Feline Oncornavirus-associated Cell Membrane Antigen Research Articles

Exposure of cats to low doses of FeLV: seroconversion as the sole parameter of infection

In felids, feline leukemia virus (FeLV) infection results in a variety of outcomes that range from abortive (virus readily eliminated and never detectable) to progressive infection (persistent viremia and viral shedding). Recently, a novel outcome was postulated for low FeLV infectious doses. Naïve cats exposed to faeces of persistently infected cats seroconverted, indicating infection, but remained negative for provirus and p27 antigen in blood. FeLV provirus was found in some tissues but not in the bone marrow, infection of which is usually considered a necessary stage for disease progression. To investigate the impact of low FeLV doses on young cats and to test the hypothesis that low dose exposure may lead to an unknown pathogenesis of infection without involvement of the bone marrow, 21 cats were infected oronasally with variable viral doses. Blood p27, proviral and viral loads were followed until week 20 post-infection. Tissue proviral loads were determined as well. The immune response was monitored by measuring FeLV whole virus and p45 antibodies; and feline oncornavirus-associated cell membrane antigen (FOCMA) assay. One cat showed regressive infection (transient antigenemia, persistent provirus-positivity, and seroconversion) with provirus only found in some organs at sacrifice. In 7 of the 20 remaining cats FOCMA assay positivity was the only sign of infection, while all other tests were negative. Overall, the results show that FeLV low dose exposure can result in seroconversion during a presumed abortive infection. Therefore, commonly used detection methods do not detect all FeLV-infected animals, possibly leading to an underestimation of the prevalence of infection.

Genetically-engineered subunit vaccine against feline leukaemia virus: protective immune response in cats

A recombinant retroviral subunit vaccine has been developed that successfully protects cats from infectious feline leukaemia virus (FeLV) challenge. The antigen used is a non-glycosylated protein derived from the envelope glycoprotein of FeLV subgroup A, expressed in Escherichia coli. This recombinant protein, rgp70D, includes the entire exterior envelope protein gp70, plus the first 34 amino acids from the transmembrane protein p15E. The vaccine consists of purified rgp70D absorbed on to aluminium hydroxide and used in conjunction with a novel saponin adjuvant. Cats immunized with this formulation developed a strong humoral immune response, including neutralizing and feline oncornavirus-associated cell membrane antigen antibodies. Vaccinated animals showed an anamnestic response upon intraperitoneal challenge with FeLV-A, and were protected from viral infection. In contrast, the control animals developed viraemia shortly after the challenge, which in most cases became chronic. Formulation of the same antigen with other widely used adjuvants elicited poor protective immune responses in cats.

Effect of diethylcarbamazine on testing serum samples for retroviral antibodies

Previous investigations have revealed that in vivo use of diethylcarbamazine ( N,N-diethyl-4-methyl-1-piperazine carboxamide; DEC), the mainstay of prevention and therapy for filariases, results in increased titers of antibody to feline oncornavirus-associated cell membrane antigen (FOCMA) in feline leukemia virus (FeLV)-infected cats.In the present study, the effect of in vitro DEC on detection of serum antibodies to retroviruses was investigated. The addition of DEC (5 mg ml −1, pH 7·3–7·4) to the serum diluent resulted in detection of antibody to FOCMA by indirect membrane immunofluorescence in five of five FeLV-infected cats, whereas samples from all five cats tested negative without the addition of DEC. In vitro DEC also increased the absorbance values generated by enzyme immunoassay human immunodeficiency virus type 1 (HIV-1) antibody testing of samples from (a) persons with AIDS-related disease; (b) persons with helper T-lymphocytes < 500 mm −3 blood; and (c) persons testing positive for antibody to HIV-1 env- but not gag-related antigens as determined by radioimmunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis ( RIP-SDS PAGE ). We conclude that in vitro as well as in vivo use of DEC may alter results of antibody testing for retroviruses.

Serological survey of feline leukemia virus infection and the outcome of antibody-positive cats

A serological survey was carried out to examine the presence of antibodies against feline leukemia virus (FeLV) and feline oncornavirus-associated cell membrane antigen (FOCMA) in 208 cat sera collected at Teikyo University School of Medicine. Seven cats (3.4%) were positive for FeLV antibodies by enzyme-linked immunosorbent assay whereas no cat was positive for FOCMA antibody by indirect membrane immunofluorescent test. Anemia, leukemia and/or lymphoma formation were not observed in these FeLV antibody-positive cats. But among these seven cats, three were positive for toxoplasma antibodies. One of them was also positive for Chlamydia psittaci antibody and it died in pneumonia. Among the four toxoplasma antibody negative cats, one was died in eosinophilic granuloma. Furthermore, two of three cats, which were used for experiments, had cold and took therapy.

Feline oncornavirus-associated cell membrane antigen: A viral and not a cellularly coded transformation-specific antigen of cat lymphomas

The feline oncornavirus-associated cell membrane antigen (FOCMA) was defined as a tumor antigen common to cat lymphomas and fibrosarcomas induced by feline leukemia virus (FeLV) and feline sarcoma virus (FeSV), respectively. The antigen was recognized by sera from cats thought to be resistant to leukemogenesis. We report here that a common denominator in the activity of naturally occurring viremic cat antisera to FOCMA is, in fact, their reactivity to FeLV C antigenic determinants. The cat antisera, monoclonal antibodies to FOCMA, and monoclonal antibodies to FeLV C, all reacted in immunofiuorescence assays with FeLV C-infected cells and immunoprecipitated a molecule electrophoretically indistinguishable from envelope glycoprotein of FeLV. Viremic cat antisera to FOCMA bound to budding virus particles of FeLV C-infected cells, even though some of them could not be absorbed by mature virion proteins. Thus, the unusual feature of cat antibodies to FOCMA is their binding to nascent but not to mature virus particles. FOCMA-positive cat lymphomas expressed antigenic determinants of FeLV-C gp70, with or without productive infection. FeLV-negative tumors not expressing FeLV C gp70 were also FOCMA negative. Furthermore, most of the viremic cat sera and the monoclonal antibodies to FOCMA did not react with FeSV-transformed nonproducer cells. The absence of FOCMA from these cells and from FeLV-negative lymphoid tumors and its presence in FeLV-C infected fibroblasts indicated that this antigen is virus encoded and not a cellular tumor-specific antigen.

Reactivation of latent feline leukaemia virus infection.

In most cats exposed to the contagious feline leukemia virus (FeLV), viral replication is contained in target haematopoietic tissues and elicits humoral immunity to FeLV and to the feline oncornavirus-associated cell membrane antigen (FOCMA). Recently, we and others have considered that these ostensibly self-limiting infections might be persistent nonproductive (latent) infections in certain haematopoietic cells. This hypothesis could account for the relapsing viraemias, protracted incubation periods, persistently high titres of antiviral and anti-FOCMA antibodies, appearance of FeLV p27 antigen in serum of otherwise non-viraemic animals and occurrence of FeLV-negative but FOCMA-positive leukaemias in naturally infected pet cats. Here we describe the reactivation of latent FeLV from myelomonocytic and lymphoid cells of cats immune to FeLV, cats bearing FeLV-negative tumours, and kittens congenitally exposed to FeLV. Furthermore, the reappearance of FeLV infection is suppressed by the Host's immune system but his can be altered by adrenal corticosteroid hormones in vivo and in vitro.

Feline leukemia - unique and cross-relating antigens on individual virus-producing tumors identified by complement-dependent antibody.

Following exposure to feline leukemia virus (FeLV), cats that resist leukemia produce complement dependent antibodies (CDA) which lyse leukemia cells in the presence of cat complement. The CDA activity has been shown to be directed to feline oncornavirus-associated cell membrane antigen (FOCMA) found on the surface of most feline leukemias. Despite the retrovirus etiology of feline leukemia, and the ubiquity of FOCMA expression by individual tumors, we found that CDA activity did not cross-react when five feline leukemia lines were compared (FL74, F422, 3272, 3281, and 79-14940). Profiles for CDA mediated lysis of the leukemia target lines were determined for sera from 149 FeLV exposed cats, and animals were identified whose sera would selectively lyse almost any possible combination of tumors from one to five lines tested. Cross absorption and cold target inhibition studies were performed on selected sera, and a spectrum of different antigens was identified which included both individual determinants and antigens shared by some of the tumor lines. Leukemias FL74 and F422 carried a cross-reactive determinant which was distinct from a second antigen shared by 3272 and 3281; lines F422, 3272 and 3281 each also carried a unique antigen. Leukemia 79-14940 did not cross-react with other target tumor lines. Targets of CDA mediated lysis on leukemia cells maybe associated with normal feline cell surface antigens, because CDA titers were sometimes reduced after serum absorption with normal cat spleen cells. The relevance of multiple FOCMA determinants is discussed in terms of tumor escape from immunity.

Characterization of clones of tumorigenic feline cells transformed by a chemical carcinogen.

Tumorigenic clonal lines derived from soft agar colonies induced by DMBA-transformed feline embryo cells were isolated and characterized. The morphologically altered clonal cells formed large aggregates, growing in this aggregate form when suspended in liquid growth medium above an agar base and forming colonies in soft agar with high efficiency. When inoculated into athymic nude mice, chemically altered clonal cells produced progressively growing sarcomas. Cells established from the tumors morphologically resembled the DMBA-transformed feline embryo cells and were characterized as cat cells by karyological analysis. The tumorigenic lines were negative for feline oncornavirus-associated cell membrane antigen (FOCMA), and for "gag-X" the transformation-related polyprotein which is encoded by the replication defective feline sarcoma virus.

Retrovirus-encoded transformation-specific polyproteins: expression coordinated with malignant phenotype in cells from different germ layers.

A transformation-associated polyprotein designated "gag-x" was previously shown to be induced by the feline sarcoma virus (FeSV) after the nonproductive transformation of rat or mink cells. We found that this protein was also expressed in cells derived from the native species (cat) with or without the production of feline leukemia helper virus (FeLV) and that cats could mount a humoral antibody response to the transformation-specific (x) portion of the molecule. Such antisera also reacted with the feline oncornavirus-associated cell membrane antigen (FOCMA) by membrane immunofluorescence. Expression of the gag-x protein was coordinated with malignant phenotype in that both transformed cat fibroblasts and cultured cells from a FeSV-induced melanoma expressed antigenically indistinguishable proteins of the same size. These cells are derived from different embryonic germ layers, suggesting that such transformation-related proteins may function in a pleiotropic manner when introduced by a virus.

Biochemical characterization of cells transformed via transfection by feline sarcoma virus proviral DNA.

Murine fibroblasts transformed by transfection with DNA from mink cells infected with the Snyder-Theilen strain of feline sarcoma virus and subgroup B feline leukemia virus were analyzed for the presence of integrated proviral DNA and the expression of feline leukemia virus- and feline sarcoma virus-specific proteins. The transformed murine cells harbored at least one intact feline sarcoma virus provirus, but did not contain feline leukemia virus provirus. The transformed murine cells expressed an 85,000-dalton protein that was precipitated by antisera directed against feline leukemia virus p12, p15, and p30 proteins. No feline oncornavirus-associated cell membrane antigen reactivity was detected on the surfaces of the transformed murine cells by indirect membrane immunofluorescence techniques. The 85,000-dalton feline sarcoma virus-specific protein was also found in feline cells transformed by transfection. However, these cells also contained env gene products. The results of this study demonstrate that the feline sarcoma virus genome is sufficient to transform murine cells and that expression of the 85,000-dalton gag-x protein is associated with transformation of both murine and feline cells transformed by transfection.

Relationship of the feline oncornavirus associated cell membrane antigen to a feline sarcoma virus encoded polyprotein.

The feline oncornavirus associated cell membrane antigen (FOCMA) is expressed on the membranes of cells transformed by infection with the feline leukemia virus (FOCMA-L) or feline sarcoma virus (FOCMA-S) (Hardy et al. 1977; Essex et al. 1977). Expression of FOCMA is transformation-specific and not dependent upon concomitant expression of antigens associated with feline leukemia virus (FeLV) or the endogenous virus RD114 (reviewed in Snyder et al., to be published a). In the natural environment FOCMA is the target for an effective immunosurveillance response: complement-dependent lytic antibodies directed to FOCMA reverse or prevent tumor development (Essex et al. 1975: Grant et al. 1977). FOCMA-L has been isolated from feline lymphosarcoma (LSA) cell membranes and has been shown to reside on a 70,000 dalton protein which is neither glycosylated nor phosphorylated (Snyder et al. 1978, to be published a). The nature of FOCMA-S is a subject of intense investigation at the present time.

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Feline embryo adherent cells were infected with the Richard or Kawakami-Theilen strains of feline leukemia virus (FeLV) and examined for feline oncornavirus-associated cell membrane antigen (FOCMA), viral group-specific antigen (gsa) production, and in vitro evidence of transformation. As early as 10 days after infection, when more than half of the infected cells were gsa positive, FOCMA was detected on 5-10 percent of the cells. Transitory morphologic alterations (epithelioid appearance and rounding) were first noted in most cultures around 20-30 days post infection. At this time, approximately 50% of the cells in infected cultures expressed FOCMA. Morphologic characteristics of transformed fibroblastic cells (rounded shape, disordered alignment, and low adhesion to substratum), as well as enhanced agglutinability by plant lectins and ability to grow in agar, were demonstrated in one of four FeLV-infected, FOCMA-positive cultures. Findings showed that FOCMA may be expressed in FeLV-infected monolayer cells independent of transformation as assessed by in vitro criteria.

Feline sarcoma virus induced in vitro progression from premalignant to neoplastic transformation of human diploid cells

Human diploid cells morphologically transformed by feline sarcoma virus were serially propagated under selective cell culture conditions. When injected into nude mice prior to passage in soft agar (0.35%), morphologically transformed cells did not produce tumors. However, when propagated under selective cell culture conditions, transformed cells grew in soft agar and, when injected subcutaneously into the subcapsular region of the n mu/n mu mice, produced neoplastic nodules histopathologically interpreted as fibromas. Karyological examination of cell populations grown out from the tumors confirmed that the tumors were composed of human cells. Examination of electron micrographs of the excised tumor tissue revealed the presence of budding virus particles. Tumor cells isolated from nude mice and morphologically transformed cells both contained the feline oncornavirus-associatied cell membrane antigen. It was concluded that expression of feline oncornavirus-associated cell membrane antigen is associated with an early stage of feline retrovirus-induced carcinogenesis, namely focus formation. In addition, it was shown that FeLV-FeSV can induce morphological transformation in human cells in vitro and that there is a requirement for the cells to passage through soft agar before subsequent tumor formation (neoplastic transformation) can be demonstrated.

Feline leukemia in Israel: prevalence of antibody to feline oncornavirus-associated cell membrane antigen in stray cats.

Feline lymphoma was recorded for the first time in pet cats in Israel. Furthermore, a serological survey in urban stray cats was carried out to determine the prevalence of antibody to feline oncornavirus-associated cell membrane antigen (FOCMA). A total of 91 cats was tested and only 5 has antibody to FOCMA. Of these 5 positive cats, 4 originated from an area where a feline leukemia virus-positive cat had been detected prior to the survey. The prevalence of stray cats with antibody to FOCMA is lower in Israel than found in similar studies in the USA and Scotland.

Monoclonal antibodies: detection of transformation-related antigens.

We have established a set of mouse hybridoma clones—using the technique of Köhler and Milstein (1975)—each producing an antibody that reacts preferentially with certain transformed cell types as opposed to their normal control cells. These clones were produced in the course of experiments designed to isolate hybridoma clones that produce antibodies to feline oncornavirus-associated cell-membrane antigen (FOCMA). FOCMA is defined by indirect immunofluorescent-antibody (IFA) assays on live FL74 cells, using FOCMA typing serum (Hardy et al. 1978); it is found either on the feline sarcoma virus (FeSV)-transformed fibroblasts (Essex et al. 1971) or in lymphomas produced presumably as a result of feline leukemia virus (FeLV) infection (Essex 1976; Hardy et al. 1978).

Isolation and characterization of proteins carrying the feline oncornavirus-associated cell-membrane antigen.

Isolation and characterization of proteins carrying the feline oncornavirus-associated cell-membrane antigen.

Restriction endonuclease mapping of unintegrated proviral DNA of Snyder-Theilen feline sarcoma virus: localization of sarcoma-specific sequences

Extrachromosomal DNA purified from mink cells acutely infected with the Snyder-Theilen strain of feline sarcoma virus (FeSV) was digested with restriction endonucleases, and the DNA fragments were electrophoretically separated, transferred to a solid substrate, and hybridized with radiolabeled DNA transcripts complementary to different portions of the FeSV RNA genome. Major DNA species 8.4 and 5.0 kilobase pairs (kbp) long represent the linear, unintegrated proviruses of Snyder-Theilen feline leukemia virus and FeSV, respectively. Transfection experiments performed with electroeluted DNAs showed that the 8.4-kbp form led to the production of replicating nontransforming virus in mink and cat cells; in contrast, the 5.0-kbp DNA produced helper virus-independent foci of transformation in mouse NIH/3T3 cells and helper virus-dependent foci in mink cells at an efficiency comparable to that obtained with unfractionated extrachromosomal DNA. Sites of restriction endonuclease cleavage for six enzymes were oriented with respect to one another within the FeSV provirus. EcoRI recognized cleavage sites at 0.3 to 0.4 kbp from each terminus of FeSV DNA, reducing the 5.0-kbp DNA to molecules 4.3 kbp long; this enzyme excised a large internal proviral DNA fragment of corresponding size from the DNA of FeSV-transformed mink nonproducer cells. By using DNA transcripts complementary to different portions of the FeSV genome, sarcoma-specific sequences (the FeSV src gene) were positioned within 2.1 and 3.4 kbp from the 5' end of the proviral DNA with respect to the viral RNA genome. The src gene is flanked at both ends by sequences shared in common with feline leukemia virus. The localization of src sequences to this region suggests that a portion of an FeSV polyprotein which contains feline oncornavirus-associated cell membrane antigen (FOCMA-S) is the major product of this gene.

Enhancement of feline leukemia virus-induced leukemogenesis in cats exposed to methylnitrosourea.

The effect of methylnitrosourea (MNU), a potent resorptive carcinogen, was evaluated for its influence on the susceptibility of adult cats to infection and induction of oncornavirus disease by feline leukemia virus (FeLV). Young adult cats at an age previously demonstrated to be highly resistant to FeLV, were injected intravenously with moderately toxic doses (15-20 mg/kg) of MNU alone or with infections FeLV (Rickard strain). Following exposure to virus and chemical, cats were monitored for antibody to the feline oncornavirus-associated cell membrane antigen (FOCMA), viremia by direct infectivity and the presence of gsa in peripheral blood leukocytes, and for toxic effects of MNU by hemogram analyses on peripheral blood. Of 8 cats injected with MNU + FeLV, 6 developed persistent viremia, 5 of which became debilitated from thymic lymphoma. Only 1 of 6 non-MNU-treated and infected cats of the same age became transiently viremic. FOCMA antibody development was markedly depressed in MNU + FeLV inoculated cats compared with cats inoculated with FeLV alone. Results show that MNU was apparently responsible for the obliteration of age-related susceptibility in cats to FeLV infection and induction of FeLV-related disease, and suggest that in nature exposure to toxic chemical carcinogens may act as factors which determine susceptibility to feline oncornaviruses in the cat.

Distribution of feline leukemia virus dna sequences in tissues of normal and leukemic domestic cats

Seroepidemiological studies indicate that leukemia and lymphoma in cats are horizontally transmitted and that feline leukemia virus (FeLV) is the agent responsible for the disease. Yet, in a significant proportion of leukemic cats, FeLV has not been identified, despite the presence of the feline oncornavirus-associated cell membrane antigen (FOCMA) and in some instances, epidemiological evidence indicating exposure to FeLV. Prompted by these so called virus negative cases of cat leukemia, we surveyed a total of 176 tissues from 50 cats for the presence of FeLV proviral DNA sequences. The animals studied included viremic and nonviremic cats that were either healthy or leukemic. We found: (1) DNA from virus-positive tissues from both healthy and leukemic cats hybridized 60–100% of 125I-FeLV (strain Rickard) RNA. (These values are normalized to hybrid yield obtained with DNA of cat cells infected with FeLV in culture, which is 55%.) (2) A few virus-negative tissues also hybridized 60–100% of FeLV RNA. These tissues were derived from lymphoma-bearing cats as well as healthy cats. (3) The majority of virus-negative tissues hybridized 30–60% of FeLV RNA, and in this group, tissues from leukemic cats did not show significantly higher hybridization. (4) The results did not define a particular tissue target site since comparable hybridization results were obtained with many different tissues. (5) A few virus-negative tissues from healthy cats hybridized only 15–30% FeLV RNA. This is considerably lower than the level found with the majority of virus-negative tissues (normal or leukemic) and may reflect the true level of endogenous FeLV related sequences in cats. If so, the higher hybridization levels (30–60% of FeLV RNA) observed with most virus-negative tissues indicates more widespread infection in cats than previously believed, and suggests integration of only partial provirus and/or infection of only a fraction of the cells in the tissue. If exogenous FeLV is involved in the leukemogenesis of “virus-negative” cats, the apparent lack of detectable proviral sequences in tissues of many of these cats over and above those of healthy cats could be interpreted in different ways, e.g., partial loss of provirus, integration of a small fragment, or presence of proviral sequences in a limited population of cells. These “virus-negative” cats may serve as a model ( S. M. Cotter and M. Essex, 1977, Amer. J. Pathol. 87, 265–268) for other animals, including man, where leukemia may sometimes be associated with certain type-C related subviral markers, but detection of provirus is unusual.

Studies on antibodies against feline leukaemia virus (FeLV) in cat sera and rabbit anti-FeLV sera: cross reaction and differences.

The indirect immunoferritin technique (IFT) that enables us to distinguish clearly whether an antibody reacts with a virus particle or only with the cell membrane, was used to study 25 cat sera and one rabbit anti-feline leukaemia virus (FeLV) serum using FL-74 cells as target. (1) All sera contained antibodies against FeLV even though 11 of the cats were viraemic at the same time; (2) from the effect of glutaraldehyde fixation of the FL-74 cells on the reaction with cat sera and the results of blocking experiments, it could be concluded that cat sera and rabbit anti-FeLV sera react partly with different antigenic specificities of FeLV, partly with the same antigens; and (3) the indirect membrane immunofluorescence test using FL-74 cells as target is not a good test to detect the presence of antibodies against feline oncornavirus-associated cell membrane antigen (FOCMA) because FL-74 cells produce a large quantity of FeLV and the fluorescence measured could be from antibodies against FeLV.