The role of in vitro-induced lymphocyte apoptosis in feline immunodeficiency virus infection: correlation with different markers of disease progression

Human immunodeficiency virus infection is characterized by a progressive decline in the number of peripheral blood CD4(+) T lymphocytes, which finally leads to AIDS. This T-cell decline correlates with the degree of in vitro-induced lymphocyte apoptosis. However, such a correlation has not yet been described in feline AIDS, caused by feline immunodeficiency virus (FIV) infection. We therefore investigated the intensity of in vitro-induced apoptosis in peripheral blood lymphocytes from cats experimentally infected with a Swiss isolate of FIV for 1 year and for 6 years and from a number of long-term FIV-infected cats which were coinfected with feline leukemia virus. Purified peripheral blood lymphocytes were either cultured overnight under nonstimulating conditions or stimulated with phytohemagglutinin and interleukin-2 for 60 h. Under stimulating conditions, the isolates from the infected cats showed significantly higher relative counts of apoptotic cells than did those from noninfected controls (1-year-infected cats, P = 0.01; 6-year-infected cats, P = 0.006). The frequency of in vitro-induced apoptosis was inversely correlated with the CD4(+) cell count (P = 0. 002), bright CD8(+) cell count (P = 0.009), and CD4/CD8 ratio (P = 0. 01) and directly correlated with the percentage of bright major histocompatibility complex class II-positive peripheral blood lymphocytes (P = 0.004). However, we found no correlation between in vitro-induced apoptosis and the viral load in serum samples. Coinfection with feline leukemia virus enhanced the degree of in vitro-induced apoptosis compared with that in FIV monoinfected cats. We concluded that the degree of in vitro-induced apoptosis was closely related to FIV-mediated T-cell depletion and lymphocyte activation and could be used as an additional marker for disease progression in FIV infection.     

Plasma viral RNA load predicts disease progression in accelerated feline immunodeficiency virus infection

Viral RNA load has been shown to indicate disease stage and predict the rapidity of disease progression in human immunodeficiency virus type 1 (HIV-1)-infected individuals. We had previously demonstrated that feline immunodeficiency virus (FIV) RNA levels in plasma correlate with disease stage in infected cats. Here we expand upon those observations by demonstrating that plasma virus load is 1 to 2 logs higher in cats with rapidly progressive FIV disease than in long-term survivors. Differences in plasma FIV RNA levels are evident by 1 to 2 weeks after infection and are consistent throughout infection. We also evaluated humoral immune responses in FIV-infected cats for correlation with survival times. Total anti-FIV antibody titers did not differ between cats with rapidly progressive FIV disease and long-term survivors. These findings indicate that virus replication plays an important role in FIV disease progression, as it does in HIV-1 disease progression. The parallels in virus loads and disease progressions between HIV-1 and FIV support the idea that the accelerated disease model is well suited for the study of therapeutic agents directed at reducing lentiviral replication.     

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.     

CXCR4 is required by a nonprimate lentivirus: heterologous expression of feline immunodeficiency virus in human, rodent, and feline cells

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors.     

Incubation time for feline immunodeficiency virus cultures

In a recent report, Fiscus et al. (S. A. Fiscus, S. L. Welles, S. A. Spector, and J. L. Lathey, J. Clin. Microbiol. 33:246-247, 1995) have shown that qualitative human immunodeficiency virus cultures can be terminated at day 21 with minimal false-negative results. We have evaluated a large number of qualitative and quantitative feline immunodeficiency virus (FIV) isolations to determine how long FIV cultures should be incubated to obtain reasonably certain results. The rate at which FIV cultures became positive was influenced by whether the cats under study were naturally or experimentally infected, the duration of in vivo infection, and the number of infected peripheral blood mononuclear cells seeded. The results show that cultures for FIV isolation should be kept for 5 to 6 weeks.     

Cytokine production by cats infected with feline immunodeficiency virus: a longitudinal study

The immune responsiveness of cats naturally or experimentally infected with feline immunodeficiency virus (FIV) was studied. Peripheral blood mononuclear cells (PBMC) from naturally infected, symptomatic animals displayed depressed proliferation and interleukin-2 (IL-2) production in response to mitogens, which was accompanied by a significant increase in IL-1, IL-6 and tumour necrosis factor (TNF) production. Longitudinal studies were performed over a period of 4 years in experimentally infected animals. The responses of cells from these cats to concanavalin A (Con A) were consistently less than those from uninfected cats throughout the period but, owing to variation between cats, were significantly lower on only a few occasions. By contrast, the responses of cells to pokeweed mitogen (PWM) were severely affected and declined progressively throughout the 4-year period. In general, responses to Con A but not PWM could be restored by the addition of exogenous IL-2. The decline in immune responsiveness was concurrent with a decline in feline (f)CD4+ cells and an inversion in the CD4:CD8 ratio. Peak production of IL-1, IL-6 and TNF coincided with periods of depressed immune responses. Additionally, immunodeficient responses and elevated levels of proinflammatory cytokines were concurrent with the presence of clinical signs. We conclude that, like human immunodeficiency virus (HIV), FIV infection results in significant perturbation of the immune response. Responses to PWM appear to correlate with disease progression which suggests that the CD3 pathway is affected in the earlier stages of the disease and that additional activation pathways such as CD2 may not be affected until the animal enters the acquired immune deficient syndrome (AIDS) stage of the disease.     

Kinetics of replication of a partially attenuated virus and of the challenge virus during a three-year intersubtype feline immunodeficiency virus superinfection experiment in cats

The effects of preinfecting cats with a partially attenuated feline immunodeficiency virus (FIV) on subsequent infection with a fully virulent FIV belonging to a different subtype were investigated. Eight specific-pathogen-free cats were preinfected with graded doses of a long-term in vitro-cultured cell-free preparation of FIV Petaluma (FIV-P, subtype A). FIV-P established a low-grade or a silent infection in the inoculated animals. Seven months later, the eight preinfected cats and two uninfected cats were challenged with in vivo-grown FIV-M2 (subtype B) and periodically monitored for immunological and virological status. FIV-P-preinfected cats were not protected from acute infection by FIV-M2, and the sustained replication of this virus was accompanied by a reduction of FIV-P viral loads in the peripheral blood mononuclear cells and plasma. However, from 2 years postchallenge (p.c.) until 3 years p.c., when the experiment was terminated, preinfected cats exhibited reduced total viral burdens, and some also exhibited a diminished decline of circulating CD4(+) T lymphocytes relative to control cats infected with FIV-M2 alone. Interestingly, most of the virus detected in challenged cats at late times p.c. was of FIV-P origin, indicating that the preinfecting, attenuated virus had become largely predominant. By the end of follow-up, two challenged cats had no FIV-M2 detectable in the tissues examined. The possible mechanisms underlying the interplay between the two viral populations are discussed.     

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.     

Apoptosis enhanced by soluble factor produced in feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV)-infected cells have been shown to undergo apoptosis by treatment with tumor necrosis factor alpha (TNF-alpha). This study detected a soluble factor which enhanced the apoptosis induced by TNF-alpha treatment. The sensitivity to TNF-alpha in the induction of apoptosis in a feline fibroblastoid cell line (CRFK) cells was significantly enhanced when the culture supernatant of FIV-infected CRFK cells or plasma samples from FIV-infected cats was added to the culture. These findings suggested that FIV infection induces production of a soluble factor which enhances CRFK cells sensitivity to TNF-alpha induction of apoptosis both in vitro and in vivo. This factor may contribute to the loss of lymphocytes in cats infected with FIV.     

Effect of dual-subtype vaccine against feline immunodeficiency virus infection

Dual-subtype feline immunodeficiency virus (FIV) vaccine, consisting of inactivated cells infected with subtypes A (Petaluma strain) and D (Shizuoka strain), was developed and tested for its vaccine efficacy against FIV infection in specific pathogen free (SPF) cats. Animals were monitored for proviral DNA by FIV-specific PCR and for FIV-specific antibody profiles by ELISA and virus-neutralization assays. In addition, blood from challenged cats was inoculated into naive SPF cats to confirm the viral status of the vaccinated cats. All cats immunized with Petaluma vaccine alone were protected against homologous Petaluma challenge, but only one of four cats was protected against heterologous Shizuoka challenge. More importantly, all cats immunized with the dual-subtype vaccine were protected against both Petaluma and Shizuoka challenges. These results suggest that a multi-subtype vaccine approach may provide the broad-spectrum immunity necessary for vaccine protection against strains from different subtypes.     

FIV vaccine studies. II. Clinical findings, hematological changes and kinetics of blood lymphocyte subsets

Five groups of cats were vaccinated with different recombinant feline immunodeficiency virus (FIV) SU vaccines expressed either in Escherichia coli or in the Baculovirus system. In Part I of this series, we described the humoral immune response and outcome of intraperitoneal FIV challenge exposure. Additionally, all cats were monitored for clinical and hematological changes and the course of blood lymphocyte subsets. These results are described in this present paper. A great increase of antibodies was found after vaccination with different recombinant FIV antigens, which did not protect the cats from intraperitoneal FIV challenge infection. This observation was paralleled by an increase of eosinophils during vaccination which was even more pronounced after challenge infection. After FIV challenge, infection lymphadenopathy, gingivitis, pharyngitis, changes in total leukocytes and neutrophils and a decrease in the CD4+:CD8+ ratio were found in cats of all groups and were considered as a sign of the FIV infection taking place, independent of vaccination. The following observations suggest that in these cats a TH2-like immune response was elicited: the high counts of eosinophils, the nature of antigen and adjuvant (aluminium hydroxide) and the high amounts of antigens used for immunization. Clearly, this type of immune response did not protect the animals from intraperitoneal FIV challenge infection.     

Point-of-care (POC) measurement of coagulation after cardiac surgery

Infection of feline immunodeficiency virus (FIV) has been shown to induce apoptosis that might be associated with the lymphocyte depletion in the infected cats. To investigate the inhibitory effect of antioxidants on FIV-induced apoptosis, we examined the effect of N-acetylcysteine (NAC) and ascorbic acid (AA) on apoptosis and virus replication in feline lymphoblastoid (Fel-039) and fibroblastoid (CRFK) cell lines infected with FIV. The treatment with NAC or AA induced a significant inhibition of viral replication and apoptosis in Fel-039 cells and tumor necrosis factor alpha (TNF-alpha)-treated CRFK cells infected with FIV. Both cell lines in the presence of noncytotoxic concentrations of NAC or AA showed in increase of intracellular glutathione (GSH) level, which might protect the cells against oxidative stresses exerted by FIV infection and TNF-alpha treatment. On the basis of these in vitro results, we suggest that antioxidant therapies aimed at restoring depleted GSH level might be effective for inhibition of viral replication and cell death associated with the development of immunodeficiency.     

Cats are protected against feline immunodeficiency virus infection following vaccination with a homologous AP-1 binding site-deleted mutant

Following establishment, via the vaginal route, of infection with an AP-1 binding-site deleted mutant (delta AP-1) of feline immunodeficiency virus (FIV), cats were challenged with a homologous intact strain (TM2) of FIV. The cats were observed for 23 weeks to evaluate the efficacy of the delta AP-1 against the homologous TM2 strain challenge. These two viruses were differentiated by Southern blotting after amplification of proviral DNA by semi-nested polymerase chain reaction in DNAs of peripheral blood mononuclear cells and tissues. A TM2-specific band was detected in one cat exposed to but not infected with delta AP-1, but not in two delta AP-1-infected. These results indicate that delta AP-1 could protect against subsequent challenge with homologous FIV TM2 strain.     

Prevalence of Dirofilaria immitis infection in cats in Saitama, Japan

To clarify Dirofilaria immitis infection among cats in Saitama Prefecture, Japan, 1,840 cats were examined postmortem for adult worms and microfilariae in the blood from 1989 to 1995. As a reference control, 500 dogs from the same area were examined in the same way and period. D. immitis worms were found in 15 cats, one of which had microfilariae in the blood. Prevalence rate of D. immitis infection was 0.8% (15/1,840) in cats and 46.8% (234/500) in dogs examined, whereas it was 4.1% and 64.6% in cats and dogs, respectively, aged 2 years and over. Worm burden per positive cat was 1.5 +/- 0.7 (mean +/- SD), the maximum number of worm was 3 in 2 cats, and 10 cats had a single worm each. All the worm-positive cats were tested for antibodies to feline immunodeficiency virus (FIV) and antigens of feline leukemia virus (FeLV) in sera. Positive rates of coinfection with D. immitis were 26.7% and 13.3% for FIV and FeLV, respectively.     

Feline immunodeficiency virus associated myopathy in the adult cat

Human immunodeficiency virus (HIV-1) associated myopathy can be a debilitating disease in humans, leading to weakness, myalgia, and muscle wasting. Subclinical neuromuscular involvement is also common. A range of histologic lesions have been described in both forms that include both inflammatory and degenerative changes. The purpose of this study was to determine whether a myopathy was present in adult cats experimentally infected with feline immunodeficiency virus (FIV). Six specific pathogen-free, laboratory-housed cats were challenged intravenously with 1000 TCID50 of the Maryland isolate of FIV (FIV-MD) at 8 months of age. The highest serum creatine kinase values were seen at 18 months postinfection (mean 9838, SD 4805 U/L) compared to preinfection (mean 950, SD 374 U/L). Needle EMG studies revealed abnormal spontaneous activity in 2 cats. All FIV-MD infected cats exhibited at least one abnormality in muscle pathology. Of the 24 muscle samples, 15 (63%) had histopathologic lesions. The predominant histologic abnormalities consisted of perivascular and pericapillary lymphocytic infiltration, and myofiber necrosis, phagocytosis, and regeneration. Lymphocytic infiltration was graded 2+ or higher in 12 of 24 muscle samples (0 = negligible; 4+ = extensive). Immunohistochemical phenotypic lymphocyte labeling in all cats demonstrated only CD8+ lymphocyte staining. This report demonstrates the presence of a FIV associated inflammatory myopathy in the adult cat. Several similarities are apparent in comparison to HIV-1 associated polymyositis reported in humans. Future studies in the cat may thus prove useful in elucidating the pathogenesis of retrovirus related myopathy in humans.     

Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture and ripening of Manchego cheese

Antiviral activity of the acyclic nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) against feline immunodeficiency virus (FIV) was investigated in field cats. The study was designed as a placebo-controlled double-blind study enrolling 27 FIV infected cats. Nine cats received PMEA at a dosage of 10 mg/kg body weight, nine cats received FPMPA at a dosage of 25 mg/kg body weight. A variety of parameters were established to evaluate the therapeutic efficacy of the compounds. The improvement was monitored by different aspects: clinical status, index of Karnofsky modified for the cat, laboratory parameters, immunological parameters, surrogate markers, and a virological parameter. Concerning the clinical and immunological parameters cats of both treatment groups disclosed a relevant improvement. Efficacy of antiviral treatment was a little bit higher in the cats treated with PMEA than in the animals injected with FPMPA. However, side effects were also more prominent in the PMEA treated group.     

Seroprevalence of Bartonella henselae and Toxoplasma gondii infections among pet cats in Kanagawa and Saitama Prefectures

Seroprevalence of Bartonella henselae and Toxoplasma gondii was investigated among 471 pet cats obtained from seven private animal hospitals in Kanagawa and Saitama Prefectures during the period from May 1994 to June 1995. 'Furthermore, 67 randomly selected from the 471 serum samples were examined for the feline immunodeficiency virus (FIV) antibody and feline leukemia virus (FeLV) antigen. The antibody to B. henselae was examined by an indirect immunofluorescent antibody test. T. gondii, FIV and FeLV infections in cats were detected with respective commercial kits. Of the cat serum samples tested, 43 (9.1%) were found to be seropositive for B. henselae and 41 (8.7%) for T. gondii. The B. henselae-positive rate (12.9%) of male cats was significantly higher than that (5.2%) of female cats. On the other hand, T. gondii-positive rate was 9.1% in male and 8.7% in female cats and there was no significant difference in the positivity between sexes. The positive rate in each hospital varied from 0 to 19.5% for B. henselae and 4.9 to 18.8% for T. gondii. The ages of B. henselae- and T. gondii-positive cats were distributed from < 1-year-old to 14-year-old and the seropositivity increased with age of cats. Of the 67 cat serum samples, 16 and 6 cases were positive for FIV and FeLV, respectively. There was no relationship between these viral and B. henselae infections in cats.     

Feline immunodeficiency virus infection--causative agent of an acquired immunodeficiency syndrome in cats

An ideal animal model for human immunodeficiency virus (HIV) infection in man should be caused by a genetically similar virus, it should cause as nearly identical clinical signs as possible, the pathogenesis of the resulting disease should be similar, and the model should be easily accessible to all researchers. Feline immunodeficiency virus (FIV) meets all these criteria. FIV is particularly useful as an animal model for investigation of the pathogenesis of lentivirus infections like cofactor studies, as well as for vaccine strategies, and drug testing in vitro and in vivo.