Real-time PCR investigation of feline leukemia virus proviral and viral RNA loads in leukocyte subsets

Cats exposed to feline leukemia virus (FeLV), a naturally occurring gammaretrovirus develop either progressive or regressive infection. Recent studies using analyses with enhanced sensitivity have correlated loads throughout FeLV with the clinical outcome, though remarkably, during the acute phase of infection, proviral and viral RNA burdens in the peripheral blood do not differ between groups. We hypothesized that viral loads in specific leukocyte subsets influence the infection outcome. Using a method established to determine the proviral and cell-associated viral RNA loads in specific leukocyte subsets, we evaluated viral loads in eleven FeLV-exposed specific pathogen-free (SPF) cats 2.5 years post-infection. Six cats had undergone regressive infection whereas five were persistently viremic. Aviremic cats had lower total proviral blood loads than the persistently infected cats and FeLV proviral DNA was shown to be integrated into genomic DNA in four out of four animals. Lymphocytes were predominantly infected vs. moncytes and granulocytes in aviremic cats. In contrast, persistently viremic cats were provirus-positive in all leukocyte subsets. The acute phase kinetics of FeLV infection were analyzed in two additional cats; an early lymphoreticular phase with productive infection in lymphocytes in both cats and in monocytes in one cat was followed by infection of the granulocytes; both cats became persistently infected. These results indicate that FeLV persistent viremia is associated with secondary viremia of bone marrow origin, whereas regressive cats only sustain a non-productive infection in low numbers of lymphocytes.     

Immunogenicity and Efficacy of a Commercial Feline Leukemia Virus Vaccine

Twenty young adult specific pathogen-free cats were randomly divided into two groups of 10 animals each. One group was vaccinated with two doses of feline leukemia virus vaccine according to the manufacturer's recommendations. All 20 cats were challenge exposed oronasally (4 times over a 1-week period), beginning 3 weeks after immunization, with a virulent subgroup A strain of FeLV (CT600-FeLV). The severity of the FeLV infection was enhanced by treating the cats with methylprednisolone acetate at the time of the last FeLV exposure. Ten of 10nonvaccinated cats became persistently viremic compared with 0/10 of the vaccinates. ELISA antibodies to whole FeLV were present at high concentrations after immunization in all of the vaccinated cats, and there was no observable anamnestic antibody response after challenge exposure. ELISA antibodies to whole FeLV appeared at low concentrations in the serum of nonvaccinated cats after infection but disappeared as the viremia became permanently established. Virus neutralizing antibodies were detected in 3/10 vaccinates and 0/10 nonvaccinates immediately before FeLV challenge exposure, and in 8/10 vaccinates and 1/10 nonvaccinates 5 weeks later. Although vaccination did not consistently evoke virus neutralizing antibodies, it appeared to immunologically prime cats for a virus-neutralizing antibody response after infection. Active FeLV infection was detected in bone marrow cells taken 14 weeks after infection from 10/10 nonvaccinates and 0/10 vaccinates. Latent FeLV infection was not detected in bone marrow cells from any of the vaccinated cats 14 weeks after challenge exposure.     

Safety and efficacy studies of live- and killed-feline leukemia virus vaccines.

The safety and the efficacy of several feline leukemia virus (FeLV) vaccines for 16-week-old kittens were determined. Vaccines were derived from an FL74 lymphoblastoid cell line that has been in continuous tissue culture passage for about 4 years. The vaccines were made from living virus, formaldehyde-inactivated whole FL74 cells, and formaldehyde-inactivated whole virus. The efficacy of each produced vaccine was determined by challenge exposure of vaccinated cats with virulent FeLV. The two formaldehyde-inactivated vaccines were found to be safe for use in kittens. Neither vaccine produce a significant feline oncornavirus-associated cell membrane antigen or virus-neutralizing antibody response, nor did they prevent infection with virulent FeLV. The inactivated whole-virus vaccine, however, did substantially decrease the proportion of kittens infected with virulent FeLV that became persistently viremic. In contrast, the whole FL74 cell vaccine did not reduce the number of infected kittens that became persistently viremic. The live-virus vaccine was found to be both safe and efficacious. About a half of the kittens vaccinated with live virus had transient bone marrow infection that lasted from 2 to 4 weeks. Viral antigen was not detected in peripheral blood, and infective virus was not shed in saliva, urine, or feces during the period that the vaccinal virus could be recovered from the bone marrow. In addition, there was no horizontal spread of vaccinal virus from vaccinated to non-vaccinated cagemates. Within several weeks, vaccinated kittens demonstrated no clinical or hematologic abnormalities and had high serum levels of feline oncornavirus-associated cell membrane antigen and virus-neutralizing antibody. Kittens vaccinated with living FeLV were resistant to infection with virulent virus.     

Efficacy and safety field trials of a recombinant DNA vaccine against feline leukemia virus infection.

A new recombinant gp70 vaccine was found to be safe and effective for prevention of infection by FeLV. The vaccine incorporates a unique purified saponin adjuvant with the recombinant antigen. Serious systemic reactions were not observed during the efficacy trial. Local reactions were transient and mild. More than 2,000 doses were administered to a cross section of household cats in a field safety trial. Only 1 cat had hypersensitivity reaction, which resolved. Among veterinarians who used the vaccine and the cat owners, the vaccine was judged satisfactory and safe. After rigorous intraperitoneal challenge exposure without use of immunosuppressants, 100% of the controls in the efficacy trial became infected, 70% of which remained persistently infected with FeLV. Among vaccinates, 45% were never viremic and 40% cleared transient infection within 12 weeks after challenge exposure. Of the 20 vaccinated cats, 3 were persistently infected. Overall, 85% of cats vaccinated with this recombinant DNA FeLV vaccine resisted persistent FeLV infection after stringent challenge exposure, which translates to preventable fraction of 78.6%.     

Year two of follow-up evaluation of a randomized, blind field trial of a commercial feline leukemia virus vaccine.

A blind randomized field trial of a commercial FeLV vaccine was conducted. Cats on study were vaccinated with either a commercial FeLV vaccine or a placebo, then housed with FeLV-positive cats in a ratio of approximately 2 study cats to 1 infected cat (results of the first 12 months of the study have been reported). All surviving placebo-treated and FeLV-vaccinated cats were re-vaccinated 1 year after initial exposure to FeLV-infected cats. Exposure continued for an additional 12 months, and the viremia status of the cats was monitored by immunofluorescent antibody (IFA) and ELISA testing at 4-month intervals. During the second year of observation, 1 additional FeLV-vaccinated cat had positive results of 2 consecutive ELISA tests, but remained IFA negative. Classifying this cat as persistently viremic reduced the estimate of the preventable fraction, but did not alter the conclusions drawn earlier, viz, that vaccination appreciably reduces the number of cats that become persistently viremic after long-term natural exposure.     

Antibody response of kittens after vaccination followed by exposure to feline leukemia virus-infected cats.

Protein (western) blot analysis and virus-neutralization assay were used to evaluate the antibody response of specific-pathogen-free kittens to FeLV vaccination and followed by natural exposure. Several kittens had barely detectable reactions to specific FeLV antigens prior to vaccination or exposure. Correlation was not found between protection against persistent viremia and antibody response after vaccination as measured by western blot analysis or virus neutralization assay. A statistically significant (P less than 0.01) difference in the antibody response against p27 antigen after natural exposure to FeLV was observed between persistently viremic kittens and transiently viremic or aviremic kittens. Measurable (P less than 0.05) virus neutralizing antibody titer after FeLV exposure was found only in a small number of kittens that were protected against persistent viremia. Lack of association between humoral response and vaccination-induced protection against persistent FeLV infection suggests an important role for cell-mediated immunity in such protection.     

Transmission of feline leukaemia virus in the milk of a non-viraemic cat

The possibility of the transmission of feline leukaemia virus (FeLV) from latently infected cats was studied. Five female cats with latent infections were examined for evidence of transmission of the virus to their kittens. One of the cats infected members of four consecutive litters of kittens which subsequently became persistently viraemic and transmitted the virus to other susceptible kittens by contact. Shortly after birth its kittens were apparently FeLV-free since neither viral antigen nor infectious virus was detected in their blood and no virus was found in cell cultures made from aspirates of bone marrow. The kittens became viraemic from 45 days of age onwards at a time when their passively acquired colostral FeLV neutralising antibodies were no longer detectable. Transmission of the virus occurred via the milk since both FeLV antigen and infectious virus were found in milk samples taken six weeks after kittening and the virus was transmitted to a fostered kitten. Eleven weeks after the birth of the fourth litter the cat became viraemic. The intermittent presence of FeLV antigens detected by the Leukassay F test, but not infectious virus, in the plasma of this cat over the previous months and a low level of serum neutralising antibodies distinguished it from four other latently infected queens which did not transmit infection to their kittens. These factors may indicate a risk of milk transmission and reactivation of latent virus.     

Long-term impact on a closed household of pet cats of natural infection with feline coronavirus, feline leukaemia virus and feline immunodeficiency virus

A closed household of 26 cats in which feline coronavirus (FCoV), feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) were endemic was observed for 10 years. Each cat was seropositive for FCoV on at least one occasion and the infection was maintained by reinfection. After 10 years, three of six surviving cats were still seropositive. Only one cat, which was also infected with FIV, developed feline infectious peritonitis (FIP). Rising anti-FCoV antibody titres did not indicate that the cat would develop FIP. The FeLV infection was self-limiting because all seven of the initially viraemic cats died within five years and the remainder were immune. However, FeLV had the greatest impact on mortality. Nine cats were initially FIV-positive and six more cats became infected during the course of the study, without evidence of having been bitten. The FIV infection did not adversely affect the cats' life expectancy.     

Exposure of cats to low doses of FeLV: seroconversion as the sole parameter of infection

In felids, feline leukemia virus (FeLV) infection results in a variety of outcomes that range from abortive (virus readily eliminated and never detectable) to progressive infection (persistent viremia and viral shedding). Recently, a novel outcome was postulated for low FeLV infectious doses. Naïve cats exposed to faeces of persistently infected cats seroconverted, indicating infection, but remained negative for provirus and p27 antigen in blood. FeLV provirus was found in some tissues but not in the bone marrow, infection of which is usually considered a necessary stage for disease progression. To investigate the impact of low FeLV doses on young cats and to test the hypothesis that low dose exposure may lead to an unknown pathogenesis of infection without involvement of the bone marrow, 21 cats were infected oronasally with variable viral doses. Blood p27, proviral and viral loads were followed until week 20 post-infection. Tissue proviral loads were determined as well. The immune response was monitored by measuring FeLV whole virus and p45 antibodies; and feline oncornavirus-associated cell membrane antigen (FOCMA) assay. One cat showed regressive infection (transient antigenemia, persistent provirus-positivity, and seroconversion) with provirus only found in some organs at sacrifice. In 7 of the 20 remaining cats FOCMA assay positivity was the only sign of infection, while all other tests were negative. Overall, the results show that FeLV low dose exposure can result in seroconversion during a presumed abortive infection. Therefore, commonly used detection methods do not detect all FeLV-infected animals, possibly leading to an underestimation of the prevalence of infection.     

Vaccination of cats experimentally infected with feline immunodeficiency virus, using a recombinant feline leukemia virus vaccine.

A group of 15 cats experimentally infected with a Swiss isolate of feline immunodeficiency virus (FIV) and a group of 15 FIV-negative control cats were inoculated with an FeLV vaccine containing recombinant FeLV-envelope. High ELISA antibody titer developed after vaccination in FIV-positive and FIV-negative cats. Vaccinated and nonvaccinated controls were later challenge exposed by intraperitoneal administration of virulent FeLV subtype A (Glasgow). Although 12 of 12 nonvaccinated controls became infected with FeLV (10 persistently, 2 transiently), only 1 of 18 vaccinated (9 FIV positive, 9 FIV negative) cats had persistent and 2 of 18 had transient viremia. From these data and other observations, 2 conclusions were drawn: In the early phase of FIV infection, the immune system is not depressed appreciably, and therefore, cats may be successfully immunized; a recombinant FeLV vaccine was efficacious in protecting cats against intraperitoneal challenge exposure with FeLV.     

FeLV-induced immunosuppression through alterations in signal transduction: down regulation of protein kinase C

Activation of protein kinase C by a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was shown to stimulate the respiratory burst in normal cat neutrophils. Neutrophils from feline leukemia virus (FeLV)-exposed viremic and nonviremic cats had significant suppression of their respiratory burst when stimulated with TPA. The addition of whole ultraviolet light-inactivated FeLV and FeLV proteins to normal cat neutrophils produced no significant suppression of the respiratory burst. These data suggest two possible mechanisms for suppression. The first is partially due to viral alterations of the neutrophil as seen in viremic cats, but, because exogenously applied FeLV or FeLV proteins had no effect on the respiratory burst, an additional mechanism is present. The second mechanism may be caused by a latent FeLV infection residing in nonviremic cat bone marrows which alters their immune system, resulting in immunosuppression.     

Detection of transient and persistent feline leukaemia virus infections

A study was made of cats persistently or transiently viraemic with feline leukaemia virus (FeLV) following experimental oronasal infection. Cats of two ages were exposed to the virus. One group was infected when eight weeks old in the expectation that most of the cats would become persistently viraemic, and the second group when 16 weeks old, so that some would show signs of a transient infection and then recover. The periods following infection when virus was detectable in the blood and in the oropharynx were determined for each group. Three methods for detecting viraemia were compared: virus isolation, immunofluorescence on blood smears and an enzyme-linked immunosorbent assay (ELISA). There was good overall agreement among the three tests in detecting virus-positive cats. Virus was found sooner after infection by virus isolation than by the other methods, and virus appeared in the blood slightly sooner in cats which developed persistent viraemia than in transiently viraemic cats. Infectious FeLV was isolated from the oropharynx of all of the persistently viraemic cats, in most cases simultaneously with virus in the plasma. Virus was also isolated from the mouth of most transiently viraemic cats. Under field conditions such transient excretion of virus lasting only a few days would rarely be detected in a single sampling. This might explain how FeLV is maintained in free range urban cats in the absence of a large number of cats with persistent active FeLV infection. For routine diagnosis, immunofluorescence would appear to offer the best chance of differentiating transient and persistent infections by FeLV.     

Possible immunoenhancement of persistent viremia by feline leukemia virus envelope glycoprotein vaccines in challenge-exposure situations where whole inactivated virus vaccines were protective

Kittens immunized with purified native FeLV-gp70 or -gp85 envelope proteins developed ELISA, but not virus neutralizing, antibodies in their serum to both whole FeLV and FeLV-gp70. Kittens vaccinated with envelope proteins and infected with feline sarcoma virus (FeSV) developed smaller tumors than nonvaccinates, but a greater incidence of persistent retroviremia. Similarly, FeLV-gp70 and -gp85 vaccinated kittens were more apt to become persistently retroviremic following virulent FeLV challenge exposure than nonvaccinates. Kittens vaccinated with inactivated whole FeLV developed smaller tumors after FeSV inoculation and had a lower incidence of persistent retroviremia than nonvaccinates. The protective effect of inactivated whole FeLV vaccine against persistent retroviremia was also seen with FeLV challenge-exposed cats. Protection afforded by inactivated whole FeLV vaccine was not associated with virus neutralizing antibodies, although ELISA antibodies to both whole FeLV and FeLV-gp70 were induced by vaccination.     

Control of feline leukaemia virus

Feline leukaemia virus (FeLV) usually occurs in its natural species, the domestic cat. FeLV is also important to human individuals as a comparative model, as it may cause a variety of diseases, some malignant and some benign, such as immunosuppression, which bears a resemblance to AIDS (acquired immune deficiency syndrome) in man. FeLV is transmitted among cats by contagion. The main sources of infection are persistently infected carrier cats which continuously excrete virus. Dissemination of FeLV among cats may be prevented by identifying infected carrier cats and removing them from contact with non-infected cats. Removal programmes using indirect immunofluorescence antibody tests were applied successfully in The Netherlands. The proportion of FeLV-positive cats decreased from 9% in 1974 to approximately 3% in 1985 during such a programme. The results of a removal programme carried out in a catbreeders' society were even better: the incidence of cats positive for FeLV decreased from 11% in 1974 to less than 2% within 4 years. None of the cats tested in this society has been found to be positive for FeLV since 1984. Besides removal programmes, other methods of control, such as pre-exposure treatment, were developed to prevent the spread of FeLV. We attempted to protect kittens against oronasal infection with FeLV by treatment with virus-neutralizing (VN) monoclonal antibodies (MoAbs) directed against an epitope on the viral glycoprotein gp70. However, no protection was achieved. It is unlikely that the amount of VN antibodies, the mode and route of their application or the infectious dose of FeLV used can account for this failure. Other possible explanations for the lack of protective effect are that (i) the restricted epitope specificity of the MoAb preparation used may have led to selection of neutralization-resistant virus mutants, or (ii) other mechanisms than virus neutralization (complement-mediated lysis, antibody-dependent cell cytotoxicity), that may be involved in protection, function less efficiently with MoAb. However, in the light of our finding that an early anti-idiotypic response is observed in all cats following administration of the MoAb preparation, the rapid clearance of anti-FeLV MoAb from the circulation is a more likely explanation. Efforts were further made to develop a vaccine for controlling FeLV infection. The immunostimulating complex vaccine (FeLV-ISCOM vaccine), a subunit vaccine in which FeLV gp70 is presented in a particular manner, looks promising. The protective effect of FeLV-ISCOM vaccine was studied by vaccinating six 8-week-old SPF cats with ISCOM, followed by oronasal challenge with FeLV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Course of feline leukemia virus infection and its detection by enzyme-linked immunosorbent assay and monoclonal antibodies

Monoclonal antibodies specific for 3 distinct epitopes of the species-specific determinants of feline leukemia virus (FeLV) p27 were used in an enzyme-linked immunosorbent assay (ELISA) for measurement of serum p27 in cats infected with FeLV. Group-specific antigen (GSA) of FeLV in peripheral blood leukocytes was also determined by an immunofluorescence assay. Antibodies to FeLV and the feline oncornavirus-associated cell membrane antigen (FOCMA) were also measured. Thirty-six cats were surveyed and assigned to 4 categories. Five developed persistent viremia (category 1), characterized by continuous expression of p27, GSA, and low antibody titers to FeLV and FOCMA. Eleven cats with transient viremia (category 2) and 13 cats that were never detectably viremic (category 3), as judged by absence of GSA and p27, developed increased antibody titers to FeLV and FOCMA. Seven cats were never viremic, as judged by the GSA in the peripheral blood leukocytes, but still had detectable serum p27 (category 4). Most category 4 cats developed high antibody titers against FOCMA and/or FeLV. Of 307 field cats examined, 7% of the healthy cats and 10% of the sick cats could be assigned to category 4. However, this difference was not significant (P greater than or equal to 0.05). Of 26 cats with neoplasms 2 (1 of 12 with lymphosarcoma) could be classified as category 4. Because virus could be isolated from 2 category 4 cats, they were considered immune carriers.     

Comparative efficacy of three commercial feline leukemia virus vaccines against methylprednisolone acetate-augmented oronasal challenge exposure with virulent virus.

Three commercial FeLV vaccines, (A, B, and C) were purchased on the open market and administered to 8- to 20-week-old specific-pathogen-free kittens, according to manufacturers' instructions. A similar group of nonvaccinated kittens served as controls. All kittens were challenge-exposed oronasally with virulent FeLV 4 weeks after the final vaccination. Serum samples were monitored for FeLV-p27 antigenemia using an ELISA at 1- to 2-week intervals for at least 16 weeks after the last day of challenge exposure. Kittens that were either transiently (1 to 4 weeks) or never viremic during this period were counted as recovered, whereas kittens that became viremic and retained viremia for at least 10 weeks were counted as persistently viremic. The 3 vaccines were found to be 39% (vaccine C), 28% (vaccine B), and 17% (vaccine A) efficacious in preventing persistent viremia in immunized, compared with nonimmunized kittens.     

Evaluation of the effect of short-term treatment with the integrase inhibitor raltegravir (Isentress™) on the course of progressive feline leukemia virus infection

Cats persistently infected with the gammaretrovirus feline leukemia virus (FeLV) are at risk to die within months to years from FeLV-associated disease, such as immunosuppression, anemia or lymphoma/leukemia. The integrase inhibitor raltegravir has been demonstrated to reduce FeLV replication in vitro. The aim of the present study was to investigate raltegravir in vivo for its safety and efficacy to suppress FeLV replication. The safety was tested in three naïve specified pathogen-free (SPF) cats during a 15 weeks treatment period (initially 20 mg then 40 mg orally b.i.d.). No adverse effects were noted. The efficacy was tested in seven persistently FeLV-infected SPF cats attained from 18 cats experimentally exposed to FeLV-A/Glasgow-1. The seven cats were treated during nine weeks (40 mg then 80 mg b.i.d.). Raltegravir was well tolerated even at the higher dose. A significant decrease in plasma viral RNA loads (∼5×) was found; however, after treatment termination a rebound effect was observed. Only one cat developed anti-FeLV antibodies and viral RNA loads remained decreased after treatment termination. Of note, one of the untreated FeLV-A infected cats developed fatal FeLV-C associated anemia within 5 weeks of FeLV-A infection. Moreover, progressive FeLV infection was associated with significantly lower enFeLV loads prior to infection supporting that FeLV susceptibility may be related to the genetic background of the cat. Overall, our data demonstrate the ability of raltegravir to reduce viral replication also in vivo. However, no complete control of viremia was achieved. Further investigations are needed to find an optimized treatment against FeLV. (250 words)     

Evaluation of efficacy and safety of an inactivated virus vaccine against feline leukemia virus infection.

An inactivated virus vaccine was developed for prevention of FeLV infection in domestic cats. When given in 2 doses, 3 weeks apart, to cats that were greater than or equal to 9 weeks old at the time of first vaccination, the vaccine prevented persistent viremia from developing in 132 of 144 (92%) vaccinates after oronasal challenge exposure with virulent FeLV. In contrast, persistent viremia developed after oronasal challenge exposure with FeLV in 39 of 45 (87%) age-matched nonvaccinated control cats. Transient viremia, indicated by early detection of p27 by ELISA in serum of cats protected from persistent viremia at 12 weeks after challenge exposure, was found in 10 of 132 (8%) vaccinates. Cats that were aviremic 12 to 16 weeks after challenge exposure were examined for reactivation of latent FeLV infection; 4 weekly doses of methylprednisolone were administered, followed by in vitro culture of bone marrow cells. Latent infection was readily reactivated in 6 of 8 (75%) nonvaccinated control cats that had been transiently viremic after challenge exposure. However, latent infection was reactivated in only 5 of 48 (10%) protected vaccinates, and in none of 38 vaccinates in which transient viremia had not been detected. In a safety field trial, only 34 mild reactions of short duration were observed after administration of 2,379 doses of vaccine to cats of various ages, breeds, and vaccination history, for a 1.43% reaction rate. Results indicate that the aforementioned inactivated virus vaccine is safe and efficacious for the prevention of infection with FeLV.     

Seroepidemiologic survey of feline immunodeficiency virus infection in cats of Wake County, North Carolina

Feline immunodeficiency virus (FIV) antibodies were detected in 9 of 123 (7.3%) cats. More clinically ill cats had titers to FIV than did healthy cats (15% vs 3.6%). Previous or current illnesses in these FIV-positive cats included urinary bladder disease, anemia, cat-bite abscesses, bacterial infections, bleeding disorders, diabetes mellitus, and chronic respiratory tract disease. All FIV-positive cats were males, with mean age of 6.0 years (range, 1 to 11 years). Half (n = 3) of the clinically ill FIV-positive cats were concurrently seropositive for FeLV antigen. Three of the ill cats were euthanatized or died 1 month after initially testing, whereas the remaining 3 ill cats and the 3 healthy FIV-positive cats were healthy 1 year after initial testing. Antibody titer to FIV persisted in 4 of 5 cats, but serotest results were equivocal in 1 cat evaluated 1 year later.