Feline oncornavirus-associated cell membrane antigen: expression in transformed nonproducer mink cells.

The feline oncornavirus-associated cell membrane antigen (FOCMA) is a target for naturally occurring immunity that protects the cat against development of fibrosarcoma and leukemia. Feline sarcoma virus-transformed "nonproducer" mink cells express high levels of FOCMA, but not the major viral structural proteins. Transformation of the same cells by murine sarcoma virus, or infection with feline leukemia virus, which is nontransforming for epithelial or fibroblastic cells, did not induce FOCMA. Thus, FOCMA expression in mind lung cells is specifically associated with transformation by feline sarcoma virus.

Establishment of two canine sarcoma cell lines: productive infection with feline leukemia virus.

Two canine sarcoma cell lines (11028, 11031) were established in vitro and have been transferred 213 and 306 times, respectively, since 1970. These cell lines had a chromosome pattern consistent with their canine origin. Both cultures were infected with feline leukemia virus (FeLV), which caused a morphologic and karyotypic changes. The cells became rounded after infection with FeLV and both cultures showed the presence of a single, large, acrocentric chromosome considered to be a marker chromosome. All tumors were transplanted into newborn beagle pups, but only the 11028-FeLV formed metastatic tumors. No helper or focus-forming activity or virus particles were found in the uninfected cultures. Helper activity and virus were demonstrated in both sarcoma lines after infection with FeLV, though no focus-forming activity was noted. Helper activity of progeny virus could be assayed on either cat or dog embryo cells.

Retrovirus receptor PiT-1 of the Felis catus.

We isolated a cDNA encoding a feline homolog of human PiT-1, a sodium-dependent phosphate symporter which is utilized by gibbon ape leukemia virus (GALV) as a receptor for entry into host cells. The overall homology between the human and feline receptors is 92 and 93% at the nucleotide and deduced amino acid levels, respectively. Hydropathy analyses implied ten potential membrane spanning regions and, in analogy to human and murine homologs, five extracellular and four intracellular loops. Strikingly, the amino acid sequence of the fourth extracellular loop, which is critical for GALV surface glycoprotein binding, has complete identity between the human and feline PiT-1s, while the mouse PiT-1, non-functional for GALV entry, is quite divergent. Ectopic expression of the feline PiT-1 in guinea pig cells, which are non-permissive to feline leukemia virus (FeLV), subgroup B virus, conferred susceptibility to FeLV-B infection confirming the functional ability of the cloned product to serve as a receptor for a natural retrovirus of the homologous species.

Isolation of feline leukemia virus from clinical specimens.

Specimens obtained from feline leukemia virus (FeLV)-positive cats were examined for infectious FeLV. Feline leukemia virus was detected by a focus-forming assay and confirmed by florescent antibody. Techniques of sample processing were evaluated and adjusted for optimum detection of FeLV. Low levels of FeLV were detected in 2 of 10 oral samples; however, the majority of these samples (17 of 27 tested) produced cytopathic effects in tissue culture which prevented Fe LV detection. Three of 24 urine samples and 1 of 20 rectal specimens were positive for FeLV. One milk sample contained high levels of FeLV.

An unusual case of feline leukemia and an associated syncytium-forming virus.

A 3-year-old female Siamese cat was admitted to a local animal hospital with a history of recent extreme lethargy and anorexia. A hemorrhagic tumor was removed from an area of oral buccal skin and diagnosed histopathologically as lymphosarcoma. Rapid physical deterioration occurred, and the cat became moribund 2 weeks after surgical operation. Necropsy revealed at least 200 spherical hemorrhagic neoplastic nodules attached to the omentum, mesentery, and peritoneum. Examination of histopathologic sections confirmed the striking characteristics of an extremely vascular and highly invasive malignant lymphoma, which was designated feline tumor No. 01 (FeT-01). There was no evidence of peripheral blood leukemia. Electron microscopic examination of tumor tissue revealed numerous viral particles having characteristics common to both feline leukemia virus (FeLV) and feline syncytium-forming virus (FeSFV). Primary cells and cultures propagated from tumor tissue were found to be negative or weakly positive for group-specific (gs) antigen by radioimmunoassay but strongly positive when assayed by indirect immunofluorescence. Co-cultivation of cells from tumor tissue, with normal prescreened feline embryo cells, revealed the presence of numerous FeSFV-like viral particles in the absence of C-type virus. A FeSFV was isolated from these passaged cells, with characteristics similar to FeSFV isolates previously described in the literature. The apparent presence of FeSFV in lymphosarcomatous tissue and the apparent absence of FeLV C-type particles in passaged cells indicate the need to make a more intensive study of the FeSFV group of viruses and the possible etiologic relationship to feline malignancies.

Feline leukaemia virus and its clinical effects in cats.

Feline leukemia virus (FeLV) infection is common among cats where contact is high. The virus can be transmitted readily between cats. It causes a variety of haemopoietic and lymphoid neoplasms; the most common types are alimentary, multicentric and thymic lymphosarcoma and lymphatic leukaemia. The virus is involved in the aetiology of certain other diseases including anaemia, glomerulonephritis and an immunosuppressive syndrome which predisposes cats to intercurrent infections. Many infected cats mount an immune response and do not suffer from any of these. The immune status is shown by serum antibody levels to feline leukaemia virus associated cell membrane antigens. Cats with a titre of 32 or more are most unlikely to suffer any ill effects and may eliminate the virus infection. The outcome of infection in an individual cat depends on the immunological competence of the cat, the dose of virus received and its ability to induce immunosuppression. FeLV infection can be detected by examination of tissues by electron microscopy, and by culture of virus from plasma and other tissues. In the United States, a method is now in use for the detection of leukaemia virus antigen in peripheral blood leukocytes; this is carried out on ordinary blood films. Successful prototype vaccines have been developed against FeLV. This paper describes the natural history of the virus, the diseases in which it is implicated and discusses recently developed diagnostic methods.

Criterios diagnósticos para la infección por el virus de la inmunodeficiencia felina y el virus de la leucemia felina en gatos domésticos de Buenos Aires, Argentina / Viral diagnostic criteria for Feline immunodeficiency virus and Feline leukemia virus infections in domestic cats from Buenos Aires, Argentina

A cross-sectional study was carried out on cats attending the Small Animal Hospital at the Faculty of Veterinary Sciences of the University of Buenos Aires to assess the prevalence and associated risk factors of Feline immunodeficiency virus (FIV) and Feline leukemia virus (FeLV) in the city of Buenos Aires, Argentina. Blood samples from 255 cats with symptoms compatible with FIV or FeLV infection, collected between 2009 and 2013 were analyzed by serology (immunochromatography, IA) and by hemi-nested PCR (n-PCR). The IA and n-PCR assays showed similar percentages of positivity for FIV while the n-PCR test was more sensitive for FeLV. Differences between the diagnostic tests and their choice according to the age of the animal are discussed. The clinical histories of ninety of the 255 cats showed blood profiles similar to others previously reported and revealed a higher risk of infection in male adult cats with outdoor access.

Para determinar la prevalencia en la ciudad de Buenos Aires del virus de la inmunodeficiencia felina (FIV) y del virus de la leucemia felina (FeLV), y analizar los factores de riesgo que pudieran estar asociados a ellos, se realizó un estudio transversal en gatos atendidos en el Hospital de Pequeños Animales de la Facultad de Ciencias Veterinarias de la Universidad de Buenos Aires. Se analizaron por serología (inmunocromatografía --#91;IA--#93;) y por hemi-nested PCR (n-PCR) 255 muestras de sangre de gatos con síntomas compatibles con infección por FIV o FeLV. La IA y la n-PCR revelaron porcentajes similares de animales positivos para FIV, mientras que para FeLV el diagnóstico por n-PCR resultó más sensible. Se discuten las diferencias halladas entre los métodos diagnósticos y su elección según la edad del animal. Las historias clínicas de 90 de los 255 gatos mostraron perfiles sanguíneos similares a otros ya reportados y revelaron el mayor riesgo de infección con ambos virus en machos adultos con acceso al exterior.

Are endogenous feline leukemia viruses really endogenous?

Full length endogenous feline leukemia virus (FeLV) proviruses exist within the genomes of many breeds of domestic cat raising the possibility that they may also exist in a transmissible exogenous form. Such viruses would share receptor usage with the recombinant FeLV-B subgroup, a viral subgroup that arises in vivo by recombination between exogenous subgroup A virus (FeLV-A) and endogenous FeLV. Accordingly, all isolates of FeLV-B made to date have contained a "helper" FeLV-A, consistent with their recombinatorial origin. In order to assess whether endogenous viruses are transmitted between cats, we examined primary isolates of FeLV for which the viral subgroup had been determined for the presence of a subgroup B virus that lacked an FeLV-A. Here we describe the identification of two primary field isolates of FeLV (2518 and 4314) that appeared to contain subgroup B virus only by classical interference assays, raising the possibility of between-host transmission of endogenous FeLV. Sequencing of the env gene and U3 region of the 3' long terminal repeat (LTR) confirmed that both viral genomes contained endogenous viral env genes. However the viral 3' LTRs appeared exogenous in origin with a putative 3' recombination breakpoint residing at the 3' end of the env gene. Further, the FeLV-2518 virions also co-packaged a truncated FeLV-A genome containing a defective env gene, termed FeLV-2518(A) whilst no helper subgroup A viral genome was detected in virions of FeLV-4314. The acquisition of an exogenous LTR by the endogenous FeLV in 4314 may have allowed a recombinant FeLV variant to outgrow an exogenous FeLV-A virus that was presumably present during first infection. Given time, a similar evolution may also occur within the 2518 isolate. The data suggest that endogenous FeLVs may be mobilised by acquisition of exogenous LTRs yielding novel viruses that type biologically as FeLV-B.

Feline leukemia virus T entry is dependent on both expression levels and specific interactions between cofactor and receptor.

Feline leukemia virus (FeLV) subgroup T uses both a multiple membrane-spanning receptor, FePit1, and a soluble cofactor, FeLIX, to enter feline cells. FeLIX is expressed from endogenous FeLV-related sequence and resembles the receptor binding domain (RBD) of the viral envelope protein. It remains unclear whether FeLV-T receptor activity requires specific residues within FePit1 and FeLIX and/or a threshold level of receptor/cofactor expression. To address this, we examined FeLV-T infection of cells expressing variable levels of FePit1 and other gammaretroviral receptors in the presence of variable amounts of soluble cofactor, either RBD or the envelope surface subunit (SU). Cofactor-receptor pairs fall into three groups with regard to mediating FeLV-T infection: those that are efficient at all concentrations tested, such as FePit1 and FeLIX; those requiring high expression of both cofactor and receptor; and those that are non-functional as receptors even at high expression. This suggests that both expression levels and specific interactions with receptor and cofactor are critical for mediating entry of FeLV-T.

Selective host range restriction of goat cells for recombinant murine leukemia virus and feline leukemia virus type A.

We isolated a strain of normal goat fibroblasts which was uniquely selective in that it allowed the replication of xenotropic murine leukemia virus but not polytropic recombinant murine leukemia virus. In addition, feline leukemia virus type A replication was severely diminished in these goat cells, whereas feline leukemia virus type B and feline endogenous RD114-CCC viruses replicated efficiently. No other known cells exhibit this pattern of virus growth restriction. These goat cells allow the study of xenotropic murine leukemia virus in mixtures which also contain recombinant murine leukemia virus and may be helpful in eliminating feline leukemia virus type which often coexists in feline sarcoma or leukemia virus mixtures with other feline leukemia virus types.

Feline leukemia virus: survival under home and laboratory conditions.

Feline leukemia virus maintained its infectiousness in cell culture medium at 37 degrees C or lower for at least 48 h. However, virus in saliva or medium was inactivated within a few hours if allowed to dry.

Influence of culture conditions of growth of FL-74 cells and feline oncornavirus cell membrane associated antigen production.

The FL-74 cell, a feline lymphoblastoid cell line derived from a tumor induced by leukemia virus, grows equally well in static suspension culture (plastic T-flask or silicone treated glass bottles) or in spinner culture. No growth was observed in unsiliconized glass bottles. Although feline leukemia virus production was nearly the same in FL-74 grown in each of the above types of vessel, the expression of the feline oncornavirus membrane associated antigen (FOCMA), as determined by membrane immunofluorescence, was more intense and more complete on cells grown in static suspension. Moreover, higher fluorescent antibody titer endpoints were observed with cells from static suspension cultures than with cells from spinner cultures, FL-74 cells grown in spinner culture, when subjected to partial synchrony by cold block or by deprivation of essential amino acids (arginine and/or isoleucine) for 12 hr, achieved a membrane fluorescent pattern for FOCMA similar to cells grown in static suspension. It is proposed that the expression of FOCMA on the cell membrane surface is cell-cycle dependent, and that the rate at which a cell passes through the cell cycle determines the pattern and intensity of the fluorescence of the cell membrane.

Pathogenic and host range determinants of the feline aplastic anemia retrovirus.

Feline leukemia virus (FeLV) C-Sarma (or FSC) is a prototype of subgroup C FeLVs, which induce fatal aplastic anemia in outbred specific-pathogen-free (SPF) cats. FeLV C isolates also possess an extended host range in vitro, including an ability, unique among FeLVs, to replicate in guinea pig cells. To identify the viral determinants responsible for the pathogenicity and host range of FSC we constructed a series of proviral DNAs by exchanging gene fragments between FSC and FeLV-61E (or F6A), the latter of which is minimally pathogenic and whose host range in vitro is restricted to feline cells. Transfer of an 886-base-pair (bp) fragment of FSC, encompassing the codons for 73 amino acids at the 3' end of pol (the integrase/endonuclease gene) and the codons for 241 amino acids of the N-terminal portion of env [the extracellular glycoprotein (gp70) gene], into the F6A genome was sufficient to confer onto chimeric viruses the ability to induce fatal aplastic anemia in SPF cats. In contrast, no chimera lacking this sequence induced disease. When assayed in vitro, all chimeric viruses containing the 886-bp fragment of FSC acquired the ability to replicate in heterologous cells, including dog and guinea pig cells. Thus, the pathogenic and the host range determinants of the feline aplastic anemia retrovirus colocalize to a 3' pol-5' env region of the FSC genome and likely reside within a region encoding 241 amino acid residues of the N terminus of the extracellular glycoprotein.

Isolation of a novel subgroup B feline leukemia virus from a cat infected with FeLV-A.

Proviruses were cloned directly from a cat that developed neurological disorders approximately 28 months after inoculation with a molecularly cloned, minimally pathogenic subgroup A feline leukemia virus (FeLV-A). In addition to FeLV-A proviruses that were nearly identical to the inoculated virus, we detected a subgroup B-like variant in brain, bone marrow, and lymph node that apparently had acquired the major portion of its extracellular envelope gene (gp70) from endogenous FeLV-related sequences. A similar recombinant was also detected, by PCR, at low levels in bone marrow from an early time postinfection (2.5 months). A full-length proviral variant with this recombinant structure cloned from brain tissue encoded a replication-defective virus. A chimera encoding the 5' gag-pol portion of FeLV-A and the 3' env-LTR portion of the defective brain-derived clone was replication-competent and had the extended host range properties of FeLV-B.

Deposition of retrovirus associated antigens (p30 and gp70) on cell membranes of feline and murine leukaemia virus infected cells.

A quantitative estimation of retrovirus associated cell membrane antigens of murine and feline cells infected with their respective type C leukosis virus is presented. Using a radio-immune assay with three broadly reactive antisera, the minimum estimated number of retrovirus associated antigenic determinants on YAC [Moloney leukaemia virus (MuLV) infected murine] and FL-74 [feline leukaemia virus (FeLV) infected feline] cells was 1.3 x 10(6) and 1.6 x10(6) determinants per cell respectively. The virus structural proteins p27-30 and gp70 were detected by three component specific antisera on murine and feline cell surfaces in amounts which varied between cell isolates. MuLV infected cells produced as many as 1.9 x 10(5) p30 antigenic determinants and 7.5 x 10(5) gp70 determinants on infected cells. FeLV infected cells (FL-74) expressed 5.6 x 10(5) p27 and 7.5 x 10(5) gp70 antigenic determinants per single cell surface. The major core protein (p27-30) and the major envelope glycoprotein (gp70) antigens are sufficiently physically separated on cell surfaces so that binding of either of the membrane antigens with component specific antibodies does not interfere with binding of antibodies specific for the other. Despite the expression of interspecies determinants for p30, gp70, and other retrovirus associated antigens detected by antibody procedures, interspecies determinants of cell mediated immunity could not be demonstrated in immune mice bearing Moloney sarcoma virus (MSV) induced tumours. Furthermore, xenogeneic immunization of mice with FL-74 cells failed to protect mice against the growth of MSV induced lymphoma or sarcoma.

Differential growth and transmission in cats of feline leukaemia viruses of subgroups A and B.

The outcome of infecting cats with FeLV of sub-groups A (FeLV-A) or B (FeLV-B) was different. After FeLV-A infection, virus was quickly recovered from the blood and oropharynx of most animals. Following infection with FeLV-B, only a small proportion of cats developed a viraemia, and this after a long interval. There was no evidence that FeLV-B was transmitted by contact. The result of infecting cats with mixtures of FeLV-A and B (FeLV-AB) was that to a large extent each virus operated independently. FeLV-A was recovered from the plasma first, in a high proportion of the cats, and FeLV-B appeared later but not in all cats. There was evidence of interaction, however, in that the proportion of cats which were viraemic with FeLV-B was greater following FeLV-AB infection than after infection with FeLV-B alone; also FeLV-B was transmitted by contact from cats excreting FeLV-AB. These results help to explain the apparent dependence of FeLV-B on FeLV-A under natural conditions and the frequency of occurrence of sugroups A and B in FeLV isolates.

Remission of leukemia and loss of feline leukemia virus in cats injected with Staphylococcus protein A: association with increased circulating interferon and complement-dependent cytotoxic antibody.

We have injected purified Staphylococcus aureus protein A intraperitoneally into leukemic cats infected with feline leukemia virus, into cats persistently infected with feline leukemia virus but without hematologic or cytologic abnormalities, and into healthy cats without feline leukemia virus infection. Pre- and post-treatment serum samples were evaluated sequentially for interferon activity and for complement-dependent cytotoxic antibody. Our results indicate that serum interferon increased dramatically (less than 3 to 324 units/ml) during treatment only in cats that responded to staphylococcal protein A therapy. Increase of interferon preceded or was closely associated with increasing levels of cytotoxic antibody, loss of viremia, and correction of cytological and hematological abnormalities of three leukemic cats. The cytotoxic antibody was shown to be specific for envelope glycoprotein gp70 of the feline leukemia virus. One persistently feline leukemia virus-infected cat without leukemia that became nonviremic also developed high levels of interferon and specific cytotoxic antibody. By contrast, interferon levels of cats not responding to treatment had levels of less than 3 to 27 units/ml. Normal healthy cats injected with staphylococcal protein A showed moderate transient increases of interferon but no detectable cytotoxic antibodies to FL-74 cells. These data suggest that interferon and cytotoxic antibody may play important, possibly complementary roles in inducing remission of leukemia and loss of viremia in cats treated with staphylococcal protein A.