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Feline leukemia virus (FeLV) occurs in nature not as a single genomic species but as a family of closely related viruses. The disease outcome of natural FeLV infection is variable and likely reflects genetic variation both in the virus and the naturally outbreeding host population. A series of studies have been undertaken with the objectives of examining natural FeLV genetic variation, the selective pressures operative in FeLV infection that lead to predominance of natural variants, and the consequences for infection and disease progression. Genetic variation among FeLV isolates was examined in a cohort of naturally infected cats with thymic lymphoma of T-cell origin, non-T-cell multicentric lymphoma, myeloproliferative disorder or anemia. The predominant isolate in the cohort, designated FeLV-945, was identified exclusively in disorders of non-T-cell origin. The FeLV-945 LTR was shown to contain a unique 21-bp repeat element, triplicated in tandem downstream of enhancer. The 21-bp triplication was shown to act as a transcriptional enhancer and to confer a replicative advantage through the assembly of a distinctive transcription factor complex. Oncogene utilization during tumor induction by FeLV-945 was studied using a recombinant Moloney murine leukemia virus containing the FeLV-945 LTR. This approach identified novel loci of common proviral integration in tumors, including the regulatory subunit of PI-3Kgamma. Mutational changes identified in FeLV-945 SU were shown not to alter receptor usage as measured by host range and superinfection interference, but to significantly increase the efficiency of receptor binding. To determine whether the unique sequence elements of FeLV-945 influence the course of infection and disease in vivo, recombinant viruses were constructed in which the FeLV-945 LTR alone, or the FeLV-945 SU gene and LTR were substituted into the prototype isolate FeLV-A/61E. Longitudinal studies of infected animals showed that substitution of the FeLV-945 LTR into FeLV-A/61E resulted in a significantly more rapid disease onset, but did not alter the tumorigenic spectrum. In contrast, substitution of both the FeLV-945 LTR and SU gene changed the disease outcome entirely. Together, these observations indicate that the distinctive LTR and SU gene of FeLV-945 mediate a rapid pathogenesis with distinctive clinical features and oncogenic mechanisms.

FeLV was discovered 40 years ago and vaccines have been commercially available for almost two decades. So far, most FeLV pathogenesis and vaccine studies were conducted assaying parameters, such as virus isolation and antigen detection. Accordingly, regressive infection was characterized by transient or undetectable viremia, while persistent viremia is typically observed in cats with progressive infection. Using real-time polymerase chain reaction assays, the spectrum of host response categories to FeLV infection was recently refined by investigating proviral and plasma viral RNA loads. Cats believed to be immune to FeLV infection were found to turn provirus-positive after virus exposure. Moreover, efficacious FeLV vaccines were found unable to prevent provirus-integration and minimal viral replication. Remarkably, no difference was found in initial proviral and plasma viral RNA loads between cats with different infection outcomes. Only subsequently, the infection outcome is associated with FeLV loads. FeLV provirus was found to persist for years; reoccurrence of viremia and disease development was observed in some cats. Thus, aviremic provirus-positive cats are FeLV carriers and, following reactivation, may act as an infection source. However, integrated viral DNA may also be essential for solid protection and long-lasting maintenance of protective immunity. In conclusion, real-time TaqMan PCR and RT-PCR assays are highly sensitive and specific. They yield a more sensitive measure for FeLV exposure than antigen detection, virus isolation or immunofluoresence assays. We recommend the use of real-time PCR assays to identify FeLV exposed cats, particularly in catteries, and investigate obscure clinical cases that may be FeLV-associated. The use of sensitive molecular methods will contribute to a more in-depth understanding of the FeLV pathogenesis.

Feline leukemia virus (FeLV), which is subclassified into three subgroups of A, B and C, is a pathogenic retrovirus in cats. FeLV-A is minimally pathogenic, FeLV-C can cause pure red cell aplasia, and FeLV-B is associated with a variety of pathogenic properties such as lymphoma, leukemia and anemia. FeLV-induced neoplasms are caused, at least in part, by somatically acquired insertional mutagenesis in which the integrated provirus may activate a proto-oncogene or disrupt a tumor suppressor gene. The common integration sites for FeLV have been identified in six loci with feline lymphomas: c-myc, flvi-1, flvi-2 (contains bmi-1), fit-1, pim-1 and flit-1. Oncogenic association of the loci includes that c-myc is known as a proto-oncogene, bmi-1 and pim-1 have been recognized as myc-collaborators, fit-1 appears to be closely linked to myb, and flit-1 insertion is shown to be associated with over-expression of a cellular gene, e.g. ACVRL1. Thus, identification of common integration sites for FeLV is a tenable model to clarify oncogenesis. Recent advances in molecular biology and cytogenetics have developed to rapidly detect numbers of retroviral integration sites by genome-wide large-scale analyses. Especially, polymerase chain reaction (PCR)-based strategies and chromosome analyses with fluorescence in situ hybridization (FISH) will be applicable for studies on FeLV.

Endogenous retroviruses are integrated in the genome of most vertebrates. They represent footprints of ancient retroviral infection and are vertically transmitted from parents to their offspring. In the genome of all domestic cats, sequences closely related to exogenous FeLV known as endogenous feline leukemia virus (enFeLV), are present. enFeLV are incapable of giving rise to infectious virus particles. However, transcription and translation of enFeLV have been demonstrated in tissues of healthy cats and in feline cell lines. The presence of enFeLV-env has been shown in specific embryonic tissues and adult thymic cells. In addition, the enFeLV-env region recombines with FeLV subgroup A giving rise to an infectious FeLV-B virus. enFeLV envelope protein, FeLIX (FeLV infectivity X-essory protein) is also involved in mediating FeLV-T infection. In order to test the hypothesis that the enFeLV loads play a role in exogenous FeLV-A infection and pathogenesis, quantitative real-time PCR and RT-PCR assays were developed. An assay, specific to U3 region of all different subtypes of exogenous FeLV, was designed and applied to quantify exogenous FeLV proviral or viral load in cats, while three real-time PCR assays were designed to quantify U3 and env enFeLV loads (two within U3 amplifying different sequences; one within env). enFeLV loads were investigated in blood samples derived from Swiss privately owned domestic cats, specific pathogen-free (SPF) cats and European wildcats (Felis silvestris silvestris). Significant differences in enFeLV loads were observed between privately owned cats and SPF cats as well as among SPF cats originating from different catteries and among domestic cats of different breeds. When privately owned cats were compared, FeLV-infected cats had higher loads than uninfected cats. In addition, wildcats had higher enFeLV loads than domestic cats. In conclusion, the quantitative real-time PCR assays described herein are important prerequisites to quantify enFeLV proviral loads in felids and thus are important tools to investigate the role of enFeLV loads in FeLV infection.

Cats exposed to feline leukemia virus (FeLV), a naturally occurring gammaretrovirus develop either progressive or regressive infection. Recent studies using analyses with enhanced sensitivity have correlated loads throughout FeLV with the clinical outcome, though remarkably, during the acute phase of infection, proviral and viral RNA burdens in the peripheral blood do not differ between groups. We hypothesized that viral loads in specific leukocyte subsets influence the infection outcome. Using a method established to determine the proviral and cell-associated viral RNA loads in specific leukocyte subsets, we evaluated viral loads in eleven FeLV-exposed specific pathogen-free (SPF) cats 2.5 years post-infection. Six cats had undergone regressive infection whereas five were persistently viremic. Aviremic cats had lower total proviral blood loads than the persistently infected cats and FeLV proviral DNA was shown to be integrated into genomic DNA in four out of four animals. Lymphocytes were predominantly infected vs. moncytes and granulocytes in aviremic cats. In contrast, persistently viremic cats were provirus-positive in all leukocyte subsets. The acute phase kinetics of FeLV infection were analyzed in two additional cats; an early lymphoreticular phase with productive infection in lymphocytes in both cats and in monocytes in one cat was followed by infection of the granulocytes; both cats became persistently infected. These results indicate that FeLV persistent viremia is associated with secondary viremia of bone marrow origin, whereas regressive cats only sustain a non-productive infection in low numbers of lymphocytes.

We previously defined four categories of feline leukemia virus (FeLV) infection, designated as abortive, regressive, latent, and progressive. To determine if detectable viral DNA is transcriptionally active in the absence of antigenemia, we developed and validated a real-time viral RNA qPCR assay. This assay proved to be highly sensitive, specific, reproducible, and allowed reliable quantitation. We then applied this methodology, together with real-time DNA qPCR and p27 capsid antigen capture ELISA, to examine cats challenged with FeLV. We found that circulating viral RNA and DNA levels were highly correlated and the assays were almost in perfect agreement. This indicates that the vast majority of viral DNA is transcriptionally active, even in the absence of antigenemia. The real-time qPCR assays are more sensitive than the most commonly used FeLV diagnostic assay, the p27 capsid antigen capture ELISA. Application of qPCR assays may add greater depth in understanding of FeLV-host relationships.

Feline retroviruses are rarely reported in lynx species. Twenty-one Iberian lynx (Lynx pardinus) blood and tissue samples collected from Doñana National Park and Los Villares (Sierra Morena) in southern Spain during 1993-2003 were analyzed by polymerase chain reaction to amplify nucleic acids from feline retroviruses. Six samples were positive for Feline leukemia virus (FeLV), but no samples tested positive for Feline immunodeficiency virus. The BLAST analysis indicated that 5 of the 6 sequences were closely related to FeLV strain Rickard subgroup A, whereas 1 sequence was identical to FeLV. To the authors' knowledge, this is the first report of FeLV in the endangered Iberian lynx.

From 2002 through 2005, an outbreak of feline leukemia virus (FeLV) occurred in Florida panthers (Puma concolor coryi). Clinical signs included lymphadenopathy, anemia, septicemia, and weight loss; 5 panthers died. Not associated with FeLV outcome were the genetic heritage of the panthers (pure Florida vs. Texas/Florida crosses) and co-infection with feline immunodeficiency virus. Genetic analysis of panther FeLV, designated FeLV-Pco, determined that the outbreak likely came from 1 cross-species transmission from a domestic cat. The FeLV-Pco virus was closely related to the domestic cat exogenous FeLV-A subgroup in lacking recombinant segments derived from endogenous FeLV. FeLV-Pco sequences were most similar to the well-characterized FeLV-945 strain, which is highly virulent and strongly pathogenic in domestic cats because of unique long terminal repeat and envelope sequences. These unique features may also account for the severity of the outbreak after cross-species transmission to the panther.

The receptor-binding domain (RBD) in the surface (SU) subunit of gammaretrovirus envelope glycoprotein is critical for determining the host receptor specificity of the virus. This domain is separated from the carboxy terminal C domain (Cdom) of SU by a proline-rich region. In this study, we show that the Cdom region in the SU from subgroup C feline leukemia virus (FeLV-C) forms a second receptor-binding domain that is distinct from its RBD, and which can independently bind to its host receptor FLVCR1, in the absence of RBD. Furthermore, our results suggest that residues located in the C2 disulfide-bonded loop in FeLV-C Cdom are critical for SU binding to FLVCR1 and for virus infection. We propose that binding of FeLV-C SU to FLVCR1 involves interaction of two receptor-binding domains (RBD and Cdom) with FLVCR1, and that this mechanism of interaction is conserved for other gammaretroviruses. Our results could have important implications for designing gammaretrovirus vectors that can efficiently infect specific target cells.

Sensitive TaqMan real-time polymerase chain reaction (PCR)-based methods have been developed recently for the detection and quantitation of feline leukemia virus (FeLV) proviral DNA in infected cats. In this chapter, we outline the design and implementation of a TaqMan real-time PCR assay to quantify total FeLV proviral and viral RNA loads in infected cats. The assay is designed to amplify all three FeLV subtypes (A-C), but not FeLV-related endogenous retroviral sequences. The system is tested and optimized using proviral DNA or viral RNA from cells infected with reference strains. The sequence used to produce the standard DNA and RNA is amplified, subcloned into a vector, and sequenced. cRNA is synthesized from the linearized plasmid DNA. Standard DNA and RNA are quantified, diluted and used to determine efficiency, sensitivity, linear amplification range, and precision of the quantitative TaqMan real-time PCR assays.

In the cat genome, endogenous feline leukemia virus (enFeLV) exists as multiple, nearly full-length proviral sequences. Even though no infectious virus is produced from enFeLV sequences, transcription and translation have been demonstrated in tissues of healthy cats and in feline cell lines. To test the hypothesis that the enFeLV loads play a role in exogenous FeLV-A infection and pathogenesis, we designed three real-time PCR assays to quantify U3 and env enFeLV loads (two within U3 amplifying different sequences; one within env). Applying these assays, we investigated the loads in blood samples derived from Swiss privately owned domestic cats, specific pathogen-free (SPF) cats and European wildcats (Felis silvestris silvestris). Significant differences in enFeLV loads were observed between privately owned cats and SPF cats as well as among SPF cats originating from different catteries and among domestic cats of different breeds. Within privately owned cats, FeLV-infected cats had higher loads than uninfected cats. In addition, higher enFeLV loads were found in wildcats compared to domestic cats. The assays described herein are important prerequisites to quantify enFeLV loads and thus to investigate the influence of enFeLV loads on the course of FeLV infection.

The efficacy of interferons (IFNs), used empirically to treat retrovirus-infected cats has been shown in vivo, but the direct effect on infected cells is largely unknown. Ten-fold serial dilutions of three recombinant IFNs available for therapy, human IFNalpha(2a), IFNalpha(A/D) and feline IFNomega were added to the chronically FeLV-infected cell line FL74. IFNs did not apparently affect viral protein expression. However, reverse transcriptase activity (RT), directly proportional to the amount of infectious free virions, decreased with increasing concentrations of IFN and longer treatment times. The induction of apoptosis by IFN was suspected. Results of its evaluation by annexin V-Fluos staining showed that IFNs decreased the viability of treated FeLV-infected cells, and increased apoptosis, but not of non-infected cells. According to the IC(50), rHuIFNalpha(A/D) appeared to be the most effective IFN in inhibiting RT.

Cats exposed to feline leukemia virus (FeLV) may develop different outcomes of the infection. However, during acute infection blood proviral and viral RNA loads of cats with progressive and regressive infection are not significantly different. Thus, not the overall loads but rather those of specific leukocyte subsets may influence the infection outcome. By combining fluorescence activated cell sorting (FACS) with sensitive real-time TaqMan PCR and reverse transcriptase (RT) PCR, we established in the present study the methods to determine FeLV proviral and viral RNA loads in specific leukocyte subsets. In addition, they were applied to analyze long-term persistently FeLV-infected (p27-positive) and FeLV exposed but nonantigenemic (p27-negative), nonviremic cats. In the latter animals, CD4(+) and B lymphocytes exhibited the highest proviral loads, whereas in p27-positive cats, all leukocyte subsets showed similar high loads. In p27-positive cats, monocytes and granulocytes bore the highest viral RNA loads, whereas only one p27-negative cat was positive for viral RNA in T lymphocytes. To our knowledge, this is the first study to investigate FeLV proviral and viral RNA loads in leukocyte subsets of FeLV exposed cats. The herein described methods are important prerequisites to gain a deeper insight into the pathogenesis of FeLV infection.

In this paper the design and use of a semi-quantitative real-time polymerase chain reaction assay (RT-PCR) for feline leukaemia virus (FeLV) provirus is described. Its performance is evaluated against established methods of FeLV diagnosis, including virus isolation and enzyme-linked immunoassay (ELISA) in a population of naturally infected cats. The RT-PCR assay is found to have both a high sensitivity (0.92) and specificity (0.99) when examined by expectation maximisation methods and is also able to detect a large number of cats with low FeLV proviral loads that were negative by other conventional test methods.

Libraries of feline leukemia virus subgroup A (FeLV-A)-derived envelope (Env) proteins with random peptides incorporated into the cell-targeting region were screened for productive gene delivery to the PC-3 human prostate cell line. In order to increase the efficiency of recovering and testing functional clones, the screen was performed in the presence of a replication-competent 4070A Env-expressing virus under conditions of viral interference. The Env proteins resulting from this library screen were able to mediate gene delivery to 4070A-infected human PC-3, DU145 prostate and TE671 rhabdomyosarcoma cells in the presence, but not absence, of 4070A helper virus. FeLV-A, FeLV-B and Moloney murine leukemia virus (Mo-MuLV) Env proteins were unable to substitute for 4070A Env. Flow cytometry and Western blot analyses indicated increased cell-surface expression and virion incorporation of library-derived Env proteins in the presence of 4070A Env. Interference assays on cells infected with both 4070A and FeLV-B are consistent with the combination of library-derived and 4070A Env proteins utilizing the Pit1 receptor.

We compared the host cell range of T-lymphotropic feline leukemia virus (FeLV-T) with that of FeLV subgroup B (FeLV-B) by pseudotype assay in the presence of FeLIX, a truncated envelope glycoprotein of endogenous FeLV. Although both viruses use Pit1 as a receptor and FeLIX does not hamper FeLV-B infection by receptor interference, the host ranges of FeLV-T and -B were not exactly the same, suggesting a different Pit1 usage at the post-binding level. A comparison of Pit1 sequences of various mammalian species indicated that extracellular loop 1 in a topology model deduced with the PHD PredictProtein algorism may be one of the regions responsible for efficient infection by FeLV-T.

Feline leukemia virus (FeLV) is a gamma retrovirus that induces fatal diseases in domestic cats. Efficacious FeLV vaccines prevent persistent viremia and development of FeLV-related disease after virus exposure, but not minimal viral replication and a provirus-positive state as recently demonstrated using sensitive real-time PCR assays. Proviral integration is an important parameter of latent infection and persistence of retroviruses in infected cells. So far, FeLV-specific real-time PCR assays could not distinguish between the integrated and episomal forms of the provirus. Thus, it was the aim of the present study to develop a rapid assay for the detection of FeLV proviral integration. The test combines conventional and quantitative real-time PCR that use virus-specific primers and primers specific for cat genomic small interspersed nuclear elements. It was applied to analyze the time course of proviral integration into the genome of a feline fibroblast cell line and detect provirus integration in peripheral white blood cells from vaccinated and unvaccinated, FeLV-exposed cats. The newly developed rapid test will essentially contribute to a better understanding of the mechanisms involved in the pathogenesis of FeLV infection and be especially useful in the development of antiretroviral vaccines and therapies aimed at the inhibition of proviral integration.

A novel diagnostic test for feline leukemia virus (FeLV) RNA in saliva from naturally infected cats is described in this study. We evaluated different diagnostic tests and compared them with the widely used enzyme-linked immunosorbent assay (ELISA) for the detection of p27 in the diagnosis of FeLV. Blood samples from 445 cats were tested for the presence of provirus by real-time PCR and plasma and saliva specimens from those cats were tested for the presence of viral RNA by real-time reverse transcription (RT)-PCR and for the presence of p27 by ELISA. In comparison to conventional ELISA, the diagnostic sensitivity and specificity of the detection of salivary FeLV RNA by real-time RT-PCR were found to be 98.1 and 99.2%, respectively. Detection of viral RNA in saliva had a positive predictive value of 94.6% and a negative predictive value of 99.7%. The kappa value was 0.96, demonstrating an almost perfect agreement between both tests. Furthermore, we confirmed previous results showing that a number of cats which tested negative for the presence of p27 in plasma were in fact positive for the presence of DNA provirus in blood specimens (5.4%). However, 96.4% of these latently infected cats did not shed viral RNA in saliva; therefore, we assume that these cats are of relatively low clinical importance at the time of testing. This study shows considerable diagnostic value of the detection of saliva FeLV RNA in naturally infected cats. This new diagnostic method has advantages over the conventional ELISA, such as less invasive sample collection and no requirement for trained personnel.

The long terminal repeat (LTR) region of leukemia viruses plays a critical role in tissue tropism and pathogenic potential of the viruses. We have previously reported that U3-LTR from Moloney murine and feline leukemia viruses (Mo-MuLV and FeLV) upregulates specific cellular genes in trans in an integration-independent way. The U3-LTR region necessary for this action does not encode a protein but instead makes a specific RNA transcript. Because several cellular genes transactivated by the U3-LTR can also be activated by NFkappaB, and because the antiapoptotic and growth promoting activities of NFkappaB have been implicated in leukemogenesis, we investigated whether FeLV U3-LTR can activate NFkappaB signaling. Here, we demonstrate that FeLV U3-LTR indeed upregulates the NFkappaB signaling pathway via activation of Ras-Raf-IkappaB kinase (IKK) and degradation of IkappaB. LTR-mediated transcriptional activation of genes did not require new protein synthesis suggesting an active role of the LTR transcript in the process. Using Toll-like receptor (TLR) deficient HEK293 cells and PKR(-/-) mouse embryo fibroblasts, we further demonstrate that although dsRNA-activated protein kinase R (PKR) is not necessary, TLR3 is required for the activation of NFkappaB by the LTR. Our study thus demonstrates involvement of a TLR3-dependent but PKR-independent dsRNA-mediated signaling pathway for NFkappaB activation and thus provides a new mechanistic explanation of LTR-mediated cellular gene transactivation.

Gammaretroviruses, including the subgroups A, B, and C of feline leukemia virus (FeLV), use a multiple-membrane-spanning transport protein as a receptor. In some cases, such as FeLV-T, a nonclassical receptor that includes both a transport protein (Pit1) and a soluble cofactor (FeLIX) is required for entry. To define which regions confer specificity to classical versus nonclassical receptor pathways, we engineered mutations found in either FeLV-A/T or FeLV-T, individually and in combination, into the backbone of the transmissible form of the virus, FeLV-A. The receptor specificities of these viruses were tested by measuring infection and binding to cells expressing the FeLV-A receptor or the FeLV-T receptors. FeLV-A receptor specificity was maintained when changes at amino acid position 6, 7, or 8 of the mature envelope glycoprotein were introduced, although differences in infection efficiency were observed. When these N-terminal mutations were introduced together with a C-terminal 4-amino-acid insertion and an adjacent amino acid change, the resulting viruses acquired FeLV-T receptor specificity. Additionally, a W-->L change at amino acid position 378, although not required, enhanced infectivity for some viruses. Thus, we have found that determinants in the N and C termini of the envelope surface unit can direct entry via the nonclassical FeLV-T receptor pathway. The region that has been defined as the receptor binding domain of gammaretroviral envelope proteins determined entry via the FeLV-A receptor independently of the presence of the N- and C-terminal FeLV-T receptor determinants.

Feline leukaemia virus (FeLV) infection in cats is not only of veterinary importance but also a well-acknowledged animal model for studying the pathogenesis of retroviral disease. After virus exposure, different courses and outcomes of FeLV infection may prevail; they have been associated with cellular and humoral immune responses and the FeLV proviral load in peripheral blood. We hypothesized that the plasma viral RNA load might be an additional relevant indicator for the infection outcome. To quantify these loads, a real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed. The assay amplifies FeLV-A, -B, and -C as some naturally infected cats could not be identified with a FeLV-A-based assay previously. The assay was applied to determine plasma FeLV RNA loads in cats infected both naturally and experimentally with FeLV. In addition, an improved real-time PCR assay for quantitation of FeLV proviral loads is described. The assays developed were more sensitive than ELISA and virus isolation in the early phase of infection. In addition, PCR allows the identification of provirus carriers that have overcome antigenaemia. Thus, for most effective detection of FeLV exposure and characterization of the infection in a cat, PCR assays are recommended as diagnostic tools.

We previously described replication-competent feline leukemia virus (FeLV) vectors with high-level and stable expression of a green fluorescent protein (GFP) or a suicide transgene in cell cultures in vitro. Considering that FeLV might potentially be used to deliver therapeutic genes in vivo, we first evaluated the expression of the GFP gene introduced in cats by the FeLV, Rickard subgroup A (FRA) construct. Eight newborn kittens were either inoculated with pFRA-GFP plasmid DNA intradermally, or challenged intraperitoneally with FRA-GFP-infected feline fibroblasts. During a 12-week observation period, five cats were shown to be progressively viremic. Quantitative PCR and RT-PCR analyses of plasma and tissue samples from these cats showed that GFP was retained in FeLV DNA or RNA to a variable degree, ranging from 0.002 to 27.890%. Tissue DNA samples were analyzed by PCR for the status of GFP and the env-transgene complex. While the proviruses carrying the GFP transgene were shown to be minor species, all tissues, however, retained the full-length GFP transgene. Despite the occurrence of predominant species with various deletions in the viral genome, approximately 1-3% of the total cell population was GFP-positive in the lymphoid tissues as visualized by laser confocal microscopy. Co-localization of immunofluorescent cells indicated that CD3-positive T cells, dendritic cells and macrophages were the major targets for GFP expression. These findings on the detectable in vivo expression of GFP for as long as a period of 3 months could be viewed positively for contemplating a therapeutic strategy for control of FeLV infection in the cats.

The outcome of feline leukemia virus (FeLV) infection in nature is variable, including malignant, proliferative, and degenerative disorders. The determinants of disease outcome are not well understood but are thought to include viral, host, and environmental factors. In particular, genetic variations in the FeLV long terminal repeat (LTR) and SU gene have been linked to disease outcome. FeLV-945 was previously identified as a natural isolate predominant in non-T-cell neoplastic and nonneoplastic diseases in a geographic cohort. The FeLV-945 LTR was shown to contain unique repeat elements, including a 21-bp triplication downstream of the enhancer. The FeLV-945 SU gene was shown to encode mutational changes in functional domains of the protein. The present study details the outcomes of infection with recombinant FeLVs in which the LTR and envelope (env) gene of FeLV-945, or the LTR only, was substituted for homologous sequences in a horizontally transmissible prototype isolate, FeLV-A/61E. The results showed that the FeLV-945 LTR determined the kinetics of disease. Substitution of the FeLV-945 LTR into FeLV-A/61E resulted in a significantly more rapid disease onset but did not alter the tumorigenic spectrum. In contrast, substitution of both the FeLV-945 LTR and env gene changed the disease outcome entirely. Further, the impact of FeLV-945 env on the disease outcome was dependent on the route of inoculation. Since the TM genes of FeLV-945 and FeLV-A/61E are nearly identical but the SU genes differ significantly, FeLV-945 SU is implicated in the outcome. These findings identify the FeLV-945 LTR and SU gene as determinants of disease.

In this study, we present enzymatic differences found between recombinant RTs derived from feline leukemia virus and feline immunodeficiency virus. Firstly, FIV RT showed low steady state K(m) values for dNTPs compared to FeLV RT. Consistent with this, FIV RT synthesized DNA more efficiently than FeLV RT at low dNTP concentrations. We observed similar concentration-dependent activity differences between other lentiviral (HIV-1 and SIV) and non-lentiviral (MuLV and AMV) RTs. Second, FeLV RT showed less efficient misincorporation with biased dNTP pools and mismatch primer extension capabilities, compared to FIV RT. In M13mp2 lacZalpha forward mutation assays, FeLV RT displayed approximately 11-fold higher fidelity than FIV RT. Finally, FeLV RT was less sensitive to 3TCTP and ddATP than FIV RT. This study represents the comprehensive enzymatic characterization of RTs from a lentivirus and a non-lentivirus retrovirus from the same host species. The data presented here support enzymatic divergences seen among retroviral RTs.

Molecularly cloned feline leukemia virus (FeLV)-clone 33 (C-33), derived from a cat with acute myelocytic leukemia (AML), was examined to assess its relation to the pathogenesis of AML and myelodysplastic syndrome (MDS). To evaluate in vitro pathogenicity of FeLV C-33, bone marrow colony-forming assay was performed on marrow cells infected with FeLV C-33 or an FeLV subgroup A strain (61E, a molecularly cloned strain with minimal pathogenicity). The myeloid colony-forming activity of feline bone marrow mononuclear cells infected with FeLV C-33 was significantly lower than that of cells infected with 61E. This suggests that FeLV C-33 has myeloid lineage-specific pathogenicity for cats, and that FeLV C-33 infection is useful as an experimental model for investigating pathogenesis of MDS and AML.

The number, chromosomal distribution, and insertional polymorphisms of endogenous feline leukemia viruses (enFeLVs) were determined in four domestic cats (Burmese, Egyptian Mau, Persian, and nonbreed) using fluorescent in situ hybridization and radiation hybrid mapping. Twenty-nine distinct enFeLV loci were detected across 12 of the 18 autosomes. Each cat carried enFeLV at only 9 to 16 of the loci, and many loci were heterozygous for presence of the provirus. Thus, an average of 19 autosomal copies of enFeLV were present per cat diploid genome. Only five of the autosomal enFeLV sites were present in all four cats, and at only one autosomal locus, B4q15, was enFeLV present in both homologues of all four cats. A single enFeLV occurred in the X chromosome of the Burmese cat, while three to five enFeLV proviruses occurred in each Y chromosome. The X chromosome and nine autosomal enFeLV loci were telomeric, suggesting that ectopic recombination between nonhomologous subtelomeres may contribute to enFeLV distribution. Since endogenous FeLVs may affect the infectiousness or pathogenicity of exogenous FeLVs, genomic variation in enFeLVs represents a candidate for genetic influences on FeLV leukemogenesis in cats.

Retroviral pseudotyping for gene transfer applications endeavors to alter vector tropism and maintain a suitable titer. We investigated the compatibility of the Jaagsiekte sheep retrovirus (JSRV) envelope glycoprotein with the feline immunodeficiency virus (FIV) vector. A construct consisting of the minimal JSRV env coding region expressed from a standard mammalian expression plasmid generated FIV vector titers of approximately 10(4) TU/ml following standard triple transfection, collection of supernatants, and concentration by centrifuge. Interestingly, retention of the native proviral 5' and 3' flanking regions surrounding the JSRV env resulted in exceptional titers of approximately 10(8) TU/ml following the same viral preparation. To discern the regions necessary to achieve this 10,000-fold increase in titer, additional constructs were designed and tested. Our results indicate that the enhanced vector titer correlates with an increase in steady-state levels of envelope RNA that results from a combination of RNA splicing and stability, leading to increased envelope protein production. Expression of four other glycoproteins in an expression plasmid retaining the enhancing elements from the JSRV proviral sequence increased FIV vector titers from 0- to 100-fold. These novel data demonstrate that optimization of the envelope expression construct can profoundly influence titers for lentivirus vectors.

The mechanisms responsible for effective vs. ineffective viral containment are central to immunoprevention and therapies of retroviral infections. Feline leukemia virus (FeLV) infection is unique as a naturally occurring, diametric example of effective vs. ineffective retroviral containment by the host. We developed a sensitive quantitative real-time DNA PCR assay specific for exogenous FeLV to further explore the FeLV-host relationship. By assaying p27 capsid antigen in blood and FeLV DNA in blood and tissues of successfully vaccinated, unsuccessfully vaccinated, and unvaccinated pathogen-free cats, we defined four statistically separable classes of FeLV infection, provisionally designated as abortive, regressive, latent, and progressive. These host-virus relationships were established by 8 weeks post-challenge and could be maintained for years. Real-time PCR methods offer promise in gaining deeper insight into the mechanisms of FeLV infection and immunity.

FeLV-945 is a representative isolate of the natural feline leukemia virus (FeLV) variant predominant in non-T-cell malignant, proliferative, and degenerative diseases in a geographic cohort. The FeLV-945 surface glycoprotein (SU) is closely related to natural horizontally transmissible FeLV subgroup A (FeLV-A) but was found to differ from a prototype to a larger extent than the members of FeLV-A differ among themselves. The sequence differences included point mutations restricted largely to the functional domains of SU, i.e., VRA, VRB, and PRR. Despite the sequence differences in these critical domains, measurements of receptor utilization, including host range and superinfection interference, confirmed the assignment of FeLV-945 to subgroup A. Other proviruses isolated from the cohort contained similar sequence hallmarks and were assigned to FeLV subgroup A. A provirus from cat 1046 contained a histidine-to-proline change at SU residue 6 within an SPHQ motif that was previously identified as a critical mediator of fusion events during virus entry. The 1046 pseudotype virus entered cells only in the presence of the soluble cofactor FeLIX provided in trans, but it retained an ecotropic host range even in the presence of FeLIX. The mutational changes in FeLV-945 were shown to confer significant functional differences compared to prototype FeLV-A viruses. The substitution of FeLV-945 envelope gene sequences for FeLV-A/61E sequences conferred a small but statistically significant replicative advantage in some feline cells. Moreover, substitution of the unique FeLV-945 long terminal repeat and envelope gene for those of FeLV-A/61E altered the disease spectrum entirely, from a thymic lymphoma of a T-cell origin to an as yet uncharacterized multicentric lymphoma that did not contain T cells.

The recombinant retrovirus, MoFe2-MuLV (MoFe2), was constructed by replacing the U3 region of Moloney murine leukemia virus (M-MuLV) with homologous sequences from the FeLV-945 LTR. NIH/Swiss mice neonatally inoculated with MoFe2 developed T-cell lymphomas of immature thymocyte surface phenotype. MoFe2 integrated infrequently (0 to 9%) near common insertion sites (CISs) previously identified for either parent virus. Using three different strategies, CISs in MoFe2-induced tumors were identified at six loci, none of which had been previously reported as CISs in tumors induced by either parent virus in wild-type animals. Two of the newly identified CISs had not previously been implicated in lymphoma in any retrovirus model. One of these, designated 3-19, encodes the p101 regulatory subunit of phosphoinositide-3-kinase-gamma. The other, designated Rw1, is predicted to encode a protein that functions in the immune response to virus infection. Thus, substitution of FeLV-945 U3 sequences into the M-MuLV long terminal repeat (LTR) did not alter the target tissue for M-MuLV transformation but significantly altered the pattern of CIS utilization in the induction of T-cell lymphoma. These observations support a growing body of evidence that the distinctive sequence and/or structure of the retroviral LTR determines its pattern of insertional activation. The findings also demonstrate the oligoclonal nature of retrovirus-induced lymphomas by demonstrating proviral insertions at CISs in subdominant populations in the tumor mass. Finally, the findings demonstrate the utility of novel recombinant retroviruses such as MoFe2 to contribute new genes potentially relevant to the induction of lymphoid malignancy.

Feline leukaemia virus (FeLV)-945 was previously identified in natural multicentric lymphomas and contains a 21 bp tandem triplication in the LTR. In the present study, FeLV LTR variation was examined in the cohort from which FeLV-945 was identified. The objectives of the study were to evaluate FeLV LTR variation within the cohort, to determine whether the FeLV-945 LTR was associated uniquely with multicentric lymphoma and to evaluate functional attributes that may have contributed selective advantage to the predominant LTR variants observed. T-cell tumours uniformly contained LTRs with duplicated enhancer sequences, although enhancer duplications conferred little transcriptional advantage. Non-T-cell malignant, proliferative and degenerative diseases contained LTRs with two, three or four tandemly repeated copies of the 21 bp sequence originally identified in FeLV-945. While the length and termini of enhancer duplications were variable, the 21 bp repeat unit was invariant. Triplication of the 21 bp repeat conferred the optimal replicative advantage in feline cells.

Background: Feline leukemia virus (FeLV) induces degenerative, proliferative and malignant hematologic disorders in its natural host, the domestic cat. FeLV-945 is a viral variant identified as predominant in a cohort of naturally infected animals. FeLV-945 contains a unique sequence motif in the long terminal repeat (LTR) comprised of a single copy of transcriptional enhancer followed by a 21-bp sequence triplicated in tandem. The LTR is precisely conserved among independent cases of multicentric lymphoma, myeloproliferative disease and anemia in animals from the cohort. The 21-bp triplication was previously shown to act as a transcriptional enhancer preferentially in hematopoietic cells and to confer a replicative advantage. The objective of the present study was to examine the molecular mechanism by which the 21-bp triplication exerts its influence and the selective advantage responsible for its precise conservation., Results: Potential binding sites for the transcription factor, c-Myb, were identified across the repeat junctions of the 21-bp triplication. Such sites would not occur in the absence of the repeat; thus, a requirement for c-Myb binding to the repeat junctions of the triplication would exert a selective pressure to conserve its sequence precisely. Electrophoretic mobility shift assays demonstrated specific binding of c-Myb to the 21-bp triplication. Reporter gene assays showed that the triplication-containing LTR is responsive to c-Myb, and that responsiveness requires the presence of both c-Myb binding sites. Results further indicated that c-Myb in complex with the 21-bp triplication recruits the transcriptional co-activator, CBP, a regulator of normal hematopoiesis. FeLV-945 replication was shown to be positively regulated by CBP in a manner dependent on the presence of the 21-bp triplication., Conclusion: Binding sites for c-Myb across the repeat junctions of the 21-bp triplication may account for its precise conservation in the FeLV-945 LTR. c-Myb binding and CBP recruitment to the LTR positively regulated virus production, and thus may be responsible for the replicative advantage conferred by the 21-bp triplication. Considering that CBP is present in hematopoietic cells in limiting amounts, we hypothesize that FeLV-945 replication in bone marrow may influence CBP availability and thereby alter the regulation of CBP-responsive genes, thus contributing to altered hematopoiesis and consequent hematologic disease.

To evaluate the presence of centrosome amplification and the resulting chromosomal instability in cat tumors, a newly established feline lymphoma cell line and four already established feline lymphoma cell lines were examined using immunohistochemical analysis of centrosomes. The number of chromosomes were subsequently counted by metaphase spread. Moreover, to explore whether mutational inactivation of the p53 gene or inactivation of the P53 protein caused by mdm2 gene overexpression, occurred in the feline lymphoma cell lines, mutational analysis of the feline p53 gene was carried out. The expression of feline mdm2 mRNA was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Centrosome amplification and chromosomal instability was observed in three out of the five feline lymphoma cell lines. Of these three feline lymphoma cell lines, one had aberrations in the P53 amino-acid sequence, whereas the others had none. There was no significant difference in the expression of mdm2 mRNA between peripheral blood mononuclear cells (PBMC) obtained from a normal cat and that of the five feline lymphoma cell lines. These findings indicate that centrosome amplification also occurs in cat tumors and is strongly correlated with chromosomal instability, suggesting that the immunostaining of centrosomes could be an alternative method for the examination of the chromosomal instability. Furthermore, this study suggests the presence of unknown mechanism that leads to the centrosome amplification in feline lymphomas.

We isolated and sequenced two complete endogenous feline leukemia viruses (enFeLVs), designated enFeLV-AGTT and enFeLV-GGAG. In enFeLV-AGTT, the open reading frames are reminiscent of a functioning FeLV genome, and the 5' and 3' long terminal repeat sequences are identical. Neither endogenous provirus is genetically fixed in cats but polymorphic, with 8.9 and 15.2% prevalence for enFeLV-AGTT and enFeLV-GGAG, respectively, among a survey of domestic cats. Neither provirus was found in the genomes of related species of the Felis genus, previously shown to harbor enFeLVs. The absence of mutational divergence, polymorphic incidence in cats, and absence in related species suggest that these enFeLVs may have entered the germ line more recently than previously believed, perhaps coincident with domestication, and reopens the question of whether some enFeLVs might be replication competent.

The host range of swinepox virus (SPV) is restricted to swine, although SPV has been shown to infect mammalian, non-swine cells, without recovery of infectious virus. SPV is a reasonable candidate for development as a non-productively replicating viral vector for use in non-swine, mammalian species, such as the cat. A novel SPV gene deletion (SPV 043) was created and found to be non-attenuating. This deletion was utilized to generate a stable recombinant virus expressing the Gag-Pro and Env proteins of feline leukemia virus (FeLV). Expression and replication of this vector was studied in embryonic swine kidney cells (ESK-4), and two feline cell lines, Crandell feline kidney cells (CRFK) and feline skin fibroblasts (FSF). Our results showed that feline cells were susceptible to infection by SPV and supported expression of foreign genes driven by synthetic poxvirus promoters, however, SPV viral DNA was not replicated in feline cells and infectious virus was not recovered. In addition, FeLV Gag virus-like particles were produced from both ESK-4 and CRFK cells and foreign antigens were incorporated into infectious SPV intracellular mature virions (IMV). These results suggest that SPV may have potential as a safe vaccine delivery vector for cats.

An important requirement for many gene therapy applications is to direct therapeutic genes specifically to target cells. Here we describe an improved vector targeting method that does not depend on the use of a known cell-targeting ligand. It entails screening a library of constitutively produced retroviruses with random amino acid substitutions in the cell-targeting region of the envelope proteins for their ability to mediate gene delivery to a target cell. By screening such a library on the ras-transformed 143B human cell line, we have isolated an envelope protein that preferentially targets 143B cells and 293T cells expressing the SV40 T antigen via a novel, unidentified receptor. Furthermore, retroviruses expressing the library-derived envelope protein can be concentrated by centrifugation. This is the first demonstration of a novel concept in vector targeting: the selection of productive retroviral entry via an alternate receptor with modified cellular tropism in the absence of a known cell-targeting moiety. The method is, in principle, applicable even to cells that have not been well characterized, and therefore potentially suitable for targeting many diverse cell types.

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.

The U3 region of the LTR of oncogenic Moloney murine leukemia virus (Mo-MuLV) and feline leukemia viruses (FeLV) have been previously reported to activate expression of specific cellular genes in trans, such as MHC class I, collagenase IV, and MCP-1, in an integration-independent manner. It has been suggested that transactivation of these specific cellular genes by leukemia virus U3-LTR may contribute to the multistage process of leukemogenesis. The U3-LTR region, necessary for gene transactivational activity, also contains multiple transcription factor-binding sites that are essential for normal virus replication. To dissect the promoter activity and the gene transactivational activity of the U3-LTR, we conducted mutational analysis of the U3-LTR region of FeLV-A molecular clone 61E. We identified minimal nucleotide substitution mutants on the U3 LTR that did not disturb transcription factor-binding sites but abrogated its ability to transactivate the collagenase gene promoter. To determine if these mutations actually have altered any uncharacterized important transcription factor-binding site, we introduced these U3-LTR mutations into the full-length infectious molecular clone 61E. We demonstrate that the mutant virus was replication competent but could not transactivate cellular gene expression. These results thus suggest that the gene transactivational activity is a distinct property of the LTR and possibly not related to its promoter activity. The cellular gene transactivational activity-deficient mutant FeLV generated in this study may also serve as a valuable reagent for testing the biological significance of LTR-mediated cellular gene activation in the tumorigenesis caused by leukemia viruses.

Feline leukemia virus (FeLV) is a type-C retrovirus associated with lymphoid and hematopoietic malignancies in cats. The FeLV-induced tumors are thought to be caused, at least in part, by somatically acquired insertional mutagenesis in which the integrated provirus may activate a proto-oncogene or disrupt a tumor suppressor gene. This study was undertaken to enumerate and map the acquired proviral insertions in the genome of a feline thymic lymphoma cell line (FT-1) infected with FeLV. Fluorescence in situ hybridization (FISH) combined with tyramide signal amplification was applied on the chromosome specimen of FT-1 cells and normal cat lymphocytes, with an entire FeLV-A genome used as a probe. Specific hybridization signals were detected from only the metaphases of the FT-1 cells, not from those of normal cat lymphocytes. Statistically based on the Poisson's distribution, at least six loci of chromosomal regions, A2p23-p22, B2p15-p14, B4p15-p14, D4q23-q24, E1p14-p13, and E2p13-p12, appeared to be positive for FeLV integration. Consistently, Southern blot hybridization analysis using an FeLV LTR-U3 probe specific for exogenous FeLV showed the integration of at least six FeLV proviral genomes in FT-1 cells. The cytogenetic technique employed here will provide valuable molecular tags to reveal unidentified tumor-associated genes in FeLV-associated tumor cells.

To evaluate host range differences between two different strains of feline leukemia virus subgroup B (FeLV-B), we compared the binding and infectivity patterns of retrovirus vectors bearing either FeLV-B-90Z or FeLV-B-GA envelopes. We report here that the ability of these envelopes to utilize different Pit1 orthologs is mediated primarily by the receptor binding domain; however, in the case of FeLV-B-90Z, the C terminus also contributes to the recognition of certain Pit1 orthologs.

Entry by retroviruses is mediated through interactions between the viral envelope glycoprotein and the host cell receptor(s). We recently identified two host cell proteins, FeLIX and Pit1, that are necessary for infection by cytopathic, T-cell-tropic feline leukemia viruses (FeLV-T). Pit1 is a classic multiple transmembrane protein used as a receptor by several other simple retroviruses, including subgroup B FeLV (FeLV-B), and FeLIX is a secreted cellular protein expressed from endogenous FeLV-related sequences (enFeLV). FeLIX is nearly identical to FeLV-B envelope sequences that encode the N-terminal half of the viral surface unit (SU), because these FeLV-B sequences are acquired by recombination with enFeLV. FeLV-B SUs can functionally substitute for FeLIX in mediating FeLV-T infection. Both of these enFeLV-derived cofactors can efficiently facilitate FeLV-T infection only of cells expressing Pit1, not of cells expressing the related transport protein Pit2. We therefore have used chimeric Pit1/Pit2 receptors to map the determinants for cofactor binding and FeLV-T infection. Three distinct determinants appear to be required for cofactor-dependent infection by FeLV-T. We also found that Pit1 sequences within these same domains were required for binding by FeLIX to the Pit receptor. In contrast, these determinants were not all required for receptor binding by the FeLV-B SU cofactors used in this study. These data indicate that cofactor binding is not sufficient for FeLV-T infection and suggest that there may be a direct interaction between FeLV-T and the Pit1 receptor.

Murine leukemia virus (MLV)-derived envelope proteins containing alterations in or adjacent to the highly conserved PHQ motif present at the N terminus of the envelope surface subunit (SU) are incorporated into vector particles but are not infectious due to a postbinding block to viral entry. These mutants can be rendered infectious by the addition of soluble receptor-binding domain (RBD) proteins in the culture medium. The RBD proteins that rescue the infectivity of these defective MLV vectors can be derived from the same MLV or from other MLVs that use distinct receptors to mediate entry. We have now constructed functional immunologically reactive gibbon ape leukemia virus (GALV) envelope proteins, tagged with a feline leukemia virus (FeLV)-derived epitope tag, which are efficiently incorporated into infectious particles. Tagged GALV envelope proteins bind specifically to cells expressing the phosphate transporter protein Pit1, demonstrating for the first time that Pit1 is the binding receptor for GALV and not a coreceptor or another type of GALV entry factor. We have also determined that GALV particles bearing SU proteins with an insertion C-terminal to the PHQ motif (GALV I(10)) bind Pit1 but fail to infect cells. Incubation with soluble GALV RBD renders GALV I(10) particles infectious, whereas incubation with soluble RBDs from MLV or FeLV-B does not. This finding is consistent with the results obtained by Lauring et al. using FeLV-T, a virus that employs Pit1 as a receptor but requires soluble FeLV RBD for entry. MLV and GALV RBDs are not able to render FeLV-T infectious (A. S. Lauring, M. M. Anderson, and J. Overbaugh, J. Virol. 75:8888-8898, 2001). Together, these results suggest that fusion-defective FeLV-T and GALV are restricted to homologous RBD rescue of infectivity.

Tissue-specific gene delivery is an important aspect of many gene therapy applications. The experiments reported here constitute the first successful demonstration that cell-specific entry can be obtained by screening a random library of retroviral envelope proteins produced from a mammalian cell system. The library consisted of 10(6) different subgroup A feline leukemia virus envelope protein variants with 10 randomly substituted amino acids in the receptor-determining region. Selecting the library for fully functional envelope proteins able to mediate stable gene transfer resulted in the identification of a single envelope protein variant (EF). Subsequent examination of the host range of EF revealed that it was highly specific for D17 canine osteosarcoma cells. This was in contrast to the host ranges of the parental subgroup A and closely related subgroup C envelope proteins. Interference assays on D17 cells further indicated that receptor usage by EF was also altered compared with the A and C envelope proteins. The EF envelope protein thus isolated should be useful for studying gene therapy treatments of osteosarcoma in a large-animal model.

We have molecularly cloned a feline leukemia virus (FeLV) (clone 33) from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV proviruses from other cats with AML, contains an unusual structure in its U3 region upstream of the enhancer (URE) consisting of three tandem direct repeats of 47 bp. To test the disease potential and specificity of this unique FeLV LTR, we replaced the U3 region of the LTR of the erythroleukemia-inducing Friend murine leukemia virus (F-MuLV) with that of FeLV clone 33. When the resulting virus, F33V, was injected into newborn mice, almost all of the mice eventually developed hematopoietic malignancies, with a significant percentage being in the myeloid lineage. This is in contrast to mice injected with an F-MuLV recombinant containing the U3 region of another FeLV that lacks repetitive URE sequences, none of which developed myeloid malignancies. Examination of tumor proviruses from F33V-infected mice failed to detect any changes in FeLV U3 sequences other than that in the URE. Like F-MuLV-infected mice, those infected with the F-MuLV/FeLV recombinants were able to generate and replicate mink cell focus-inducing viruses. Our studies are consistent with the idea that the presence of repetitive sequences upstream of the enhancer in the LTR of FeLV may favor the activation of this promoter in myeloid cells and contribute to the development of malignancies in this hematopoietic lineage.

Feline retrovirus infections have been extensively studied for more than 30 years as an animal model for the persistent infections and pathogenesis caused by retroviruses in general. Two retroviruses, feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV), have been recognized as causative agents of a variety of diseases including proliferative and degenerative diseases. Recent studies revealed the receptors of FeLV, its variants and FIV. FeLVs utilize at least three distinct receptors, two of which have been successfully cloned and characterized. Furthermore, an FeLV variant which induces severe immunodeficiency, utilizes a truncated envelope of the endogenous FeLV as coreceptor or cofactor for viral entry. FIV utilizes as receptor one of the chemokine receptors, CXCR4 which also is a coreceptor for the T-lymphotropic human immunodeficiency virus. This review provides an overview to the infections of FeLV and FIV, specifically focuses on the viral genomic structures, FeLV variants, the immune responses and recent findings on the receptors for FeLV and FIV. Better understanding of retroviral persistence and pathogenesis will aid the development of prophylactic vaccines and therapeutic medicine to interfere with retrovirus infections.

Objective: To determine whether feline vaccine site-associated sarcomas (VSS) contain a higher amount of endogenous FeLV (enFeLV) RNA, compared with feline nonvaccine site-associated sarcomas (non-VSS)., Sample Population: Formalin-fixed paraffin-embedded (FFPE) tissues from 50 VSS and 50 cutaneous non-VSS., Procedure: RNA was extracted from FFPE sections of each tumor, and regions of the long terminal repeat (LTR) and envelope (env) gene of enFeLV were amplified by use of reverse transcriptase-polymerase chain reaction (RT-PCR). The density of each RT-PCR product band for enFeLV was compared with that of a constitutively expressed gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An integrated density value (IDV) was determined by use of densitometry, and the IDV ratio for enFeLV to GAPDH was calculated for each enFeLV primer set., Results: The median (interquartile range) of the IDV ratio for the enFeLV LTR primer set was 0.52 (0.26 to 1.17) for the VSS group and 0.84 (0.21 to 1.53) for the non-VSS group. The median (interquartile range) of the IDV ratio for the enFeLV env primer set was 0.60 (0.37 to 0.91) for the VSS group and 0.59 (0.36 to 1.09) for the non-VSS group., Conclusions: Because the amount of enFeLV RNA within the LTR and env gene was not significantly different between the VSS and non-VSS groups, enFeLV replication or expression is unlikely to be involved in VSS development.

The purpose of this study was to determine if polymerase chain reaction (PCR) could be used to detect FeLV proviral DNA in bone marrow samples of cats with varying suspicion of latent infection. Blood and bone marrow samples from 50 cats and bone marrow from one fetus were collected, including 16 cats with diseases suspected to be FeLV-associated. Serum enzyme-linked immunosorbent assay (ELISA), blood and bone marrow immunofluorescent antibody test (IFA), and blood and bone marrow PCR were performed on each cat, and IFA and PCR on bone marrow of the fetus. Forty-one cats were FeLV negative. Five cats and one fetus were persistently infected with FeLV. Four cats had discordant test results. No cats were positive on bone marrow PCR only. It appears persistent or latent FeLV infection is not always present in conditions classically associated with FeLV., (Copyright 2001 European Society of Feline Medicine.)