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.     

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.     

Gastrointestinal T lymphocytes retain high potential for cytokine responses but have severe CD4(+) T-cell depletion at all stages of simian immunodeficiency virus infection compared to peripheral lymphocytes

Gastrointestinal complications in human immunodeficiency virus (HIV) infection are indicative of impaired intestinal mucosal immune system. We used simian immunodeficiency virus (SIV)-infected rhesus macaques as an animal model for HIV to determine pathogenic effects of SIV on intestinal T lymphocytes. Intestinal CD4(+) T-cell depletion and the potential for cytokine responses were examined during SIV infection and compared with results for lymphocytes from lymph nodes and blood. Flow cytometric analysis demonstrated severe depletion of CD4(+)CD8(-) single-positive T cells and CD4(+)CD8(+) double-positive T cells in intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) during primary SIV infection which persisted through the entire course of SIV infection. In contrast, CD4(+) T-cell depletion was gradual in peripheral lymph nodes and blood. Flow cytometric analysis of intracellular gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production following short-term mitogenic activation revealed that LPL retained same or higher capacity for IFN-gamma production in all stages of SIV infection compared to uninfected controls, whereas peripheral blood mononuclear cells displayed a gradual decline. The CD8(+) T cells were the major producers of IFN-gamma. There was no detectable change in the frequency of IL-4-producing cells in both LPL and peripheral blood mononuclear cells. Thus, severe depletion of CD4(+) LPL and IEL in primary SIV infection accompanied by altered cytokine responses may reflect altered T-cell homeostasis in intestinal mucosa. This could be a mechanism of SIV-associated enteropathy and viral pathogenesis. Dynamic changes in intestinal T lymphocytes were not adequately represented in peripheral lymph nodes or blood.     

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.     

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.     

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.     

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.     

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.     

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.     

DNA vaccination affords significant protection against feline immunodeficiency virus infection without inducing detectable antiviral antibodies

To test the potential of a multigene DNA vaccine against lentivirus infection, we generated a defective mutant provirus of feline immunodeficiency virus (FIV) with an in-frame deletion in pol (FIVDeltaRT). In a first experiment, FIVDeltaRT DNA was administered intramuscularly to 10 animals, half of which also received feline gamma interferon (IFN-gamma) DNA. The DNA was administered in four 100-microg doses at 0, 10, and 23 weeks. Immunization with FIVDeltaRT elicited cytotoxic T-cell (CTL) responses to FIV Gag and Env in the absence of a serological response. After challenge with homologous virus at week 26, all 10 of the control animals became seropositive and viremic but 4 of the 10 vaccinates remained seronegative and virus free. Furthermore, quantitative virus isolation and quantitative PCR analysis of viral DNA in peripheral blood mononuclear cells revealed significantly lower virus loads in the FIVDeltaRT vaccinates than in the controls. Immunization with FIVDeltaRT in conjunction with IFN-gamma gave the highest proportion of protected cats, with only two of five vaccinates showing evidence of infection following challenge. In a second experiment involving two groups (FIVDeltaRT plus IFN-gamma and IFN-gamma alone), the immunization schedule was reduced to 0, 4, and 8 weeks. Once again, CTL responses were seen prior to challenge in the absence of detectable antibodies. Two of five cats receiving the proviral DNA vaccine were protected against infection, with an overall reduction in virus load compared to the five infected controls. These findings demonstrate that DNA vaccination can elicit protection against lentivirus infection in the absence of a serological response and suggest the need to reconsider efficacy criteria for lentivirus vaccines.     

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.     

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.     

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.     

Feline immunodeficiency virus (FIV)-associated lymphoma: a potential role for immune dysfunction in tumourigenesis

To determine the potential role of immune dysfunction in feline immunodeficiency virus (FIV)-associated lymphomagenesis, we present the results of immunological monitoring during the chronic phase of experimental FIV infection in two cats which subsequently developed lymphoma. In one cat, C1, cell-mediated immunity was depressed throughout the monitoring period but particularly from 125-200 weeks post-infection (pi), when this cat demonstrated profoundly impaired lymphocyte blastogenesis and markedly increased interleukin-1 (IL-1) production compared to age-matched, uninfected control cats. Lymphocyte function in the other cat, C2, was preserved to a greater degree. Alterations in the levels of immunoglobulin isotypes M, A and G in CD4+-, CD8+- and CD21+-lymphocyte sub-sets were demonstrated in both cats. Southern blot analysis revealed the presence of integrated FIV-provirus in tumour DNA from C2 but not C1 indicating a possible direct role for the virus in the former case only. In this study we have characterised, for the first time, the FIV-induced immune dysfunction in cats which developed lymphoma, demonstrating potential indirect mechanisms of tumourigenesis.     

Feline immunodeficiency virus (FIV) infection leads to increased incidence of feline odontoclastic resorptive lesions (FORL)

Feline odontoclastic resorptive lesions (FORL), previously known as 'neck lesions,' are commonly known in domestic, but also in non-domestic cats. They are characterized by odontoclastic resorptive processes, which take place at the dental root and at the periodontium. Chronic inflammation of gingiva and periodontium is believed to be an important etiological factor in the development of FORL. In this context, various feline viruses have been discussed to play a relevant role in the pathogenesis of these lesions. The aim of this project was to determine in a blinded study the incidence of FORL in 10 cats which were infected for several years with feline immunodeficiency virus (FIV), but were otherwise free of feline viral infections (feline leukemia virus, feline calicivirus, feline herpesvirus, feline parvovirus, feline coronavirus, feline syncytium-forming virus). Nine age-matched controls were kept under identical conditions, but free of FIV. Subgingival resorptive lesions were found in six of 10 FIV-positive cats, but in three of nine controls only. FIV-positive cats had significantly more often gingivae with an increased tendency for bleeding upon probing than FIV-negative cats (p=0.0055), and they had slightly more often hyperplastic gingivae (p=0.0867). In conclusion, signs characteristic of FORL such as subgingival lesions, granulomatous or hyperplastic gingivae with a tendency for bleeding, were found significantly more often in FIV-positive cats than in the controls (p=0.0198). Therefore, it was concluded that FIV infection is an important factor for the occurrence of FORL, possibly through immune suppression or changes of the (sub)gingival micro-environment. However, non-infected control cats also showed some evidence of FORL in the absence of all tested viral infections. Therefore, factors other than viral infections must also play a role in the development of FORL in cats.     

Loss of CD4+ T cells in human immunodeficiency virus type 1-infected chimpanzees is associated with increased lymphocyte apoptosis

Supportive evidence that apoptosis contributes to loss of CD4+ lymphocytes in human immunodeficiency virus type 1 (HIV-1)-infected humans comes from an apparent lack of abnormal apoptosis in apathogenic lentivirus infections of nonhuman primates, including HIV-1 infection of chimpanzees. Two female chimpanzees were inoculated, one cervically and the other intravenously, with HIV-1 derived from the LAI/LAV-1b strain, which was isolated from a chimpanzee infected with the virus for 8 years. Within 6 weeks of infection, both recipient chimpanzees developed a progressive loss of CD4+ T cells which correlated with persistently high viral burdens and increased levels of CD4+ T-cell apoptosis both in vitro and in vivo. Lymph nodes from both animals also revealed evidence of immune hyperactivation. Intermediate levels of T-cell apoptosis in both peripheral blood and lymph nodes were seen in a third chimpanzee that had been infected with the LAI/LAV-1b strain for 9 years; this animal has maintained depressed CD4/CD8 T-cell ratios for the last 3 years. Similar analyses of cells from 4 uninfected animals and 10 other HIV-1-infected chimpanzees without loss of CD4+ cells revealed no difference in levels of apoptosis in these two control groups. These results demonstrate a correlation between immune hyperactivation, T-cell apoptosis, and chronic loss of CD4+ T cells in HIV-1-infected chimpanzees, providing additional evidence that apoptosis is an important factor in T-cell loss in AIDS. Furthermore, the results show that some HIV-1 strains are pathogenic for chimpanzees and that this species is not inherently resistant to HIV-1-induced disease.     

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.