Feline Leukemia Virus Provirus Research Articles

Xenotransfusion of Blood from Dog to Cat: Should Canine Blood Be Our First Choice for Feline Transfusion in Emergency Situations?

Despite the ability to determine feline blood types, the transfusion of canine blood to cats is still practiced in some countries. Xenotransfusion is effective—even if its effects only last for a few days—and is not associated with serious adverse effects. It avoids the need for blood typing, and most importantly, it avoids the transmission of intraspecific infectious agents, notably the feline leukemia virus (FeLV). Transfusion with canine blood is easier, quicker and less costly than transfusion with feline blood; it is less disagreeable for the donor. In the light of these arguments, when feline blood collected according to current guidelines is not available, in particular when the donor is not confirmed to be negative for the FeLV provirus, the authors consider it to be judicious to use canine blood for feline transfusion in emergency situations; this practice is preferable to inaction and to the inoculation of an infectious agent. Allotransfusion remains preferable in non-emergency situations as a treatment of chronic compensated anaemiae or if an appropriate donor (negative for FeLV provirus) is available. However, 2–4 days after a xenotransfusion, if a clinical alteration and a significant decrease in haematocrit are observed, a transfusion with cat’s blood confirmed to be negative for FeLV provirus should be performed. Xenotransfusion should never be used twice.

Adeno-Associated Vector-Delivered CRISPR/SaCas9 System Reduces Feline Leukemia Virus Production In Vitro.

Feline leukemia virus (FeLV) is a retrovirus of cats worldwide. High viral loads are associated with progressive infection and the death of the host, due to FeLV-associated disease. In contrast, low viral loads, an effective immune response, and a better clinical outcome can be observed in cats with regressive infection. We hypothesize that by lowering viral loads in progressively infected cats, using CRISPR/SaCas9-assisted gene therapy, the cat’s immune system may be permitted to direct the infection towards a regressive outcome. In a step towards this goal, the present study evaluates different adeno-associated vectors (AAVs) for their competence in delivering a gene editing system into feline cells, followed by investigations of the CRISPR/SaCas9 targeting efficiency for different sites within the FeLV provirus. Nine natural AAV serotypes, two AAV hybrid strains, and Anc80L65, an in silico predicted AAV ancestor, were tested for their potential to infect different feline cell lines and feline primary cells. AAV-DJ revealed superior infection efficiency and was thus employed in subsequent transduction experiments. The introduction of double-strand breaks, using the CRISPR/SaCas9 system targeting 12 selected FeLV provirus sites, was confirmed by T7 endonuclease 1 (T7E1), as well as Tracking of Indels by Decomposition (TIDE) analysis. The highest percentage (up to 80%) of nonhomologous end-joining (NHEJ) was found in the highly conserved gag and pol regions. Subsequent transduction experiments, using AAV-DJ, confirmed indel formation and showed a significant reduction in FeLV p27 antigen for some targets. The targeting of the FeLV provirus was efficient when using the CRISPR/SaCas9 approach in vitro. Whether the observed extent of provirus targeting will be sufficient to provide progressively FeLV-infected cats with the means to overcome the infection needs to be further investigated in vivo.

Transfusion transmissible pathogens are prevalent in healthy cats eligible to become blood donors.

This study aims to determine the prevalence of subclinical infectious agents considered core pathogens for worldwide screening in healthy, client-owned, indoor cats eligible to become blood donors in Spain and Portugal. Blood samples of healthy, indoor, domestic cats selected to be potential blood donors were tested for feline leukaemia virus antigens, feline immunodeficiency virus antibodies and polymerase chain reactions for Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum, Candidatus Mycoplasma turicensis, feline leukaemia virus provirus, Leishmania spp. and Bartonella spp. Not all donors were tested for all agents. Overall, 5105 healthy indoor cats were tested and 8.1% (414/5105) had at least one subclinical infectious agent that is transmissible through blood product transfusion. 1.5% (77/5105) were positive for feline leukaemia virus antigens and 2.9% (148/5105) were positive for feline immunodeficiency virus antibodies, therefore they were excluded as donors. The overall prevalence of haemoplasmas in feline leukaemia virus and feline immunodeficiency virus SNAP-negative feline blood donors was 3.7% (181/4880) [1.3% (63/4880) were positive for Mycoplasma haemofelis; 2.3% (112/4880) to Candidatus Mycoplasma haemominutum and 0.12% (6/4880) to Candidatus Mycoplasma turicensis]. The prevalence of feline leukaemia provirus was 5.2% (9/173) and of Bartonella spp. was 0.2% (2/1122). None of the 173 cats were positive for Leishmania spp. The prevalence of many transfusion transmissible pathogens was relatively high in this healthy, client-owned, indoor cats eligible to become blood donors. Performing an extended screening panel that includes at least polymerase chain reactions for Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum, Candidatus Mycoplasma turicensis, feline leukaemia virus provirus, and Bartonella spp., in addition to feline leukaemia virus antigens and feline immunodeficiency virus antibodies, is recommended in pet blood banks from analogous regions.

Linfoma multicéntrico en felino doméstico: Reporte de caso

Lymphomas are neoplasms characterized by the clonal proliferation of malignant lymphocytes, originating mainly from lymphoid organs, such as bone marrow, spleen and lymph nodes. According to human terminology, lymphomas that occur in domestic animals are known as non-Hodgkin, whose biological behavior, epidemiology, cell morphology and phenotyping are quite similar between humans and small animals. Lymphoma is the most common feline neoplasia, comprising more than 50% of all hemolymphatic tumors. The precise etiology of feline lymphoma is not known in many cases; however, viral causes are well defined, with infections by both feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). When lymphoma is caused by FeLV, it is usually associated with progressive infection, but regressive infections can also be involved in tumor formation, whose virus is not detected by the ELISA test, but in PCR (peripheral blood or bone marrow) is found FeLV provirus DNA, this suggests that the virus may be associated with a higher proportion of lymphomas than expected. Such neoplasms can present in different anatomical locations being classified into multicentric, mediastinal (or thymic), alimentary and extranodal (cutaneous, nasal, renal and nervous system lymphoma). In cats, mediastinal and alimentary forms are more common than multicentric and extranodal forms. The present work aims to present the case report of a domestic feline, male, SRD, 1 year old and FIV / FeLV negative in the ELISA, showing an increase in peripheral lymph nodes (submandibular, inguinal and popliteal) and an increase in liver lymph nodes, pancreatoduodenal, medial, colic and mesenteric iliac. Submandibular lymph node cytology was performed where immunoblastic lymphoma was diagnosed. The patient was submitted to the CHOP chemotherapy protocol (Cyclophosphamide, Doxorubicin, Vincristine and Prednisolone) for 24 weeks, being discharged after this period.

Retroviral DNA--the silent winner: blood transfusion containing latent feline leukemia provirus causes infection and disease in naïve recipient cats.

Background The feline leukemia virus (FeLV) is a gamma-retrovirus of domestic cats that was discovered half a century ago. Cats that are infected with FeLV may develop a progressive infection resulting in persistent viremia, immunodeficiency, tumors, anemia and death. A significant number of cats mount a protective immune response that suppresses viremia; these cats develop a regressive infection characterized by the absence of viral replication and the presence of low levels of proviral DNA. The biological importance of these latter provirus carriers is largely unknown.ResultsHere, we demonstrate that ten cats that received a transfusion of blood from aviremic provirus carriers developed active FeLV infections, some with a progressive outcome and the development of fatal FeLV-associated disease. The infection outcome, disease spectrum and evolution into FeLV-C in one cat mirrored those of natural infection. Two cats developed persistent antigenemia; six cats were transiently antigenemic. Reactivation of infection occurred in some cats. One recipient developed non-regenerative anemia associated with FeLV-C, and four others developed a T-cell lymphoma, one with secondary lymphoblastic leukemia. Five of the ten recipient cats received provirus-positive aviremic blood, whereas the other five received provirus- and viral RNA-positive but aviremic blood. Notably, the cats that received blood containing only proviral DNA exhibited a later onset but graver outcome of FeLV infection than the cats that were transfused with blood containing proviral DNA and viral RNA. Leukocyte counts and cytokine analyses indicated that the immune system of the latter cats reacted quicker and more efficiently.ConclusionsOur results underline the biological and epidemiological relevance of FeLV provirus carriers and the risk of inadvertent FeLV transmission via blood transfusion and demonstrate the replication capacity of proviral DNA if uncontrolled by the immune system. Our results have implications not only for veterinary medicine, such as the requirement for testing blood donors and blood products for FeLV provirus by sensitive polymerase chain reaction, but are also of general interest by revealing the importance of latent retroviral DNA in infected hosts. When aiming to eliminate a retroviral infection from a population, provirus carriers must be considered.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0231-z) contains supplementary material, which is available to authorized users.

Long-term follow up of feline leukemia virus infection and characterization of viral RNA loads using molecular methods in tissues of cats with different infection outcomes

It is a remarkable feature for a retrovirus that an infection with feline leukemia virus (FeLV) can result in various outcomes. Whereas some cats contain the infection and show a regressive course, others stay viremic and succumb to the infection within a few years. We hypothesized, that differences in the infection outcome might be causally linked to the viral RNA and provirus loads within the host and these loads therefore may give additional insight into the pathogenesis of the virus. Thus, the goals of the present study were to follow-up on experimentally infected cats and investigate tissues from cats with different infection outcomes using sensitive, specific TaqMan real-time PCR and reverse transcriptase (RT)-PCR.Nineteen experimentally FeLV-A/Glasgow-1-infected cats were categorized into having regressive, progressive or reactivated FeLV infection according to follow-up of FeLV p27 antigen detection in the blood. Remarkably, regressively infected cats showed detectable provirus and viral RNA loads in almost all of the 27 tested tissues, even many years after virus exposure. Moreover, some regressively infected cats reactivated the infection, and these cats had intermediate to high viral RNA and provirus tissue loads. The highest loads were found in viremic cats, independent of their health status. Tissues that represented sites of virus replication and shedding revealed the highest viral RNA and provirus loads, while the lowest loads were present in muscle and nerve tissues.A supplementary analysis of 20 experimentally infected cats with progressive infection revealed a median survival time of 3.1 years (range from 0.6 to 6.5 years); ∼70% (n=14) of these cats developed lymphoma, while leukemia and non-regenerative anemia were observed less frequently.Our results demonstrate that the different infection outcomes are associated with differences in viral RNA and provirus tissue loads. Remarkably, no complete clearance of FeLV viral RNA or provirus was detected in cats with regressive infection, even up to 12 years after exposure. In several cases FeLV reactivation could be observed. Thus, retroviruses integrated as provirus into the host's genome, could not be eliminated completely by the host and maintained their full potential for replication and reactivation.

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.

Incidence of persistent viraemia and latent feline leukaemia virus infection in cats with lymphoma

In the past, feline leukaemia virus (FeLV) infection, and also latent FeLV infection, were commonly associated with lymphoma and leukaemia. In this study, the prevalence of FeLV provirus in tumour tissue and bone marrow in FeLV antigen-negative cats with these tumours was assessed. Seventy-seven diseased cats were surveyed (61 antigen-negative, 16 antigen-positive). Blood, bone marrow, and tumour samples were investigated by two polymerase chain reaction (PCR) assays detecting deoxyribonucleic acid (DNA) sequences of the long terminal repeats (LTR) and the envelope (env) region of the FeLV genome. Immunohistochemistry (IHC) was performed in bone marrow and tumour tissue. None of the antigen-negative cats with lymphoma was detectably infected with latent FeLV. The prevalence of FeLV viraemia in cats with lymphoma was 20.8%. This suggests that causes other than FeLV play a role in tumorigenesis, and that latent FeLV infection is unlikely to be responsible for most feline lymphomas and leukaemias.

Prevalence of feline leukaemia provirus DNA in feline lymphomas

A significant drop in the prevalence of feline leukaemia virus (FeLV) antigenaemic cats and antigen-associated lymphomas has been observed after the introduction of FeLV vaccination and antigen-testing with removal of persistently antigenaemic cats. However, recent reports have indicated that regressively infected cats may contain FeLV provirus DNA and that lymphoma development may be associated with the presence of provirus alone. In the present study, we investigated the presence of FeLV antigen and provirus DNA in 50 lymphomas by immunohistochemistry and semi-nested polymerase chain reaction, respectively. Interestingly, almost 80% of T-cell lymphomas and 60% of B-cell lymphomas contained provirus DNA while only 21% of T-cell lymphomas and 11% of B-cell lymphomas expressed FeLV antigen. In conclusion, our results support previous hypotheses that vaccination and removal of persistently antigenaemic cats have led to a drop in FeLV antigen-expressing lymphomas. However, FeLV provirus DNA is still present in a high percentage of feline lymphomas.

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.

Exposure of cats to low doses of FeLV: seroconversion as the sole parameter of infection

In felids, feline leukemia virus (FeLV) infection results in a variety of outcomes that range from abortive (virus readily eliminated and never detectable) to progressive infection (persistent viremia and viral shedding). Recently, a novel outcome was postulated for low FeLV infectious doses. Naïve cats exposed to faeces of persistently infected cats seroconverted, indicating infection, but remained negative for provirus and p27 antigen in blood. FeLV provirus was found in some tissues but not in the bone marrow, infection of which is usually considered a necessary stage for disease progression. To investigate the impact of low FeLV doses on young cats and to test the hypothesis that low dose exposure may lead to an unknown pathogenesis of infection without involvement of the bone marrow, 21 cats were infected oronasally with variable viral doses. Blood p27, proviral and viral loads were followed until week 20 post-infection. Tissue proviral loads were determined as well. The immune response was monitored by measuring FeLV whole virus and p45 antibodies; and feline oncornavirus-associated cell membrane antigen (FOCMA) assay. One cat showed regressive infection (transient antigenemia, persistent provirus-positivity, and seroconversion) with provirus only found in some organs at sacrifice. In 7 of the 20 remaining cats FOCMA assay positivity was the only sign of infection, while all other tests were negative. Overall, the results show that FeLV low dose exposure can result in seroconversion during a presumed abortive infection. Therefore, commonly used detection methods do not detect all FeLV-infected animals, possibly leading to an underestimation of the prevalence of infection.

Fecal shedding of infectious feline leukemia virus and its nucleic acids: A transmission potential

Although it is assumed that fecal shedding of feline leukemia virus (FeLV) constitutes a transmission potential, no study has been performed showing that feces of infected cats can be a source of infection. In this study, we investigated fecal viral shedding of FeLV and its role in viral pathogenesis with the goal to improve infection control. FeLV RNA and DNA levels were determined in rectal swabs of experimentally infected cats by real-time PCR, and the results were correlated with proviral and viral loads in whole blood and plasma, respectively, and plasma p27 levels. All antigenemic cats shed FeLV RNA and DNA in feces. To determine whether the viral RNA detected was infectious, virus isolation from feces was also performed. Infectious virus was isolated from feces of antigenemic cats, and these results perfectly correlated with the isolation of virus from plasma. Naïve cats exposed to these feces seroconverted, showing that infection through feces took place, but remained negative for the presence of FeLV provirus and p27 in blood, an outcome so far not described. Some of the organs collected after euthanasia were provirus positive at low copy numbers. From these results it is concluded that fecal shedding of FeLV plays a role in transmission, but it is probably of secondary importance in viral pathogenesis. Nevertheless, sharing of litter pans by susceptible and viremic cats could increase the environmental infectious pressure and appropriate measures should be taken to avoid unnecessary viral exposure.

Rapid detection of feline leukemia virus provirus integration into feline genomic DNA

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.

P.3.022 Antistressory effect of mexidol in inbredC57BL/6 and BALB/C mice

In the past, feline leukaemia virus (FeLV) infection, and also latent FeLV infection, were commonly associated with lymphoma and leukaemia. In this study, the prevalence of FeLV provirus in tumour tissue and bone marrow in FeLV antigen-negative cats with these tumours was assessed. Seventy-seven diseased cats were surveyed (61 antigen-negative, 16 antigen-positive). Blood, bone marrow, and tumour samples were investigated by two polymerase chain reaction (PCR) assays detecting deoxyribonucleic acid (DNA) sequences of the long terminal repeats (LTR) and the envelope (env) region of the FeLV genome. Immunohistochemistry (IHC) was performed in bone marrow and tumour tissue. None of the antigen-negative cats with lymphoma was detectably infected with latent FeLV. The prevalence of FeLV viraemia in cats with lymphoma was 20.8%. This suggests that causes other than FeLV play a role in tumourgenesis, and that latent FeLV infection is unlikely to be responsible for most feline lymphomas and leukaemias.

Analysis of the disease potential of a recombinant retrovirus containing Friend murine leukemia virus sequences and a unique long terminal repeat from feline leukemia virus.

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.

Transduction of Notch2 in feline leukemia virus-induced thymic lymphoma.

Feline leukemia virus (FeLV) is thought to induce neoplastic diseases in infected cats by a variety of mechanisms, including the transduction of host proto-oncogenes. While FeLV recombinants that encode cellular sequences have been isolated from tumors of naturally infected animals, the acquisition of an unrelated host gene has never been documented in an experimental FeLV infection. We isolated recombinant FeLV proviruses encoding feline Notch2 sequences from thymic lymphoma DNA of two cats inoculated with the molecularly cloned virus FeLV-61E. Four recombinant genomes were identified, three in one cat and one in the other. Each had similar but distinct transduction junctions, and in all cases, the insertions replaced most of the envelope gene with a region of Notch2 that included the intracellular ankyrin repeat functional domain. The product of the FeLV/Notch2 recombinant provirus was a novel, truncated 65- to 70-kD Notch2 protein that was targeted to the cell nucleus. This virally encoded Notch2 protein, which resembles previously constructed, constitutively activated forms of Notch, was apparently expressed from a subgenomic transcript spliced at the normal envelope donor and acceptor sequences. The data reported here implicate a nuclear, activated Notch2 protein in FeLV-induced leukemogenesis.

Genetic Determinants of Feline Leukemia Virus-Induced Multicentric Lymphomas

Three discrete forms of feline leukemia virus (FeLV)-associated lymphoma have been described clinically: (1) thymic, (2) alimentary, and (3) multicentric. The most common and best-characterized lymphomas are of T-cell origin, generally occurring in the thymus. These tumors typically contain mature T-cells, involve the activation of a distinctive set of proto-oncogenes, and contain FeLV proviruses whose long terminal repeat (LTR) sequences contain tandemly repeated enhancers. Previous studies of a small group of extrathymic, multicentric lymphomas implicated a different set of genetic determinants. The present study expands those observations by examining the lineage of origin, the involvement of proto-oncogenes, and the structure of LTR andenvgene sequences in a set of 11 natural, extrathymic lymphomas of the multicentric type. A pattern of genetic events associated with FeLV-positive multicentric lymphomas emerges from this analysis that is clearly distinct from the pattern associated with thymic lymphomas. The tumors do not contain T-cells or B-cells, as evidenced by the germ line organization of TCRβ and IgH loci. Proto-oncogenes strongly implicated in T-cell lymphomagenesis are not involved in these tumors. Rather, a distinct set of proto-oncogenes may be involved. Most striking is the repeated occurrence of an FeLV isolate whose LTR andenvgene bear unique sequence elements.

Abnormal Processing of a Recombinant Feline Leukemia Virus Envelope Polyprotein and Its Interference with Subgroup C Virus Infection

Processing of the env polyprotein of a noninfectious feline leukemia virus (FeLV) recombinant, named r6gp, was examined in human-transfected cells. The r6gp provirus was previously generated in the frame of FeLV, subgroup B, GA clone with substitution of all but 40 C-terminal amino acid sequences of the surface glycoprotein (SU) from an endogenous FeLV provirus element (CFE-6). Although r6gp produced a normal size (85 kDa) env glycoprotein precursor, the product, unlike the precursor of the parental virus, was neither additionally glycosylated nor further processed into mature env proteins. Biochemical observations were consistent with the idea that the chimeric env polyprotein was trapped in the endoplasmic reticulum (ER) and were directly supported by immunofluorescence microscopy analyses. Interestingly, the residence of the chimeric protein in the ER specifically interfered with FeLV, subgroup C (Sarma) virus infection but not the parental FeLV-B virus infection. Since FeLV-C provirus sequences could be readily detected in the infected cells, it appeared that r6gp env expression did not block entry of the challenge virus. While FeLV-B and CFE-6 env genes share an extensive overall sequence homology, a variable region (region VI) of CFE-6 near the C-terminus of SU, which was retained in the r6gp construct, exhibits a considerably higher degree of homology to FeLV-C than FeLV-B. Thus, we propose that region VI is involved in conferring specificity for the env polyprotein oligomerization in the ER, and that co-oligomerization of the trapped r6gp env with FeLV-C is the reason for specific interference with FeLV-C infection. The results also demonstrate for the first time a functional abnormality of a recombinant FeLV env gene which is structurally similar to those commonly detected in FeLV-induced feline lymphosarcomas.

Coincident Involvement of flvi-2, c- myc, and Novel env Genes in Natural and Experimental Lymphosarcomas Induced by Feline Leukemia Virus

The flvi-2 locus is a target of insertional mutagenesis in thymic lymphosarcomas induced by feline leukemia virus (FeLV). flvi-2 encodes the gene bmi-1, whose product is implicated as a myc-collaborator in the induction of B- and T-cell lymphoma. We have examined the involvement of flvi -2 and myc in natural and experimentally induced FeLV-positive feline lymphosarcomas which are heterogeneous in anatomical origin, geographic origin, and strain of FeLV involved. We further compared these findings with previous reports of novel FeLV env genes in the same tumors. The results show that proviral insertion at flvi-2 occurs commonly in natural and experimental feline thymic lymphosarcomas of diverse origins [52% overall], and that alterations in c- myc commonly accompany insertional mutagenesis of flvi-2 [54% overall]. However, 46% of tumors with flvi-2 insertions apparently lack involvement of c- myc. These observations support the hypothesis that interruption of flvi-2 may be an early event in a multistep cascade, one possibility for completion of which is activation of c- myc. Interruption of flvi-2 was not observed in nonthymic lymphosarcomas of alimentary or multicentric origin, although c- myc may be involved. A proportion of both thymic and nonthymic tumors have been shown previously to contain FeLV proviruses with recombinant or mutant env genes. Our findings strongly implicate the insertional mutagenesis of flvi-2, the activation of c- myc, and the emergence of novel env genes in FeLV-mediated lymphomagenesis, particularly in the induction of thymic lymphosarcoma. The data show that these events may overlap, but do not necessarily occur concurrently.

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.