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Endogenous retroviruses (ERV) are indicators of vertebrate evolutionary history and play important roles as homeostatic regulators. ERV long terminal repeat (LTR) elements may act as cis-activating promoters or trans-activating enhancer elements modifying gene transcription distant from LTR insertion sites. We previously documented that endogenous feline leukemia virus (FeLV)-LTR copy number variation in individual cats tracks inversely with susceptibility to virulent FeLV disease. To evaluate FeLV-LTR insertion characteristics, we assessed enFeLV-LTR integration site diversity in 20 cats from three genetically distinct populations using a baited linker-mediated PCR approach. We documented 765 individual integration sites unequally represented among individuals. Only three LTR integration sites were shared among all individuals, while 412 sites were unique to a single individual. When primary fibroblast cultures were challenged with exogenous FeLV, we found significantly increased expression of both exogenous and endogenous FeLV orthologs, supporting previous findings of potential exFeLV-enFeLV interactions; however, viral challenge did not elicit transcriptional changes in genes associated with the vast majority of integration sites. This study assesses FeLV-LTR integration sites in individual animals, providing unique transposome genotypes. Further, we document substantial individual variation in LTR integration site locations, even in a highly inbred population, and provide a framework for understanding potential endogenous retroviral element position influence on host gene transcription.

Oncogene-containing retroviruses are generated by recombination events between viral and cellular sequences, a phenomenon called “oncogene capture”. The captured cellular genes, referred to as “v- onc ” genes, then acquire new oncogenic properties. We report a novel feline leukemia virus (FeLV), designated “FeLV– AKT ”, that has captured feline c- AKT1 in feline lymphoma. FeLV– AKT contains a gag–AKT fusion gene that encodes the myristoylated Gag matrix protein and the kinase domain of feline c-AKT1, but not its pleckstrin homology domain. Therefore, it differs structurally from the v- Akt gene of murine retrovirus AKT8. AKT may be involved in the mechanisms underlying malignant diseases in cats.

BACKGROUND: Cats infected with exogenous feline leukemia virus (exFeLV) have a higher chance of lymphoma development than uninfected cats. Furthermore, an increased exFeLV transcription has been detected in lymphomas compared to non-malignant tissues. The possible mechanisms of lymphoma development by exFeLV are insertional mutagenesis or persistent stimulation of host immune cells by viral antigens, bringing them at risk for malignant transformation. Vaccination of cats against exFeLV has in recent years decreased the overall infection rate in most countries. Nevertheless, an increasing number of lymphomas have been diagnosed among exFeLV-negative cats. Endogenous feline leukemia virus (enFeLV) is another retrovirus for which transcription has been observed in cat lymphomas. EnFeLV provirus elements are present in the germline of various cat species and share a high sequence similarity with exFeLV but, due to mutations, are incapable of producing infectious viral particles. However, recombination between exFeLV and enFeLV could produce infectious particles. RESULTS: We examined the FeLV expression in cats that have developed malignant lymphomas and discussed the possible mechanisms that could have induced malignant transformation. For expression analysis we used next-generation RNA-sequencing (RNA-Seq) and for validation reverse transcription quantitative PCR (RT-qPCR). First, we showed that there was no expression of exFeLV in all samples, which eliminates the possibility of recombination between exFeLV and enFeLV. Next, we analyzed the difference in expression of three enFeLV genes between control and lymphoma samples. Our analysis showed an average of 3.40-fold decreased viral expression for the three genes in lymphoma compared to control samples. The results were confirmed by RT-qPCR. CONCLUSIONS: There is a decreased expression of enFeLV genes in lymphomas versus control samples, which contradicts previous observations for the exFeLV. Our results suggest that a persistent stimulation of host immune cells is not an appropriate mechanism responsible for malignant transformation caused by feline endogenous retroviruses.

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34+ peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34+ cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34+ cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45+ cells in total bone marrow were comparable to that of the control, mock-transduced group (37–45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the γ-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the γ-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.

Adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration. The integration profiles of vectors based on MLV, ASLV, SIV and HIV have all been shown to be non-random, and novel vectors with a safer integration pattern have been sought. Recently, we developed a producer cell line called CatPac that packages standard MoMLV vectors with feline leukemia virus (FeLV) gag , pol and env gene products. We now report the integration profile of this vector, asking if the FeLV integrase and capsid proteins could modify the MoMLV integration profile, potentially resulting in a less genotoxic pattern. We transduced rhesus macaque CD34+ hematopoietic progenitor cells with CatPac or standard MoMLV vectors, and determined their integration profile by LAM-PCR. We obtained 184 and 175 unique integration sites (ISs) respectively for CatPac and standard MoMLV vectors, and these were compared with 10 000 in silico -generated random IS. The integration profile for CatPac vector was similar to MoMLV and equally non-random, with a propensity for integration near transcription start sites and in highly dense gene regions. We found an IS for CatPac vector localized 715 nucleotides upstream of LMO-2, the gene involved in the acute lymphoblastic leukemia developed by X-SCID patients treated by gene therapy using MoMLV vectors. In conclusion, we found that replacement of MoMLV env , gag and pol gene products with FeLV did not alter the basic integration profile. Thus, there appears to be no safety advantage for this packaging system. However, considering the stability and efficacy of CatPac vectors, further development is warranted, using potentially safer vector backbones, for instance those with a SIN configuration.

Supernatants from cultures of normal feline lymphocytes stimulated with Staphylococcus enterotoxin A showed antiviral activity, characterized as a gamma-like interferon. With the addition of inactivated feline leukemia virus, markedly less interferon was produced. The reduction in interferon production was not attributable to lowered lymphocyte viability or reduced mitogenic properties of Staphylococcus enterotoxin A and appears to be a direct retroviral effect. This finding may reflect clinically relevant events that may contribute to the development of the feline or human states of acquired immunodeficiency.

The 2′-azido analogs of poly(U) and poly(C), poly(dUz) [poly(2′-azido-2′-deoxyuridylic acid)], and poly(dCz) [poly(2′-azido-2′-deoxycytidylic acid)], were found to inhibit the RNA-directed DNA polymerase (reverse transcriptase) activity of murine leukemia (Moloney, Rauscher) and sarcoma (Moloney) virus, and feline leukemia (Theilen) and sarcoma (Gardner) virus, while under the same conditions the unsubstituted parent compounds failed to do so. In addition, poly(dUz) and poly(dCz) inhibited the replication of exogenous murine sarcoma virus (Moloney) in nontransformed cells (as assessed by an infectious center assay), but poly(dUz) failed to suppress the formation of endogenous sarcoma and leukemia viruses in transformed cell lines (MO-P, JLSV5). In these same cells, poly(dUz) failed to inhibit the multiplication of vesicular stomatitis virus. These data add further strength to the contention that reverse transcriptase is necessary for the productive infection and transformation of normal cells by oncornaviruses but is not essential for the maintenance of this transformed state and the continuous production of new viruses particles by these transformed cells.

Feline sporotrichosis due to Sporothrix brasiliensis is frequently severe and often correlated to zoonotic transmission. Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) cause immunodeficiency in cats; no association has been identified with critical cases of sporotrichosis. Moreover, the cytokine profile in Sporothrix-infected cats and a potential impact of retrovirus co-infections on their immunity is unknown. This study assessed immunological parameters in cats with sporotrichosis with and without FIV or FeLV co-infection. FeLV infection was detected by antigen ELISA and by provirus PCR. FIV infection was investigated through ELISA and Western blot. Cytokine transcription (IFN-γ, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α) was quantified using RT-qPCR and lymphocyte subpopulations (CD4, CD8, CD5 and CD21) were assessed by flow cytometry. Thirty cats with sporotrichosis were recruited to the study, including three FIV-positive and five FeLV-positive (progressive infection) cats. One cat with regressive FeLV infection was excluded from statistics. In comparison to retrovirus-negative cats, FIV-positive cats and FeLV-positive cats had higher IL-10 levels, FeLV-positive cats had lower IL-4 levels and FIV-positive cats had lower IL-12 levels and a lower CD4+/CD8+ ratio. Remarkably, all cats with poor general condition were FeLV (progressive infection) or FIV-positive, but the retrovirus status was not associated with the sporotrichosis treatment length or outcome. The immunological changes and the more severe clinical presentation observed in cats with retrovirus co-infections encourage future prospective studies that address the impact of these changes on prognostic determinants of feline sporotrichosis and the development of new therapy strategies that control disease spread.

Longitudinal studies of cats naturally infected with feline leukemia virus (FeLV) are important for understanding disease outcomes. Levels of p27 antigen and copy numbers of proviral DNA have been associated with FeLV-infection courses. The purpose of this prospective study was to establish cutoff values for p27 antigen concentration and proviral DNA load that distinguished high positive from low positive groups of cats and to evaluate an association with survival. At enrollment, 254 cats were tested by point-of-care and microtiter plate enzyme-linked immunosorbent assays (ELISAs) for p27 antigen and real-time polymerase chain reaction (PCR) for proviral DNA. The 127 positive cats were retested monthly for six months and monitored for survival over the four-year study. A receiver operating characteristic-based analysis of samples with concordant or discordant qualitative results for p27 antigen and proviral DNA was used to establish cutoff values, and when applied to test results at enrollment for classifying cats as high positive or low positive, a significant difference in survival was observed. High positive cats had a median survival of 1.37 years (95% CI 0.83–2.02) from time of enrollment, while most low positive cats were still alive (93.1% survival). Quantitative results for p27 antigen concentration and proviral DNA load were highly correlated with survival times in FeLV-infected cats.

Specific treatments for the long-life infections by feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are either toxic, expensive or not too effective. Interferon α (IFN-α) is an immunomodulatory molecule which has been shown in vitro to decrease the release of infective particles. The aim of this study was to follow the progress of the clinical score and viral parameters of FeLV- and FIV-naturally infected privately owned cats treated with recombinant human IFN-α (rHuIFN-α, Roferon-A). Twenty-seven FeLV-infected cats (FeLV+) and 31 FIV-infected cats (FIV+) were enrolled in the study. Owners were instructed to orally administer 1 mL/day of 60 IU rHuIFN-α/mL in alternating weeks for four months. Blood samples were taken at the beginning of the study (M0), mid-treatment (M2), end of treatment (M4), and 6–10 months later (M10). Clinical status at these time points improved notably with rHuIFN-α treatment, regardless of the initial severity of the disease, an effect which lasted throughout the study in most animals (15 of the 16 FeLV+ symptomatic cats; 20 of the 22 FIV+ symptomatic cats) improved markedly their clinical situation. In FeLV+ cats plasma antigenemia (p27CA), reverse transcriptase (RT) activity, and proviral load decreased at M2 and M4 but increased again at M10 (“rebound effect”). The level of antigenemia or RT activity was below the detection limits in FIV+ cats, and the effect on proviral load was less marked than in FeLV+ cats. Taken together, these results indicate that rHuIFN-α is a good candidate for treating FeLV+ cats, but the “rebound effect” seen when treatment was discontinued suggests that additional studies should be conducted to clarify its effect on progression of the infection in cats.