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.

Antiviral therapy reduces viral burden but does not prevent thymic involution in young cats infected with feline immunodeficiency virus.

The thymus is a major target organ in human immunodeficiency virus type 1 (HIV-1)-infected children and feline immunodeficiency virus (FIV)-infected young cats (G. A. Dean and N. C. Pedersen, J. Virol. 72:9436-9440, 1998; J. L. Heeney, Immunol. Today 16:515-520, 1995; S. M. Schnittman et al., Proc. Natl. Acad. Sci. USA 87:7727-7731, 1990; T. A. Seemayer et al., Hum. Pathol. 15:469-474, 1984; H.-J. Shuurn et al., Am. J. Pathol. 134:1329-1338, 1989; J. C. Woo et al., J. Virol. 71:8632-8641, 1997; J. C. Woo et al., AIDS Res. Hum. Retrovir. 15:1377-1388, 1999). It is likely that the accelerated disease process in children and cats is due to infection of the thymus during the time when generation of naive T lymphocytes is needed for development of the mature immune system. Zidovudine (ZDV) monotherapy, which is used to prevent and treat perinatal HIV-1 infection (R. Sperling, Infect. Dis. Obstet. Gynecol. 6:197-203, 1998), previously had been shown to reduce viral burden in FIV-infected young cats (K. A. Hayes et al., J. Acquir. Immune Defic. Syndr. 6:127-134, 1993). The purpose of this study was to evaluate the effect of drug-induced reduction of viral burden in the thymus on virus-mediated thymic involution and peripheral blood CD4 decline using FIV-infected cats as a model for pediatric HIV-1 infection. Eight-week-old cats were randomly assigned to uninfected, saline-treated; uninfected, ZDV-treated; FIV-infected, saline-treated; and FIV-infected, ZDV-treated groups. Parameters measured included blood lymphocyte numbers, viral load in blood and thymic tissue, and thymic histopathology. While the viral burden was significantly reduced by ZDV monotherapy in peripheral blood lymphocytes, plasma, and thymus, thymic lesions were similar for the treated and untreated FIV-infected cats. Further, markedly lowering the viral burden did not increase blood CD4 lymphocyte numbers or prevent their decline. The data suggest that an inflammatory process continued in spite of reduced virus replication. These observations imply that reducing virus load and limiting thymic inflammation are separate factors that must be addressed when considering therapeutic strategies aimed at preserving thymic function.

Racial disparity in the frequency of PTEN mutations, but not microsatellite instability, in advanced endometrial cancers.

Survival of African Americans with endometrial cancer is significantly worse than that of whites. Mutation of the PTEN tumor suppressor gene and microsatellite instability occur in some endometrial cancers, and they are associated with favorable prognostic features. The aim of this study was to determine whether there is a racial disparity in the frequency of these molecular alterations that contributes to differences in outcome in advanced endometrial cancer. We screened 140 stage III/IV endometrial adenocarcinomas (78 Caucasian, 62 African American) for mutations in the PTEN gene. Paired DNA samples were available in 100 cases and were analyzed for microsatellite instability using three polymorphic markers. African-American women had cancers with significantly higher stage and grade that were more often nonendometrioid. In addition, median survival of African Americans (1.0 years) was worse than that of whites (2.5 years; P = 0.02). PTEN mutation was seen in 20 of 140 (14%) cancers and was associated with endometrioid histology and more favorable survival. The frequency of PTEN mutations was significantly higher in whites (17 of 78; 22%) than in African Americans (3 of 62; 5%; P = 0.006). Microsatellite instability was found in 15% of cancers, exclusively in endometrioid cases, and was associated with favorable survival (P = 0.01). There was no racial difference in the frequency of microsatellite instability. We conclude that mutation of the PTEN tumor suppressor gene is associated with favorable survival in advanced endometrial cancer and is 4-fold more frequent in Caucasians relative to African Americans. This suggests that differences in the frequency of PTEN mutations contribute to the racial disparity in endometrial cancer survival.

Effect of zidovudine on the primary cytolytic T-lymphocyte response and T-cell effector function.

Azidothymidine (AZT) and other nucleoside analogues, used to treat AIDS, can cause severe clinical side effects and are suspected of suppressing immune cell proliferation and effector immune cell function. The purpose of the present study was to quantitatively measure the effects of AZT on cytotoxic T-lymphocyte (CTL) priming and to determine if the major histocompatibility complex-restricted CTL killing was affected by AZT exposure. For this purpose, we employed a murine alloantigen model and limiting-dilution analysis (LDA) to estimate cytotoxic effector cell frequencies of alloreactive splenocytes treated with drug during antigen sensitization. This noninfectious model was chosen to avoid analysis of a virus-compromised immune system. Exposure of splenocytes to therapeutic concentrations of AZT (2 to 10 microM) caused a two- to threefold dose-dependent reduction in CLT precursor frequency. This reduction was caused by decreased proliferation of alloantigen-specific CTLs rather than loss of function, because full cytolytic function could be restored by adjusting the AZT-treated effector/target cell ratios to that of untreated cells. In addition, when AZT was added to the assay system at various times during antigen sensitization there was a time-related loss of the suppressive effect on the generation of cytolytic effector function, suggesting that functional CTLs are not affected by even high doses of AZT. Taken together, the data indicate that the reduction of CTL function associated with AZT treatment is due to a quantitative decrease of effector cell precursor frequency rather than to direct drug cytotoxicity or interference with mediation of cytolysis. Furthermore, antigen-naive immune cells were most sensitive to this effect during the first few days following antigen encounter.

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.

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.

Frontal lobe neuronal injury correlates to altered function in FIV-infected cats.

Six cats infected intravenously at 8 weeks of age with feline immunodeficiency virus Maryland isolate (FIV-MD), were evaluated at 8 and 14 months of age (6 months and 12 months postinfection, respectively) with high spatial resolution proton magnetic resonance spectroscopy (MRS) of the frontal cortex. Two separate control cat groups were evaluated at 8 months and 16 months of age. Single voxel two-dimensional high-resolution proton magnetic resonance imaging was performed using the PRESS sequence by selecting a 0.125 ml volume of interest in the medial frontal cortex. A significant reduction in both N-acetylaspartate (NAA) and NAA: choline ratio was found in the FIV 14-month-old group compared with FIV 8-month-old cats, and to the respective age-matched control 16-month-old cats. A negative correlation between NAA and CD4 lymphocyte count was seen in the FIV-14 group only. This group of FIV cats also exhibited a higher proportion of quantitative electroencephalographic relative slow wave activity (RSWA) that correlated to lower NAA content in the frontal cortical voxel. Although peripheral blood proviral load increased over time of infection, no correlation was found between proviral blood or lymph node load and NAA values, CD4 lymphocyte counts, or frontal cortical RSWA. Thus, this study demonstrated that neurologic functional disruption of the frontal cortex correlated strongly with neuronal injury and/or loss in FIV-MD-infected cats independent of peripheral proviral load. The ability to define in vivo neurodegeneration further in this animal model helps in understanding the neuropathogenesis of lentivirus infection, and possibly, a means to follow progression and reversibility during the initial stages of brain infection as therapeutic agents are identified.

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.

The interactive patient: a multimedia interactive educational tool on the World Wide Web.

To develop a physician-friendly continuing medical education tool for use on the World Wide Web, and to improve physicians' clinical and history taking skills, developers at Marshall University designed a multimedia interactive patient encounter for a Web server. Any physician with access to the Internet can use this program to take a history, perform a physical examination, order laboratory and radiologic studies, and submit a diagnosis and treatment plan. The system evaluates the user's performance electronically and delivers CME credits by mail. The Interactive Patient has been embraced by the medical community, with many institutions providing links to this new tool and hundreds of physicians using it. The enthusiasm of most users demonstrates that combining education with fun enhances the learning experience.

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.

Early suppression of viremia by ZDV does not alter the spread of feline immunodeficiency virus infection in cats.

Prophylactic zidovudine (ZDV) therapy in feline immunodeficiency virus (FIV) inoculated cats was evaluated for 12 months postinfection (pi) and 11 months post drug treatment. Plasma FIV antigenemia was prevented in six of six ZDV-treated and none of six untreated cats during the initial phase of infection. The present study is a continuation of that earlier work. CD4 lymphocyte numbers from ZDV-treated cats were higher than in the untreated cats. CD8 lymphocytes numbers were maintained within control limits in the ZDV-treated cats, while they declined in the untreated cats. Anti-FIV antibody titers were comparable between the ZDV-treated and the untreated cats. Histologically, lymphoid tissues for the untreated and ZDV-treated cats were unremarkable and similar to those of the uninfected control cats. Low-level FIV antigen was detected by enzyme-linked immunosorbent assay in thymus or lymph node homogenates from 3 of 11 cats tested. Polymerase chain reaction analysis showed FIV DNA in blood, lymph node, bone marrow, spleen, thymus, and brain from FIV-inoculated cats irrespective of ZDV treatment. Therefore, while prophylactic ZDV treatment prevented detectable plasma antigenemia and FIV-induced CD8 lymphocyte decline, it did not slow infection of tissues and blood cells of FIV-inoculated cats.

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.

In vivo disposition of caffeine predicted from hepatic microsomal and hepatocyte data.

The kinetics of caffeine metabolism has been investigated in freshly isolated hepatocytes, hepatic microsomes, and in vivo in male Sprague-Dawley rats. A simple Michaelis-Menten model provides an adequate description of each of the three sets of data. There is reasonable agreement between the KM values for the three systems (56-200 microM). Vmax values for hepatocytes and microsomes show good agreement when expressed in the same units using scaling factors for hepatic cellularity and microsomal protein yield [315 and 420 nmol/min/standard rat weight (SRW), respectively]. Both values slightly exceed the in vivo-determined Vmax (190 nmol/min/SRW). Taking the Vmax/KM ratio (intrinsic clearance) as the basis for scaling, the in vitro data from both the hepatocyte (2.6 ml/min/SRW) and microsomal (2.7 ml/min/SRW) studies provide a good prediction of the in vivo total body clearance (3.4 ml/min/SRW).

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.

Fusion activity dissociated from replication ability in feline immunodeficiency virus (FIV) in human cells.

Multinucleated-giant-cell formation followed by cell killing was observed after cocultivation of the feline immunodeficiency virus (FIV)-producing feline T-cell line 3201/FIV with various human cells, including T-cell lines carrying human T-cell lymphotropic virus type I (HTLV-I). The susceptibility to giant cell formation varied with the cell lines tested. Cocultivation of irradiated 3201/FIV cells with MT-2 cells resulted in giant cell formation as early as 2 h in culture, with striking resemblance to that induced by human immunodeficiency virus (HIV). MT-4 cells (HTLV-I positive) and H9 cells (HTLV-I negative) were less susceptible than MT-2 to the induction of syncytia. MOLT-4 cells (HTLV-I negative) had intermediate sensitivity to syncytia formation. No syncytia were observed in the monocytic cell line U-937 (HTLV-I negative). Syncytia formation between 3201/FIV and MT-2 cells was inhibited by polyclonal cat anti-FIV antisera but not polyclonal cat anti-feline leukemia virus (FeLV) antisera, goat anti-FeLV, uninfected specific-pathogen-free cat serum, human anti-HTLV-I antisera, or normal human and goat serum. Concentrated cell-free FIV supernatant from 3201/FIV also induced giant cells of MT-2 cells that were indistinguishable from those induced by cocultivation. Giant cells and extensive cell killing associated with giant cell formation declined and disappeared within 10 days. Surviving cells appeared to be of normal size and grew continuously without expressing FIV antigen or releasing infective virus. Although Southern blot analysis using probes specific for FIV could not detect proviral DNA in any of the five human cell lines cocultured with irradiated 3201/FIV cells, the polymerase chain reaction (PCR) technique detected FIV-specific DNA in MOLT-4 cells. DNA from the FIV-PCR positive MOLT-4 cells was PCR negative for endogenous FeLV-specific sequences, indicating that the MOLT-4 cell DNA was not contaminated with DNA from feline cells (i.e., 3201 cells). The FIV-MOLT-4 cells remained PCR positive for FIV after 40 passages, suggesting stable integration in the human cell line. These findings indicate that FIV is capable of forming proviral DNA in human T-lymphoid cells by cocultivation, although this FIV-carrying human cell line failed to produce replication-competent viruses.

Prophylactic ZDV therapy prevents early viremia and lymphocyte decline but not primary infection in feline immunodeficiency virus-inoculated cats.

Prophylactic zidovudine (ZDV) therapy was evaluated in the feline immunodeficiency virus (FIV)-inoculated cat model for HIV-1 infection in humans. ZDV treatment (30 mg/kg/day via continuous subcutaneous infusion) was initiated 48 h prior to virus inoculation and continued for 28 days. Transient plasma antigenemia evident in six of six untreated cats at week 2 post-inoculation (pi) was absent in the ZDV-treated cats although at 10 and 14 weeks pi (6 and 10 weeks after drug treatment), one of the ZDV-treated cats had low-level antigenemia. Both CD4 and CD8 lymphocyte numbers were consistently higher in the ZDV-treated cats when compared to both the FIV-inoculated untreated cats and the virus-naive, age-matched controls. CD4:CD8 ratios were lower for the ZDV-treated cats than either the FIV-inoculated untreated or virus-naive, control cats. The decreased CD4:CD8 ratios were the result of an increase in CD8 lymphocytes in the ZDV-treated cats while decreased ratios in the FIV-inoculated untreated cats were due to cell loss. Both ZDV-treated and untreated cats showed nearly identical FIV-specific antibody responses beginning 2 weeks pi. Polymerase chain reaction (PCR) results from blood lymphocytes showed that six of six ZDV-treated and six of six untreated cats were positive for FIV-specific gag sequences. Although primary infection was not prevented, these results suggest that prophylactic ZDV therapy deterred early systemic spread of infection mediated by viremia and delayed absolute CD4 and CD8 lymphocyte decline.