Feline cytotoxic immune mechanisms against virus-associated leukemia and fibrosarcoma

Humoral and cellular cytotoxic immune mechanisms of cats were compared against feline leukemia virus (FeLV)- and feline sarcoma virus (FeSV)-transformed cells. The groups of animals studied were nonexposed control cats; FeLV-infected immune or viremic tumor-bearing cats; FeSV-inoculated tumor progressor or regressor cats, and cats immunized with FeSV-transformed autochthonous fibroblasts (ATF). Sera containing complement-dependent antibodies (CDA), which lysed FeLV-producer lymphoma lines, had no cytotoxic effects when tested against FeLV-producer FeSV-transformed fibroblasts. Sera with lytic CDA activity were also tested for antibody-dependent cellular cytotoxic (ADCC) effects with peripheral blood lymphocytes (PBL) from nonimmune cats. No ADCC activity was detected against either lymphoid or fibroblast target lines. To demonstrate that cat PBL contained ADCC effector cells, antibody-coated murine target cells were employed and positive results obtained. Natural killer (NK) assays were performed using PBL from normal and tumor-bearing cats. Cytotoxic effects were only detectable to FeLV-producer lymphomas, and comparable levels of NK activity were found in normal and lymphoid tumor-bearing animals. In cats immunized with ATF, a population of effector cells was found in peripheral blood which had functional characteristics of cytotoxic T lymphocytes (CTL). The killing of ATF by CTL-like cells was not inhibited by FeLV/FeSV immune sera or by sera from autochthonous immune cats. The comparative importance of humoral and cellular cytotoxic mechanisms against FeLV- and FeSV-induced tumors is discussed.     

Isolation and characterization of circulating feline leukemia virus-immune complexes from plasma of persistently infected pet cats removed by ex vivo immunosorption

IgG and circulating IgG immune complexes (CIC) were purified from plasma of three pet cats persistently infected with feline leukemia virus (FeLV) by adsorption to, and elution from, Staphylococcus aureus Cowan I. CIC were then separated from free IgG by sucrose gradient ultracentrifugation and were analyzed for the presence of FeLV structural proteins and corresponding specific antibodies. Radioimmunoprecipitation analysis indicated that FeLV envelope (gp70) and major core (p30) proteins, along with cat IgG heavy and light chains, were present in the CIC from all three cats. Further analysis of the CIC from one of the cats also revealed the presence of FeLV core proteins p15 and p12. IgG purified from isolated CIC was also shown to bind specifically to purified FeLV gp70, p30, and p15. These data provide direct evidence for FeLV-specific CIC in the plasma of persistently viremic pet cats, and suggest these animals are immunologically response to the virus even though free antibodies against the major structural proteins cannot be demonstrated in standard assays.     

Viral inactivation in blood and red cell concentrates with benzoporphyrin derivative.

Using both the vesicular stomatitis virus (VSV) and feline leukemia virus (FeLV) as models we have shown that the photosensitizer benzoporphyrin derivative ring A (BPD), when activated with red light (600-700 nm), is effective in eliminating both free virus and virally infected cells from spiked blood products and whole blood drawn from viremic cats experimentally infected with FeLV, under conditions which appear to share red blood cells. The effect of photodynamic therapy on infected lymphocytes, as visualized by scanning electron microscopy, initially appeared as a limited area of tiny holes in the membrane. These holes were subsequently seen to increase in size until the membrane appeared completely decomposed. The red cell membranes however, seem to be undamaged by such photodynamic treatment.     

Induction of protective immune response in cats by vaccination with feline leukemia virus iscom

An effective candidate subunit vaccine consisting of the gp 70/85 of feline leukemia virus (FeLV) was prepared by using the immunostimulating complex (iscom) method for the presentation of membrane proteins of enveloped viruses. Two 32-wk-old specific pathogen-free (SPF) cats were immunized with a FeLV iscom vaccine prepared from the supernatant fluid of the FL74 tumor cell line without adjuvant. Both cats developed FeLV serum antibodies, as measured in an enzyme-linked immunosorbent assay (ELISA) and in a virus neutralization test. A proportion of the antibodies were directed to an epitope located on gp70/85, which was shown in competition ELISA with a peroxidase-labeled virus-neutralizing monoclonal antibody to be shared by all three subtypes of FeLV. The protective effect of FeLV iscom was studied by vaccinating six 8-wk-old SPF cats with iscom prepared from cell culture supernatant of another tumor cell line F422, followed by oronasal challenge with 10(6) ffu FeLV-A (strain Glasgow-1). Six unvaccinated cats were also challenged with the same dose of FeLV. The vaccinated cats developed FeLV serum antibodies, some of which were directed to the shared epitope on gp70/85. At 10 wk after challenge, none was viremic, whereas three of the control cats had developed FeLV viremia. The potential of FeLV iscom as a vaccine against FeLV-associated disease in cats, and of iscom vaccines for protection against mammalian retrovirus infections, is discussed.     

Feline leukemia virus infection as a potentiating cofactor for the primary and secondary stages of experimentally induced feline immunodeficiency virus infection.

Preexistent feline leukemia virus (FeLV) infection greatly potentiated the severity of the transient primary and chronic secondary stages of feline immunodeficiency virus (FIV) infection. Of 10 FeLV-FIV carrier cats, 5 died of experimentally induced FIV infection, compared with 2 deaths in 10 cats infected only with FeLV and 1 death in 7 cats infected only with FIV. FIV-infected cats with preexistent FeLV infections developed severe depression, anorexia, fever, diarrhea, dehydration, weight loss, and leukopenia 4 to 6 weeks after infection and were moribund within 2 weeks of the onset of signs, whereas cats infected only with FIV developed much milder self-limiting gross and hematologic abnormalities. Pathologic findings in dually infected cats that died were similar to those observed previously in cats dying from uncomplicated primary FIV infection but were much more widespread and severe. Coinfection of asymptomatic FeLV carrier cats with FIV did not increase the levels of FeLV p27 antigen present in their blood over that seen in cats infected with FeLV alone. The amount of proviral FIV DNA was much higher, however, in dually infected cats than in cats infected only with FIV; there was a greater expression of FIV DNA in lymphoid tissues, where the genome was normally detected, and in nonlymphoid tissues, where FIV DNA was not usually found. Dually infedted cats that recovered from the primary stage of FIV infection remained more leukopenic than cats infected with FIV or FeLV alone, and their CD4+/CD8+ T-lymphocyte ratios were inverted. One of these cats developed what was considered to be an opportunistic infection. It was concluded, therefore, that a preexistent FeLV infection in some way enhanced the expression and spread of FIV in the body and increased the severity of both the resulting transient primary and chronic secondary stages of FIV infection. This study also demonstrated the usefulness of the FIV model in studying the role of incidental infectious diseases as cofactors for immunodeficiency-causing lentiviruses.     

Epizootiology and management of feline leukemia virus in the Florida puma

Feline leukemia virus (FeLV) was not detected in Florida pumas (Puma concolor coryi) in almost 20 yr of surveillance; however, the finding of two FeLV antigen-positive pumas during the 2002-2003 capture season led to an investigation of FeLV in the population. Between January 1990 and April 2007, the proportion of pumas testing FeLV antibody positive increased, with antibody-positive pumas concentrated in the northern portion of puma range. Five of 131 (4%) pumas sampled between July 2000 and April 2007 were viremic, with all cases clustered in Okaloacoochee Slough (OKS). Clinical signs and clinical pathology at capture were absent or included lymphadenopathy, moderate-to-severe anemia, and lymphopenia. All viremic pumas died; causes of death were septicemia (n=2), intraspecific aggression (n=2), and anemia/dehydration (n=1). Outcome after FeLV exposure in pumas was similar to that in domestic cats, with evidence of regressive, latent, and persistent infections. Management of the epizootic included vaccination, and as of April 2007, 52 free-ranging pumas had received one or more inoculations. Vaccinations were concentrated in OKS and in a band between OKS and the remainder of the puma population. There have been no new cases since July 2004; however, the potential for reintroduction of the virus remains.     

Longitudinal analysis of feline leukemia virus-specific cytotoxic T lymphocytes: correlation with recovery from infection

Feline leukemia virus (FeLV) is a common naturally occurring gammaretrovirus of domestic cats that is associated with degenerative diseases of the hematopoietic system, immunodeficiency, and neoplasia. Although the majority of cats exposed to FeLV develop a transient infection and recover, a proportion of cats become persistently viremic and many subsequently develop fatal diseases. To define the dominant host immune effector mechanisms responsible for the outcome of infection, we studied the longitudinal changes in FeLV-specific cytotoxic T lymphocytes (CTLs) in a group of na?ve cats following oronasal exposure to FeLV. Using (51)Cr release assays to measure ex vivo virus-specific cytotoxicity, the emerging virus-specific CTL response was correlated with modulations in viral burden as assessed by detection of infectious virus, FeLV p27 capsid antigen, and proviral DNA in the blood. High levels of circulating FeLV-specific effector CTLs appeared before virus neutralizing antibodies in cats that recovered from exposure to FeLV. In contrast, persistent viremia was associated with a silencing of virus-specific humoral and cell-mediated host immune effector mechanisms. A single transfer of between 2 x 10(7) and 1 x 10(8) autologous, antigen-activated lymphoblasts was associated with a downmodulation in viral burden in vivo. The results suggest an important role for FeLV-specific CTLs in retroviral immunity and demonstrate the potential to modulate disease outcome by the adoptive transfer of antigen-specific T cells in vivo.     

Tumorigenic potential of a myc-containing strain of feline leukemia virus in vivo in domestic cats.

The oncogenic capacity of a myc-containing strain of feline leukemia virus (FeLV), termed LC-FeLV, has been examined after inoculation of the virus into neonatal kittens. Like other myc-containing strains of FeLV, LC-FeLV may induce with relatively short latency, but does not necessarily induce, thymic lymphosarcoma in viremic animals. Naturally occurring and experimentally induced tumors are T-cell lymphomas which contain clonally integrated LC-FeLV proviral DNA and which cannot readily be cultivated in vitro in the presence or absence of exogenously supplied interleukin-2. Acquisition of myc by FeLV decreases the period of latency before the appearance of tumors but does not expand the spectrum of tumors induced by FeLV alone.     

Management measures to control a feline leukemia virus outbreak in the endangered Iberian lynx

The feline leukemia virus (FeLV) is a retrovirus that affects domestic cats all over the world. Its pathogenic effects generally include anemia, immunosuppression or tumors. Dissemination over populations is linked to cat sociality, because the virus is transmitted by direct contact. Although the domestic cat is its common host, FeLV infection has also been described in some wild felids. In the Iberian lynx Lynx pardinus , some sporadic FeLV infection cases have been reported since 1994, but an outbreak with the involvement of several animals has never been described until now. During spring 2007, an FeLV outbreak hit the Doñana (SW Spain) population. The infection rapidly spread throughout the densest subpopulation throughout Doñana. Infected animals showed very acute anemic disease, most of them dying in <6 months. To avoid FeLV dissemination, a control program was carried out that included removal of viremic lynxes, vaccination of negative individuals and reduction of the feral cat population. The program was implemented both in Doñana and in Sierra Morena populations. In Doñana, around 80% of the total lynx population and 90% of the outbreak focus subpopulation were evaluated. Seven out of the 12 infected individuals found died and two reverted to latency; the remaining viremic animals have been kept in captivity. The outbreak appears to have been successfully confined to the subpopulation where the virus appeared and no more cases have been found since August 2007. In the larger Sierra Morena population, 8% of the lynx population was surveyed. Thirty-four uninfected Iberian lynxes were vaccinated at least once. The FeLV prevalence was found to be 27% in the Doñana population and 0% in the Sierra Morena population.     

Retrovirus-induced feline pure red cell aplasia: the kinetics of erythroid marrow failure

Cats viremic with feline leukemia virus subgroup C (FeLV-C) develop pure red cell aplasia (PRCA) characterized by the loss of detectable late erythroid progenitors (CFU-E) in marrow culture. Normal numbers of early erythroid progenitors (BFU-E) and granulocyte-macrophage progenitors (CFU-GM) remain, suggesting that the maturation of BFU-E to CFU-E is impaired in vivo. We have examined the cell cycle kinetics of BFU-E and their response to hematopoietic growth factor(s) to better characterize erythropoiesis as anemia develops. Within 3 weeks of FeLV-C infection, yet 6-42 weeks before anemia, the traction of BFU-E in DNA synthesis as determined by tritiated thymidine suicide increased to 43 +/- 4% (normal 23 +/- 2%) while there was no change in the cell cycle kinetics of CFU-GM. In additional studies, we evaluated the response of marrow to the hematopoietic growth factor(s) present in medium conditioned by FeLV-infected feline embryonic fibroblasts (FEA/FeLV CM). With cells from normal cats or cats viremic with FeLV-C but not anemic, a 4-fold increase in erythroid bursts was seen in cultures with 5% FEA/FeLV CM when compared to cultures without CM. However, just prior to the onset of anemia, when the numbers of detectable CFU-E decreased, BFU-E no longer responded to FEA/FeLV CM in vitro. BFU-E from anemic cats also required 10% cat or human serum for optimal in vitro growth. These altered kinetics and in vitro growth characteristics may relate to the in vivo block of BFU-E differentiation and PRCA. Finally, when marrow from cats with PRCA was placed in suspension culture for 2 to 4 days in the presence of cat serum and CM, the numbers of BFU-E increased 2- to 4-fold although no CFU-E were generated. By 4 to 7 days, CFU-E were detected, suggesting that conditions contributing to the block of erythroid maturation did not persist. The suspension culture technique provides an approach to study further the defect in erythroid differentiation characteristic of feline PRCA.     

Multicentric T-cell lymphoma associated with feline leukemia virus infection in a captive namibian cheetah (Acinonyx jubatus)

This case report describes a multicentric lymphoma in a 4 yr old female wildborn captive cheetah (Acinonyx jubatus) in Namibia after being housed in an enclosure adjacent to a feline leukemia virus (FeLV) infected cheetah that had previously been in contact with domestic cats. The year prior to the onset of clinical signs, the wild-born cheetah was FeLV antigen negative. The cheetah subsequently developed lymphoma, was found to be infected with FeLV, and then rapidly deteriorated and died. At necropsy, the liver, spleen, lymph nodes, and multiple other organs were extensively infiltrated with neoplastic T-lymphocytes. Feline leukemia virus DNA was identified in neoplastic lymphocytes from multiple organs by polymerase chain reaction and Southern blot analysis. Although the outcome of infection in this cheetah resembles that of FeLV infections in domestic cats, the transmission across an enclosure fence was unusual and may indicate a heightened susceptibility to infection in cheetahs. Caution should be exercised in holding and translocating cheetahs where contact could be made with FeLV-infected domestic, feral, or wild felids.     

Novel Feline Leukemia Virus Interference Group Based on the env Gene

Feline leukemia virus (FeLV) subgroups have emerged in infected cats via the mutation or recombination of the env gene of subgroup A FeLV (FeLV-A), the primary virus. We report the isolation and characterization of a novel env gene, TG35-2, and report that the TG35-2 pseudotype can be categorized as a novel FeLV subgroup. The TG35-2 envelope protein displays strong sequence identity to FeLV-A Env, suggesting that selection pressure in cats causes novel FeLV subgroups to emerge.     

Humoral immune response of asymptomatic cats naturally infected with feline leukemia virus

The humoral immune response of cats that were naturally infected with the feline leukemia virus (FeLV) was examined after antigenic stimulation with the synthetic antigen poly(L-Tyr, L-Glu)-poly(DL-Ala)-poly(L-Lys). The primary humoral antibody response in FeLV-infected cats was both delayed and greatly reduced, compared with that seen in uninfected control cats. A similar discordance was observed after secondary stimulation with the antigen, in the FeLV-infected cats had both a delayed response and a reduced response, compared with uninfected cats. The levels of total immunoglobulins of the immunoglobulin G and immunoglobulin M classes in the sera of FeLV-infected cats were significantly higher (two- and threefold, respectively) than were those of the uninfected control animals. The presence of an impaired humoral immune response to newly presented antigens in the presence of elevated immunoglobulin levels has been thoroughly documented in the case of people with the acquired immunodeficiency syndrome. This further emphasizes the potential value of FeLV-infected cats as a model for human acquired immunodeficiency syndrome.     

Phylogenetic and Structural Diversity in the Feline Leukemia Virus Env Gene

Feline leukemia virus (FeLV) belongs to the genus Gammaretrovirus, and causes a variety of neoplastic and non-neoplastic diseases in cats. Alteration of viral env sequences is thought to be associated with disease specificity, but the way in which genetic diversity of FeLV contributes to the generation of such variants in nature is poorly understood. We isolated FeLV env genes from naturally infected cats in Japan and analyzed the evolutionary dynamics of these genes. Phylogenetic reconstructions separated our FeLV samples into three distinct genetic clusters, termed Genotypes I, II, and III. Genotype I is a major genetic cluster and can be further classified into Clades 1–7 in Japan. Genotypes were correlated with geographical distribution; Genotypes I and II were distributed within Japan, whilst FeLV samples from outside Japan belonged to Genotype III. These results may be due to geographical isolation of FeLVs in Japan. The observed structural diversity of the FeLV env gene appears to be caused primarily by mutation, deletion, insertion and recombination, and these variants may be generated de novo in individual cats. FeLV interference assay revealed that FeLV genotypes did not correlate with known FeLV receptor subgroups. We have identified the genotypes which we consider to be reliable for evaluating phylogenetic relationships of FeLV, which embrace the high structural diversity observed in our sample. Overall, these findings extend our understanding of Gammaretrovirus evolutionary patterns in the field, and may provide a useful basis for assessing the emergence of novel strains and understanding the molecular mechanisms of FeLV transmission in cats.     

Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5

Human SERINC5 (SER5) protein is a recently described restriction factor against human immunodeficiency virus-1 (HIV-1), which is antagonized by HIV-1 Nef protein. Other retroviral accessory proteins such as the glycosylated Gag (glycoGag) from the murine leukemia virus (MLV) can also antagonize SER5. In addition, some viruses escape SER5 restriction by expressing a SER5-insensitive envelope (Env) glycoprotein. Here, we studied the activity of human and feline SER5 on HIV-1 and on the two pathogenic retroviruses in cats, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). HIV-1 in absence of Nef is restricted by SER5 from domestic cats and protected by its Nef protein. The sensitivity of feline retroviruses FIV and FeLV to human and feline SER5 is considerably different: FIV is sensitive to feline and human SER5 and lacks an obvious mechanism to counteract SER5 activity, while FeLV is relatively resistant to SER5 inhibition. We speculated that similar to MLV, FeLV-A or FeLV-B express glycoGag proteins and investigated their function against human and feline SER5 in wild type and envelope deficient virus variants. We found that the endogenous FeLV recombinant virus, FeLV-B but not wild type exogenous FeLV-A envelope mediates a strong resistance against human and feline SER5. GlycoGag has an additional but moderate role to enhance viral infectivity in the presence of SER5 that seems to be dependent on the FeLV envelope. These findings may explain, why in vivo FeLV-B has a selective advantage and causes higher FeLV levels in infected cats compared to infections of FeLV-A only.     

Endogenous RD-114 virus genome expression in malignant tissues of domestic cats.

Endogenous xenotropic cat type C virus (RD-114)- and infectious feline leukemia virus (FeLV)-specific gene expressions were measured in spontaneous sarcomas carcinomas, and nonmalignant cat tissues by molecular hybridization for virus-specific RNA and competition radio-immunoassays for the major internal protein (p30) of these two viruses. The results indicate that RD-114 gene expression in sarcomas and carcinomas at both RNA and p30 levels is significantly higher than histologically normal tissues from cats free of cancer. In contrast, the levels of FeLV viral RNA and p30 are fount to be low or undetectable in the majority of these tumored and normal tissues examined. Whereas variability in the amounts of RD-114 OR FeLV RNA and p30 expressed is found in tissues from different cats, their expression is fairly uniform in multiple malignant tissues of the same cat. The finding of widespread occurrence of elevated RD-114 gene expression in sarcomas and carcinomas is consistent with our similar observation with natural lymphomas of domestic cats and suggests that expression of certain functions of this endogenous virus may be etiologically involved in the development of many different spontaneous neoplasms of cats.     

Protection of cats against feline leukemia virus by vaccination with a canarypox virus recombinant, ALVAC-FL.

Two ALVAC (canarypox virus)-based recombinant viruses expressing the feline leukemia virus (FeLV) subgroup A env and gag genes were assessed for their protective efficacy in cats. Both recombinant viruses contained the entire gag gene. ALVAC-FL also expressed the entire envelope glycoprotein, while ALVAC-FL(dl IS) expressed an env-specific gene product deleted of the putative immunosuppressive region. Although only 50% of the cats vaccinated with ALVAC-FL(dl IS) were protected against persistent viremia after oronasal exposure to a homologous FeLV isolate, all cats administered ALVAC-FL resisted the challenge exposure. Significantly, protection was afforded in the absence of detectable FeLV-neutralizing antibodies. These results represent the first effective vaccination of cats against FeLV with a poxvirus-based recombinant vector and have implications that are relevant not only to FeLV vaccine development but also to developing vaccines against other retroviruses, including human immunodeficiency virus.     

Pathogenesis of feline leukemia virus T17: contrasting fates of helper, v-myc, and v-tcr proviruses in secondary tumors.

A naturally occurring feline thymic lymphosarcoma (T17) provided the unique observation of a T-cell antigen receptor beta-chain gene (v-tcr) transduced by a retrovirus. The primary tumor contained three classes of feline leukemia virus (FeLV) provirus, which have now been characterized in more detail as (i) v-tcr-containing recombinant proviruses, (ii) v-myc-containing recombinant proviruses, and (iii) apparently full-length helper FeLV proviruses. The two transductions appear to have been independent events, with distinct recombinational junctions and no sequence overlap in the host-derived inserts. The T17 tumor cell line releases large numbers of FeLV particles of low infectivity; all three genomes are encapsidated, but passage of FeLV-T17 on feline fibroblast and lymphoma cells led to selective loss of the recombinant viruses. The oncogenic potential of the T17 virus complex was, therefore, tested by infection of neonatal cats with virus harvested directly from the primary T17 tumor cell line. A single inoculation of FeLV-T17 caused persistent low-grade infection culminating in thymic lymphosarcoma and acute thymic atrophy, which was accelerated by coinfection with the weakly pathogenic FeLV subgroup A (FeLV-A)/Glasgow-1 helper. Molecularly cloned FeLV-tcr virus (T-31) rescued for replication by a weakly pathogenic FeLV-A/Glasgow-1 helper virus was similarly tested in vivo and induced thymic atrophy and thymic lymphosarcomas. Most FeLV-T17-induced tumors manifested either v-myc or an activated c-myc allele and had undergone rearrangement of endogenous T-cell antigen receptor beta-chain genes, supporting the proposition that the oncogenic effects of c-myc linked to the FeLV long terminal repeat are targeted to a specific window in T-cell differentiation. However, neither the FeLV-T17-induced tumors nor the T-31 + FeLV-A-induced tumors contained clonally represented v-tcr sequences. Only one of the FeLV-T17-induced tumors contained detectable v-tcr proviruses, at a low copy number. While v-tcr does not have a readily transmissible oncogenic function, a more restricted role is not excluded, perhaps involving antigenic peptide-major histocompatibility complex recognition by the T-cell receptor complex. Such a function could be obscured by the genetic diversity of the outbred domestic cat host.     

Prevalence of feline leukemia virus infection in random source laboratory cats

Three years of quarantine data involving the prevalence of Feline Leukemia Virus (FeLV) in laboratory cats (Felis catus) was evaluated. Testing was performed using a commercially available ELISA system. Annual prevalence of infection ranged from 5.4 to 10.7% of the 937 cats tested. Results obtained with the enzyme linked immunosorbent assay (ELISA) system compared well with tests performed using an immunofluorescent antibody assay system (IFA). Seasonal periodicity was noted with higher infection rates among animals received in April. Holding period at the supplier and gender did not influence disease prevalence. The availability of a simple test system, the unacceptability of FeLV infected cats for research, the danger of transmission and a higher prevalence of infection than earlier reports indicated that supplier and user evaluation of research cats for FeLV would be prudent.