Pathogenesis of oral FIV infection.

Feline immunodeficiency virus (FIV) is the feline analogue of human immunodeficiency virus (HIV) and features many hallmarks of HIV infection and pathogenesis, including the development of concurrent oral lesions. While HIV is typically transmitted via parenteral transmucosal contact, recent studies prove that oral transmission can occur, and that saliva from infected individuals contains significant amounts of HIV RNA and DNA. While it is accepted that FIV is primarily transmitted by biting, few studies have evaluated FIV oral infection kinetics and transmission mechanisms over the last 20 years. Modern quantitative analyses applied to natural FIV oral infection could significantly further our understanding of lentiviral oral disease and transmission. We therefore characterized FIV salivary viral kinetics and antibody secretions to more fully document oral viral pathogenesis. Our results demonstrate that: (i) saliva of FIV-infected cats contains infectious virus particles, FIV viral RNA at levels equivalent to circulation, and lower but significant amounts of FIV proviral DNA; (ii) the ratio of FIV RNA to DNA is significantly higher in saliva than in circulation; (iii) FIV viral load in oral lymphoid tissues (tonsil, lymph nodes) is significantly higher than mucosal tissues (buccal mucosa, salivary gland, tongue); (iv) salivary IgG antibodies increase significantly over time in FIV-infected cats, while salivary IgA levels remain static; and, (v) saliva from naïve Specific Pathogen Free cats inhibits FIV growth in vitro. Collectively, these results suggest that oral lymphoid tissues serve as a site for enhanced FIV replication, resulting in accumulation of FIV particles and FIV-infected cells in saliva. Failure to induce a virus-specific oral mucosal antibody response, and/or viral capability to overcome inhibitory components in saliva may perpetuate chronic oral cavity infection. Based upon these findings, we propose a model of oral FIV pathogenesis and suggest alternative diagnostic modalities and translational approaches to study oral HIV infection.

Emergence of CD134 cysteine-rich domain 2 (CRD2)-independent strains of feline immunodeficiency virus (FIV) is associated with disease progression in naturally infected cats.

BACKGROUND: Feline immunodeficiency virus (FIV) infection is mediated by sequential interactions with CD134 and CXCR4. Field strains of virus vary in their dependence on cysteine-rich domain 2 (CRD2) of CD134 for infection. FINDINGS: Here, we analyse the receptor usage of viral variants in the blood of 39 naturally infected cats, revealing that CRD2-dependent viral variants dominate in early infection, evolving towards CRD2-independence with disease progression. CONCLUSIONS: These findings are consistent with a shift in CRD2 of CD134 usage with disease progression.

Feline glycoprotein A repetitions predominant anchors transforming growth factor beta on the surface of activated CD4(+)CD25(+) regulatory T cells and mediates AIDS lentivirus-induced T cell immunodeficiency.

Using the feline immunodeficiency virus (FIV) model for AIDS-lentivirus infection, our laboratory has previously demonstrated that T regulatory (Treg) cell-mediated immune T and B cell dysfunction contributes to lentivirus persistence and chronic disease through membrane bound transforming growth factor beta (mTGFb). Studying Treg cells in the context of infection has been problematic as no inducible marker for activated Treg cells had been identified. However, recent reports in human Treg studies have described a novel protein, glycoprotein A repetitions predominant (GARP), as a unique marker of activated human Treg cells that anchors mTGFb. Herein we extend these studies to the feline Treg system, identifying feline GARP and demonstrating that human and feline GARP proteins are homologous in structure, expression pattern, and ability to form a complex with TGFb. We further demonstrate that GARP and TGFb form a complex on the surface of activated Treg cells and that these GARP(+)TGFb(+) Treg cells are highly efficient suppressor cells. Analysis of expression of this Treg activation marker in the FIV-AIDS model reveals an up-regulation of GARP expressing Treg cells during chronic FIV infection. We demonstrate that the GARP(+) Treg cells from FIV-infected cats suppress T helper cells in vivo and that blocking GARP or TGFb eliminates this suppression. These data suggest that GARP is expressed in complex with TGFb on the surface of activated Treg cells and plays an important role in TGFb(+) Treg-mediated T cell immune suppression during lentivirus infection.

Partial regulatory T cell depletion prior to acute feline immunodeficiency virus infection does not alter disease pathogenesis.

Feline immunodeficiency virus (FIV) infection in cats follows a disease course similar to HIV-1, including a short acute phase characterized by high viremia, and a prolonged asymptomatic phase characterized by low viremia and generalized immune dysfunction. CD4(+)CD25(hi)FoxP3(+) immunosuppressive regulatory T (Treg) cells have been implicated as a possible cause of immune dysfunction during FIV and HIV-1 infection, as they are capable of modulating virus-specific and inflammatory immune responses. Additionally, the immunosuppressive capacity of feline Treg cells has been shown to be increased during FIV infection. We have previously shown that transient in vivo Treg cell depletion during asymptomatic FIV infection reveals FIV-specific immune responses suppressed by Treg cells. In this study, we sought to determine the immunological influence of Treg cells during acute FIV infection. We asked whether Treg cell depletion prior to infection with the highly pathogenic molecular clone FIV-C36 in cats could alter FIV pathogenesis. We report here that partial Treg cell depletion prior to FIV infection does not significantly change provirus, viremia, or CD4(+) T cell levels in blood and lymphoid tissues during the acute phase of disease. The effects of anti-CD25 mAb treatment are truncated in cats acutely infected with FIV-C36 as compared to chronically infected cats or FIV-naïve cats, as Treg cell levels were heightened in all treatment groups included in the study within two weeks post-FIV infection. Our findings suggest that the influence of Treg cell suppression during FIV pathogenesis is most prominent after Treg cells are activated in the environment of established FIV infection.

Feline immunodeficiency virus neuropathogenesis: from cats to calcium.

Invasion of human immunodeficiency virus (HIV) into the central and peripheral nervous system produces a wide range of neurological symptoms, which continue to persist even with adequate therapeutic suppression of the systemic viremia. The development of therapies designed to prevent the neurological complications of HIV require a detailed understanding of the mechanisms of virus penetration into the nervous system, infection, and subsequent neuropathogenesis. These processes, however, are difficult to study in humans. The identification of animal lentiviruses similar to HIV has provided useful models of HIV infection that have greatly facilitated these efforts. This review summarizes contributions made from in vitro and in vivo studies on the infectious and pathological interactions of feline immunodeficiency virus (FIV) with the nervous system. In vivo studies on FIV have provided insights into the natural progression of CNS disease as well as the contribution of various risk factors. In vitro studies have contributed to our understanding of immune cell trafficking, CNS infection and neuropathogenesis. Together, these studies have made unique contributions to our understanding of (1) lentiviral interactions at the blood-cerebrospinal fluid (CSF) barrier within the choroid plexus, (2) early FIV invasion and pathogenesis in the brain, and (3) lentiviral effects on intracellular calcium deregulation and neuronal dysfunction. The ability to combine in vitro and in vivo studies on FIV offers enormous potential to explore neuropathogenic mechanisms and generate information necessary for the development of effective therapeutic interventions.

Prevalence of feline immunodeficiency virus infection in domesticated and feral cats in eastern Australia.

Serum samples from 340 pet cats presented to three inner city clinics in Sydney Australia, 68 feral cats from two separate colonies in Sydney, and 329 cattery-confined pedigree and domestic cats in eastern Australia, were collected over a 2-year period and tested for antibodies directed against feline immunodeficiency virus (FIV) using immunomigration (Agen FIV Rapid Immunomigration test) and enzyme-linked immunosorbent assay methods (Snap Combo feline leukaemia virus antigen/FIV antibody test kit, IDEXX Laboratories). Western blot analysis was performed on samples in which there was discrepancy between the results. Information regarding breed, age, gender, housing arrangement and health status were recorded for all pet and cattery-confined cats, while the estimated age and current physical condition were recorded for feral cats. The FIV prevalence in the two feral cat populations was 21% and 25%. The majority of FIV-positive cats were male (60-80%). The FIV prevalence in cattery-confined cats was nil. The prevalence of FIV in the pet cat sample population was 8% (27/340) with almost equal prevalence in 'healthy' (13/170) and 'systemically unwell' (14/170) cats. The age of FIV-positive pet cats ranged from 3 to 19 years; all FIV-positive cats were domestic shorthairs with outside access. The median age of FIV-positive pet cats (11 years) was significantly greater than the median age of FIV-negative pet cats (7.5 years: P<0.05). The prevalence of FIV infection in male pet cats (21/172; 12%) was three times that in female pet cats (6/168; 4%; P<0.05). With over 80% of this pet cat population given outside access and continued FIV infection present in the feral population, this study highlights the need to develop rapid, accurate and cost-effective diagnostic methods that are not subject to false positives created by concurrent vaccination against FIV. This is especially important in re-homing stray cats within animal shelters and monitoring the efficacy of the new vaccine, which has not been challenged against Australian strains. The absence of FIV within cattery-confined cats highlights the value in routine screening and indoor lifestyles. This study provides cogent baseline FIV prevalences in three cat subpopulations which can be used for appraising potential disease associations with FIV in Australia.

Expression of CXCR4 on feline peripheral blood mononuclear cells: effect of feline immunodeficiency virus infection.

CXCR4 expression on feline peripheral blood mononuclear cells (PBMC) was analyzed. While monocytes and B lymphocytes expressed CXCR4, no CXCR4 was detected on T lymphocytes, in stark contrast to the expression pattern on T lymphocytes from humans. In spite of the important role that CXCR4 plays in infection with feline immunodeficiency virus, expression on PBMC in vivo was unaffected by infection with either a primary or a cell culture-adapted virus strain.

Unique susceptibility of the fetal thymus to feline immunodeficiency virus infection: an animal model for HIV infection in utero.

PROBLEM: Human infants infected in utero with HIV develop thymus insufficiency and progress to AIDS sooner than infants infected peripartum. However, direct analysis of the thymus is difficult due to limited tissue access and variable timing of vertical transmission. METHOD OF STUDY: Fetal and neonatal cats were inoculated with feline immunodeficiency virus (FIV) at an equivalent infectious dose. The thymus, blood, and lymph nodes were harvested and compared at 23 and 46 days post-inoculation (p.i.) and also compared to sham-inoculated, age-matched controls. Lymphocyte phenotypes were analyzed by flow cytometry and virus burden was quantified in histologic sections and by virus isolation from plasma. RESULTS: Fetal cats inoculated with FIV had acute thymus atrophy at birth, which coincided with peak viremia. At 46 days p.i., thymus size and cell composition rebounded and supported increased productive infection. In contrast, neonatal cats inoculated with FIV developed chronic thymus atrophy and degeneration, which was associated with decreasing productive infection and low-level viremia. CONCLUSIONS: The fetal thymus is uniquely vulnerable to acute, transient depletion and high-level productive infection. The neonatal thymus is less vulnerable to acute changes, and responds through progressive atrophy and declining productive infection. Reduced immune competence, as reflected by the failure to control virus replication, may contribute to the accelerated progression of FIV and HIV infections in utero.

Cytokine response in multiple lymphoid tissues during the primary phase of feline immunodeficiency virus infection.

Type 1 and 2 cytokine mRNA responses were measured at various time periods and in various lymphoid compartments during the acute stage (first 4 months) of feline immunodeficiency virus (FIV) infection in laboratory cats. Cytokine responses were correlated with virus replication. Virus was detected in plasma and tissue from day 14 postinfection (p.i.) onward, peaked at 56 to 70 days, and declined greatly by 70 days. Virus replication was highest in the thymus, followed by spleen, mesenteric lymph nodes, and cervical lymph nodes. Baseline cytokine levels were highest in the mesenteric lymph nodes and lowest in the cervical lymph nodes. Cytokine upregulation after FIV infection was most dramatic in the cervical lymph nodes, with the greatest increase in interleukin-10 (IL-10) and gamma interferon (IFN-gamma). Cytokine transcription in the mesenteric lymph node increased above baseline by day 14 p.i. for IFN-gamma, IL-12p40, IL-4, and IL-10, while elevations in the spleen were mainly for IFN-gamma, IL-12p40 and IL-10. An increase in IFN-gamma, IL-10, and IL-12p40 occurred in the thymus at day 56 p.i., concomitant with the onset of thymitis. In general, type 2 cytokines (IL-4 and IL-10) were increased greater than 1 log over baseline, while the elevations in type 1 cytokines were less than 1 log. In the tissues tested, CD4(+) cells were the primary source of IL-2, IL-4, and IL-10. Both CD4(+) and CD8(+) cells produced IFN-gamma, while no cytokine mRNA was detected in B cells. These results demonstrate the presence of a heterogeneous cytokine response in lymphoid tissues during the primary stage of FIV infection. The nature and intensity of the response differed from one compartment to the other and, in the case of the thymus, also with inflammatory changes. Although limited in scope, the present study confirms the usefulness of the FIV infection model in studying early cytokine events that lead to the secondary subclinical carrier state typical of most lentivirus infections.

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.

Parameters of disease progression in long-term experimental feline retrovirus (feline immunodeficiency virus and feline leukemia virus) infections: hematology, clinical chemistry, and lymphocyte subsets.

After several years of latency, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) cause fatal disease in the cat. The aim of this study was to determine laboratory parameters characteristic of disease progression which would allow a better description of the asymptomatic phase and a better understanding of the pathogenesis of the two infections. Therefore, experimentally infected cats (FIV and/or FeLV positive) and control animals were observed over a period of 6.5 years under identical conditions. Blood samples were analyzed for the following: complete hematology, clinical chemistry, serum protein electrophoresis, and determination of CD4+ and CD8+ lymphocyte subsets. The following hematological and clinical chemistry parameters were markedly changed in the FIV-infected animals from month 9 onwards: glucose, serum protein, gamma globulins, sodium, urea, phosphorus, lipase, cholesterol, and triglyceride. In FeLV infection, the markedly changed parameters were mean corpuscular volume, mean corpuscular hemoglobin, aspartate aminotransferase, and urea. In contrast to reports of field studies, neither FIV-positive nor FeLV-positive animals developed persistent leukopenia, lymphopenia, or neutropenia. A significant decrease was found in the CD4+/CD8+ ratio in FIV-positive and FIV-FeLV-positive animals mainly due to loss of CD4+ lymphocytes. In FeLV-positive cats, both CD4+ and, to a lesser degree, CD8+ lymphocytes were decreased in long-term infection. The changes in FIV infection may reflect subclinical kidney dysfunction, changes in energy and lipid metabolism, and transient activation of the humoral immune response as described for human immunodeficiency virus (HIV) infections. The changes in FeLV infection may also reflect subclinical kidney dysfunction and, in addition, changes in erythrocyte and immune function of the animals. No severe clinical signs were observed in the FIV-positive cats, while FeLV had a severe influence on the life expectancy of persistently positive cats. In conclusion, several parameters of clinical chemistry and hematology were changed in FIV and FeLV infection. Monitoring of these parameters may prove useful for the evaluation of candidate FIV vaccines and antiretroviral drugs in cats. The many parallels between laboratory parameters in FIV and HIV infection further support the importance of FIV as a model for HIV.

Maintenance of high virus load even after seroconversion in newborn cats acutely infected with feline immunodeficiency virus.

The viral loads in adult and newborn cats have been compared following injection with feline CD4+ FeL-039 line cells acutely infected with feline immunodeficiency virus (FIV). The level of virus genome in peripheral blood mononuclear cells (PBMC) increased progressively despite seroconversion in the newborn cats, whereas the virus genome was apparently cleared after seroconversion in the adult cats. Immunohistochemical staining of thymus of the FIV-infected newborn cats showed clusters of viral antigen-positive cells. These results indicate that FIV infection of the newborn cat results in higher virus loads than infection of the adult cat. We discuss these findings in relation to FIV as a model system for studies of the infection of neonates with an immunosuppressive retrovirus.

Early events in lymph nodes during infection with SIV and FIV.

To elucidate the initial pathogenic events in lymphoid organs, the major reservoir of virus in HIV infection, follow-ups of viral load, pathological changes and target cells were performed in the rhesus macaque SIVmac251 model and in the cat FIV model. Lymph nodes (LN) obtained from animals sacrificed at early time points following experimental inoculation were analysed by in situ hybridization for virus load and by combined immunohistochemistry and in situ hybridization for virus cellular tropism. In the SIV model, the LN presented a high viral load at 7 days post inoculation (p.i.); at this stage, macrophages and T4 lymphocytes were identified as the target cells of the virus. A shift in the pattern of viral infection was observed at 2 weeks p.i., with a concentration of viral RNA in follicular dendritic cells (FDC) in the germinal centres of the developing lymphoid follicles. This FDC-associated virus persisted at high levels for 2 months p.i. in the FIV model, the number of infected cells detected in LN was very low compared with that found in the SIV model, and a similar role played by FDC was found.

Contribution of the feline Langerhans cell to the FIV model.

In order to identify an equivalent of Langerhans cells in cat stratified epithelia, we used a panel of monoclonal antibodies known to be reactive with membrane antigens present on human Langerhans cells. The labelling was carried out by immunoperoxidase staining, for examination by light microscope, and by immunogold labelling, for electron microscopy. Out of 18 antibodies tested, only one, MHM23 antibody, specific against CD18 antigen, presented reactivity with dendritic epithelial cells on either frozen sections, epidermal sheets or cell suspension cytospins. On the ultrastructural level, these clear, dendritic, CD18+ cells showed "zipper-like" shapes similar to Birbeck granules, which are characteristic ultrastructural markers of Langerhans cells. This observation favours the hypothesis that these CD18+ cells in cat stratified epithelia are the equivalent of human Langerhans cells. These labelled cells were found in all epidermal locations and in the mucous membranes (oral, vaginal, rectal and oesophageal membranes). As feline immunodeficiency virus (FIV) transmission may occur through these membranes, the involvement of these feline Langerhans cells was studied in cats seropositive for FIV.

Early and progressive helper T-cell dysfunction in feline leukemia virus-induced immunodeficiency.

Cats infected with the highly pathogenic feline leukemia virus isolate FeLV-FAIDS develop an immunodeficiency syndrome characterized by progressive loss of CD4+ T cells and eventual pan-lymphocyte depletion. Prior to the decline in circulating CD4+ cells, infected cats are unable to mount primary antibody responses to T-dependent antigens. We investigated whether the altered ability of helper T cells to produce cytokines necessary for B cell growth and differentiation might be a primary event in the pathogenesis of FeLV-FAIDS infection. We found that as early as 9 weeks after infection, lymphocytes from cats with normal CD4+ cell numbers produced significantly lower levels of B-cell stimulatory factors. This deficit became progressively more severe with time. By contrast, resting B cells isolated from infected cats did not differ from controls in the ability to undergo activation and differentiation to antibody-secreting cells. The results suggest that a progressive defect in Th function occurs prior to CD4+ T-cell depletion early in the course of FeLV-FAIDS induced immunodeficiency.