Feline oncovirus-associated cell membrane antigen and feline leukemia virus group-specific antigen expression in bone marrow and serum.

Feline retrovirus-induced possible preneoplastic lesions were identified by detection of feline oncovirus-associated cell membrane antigen (FOCMA) in bone marrow (BM) and mesenteric lymph nodes of noninbred, domestic short- or long-hair kittens. Cell-bound FOCMA was detected 1-3 weeks after feline leukemia virus (FeLV) infection and at least 6 months before a tumor was evident. BM FOCMA was detected earlier and reached higher levels at early periods in cats that became persistently viremic. Serum FeLV group-specific antigen (gsa) increased more rapidly and reached a higher level in cats that became persistently viremic, but nonviremic cats still had detectable gsa as late as 7-9 months after FeLV infection, even in the presence of high levels of viral antibody. The presence in nonviremic cats of FeLV gsa in sera and FOCMA in BM and sera suggests that these cats may have a persistent, low-level FeLV infection.

Feline oncornavirus-associated cell membrane antigen. V. Humoral immune response to virus and cell membrane antigens in cats inoculated with Gardner-Arnstein feline sarcoma virus.

Tumor growth responses in 5- to 6-week-old kittens inoculated with the Gardner-Arnstein strain of feline sarcoma virus exhibited three distinct pattern: 1) complete tumor regression or no detectable tumor growth in approximately one-third of 43 inoculated kittens, 2) rapid tumor progression which led to debilitation and death within 16.2 +/- 4.2 weeks following infection in an additional one-third, and 3) slow tumor growth or temporary regressions in the remaining third. The feline oncornavirus-associated cell membrane antigen (FOCMA) antibody response was closely correlated with tumor progression; rapid progressors had the lowest antibody titers, whereas those in the "no tumor or permanent regression" categories had the highest titers. These results agreed with those previously observed with another virus strain, the Snyder-Theilen feline sarcoma virus. Cats in the intermediate categories of tumor growth also had intermediate levels of FOCMA antibody. The presence of virus-neutralizing (VN) activity was not always correlated with anti-FOCMA activity. Animals in the rapid-progressor category, compared to the regressors or slow progressors, were more likely to have detectable VN antibody during early periods. Conversely, animals in the regressor group or group with no tumors were more likely to show an early rise in detectable anti-FOCMA activity than animals in either of the progressor groups.

Determinants of susceptibility and resistance to feline leukemia virus infection. I. Role of macrophages.

The role of autochthonous peritoneal feline macrophages (M theta) in the age-related resistance of cats to feline leukemia virus (FeLV) was investigated by a study of the functional properties and FeLV susceptibility of M theta from kittens and adult cats and the effect of hydrocortisone (HC) and silica on M theta-FeLV interactions. Although the phagocytic functions of isolated M theta from kittens and adults were equivalent, the mean FeLV susceptibility of M theta from kittens was five times that of M theta from adult cats, thus establishing a direct correlation between the age-related susceptibility of cats and M theta from cats to FeLV. M theta of viremic cats were found to be infected with FeLV in vivo; virus titers were slightly higher than those obtained after in vitro infection of M theta, M theta from cats that had experienced regressive FeLV infection were not significantly more resistant to FeLV infection in vitro than were M theta from naive adult specific-pathogen-free cats. HC, which has been shown to enhance the in vivo FeLV susceptibility of cats, also enhanced the permissiveness of M theta from cats to FeLV in vitro (600-fold for M theta from adult cats and 200-fold for M theta) from kittens. M theta permissiveness to FeLV was highly sensitive to HC and occurred in M theta infected in vivo or in vitro. In parallel with the effect of HC on the natural resistance of cats to FeLV, administration of silica before virus inoculation also markedly enhanced the FeLV susceptibility of adult cats. Silica was toxic for isolated M theta but not for lymphocytes in vitro, and silica produced monocytopenia and neutrophilia, delayed skin allograft rejection, and augmented feline oncovirus-associated cell membrane antigen antibody responses in vivo. These experiments indicate that M theta were linked to the natural resistance of cats to FeLV and that the temporary elimination of M theta functions (e.g., by silica) and/or the conversion of the M theta-FeLV relationship from a nonpermissive to a permissive state (e.g., by corticosteroids) resulted in failure of early virus containment, in persistent virus amplification in hemolymphatic tissues, and in subsequent FeLV-related proliferative or antiproliferative disease.

Determinants of susceptibility and resistance to feline leukemia virus infection. II. Susceptibility of feline lymphocytes to productive feline leukemia virus infection.

The interplay between feline leukemia virus (FeLV) and feline lymphocytes (lc) infected in vitro or in vivo was investigated. Surface marker analysis and viral infectivity (VI) assays of lc populations were used to determine susceptibility of lc subsets to FeLV. The principal FeLV-replicating cell in the mesenteric lymph node of persistently infected, preleukemic cats was a nonadherent, complement receptor (CR)-bearing lc (B-cell). The lymph nodes of preleukemic cats also had increased numbers of uninfected T-cells [cells forming rosettes with guinea pig erythrocytes (GPE)] and cells with receptors for the Fc portion of 7S IgG (Fc gamma R cells) as compared with lymph nodes of age-matched specific-pathogen-free (SPF) cats. The induction of productive infection of feline peripheral blood mononuclear leukocytes (PBL) in vitro depended on a 48-hour in vitro preincubation period before virus exposure. The equivalent susceptibility of whole and adherent cell-depleted PBL to productive infection and the failure of hydrocortisone to enhance viral infection were compatible with identification of the FeLV-replicating cell as an lc. Furthermore, lc from susceptible SPF kittens replicated 50 times as much FeLV as did lc from resistant adult SPF cats. The Ic productively infected with FeLV after in vitro exposure were more precisely identified with the use of Ficoll-Isopaque density gradient separations of rosetted and nonrosetted lc. Whole PBL, GPE rosette-positive PBL (T-cells), and CR-positive PBL (B-cells) were permissive to FeLV infection, and maximal VI was evident at 14 days after exposure. The substantial (1,325-fold) increment in VI found in the Fc gamma R-depleted PBL suggested a role for Fc gamma R cells in the containment of FeLV infection. Unstimulated mononuclear leukocytes from blood, spleen, lymph node, thymus, and marrow were susceptible to productive FeLV infection after in vitro exposure. The degree of spontaneous DNA synthesis in marrow, thymus, and spleen but not lymph node or PBL was inversely related to permissiveness to viral replication. Mitogen activation of lc was associated with decreased viral replication when either T-cell mitogens (concanavalin A, phytohemagglutinin, or pokeweed mitogen) or a B-cell mitogen (dextran sulfate) was used. Virus production by spleen cells and PBL was enhanced twofold to tenfold by prior lc stimulation by the B-cell mitogen, lipopolysaccharide, or protein A-bearing Staphylococcus aureus, a mitogen for feline T-cells with Fc gamma R. Both productively infected (preincubated) and nonproductively infected (freshly isolated) PBL transferred infectious FeLV to autochthonous peritoneal macrophages (M theta); most of the virus in PBL-peritoneal M theta cocultures was produced by adherent cells, irrespective of whether the adherent or nonadherent cell population was inoculated originally.

Horizontal transmission of feline leukemia virus under experimental conditions.

Thirty-seven specific-pathogen-free (SPF) cats ranging from newborn to 1 year were inoculated with the Rickard strain of feline leukemia virus (FeLV). Each inoculated cat shared a cage with a control SPF cat for 40 weeks post inoculation. After 4-5 weeks, 20 of the inoculated cats became group-specific antigen (gsa)-positive; the other 17 remained gsa-negative but developed virus neutralizing and feline oncornavirus cell membrane-associated antigen antibody titers. Seventeen of the control cats in contact with the gsa-positive cats developed evidence of FeLV infection 4-18 weeks after virema was detected in their inoculated cage mates. Of the control cats in contact with inoculated cats that remained gsa-negative, none developed evidence of FeLV infection. Data indicated that the gsa-positive state in cats inoculated with FeLV correlated with the capacity for horizontal transmission of the virus.

Feline leukemia virus infection: age-related variation in response of cats to experimental infection.

Sixty-seven specific-pathogen-free cats of various ages (newborn, 2 wk, 1 mo, 2 mo, 4 mo, and 1 yr) were inoculated ip with either the Rickard (R) or the Kawakami-Theilen (KT) strain of feline leukemia virus (FeLV). Susceptibility to FeLV was judged by induction of a) FeLV group-specific antigens (gsa) in leukocytes, b) FeLV-related disease, c) antibody to feline oncornavirus-associated cell membrane antigen (FOCMA), and d) virus-neutralizing (VN) antibody. Susceptibility to FeLV-decreased with age. Persistent viremia and FeLV-related disease developed in 100% of cats inoculated as newborns, in 85% of cats inoculated at 2 weeks to 2 months of age, and in 15% of cats inoculated at 4 months or 1 year of age. Cats susceptible to FeLV leukemogenesis became persistently FeLV gsa-positive (viremic) at 4 weeks post inoculation and thereafter and produced little or no FOCMA or VN antibody. Cats that resisted leukemogenesis by FeLV all developed persistent FOCMA and VN titers and never became FeLV gsa-positive. The disease in inoculated cats was influenced by virus strain; FeLV-R induced predominantly thymic lymphosarcoma, whereas FeLV-KT caused fatal nonregenerative anemia without concurrent neoplasia.

Characterization of feline T-and B-lymphocytes and identification of an experimentally induced T-cell neoplasm in the cat.

Membrane markers of feline T- and B-lymphocytes were identified for further investigation of leukemogenesis in the cat. Feline T-cells formed spontaneous erythrocyte rosettes with guinea pig (GPE) and rat erythrocytes (RE). The receptors for GPE and RE were separate entities and expressed independently on lymphoid cell membranes. The RE receptor appeared to be present only on more mature or differentiated T-cells, whereas the GPE receptor reacted with a broader population that included less differentiated T-cells. Feline B-cells bore a complement receptor that was detected by adherence of SE coated with antibody and complement. Malignant lymphoblasts obtained from thoracic fluid of cats with feline leukemia virus (FeLV)-induced thymic lymphosarcomas, as well as FL-74 cells (a FeLV-transformed feline lymphoblastoid cell line) expressed T-cell markers. These results provided definitive evidence for markers of feline T- and B-cells and identified an experimentally induced T-cell lymphosarcoma.

Lymphocyte mitogen reactivity and enumeration of circulating B- and T-cells during feline leukemia virus infection in the cat.

Mitogen-induced blast transformation of peripheral blood lymphocytes and quantitative changes in circulating T- and B-cells were studied serially in cats inoculated with feline leukemia virus (FeLV). Concanavalin A-induced blast transformation sharply declined beginning at 5 weeks post inoculation (Pl) in FeLV-infected cats when compared to age-matched uninfected control cats. Similar but less consistent changes were seen in responses to pokeweed mitogen-induced stimulation. In most infected kittens this defect persisted until they died from thymic lymphosarcoma, 15-24 weeks Pl. An early lymphopenia, due primarily to a decrease in circulating B-cells, occurred in infected cats 5-8 weeks Pl. Following a return of total and B-lymphocytes to control values, infected cats developed increased numbers of T-cells at 16 or more weeks Pl, which correlated with circulating lymphoblastic lymphocytes bearing T-cell markers. These results correlated neoplasia arising in a thymus-derived lymphocyte population with mitogenic hyporeactivity in the preneoplastic period and suggested that FeLV-induced immune alterations may be a necessary antecedent of leukemogenesis in the cat.

Feline oncornavirus-associated cell membrane antigen. VI. Cytotoxic antibody in cats exposed to feline leukemia virus.

Feline leukemia virus (FeLV)-infected specific pathogen-free (SPF) cats, normal uninfected SPF cats, and healthy cats from leukemic households were tested for antibody reactive to the feline oncornavirus-associated cell membrane antigen (FOCMA)-containing target cell line FL-74 by microcytotoxicity and indirect membrane immunofluorescence. Of the infected SPF animals, 81% showed concordant reactivity for the two tests. In contrast, only 55% of the healthy cats known to be naturally exposed to FeLV for long periods showed such concordance. FOCMA antibody could not be detected in normal SPF cats by either indirect membrane immunofluorescence or microcytotoxicity. Most cats in the FeLV-infected SPF group developed antibody detectable by both procedures by the fifth week post inoculation. Antibody detectable by membrane immunofluorescence persisted in a high percentage (75-90%) of the animals throughout the observation period of 19 weeks; after 9 weeks, fewer cats had antibody that was also detectable by microcytotoxicity.

Preparation of cell-free feline oncornavirus-associated cell membrane antigen.

Cell-free feline oncornavirus-associated cell membrane antigen (FOCMA) was prepared from a feline lymphoblastoid cell line of tumor origin (FL-74). Membrane fractions, separated on sucrose density gradients, and papain-solubilized products were found to contain FOCMA as determined by their capacity to inhibit reference cytotoxic cat antisera.

Feline oncornavirus-associated cell membrane antigen. IV. Antibody titers in cats with naturally occurring leukemia, lymphoma, and other diseases.

Cats with naturally occurring leukemia and lymphoma had low or negative humoral antibody titers to the feline oncornavirus-associated cell membrane antigen (FOCMA). Geographic differences were seen in the relative frequencies of various forms of lymphoproliferative neoplasms. Lymphatic leukemia and thymic lymphoma were most common in Boston, whereas alimentary lymphoma was most frequent in Glasgow. No significant differences were found in geometric mean FOCMA antibody titers for the various forms of leukemia-lymphoma or for feline leukemia virus (FeLV)-positive as compared to FeLV-negative cats. Approximately 70% of 76 Boston cats with nonregenerative anemias were FeLV gs antigen (gsa) positive; this was similar to the percentage with leukemia-lymphoma from the same population that was positive. Fifty-five to 62% of the Boston cats with other infectious diseases, such as peritonitis and septicemia, were gsa positive. We postulate that this is due to a predisposition to infectious diseases by the immunosuppressive action of FeLV. Young cats from the Boston population that developed lymphoma, infectious peritonitis, and certain other diseases were more likely to be FeLV gsa positive than older cats with the same diseases.

Infection of feline embryo adherent cells with feline leukemia virus: feline oncornavirus-associated cell membrane antigen expression and morphologic transformation.

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.

Antibody in cats to mammalian RNA tumor virus interspecies antigens.

The studies were undertaken to determine whether the cat, a mammalian species that carries xenotropic endogenous C-type virus(es) and in addition undergoes horizontally transmitted oncogenic C-type RNA tumor virus infections, responds immunologically to the mammalian C-type virus interspecies antigens. Sera from normal cats and from cats with spontaneous or virus-induced neoplasms were examined for antibodies to interspecies antigen antigen by complement-fixation inhibition, by inhibition of the paired radioiodine-labeled antibody technique (PRILAT inhibition), and by two-step radioimmunoelectrophoresis. Using three separate complement-fixation inhibition systems designed to detect antibodies to interspecies antigen(s), 23 of 23 sera from tumor-bearing cats and 24 of 31 sera from normal cats were positive in both systems. The negative sera were from germ-free cats. Among the 49 positive sera, 47 yielded titers of 1:16 or greater by one or more complement-fixation inhibition tests. Of these 47 sera, 42 were positive by the paired radioiodine-labeled antibody technique inhibition test; the 5 paired radioiodine-labeled antibody technique-negative sera were from normal specific-pathogen-free cats. Direct reaction with the interspecies determinant on the p30 protein from Rauscher murine leukemia virus by immunoglobulin from cats immunized with feline leukemia virus was shown by two-step radioimmunoelectrophoresis. The feline antibody was also identified as an immunoglobulin by column chromatography and two-step radioimmunoelectrophoresis. These antibodies did not fix guinea pig complement during reaction with the interspecies antigen. That other mammals may produce similar noncomplement-fixing (guinea pig) antibodies to RNA tumor virus antigens is likely.

Inhibition of concanavalin A stimulation of feline lymphocytes by inactivated feline leukemia virus.

In a lymphocyte blast transformation assay, the response of feline lymphocytes to concanavalin A was suppressed 20 to 65 percent in the presence of inactivated feline leukemia virus. The decrease was not due to viral cytotoxicity, as determined by trypan blue viability counts, nor was the virus binding the concanavalin A and interfering with its mitogenic stimulation. The virus may be biochemically repressive in itself, interfering with cell-mediated immunity within the feline system.

Inhibition of human lymphocyte mitogen and antigen response by a 15,000-dalton protein from feline leukemia virus.

Peripheral blood lymphocyte response of normal human subjects to mitogens and antigens was suppressed by a 15,000-dalton protein (p15) from a C-type feline leukemia virus. Four of six subjects were suppressed 70 to 96% when responding to concanavalin A or phytohemagglutinin in the presence of 5.0 microgram of p15. The three subjects who responded to streptokinase-streptodornase and Candida were suppressed 68 to 91% when cultured with 5.0 microgram of p15. The subviral protein did not appear to be cytotoxic at doses reported. Lymphocyte membrane studies with fluorescein isothiocyanate-concanavalin A revealed a reduction in concanavalin A-induced cap formation of 51 to 91% in the presence of the same dose of p15. These results demonstrate that in vitro immunological dysfunction in human lymphocytes can be induced by a C-type virion protein.

Inactivation of tumor cell-associated feline oncornavirus for preparation of an infectious virus-free tumor cell immunogen.

Ultraviolet (UV) and thermal methods of inactivating the oncogenic potential of C-type particle-producing feline oncornavirus-induced tumor cells were developed. The techniques were evaluated by several parameters for their use in preparation of cellular immunogens. The UV inactivation dose required to reduce the number of focus-forming units per ml by 1 log10 for FL-74 lymphoblastoid cell-associated feline leukemia virus was 44,000 ergs/sq mm, and the thermal inactivation dose required to reduce the number of focus-forming units per ml by 1 log10 at 45 degrees was 16 min. Inactivation of greater than 6 log10 of virus per ml associated with 4 x 10(8) cells required a UV dose of 270,000 ergs/sq mm, 100 min at 45 degrees or 3 min at 56 degrees. All three treatments concomitantly destroyed the replicating potential of FL-74 cells as shown by their inability to propagate under normal growth conditions and to incorporate [3H]thymidine into nuclear DNA. UV inactivation and thermal inactivation at 45 degrees allowed the best retention of feline oncornavirus-associated cell membrane antigen. A 50% loss in antigenic activity was observed as a result of 56 degrees treatment, but this method was the only one that did not destroy the surface structural integrity of FL-74 cells.

Relationship between feline leukemia virus antigen expression and viral infectivity in blood, bone marrow, and saliva of cats.

Correlation was greater than 90% between feline leukemia virus (FeLV), group-specific antigen (GSA) in leukocytes, and viral infectivity (VI) in serum or plasma from 132 cats infected with either the Rickard strain of FeLV, the Snyder-Theilen strain of feline sarcoma virus, or field strains of FeLV. Detection of GSA in blood cells was at least as sensitive as detection of VI in serum. In 45% of FeLV GSA-positive cats inoculated with FeLV-Rickard strain, VI was detected in saliva. No saliva samples from GSA-negative cats had VI. Sequential bone marrow biopsies from 34 cats inoculated with Snyder-Theilen feline sarcoma virus indicated that the correlation between FeLV GSA in bone marrow cells and blood cells was virtually 100%. FeLV GSA appeared in bone marrow leukocyte precursors 1 week before its appearance in peripheral blood leukocytes in 50% of the cats. The FeLV GSA-positive state was transient (3 to 6 weeks) in 34% of the Snyder-Theilen feline sarcoma virus-inoculated cats.

Influence of adrenal corticosteroids on the susceptibility of cats to feline leukemia virus infection.

The natural resistance of adult specific-pathogen-free cats to feline leukemia virus (FeLV) was abrogated by treatment with various doses of a synthetic corticosteroid, methylprednisolone acetate (MPA), prior to either oral-nasal or i.p. inoculation of FeLV. Persistent viremia was induced in 82% (18 of 22) of MPA-treated cats versus 11% (1 of 9) of age-matched control cats. MPA-treated FeLV-inoculated cats developed prolonged lymphopenia (2 to 8 weeks postinoculation) and a delayed antibody response to the feline oncornavirus-associated cell membrane antigen. The distribution of FeLV group specific antigen in tissues of MPA-treated, FeLV-inoculated cats suggested that corticosteroids enhanced susceptibility to FeLV by impairing early viral containment in the reticuloendothelial and lymphoid tissues.

Mobility of lymphocyte surface membrane concanavalin A receptors of normal and feline leukemia virus-infected viremic felines.

The virus-associated depression of concanavalin A mitogenesis which accompanies feline leukemia virus-induced cat lymphoma was investigated by comparing lymphocyte surface receptor mobility of normal cats to that of viremic diseased animals. The mechanics of feline lymphocyte receptor mobility were studied using fluorescein-conjugated concanavalin A to quantitate lymphocyte capping. The results of a study of 21 disease-free animals showed that cat lymphocytes undergo appreciable concanavalin A capping, with a mean capping rate of 17% under conditions developed in this study. In contrast, morphologically normal peripheral blood lymphocytes of six feline leukemia virus-infected viremic cats, with or without lymphoma, exhibited a mean capping of only 7%, significantly less than that of the control animals (p less than 0.005). These findings suggest that a membrane-related lymphocyte deficiency accompanies the development of virus-induced lymphoma in the cat.

Increased susceptibility to feline leukemia virus infection in cats exposed to methylnitrosourea.

Exposure of adult specific-pathogen-free cats to methylnitrosourea resulted in increased susceptibility to infection by feline leukemia virus. A greater proportion of cats exposed to methylnitrosourea and feline leukemia virus (69%) became persistently viremic than those exposed to feline leukemia virus alone (17%). Segmented neutrophils were reduced by 90 to 99% within 3 days following exposure to methylnitrosourea, (15 to 20 mg/kg) whereas the effects on lymphocytes and erythrocytes, although less obvious, were also detected.