Influence of bursectomy on bone growth and anemia induced by avian osteopetrosis viruses.

This study examines the contribution of the bursa of Fabricius to the pathogenic manifestations of two myeloblastosis-associated viruses which primarily cause osteopetrosis [MAV-1(O) and MAV-2(O)]. MAV-2(O) infection of surgically bursectomized 1-month-old chicks resulted in a rapidly fatal anemia whereas infection of untreated chicks of the same age resulted in a transient drop in hematocrit. Surgical bursectomy of embryos before or after embryonal infection with MAV-2(O) did not alter the course of osteopetrosis, indicating that the bursa was not a source of target cells. Bursectomy prolonged the period of susceptibility to MAV-2(O) induced osteopetrosis until one day posthatching; untreated chicks were not susceptible to osteopetrosis induction at that age. MAV-1(O) infection of eight-day-old bursectomized chicks resulted in osteopetrosis in the absence of anemia; untreated eight-day-old chicks infected with MAV-1(O) showed no effects of virus infection. A role for the bursa in MAV-2(O) infection was found in the participation of neutralizing antibodies in the recovery from anemia. A single dose of antiviral antibody was found to prevent the appearance of anemia. The protective effect of antiviral antibody was dose dependent, and antiserum administration had to be initiated within three days after virus in order to be effective. Antiviral antibody against MAV-1(O) did not protect against MAV-2(O)-induced anemia, suggesting subgroup specificity. These results suggest that the bursa does not provide a stem cell which participates in the bone hyperplasia induced by MAV-1(O) and MAV-2(O). Rather, the humoral antibodies provided by cells derived from the bursa may serve to eliminate viremia and limit virus-specific cytopathogenic effects.

Association of persistent synthesis of viral DNA with macrophage accessory cell dysfunction induced by avian retrovirus myeloblastosis-associated virus of subgroup B inducing osteopetrosis in chickens.

This investigation concentrates on a regenerative anemia and immunosuppression occurring in the absence of osteopetrosis. Polyclonal activation of T-cells was used as an in vitro test system to study immunosuppression induced by the avian myeloblastosis-associated virus of Subgroup B inducing osteopetrosis [MAV-2(O)]. T-cell unresponsiveness in vitro was attributed to a defect in an accessory cell function of the macrophage. Counterflow centrifugation fractionation followed by mixing experiments indicated that the T-cell population from immunosuppressed chickens responded to mitogen stimulation when added to control macrophage cultures. In addition, lymphocyte fractions from uninfected chickens were unresponsive when added to macrophage cultures isolated from MAV-2(O)-infected chickens. Cultured splenic macrophages isolated from infected chickens contained high levels of both integrated and unintegrated viral DNA and formed syncytia by 21 days in culture. The macrophages remained viable and exhibited mature functional characteristics during mitogen stimulation assays. Therefore, it was speculated that the persistent synthesis of retrovirus DNA might be involved in the inability of infected macrophages to function as accessory cells.

Retroviral expression in mononuclear blood cells isolated from a patient with osteopetrosis (Albers-Schönberg disease).

We report the presence of reverse transcriptase activity in the supernatant of long-term culture of mononuclear blood cells (monocytes and lymphocytes) isolated from a 27-year-old patient suffering from benign osteopetrosis. The enzyme was purified to homogeneity according to the technique of Chandra and Steel, by chromatography, first on DEAE-cellulose (DE 52) and then on phosphocellulose (P11). After purification, the enzyme was characterized biochemically for its template specificity and ionic requirements. The purified enzyme was able to transcribe poly(rA).(dT)12-18 and poly(rC).(dG)12-18 very efficiently and had a marked preference for Mg2+ ions over Mn2+ ions. The pattern of ionic dependency for this enzyme is similar to that of reverse transcriptases purified from human lymphotropic viruses. The patient was tested and found sero-negative for HIV-1, HIV-2, and HTLV-I and seropositive (immunoglobulin G) for cytomegalovirus. Epstein-Barr virus nuclear antigens (EBNA) were detected in the patient's B lymphocytes. Since reverse transcriptase is the hallmark of retroviruses, we suggest that a retrovirus may be involved in the etiology of osteopetrosis.

Osteoclasts in human osteopetrosis contain viral-nucleocapsid-like nuclear inclusions.

We report the discovery of nuclear inclusions in the osteoclasts of three unrelated patients with benign osteopetrosis that resemble the osteoclast inclusions characteristic of Paget's disease of bone. These inclusions are morphologically and dimensionally identical to the nucleocapsids of a virus of the Paramyxoviridae family. Supporting a possible viral association with benign osteopetrosis in the observation of the presence of antigens of respiratory syncytial virus, measles virus, and/or mumps virus in the cells of all five patients whose paraffin-embedded bone specimens were tested. These included two patients whose osteoclasts contained nuclear inclusions. No patients with the malignant form of the disease have been studied. There is as yet no proof that a virus is causally related to human osteopetrosis even though a virus can produce an avian form of the disease.

A sequential study of bone lesions caused by isolates of an avian osteopetrosis virus, MAV-2(0).

The pathogenesis of avian osteopetrosis caused by rapid and slow-onset isolates of myeloblastosis associated virus, MAV-2(0), was studied by inoculation of 10-day-old chick embryos with virus. Femur and calvarium were examined at 15, 17 and 19 days in ovo and 7 and 25 days after hatching by histologic and immunoperoxidase techniques. Femur and calvarium were also examined by electron microscopy at 17 and 19 days in ovo and at 7 days after hatching. Avian osteopetrotic bone lesions were characterized by exuberant periosteal proliferation; the time of onset varied with different virus isolates. In the femur virus was first associated with osteoprogenitor cells, then with osteoblasts and finally with osteocytes as the cells progressed through normal sequences of differentiation. The amount of virus produced by these cells did not correlate with onset of periosteal proliferation. Slow onset isolates provoked early virus production, but proliferative lesions did not develop until later. Conversely, the rapid onset isolate induced little early virus production, although lesions were present. Periosteal proliferation was associated with and preceded by perivascular edema and perivascular cell necrosis within the bone cortex following infection by all isolates. However, the rapid onset isolate caused more severe lesions than other isolates. These lesions included vascular thrombosis, capillary necrosis and focal bone necrosis. The relationship between early vascular lesions and late periosteal proliferation seen with the slow onset isolates is not as clear as with the rapid onset isolate. Calvarial bone, a representative flat bone, was found to have virus present, but at a level less than the femur. Vascular lesions were rarely seen in the calvarium and bone proliferation did not occur at this site.

Endogenous murine leukemia viruses: frequency of radiation-activation and novel pathogenic effects of viral isolates.

Female C57BL/6 and BALB/c mice were injected i.p. with 0.06 microCi/kg or 0.5 microCi/kg of the short-lived alpha-emitting radionuclide 224radium at 3-day intervals. Infectious N-ecotropic XC+, and xenotropic C-type retroviruses were activated in several tissues in both strains. In C57BL/6 mice the activation of ecotropic and xenotropic virus was dose-dependent as observed 4 weeks after the start of irradiation. In BALB/c mice a few animals showed activation of ecotropic virus after four weeks of irradiation. The expression of xenotropic virus was similar in irradiated mice and controls. Viral antigen, indicative for viraemia, was not detected in irradiated or control animals. Antiviral antibodies were found in both control and irradiated mice but higher titers were found in the irradiated mice. Bone tissue-derived N-tropic XC+ virus isolates were found to be non-oncogenic in newborn mice of the parental strain. In contrast, the same virus isolates induced a novel pattern of disease, such as osteopetrosis and osteomas together with malignant lymphomas in NMRI mice. The data indicate that the pattern of endogenous murine leukemia virus activation by internal alpha-irradiation is dependent on the dose rate, and on the genetics of the mouse strain.

A new approach to the study of the origin of genetic diseases: retroviral etiology of osteopetrosis.

The recent evidence of retroviral activity in a patient suffering from an atypical form of benign osteopetrosis suggests that it may represent the ancestral infectious form of a disease recognized until now only as a genetic disease. Therefore, making c-DNA probes from that virus should evidence retroviral-like sequences integrated in the genome of patients suffering from the inherited form of the disease. One of the goals of these investigations is to use these probes to map human "osteopetrosis" mutation(s) in the genome. A new approach is therefore defined for the study of these diseases, which although similar in their clinical manifestations, appear either as "sporadic" or "familial" cases: a retroviral etiology can account for this discrepancy. Another approach is also suggested for these kinds of disease, consisting of the search for retroviral sequences in a gene coding for an enzyme, when the main disease is associated with an apparently unrelated enzymatic deficiency. The insertion of retroviral sequence in a gene coding for an enzyme may result in both the disease and the inactivation of the gene. By comparing the genomic sequence of the enzyme in normal and pathological cases, the integrated retroviral gene(s) sequence will be available.

Retroviruses, immunosuppression and osteopetrosis.

Since decades, retroviruses are known to induce the formation of dense bones (osteopetrosis) in animals, either by increasing osteoblastic proliferation (ALVs viruses) or by decreasing osteoclastic bone resorption (FeLV virus). The latter is a good model of the inherited disease since neonatally infected cats die from wasting disease, like mutant op/op rats and children suffering from juvenile-malignant osteopetrosis. These similarities have prompted this review of retrovirus-induced osteopetrosis in animals. We suggest the possibility that retroviruses might be involved, at least in part, in the induction of the human disease.

Sequential study of visceral lesions caused by isolates of an avian osteopetrosis virus (myeloblastosis-associated virus).

Ten-day-old chicken embryos were inoculated with isolates of myeloblastosis-associated virus that induced osteopetrosis of slow or rapid onset. Bursa of Fabricius, thymus, spleen, bone marrow, kidney, liver, and lung were examined at 15, 17, and 19 days in ovo and at 7 and 25 days after hatching by histologic and immunoperoxidase techniques. Tissues from 19-day-old in ovo embryos also were examined by electron microscopy. The lymphoid organs of embryos inoculated with all isolates manifested changes suggesting inhibited development. Virus was most often associated with macrophages, heterophils, and nonlymphoid stromal cells in these organs. Viral particles and antigen were abundant in tissues from embryos inoculated with slow-onset isolates, but cell necrosis was infrequent. The kidney and bursa had especially abundant viral particles and antigen. Conversely, viral particles and antigen were minimal in tissues from embryos inoculated with the rapid-onset isolate, yet intravascular cellular thrombi, substantial cell necrosis, and increased heterophils and hemocytoblasts were found.

Persistent viral DNA synthesis associated with an avian osteopetrosis-inducing virus.

Bone and red blood cell DNA was obtained from MAV-2(O) infected, osteopetrotic chickens and analyzed for the presence of integrated and unintegrated viral DNA. Results indicate that unintegrated MAV-2(O) DNA did not appear until after osteopetrotic lesions were well established. These observations lead us to conclude that, unlike some retroviral diseases, unintegrated viral DNA may not play a significant role in the pathogenesis of MAV-2(O) osteopetrosis. In addition, there was no evidence of a clonally derived tumor in the bone.

Pathogenic potential of myeloblastosis-associated virus: implication of env proteins for osteopetrosis induction.

To identify the nucleotide sequences responsible for the tumorigenic specificity of myeloblastosis-associated virus (MAV) we have established the complete nucleotide sequences of three infectious clones inducing either both osteopetrosis and nephroblastoma [MAV2(O)/2 and MAV2(O)p9] or only nephroblastoma [MAV1(N)], and compared their biological properties in the same chicken host strain. The MAV2(O)p9 originally described as a type 2 strain was found to carry a hybrid env gene containing sequences of both the types 1 and 2, and it induced milder and less rapid osteopetrosis than the original MAV2(O) clone when injected into Brown Leghorn chickens. These results, together with sequence comparisons between the MAV strains examined, suggest that subtle changes in the primary structure of the TM env protein's extracellular domain are likely to affect the tumorigenic potential of MAV.

Sequences from myeloblastosis-associated virus MAV-2(O) and UR2AV involved in the formation of plaques and the induction of osteopetrosis, anemia, and ataxia.

Recombinant viruses were made between myeloblastosis-associated virus MAV-2(O) and UR2AV to examine the relationship between regions of the MAV-2(O) genome and disease induction. The env-long terminal repeat (LTR) portion of MAV-2(O), when substituted into UR2AV, was sufficient to induce osteopetrosis identical to that caused by the parent MAV-2(O). When this region was reduced to the gp37 and LTR of MAV-2(O), osteopetrosis more severe than that caused by the parent virus was induced. Recombinant viruses that contained all or part of the MAV-2(O) env gene in the absence of the MAV-2(O) LTR induced a severe, chronic anemia and late-onset osteopetrosis, leading to the conclusion that the MAV-2(O) LTR, in addition to env, was required for rapid induction of osteopetrosis. A viral recombinant, pEU, which contained the gp85 segment of UR2AV substituted into MAV-2(O), induced an ataxia/cerebellar dysfunction not seen during infection with the other chimeric or parent viruses. In vitro studies of the parent and recombinant viruses demonstrated that the ability to form plaques on chicken embryo fibroblasts correlated with the presence of the MAV-2(O) gp37 and LTR except for construct pEU. When the viruses were inoculated into 10-day-old chickens, chimeras containing the env-LTR of gp37-LTR region of MAV-2(O) induced severe regenerative anemia similar to that induced by MAV-2(O). pEU was the exception, suggesting that the unique configuration of this chimera is responsible for its unusual pathogenic properties.

Sequences near the 5' long terminal repeat of avian leukosis viruses determine the ability to induce osteopetrosis.

Avian leukosis virus (ALV)-induced osteopetrosis is associated with the accumulation of unintegrated viral DNA in osteoblasts. Viruses constructed from the DNAs of an osteopetrosis-inducing ALV (Br21) and a non-osteopetrosis-inducing ALV (RAV-0) have been used to test for the role of viral genes in the induction of osteopetrosis. Our results map osteopetrotic potential to a 1,400-base-pair region near the 5' long terminal repeat. This region contains signals for the splicing, translation, and packaging of viral RNAs and coding sequences for the gag proteins p19 and p10 and the N terminus of p27.

MAV-2-O replicates at a reduced rate in cells from the osteopetrosis resistant G-B1 chicken.

The replication of the avian osteopetrosis virus MAV-2-O was compared in chick embryo fibroblasts from two strains of chicken. These were G-B1 which is relatively resistant to MAV-2-O and CB which is susceptible. The production of MAV-2-O was delayed in G-B1 cells (compared with CB cells). The same result was observed after infection with Rous sarcoma viruses of subgroups B, C, and D. In addition, the transforming viruses induced foci on G-B1 fibroblasts 24 to 48 hours later than on CB fibroblasts. In G-B1 cells there was also a delayed kinetics of intracellular viral RNA production. Integrated and linear unintegrated MAV-2-O DNA species were also present in lower amounts in G-B1 than in CB fibroblasts at 3 days postinfection. In vivo studies confirmed the in vitro situation. There was a marked difference in the amount of virus present in the osteoid bone matrix and the osteocytic lacunae of osteopetrotic bones from susceptible and G-B1 chickens. In contrast to the bone lesions from susceptible animals, budding virus particles were not detectable in lesions from G-B1 chickens. There was no difference in the amount of virus in osteopetrotic and non-osteopetrotic bone of susceptible chickens suggesting that virus replication alone is not sufficient for induction of osteopetrosis and that an additional specific virus-cell interaction is required. The relative resistance of strain G-B1 may therefore, be a consequence of a reduced frequency of this interaction. Its basis may be the lower amount of integrated, as well as unintegrated, viral DNA.

5' avian leukosis virus sequences and osteopetrotic potential.

Recombinants of Rous-associated virus-0 and Br21 have been used to localize 5' viral sequences that affect the osteopetrotic potential of avian leukosis viruses. Rous-associated virus-0 is a benign subgroup E virus of endogenous origin that does not cause osteopetrosis. Br21 is a constructed subgroup E virus with high osteopetrotic potential. 5' sequences that affected osteopetrotic potential resided in an 834-bp region near the 5' LTR. Sequence analysis of this region revealed differences between Br21 and RAV-0 in the mRNA leader and codons for MA.

Avian leukosis virus-induced osteopetrosis is associated with the persistent synthesis of viral DNA.

DNAs from 19 cases of avian leukosis virus-induced osteopetrosis have been analyzed for viral sequences. Among these were instances of rapid, intermediate, and slow onset osteopetrosis. The DNAs from osteopetrotic bone contained no evidence for osteopetrosis being caused by proviral insertions into or viral transductions of a host protooncogene. Instead, DNAs from osteopetrotic bone displayed evidence for osteopetrosis being associated with the persistent synthesis of viral DNA. Each of the 19 DNAs contained unintegrated as well as integrated viral DNA. Rapid onset osteopetrosis contained about 3X more viral and proviral DNA than intermediate or late onset osteopetrosis. Unintegrated viral DNA could not be detected in DNAs extracted from the bursa bone marrow of osteopetrotic chickens or in DNA extracted from the normal bones of an avian leukosis virus-infected chicken. Thus, the persistent synthesis of unintegrated viral DNA was observed in osteopetrotic but not normal tissues of avian leukosis virus-infected chickens.

Effect of avian osteopetrosis virus infection on cells and their collagen synthesis in vitro.

Primary avian tendon fibroblasts and calvarial osteoblasts were infected with the avian osteopetrosis virus MAV.2-O, in vitro. The infected tendon cells could be cloned in soft agar and kept in culture for at least 25 passages, a number not reached by uncloned infected cells. In contrast to many other virus-transformed fibroblasts, these cells continued making collagen and fibronectin, and there were no gross morphological changes as observed in the light microscope. Changes were seen in their cytoskeletal structure, however, as observed by immunofluorescence. The cloned cells were not tumorigenic in nude mice, nor had they an altered pattern of protein phosphorylation. MAV.2-O-infected fibroblasts and the cloned cells synthesized 2-3 times more collagen type I, the main product of their biosynthetic machinery, than control cells. The proportion of the total cellular RNA consisting of specific mRNAs for the precursor of collagen, procollagen pro-alpha 1 and pro-alpha 2 chains, was higher in the infected cells than in normal fibroblasts. Southern blotting experiments indicated that there was no rearrangement of the collagen genes after infection with this virus. Furthermore, large viral DNA fragments were not integrated into the immediate vicinity of the 5' end of the alpha 2-collagen gene.

Sequences in the gag-pol-5'env region of avian leukosis viruses confer the ability to induce osteopetrosis.

DNA sequences encoding the genomes of three subgroup E avian leukosis viruses have been molecularly cloned. Virus recovered from one of these cloned DNAs (pRAV-0) caused no osteopetrosis while virus recovered from the second (lambda NY203) caused late onset osteopetrosis and virus recovered from the third (lambda NTRE-2) caused intermediate onset osteopetrosis. Restriction endonuclease fragments of the cloned viral DNAs were used to construct recombinant viruses that could be used to test for the role of gag-pol-5'env and 3'env-LTR sequences in the induction of osteopetrosis. The results of the pathogenicity tests indicate that gag-pol-5'env sequences confer the ability to induce osteopetrosis while 3'env-LTR sequences influence the time of onset and the severity of osteopetrosis.