens. However, postinoculation bactericidal ability was similar in both groups. Avian leukosis virus subgroup J provirus was present in heterophils isolated from congenitally infected chickens. Heterophils isolated from broiler chickens congenitally infected with avian leukosis virus subgroup J exhibit no significant functional deficits, and infected and uninfected chickens exhibit similar susceptibility to staphylococcal infection.

Congenital transmission of avian leukosis viruses (ALV) occurs readily through the egg, but transmission of ALV through male seminal fluid is considered to be nonexistent or rare. Progeny from mating endogenous late-feathering (LF), K/k+ males carrying an endogenous virus gene (ev21) with virgin early-feathering (EF) k+/w females were examined for the presence of infectious endogenous virus EV21 using an enzyme-labeled immunoassay for viral capsid antigen p27. All 177 LF chicks expressed EV21, p27, and 171 of 175 EF chicks did not express p27. Blood from the four p27-positive EF chicks revealed only infectious Subgroup E ALV as determined by subgroup-specific virus assays. Southern blot DNA hybridizations, however, ruled out germline integration of EV21 among the four infected EF progeny. Virus EV21 was not shed in albumens of the dams. Moreover, antibodies against ALV Subgroups A and E were not detected in dams 17 wk after the first insemination. Chicks infected with EV21 were found only in the first two of six hatches. Data suggested direct infection of the embryos from viremic semen rather than congenital infection through infected hens. Direct male transmission of EV21 to progeny may be the basis for persistence of refractory lines noted in some ALV eradication programs. Based on the absence of recombinants among 352 progeny, ev21 and K appear to be less than .3 cM apart.

The 60 S viral RNA complex isolated from leukaemic plasma of chicken infected by avian myeloblastosis virus (AMV) was denatured, the poly(A)-RNA selected and centrifuged in a linear sucrose density gradient. RNA from each fraction was translated in vitro and the products were analyzed by slab polyacrylamide gel electrophoresis (PAGE). Unprocessed primary translation product (p64env) of MAV env gene from 21 S RNA fraction was immunoprecipitated by anti-gp85 serum. If, however, this RNA was translated in the presence of dog pancreas microsomal membranes (DPM), the processed 92 K MAV glycoprotein precursor (p92env) was immunoprecipitated by anti-gp85 serum. This precursor, unlike p64env was resistant to exogenous protease.

We previously reported that serial passaging of Rous-associated virus type 1 in nondividing chicken embryo neuroretina cells leads to reproducible generation of acutely mitogenic retroviruses that transduced the catalytic domain of c-mil/c-raf or c-Rmil/B-raf. On the basis of structural analysis of several retroviruses, we proposed that the early step of oncogene transduction is the constitution of alternatively spliced leader-delta onc-poly(A) transcripts. Here, we show that neuroretina cells do synthesize hybrid leader-delta mil and leader-delta Rmil RNAs and that these RNAs exhibit mitogenic properties and serve as templates for the generation of transducing retorviruses.

Structural properties and length distribution profile of 7 S avian myeloblastosis virus (AMV) DNA were studied by means of electron microscopy using two different techniques. This DNA represents mostly double strands, the single strands being in minority. We have shown directly that this DNA forms a bent structure typical of the majority of molecules. These bends are sensitive to the distamycin treatment which stretches most of the bent molecules. Some amount (up to 30%) of circular DNA molecules was detected also in DNA preparations, the nature and the size of which are reminiscent of electron microscopic data on microbubbles of replicating DNA. No specific AMV DNA structural features were found using osmium-tetroxide treatment. The basic size of AMV DNA was estimated to be approximately 150 bp, but its multimers were also detected. Their presence and significance is discussed.

We previously demonstrated that Avian Leukemia Viruses (ALV) carrying the v-myc gene specifically induce two types of tumors, cardiomyocytic tumors when the virus is injected before embryonic day 3 (E3), skin tumors when the virus is injected at E3 or E5. Aiming to elucidate the mechanisms which determine this time-dependent change in target, we infected chick and quail embryos at E3 and E5 with replication-deficient, lacZ gene-carrying, ALV-based viruses produced by a packaging cell line. Three constructs driven by 3 different Long Terminal Repeats (LTRs) were tested and yielded similar results. When the constructs were inoculated at E3 and the lacZ gene product revealed 5 days later, around 70% of the embryos carried lacZ+ clones in the heart, around 50% had positive clones in the skin anywhere on the body, while a few embryos displayed clones in internal organs (liver, stomach, lungs). Immunocytological identification of the heart cell type(s) expressing the virus revealed that the only cells infected were cardiomyocytes. When the constructs were inoculated at E5, no lacZ+ clones appeared in the heart but all were located in the cephalic skin. In order to examine the relationship between viral integration and expression, DNA of different organs or tissues from lacZ stained embryos was analyzed by PCR. A tight correlation between integration and expression in the heart and in the skin was revealed in most cases. In contrast, a significant PCR signal was often detected in the liver or the stomach despite weak or absent expression as revealed by lacZ+ clones. We then investigated the influence of envelope glycoprotein subgroups on the tropism of these constructs. The lacZ vector driven by RAV-2 LTRs was packaged as subgroups A, B or E viral particles. The A subgroup, used in the part of the study described above, infects both chick and quail while the B and E subgroups are specific for chick or quail respectively. These B and E subgroups induced lacZ+ clones in the heart (after E3 injection) while no clones or only a few were detected in the skin either after E3 or E5 injection. The following conclusions can be drawn: 1) cardiomyocytes are at E3 the major target for integration and expression of ALV-derived viruses in vivo; 2) targets change rapidly with embryonic age; and 3) tissue-specific infections depend on the envelope subgroup, thus presumably on the presence of the cognate receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Viruses rapidly able to transform cultured chicken bone marrow cells have been isolated from cases of myelocytic myeloid leukosis (MML) induced experimentally by the HPRS-103 strain of avian leukosis virus, and from field cases of MML. HPRS-103 virus itself did not acutely transform cultured bone-marrow cells. These findings suggest that during myeloid leukemogenesis by HPRS-103 virus, recombinant viruses are generated with transduced cellular oncogenes. The transformed cell appeared to be a macrophage precursor cell. Transformed cells in culture lost their proliferative capacity after a few weeks and then tended to resemble more differentiated macrophages. This change could be reversed temporarily by addition of a myelomonocytic growth factor, cMGF, to the culture medium. In oncogenicity tests, a selection of the virus strains induced MML, nephroblastomas, renal adenomas/adenocarcinomas, and other tumors in line 21 meat-type chickens but not in line 0 chickens. This difference may have been related to a propensity for the virus strains to induce persistent tolerant viremic infections in the line 21 chickens following infection at 1 day of age. The oncogenic pattern was not clearly related to the ability of the viruses to transform cultured bone-marrow cells. The generation of acutely transforming viruses during myeloid leukemogenesis may be relevant to the occurrence of MML in the field.

Analyses of six tissues from Rous-associated virus type 1-infected chickens have revealed a number of tissue-specific differences in virus synthesis and distribution. Chicks were infected at 1 day of age. Tissue (bursa, thymus, liver, kidney, muscle, and brain) were harvested 1 month later. Each of the tissues contained an average of 1.8 to 4 copies of viral DNA per cell. Most of this DNA was integrated. In brain, about one-third of the total viral DNA was in an unintegrated, linear form. Bursa, thymus, liver, and kidney expressed both Gag and Env proteins. In contrast, muscle expressed more Gag than Env, and brain expressed neither Gag nor Env. Tissues that were producing both Gag and Env contained higher levels of mature virus particles than tissues that were not producing both of these proteins.

The HPRS-103 strain of avian leukosis virus (ALV) was isolated recently from meat-type chickens and represents a new envelope subgroup. Its oncogenicity has been studied in three meat-type and five Leghorn strains of chickens. In the meat-type strains, the virus, following embryonal inoculation, induced an overall incidence of 27% myelocytic myeloid leukosis (myelocytomatosis) and 12% renal adenomas, with long median latent periods. Amongst the Leghorn lines, these tumors occurred with similar incidence in line 0, but with lower or zero incidences in the other lines. A variety of other tumours occurred with low incidence. Embryonal infection resulted in a permanently tolerant viraemic state with shedding of ALV group specific (gs)-antigen to egg albumen; contact infection resulted mainly in the development of non-shedder birds with serum virus-neutralising antibodies. Contact infection in a meat-type line was associated with the development of transient or permanent viraemia in some birds, and a low tumour incidence. A viraemic phase was not detected following contact infection in a Leghorn line and no tumours developed. The long latent period between embryo infection and tumour mortality, apparently differing from the consequences of infection with acutely transforming ALVs, and the inability of HPRS-103 ALV to transform cultured bone marrow cells, suggests that this virus may lack a viral oncogene and exert its oncogenic properties by some other mechanism such as promoter insertion activation of a cellular oncogene.

In order to enhance the outcome of high-quality reverse transcriptase enzyme, an efficient biotechnology was developed of accumulating and isolating the avian myeloblastosis virus (AMV) in high titres from blood plasma of leukosis-free chickens. When commercial chickens are infected in most sensitive one-day age, the virus titre does not exceed the value of 10(12) particles per 1 ml of plasma. We used 3-4-day old leukosis free chickens and achieved a stable average titre of the virus of 5.10(12) particles/ml due to adaptation of the virus to such chickens and their selection for a high sensitivity to AMV.

We have previously reported that infection of 9- to 13-day-old chicken embryos with RAV-1 results in rapid development of a novel B-cell lymphoma in which proviral insertion has activated expression of the c-myb gene (E. Pizer and E. H. Humphries, J. Virol. 63:1630-1640, 1989). The biological properties of these B-cell lymphomas are distinct from those associated with the B-cell lymphomas that develop following avian leukosis virus proviral insertion within the c-myc locus. In an extension of this study, more than 200 chickens, infected as 10- to 11-day-old embryos, were examined for development of lymphomas that possess disrupted c-myb loci. Fourteen percent developed disseminated B-cell lymphoma. In the majority of these tumors, the RAV-1 provirus had inserted between the first and second exons that code for p75c-myb. However, insertions between the second and third exons and between the third and fourth exons were also detected. In situ analysis of myb protein expression in tumor tissue revealed morphological features suggesting that the tumor originates in the bursa. Within the bursa, the lymphoma appeared to spread from follicle to follicle without compromising the structural integrity of the organ. Tumor masses in liver demonstrated heterogeneous levels of myb protein suggestive of biologically distinct subpopulations. In contrast to the morbidity data, immunohistological analysis of bursae from 4- to 6-week-old chickens at risk of developing lymphomas bearing altered c-myb loci revealed lesions expressing elevated levels of myb in 16 of 19 birds. The activated myb lymphoma displayed very poor capacity to proliferate outside its original host. Only 1 of 33 in vivo transfers of tumor to recipient hosts established a transplantable tumor. None of the primary tumor tissue nor the transplantable tumor exhibited the capacity for in vitro proliferation. Similar experimental manipulation has yielded in vitro lines established from avian B-cell lymphomas expressing elevated levels of c-myc or v-rel. The dependence on embryonic infection for development of activated-myb lymphoma suggests a requirement for a specific target cell in which c-myb is activated by proviral insertion. It is likely, moreover, that continued tumor development requires elevated expression of myb proteins within a specific cell population in a restricted stage of differentiation.

Infection by exogenous avian leukosis viruses (ALVs) causes economic loss from neoplastic mortality and from impaired performance of subclinically infected chickens. This paper reviews progress in research related to natural infection and its control. Subgroup A ALVs causing lymphoid leukosis are the most common viruses in the field, but variant viruses can arise and cause losses. In Israel in recent years, epidemic outbreaks of haemangiosarcomas caused by a virus of unusual cytopathogenicity have occurred. In the UK, an ALV belonging to a new subgroup for chickens has been recently isolated; this virus is able to cause myeloid leukosis and nephromas. ALV infection of commercial stock is controlled by virus eradication schemes that prevent vertical transmission of ALV from one generation to the next. In this regard, endogenous leukosis viruses and ev loci, notably ev21 linked to the K slow-feathering gene, have been shown to have a detrimental influence on responses to infection by exogenous ALVs, and on the success of eradication schemes. Tolerogenic properties of the ev loci are involved. Attention has also been directed to whether ev loci have any direct influence on performance traits. Although eradication provides the main means of controlling ALV, the development of transgenic techniques in chickens has renewed interest in genetic resistance, and some progress has also been made in developing recombinant vaccines.

The highly leukemogenic avian retrovirus E26 expresses the two transcriptional activator-type oncogenes v-myb and v-ets as a nuclear fusion protein. Previous studies have shown that both oncogenes cooperate in the transformation of erythroid cells in vitro and that the phenotypes of transformed cells differ, depending on whether the oncogenes are coexpressed as separate proteins or as a fusion protein. Here we show that virus constructs encoding either v-Myb or v-Ets as their only oncoprotein are nonleukemogenic and that constructs coexpressing nonfused v-Myb and v-Ets proteins appear to be weakly leukemogenic. Surprisingly, leukemic animals injected with the latter contain highly leukemogenic variant viruses that exhibit internal deletions in their genome, resulting in the synthesis of novel Myb-Ets fusion proteins. These results show that v-Myb and v-Ets must be fused to cause leukemia and establish a new mechanism of oncogene activation and cooperation.

Retroviral vectors carrying either the v-jun and v-erbB sequences or the v-jun gene linked to the neomycin resistance gene were constructed on the basis of the structural genome organization of avian erythroblastosis virus (AEV). These viruses, called JB and JN, respectively, were rescued as Rous-associated virus-1 pseudotypes, and they were shown to successfully transform chicken embryo fibroblasts in vitro. However, in agar, colonies developed from JB-infected fibroblasts were three to five times larger than those obtained after infection with JN or with AEV Pst124 carrying only a functional v-erbB gene. In vivo, on chorioallantoic membrane (CAM) assays, JB produced fibrosarcomas that were more rapidly growing and much larger than those induced by JN or AEV Pst124. Moreover, in chickens infected in ovo with JB, multiple fibrosarcomas arose in different organs a few days after birth, whereas no tumor could be detected in parallel experiments in either JN- or AEV Pst124-infected animals. These results demonstrate that in embryo fibroblast cells, v-jun and v-erbB can act synergistically to enhance the transformation potential of either oncogene alone both in vitro and in vivo.

The influence of the endogenous virus ev6 on congenital transmission of EV21, the infectious viral product encoded by locus ev21, and the immune response to exogenous avian leukosis virus (ALV) infection was studied in rapid-feathering (RF) female progeny from four classes of slow-feathering (SF) (ev21+ and RF (ev21-) dams with and without ev6. Apart from transmitting infectious EV21 and ev6 to progency, dam ev genotype did not influence the immune response or shedding of RPL-40. The endogenous virus envelope glycoprotein encoded by ev6, however, completely restricted shedding and congenital transmission of infectious endogenous virus EV21, from SF dams. After 19 wk of exposure to ALV strain RPL-40 infected cage mates, only 11% of the congenitally infected female progeny mounted neutralizing antibodies against RPL-40, whereas 73% of their noncongenitally infected sisters seroconverted. More ev6+ female progeny, however, were shedders of RPL-40 and developed tumors than ev6- sisters. Among progeny from the four classes of dams, EV21 congenitally infected hens had the highest incidence (31%) of RPL-40-induced tumors.

The ability of an avian retrovirus to cause central nervous system (CNS) disease was investigated in chickens infected in ovo with Rous associated virus-1. Viral envelope and core proteins and mature virions were found throughout CNS parenchyma, with the highest amounts localized in the granular layer of the cerebellum, in blood vessel endothelium, and the choroid plexus. This distribution was established by the time of hatching and persisted throughout the 14 weeks of observation. The highest levels of integrated proviral DNA and viral mRNA, were present in the cerebellum, consistent with the distribution of viral antigens. Mononuclear cell infiltrates were evident throughout the CNS, consistent with an inflammatory process. However, demyelination or vacuolar changes, as observed in other retroviral-induced CNS diseases, were not detected. Clinical symptoms of progressive neurologic dysfunction, i.e., weakness or paralysis of the hindlimbs, imbalance, and ataxia, were present in 7 of 38 infected chickens before termination of the experiment at 14 weeks posthatch. Viral antigens or lymphocyte infiltration were not detected in peripheral nerves. These findings suggest that the avian system may provide a valuable model to analyze the mechanisms governing retroviral induced CNS disease.

Organisms from human to Drosophila have been found to contain cellular sequences and transcripts that are homologous to the ets region of the avian retrovirus, E26. Ets-related sequences are present on at least two distinct functional loci in chickens and mammals, and have been designated ets-1 and ets-2. The E26 virus transduced sequences from the chicken ets-1 locus, which encompasses over 60 kb of DNA. The ets genes characterized so far from sea urchin and Drosophila are most closely related to the 3' end of the known ets genes. The predicted viral and avian ets proteins are very similar, except at the termini. The similarity between the predicted ets proteins so far described is discussed. The ets proteins have been identified and localized by immunoprecipitation and immunofluorescence. While the ets-1 proteins are found in the nuclear and cytoplasmic fractions, the viral gag-myb-ets protein (p135) and the ets-2 proteins are nuclear. The ets-1 and ets-2 genes are differentially regulated in different cell types, probably reflecting unique controlling elements. Because chromosomal translocations have been associated with different human leukemias, studies addressing the possible association with the ETS1 (11q23) or ETS2 (21q22.3) loci are reviewed.

Each of twelve tumors induced by either Rous-associated virus-1 or -2 (RAV-1 or RAV-2) contained a predominant population of cells with ALV proviruses integrated at common sites, consistent with a clonal origin. Seven of nine RAV-2-induced bursal tumors contained single proviruses, and all seven solitary proviruses had suffered deletions. The detailed structures of four of these proviruses show that major deletions had occurred near or at the 5' ends, spanning sequences potentially important in the production of viral RNA. One provirus also lacked most of the information coding for the replicative functions of the virus. Restriction maps suggest that these four proviruses were inserted in similar regions of the host genome. We have studied virus-specific RNA in four bursal tumors and four cell lines derived from bursal tumors. No normal viral RNA species were detectable in three tumors containing single aberrant proviruses. However, transcripts of 2.2. kb which reacted only with a hybridization probe specific for the 5' end of viral RNA were observed in one of these three tumors. Analogous species, varying in length from 1.5 to 6.0 kb, were observed in a fourth bursal tumor with multiple proviruses and in all four cell lines. (This tumor and the cell lines also contained normal species of ALV mRNA and apparently normal proviral DNA). The structures of the aberrant proviruses and the absence of normal viral RNA in some tumors indicate that expression of viral genes is not required for maintenance of the tumor phenotype. In at least some cases, the mechanism of oncogenesis may involve stimulation of transcription of flanking cellular sequences by a viral promoter.

We have isolated a new acute avian transforming virus which contains the oncogene myc. This virus, designated FH3, was isolated after injection of a 10-day-old chick embryo with avian leukosis virus. While FH3 shares many properties with other v-myc-containing avian retroviruses, it also has several unique properties. The primary target for transformation in vitro is chicken macrophages; infection of chicken fibroblasts does not lead to complete morphological transformation. FH3 also exhibits a limited host range, in that Japanese quail macrophages and fibroblasts are infected but are not completely transformed. FH3 induces in vivo a limited tumor type if injected into 10-day-old chick embryos; only a cranial myelocytoma, which does not appear to be metastatic, can be detected. The v-myc gene of FH3 is expressed predominantly as a P145 Gag-Myc protein which is encoded by a ca. 8-kilobase genomic RNA. This FH3-encoded polyprotein is localized in the nucleus of all infected cells, whether or not they are transformed.

Five lines of chickens maintained as specific pathogen-free flocks in Australia were characterized in relation to endogenous antigens and endogenous avian leukosis virus expression. Embryos of line N were predominantly of C/E phenotype, uniformly positive for group-specific antigen and chick helper factor (gs+chf+) and 38% expressed endogenous virus at a very low titre. Embryos of line M4 were uniformly of C/ABE phenotype and were either gs+chf+ or gs-chf+. Line W19 embryos segregated for susceptibility to viruses of subgroup A, B and D and were either of C/E or C/ABE phenotype. The majority of W19 embryos were gs+chf+ with a small proportion being gs+chf-. Line I13 embryos were either of C/0 or C/ABE phenotype, uniformly gs-chf- and 44% of embryos expressed endogenous virus at a low titre. Line S segregated for susceptibility to subgroup E virus and embryos were either of C/E or C/0 phenotype, while the majority of embryos from line S were gs-chf- with some embryos being gs+chf+ or gs-chf+. The degree of interference of gs+chf+ and gs-chf+ phenotypes with subgroup E virus infection was identical with the interference patterns of classical gs+chf+ and gs-chf+ phenotypes. The resistance to infection with avian sarcoma viruses of subgroups E in lines N and M4, and to a degree in line W19, was highly associated with the presence of chf. Resistance to subgroup E virus was independent of chf in lines S and I13, probably being under the control of an independent locus. Cellular restriction of endogenous virus replication existed in all subgroup E virus-susceptible cells of line I13 in contrast to cells of line S which supported replication of endogenous virus. The phenotype of chicken cells for the expression of endogenous gs antigen and chf could accurately be predicted from the test performed on whole blood cells.

A microplate enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to avian leukosis virus (ALV) of subgroups A and B in infected chickens was developed with the use of Rous-associated virus (RAV)-1 (subgroup A) and RAV-2 (subgroup B) antigens purified by sucrose-gradient centrifugation. The antigen was used for ELISA after treatment with Triton X-100. In the ELISA, the subgroup viral antigen reacted strongly with homologous antiserum but also reacted with heterologous antiserum. Tests with serum absorbed with purified homologous and heterologous virus and tests for antigen-blocking by group-specific antibodies to ALV revealed that the reaction was caused mainly by subgroup-specific antibodies. The ELISA was 8 to 32 times more sensitive than the virus-neutralization (VN) test and detected antibodies to ALV earlier than the VN test in chickens infected experimentally with RAV-1 and RAV-2. In field application of the ELISA, 44.2% of 484 chicken sera were positive for RAV-1 and/or RAV-2 antigen, and 80.4% of flocks were positive. These findings indicate that ELISA is superior to the VN test in sensitivity, simplicity, rapidity, and applicability for large-scale field surveys for ALV infection.

The tumours associated to lymphoid leucosis and Marek's disease are compared with lymphoproliferative diseases of man and other species, by morphological, immunomorphological, and aetiological criteria. Morphological and immunomorphological similarities were found to exist between lymphoid leucosis and malignant lymphomas of the B-cell system in man, more particularly, follicular lymphoma, lamphoblastic lymphosarcoma of the B-cell type, Burkitt's lymphoma, and immunoblastic lymphoma. Equivalent similarities with Marek's disease are relating particularly to Hodgkin's disease, infectious mononucleosis, and malignant lymphomas of the T-cell system. Aetiological relations were found to exist between lymphoid leucosis and the oncorna-virus-dependent lymphatic tumours in mammals, while similarities with Marek's disease were relating to infectious mononucleosis, Burkitt's lymphoma, and herpes-virus-dependent lymphatic tumours in monkey. Reference is made to the model nature of both lymphoid leucosis and Marek's disease and the resulting relevance to tumour research.

The presence of avidin, a progesterone-dependent oviductal glycoprotein, was studied in viral tumors. Newborn chickens were infected with the acute leukemia virus OK 10, and the first tumors occurred within 2-3 weeks. Avidin was assayed using a [14C]biotin-binding method and radioimmunoassay. In the control chickens, avidin concentrations were less than 0.3 microgram/g in the plasma and less than 1.5 microgram/g in various tissues including the immature oviduct. In the OK 10 virus-infected chickens, no significant induction was observed during the acute infection or any time thereafter if no tumors were seen. In chickens that developed tumors, avidin concentrations were significantly increased in tumorous tissue only located in the mesenterium and occasionally in the oviduct. In tumors occurring elsewhere (kidney, ovary, muscle, testis, liver, colon) avidin concentrations were not elevated. Tumor-associated avidin had extraordinary biotin-binding capacity after treatment at +90 degrees C similar to the progesterone-dependent avidin, whereas antibody-binding properties suggested that tumor-associated avidin may have a somewhat altered antigenic structure.

Three methods were used in attempts to obtain non-oncogenic avian leukosis virus for possible use as an immunoprophylactic agent for the control of lymphoid leukosis in chickens. These were: 1) isolate a nononcogenic virus from commercial breeder flocks experiencing very little or no lymphoid leukosis; 2) obtain a non-oncogenic recombinant from mixed infection of a strain with low oncogenicity, Rous-associated virus-60 (RAV-60), with RAV-1 or RAV-2 in cell culture; and 3) attempt to attenuate subgroup A avian leukosis virus by serial passage in avian cell culture. Of 43 isolates obtained from field sources, all were pathogenic except one, and its pathogenicity was questionable because of the low amount of virus tested. All 42 clones from mixed infection of highly oncogenic and poorly oncogenic virus and all clones passaged serially in cell culture were oncogenic.

Avian erythroblastosis virus (AEV) is an acutely transforming retrovirus whose putative oncogenes (v-erb-A and v-erb-B) encode the proteins P74gag-erb-A and P61-68erb-B. The existence of these two gene products has prompted the question of whether one or both proteins are required in the transformation of erythroblasts and fibroblasts by AEV. In the accompanying manuscript, we describe the use of site-specific mutagenesis to generate mutants of AEV unable to synthesize P61-68erb-B. Here we present our analysis of the oncogenic potential of an AEV mutant unable to synthesize P74gag-erb-A due to a large deletion encompassing both gag and v-erb-A sequences. The erb-A-mutant retrovirus propagated quite poorly on fibroblasts in culture; however, fibroblasts harboring the erb-A mutant genome were transformed in the absence of P74gag-erb-A expression. The mutant virus failed to induce erythroleukemias in chickens, but the validity of this finding is compromised by the poor replicative capacity of the mutant. The results presented in this and the preceding manuscript indicate that P61-68erb-B is both necessary and sufficient for neoplastic transformation of fibroblasts by AEV; by contrast, a role for p74gag-erb-A in leukemogenesis by AEV has not yet been rigorously excluded.

Marek's disease herpesvirus (MDHV) isolated from chickens free of naturally occurring avian leukosis virus (ALV) infection produced characteristic foci in both chicken embryo fibroblast (CEF) and chicken kidney cell (CKC) cultures. MDHV-A, which was extracted from the feather follicle epithelium of chickens naturally infected with ALV, did not induce cytologic changes in CEF cultures, but did cause focus formation in CK cultures. ALV was detected in MDHV-A, but not in MDHV preparations. MDHV-A (reconstructed in vivo) and MDHV were further distinguished from one another by inoculation of ALV-free LSI-SPF chickens. MDHV-A elicited a high incidence of early mortality which was not accompanied by the gross tumor spectrum characteristic of Marek's disease, although extensive histologic lesions were present. The differences between MDHV and MDHV-A were not as striking in another line of ALV-free chickens (SPAFAS). By contrast, among conventional chickens with naturally occurring ALV infection, neither MDHV-A nor MDHV caused appreciable early mortality although both were highly oncogenic (gross tumor development). These observations demonstrate that the presence of an oncornavirus (ALV), detected by radioimmune but not complement fixation assays, can influence the in vitro and in vivo characteristics of an oncogenic herpesvirus (MDHV). The observations recorded here resolve some of the inconsistencies reported in the literature. Thus, the apparent failure by some to find interactions between MDHV and oncornaviruses can be ascribed to the comparatively limited sensitivity of the complement fixation assay used to detect oncornaviruses (ALV). We have shown that the presence of oncornavirus detectable by radioimmune assay, but not by complement fixation, can influence the in vitro and in vivo responses of an oncogenic herpesvirus (MDHV). Our observations relating to viral interaction do not imply that MDHV required the presence of ALV to produce disease.

A spontaneous tumor was obtained from a hen of White-Leghorn stock raised in a local farm in Yamaguchi City, in December 1973. The tumor had characteristics of fibro- or myxofibro-sarcoma and could be maintained by transplantation in chickens of similar flocks. This tumor was proved to produce sarcoma virus at 33rd passage and the transforming virus was designated as Y73 sarcoma virus. Biological studies suggested that the virus recovered from the tumor belonged to avian leukosis-sarcoma complex having subgroup A specificity. The possibility of defectiveness of this virus will be discussed.

Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds. Two domains within its replication-defective genome, erb-A and erb-B, have been implicated in AEV-mediated oncogenesis. An efficient transfection system for generating infectious, transforming virus from molecular clones of AEV and RAV-1 (helper virus) was combined with the techniques of site-specific mutagenesis to investigate the contribution of erb-B to the two forms of oncogenesis induced by AEV. Deletion and frameshift mutations were constructed in the erb-B locus of cloned AEV DNA in vitro. Infectious retroviruses harboring these mutations were recovered and their ability to transform fibroblasts in vitro or induce erythroleukemia in vivo was assessed. The presence of mutant viral genomes in chick embryo fibroblasts or erythroblasts of infected birds was confirmed by suitable biochemical analyses. Expression of viral genes in cells infected with AEV mutants was examined by immunoprecipitation with antisera to erb-A and erb-B proteins. It was found that the product of erb-B is necessary for transformation of fibroblasts and induction of erythroblastosis by AEV, although a small portion of this protein at the carboxy terminus is dispensable.

We previously reported our characterizations of the B-lymphoma tumors induced in a highly susceptible line of chickens (15I5 X 7(2)) by the avian leukosis virus RAV-1 (Proc. Natl. Acad. Sci. 78:3418-3422, 1981). We demonstrated that in greater than 90% of the tumors, the RAV-1 provirus is integrated near a cellular oncogene, c-myc. In the present study, we devised a simple approach, relying on SalI digestion, for further defining the locations and orientations of the proviruses with respect to the c-myc gene. We report here that in the great majority of cases the provirus is situated upstream from and in the same transcriptional direction as the c-myc gene--a configuration compatible with the promoter-insertion model proposed by Hayward et al. (Nature [London] 290:475-480, 1981).

Infection of susceptible chickens with avian leukosis virus (ALV) results in the development of bursal lymphomas. These neoplasms develop within the bursa of Fabricius following a latent period of several months. The response exhibited by two previously uncharacterized chicken lines to ALV infection has been examined. The two lines, Hyline SC and FP, responded differently to ALV infection. During a 24-week period following intravenous ALV infection, 27 of 50 SC chickens developed bursal lymphomas. No lymphomas developed in the 36 FP chickens tested. A majority of the SC chickens that developed lymphomas also exhibited widespread metastasis to the liver, spleen, and kidneys. Analysis of cellular DNA from the primary and metastatic tumors demonstrated the clonal nature of these neoplasms and revealed altered c-myc loci, as reported in other studies, suggesting the importance of this locus in the development of these tumors. Further characterization of the ALV infection of SC and FP chickens will provide an opportunity to analyze the mechanism of resistance and to contribute to the understanding of the tumorigenic process.

Proviruses of avian leukosis virus (ALV) are located in the vicinity of a putative cellular oncogene (c-myc) in ALV-induced bursal lymphomas. Enhanced expression of c-myc occurs in association with proviruses found in any of three configurations: (I) on the 5' side ('upstream') of c-myc in the same transcriptional orientation; (II) on the 3' side ('downstream') of c-myc in the same orientation; (III) upstream, in the transcriptional orientation opposite to that of c-myc. Thus, activation of adjacent cellular genes by retroviral DNA can involve mechanisms other than provision of a transcriptional promoter.

30 one-day-old chickens of the commercial HX-SL hybrid from the incubator house at Premyslovice were infected with tissue culture supernatant of clone LSCC-H32. This tissue culture produced the virus of avian lymphoid leucosis of subgroups A and B. After killing at the age of 21-37 weeks, samples of chick pancreas were examined histopathologically and by electron microscopy. Serological examination of serum and feather pulp for presence of avian sarcoma-leucosis gs antigen was simultaneously performed, using the ELISA test. In the pancreas of the experimental chickens both complete and incomplete viral particles of avian leucosis were found. Most frequently, they were localised in the electron-dense secretions of the lumen of pancreatic acini at apical plasma membranes of acinar cells but also in zymogenic granules and in intercellular spaces. The significance of these findings to possible horizontal transmission is discussed.

A phenotypic mixing (PM) test for detecting and assaying avian leukosis viruses (ALV) of the A, B, C, and D subgroups is described. An ALV and Rous sarcoma virus RSV-0) are phenotypically mixed by co-cultivating on C/O (cells susceptible to all subgroups of ALV) cells for a certain period. Then the RSV with the new virus property is assayed on C/E cells (cells resistant to infection with subgroup E leukosis/sarcoma viruses). The test is relatively simple and rapid, and its results are unequivocal. It is as sensitive as the more lengthy complement-fixation test (COFAL). The system is suitable for detecting avian leukosis viruses in samples such as heparinized blood, plasma, and embryo extracts.

The proviral DNA of chicken peripheral blood leukemic myeloblasts was analyzed by restriction endonuclease digestion and Southern blotting. Two restriction endonuclease-generated fragments, an EcoRI 2.2-megadalton (Md) and a HindIII 2.6-Md fragment, were present upon enzyme cleavage of all leukemic myeloblast DNA preparations in addition to endogenous or helper-specific fragments. Neither of these fragments was derived from viral DNA of the two known myeloblastosis-associated viruses (MAV-1 and MAV-2). In contrast, DNA similarly treated from the erythrocytes of leukemic chickens showed only small amounts of the two avian myeloblastosis virus-specific fragments, whereas the helper virus-specific fragments were present in the amount seen in MAV-producing chicken embryo fibroblasts. The appearance of the EcoRI 2.2-Md and HindIII 2.6-Md specific fragments in all leukemic myeloblast DNA preparations indicates they are presumably part of the leukemogenic genome that must be present to induce acute myeloblastic leukemia.

Two trials were conducted to determine whether lymphoid leukosis virus (LLV) could be eradicated from chicken breeder stocks in one generation. Dams were selected as potentially virus-free parents on the basis of negative tests for virus in progeny embryos (trial 1) or in vaginal-cloacal swabs (VCS) (trial 2) of the dams. In trial 1, 8 of 12 groups of chickens hatched from selected breeders remained free of LLV infection through 36 weeks of life. In trial 2, VCS appeared to be more efficient in detecting possible shedder dams, and only 1 of 72 groups of chickens showed evidence of infection at 14 weeks after hatching. Within that positive group, a single chicken was shown to be the possible cause of the infection. The results show that eradication of LLV can be accomplished in one generation by: 1) virological examination of dams for shedding; 2) elimination of the shedder dams; and 3) small-group rearing of the progeny chicks.

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.

Eleven recently isolated erbB-transducing viruses as well as avian erythroblastosis virus (AEV)-R (ES4) and AEV-H have been characterized for the type of disease they cause, their ability to transform fibroblasts in culture, their ability to cause disease in pedigrees of chicken that differ in susceptibility to erbB-induced erythroblastosis, and the structure of their erbB genes. Differences in each of the biological parameters correlated with differences in erbB sequences encoding the C-terminal domain of the epidermal growth factor receptor (EGFR). Seven viruses were strain restricted in their ability to induce erythroblastosis and did not transform fibroblasts. These seven viruses contained v-erbB genes encoding the complete C terminus of the EGFR. AEV-R and AEV-H were not pedigree restricted in their ability to induce erythroblastosis and could transform fibroblasts. These viruses contain v-erbB genes that lack codons for the immediate C terminus of the EGFR. Three viruses caused angiosarcoma and one caused fibrosarcoma. The angiosarcoma and fibrosarcoma-inducing viruses were not strain restricted and did not cause erythroblastosis. The v-erbB genes of each of these viruses contained extensive internal deletions or 3' truncations in sequences encoding the C-terminal domain of the EGFR.

The genome structure of a newly isolated avian erythroblastosis virus, AEV-H, was analyzed. Using DNA probes specific for the LTR sequence of SR-RSV-A, and for the erbA gene and the erbB gene of the ES4 strain of AEV, we have shown that the genome of AEV-H is 35S in size and carries the erbB gene but not the erbA gene. Comparison of the restriction sites of molecularly cloned AEV-H DNA with that of cloned DNA of the associated virus revealed that the env gene of the associated virus was replaced with the erbB gene to generate AEV-H. The genome structure of AEV-H is, therefore, determined to be 5'-gag-pol-erbB-3'. Moreover, we have isolated a mutant of AEV-H, td-130, that can induce sarcomas but not erythroblastosis in chickens. The restriction analysis of proviral DNA of the td-130 showed that it carries a deletion of about 150 to 200 nucleotides in the erbB gene. These data indicate that the erbB protein is responsible for both erythroblastosis and sarcomas.

We isolated molecular clones of the provirus-host cell junctions (tumor junction fragments) from two avian leukosis virus-induced lymphomas and compared the structures of these clones with a clone of the normal c-myc gene. Restriction mapping and DNA sequencing demonstrated that normal proviral integration events occurred adjacent to c-myc in both tumors, without gross structural alteration of c-myc. The right long terminal repeat of an avian leukosis virus provirus is integrated upstream from the bulk of the c-myc coding sequences and oriented such that transcription can initiate within the long terminal repeat and proceed downstream into c-myc. A comparison of a tumor junction fragment with the v-myc gene showed that there are two regions of v-myc-related sequences (which are probably exons) separated by 1 kilobase of sequences unrelated to v-myc (probably an intron). A DNA sequence analysis of the tumor junction fragments suggested that integration had occurred in exons adjacent to splice donor sites. This suggests that there are additional exons and introns in c-myc. Based on these findings, a model is proposed for the genesis of the tumor-specific RNAs containing viral-5' and c-myc information in avian leukosis virus-induced lymphomas.

Monospecific antiserum obtained from rabbits hyperimmunized against homogeneous p27 group specific protein purified from avian myeloblastosis virus was commercially procured and was then conjugated with fluorescein isothiocyanate. The conjugate was applied to spleens from naturally or experimentally infected chickens that had no evidence of lymphoid tumors. Fluorescence was usually localized in connective tissue of sheathed capillaries giving it a ring-like appearance. Sites of fluorescence corresponded to sites of greatest virus concentration as detected by electron microscopy, indicating that in such cases the group specific antigen may have been associated with virus particles. The group specific antigen could also be detected in the spleen by complement fixation and results of this test usually agreed with the immunofluorescent test and with the phenotypic mixing test which detects exogenous lymphoid leukosis virus.

ALV-induced erythroblastosis results from the specific interruption of the host oncogene, c-erbB, by the insertion of an intact provirus. Integrated proviruses are oriented in the same transcriptional direction as c-erbB, and expression of truncated c-erbB transcripts is observed. Evidence, including sequence analysis of cDNA clones, indicates that transcription of truncated c-erbB mRNA is initiated in the 5' LTR of the integrated provirus. This transcript is processed through a series of remarkable splicing reactions to yield viral gag and env sequences fused to erbB sequences. These results establish a novel pathway of promoter insertion oncogenesis that stands in contrast to the pathways used in the activation of c-myc in B lymphomas.

Subgroup F avian leukosis viruses, such as RAV-61 and ring-necked pheasant virus, are recombinants between exogenous chicken retroviruses and endogenous pheasant viruses and contain new envelope (env) genes. Chickens infected as 10-day-old embryos with subgroup F viruses develop fibrosarcomas, nephroblastomas, osteopetrosis, B-cell lymphomas, and a high incidence of a proliferative disorder involving the lung. Fibrosarcomas, nephroblastomas, and lymphomas appear after long latent periods (3 to 12 months). They contain discrete virus-cell junction fragments and are therefore clonal outgrowths of a single infected cell. Two ring-necked pheasant virus-induced B-cell lymphomas and an adenocarcinoma of the abdomen contained proviruses integrated at the c-myc locus and elevated levels of myc mRNA. At least four of the fibrosarcomas appeared to contain proviruses integrated at a common site, suggesting that a specific cellular gene may be involved in these tumors. The host gene has not been identified, however; 16 different oncogene probes failed to hybridize to fibrosarcoma junction fragments. In contrast to these neoplasms, lung lesions appeared rapidly (4 to 5 weeks), showed no evidence of clonality, and lacked long terminal repeat-initiated transcripts other than viral 35S and 21S mRNA. We conclude, therefore, that subgroup F retroviruses induce the proliferative disorder of the lung by a different mechanism.

The effect of glutaurine (gamma-L-glutamyl-taurine, Litoralon) on the take and development of hepatoma and acute leukaemia induced by MC29/L avian oncorna-virus has been investigated in turkey poults. Glutaurine significantly decreased the incidence of hepatoma, but had no significant effect on the lethality of MC29/L infected birds. The number of primitive myeloid cells was lower in the peripheral blood of glutaurine treated birds than in the untreated controls. Reverse transcriptase determinations in turkey fibroblast cell cultures indicated that glutaurine delays MC29/L virus expression.