Fozivudine tidoxil as single-agent therapy decreases plasma and cell-associated viremia during acute feline immunodeficiency virus infection.

BACKGROUND: Feline immunodeficiency virus (FIV) is a lentivirus that infects domestic and wild felidae and the course of disease is similar to that of human immunodeficiency virus infection. The thymidine nucleoside analog fozivudine (FZD) tidoxil is a lipid-zidovudine (ZDV) conjugate and member of the family of nucleoside reverse transcriptase (RT) inhibitors (NRTIs). HYPOTHESIS: FZD administration to cats during acute FIV infection produces antiviral activity with fewer adverse effects than its parent compound ZDV (AZT). ANIMALS: Male, neutered cats approximately 7 months of age (n = 12). METHODS: FZD (45 mg/kg q12h, n = 6) or placebo (n = 6) was administered PO in a nonblinded trial for 6 weeks to cats infected with the NCSU(1) isolate of FIV. Peripheral blood was collected preinfection and at 2, 4, and 6 weeks postinfection for CBC, evaluation of CD4(+) and CD8(+) cell counts by flow cytometry, and quantification of plasma and cell-associated viremia by real time RT-PCR. RESULTS: Treatment of cats with FZD during the acute stage of FIV infection decreased plasma and cell-associated viremia during the first 2 weeks of infection, but was not protective against FIV, as all cats were infected by 6 weeks. CONCLUSIONS: At the dosage used in this study, treatment with FZD results in a short-term decrease in viral load with no adverse effects. Further investigation of FZD is warranted to assess pharmacokinetics, optimal dosage, and to directly compare the antiviral activity of FZD to ZDV in naturally infected cats.

C-Terminal gp40 peptide analogs inhibit feline immunodeficiency virus: cell fusion and virus spread.

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1), gp160, is synthesized as a protein precursor that when proteolytically cleaved yields two subunits, gp120 and gp41. gp120 is the surface glycoprotein on HIV-1 responsible for binding to CD4, and gp41 is the transmembrane glycoprotein involved in the membrane fusion process. gp41 is divided into the N-terminal fusion peptide, the heptad repeat 1 (HR1) and HR2 regions, and the C-terminal transmembrane region, which are collectively responsible for virus fusion and entry into the cell. Synthetic peptides derived from the HR2 and HR1 regions of HIV-1(LAI) have been shown to prevent virus-cell fusion and infection in vitro. In phase II clinical trials in HIV patients, data revealed that T20 has antiviral efficacy and is well tolerated. Similar results were obtained in vitro with HIV-2 and simian immunodeficiency virus, supporting the conservation of the gp41 ectodomain among lentiviruses. Feline immunodeficiency virus (FIV) infection in the cat has been used as a model to develop potential antivirals for HIV. To determine if synthetic gp40 analogs capable of inhibiting FIV infection could be identified, 15 overlapping 35-amino-acid peptides derived from the C-terminal HR2 domain of FIV gp40 were synthesized. These peptides were tested for efficacy against FIV in a syncytium-forming assay with FIV-infected CrFK cells and HeLa cells expressing the FIV receptor CXCR4. Several peptides exhibited activity at the nanogram level. Antiviral activity was confirmed by suppression of reverse transcriptase in a FIV feline CD4(+)-T-cell (FCD4-E) acute-infection assay. These data demonstrate that synthetic peptides derived from the HR2 domain of the FIV gp41 protein are effective inhibitors of FIV infection.

Infection of the choroid plexus by feline immunodeficiency virus.

The human, simian, and feline immunodeficiency viruses rapidly penetrate into the brain and trigger an inflammatory process that can lead to significant neurologic disease. However, the mechanisms that permit efficient trafficking of macrophage-tropic and the more neurotoxic lymphocytotropic isolates are still poorly understood. One potential source of virus entry may be the blood-CSF barrier provided by the choroid plexus. Infected cells are often detected within the choroid plexus but it is unclear whether this reflects trafficking cells or infection of the large macrophage population within the choroidal stroma. To address this issue, we cultured fetal feline choroid plexus and evaluated the ability of feline immunodeficiency virus (FIV) to establish a primary infection. Significant provirus was detected in macrophage-enriched choroid plexus cultures as well as in the choroid plexus of cats infected in vivo. FIV p24 antigen production in vitro was very low but detectable. Addition of a feline T-cell line to macrophages inoculated with FIV resulted in a dense clustering of the T cells over macrophages with dendritic cell-like morphologies and a robust productive infection. The direct infection of choroid plexus macrophages with FIV, the efficient transfer of the infection to T cells indicate that the choroid plexus can be a highly efficient site of viral infection and perhaps trafficking of both macrophage-tropic and T-cell-tropic viruses into the CNS.

Constitutive expression of types 1 and 2 cytokines by alveolar macrophages from feline immunodeficiency virus-infected cats.

Evidence suggests that feline immunodeficiency virus (FIV), causes pulmonary immunodeficiency. The overall objective of this study was to explore FIV-induced alterations in cell counts and cytokine gene expression in the pulmonary compartment during the acute stage infection. Bronchoalveolar lavage (BAL) cells were collected from FIV-infected and control cats at 0, 4, 10, and 16 weeks post-FIV infection for phenotype and cytokine analysis. The major change in BAL cellular populations following FIV-infection was the development of a neutrophilia. Total BAL cell counts and relative numbers of alveolar macrophages (AM), eosinophils, and lymphocytes remained similar in both groups. The RT-qcPCR analyses of AM purified from BAL showed constitutive expression of TNFalpha, IL6 and IL10 mRNAs that peaked during the acute stage of infection then declined. The TNFalpha and IL6 bioactive protein secretion showed a similar response. In contrast, IFNgamma expression increased progressively with time after infection and paralleled a progressive increase in FIV-gag mRNA in AM. The IL12 p40 expression also differed from the other cytokines in that there was a progressive decrease in the number of cats with AM IL12 expression following FIV infection. Infection of AM in vitro with FIV also caused an increase in TNFalpha and IL6 mRNA and bioactive protein suggesting that the increased cytokine response by AM following infection of cats with FIV is an intrinsic characteristic of FIV-infected AM. In summary, pulmonary immune changes seen in FIV-infected cats are similar to those seen in HIV-infected human patients.

Cloning and sequencing hybrid striped bass (Morone saxatilis x M. chrysops) transforming growth factor-beta (TGF-beta), and development of a reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay to measure TGF-beta mRNA of teleost fish.

A transforming growth factor (TGF)-beta was isolated and cloned from hybrid striped bass (Morone saxatilis x M. chrysops) anterior kidney mononuclear cells. This isolate (Genbank accession number AF140363) contains an open reading frame of 1146 bases coding for a 382 amino acid protein most similar to rainbow trout TGF-beta (57.3 and 78.6% identity with precursor and active protein, respectively) and rat TGF-beta 1 (41.1 and 68.8% identity with precursor and active protein, respectively). Consensus primers were demonstrated to amplify specifically by polymerase chain reaction (PCR), a TGF-beta segment from 14 species of teleost fish comprising 10 taxonomic families in 7 orders. A reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay was devised to measure TGF-beta mRNA expression in teleost fish. Higher levels of TGF-beta mRNA expression were detected in mononuclear cells of peripheral blood than from spleen or anterior kidney.

T cells overexpressing interferon-gamma and interleukin-10 are found in both the thymus and secondary lymphoid tissues of feline immunodeficiency virus-infected cats.

Similar to human immunodeficiency virus type 1, feline immunodeficiency virus (FIV) replicates in the thymus of infected animals, causing marked alteration in thymic lymphocyte subpopulations. The immune phenotype and cytokine patterns in the thymus and secondary lymphoid tissues of FIV-infected cats were investigated. FIV infection caused an acute-stage transient reduction in CD4CD8 double-positive thymocytes, a marked increase in CD8 single-positive thymocytes, and formation of thymic B cell lymphoid follicles. Interferon (IFN)-gamma and interleukin (IL)-10 mRNA were up-regulated in both the thymus and lymph nodes of FIV-infected cats. Analysis of purified CD4 and CD8 cells revealed that CD4 cells produced most of the IL-10, whereas IFN-gamma was produced by both subsets. Quantitative-competitive reverse-transcription polymerase chain reaction analysis revealed that thymocytes, especially CD4CD8 thymocytes, had much greater levels of gag mRNA than did lymph node T cells. Thus, overexpression of IFN-gamma and IL-10 is a feature of the thymus and secondary lymphoid tissues of FIV-infected cats.

Progressive expansion of an L-selectin-negative CD8 cell with anti-feline immunodeficiency virus (FIV) suppressor function in the circulation of FIV-infected cats.

The acute stage of feline immunodeficiency virus (FIV) infection is characterized by the appearance of a major CD8 subpopulation with reduced expression of the CD8 beta chain (CD8alpha+betalo). CD8 antiviral activity was subsequently shown to be mediated by the CD8alpha+betalo phenotype, which is the dominant CD8 phenotype in long-term infected cats. Two- and three-color flow cytometric analysis demonstrated that the CD8alpha+betalo subset is L-selectin negative (CD62L-) and has increased expression of CD44, CD49d, and CD18, consistent with an activation phenotype. The CD8alpha+betaloCD62L- cells but not the CD8alpha+betahiCD62L+ cells demonstrated strong antiviral activity in the FIV acute-infection assay. The progressive expansion of the CD8alpha+betaloCD62L- effector subset cells in FIV-infected cats parallels that seen in human immunodeficiency virus (HIV)-infected patients, suggesting that failure in homeostatic mechanisms regulating lymphocyte activation or trafficking (or both) may be a consequence of both HIV and FIV infections.

Immunomodulation and therapeutic effects of the oral use of interferon-alpha: mechanism of action.

It is now well accepted that type 1 interferons (IFNs), IFN-alpha and IFN-beta, in addition to being molecules with powerful antiviral activity, play a critical role in modulating immune responses to foreign and self-antigens. This review of the literature documents the immunomodulatory effects of IFN-alpha and discusses its position and importance in the cytokine cascade. In addition, this review attempts to organize the literature describing local and systemic immunomodulatory effects of orally administered low doses of IFN-alpha, and provide a physiological explanation for the mechanism of action. Evidence suggests that, early in the process of antigen presentation to T helper (Th) cells, IFN-alpha derived principally from the antigen-presenting cells (APC) provides an important signal for Th precursor differentiation in favor of a Th1 immune response. IFN-alpha, perhaps via upregulation of the high-alphaffinity interleukin-12beta1/beta2 (IL-12beta1/beta2) receptor, renders Th1 cells responsive to IL-12 resulting in production of high levels of IFN-gamma crucial to the development of Th1 immune responses. In addition to being instrumental in the development of Th1 immune responses, IFN-alpha appears to be the major cytokine responsible for the amplification of the CD8+ T cell response and resistance to viral infections. Orally administered IFN-alpha induces similar Th1 cytokine responses in buccal mucosal lymph nodes (LN), including upregulation of IFN-gamma expression and downregulation of IL-4. Moreover, reports of systemic immune effects such as decreased autoimmune responses, increased antiviral and antibacterial responses, and generalized immune function changes after oral IFN-alpha administration are consistent with the known immunomodulatory role of IFN-alpha in a physiological setting. Responses to orally administered low doses of IFN-alpha also adhere to the principle of low-dose priming and high-dose anergy that dictates the cellular and cytokine responses to exogenously added cytokines both in vivo and in vitro. These observations collectively suggest that IFN-alpha administered to mucosal-associated immune tissue replicates the known physiological role of IFN-alpha, including regulation of CD4+ Th1 immunomodulatory cells and activation of CD8+ effector cells, which are both crucial to development of protective immune responses. What remains to be determined is how local mucosal immune responses to IFN-alpha given orally are translated into systemic immune responses and resistance to disease. This important question, the answer to which will have profound implications for new immunotherapies for immune-based diseases, is the focus of current research.

Shedding of feline immunodeficiency virus in semen of domestic cats during acute infection.

OBJECTIVE: To examine shedding of cell-free and cell-associated feline immunodeficiency virus (FIV) in semen of domestic cats during acute infection. ANIMALS: 7 specific-pathogen-free sexually intact male cats. PROCEDURE: 6 cats were inoculated IV with 5 x 10(6) 50% tissue culture infective doses of FIV-NCSU1, and 1 cat served as an uninfected (control) cat. Infection was confirmed in the 6 cats. Periodically for up to 16 weeks after inoculation, cats were anesthetized and ejaculates obtained by use of electroejaculation. Virus was isolated from filtered seminal plasma and washed seminal cells by co-cultivation with a feline CD4+ T-cell line. Seminal cell lysates were also examined for a 582-base pair segment of FIV gag provirus DNA, using a nested polymerase chain reaction amplification. RESULTS: During the acute phase of FIV infection, virus was evident in semen of 5 inoculated cats. Five cats had virus-positive seminal plasma and 3 had virus-positive cellular constituents during the study. Virus was isolated from 8/22 (36%) seminal plasma samples and 2/17 (18%) seminal cell specimens. Provirus DNA was detected in 5/24 (21%) seminal cell lysates. Cell-free virus was isolated as early as 6 weeks after inoculation, whereas cell-associated virus was isolated as early as 12 weeks after inoculation. Provirus DNA was detected in seminal cells from one cat as early as 1 week after inoculation. CONCLUSIONS AND CLINICAL RELEVANCE: Cell-free and cell-associated FIV are shed in semen of cats early during the course of infection. Samples obtained before seroconversion may contain virus. Virus shedding in ejaculates varies between and within cats during acute infection.

The CD8+ cell phenotype mediating antiviral activity in feline immunodeficiency virus-infected cats is characterized by reduced surface expression of the CD8 beta chain.

The acute stage of feline immunodeficiency virus (FIV) infection is characterized by a CD8+ anti-FIV response that parallels the appearance of a CD8+ subpopulation with reduced expression of the beta chain (CD8 alpha + beta lo). The relationship between the CD8 alpha + beta lo phenotype and CD8+ anti-FIV activity was examined. Flow cytometric analysis of peripheral blood mononuclear cells with anti-CD8 beta chain monoclonal antibody 117 revealed that the CD8 alpha + beta lo phenotype expanded throughout the asymptomatic infection, constituting 80%-90% of the CD8 beta + cells in long-term-infected cats. Purified CD8 alpha + beta hi and CD8 alpha + beta lo subpopulations were analyzed for anti-FIV activity in an acute infection assay. Anti-FIV activity resided principally in the CD8 alpha + beta lo population and was demonstrated in acute FIV infections, as well as in long-term asymptomatic infections. These data suggest that a unique CD8 alpha + beta lo anti-FIV phenotype arises early in infection and may play a major role in eliminating virus and maintaining the asymptomatic infection.

Elevated interleukin-10-to-interleukin-12 ratio in feline immunodeficiency virus-infected cats predicts loss of type 1 immunity to Toxoplasma gondii.

Similar to human immunodeficiency virus, feline immunodeficiency virus (FIV) induces immunodeficiency and enhanced susceptibility to secondary pathogens. To explore cytokine alterations in lentivirus immunodeficiency, constitutive mRNA expression was measured in lymph nodes of healthy and FIV-infected cats before and after challenge with Toxoplasma gondii. Cytokine mRNA expression was similar in control and FIV-infected cats during the first 10 weeks after infection. At 16 weeks, interferon (IFN)-gamma, tumor necrosis factor-alpha, and interleukin (IL)-10 mRNA were increased in FIV-infected cats. Challenge with T. gondii induced an increase in IL-2, IFN-gamma, and IL-12 in the lymph nodes of control cats, whereas IFN-gamma and IL-10 but not IL-2 or IL-12 increased in the lymph nodes of FIV-T. gondii coinfected cats. These results indicate that FIV immunodeficiency may derive from a failure to generate an IL-12-dependent type 1 response and that an elevated level of IL-10 mRNA expression is a predictor of lentivirus immunodeficiency.

Feline immunodeficiency virus is shed in semen from experimentally and naturally infected cats.

Although a laboratory isolate of feline immunodeficiency virus (FIV), FIV-NCSU1, has been transmitted by artificial insemination in domestic cats, transmission by naturally infected males during mating has not been reported. In order to determine whether virus shedding in semen is unique to the NCSU1 isolate, we analyzed electroejaculates from four specific-pathogen-free males infected with another laboratory strain, FIV-Petaluma, and eight random source males with naturally acquired infections. Seminal cell lysates from the cats infected with the Petaluma isolate were screened by nested polymerase chain reaction amplification for FIV gag DNA. Seminal cells and seminal plasma from these FIV-Petaluma cats were further analyzed for the presence of virus by cocultivation with a feline CD4+ T cell line and Crandell feline kidney cells. Electroejaculates from the naturally infected cats were cocultivated with the T cell line. Our results demonstrated that cell-free FIV was present in seminal plasma from two FIV-Petaluma cats and two naturally infected cats. Cell-associated seminal virus was detected in all of the FIV-Petaluma infected cats and one naturally infected cat. Secretion of viral gag p26 antigen, an indication of active viral replication, was evident in cocultures containing motile sperm purified by a swim-up procedure from a FIV-Petaluma cat. These results confirm that FIV shedding in semen is not restricted to a specific virus isolate. Furthermore, swim-up sperm from FIV-infected cats may be infectious in vitro.

Biphasic thymus response by kittens inoculated with feline immunodeficiency virus during fetal development.

The objective of this study was to assess the response of the feline thymus to fetal infection with feline immunodeficiency virus (FIV), an animal model for human immunodeficiency virus infection. Thirteen feline embryos from four litters were directly inoculated with FIV during the sixth week postbreeding, a period corresponding to the late second trimester of pregnancy. Thymus tissue was collected and analyzed from randomly selected kittens at 2, 4, and 16 weeks postinoculation (PI) and compared to age-matched control kittens that did not receive fetal inoculations. Of three kittens evaluated at 2 weeks PI (week 8 of gestation), neither thymus:body weight ratio nor histologic structure differed from five age-matched control animals. However, analysis of thymocyte subpopulations by flow cytometry revealed a significant (P = 0.011) reduction in the percentage of cluster of differentiation (CD)4+/CD8+ cells from an average of 66% in control fetuses to 45% in infected fetuses. FIV RNA transcription, assessed by in situ hybridization using an FIVgag RNA probe, was widely distributed throughout the thymus in patterns suggestive of both stromal and parenchymal infection. By 4 weeks PI (week 1 postpartum), the thymus:body weight ratio was significantly reduced (P = 0.007) from 0.36% in five control kittens to 0.13% in four fetal inoculates. Severely atrophied thymus lobules supported minimal virus transcription and mean CD4+/CD8+ thymocyte percentages were lower (P = 0.021) in infected kittens (15%) compared to age-matched controls (66%). By 16 weeks PI (week 12 postpartum), thymus:body weight ratios of six inoculated kittens were not significantly different from six age-matched controls, suggesting that partial postnatal thymus regeneration had occurred. However, despite similar size, the regenerative thymus contained reduced percentages of CD4+/CD8+ thymocytes (infected: 40% versus control: 76%; P = 0.009) and increased percentages of CD4+/CD8- (11% versus 5%; P = 0.002) and CD4-/CD8+ (16% versus 9%; P = 0.035) lymphocytes. These changes were associated with widespread FIV transcription within thymic lymphocytes. Thus, the thymus of kittens infected with FIV during late fetal development is characterized by two distinct changes: neonatal atrophy and postnatal regeneration. Despite a recovery in thymus weight, thymus regeneration ineffectively restores the normal phenotypic distribution of thymocytes and supports FIV transcription.

Mucosally transmitted feline immunodeficiency virus induces a CD8+ antiviral response that correlates with reduction of cell-associated virus.

Intravaginal inoculation of cats with feline immunodeficiency virus (FIV) results in acute systemic infection accompanied by a strong CD8+ immune response that inhibits viral replication. CD8+ anti-FIV activity, revealed by increased FIV replication in peripheral blood mononuclear cells (PBMC) depleted of CD8+ lymphocytes, was detected by 6 weeks after inoculation and correlated with reduced PBMC-associated virus at 12, 16, and 32 weeks after inoculation. Some cats with strong CD8+ anti-FIV activity during acute infection did not seroconvert and yielded no evidence of FIV infection at later times. These data suggest that CD8+ immunity may play a major role in eliminating virus during primary transmucosal FIV infection and may down-regulate viral replication during asymptomatic infection.

Horizontal transmission of feline immunodeficiency virus with semen from seropositive cats.

The AIDS virus of cat species, feline immunodeficiency virus (FIV), has been used extensively as an animal model of HIV-1 infection. This felid lentivirus shares many molecular and biochemical traits with HIV-1 and causes similar immunologic and clinical perturbations, most notably CD4+ cell loss, impaired cell-mediated immunity and increased susceptibility to opportunistic pathogens. Previous reports have shown that FIV is transmitted horizontally by biting and vertically in utero and through nursing. Our objective was to determine whether FIV could be venereally transmitted in domestic cats. In the first experiment, susceptibility of the female reproductive tract to mucosal transmission of the FIV isolate, NCSU1, was demonstrated via intravaginal inoculation with infected cultured cells. We next identified virus in electroejaculates from asymptomatic, chronically FIV-NCSU1-infected, adult males. A fragment of FIV gag provirus DNA was detected by nested polymerase chain reaction (PCR) in nonfractionated seminal cells and in swim-up sperm preparations. Additionally, replication-competent virus was isolated from cell-free seminal plasma and seminal cells by co-cultivation with a feline CD4+ T-cell line. In the third study, queens were artificially inseminated via an intrauterine laparoscopic technique with electroejaculates from FIV-NCSU1-infected males. Of six inseminations carried out with fresh semen, three resulted in infection of queens. Lastly, immunohistochemical studies identified potential virus target cell populations in normal female reproductive tissues. In conclusion, these experiments indicate that FIV infection in domestic cats may provide a unique small animal model of sexual transmission of HIV-1.

Transmission of feline immunodeficiency virus in domestic cats via artificial insemination.

The objective of this study was to determine whether semen from male domestic cats infected with feline immunodeficiency virus (FIV) can transmit virus to females. Twelve inseminations were performed by an intrauterine laparoscopic technique with fresh or cryopreserved electroejaculates from asymptomatic males chronically infected with the NCSU1 strain of FIV. Of six inseminations performed with fresh semen, three resulted in infection of queens, as indicated by seroconversion, expression of FIV gag provirus in peripheral blood leukocytes, and reduced peripheral CD4+/CD8+ T-lymphocyte ratios. None of the six inseminates with thawed cryopreserved semen resulted in infection. Two infected queens and one uninfected queen became pregnant. Virus was not evident in the seven offspring. We conclude that FIV can be transmitted horizontally by artificial insemination with fresh semen.

Changes in lymphocyte subsets with age in perinatal cats: late gestation through eight weeks.

The age-related changes in numbers and proportions of peripheral blood lymphocyte subsets (pan-T, CD4, CD8, Ig, and null) were evaluated by two-color flow cytometry in 16 feline fetuses (56-58 days gestational age) and 21 kittens from birth through 8 weeks of age. Populations of pan-T+, CD4+, and CD8+ cells increased in total numbers as a function of increases in total lymphocyte numbers while proportions of these subsets remained relatively static. In contrast, both the total number and proportion of Ig+ cells increased from birth to 4 weeks of age, after which there were essentially no changes. Null (pan-T-, Ig-) cells were highest during late gestation and declined steadily thereafter to become a minimal component of the peripheral lymphocyte subsets. Compared with normal adult values, CD4/CD8 ratios were high throughout the 8 week study period. These results illustrate that the neonatal cat blood lymphocyte profile undergoes maturational changes and emphasize the importance of evaluating age-matched controls in studies of conditions that may alter feline lymphocyte subsets.

Effect of FIV infection on lung inflammatory cell populations recovered by bronchoalveolar lavage.

Human immunodeficiency virus (HIV) and ovine progressive pneumonia virus have been associated with lymphocytic pneumonitis. Pulmonary cell populations in cats infected with feline immunodeficiency virus (FIV) were evaluated by bronchoalveolar lavage (BAL) to identify changes associated with lentivirus infection in this species. Bronchoalveolar lavage was performed through an endotracheal tube using 15 ml kg-1 body weight of sterile 0.9% sodium chloride solution. Results of BAL fluid cytologic analysis from 19 cats experimentally infected with FIV for at least 8 months were compared with results from 34 uninfected cats. Infected cats had significantly higher total cell counts and relative neutrophil counts (P < 0.01). Lymphocytosis did not occur. Bronchoalveolar lavage fluid was collected from nine additional cats prior to, and 2, 6, and 17-18 weeks following infection with FIV. Neither neutrophilia nor lymphocytosis was associated with FIV infection in these cats.

Molecularly cloned feline immunodeficiency virus NCSU1 JSY3 induces immunodeficiency in specific-pathogen-free cats.

A full-length feline immunodeficiency virus NCSU1 (FIV-NCSU1) genome (JSY3) was cloned directly from FIV-NCSU1-infected feline CD4+ lymphocyte (FCD4E) genomic DNA and identified by PCR amplification with 5' long terminal repeat, gag, env, and 3' long terminal repeat primer sets. Supernatant from FCD4E cells cocultured with JSY3-transfected Crandell feline kidney (CrFK) cells was used as an inoculum. Cell-free JSY3 virus was cytopathogenic for FCD4E lymphocytes but did not infect CrFK cells in vitro. To determine in vivo infectivity and pathogenesis, six young adult specific-pathogen-free cats were inoculated with cell-free JSY3 virus. Provirus was detected at 2 weeks postinfection (p.i.) and was still detectable at 25 weeks p.i. as determined by gag region PCR-Southern blot analysis of peripheral blood mononuclear cell lysates. Infectious virus was recovered from peripheral blood mononuclear cells at 6 and 25 weeks p.i., and an antibody response to FIV was detected by 4 weeks. In the acute phase of infection, JSY3 provirus was found only in the CD4+ lymphocyte subset; however, by 14 weeks p.i., the greatest provirus burden was detected in B lymphocytes. All six cats were panlymphopenic at 2 weeks p.i., CD4+/CD8+ ratios were inverted by 6 weeks p.i., and five of the six cats developed lymphadenopathy by 10 weeks p.i. To determine if the JSY3 molecular clone caused immunodeficiency similar to that of the parental wild-type FIV-NCSU1, the cats were challenged with the low-virulence ME49 strain of Toxoplasma gondii at 29 weeks p.i. Five of six cats developed clinical signs consistent with generalized toxoplasmosis, and three of six cats developed acute respiratory distress and required euthanasia. Histopathologic examination of the severely affected cats revealed generalized inflammatory reactions and the presence of T. gondii tachyzoites in multiple tissues. None of the six age- and sex-matched specific-pathogen-free cats inoculated with only T. gondii developed clinical disease. Our results suggest that the pathogenesis of the molecularly cloned NCSU1 JSY3 is similar to that of wild-type FIV-NCSU1.