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.

Presence of Endogenous Viral Elements Negatively Correlates with Feline Leukemia Virus Susceptibility in Puma and Domestic Cat Cells.

While feline leukemia virus (FeLV) has been shown to infect felid species other than the endemic domestic cat host, differences in FeLV susceptibility among species has not been evaluated. Previous reports have noted a negative correlation between endogenous FeLV (enFeLV) copy number and exogenous FeLV (exFeLV) infection outcomes in domestic cats. Since felids outside the genus Felis do not harbor enFeLV genomes, we hypothesized absence of enFeLV results in more severe disease consequences in felid species lacking these genomic elements. We infected primary fibroblasts isolated from domestic cats (Felis catus) and pumas (Puma concolor) with FeLV and quantitated proviral and viral antigen loads. Domestic cat enFeLV env and long terminal repeat (LTR) copy numbers were determined for each individual and compared to FeLV viral outcomes. FeLV proviral and antigen levels were also measured in 6 naturally infected domestic cats and 11 naturally infected Florida panthers (P. concolor coryi). We demonstrated that puma fibroblasts are more permissive to FeLV than domestic cat cells, and domestic cat FeLV restriction was highly related to enFeLV-LTR copy number. Terminal tissues from FeLV-infected Florida panthers and domestic cats had similar exFeLV proviral copy numbers, but Florida panther tissues have higher FeLV antigen loads. Our work indicates that enFeLV-LTR elements negatively correlate with exogenous FeLV replication. Further, Puma concolor samples lacking enFeLV are more permissive to FeLV infection than domestic cat samples, suggesting that endogenization can play a beneficial role in mitigating exogenous retroviral infections. Conversely, presence of endogenous retroelements may relate to new host susceptibility during viral spillover events.IMPORTANCE Feline leukemia virus (FeLV) can infect a variety of felid species. Only the primary domestic cat host and related small cat species harbor a related endogenous virus in their genomes. Previous studies noted a negative association between the endogenous virus copy number and exogenous virus infection in domestic cats. This report shows that puma cells, which lack endogenous FeLV, produce more virus more rapidly than domestic cat fibroblasts following cell culture challenge. We document a strong association between domestic cat cell susceptibility and FeLV long terminal repeat (LTR) copy number, similar to observations in natural FeLV infections. Viral replication does not, however, correlate with FeLV env copy number, suggesting that this effect is specific to FeLV-LTR elements. This discovery indicates a protective capacity of the endogenous virus against the exogenous form, either via direct interference or indirectly via gene regulation, and may suggest evolutionary outcomes of retroviral endogenization.

Cerebral toxoplasmosis in a cat with feline leukemia and feline infectious peritonitis viral infections.

A diarrheic young cat died after neurological involvement. Biochemistry pointed to feline infectious peritonitis (FIP). The final diagnosis was severe multifocal meningoencephalitis due to Toxoplasma gondii. The presence of the parasite in the brain was confirmed using immunohistochemical staining. Concomitant feline leukemia virus (FeLV) and FIP were possible contributors to the clinical, fatal outcome.

Toxoplasmose cérébrale chez un chat atteint des infections virales de leucémie féline et de péritonite infectieuse féline. Un jeune chat diarrhéique est mort après des symptômes neurologiques. La biochimie a signalé une péritonite infectieuse féline (FIP). Le diagnostic final a été une méningo-encéphalite multifocale grave causée par Toxoplasma gondii. La présence du parasite dans le cerveau a été confirmée à l'aide de la coloration immunohistochimique. La présence concomitante du virus de la leucémie féline (FeLV) et de la FIP sont des facteurs possibles ayant contribué au résultat clinique mortel.(Traduit par Isabelle Vallières).

Polymerase chain reaction-based detection of myc transduction in feline leukemia virus-infected cats.

Feline lymphomas are associated with the transduction and activation of cellular proto-oncogenes, such as c-myc, by feline leukemia virus (FeLV). We describe a polymerase chain reaction assay for detection of myc transduction usable in clinical diagnosis. The assay targets c-myc exons 2 and 3, which together result in a FeLV-specific fusion gene following c-myc transduction. When this assay was conducted on FeLV-infected feline tissues submitted for clinical diagnosis of tumors, myc transduction was detected in 14% of T-cell lymphoma/leukemias. This newly established system could become a useful diagnostic tool in veterinary medicine.

[FeLV infection in the cat: clinically relevant aspects]. / Die FeLV-Infektion der Katze: Praxis-relevante Aspekte.

The feline leukemia virus (FeLV) is a retrovirus of the domestic cat that was described almost 50 years ago. The FeLV-infection may lead to fatal diseases in domestic and small wild cats. The use of efficacious diagnostics assays and vaccines led to a reduction of the FeLV prevalence; however, FeLV still poses a problem for the cat presented with the infection. This article aims to describe recent developments in diagnostics and findings in the infection pathogenesis that are clinically relevant.

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

Clinical aspects of feline immunodeficiency and feline leukemia virus infection.

Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are retroviruses with a global impact on the health of domestic cats. The two viruses differ in their potential to cause disease. FIV can cause an acquired immunodeficiency syndrome that increases the risk of developing opportunistic infections, neurological diseases, and tumors. In most naturally infected cats, however, FIV itself does not cause severe clinical signs, and FIV-infected cats may live many years without any health problems. FeLV is more pathogenic, and was long considered to be responsible for more clinical syndromes than any other agent in cats. FeLV can cause tumors (mainly lymphoma), bone marrow suppression syndromes (mainly anemia) and lead to secondary infectious diseases caused by suppressive effects of the virus on bone marrow and the immune system. Today, FeLV is less important as a deadly infectious agent as in the last 20 years prevalence has been decreasing in most countries.

Myeloma-related disorder with leukaemic progression in a cat.

A 10-year-old American Shorthair cat with nasal discharge, anorexia, and weight loss was found to have pancytopenia and hyperproteinaemia. Bone marrow aspiration revealed atypical plasma cells that totalled 50% of the nucleated bone marrow cells. The number of atypical plasma cells progressively increased in the peripheral blood during the observation period of 64 days. The cat did not respond to treatments with melphalan, chlorambucil, and prednisolone, and died 71 days after the initial presentation. Clinical, cytological, histopathological, and immunohistochemical findings in this case supported the diagnosis of myeloma-related disorder (MRD) with leukaemic progression.

Myelodysplastic syndromes and acute myeloid leukemia in cats infected with feline leukemia virus clone33 containing a unique long terminal repeat.

Feline leukemia virus (FeLV) clone33 was obtained from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV from other cats with AML, differs from the LTRs of other known FeLV in that it has 3 tandem direct 47-bp repeats in the upstream region of the enhancer (URE). Here, we injected cats with FeLV clone33 and found 41% developed myelodysplastic syndromes (MDS) characterized by peripheral blood cytopenias and dysplastic changes in the bone marrow. Some of the cats with MDS eventually developed AML. The bone marrow of the majority of cats with FeLV clone33 induced MDS produced fewer erythroid and myeloid colonies upon being cultured with erythropoietin or granulocyte-macrophage colony-stimulating factor (GM-SCF) than bone marrow from normal control cats. Furthermore, the bone marrow of some of the cats expressed high-levels of the apoptosis-related genes TNF-alpha and survivin. Analysis of the proviral sequences obtained from 13 cats with naturally occurring MDS reveal they also bear the characteristic URE repeats seen in the LTR of FeLV clone33 and other proviruses from cats with AML. Deletions and mutations within the enhancer elements are frequently observed in naturally occurring MDS as well as AML. These results suggest that FeLV variants that bear URE repeats in their LTR strongly associate with the induction of both MDS and AML in cats.

Chronic eosinophilic leukemia in a cat: cytochemical and immunophenotypical features.

A 3-year-old, male, domestic shorthaired cat was presented with a 3-day history of anorexia and depression. The cat was moderately dehydrated, had pale, slightly icteric, mucous membranes, oral ulcerations, and mild hepatosplenomegaly. A feline leukemia virus (FeLV) antigen test was positive. CBC results obtained at initial presentation included severe normocytic, normochromic, nonregenerative anemia, severe thrombocytopenia, and marked leukocytosis (>100,000/microL) with 77% eosinophils. After 15 days of treatment with prednisone and doxycycline, the cat had persistent severe nonregenerative anemia (HCT 3.4%), thrombocytopenia (28,000/microL), and extreme eosinophilia (total eosinophils, 123.1 x 10(3)/microL; segmented 103.0 x 10(3)/microL; immature 20.1 X 10(3)/microL). Cytologic examination of aspirates from bone marrow, liver, lymph nodes, and spleen revealed a predominance of mature and immature eosinophils, many with dysplastic changes. The M:E ratio was 96.4. On histopathologic examination, multiple organs were infiltrated by eosinophilic granulocytes. Neoplastic cells in blood and bone marrow stained positive for alkaline phosphatase and were negative for myeloperoxidase, chloroacetate esterase, and alpha-naphthyl acetate esterase. On flow cytometric analysis of peripheral blood, the neoplastic cells were positive for CD11b and CD14. These findings were consistent with chronic eosinophilic leukemia. To our knowledge, this is the first report of chronic eosinophilic leukemia in a cat associated with naturally acquired FeLV infection, in which flow cytometry was used to characterize the neoplastic cells.

Effect of preexisting FeLV infection or FeLV and feline immunodeficiency virus coinfection on pathogenicity of the small variant of Haemobartonella felis in cats.

OBJECTIVE: To investigate the effects of preexisting FeLV infection or FeLV and feline immunodeficiency (FIV) coinfection on the pathogenicity of the small variant of Haemobartonella felis (Hfsm, California variant) in cats. ANIMALS: 20 FeLV infected, 5 FeLV-FIV coinfected, and 19 retrovirus-free cats. PROCEDURES: A client-owned cat, coinfected with FeLV and Hfsm, was the source for Hfsm. Inoculum 1 (FeLV free) was obtained by passage of source Hfsm through 4 FeLV-resistant cats. Inoculum 2 was obtained by further passage of Hfsm (inoculum 1) through 2 specific pathogen-free cats. RESULTS: A mild-to-moderate anemia started 21 days after inoculation, with its nadir occurring at 35 to 42 days after inoculation. Infection with Hfsm induced greater decrease in hemoglobin concentration in FeLV infected cats, compared with retrovirus free cats. Reticulocytosis, macrocytosis, and polychromasia of erythrocytes developed in anemic cats regardless of retrovirus infection status. Mean neutrophil counts decreased during the hemolytic episode. For most cats, the anemia was transient. Four FeLV infected cats, 1 of which was also FIV infected, developed fatal FeLV-associated myeloproliferative diseases. Of the surviving cats, 8 died over the next 24 months from other FeLV-related diseases. Hemolysis did not recur after the initial episode. Inoculum 1 induced more severe anemia than inoculum 2. CONCLUSIONS AND CLINICAL RELEVANCE: Our results support the clinical observation that cats coinfected with FeLV and H felis develop more severe anemia than cats infected with H felis alone. Infection with Hfsm may induce myeloproliferative disease in FeLV infected cats. The small variant of H felis may lose pathogenicity by passage through FeLV-free cats.

Detection of feline leukaemia virus in blood and bone marrow of cats with varying suspicion of latent infection.

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.

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.

Feline leukemia virus and feline immunodeficiency virus.

Ophthalmic manifestations of FeLV or FIV infection can occur in all ocular tissues and may be manifestations of direct viral effects or secondary to viral-related malignant transformation. Additionally, the manifestations of common feline ophthalmic pathogens may be more severe and poorly responsive to therapy because of the immunosuppressive effects of FeLV or FIV infection. Prompt diagnosis of underlying viral infection in cats with ophthalmic disease is paramount for accurate diagnosis and prognosis and is required for appropriate therapeutic decision making.

Expression of viral proteins in feline leukemia virus-associated enteritis.

Fourteen cases of feline leukemia virus (FeLV)-associated enteritis were immunohistologically examined for the expression of FeLV proteins gp70, p27, and p15E in the jejunum, mesenteric lymph nodes, spleen, and bone marrow. Results were compared with those of FeLV-infected cats without intestinal alterations. Other viral infections and specific bacterial, fungal, and parasitic infections were excluded by standard microbiologic methods, histopathology, immunohistology, and in situ hybridization. In FeLV-associated enteritis, FeLV gp70 and p15E were strongly expressed in intestinal crypt epithelial cells. In contrast, FeLV-positive cats without intestinal alterations showed only faint staining for gp70 and p15E and comparatively strong p27 expression in these cells. Findings suggest a direct relation between FeLV infection and alterations in intestinal crypt epithelial cells that may be attributed to the envelope proteins gp70 and p15E and/or their precursor protein. Distinct similarities to the intestinal changes in the experimentally induced FeLV-feline AIDS syndrome are obvious, suggesting that naturally occurring feline AIDS variants may be responsible for FeLV-associated enteritis.

Acute feline leukemia virus infection causes altered energy balance and growth inhibition in weanling cats.

Naturally occurring retroviral infections cause progressive weight loss, immune suppression, invasion by opportunistic organisms, and eventual death. Feline leukemia virus (FeLV) inhibited growth and decreased energy intake in seven experimentally infected weanling cats compared with age- and sex-matched controls. Remarkably, changes in energy intake, energy expenditure, and weight gain occurred in the acute phase of infection prior to the systemic/productive bone marrow phase of FeLV infection. In other words, growth inhibition developed before FeLV infection was clinically detectable with use of standard enzyme-linked immunosorbent assay or fixed-cell immunofluorescence assays of circulating neutrophils and platelets. Acutely infected, previremic cats consumed 25% less energy [Day 4 postinoculation to Day 16 postinoculation (p < 0.05)] and expended 20% less energy [Day 8 postinoculation to Day 18 postinoculation (p < 0.05)] compared with control cats. Growth stunting of inoculated cats began by Day 11 postinoculation (p < 0.05) and was not corrected during the remaining 4 months of the study. Thus, experimental FeLV infection causes perturbations of metabolism and energy balance resulting in permanent growth impairment. Secondly, detrimental metabolic effects begin in the acute phase of retroviral infection, prior to the clinically detectable phase of FeLV infection.

Erythroleukemia in two cats naturally infected with feline leukemia virus in the same household.

Erythroleukemia was observed in two unrelated cats infected with feline leukemia virus (FeLV) from the same household. Case 1, a 1-year-old neutered male cat developed erythroleukemia (M6) after a diagnosis of myelodysplastic syndrome (MDS-Er) on the criteria of FAB classification of acute leukemias. Case 2, a 1-year-old neutered female cat, which had close contact with Case 1, also developed erythroleukemia (M6Er). In both cases, marked proliferation of erythroid progenitor cells with disproportionally large numbers of immature forms was observed in the bone marrow. In Case 1, neoplastic proliferation of myeloid cells in the bone marrow was also noted at the terminal stage. Combination chemotherapy with daunomycin was partially effective for treatment of these erythroid neoplasias, but did not induce complete remission. Southern blot analysis using exogenous FeLV-specific probes indicated the clonal origin of these hematopoietic tumor cells. Furthermore, the erythroid and myeloid tumor cells in Case 1 were shown to be derived from independent transformed clones. A variant FeLV was shown to be integrated into the tumor cells in Case 1, while a full-length FeLV was found in both cases. Because these erythroid neoplastic diseases occurred in two unrelated cats kept in the same household and these diseases are rare, they may both have been associated with the same FeLV strain.

Chromosome abnormalities and oncogenesis in cat leukemias.

Chromosome abnormalities are found in feline leukemia virus (FeLV)-infected tumor cells as well as in tumor cells free of the virus. Three cell lines derived from tumors in the domestic cat (Felis catus), two of thymic origin and one of multicentric lymphoma origin, were analyzed cytogenetically to determine whether the FeLV virus was associated with chromosomal abnormalities in these tumor cell lines. One thymic tumor and the multicentric lymphoma were FeLV infected. The other thymic tumor cell line was FeLV-free. The normal diploid number in the domestic cat is 38. All three cell lines had numerical chromosome abnormalities with modal numbers of 37, 38 (pseudodiploid), and 39, respectively and had consistent structural chromosome abnormalities. Three markers in the virus-free cell line (S markers) were shared with one or the other of the virus-positive cell lines. The two FeLV-positive cell lines did not have S markers in common. The finding of chromosome abnormalities in both the virus-infected and the virus-free cell lines suggests that these abnormalities may be important in oncogenesis. The FeLV virus could not be considered the only causative agent of the abnormalities observed.

Lymphocytotoxic strains of feline leukemia virus induce apoptosis in feline T4-thymic lymphoma cells.

Feline leukemia retrovirus (FeLV) strains with subgroup C env genes kill feline T4 lymphoma 3201 cells by 7 to 12 days after in vitro inoculation, whereas FeLV strains with subgroup A env genes do not. Neither FeLV-A nor FeLV-C kill feline fibroblasts. FeLV-C, but not FeLV-A, is replicated to higher titer by 3201 cells and productive infection precedes death by 3 to 7 days. Transcriptional activity of the FeLV-C long terminal repeat, as assessed by chloramphenicol acetyltransferase activity, is high in feline lymphoid cells but low in feline fibroblasts. Activity of the FeLV-A long terminal repeat is moderate in both cell types. FeLV-C-infected cells form aggregates 1 to 4 days before dying; ultrastructurally, virus particles can be seen approximating the clustered cells. Dying cells demonstrate nuclear condensation, surface blebbing, and fragmentation. DNA fragmentation and laddering compatible with apoptosis occur 1 to 2 days before massive cell death. In FeLV-C-infected 3201 cells, a shift from phospholipid to neutral lipid incorporation of [14C]oleic acid, increases in palmitic acid proportions and decreases in linoleic acid proportions occur 1 to 2 days before peak killing. Exposure of 3201 cells to ultraviolet-inactivated FeLV-KT (200-800 micrograms/10(6) cells) causes cytostasis within 2 days and death within 4 days. Blebbing and nuclear condensation occur but clusters do not form. The induction of programmed cell death in feline thymic lymphoma cells by subgroup C feline retroviruses may be relevant to the pathogenesis of FeLV-induced thymic atrophy, paracortical lymphoid depletion and acquired immunodeficiency in vivo.