Dominance of highly divergent feline leukemia virus A progeny variants in a cat with recurrent viremia and fatal lymphoma

ABSTRACT: BACKGROUND: In a cat that had ostensibly recovered from feline leukemia virus (FeLV) infection, we observed the reappearance of the virus and the development of fatal lymphoma 8.5 years after the initial experimental exposure to FeLV-A/Glasgow-1. The goals of the present study were to investigate this FeLV reoccurrence and molecularly characterize the progeny viruses. RESULTS: The FeLV reoccurrence was detected by the presence of FeLV antigen and RNA in the blood and saliva. The cat was feline immunodeficiency virus positive and showed CD4+ T-cell depletion, severe leukopenia, anemia and a multicentric monoclonal B-cell lymphoma. FeLV-A, but not -B or -C, was detectable. Sequencing of the envelope gene revealed three FeLV variants that were highly divergent from the virus that was originally inoculated (89-91% identity to FeLV-A/Glasgow-1). In the long terminal repeat 31 point mutations, some previously described in cats with lymphomas, were detected. The FeLV variant tissue provirus and viral RNA loads were significantly higher than the FeLV-A/Glasgow-1 loads. Moreover, the variant loads were significantly higher in lymphoma positive compared to lymphoma negative tissues. An increase in the variant provirus blood load was observed at the time of FeLV reoccurrence. CONCLUSIONS: Our results demonstrate that ostensibly recovered FeLV provirus-positive cats may act as a source of infection following FeLV reactivation. The virus variants that had largely replaced the inoculation strain had unusually heavily mutated envelopes. The mutations may have led to increased viral fitness and/or changed the mutagenic characteristics of the virus

Foodborne Transmission of Bovine Spongiform Encephalopathy to Nonhuman Primates

Risk for human exposure to bovine spongiform encephalopathy (BSE)–inducing agent was estimated in a nonhuman primate model. To determine attack rates, incubation times, and molecular signatures, we orally exposed 18 macaques to 1 high dose of brain material from cattle with BSE. Several macaques were euthanized at regular intervals starting at 1 year postinoculation, and others were observed until clinical signs developed. Among those who received ≥5 g BSE-inducing agent, attack rates were 100% and prions could be detected in peripheral tissues from 1 year postinoculation onward. The overall median incubation time was 4.6 years (3.7–5.3). However, for 3 macaques orally exposed on multiple occasions, incubation periods were at least 7–10 years. Before clinical signs were noted, we detected a non-type 2B signature, indicating the existence of atypical prion protein during the incubation period. This finding could affect diagnosis of variant Creutzfeldt-Jakob disease in humans and might be relevant for retrospective studies of positive tonsillectomy or appendectomy specimens because time of infection is unknown

Female cats have lower rates of apoptosis in peripheral blood lymphocytes than male cats: correlation with estradiol-17beta, but not with progesterone blood levels

During earlier study, we quantified by flow cytometry the rate of apoptotic feline lymphocytes after overnight culture. We found evidence that the sex of the animals influences the rate of apoptosis, intact females showed lower rates of apoptosis in lymphocytes cultured overnight than castrated male cats. This observation was also confirmed for cats that were previously experimentally infected with the feline immunodeficiency virus (FIV). In an attempt to find an explanation for these sexually determined differences, plasma estradiol-17beta and progesterone levels were measured by radio-immuno assay in the blood of these cats. The hormone levels were analyzed with respect to the rate of lymphocyte apoptosis. As expected, castrated males had lower blood levels of estradiol and progesterone than females. However, no overall correlation was found between hormone blood levels and rate of apoptosis under non-stimulating conditions. Interestingly, the rate of apoptosis found in lymphocytes collected from females and stimulated overnight in phytohaemaglutinin-containing medium, showed a strong negative correlation with the estradiol levels in the blood of these cats. To our knowledge, this is the first confirmation that estradiol in physiological concentrations may protect peripheral lymphocytes from apoptosis after stimulation. No correlation was found in male cats. In conclusion, these observations broaden the list of sexually determined differences of the immune system, sex and sex hormones predispose males and females for certain immune responses and dysfunctions. The present observations have to be taken into account when designing or interpreting experiments on apoptosis and, for example, evaluating the influence of a preexisting FIV infection on the rate of apoptosis. It would be highly advisable to include only spayed cats in studies on the immune system so as to minimize the influence of sex hormones

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

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

Recombinant FeLV vaccine: long-term protection and effect on course and outcome of FIV infection

The efficacy and the long-term protection of a recombinant feline leukemia virus (FeLV) vaccine were determined in 30 specified pathogen free cats for over 3 years. At the same time, in order to specify the effects of feline immunodeficiency virus (FIV) on the immune system, one half of the cats (n = 15) were previously infected with the Swiss isolate FIV Zurich 2. The second half of the animals (n = 15) served as non-infected controls. Eighteen (nine FIV-negative, nine FIV-positive) vaccinated and 12 (six FIV-negative, six FIV-positive) non-vaccinated cats were intraperitoneally challenged with FeLV A. Seventeen of 18 vaccinated cats were protected against persistent viremia, while ten of 12 non-vaccinated controls became infected. An increase of antibodies against FeLV SU was found in all protected cats after the challenge exposure. No difference in vaccine efficacy was found between FIV-negative and FIV-positive animals. The whole group of cats was observed for over 3 years. There were no further vaccinations during this period. CD4+ and CD8+ cell subsets, clinical outcome and time of survival of the cats were recorded. FIV-negative and FIV-positive animals were kept in two different rooms. However, FeLV-negative and FeLV viremic cats were housed together in both rooms in order to imitate a natural FeLV exposure situation. Anti-recombinant FeLV SU antibodies were measured by enzyme-linked immunosorbent assay. Although a continuous decline of antibodies was found in FeLV vaccinated cats, they remained protected against constant FeLV challenge for over 3 years. FIV infection had a stronger effect on the depression of the CD4+:CD8+ ratio than FeLV infection. Within the group of FIV-positive cats, the FeLV-vaccinated animals had significantly better survival rates as well as better clinical and laboratory parameters. FIV- and FeLV-coinfected cats showed the lowest CD4+:CD8+ ratio, mainly caused by decreased CD4+ lymphocyte counts. CD8+ lymphocytes with strong fluorescence (CD8(high)) disappeared and cells with weak fluorescence (CD8(low)) appeared instead. Prevention of coinfection by immunizing FIV-positive cats against FeLV infection improved the clinical outcome and prolonged the cat's life expectancy

Vaccination with feline immunodeficiency virus induces CD4 epitope masking by soluble factors

Soluble factors are important effector mechanisms to control for lentiviral replication. Vaccination of cats with recombinant outer surface proteins (SU) of the FIV envelope protein in combination with complete Freund adjuvant (CFA) and rabies nucleocapsid (NC) protein led to significantly reduced viral loads [Leutenegger, C.M., Hofmann-Lehmann, R., Holznagel, E., Cuisinier, A.M., Wolfensberger, C., Duquesne, V., Cronier, J., Allenspach, K., Aubert, A., Ossent, P. , Lutz, H., 1998. AIDS Res. Hum. Retroviruses, 14(3) 275-283]. Lymphocytes from vaccinated and non-vaccinated cats were stained with two monoclonal antibodies, Fel7 and CAT30A, directed against the feline CD4 antigen. Peripheral blood lymphocytes from cats vaccinated with the SU glycoprotein, CFA and rabies NC protein showed a significantly reduced number of cells after staining with CAT30A, while the number in Fel7 positive lymphocytes remained unchanged. This decreased CAT30A fluorescent staining could be reproduced in vitro by pre-incubating FIV-negative lymphocytes with immune sera from cats in which reduced CAT30A staining was detected. Neither experimental infection nor vaccination with the unglycosylated SU protein alone resulted in this epitope masking. Furthermore, this masking phenomenon was negatively correlated with a decreased susceptibility to activation-induced cell death (AICD). These findings will be discussed based on the current knowledge of CD8(+) T-cell antiviral factors and their involvement in lentiviral infection and/or replication

Foodborne Transmission of Bovine Spongiform Encephalopathy to Nonhuman Primates

Risk for human exposure to bovine spongiform encephalopathy (BSE)–inducing agent was estimated in a nonhuman primate model. To determine attack rates, incubation times, and molecular signatures, we orally exposed 18 macaques to 1 high dose of brain material from cattle with BSE. Several macaques were euthanized at regular intervals starting at 1 year postinoculation, and others were observed until clinical signs developed. Among those who received ≥5 g BSE-inducing agent, attack rates were 100% and prions could be detected in peripheral tissues from 1 year postinoculation onward. The overall median incubation time was 4.6 years (3.7–5.3). However, for 3 macaques orally exposed on multiple occasions, incubation periods were at least 7–10 years. Before clinical signs were noted, we detected a non-type 2B signature, indicating the existence of atypical prion protein during the incubation period. This finding could affect diagnosis of variant Creutzfeldt-Jakob disease in humans and might be relevant for retrospective studies of positive tonsillectomy or appendectomy specimens because time of infection is unknown