Exogenous Feline Leukemia Virus Research Articles

Endogenous feline leukemia virus long terminal repeat integration site diversity is highly variable in related and unrelated domestic cats

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.

Progressive and regressive infection with feline leukemia virus (FeLV) in cats in southern Brazil: Prevalence, risk factors associated, clinical and hematologic alterations.

Enzyme-linked immunosorbent assay (ELISA) for viral antigen is commonly used for the diagnosis of progressive feline leukemia virus (FeLV) infection but is not able to determine the true prevalence of infection when used as the sole test. Additional testing to detect proviral DNA will identify regressive (antigen negative) FeLV infections as well as progressive infections. Therefore, this study aimed to determine the prevalence of progressive and regressive FeLV infection, outcome-associated factors, and hematologic changes. A cross-sectional study was performed on 384 cats selected from routine hospital care. Blood samples were subjected to complete blood count, ELISA for FeLV antigen and FIV antibody, and nested PCR amplifying the U3- LTR region and gag gene, which are conserved in most exogenous FeLV. The prevalence of FeLV infection was 45.6% (CI95% 40.6-50.6%). The prevalence of progressive infection (FeLV+P) was 34.4% (CI95% 29.6-39.1%), that of regressive infection (FeLV+R) was 10.4% (CI95% 7.4-13.4%), for discordant but positive results 0.8% (CI95% 0.75-0.84%), for FeLV+P coinfected with FIV 2.6% (CI95% 1.2-4.0%), and FeLV+R coinfected with FIV 1.5% (CI95% 0.3-2.7%). Male cats were three times more likely to be in the FeLV+P group. Cats coinfected with FIV were 4.8 times more likely to belong to the FeLV+R group. In the FeLV+P group, the main clinical changes were lymphoma (38.5%), anemia (24.4%), leukemia (17.9%), concomitant infections (15.4%), and feline chronic gingivostomatitis - FCGS (3.8%). In the FeLV+R group, the main clinical signs were anemia (45.4%), leukemia (18.2%), concomitant infections (18.2%), lymphoma (9.1%), and FCGS (9.1%). Cats in the FeLV+P and FeLV+R groups showed mainly thrombocytopenia (56.6% and 38.2%), non-regenerative anemia (32.8% and 23.5%), and lymphopenia (33.6% and 20.6%). Hemoglobin concentration, packed cell volume (PCV), platelet count, lymphocytes, and eosinophils in the FeLV+P and FeLV+R groups had lower medians than the control group (FeLV/FIV-uninfected, healthy). Erythrocyte and eosinophil counts were statistically different among the three groups, with the medians of the FeLV+P and FeLV+R groups being lower than those of the control group. In addition, the median PCV and band neutrophil counts were higher in FeLV+P than in FeLV+R. Our results show a high prevalence of FeLV, different factors associated with the course of infection, and more frequent and severe hematologic changes in progressive infections compared with regressive infections.

Detection and genetic characterization of feline retroviruses in domestic cats with different clinical signs and hematological alterations.

Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are globally distributed retroviruses that infect domestic cats and cause various syndromes that can lead to death. The aim of this study was to detect and genotype feline retroviruses in Mexican domestic cats. We used PCR assays to identify proviral DNA and viral RNA in 50 domestic cats with different clinical signs and hematological alterations. Endogenous FeLV (enFeLV) was identified in the genomic DNA of all cats in the study, and we detected transcripts of the LTR region of enFeLV in 48 individuals. Exogenous FeLV (exFeLV) was found in 13 cats. Furthermore, we detected FIV proviral DNA in 10 cats. The enFeLV sequences were shown to be the most variable, while the exFeLV sequences were highly conserved and related to previously reported subgroup A sequences. Sequencing of the FIV gag gene revealed the presence of subtype B in the infected cats.

Could Phylogenetic Analysis Be Used for Feline Leukemia Virus (FeLV) Classification?

The surface envelope (SU) protein determines the cell tropism and consequently the pathogenesis of the feline leukemia virus (FeLV) in felids. Recombination of exogenous FeLV (exFeLV) with endogenous retroviruses (enFeLV) allows the emergence of more pathogenic variants. Currently, phenotypic testing through interference assays is the only method to distinguish among subgroups—namely, FeLV-A, -B, -C, -E, and -T. This study proposes a new method for FeLV classification based on molecular analysis of the SU gene. A total of 404 publicly available SU sequences were used to reconstruct a maximum likelihood tree. However, only 63 of these sequences had available information about phenotypic tests or subgroup assignments. Two major clusters were observed: (a) clade FeLV-A, which includes FeLV-A, FeLV-C, FeLV-E, and FeLV-T sequences, and (b) clade enFeLV, which includes FeLV-B and enFeLV strains. We found that FeLV-B, FeLV-C, FeLV-E, and FeLV-T SU sequences share similarities to FeLV-A viruses and most likely arose independently through mutation or recombination from this strain. FeLV-B and FeLV-C arose from recombination between FeLV-A and enFeLV viruses, whereas FeLV-T is a monophyletic subgroup that has probably originated from FeLV-A through combined events of deletions and insertions. Unfortunately, this study could not identify polymorphisms that are specifically linked to the FeLV-E subgroup. We propose that phylogenetic and recombination analysis together can explain the current phenotypic classification of FeLV viruses.

Rapid characterization of feline leukemia virus infective stages by a novel nested recombinase polymerase amplification (RPA) and reverse transcriptase-RPA

Feline leukemia virus (FeLV) is a major viral disease in cats, causing leukemia and lymphoma. The molecular detection of FeLV RNA and the DNA provirus are important for staging of the disease. However, the rapid immunochromatographic assay commonly used for antigen detection can only detect viremia at the progressive stage. In this study, nested recombinase polymerase amplification (nRPA) was developed for exogenous FeLV DNA provirus detection, and reverse transcriptase polymerase amplification (RT-RPA) was developed for the detection of FeLV RNA. The approaches were validated using 108 cats with clinicopathologic abnormalities due to FeLV infection, and from 14 healthy cats in a vaccination plan. The nRPA and RT-RPA assays could rapidly amplify the FeLV template, and produced high sensitivity and specificity. The FeLV detection rate in regression cats by nRPA was increased up to 45.8% compared to the rapid immunochromatographic assay. Hence, FeLV diagnosis using nRPA and RT-RPA are rapid and easily established in low resource settings, benefiting FeLV prognosis, prevention, and control of both horizontal and vertical transmission.

Endogenous Feline Leukemia Virus (FeLV) siRNA Transcription May Interfere with Exogenous FeLV Infection.

Endogenous retroviruses (ERVs) are increasingly recognized for biological impacts on host cell function and susceptibility to infectious agents, particularly in relation to interactions with exogenous retroviral progenitors (XRVs). ERVs can simultaneously promote and restrict XRV infections using mechanisms that are virus and host specific. The majority of endogenous-exogenous retroviral interactions have been evaluated in experimental mouse or chicken systems, which are limited in their ability to extend findings to naturally infected outbred animals. Feline leukemia virus (FeLV) has a relatively well-characterized endogenous retrovirus with a coexisting virulent exogenous counterpart and is endemic worldwide in domestic cats. We have previously documented an association between endogenous FeLV (enFeLV) long terminal repeat (LTR) copy number and abrogated exogenous FeLV in naturally infected cats and experimental infections in tissue culture. Analyses described here examine limited FeLV replication in experimentally infected peripheral blood mononuclear cells, which correlates with higher enFeLV transcripts in these cells compared to fibroblasts. We further examine NCBI Sequence Read Archive RNA transcripts to evaluate enFeLV transcripts and RNA interference (RNAi) precursors. We find that lymphoid-derived tissues, which are experimentally less permissive to exogenous FeLV infection, transcribe higher levels of enFeLV under basal conditions. Transcription of enFeLV-LTR segments is significantly greater than that of other enFeLV genes. We documented transcription of a 21-nucleotide (nt) microRNA (miRNA) just 3' to the enFeLV 5'-LTR in the feline miRNAome of all data sets evaluated (n = 27). Our findings point to important biological functions of enFeLV transcription linked to solo LTRs distributed within the domestic cat genome, with potential impacts on domestic cat exogenous FeLV susceptibility and pathogenesis. IMPORTANCE Endogenous retroviruses (ERVs) are increasingly implicated in host cellular processes and susceptibility to infectious agents, specifically regarding interactions with exogenous retroviral progenitors (XRVs). Exogenous feline leukemia virus (FeLV) and its endogenous counterpart (enFeLV) represent a well-characterized, naturally occurring XRV-ERV dyad. We have previously documented an abrogated FeLV infection in both naturally infected cats and experimental fibroblast infections that harbor higher enFeLV proviral loads. Using an in silico approach, we provide evidence of miRNA transcription that is produced in tissues that are most important for FeLV infection, replication, and transmission. Our findings point to important biological functions of enFeLV transcription linked to solo-LTRs distributed within the feline genome, with potential impacts on domestic cat exogenous FeLV susceptibility and pathogenesis. This body of work provides additional evidence of RNA interference (RNAi) as a mechanism of viral interference and is a demonstration of ERV exaptation by the host to defend against related XRVs.

Decreased expression of endogenous feline leukemia virus in cat lymphomas: a case control study.

BackgroundCats 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.ResultsWe 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.ConclusionsThere 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0378-9) contains supplementary material, which is available to authorized users.

Establishment of a novel feline leukemia virus (FeLV)-negative B-cell cell line from a cat with B-cell lymphoma

We established a novel feline B-cell line, MS4, from the neoplastic pleural effusion of a cat with cutaneous B-cell lymphoma. Immunophenotype staining of the MS4 cells was positive for CD20, CD79α, and IgA and negative for CD3, CD4, CD5, CD8α, CD18, CD21, CD22, IgM, IgG, Ig light chain, and MHC class II. PCR analysis for immunoglobulin heavy chain gene rearrangements revealed a monoclonal rearrangement, whereas no clonal rearrangement of the T-cell receptor γ gene was detected. Southern blotting with an exogenous feline leukemia virus (FeLV) U3 probe revealed no integration of exogenous FeLV provirus. The MS4 cell line is the first FeLV-negative feline B-cell lymphoma cell line, and may be used to investigate the pathogenesis of spontaneously occurring feline lymphoma and the development of new therapies.

High prevalence of non-productive FeLV infection in necropsied cats and significant association with pathological findings

Applying a combination of semi-nested PCR and immunohistology (IHC), the presence of exogenous feline leukemia virus infection was studied in 302 necropsied cats with various disorders. 9% showed the classical outcome of persistent productive FeLV infection which was represented by FeLV antigen expression in different organs. 152 cats (50%) harboured exogenous FeLV-specific proviral sequences in the bone marrow but did not express viral antigen. These cats were considered as horizontally but non-productively infected. Statistical evaluation showed a significant association of non-productive horizontal FeLV infection with a variety of parameters. Non-productively infected cats were statistically significantly older and more often originated from animal shelters than cats without exogenous FeLV infection. Furthermore, some pathological disorders like anemia, panleukopenia, and purulent inflammation showed significant association with non-productive FeLV infection. No significant association was found with lymphosarcoma, known for a long time to be induced by productive FeLV infection.

Molecular cytogenetic analysis of feline leukemia virus insertions in cat lymphoid tumor cells

This study was conducted to map the acquired proviral insertions in the chromosomal genome of feline lymphoid tumors induced by feline leukemia virus (FeLV). Chromosome specimens of the lymphoid tumor-derived cell lines and normal cat lymphocytes were subjected to fluorescence in situ hybridization and tyramide signal amplification, using an exogenous FeLV-A genome as a probe. Specific hybridization signals were detected only on the metaphase chromosomes of the tumor cells. Poisson's distribution-based statistics indicated that 6 chromosomal loci in each cell line showed FeLV integration. In the examination of metaphase chromosomes of FL-74, FT-1 and KO-1 cells, significant signals were detected on B2p15-p14, B2q11, D1p14, E1p14-p13, E1q12 and F2q16; A2p23-p22, B2p15-p14, B4p15-p14, D4q23-q24, E1p14-p13 and E2p13-p12; and A2p22, A3q22, B1p13, B1q13, D1p13 and D3p15-p14, respectively. Consistently, Southern blot hybridization using an FeLV LTR-U3 probe specific for exogenous FeLV revealed the presence of at least 6 copies of exogenous FeLV proviruses at different integration sites in each cell line. These results indicate that there may be common FeLV integration sites at least in A2p22 and B2p15-p14. The cytogenetic analysis used in this study can promptly screen FeLV insertions and provide tags for identifying the novel common integration site.

Naturally occurring feline leukemia virus subgroup A and B infections in urban domestic cats

A nested-PCR (n-PCR) was used to detect feline leukemia virus (FeLV) proviral DNA in blood samples from 464 sick and 608 healthy domestic cats (Felis catus) selected by convenience, and a significantly high prevalence of FeLV infection was observed. n-PCR results revealed the presence of FeLV proviral DNA in 47.2 % of sick cats and 47.4 % of healthy cats. Phylogenetic analysis revealed that FeLV samples from healthy or sick cats were grouped into separate clades. We determined FeLV subgroups by an n-PCR based on the envelope (env) gene. The partial env gene of FeLV Minas Gerais (MG) samples were compared to various exogenous FeLV isolates and endogenous (enFeLV) provirus from the same region. FeLV-B MG samples were more similar to endogenous sequences and to natural FeLV-B isolates than to either FeLV-A or FeLV-C. The results revealed the circulation of FeLV-B in large populations of urban domestic cats in Brazil.

Association between endogenous feline leukemia virus loads and exogenous feline leukemia virus infection in domestic cats

Recently, we demonstrated that endogenous feline leukemia virus (enFeLV) loads may vary among cats of different populations and that FeLV-infected cats have higher enFeLV loads than uninfected cats. Thus, we hypothesized that enFeLV might influence the pathogenesis and outcome of FeLV infection. No significant difference in the infection outcome (regressive versus progressive infection) was observed between groups of cats with high or low enFeLV loads following FeLV-A challenge. However, cats with high enFeLV loads showed higher viral replication (plasma viral RNA and p27 antigen levels) than cats with low enFeLV loads in the early phase of the infection. The enFeLV transcription level varied at different time points, but no clear-cut pattern was observed. In conclusion, our results demonstrated an association between enFeLV loads and FeLV replication but not outcome of infection. enFeLV should be considered as an important confounder in experimental FeLV infection or vaccination studies.

Quantification of endogenous and exogenous feline leukemia virus sequences by real-time PCR assays

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.

Re-examination of feline leukemia virus: host relationships using real-time PCR

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.

Detection of the integrated feline leukemia viruses in a cat lymphoid tumor cell line by fluorescence in situ hybridization.

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.

Inhibition of Feline Leukemia Virus Subgroup A Infection by Coinoculation with Subgroup B

Feline leukemia virus (FeLV) subgroup B arises de novo through recombination between the env genes of exogenous FeLV subgroup A and endogenous FeLV-like sequences. FeLV-B, which by itself is poorly infectious, will increase to high titer in the presence of FeLV-A, and is associated with FeLV-related neoplastic disease. Although the participation of FeLV-B in disease progression has not been definitively proven, circumstantial evidence supports the hypothesis that the generation of FeLV-B is linked to disease progression. The present study was designed to evaluate whether increasing the levels of FeLV-B early in FeLV-A infection could result in reduction of the incubation period for development of neoplastic disease. For this study, an isolate of FeLV-B, designated FeLV-1B3, was biologically cloned, partially sequenced, and subgroup typed. In in vivo studies, none of the neonatal cats inoculated with FeLV-1B3 alone converted to viremia positive, and all remained healthy throughout the observation period. All of the kittens inoculated with FeLV-A alone became chronically viremic, and those held for long-term observation all developed either neoplastic disease or anemia. However, kittens inoculated with the combination of FeLV-1B3 and FeLV-A showed attenuated infections whereby the majority of cats failed to develop chronic viremia. The apparent interference of FeLV-A infection by FeLV-B was time and titer dependent. This unexpected result suggests that FeLV-B may act as an attenuated virus, causing inhibition of FeLV-A possibly through an immune-mediated mechanism. Partial support for this view was provided by postmortem examination of cats inoculated with FeLV-1B3 alone. Even though none of these cats became viremic, FeLV antigen was detected as focal infections in select tissues, especially salivary gland epithelium, where enough antigen may be expressed to provide an immunizing dose against gag and pol cross-reacting antigens. This work may also provide another approach to vaccine development based on endogenous retrovirus vector systems.

A novel truncated env gene isolated from a feline leukemia virus-induced thymic lymphosarcoma.

We PCR amplified the exogenous feline leukemia virus (FeLV)-related env gene species from lymphosarcomas induced by intradermally administered plasmid DNA of either the prototype FeLV, subgroup A molecular clone, F6A, or a new molecular clone, FeLV-A, Rickard strain (FRA). Of the nine tumors examined, six showed the presence of deleted env species of variable sizes in the tumor DNA. One env mutant, which was detected in a FRA-induced thymic lymphosarcoma, had a large internal deletion beginning from almost the N-terminal surface glycoprotein (SU) up to the middle region of the transmembrane (TM) protein of the env gene. The deduced polypeptide of this truncated env (tenv) retained the complete signal peptide and seven amino acids of the N-terminal mature SU of FRA env gene, followed by eight amino acids from the frameshift in the TM region. To study the biological function of tenv, we used a murine retrovirus vector to produce amphotropic virions. Infection of feline fibroblasts (H927), human fibrosarcoma cells (HT1080), or human B-lymphoma cells (Raji) led to pronounced cytotoxicity, while the tenv virus did not induce significant cytotoxicity to feline T-lymphoma cells (3201B) or human T-lymphoma cells (CEM). Together, these results convincingly demonstrated that the genetic events that led to truncation in the env gene occurred de novo in FeLV lymphomagenesis and that such a product, tenv could induce cytotoxicity to fibroblastic and B-lymphoid cells but not to T-lymphoid tumor cells. This type of selective toxicity might be potentially important in the development of the neoplastic disease.

Clonality analysis of various hematopoietic disorders in cats naturally infected with feline leukemia virus.

The clonality analysis of the bone marrow cells was carried out by detecting the integrated proviruses of feline leukemia virus (FeLV) to understand the pathogenesis of FeLV-associated hematopoietic disorders in cats. Bone marrow cells from 4 cases with acute myeloid leukemia (AML), 9 cases with myelodysplastic syndromes (MDS), 2 cases with pure red cell aplasia (PRCA) and 3 healthy carriers infected with FeLV were subjected to Southern blot analyses using an exogenous FeLV probe. Clonal hematopoiesis was found in all the cases with AML and in 6 of the 9 cases with MDS, but not in the cases with both PRCA and healthy carriers infected with FeLV. In the 2 cases with MDS, it was thought that the same clones of the hematopoietic cells might proliferate before and after the progression of the disease irrespective of the changes of the hematological diagnoses by cytological examination. This study indicates that MDS in cats is a disease manifestation as a result of clonal proliferation of hematopoietic cells and can be recognized as a pre-leukemic state of AML.

Defective endogenous proviruses are expressed in feline lymphoid cells: evidence for a role in natural resistance to subgroup B feline leukemia viruses.

Endogenous feline leukemia virus (FeLV)-related sequences (enFeLV) are a family of proviral elements found in domestic cats and their close relatives. These elements can recombine with exogenous, infectious FeLVs of subgroup A (FeLV-A), giving rise to host range variants of FeLV-B. We found that a subset of defective enFeLV proviruses is highly expressed in lymphoma cell lines and in a variety of primary tissues, including lymphoid tissues from healthy specific-pathogen-free cats. At least two RNA species were detected, a 4.5-kb RNA containing gag, env, and long terminal repeat sequences and a 2-kb RNA containing env and long terminal repeat sequences. Cloning of enFeLV cDNA from two FeLV-free lymphoma cell lines (3201 and MCC) revealed a long open reading frame (ORF) encoding a truncated env gene product corresponding to the N-terminal portion of gp70env. Interestingly, all of three natural FeLV-B isolates include 3' env sequences which are missing from the highly transcribed subset and hence must be derived from other enFeLV elements. The enFeLV env ORF cDNA clones were closely similar to a previously characterized enFeLV provirus, CFE-16, but were polymorphic at a site corresponding to an exogenous FeLV neutralization epitope. Site-specific antiserum raised to a C-terminal 30-amino-acid peptide of the enFeLV env ORF detected an intracellular product of 35 kDa which was also shed from cells in stable form. Expression of the 35-kDa protein correlated with enFeLV RNA levels and was negatively correlated with susceptibility to infection with FeLV-B. Cell culture supernatant containing the 35-kDa protein specifically blocked infection of permissive fibroblast cells with FeLV-B isolates. We suggest that the truncated env protein mediates resistance by receptor blockade and that this form of enFeLV expression mediates the natural resistance of cats to infection with FeLV-B in the absence of FeLV-A.

Biologically selected recombinants between Feline Leukemia Virus (FeLV) subgroup a and an endogenous FeLV element

In efforts to elucidate the proximal leukemogens that might be produced during a feline leukemia virus (FeLV) infection of cats, homologous recombinations between molecularly cloned exogenous and endogenous FeLV proviruses of known sequences were examined in cell cultures in vitro. A plasmid containing an infectious member of the most commonly occurring FeLV subgroup (FeLV subgroup A or FeLV-A) was coexpressed with noninfectious constructs containing the envelope ( env) gene of an endogenously inherited FeLV-like feline genomic element in transfected feline fibroblasts. The viruses generated were selected for their ability to propagate in human cells which are resistant to infection by the parental ecotropic FeLV-A or the noninfectious endogenous constructs. An analysis of the recombinants thus derived identified a limited number of sites in the env gene which were preferentially utilized in the generation of recombinant FeLVs under the selection conditions used. These sites were clustered in the surface glycoprotein (SU) moiety of the env gene, and it appeared that most, but not all, of the SU gene product of FeLV-A, beginning from the N-terminus, can be replaced by sequences from an endogenous element, still allowing the virus to be biologically viable. In fact, these substitutions in the env gene expanded infectivity of the parental FeLV-A from ecotropic to polytropic cell tropism. Additionally, substitutions in the SU region yielded many recombinants in which a primary neutralizing pentapeptide epitope of FeLV-A was altered because of its variance in the endogenous element. In several of the recombinants, this sequence was also found to be frequently mutated. Consistent with the changes identified in this antibody-binding domain, the recombinant viruses were only weakly inhibited by a monoclonal antibody directed against this epitope, while FeLV-A was highly sensitive to neutralization.