SV40 Immortalization of feline fibroblasts as targets for MHC-restricted cytotoxic T-cell assays.

CTL assays in outbred cats have been difficult to perform because of a lack of a good source of syngeneic target cell. Primary fibroblasts from cats are widely used as target cells for MHC-restricted cytotoxic T-cell (CTL) assays, but their limited life-spans of 8-10 culture passages can be problematic for longitudinal studies. To circumvent the life-span limitations of primary fibroblast cultures, we developed a procedure for immortalizing feline primary fibroblast cells by transfection with a molecular clone of simian virus 40 (SV40). Fibroblast cultures from skin biopsies of 28 cats were immortalized using this procedure and have been passaged for longer than 6 months without showing any phenotypic difference from the original primary cells. Non-SV40 transfected feline fibroblasts from a selection of animals in the same group survived for only 6-8 weeks before reaching senescence. The immortalized fibroblasts expressed SV40 T-antigen and Class I MHC protein, and were successfully used as target cells in 51Cr release CTL assays in feline immunodeficiency virus (FIV)-infected cats and in vitro stimulated allogeneic T-cell cultures.

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.

Differential pathogenicity of two feline leukemia virus subgroup A molecular clones, pFRA and pF6A.

F6A, a molecular clone of subgroup A feline leukemia virus (FeLV) is considered to be highly infectious but weakly pathogenic. In recent studies with a closely related subgroup A molecular clone, FRA, we demonstrated high pathogenicity and a strong propensity to undergo recombination with endogenous FeLV (enFeLV), leading to a high frequency of transition from subgroup A to A/B. The present study was undertaken to identify mechanisms of FeLV pathogenesis that might become evident by comparing the two closely related molecular clones. F6A was shown to have an infectivity similar to that of FRA when delivered as a provirus. Virus load and antibody responses were also similar, although F6A-infected cats consistently carried higher virus loads than FRA-infected cats. However, F6A-infected cats were slower to undergo de novo recombination with enFeLV and showed slower progression to disease than FRA-infected cats. Tumors collected from nine pF6A- or pFRA-inoculated cats expressed lymphocyte markers for T cells (seven tumors) and B cells (one tumor), and non-T/B cells (one tumor). One cat with an A-to-A/C conversion developed erythrocyte hypoplasia. Genomic mapping of recombinants from pF6A- and pFRA-inoculated cats revealed similar crossover sites, suggesting that the genomic makeup of the recombinants did not contribute to increased progression to neoplastic disease. From these studies, the mechanism most likely to account for the pathologic differences between F6A and FRA is the lower propensity for F6A to undergo de novo recombination with enFeLV in vivo. A lower recombination rate is predicted to slow the transition from subgroup A to A/B and slow the progression to disease.

Transfer of the feline erythropoietin gene to cats using a recombinant adeno-associated virus vector.

Chronic renal failure and the associated erythropoietin-responsive anemia afflicts over 2 million domestic cats in the United States, resulting in morbidity that can affect the owner-pet relationship. Although treatment of cats with recombinant human erythropoietin (Epo) protein can be effective, response to the drug often dissipates over time, probably due to the development of antibodies reactive with the human protein. As an alternate approach to the treatment of this disease, we have developed a recombinant adeno-associated virus vector containing the feline erythropoietin gene (rAAV/feEpo). This vector, when administered intramuscularly to normal healthy cats, caused a dose-related increase in hematocrit over a 7-week period after injection. Thus, the rAAV/feEpo vector holds promise as a simple, safe and effective therapy for the anemia of chronic renal failure in domestic cats.

Neurophysiologic and immunologic abnormalities associated with feline immunodeficiency virus molecular clone FIV-PPR DNA inoculation.

Although direct feline immunodeficiency virus (FIV) proviral DNA inoculation has been shown to be infectious in cats, long-term studies to assess the pathogenic nature of DNA inoculation are lacking. We have recently reported that direct feline leukemia virus (FeLV) DNA inoculation resulted in infection and the development of FeLV-related disease end points with similar temporal expression and virulence to that of cats infected with whole virus. We show in this study that pFIV-PPR DNA inoculation resulted in infection of cats and the development of FIV-related immunologic and neurologic abnormalities. Infected cats demonstrated progressive loss of CD4+ lymphocytes resulting in decreased CD4:CD8 ratios. Neurologic dysfunction was demonstrated by increased bilateral frontal lobe slow-wave activity. Prolongation of the visual evoked potential peak latency onset response pattern also supported a similar progression of abnormal cortical response. Furthermore, histopathologic examination revealed lesions attributed to FIV infection in lymph node, thymus, brain, and lung. Finally, nested polymerase chain reaction detected FIV provirus in brain, bone marrow, mesenteric lymph node, thymus, spleen, tonsil, and liver. These results confirm that FIV DNA inoculation is an efficient model for study of the pathogenic nature of molecular clones in vivo and offers the opportunity to measure temporal genomic stability of a homogeneous challenge material.

Recombinant feline leukemia virus (FeLV) variants establish a limited infection with altered cell tropism in specific-pathogen-free cats in the absence of FeLV subgroup A helper virus.

Feline leukemia virus subgroup B (FeLV-B) is commonly associated with feline lymphosarcoma and arises through recombination between endogenous retroviral elements inherited in the cat genome and corresponding regions of the envelope (env) gene from FeLV subgroup A (FeLV-A). In vivo infectivity for FeLV-B is thought to be inefficient in the absence of FeLV-A. Proposed FeLV-A helper functions include enhanced replication efficiency, immune evasion, and replication rescue for defective FeLV-B virions. In vitro analysis of the recombinant FeLV-B-like viruses (rFeLVs) employed in this study confirmed these viruses were replication competent prior to their use in an in vivo study without FeLV-A helper virus. Eight specific-pathogen-free kittens were inoculated with the rFeLVs alone. Subsequent hematology and histology results were within normal limits, however, in the absence of detectable viremia, virus expression, or significant seroconversion, rFeLV proviral DNA was detected in bone marrow tissue of 4/4 (100%) cats at 45 weeks postinoculation (pi), indicating these rFeLVs established a limited but persistent infection in the absence of FeLV-A. Altered cell tropism was also noted. Focal infection was seen in T-cell areas of the splenic follicles in 3/4 (75%) rFeLV-infected cats analyzed, while an FeLV-A-infected cat showed focal infection in B-cell areas of the splenic follicles. Nucleotide sequence analysis of the surface glycoprotein portion of the rFeLV env gene amplified from bone marrow tissue collected at 45 weeks pi showed no sequence alterations from the original rFeLV inocula.

Pathogenicity induced by feline leukemia virus, Rickard strain, subgroup A plasmid DNA (pFRA).

A new provirus clone of feline leukemia virus (FeLV), which we named FeLV-A (Rickard) or FRA, was characterized with respect to viral interference group, host range, complete genome sequence, and in vivo pathogenicity in specific-pathogen-free newborn cats. The in vitro studies indicated the virus to be an ecotropic subgroup A FeLV with 98% nucleotide sequence homology to another FeLV-A clone (F6A/61E), which had also been fully sequenced previously. Since subgroup B polytropic FeLVs (FeLV-B) are known to arise via recombination between ecotropic FeLV-A and endogenous FeLV (enFeLV) env elements, the in vivo studies were conducted by direct intradermal inoculation of the FRA plasmid DNA so as to eliminate the possibility of coinoculation of any FeLV-B which may be present in the inoculum prepared by propagating FeLV-A in feline cell cultures. The following observations were made from the in vivo experiments: (i) subgroup conversion from FeLV-A to FeLV-A and FeLV-B, as determined by the interference assay, appeared to occur in plasma between 10 and 16 weeks postinoculation (p.i.); (ii) FeLV-B-like recombinants (rFeLVs), however, could be detected in DNA isolated from buffy coats and bone marrow by PCR as early as 1 to 2 weeks p.i.; (iii) while a mixture of rFeLV species containing various amounts of N-terminal substitution of the endogenous FeLV-derived env sequences were detected at 8 weeks p.i., rFeLV species harboring relatively greater amounts of such substitution appeared to predominate at later infection time points; (iv) the deduced amino acid sequence of rFeLV clones manifested striking similarity to natural FeLV-B isolates, within the mid-SU region of the env sequenced in this work; and (v) four of the five cats, which were kept for determination of tumor incidence, developed thymic lymphosarcomas within 28 to 55 weeks p.i., with all tumor DNAs harboring both FeLV-A and rFeLV proviruses. These results provide direct evidence for how FeLV-B species evolve in vivo from FeLV-A and present a new experimental approach for efficient induction of thymic tumors in cats, which should be useful for the study of retroviral lymphomagenesis in this outbred species.

In vivo evolution and selection of recombinant feline leukemia virus species.

Ecotropic feline leukemia viruses subgroup A (FeLV-A) is known to recombine with endogenous FeLV (enFeLV) env elements yielding polytropic FeLV-B viruses. However, scattered nucleotide differences exist between enFeLV env elements and corresponding sequences of exogenous FeLV-B isolates. To address this disparity, we examined recombinant FeLV (rFeLV) viruses obtained from three experimentally-induced feline thymic tumors, along with rFeLVs derived from one naturally-occurring thymic tumor. Two of the three experimental cats were challenged with a FeLV-A/Rickard preparation, while one cat received this FeLV-A along with a mixture of in vitro-generated rFeLVs. The FeLV-A/Rickard preparation employed in this study was shown to be free of detectable rFeLVs since no recombinant products were observed in this preparation following nested PCR analyses. For each of the four tumor DNAs, nucleotide sequence analysis was performed on multiple clones of rFeLV-specific PCR products derived from the surface glycoprotein (SU) portion of the recombinant proviral env gene. Relative to the parental enFeLV sequence used to generate the rFeLVs, a total of 19 nucleotide differences were found scattered within the SU region of the env gene in these in vivo-derived rFeLV clones. Most interestingly, this set of 19 differences led to complete sequence identity with natural FeLV-B isolates. Our results indicate these differences are present early in the in vivo evolution of recombinant viruses, suggesting that rFeLVs harboring these differences are strongly selected. We also present evidence indicating an in vivo selection pattern exists for specific recombinant species containing relatively greater amounts of enFeLV-derived SU sequence. This in vivo selection process appears to be gradual, occurring over the infection timecourse, yielding rFeLV species which have recombination structural motifs similar to those seen in natural FeLV-B isolates.

Evidence that high-dosage zidovudine at time of retrovirus exposure reduces antiviral efficacy.

The antiviral efficacy of prophylactic 3'-azido-3'-deoxythymidine (AZT) therapy administered by continuous infusion or intermittent injection was compared in pediatric cats infected with feline leukemia virus. A 4-week treatment regimen of AZT was initiated at -48, 8, or 96 h postinfection (p.i.). For AZT therapy begun at -48 h p.i., significant efficacy was attained when therapy was given by continuous infusion but not by intermittent injection. However, when AZT therapy was delayed until 96 h p.i., both continuous infusion and intermittent injection gave complete protection. The results suggest that intermittent AZT administration is less efficacious than continuous infusion. Higher peak AZT concentrations in plasma associated with intermittent injection compared with those associated with continuous infusion may be immunotoxic, thus reducing the drug-induced vaccine effect. Furthermore, AZT toxicity seemed to be restricted to a window of sensitivity close to the time of virus challenge because delaying the start of AZT therapy until 96 h p.i. was highly efficacious, regardless of the method of administration.

Feline leukemia virus variants in experimentally induced thymic lymphosarcomas.

This study was initiated to evaluate the in vivo infectivity and pathogenicity of a group of recombinant feline leukemia viruses (rFeLVs) previously generated by in vitro forced recombination between a FeLV subgroup A virus (FeLV-A) and an endogenous FeLV (enFeLV) envelope (env) element (Sheets et al., 1992, Virology 190, 849-855). To determine infectivity of rFeLVs, neonatal cats were inoculated with rFeLVs alone or in combination with FeLV-A. The recombinant viruses were able to replicate efficiently in vivo only when administered along with FeLV-A. Of six co-infected cats, three developed thymic lymphosarcomas, one severe aplastic anemia, and two cachexia and depression; all were viremic and seroconverted shortly after inoculation. While both virus types were detected in virtually all tissues examined from these tumor-bearing cats, there was a particularly noteworthy sequence reversion in the rFeLVs. It is known that exogenous FeLV isolates carry a conserved neutralizing MGPNL epitope in the middle of the surface glycoprotein domain of the env gene. In contrast, the parental recombinant viruses used to inoculate these cats harbored the enFeLV-derived MGPNP sequence at this position. However, all in vivo-propagated recombinants displayed the MGPNL sequence, while the env-encoded backbone flanking the MGPNL sequence was that of the parental recombinant virus. These results suggest that viruses with the MGPNL epitope have an in vivo proliferative advantage. The data also provide an explanation for the conservation of this epitope in exogenous FeLVs despite the existence of variant forms in enFeLV proviral elements with which they can recombine.

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.

Differential antiviral activities and intracellular metabolism of 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine in human cells.

Dideoxynucleosides such as 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI) can effectively inhibit the replication of human immunodeficiency virus (HIV) in T lymphoid cells. There is evidence that HIV can infect and replicate in other cells including monocytoid cells and macrophages. The present study compared the antiretroviral activities of ddI and AZT in three lineages of human cells, i.e., MOLT4 (T lymphocytoid, CD4+), U937 (monocytoid, CD4+), and HT1080 (fibroblastoid, CD4-) cells. Feline leukemia virus, a retrovirus that causes immunodeficiency in cats, was used to infect the cells. The drug concentrations needed to reduce the viral p27 antigen titers in cell lysates by 50% (IC50s) were determined. The data show that AZT and ddI inhibited viral replication in all three cell lines. The IC50s of AZT were 0.02, 1.75, and 2.31 microM in MOLT4, HT1080, and U937 cells, respectively. For ddI, the IC50s were 4.31, 9.52, and 43.5 microM, respectively. These data indicate differential antiviral activities of ddI and AZT in the different cells with the following rank order of drug sensitivity: MOLT4 > HT1080 > U937. A study of the intracellular metabolism of [3H]AZT and [3H]ddI shows that the antiretroviral activities of AZT and ddI in the three cell lines correlated with the levels of their intracellular triphosphate metabolites.

Effects of whole blood lysis and fixation on the infectivity of human T-lymphotropic virus type 1 (HTLV-I).

Whole blood lysis and fixation methods for flow cytometric (FCM) analysis were tested for their ability to reduce the infectivity of human T-lymphotropic virus type 1 (HTLV-I). Our goals were to: (1) determine the effects of 1.0 and 2.0% paraformaldehyde (PF) fixation on HTLV-I infected cell lines and (2) assess the infectivity of blood samples containing HTLV-I-infected cells following processing with 5 commercially available products (Immuno-lyse, ImmunoPrep/Q-Prep, FACS lysis solution, GenTrak lyse and fix reagent, and Ortho-mune lysing reagent) compared to ammonium chloride lysis with either 0.1 or 1.0% PF fixation. Infectivity was determined by monitoring HTLV-I p24 antigen production in cocultures of treated leukocytes with uninfected peripheral blood mononuclear cells (PBMC). Each method effectively reduced the viability of treated leukocytes. Commercial lysis/fixation methods significantly reduced HTLV-I infectivity compared to prepared ammonium chloride/PF-based methods. For all preparations, increasing the time of fixation (e.g., 60 min) effectively reduced viral infectivity. Taken together, these data suggest that commercially available fixatives greatly reduce, but do not eliminate the risk of HTLV-I infection during processing of viral-infected cells for FCM analysis.

Recombination between feline exogenous and endogenous retroviral sequences generates tropism for cerebral endothelial cells.

Brain tissues of domestic cats that died of aplastic anemia from infection with either parental feline leukemia virus (FeLV), subgroup C, or a mixture of FeLV-C and recombinants between FeLV-C and an endogenous FeLV provirus were examined by the immunoperoxidase staining technique using a monoclonal antibody (C11D8) directed against an epitope of the viral surface glycoprotein (SU). Positive staining of the central nervous system (CNS) capillary endothelial cells with no labeling on neuronal or glial cells was observed in cats that were inoculated with the virus mixture. This was in contrast to brain tissue of cats infected with FeLV-C alone, which showed no such staining. While non-CNS endothelial cells derived from human umbilical vein (HUVEC) could be readily infected in culture by FeLV-C, endothelial cells derived from human retina (REC) or brain (BEC) were resistant to infection by this parental virus. These latter cells in culture, however, could be infected by the viral mixture. The data suggested that at least one or more of the presumptive recombinant viruses could specifically infect CNS-derived endothelial cells. Using polymerase chain reaction and DNA sequencing strategies to amplify and analyze DNA fragments of the proviral SU region from cells infected with REC-selected viruses, we found the occurrence of a single recombinant in which two-thirds of the SU gene from the N-terminus of FeLV-C was replaced by the endogenous FeLV element. This recombinant virus, when molecularly cloned, should be useful in determining its potential in vivo neuropathogenicity.

Pathogenicity of a subgroup C feline leukemia virus (FeLV) is augmented when administered in association with certain FeLV recombinants.

There is evidence to suggest that infectious feline leukemia viruses (FeLVs) may be altered biologically because of homologous recombination with non-infectious endogenous FeLV (enFeLV) sequences in the infected cells. To evaluate the role of such recombination events in FeLV pathogenesis, a molecular clone of subgroup C FeLV, Sarma strain (FSC), was tested for induction of aplastic anemia in the absence or presence of mixtures of recombinants between FSC and an enFeLV element. In the recombinants, FSC sequences in the viral surface glycoprotein (SU) protein were variably replaced by the corresponding sequences of the enFeLV. The results showed that the virus mixtures varied in their infectivity to neonatal specific pathogen-free cats. One group of mixtures, although exhibiting relatively reduced infectivity, represented the most acute disease-inducing agents. The presence of recombinants in this mixture significantly accelerated the development of erythrocyte aplasia compared to cats infected with FSC alone. In addition, infected cells appeared to be distributed differently in various hematopoietic organs with respect to infection with FSC versus viral mixture. Viral recombinants which were present in this inoculum mixture, however, could not be detected in the plasma or infected tissues of the cats at the end stage of the disease, although their presence in the plasma at the early stages could be detected. Clearly, parental FSC outgrew the recombinants in the infected animals, since its detection was prominent at all stages of the progression of the disease. Therefore, we hypothesize that recombinants initially present in the infected animals, while only poorly replicated compared to FSC in the host, might have had the opportunity to infect certain target cells (potentially erythroid progenitor cells) and then disappeared with the associated cytopathic effect.

Loss of neutrophil and natural killer cell function following feline immunodeficiency virus infection.

Experimental infection with the Mt. Airy isolate of feline immunodeficiency virus (FIVMA), a lentivirus isolated from a domestic cat exhibiting signs of an immunodeficiency-like syndrome, results in transient lymphadenopathy, fever, stomatitis, enteritis, neurologic abnormalities, and immunosuppression. The effects of FIVMA infection on neutrophil and natural killer cell (NK) function were examined in vitro. Suppression of neutrophil chemiluminescence (CL) responses, as well as reduction in NK-mediated cytotoxicity were demonstrated. Neutrophil CL was decreased by 50% in infected cats when compared to control values. This loss of CL was present through 6 months after infection. In addition, NK-mediated cytotoxicity was approximately 50% less in FIVMA infected cats than in controls. Loss of innate immunity was paralleled with inversion in feline CD4/CD8 lymphocyte ratios and decreases in lymphocyte mitogenesis seen as early as 5 weeks after infection. These results suggest that FIVMA infection induces an immunodeficiency disorder in infected cats similar to that seen in human immunodeficiency virus infections.

Pre- and postexposure chemoprophylaxis: evidence that 3'-azido-3'-dideoxythymidine inhibits feline leukemia virus disease by a drug-induced vaccine response.

The benefits of postexposure 3'-azido-3'-dideoxythymidine (AZT) prophylaxis following human immunodeficiency virus exposure are unknown. We describe a comprehensive assessment of pre- and postexposure AZT therapy in the feline leukemia virus (FeLV)-cat model for AIDS which included in vitro testing, an in vivo dose-response titration, a postexposure treatment study, plasma drug concentration determinations, and evaluation of the immune response to FeLV. In in vitro studies, AZT prevented FeLV infection of a feline T-lymphoid cell line, giving 50 and 90% inhibition concentrations of 4.6 and 11.1 mM, respectively. In all of the in vivo efficacy studies, AZT was administered by continuous subcutaneous infusion for 28 days. AZT toxicity was excessive at a dosage of 120 mg/kg of body weight per day, causing acute anemia, but AZT was tolerable at 60 mg/kg/day. In preexposure studies, AZT was efficacious in preventing chronic antigenemia at a dosage of > or = 15 mg/kg/day, at which plasma AZT concentrations averaged between 0.51 and 0.81 micrograms/ml (2.13 and 3.03 microM). As a postexposure treatment, at 60 mg/kg/day, AZT prevented chronic FeLV antigenemia when treatment was started up to 96 h post-virus inoculation (p.i.), but not when treatment was started at 192 h p.i. The 4-day period between 96 and 192 h p.i. appears to be critical for establishing chronic viremia. It is presumed that the increase in virus load between 4 and 8 days p.i. was able to overwhelm the immunologic functions responsible for containment of FeLV infection, even though AZT therapy effectively controlled viremia during the treatment period. The antibody response to FeLV varied depending on the time of AZT treatment initiation relative to virus challenge. When AZT treatment was started 48 h before or 8 h after FeLV challenge, antibodies to FeLV were not detected until after AZT treatment was discontinued at 28 days p.i. Following AZT treatment, however, antibody titers rapidly increased at a rate suggestive of a secondary immune response. When AZT treatment was initiate at later time points relative to virus challenge (24, 48, and 96 h p.i.), antibodies to FeLV became detectable during the treatment period. These results indicate that AZT treatment does not completely prevent FeLV infection, even when treatment begins before virus challenge, and that immune sensitization to FeLV proceeds during the prophylactic drug treatment period.

Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes.

Feline leukaemia viruses (FeLVs) are classified into subgroups A, B and C by their use of different host cell receptors on feline cells, a phenotype which is determined by the viral envelope. FeLV-A is the ubiquitous, highly infectious form of FeLV, and FeLV-C isolates are rare variants which are invariably isolated along with FeLV-A. The FeLV-C isolates share the capacity to induce acute non-regenerative anaemia and the prototype, FeLV-C/Sarma, has strongly age-restricted infectivity for cats. The FeLV-C/Sarma env sequence is closely related to that of common, weakly pathogenic FeLV-A isolates. We now show by construction of chimeric viruses that the receptor specificity of FeLV-A/Glasgow-1 virus can be converted to that of FeLV-C by exchange of a single env variable domain, Vr1, which differs by a three codon deletion and nine adjacent substitutions. Attempts to dissect this region further by directed mutagenesis resulted in disabled proviruses. Sequence analysis of independent natural FeLV-C isolates showed that they have unique Vr1 sequences which are distinct from the conserved FeLV-A pattern. The chimeric viruses which acquired the host range and subgroup properties of FeLV-C retained certain FeLV-A-like properties in that they were non-cytopathogenic in 3201B feline T cells and readily induced viraemia in weanling animals. They also induced a profound anaemia in neonates which had a more prolonged course than that induced by FeLV-C/Sarma and which was macrocytic rather than non-regenerative in nature. Although receptor specificity and a major determinant of pathogenicity segregate with Vr1, it appears that sequences elsewhere in the genome influence infectivity and pathogenicity independently of the subgroup phenotype.

Anti-human immunodeficiency virus (HIV) agents are also potent and selective inhibitors of feline immunodeficiency virus (FIV)-induced cytopathic effect: development of a new method for screening of anti-FIV substances in vitro.

The ability of several known anti-HIV substances to inhibit feline immunodeficiency virus (FIV) was tested. The results showed that FIV infection of feline T-cells was almost completely blocked in the presence of all of the agents tested. However, FIV-induced syncytium formation between a human T-cell line (MT-2 cells) and a FIV-infected feline lymphocyte cell line (3201/FIV) was inhibited only by dextran sulfate and pradimicin A. The assay used to measure syncytium inhibition was rapid and did not use potentially hazardous human immunodeficiency virus (HIV)-infected cells. The efficacy results coincided with those of HIV studies.

Incidence of localized feline leukemia virus infection in cats.

Anecdotal descriptions of atypical FeLV infections, wherein standard clinical ELISA or immunofluorescence testing fails to detect active infections, suggest that an unknown proportion of FeLV-infected cats may go undetected. In this study, 127 viremic and nonviremic cats experimentally inoculated with FeLV were evaluated at necropsy for atypical expression of FeLV antigen. Results from viremic cats were in accordance with results of earlier studies on the pathogenesis of FeLV infection in cats, wherein antigen was found in lymphoid and epithelial tissues. Differences in time course or tissue distribution of viral antigen in some cats appeared to be attributable to the challenge virus preparations, consisting of cell-free tumor homogenate or infectious plasma. It was discovered that 5 of 19 of the FeLV challenge-exposed cats that were nonviremic had FeLV-specific antigens in select tissues (bone marrow, spleen, lymph node, and small intestine) 6 to 75 weeks after inoculation. These results indicated an additional category of possible outcomes for cats exposed to FeLV. Localized FeLV infection, as described here, may explain the discordance between clinical disease and laboratory testing for FeLV.