Heterophil function and resistance to staphylococcal challenge in broiler chickens naturally infected with avian leukosis virus subgroup J.

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.

Male-mediated venereal transmission of endogenous avian leukosis virus.

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.

In vitro translation of fractionated virus-specific RNA isolated from plasma of chicken infected by avian myeloblastosis virus. Unprocessed and processed myeloblastosis-associated virus env-polypeptide precursors.

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.

Occurrence of alternatively spliced leader-delta onc-poly(A) transcripts in chicken neuroretina cells infected with Rous-associated virus type 1: implication in transduction of the c-mil/c-raf and c-Rmil/B-raf oncogenes.

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.

Avian myeloblastosis virus core-bound 7 S DNA, highly bent minute structures with sequence-directed curvature.

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.

Patterns of integration and expression of retroviral, non-replicative vectors in avian embryos: embryo developmental stage and virus subgroup envelope modulate tissue-tropism.

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)

Recovery of acutely transforming viruses from myeloid leukosis induced by the HPRS-103 strain of avian leukosis virus.

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.

Tissue tropism of avian leukosis viruses: analyses for viral DNA and proteins.

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.

Myeloid leukaemogenicity and transmission of the HPRS-103 strain of avian leukosis virus.

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.

[The technology for isolating the avian myeloblastosis virus in high titers from leukosis-free chicks]. / Tekhnologiia polucheniia virusa mieloblastoza ptits v vysokikh titrakh na bezleikoznykh tsypliatakh.

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.

Activation of the c-myb locus is insufficient for the rapid induction of disseminated avian B-cell lymphoma.

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.

Developments in avian leukosis research.

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.

Fusion of the nuclear oncoproteins v-Myb and v-Ets is required for the leukemogenicity of E26 virus.

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.

Cooperation of v-jun and v-erbB oncogenes in embryo fibroblast transformation in vitro and in vivo.

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.

Interactions between endogenous virus loci ev6 and ev21. 2. Congenital transmission of EV21 viral product to female progency from slow-feathering dams.

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.

In ovo infection with the avian retrovirus RAV-1 leads to persistent infection of the central nervous system.

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.

Molecular analysis of the ets genes and their products.

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.