Synergy of subgroup J avian leukosis virus and Eimeria tenella to increase pathogenesis in specific-pathogen-free chickens.

To investigate the effects of co-infections of subgroup J avian leukosis virus (ALV-J) and Eimeria tenella on the pathogenesis in specific-pathogen-free (SPF) white leghorn chickens, groups of chickens were infected with ALV-J strain NX0101 at one day of age or with E. tenella at 14 days of age or both. The control group was left uninfected and was mock-inoculated with phosphate buffer saline (PBS). Mortality rates, body weights, cecal lesions, and viremia of infected chickens in each group were evaluated. Immune status was evaluated by measuring several parameters: immune organ weight/body weight index, specific humoral responses to inactivated NDV vaccine and to inoculated E. tenella, proportions of blood CD3+CD4+ and CD3+CD8α+ lymphocytes and transcriptional levels of cytokines in blood and cecal tonsils. The results show that co-infections of ALV-J and E. tenella induced a higher mortality rate and a lower body weight in SPF chickens compared to single-pathogen infection. In co-infected chickens, ALV-J accelerated the disease symptoms induced by E. tenella, and the E. tenella extended the ALV-J viremia. Thymus atrophy, decrease in the humoral response levels to pathogens and the NDV vaccine, modifications in the blood lymphocyte sub-populations and transcriptional cytokine disorders were found in co-infected chickens compared to chickens infected with one pathogen alone and to controls. We underline a synergy between ALV-J and E. tenella that results in increasing pathogenesis in SPF chickens.

E (XSR) element contributes to the oncogenicity of Avian leukosis virus (subgroup J).

Among the six subgroups of Avian leukosis virus (ALV) that infect chickens, subgroup J (ALV-J) was isolated from meat-type chickens where it predominantly induces myeloid leukosis (ML) and erythroblastosis (EB). The sequence of HPRS-103, the ALV-J prototype virus, shows several distinct features, one of which is the presence of a distinct hairpin stem-loop structure called the E (also called XSR) element in the 3' untranslated region. In order to determine the role of the E element in ALV-induced pathogenicity, a comparison was made of the oncogenicity of viruses derived from the provirus clones of parental and E element-deleted HPRS-103 viruses in two genetically distinct lines of birds. In line 15I birds, deletion of the E element had profound effects on virus replication in vivo, as only 55 % of birds showed evidence of infection, compared with 100 % infection by the parental virus. Furthermore, none of the line 15I birds infected with this virus developed tumours, indicating that the E element does contribute to the oncogenicity of the virus. On the other hand, deletion of the E element had only a marginal effect on the incidence of tumours in line 0 birds. These results indicate that, although the E element per se is not absolutely essential for tumour induction by this subgroup of viruses, it does contribute to oncogenicity in certain genetic lines of chicken.

Silent point mutation in an avian retrovirus RNA processing element promotes c-myb-associated short-latency lymphomas.

The avian leukosis virus DeltaLR-9 causes a high frequency of B-cell lymphomas within weeks after injection into 10-day-old chicken embryos. These lymphomas result from proviral integrations into the oncogene c-myb. In contrast, LR-9, which lacks the 42-nucleotide gag gene deletion of DeltaLR-9, does not cause a high frequency of c-myb-associated short-latency lymphomas. Although viral replication rates and spliced env mRNA levels were found to be similar for both viruses, DeltaLR-9 exhibited an increase in readthrough transcription compared to LR-9. The DeltaLR-9 deletion is located in the region of the gag gene corresponding to the matrix (MA) protein as well as in the negative regulator of splicing (NRS) element. To test whether disruption of the NRS or of the MA protein was responsible for inducing short-latency lymphomas, we generated viruses with NRS point mutations that maintained the wild-type Gag amino acid sequence. One of the mutant viruses induced an even higher incidence than DeltaLR-9 of short-latency lymphomas with viral integrations into c-myb. Thus, we propose that disruption of the NRS sequence promotes readthrough transcription and splicing to the downstream myb gene, causing overexpression of a slightly truncated Myb protein, which induces short-latency tumors.

The Myb leucine zipper is essential for leukemogenicity of the v-Myb protein.

The AMV v-Myb oncoprotein causes oncogenic transformation of myelomonocytic cells in vivo and in vitro. Its transforming capacity is strictly dependent upon the N-terminal DNA binding domain, the central transactivation region, and on the C-terminal domain containing a putative leucine zipper motif. Here we show that the v-MybL3,4A mutant, in which Leu325 and Leu332 of the leucine zipper have been replaced by alanines, failed to induce leukemia in virus infected chicken. This demonstrates that the leucine zipper domain is indispensable for v-myb induced leukemogenesis in vivo. v-MybL3,4A was, however, still able to transform myelomonocytic cells from chicken bone marrow in vitro. Yet, while v-mybL3,4A transformed cells were impaired in growth at 37 degrees C, they failed to grow at 42 degrees C, the physiological body temperature of avian species. This might explain the loss of v-MybL3,4A leukemogenic potential in vivo. We also demonstrate that the v-Myb leucine zipper domain interacts in vitro with two host cell proteins, p26 and p28. This interaction is compromised in v-MybL3,4A indicating that binding of v-Myb to p26 and p28 might be important for the leukemogenic potential of v-Myb.

Augmentation of retrovirus-induced lymphoid leukosis by Marek's disease herpesviruses in White Leghorn chickens.

Our objective was to determine whether the cell-associated herpesvirus vaccines used in chickens to control Marek's disease tumors can augment development of lymphoid leukosis (LL) induced by exogenous avian leukosis virus (ALV). Various single or mixed Marek's disease vaccines were inoculated at day 1, and ALV was injected at 1 to 10 days, with chickens of several experimental or commercial strains. Development of LL was monitored at 16 to 48 weeks in various experiments. In several strains of chickens we repeatedly found that the widely used serotype 3 turkey herpesvirus vaccine did not augment LL in comparison with unvaccinated controls. However, LL development and incidence were prominently augmented in several chicken strains vaccinated with serotype 2 vaccines, used alone or as mixtures with other serotypes. In one chicken strain, augmentation was demonstrated after natural exposure to ALV or serotype 2 Marek's disease virus viremic shedder chickens. Augmentation of LL by virulent or attenuated Marek's disease viruses of serotype 1 was intermediate in effect. Serotype 2 Marek's disease virus augmentation of LL was prominent in three laboratory lines and one commercial strain of White Leghorns, but it was not observed in an LL-resistant laboratory line or four commercial strains susceptible to ALV infection. Chickens developed similar levels of viremia and neutralizing antibodies to ALV regardless of the presence of augmentation of LL, suggesting that the mechanism of enhanced LL did not result from differences in susceptibility or immune response to ALV. We postulate that the serotype 2 herpesviruses may augment LL through one of several possible influences on bursal cells that are subsequently transformed by exogenous ALV.

[Retroviruses with 2 oncogenes]. / Retrowirusy majace dwa onkogeny.

Less than a decade ago, some acute transforming avian leukemia retroviruses that transduced not one but two distinct cellular genes that could cooperate in the viral-proned transformation processes was discovered. In this paper three examples of such retroviruses are presented. This are: avian erythroblastosis virus ES 4 (AEV-ES 4), avian erythroblastosis virus E 26 (AEV-E 26), avian Mill-Hill-2 myelocytomatosis virus (MH 2).

[Effect of a mixed viral infection on the development of neoplasms induced by oncogenic viruses]. / Vliianie smeshannoi virusnoi infektsii na razvitie neoplazii, indutsirovannykh onkogennymi virusami.

Data are presented concerning the stimulating effect of vaccinia and herpes simplex type 2 viruses on the development of leukemia in BALB/C, C57BL/6, and AKR mice. Mixed infection with PAB-49 and Marek disease virus of brown leghorn chickens was shown to increase the frequency of lymphomas development.

[Oncogenes and the origin of leukemia. Acute avian leukemia viruses]. / Onkogene und Leukämieentstehung. Untersuchungen an akuten Hühner-Leukämieviren.

Oncogenes have been intimately associated with the genesis of human neoplasms. A particularly useful system to study the mechanism of tumorigenesis is a small group of avian retroviruses that carry two oncogenes. These viruses causes acute leukemias and can transform hematopoietic cells in vitro. The mechanisms by which viral oncogenes affect the growth control and differentiation of their target cells is now understood in fair detail for two of these virus strains. In the avian erythroblastosis virus AEV, the v-erbB oncogene deregulates the growth control of erythroid precursors, while verbA blocks their terminal differentiation into erythrocytes. Based on the findings that v-erbB oncogene corresponds to a mutated growth factor receptor gene and that v-erbA corresponds to a mutated hormone receptor gene, models have been developed that explain the function of these two oncogenes on a molecular basis. The myelomonocytic leukemia virus MH2 acts by a completely different mechanism. In this case, the v-myc oncogene stimulates the proliferation of macrophage-like cells, while the v-mil gene stimulates them to produce their own growth factor, thus leading to autocrine growth. It will be interesting to determine whether the type of mechanisms of oncogene cooperativity elucidated for acute leukemia viruses are also operative during leukemogenesis in humans.

[Induction of tumors of the meningeal membranes in chicks by the avian myeloblastosis virus]. / Induktsiia opukholei obolochek golovnogo mozga u tsypliat virusom mieloblastoza ptits.

The meningiomas could be easily produced by intracerebral injection of the avian myeloblastosis virus (0.01 ml of virus suspension with titer 10(9)-10(12) PFU/ml) into highly susceptible (White Leghorn Anya cross) or randomly bred newborn (24-48 hours) chicken. Tumours were observed in 66.7% highly susceptible chickens and in 33.73% of randomly bred chickens. First clinical signs of disease appeared on the 7-9th days after infection. Death occurred on the 3-4th days after beginning of the disease. Five types of tumours were detected: meningotheliomatous, fibroblastic, angiomatous, mixed and anaplastic. Our data are first description of the tumours of CNS induced by the avian myeloblastosis virus.

High-frequency transduction of c-erbB in avian leukosis virus-induced erythroblastosis.

Twenty-one cases of Rous-associated virus type 1-induced erythroblastosis have been analyzed for novel restriction endonuclease fragments of c-erbB. Twenty of the erythroleukemias contained novel c-erbB fragments; 10 of these were found to contain a proviral insertion in c-erbB, and 10 were found to have a new transduction of c-erbB. Each of the proviral insertions was in the same transcriptional orientation as c-erbB, and most appeared to have retained both long terminal repeats as well as 5' viral sequences that signal packaging of RNA into virions. Each of the new c-erbB transducing viruses had a characteristic EcoRI fragment that contained a spliced form of c-erbB sequences. When inoculated into 1-week-old chickens, the new transducing viruses caused rapid-onset erythroblastosis.

Induction of angiosarcoma by a c-erbB transducing virus.

Recently, 12 new transductions of c-erbB have been identified in a series of Rous-associated virus type 1-induced erythroleukemias. During the passage of these new transducing viruses it has become apparent that the erythroleukemia in chicken 5005 contained two different c-erbB transducing viruses. One induces erythroblastosis, whereas the second induces angiosarcoma. The angiosarcoma- and erythroblastosis-inducing viruses appear to have had a common ancestor, since tumors induced by each contain a novel, 4.3-kilobase c-erbB-related EcoRI fragment. The angiosarcoma-inducing virus has been named avian angiosarcoma virus and is designated for the chicken in which it originated.

Transformation of erythroid cells by Rous sarcoma virus (RSV).

RSV transforms several nonhematopoietic cell types and as reported here also has the capacity to transform hematopoietic cells of the erythroid lineage. In vitro, the three RSV isolates tested induced erythroblast-like colonies in infected bone marrow cells that were distinguishable by size and cell arrangement from those induced by avian erythroblastosis virus (AEV). Also in contrast to AEV-transformed erythroblast cultures, isolated cell colonies induced by RSV required complex growth conditions in liquid medium similar to the in vitro conditions necessary for erythroblasts transformed by the acute leukemia virus E26. Temperature-shift experiments using temperature-sensitive (ts) NY68 RSV revealed that when grown at the nonpermissive temperature (42 degrees), mutant-infected cells became benzidine positive and partially differentiated into erythrocytes. Wild-type (wt) RSV-transformed cells did not undergo similar changes. However, both wt RSV-, and to a greater extent, ts RSV-transformed cultures at the permissive temperature (37 degrees) did contain populations of spontaneously differentiating erythroid cells signifying that the transforming activity of the virus did not fully arrest erythroid maturation. In addition, the RSV-transformed cells did express tyrosine kinase activity. When injected intravenously into birds, RSV induced an erythroblastosis-like disease similar to AEV but also caused fibrosarcomas and leg paralysis. These results show that RSV can alter the pattern of erythroid differentiation in a manner similar to, but distinct from, AEV and indicate that the tyrosine-specific pp60src kinase is involved in erythroid cell transformation. Since the src and erb B proteins share a significant amino acid homology, these data suggest that both may also share a common functional homology.

[Mechanism of virus-induced leukemogenesis in an animal model system]. / Uber den Mechanismus der virus-induzierten Leukämieentstehung in einem tierischen Modellsystem.

Retroviruses can cause different types of leukemias in chickens. A small group of virus strains induce acute leukemias and transform hematopoietic cells in vitro. We have shown that cells transformed in vitro by erythroblastosis and myeloblastosis viruses resemble in vivo transformed cells in their phenotype of differentiation. The erythroblastosis virus AEV carries two cell-derived oncogenes termed erbA and erbB. The analysis of AEV using deletion mutants showed that erbB is the main oncogene which is capable of inducing a "mild" form of erythroleukemia. The added action of the erbA oncogene leads to an aggressive, more "advanced" form of erythroleukemia. The erbA oncogene itself, however, does not exhibit any detectable biological activity. Using temperature sensitive mutants of AEF we could also show that erbB, together with erbA, blocks the differentiation of hematopoietic precursor cells. The continued synthesis of a functional erbB-encoded protein is necessary to maintain the "blocked", i.e. leukemic state of the infected cells.

Site-specific mutagenesis of avian erythroblastosis virus: v-erb-A is not required for transformation of fibroblasts.

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.

Site-specific mutagenesis of avian erythroblastosis virus: erb-B is required for oncogenicity.

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.