Evaluation of an indirect enzyme-linked immunosorbent assay for the detection of feline lentivirus-reactive antibodies in wild felids, employing a puma lentivirus-derived synthetic peptide antigen.

An enzyme-linked immunosorbent assay (ELISA) using a puma lentivirus-derived synthetic peptide as coating antigen was evaluated as a diagnostic test for infection with feline immunodeficiency virus (FIV) or related lentiviruses in free-ranging lions. The sensitivity and specificity of the ELISA was determined using two approaches. In the first approach, the results were standardized according to certain statistical criteria, and in the second, the puma lentivirus western blot was used as the gold standard. The sensitivity of the test when compared with the standardized results was 85.4% and the specificity 100%. The sensitivity of the test when using the western blot as the gold standard was 78.6% and the specificity 100%. The test would therefore be well-suited to the screening of populations of wild felids in which FIV or related lentiviruses are endemic. The results also indicate that in spite of genetic divergence between lentiviruses isolated from Panthera and Felis spp., puma lentivirus-derived antigens can be used in immunoassays for the detection of antibodies in Panthera spp. reactive to FIV or related lentiviruses. The results also indicate that the lion population in the Hluhluwe-Umfolozi Game Reserve, South Africa is lentivirus negative.

Growth of lion and puma lentiviruses in domestic cat cells and comparisons with FIV.

Feline immunodeficiency virus (FIV-Fca) is a lentivirus that causes gradual immunological deterioration in domestic cats. Lentiviruses related to FIV have been detected in several nondomestic feline species; the biologic significance of these viruses remains to be defined. To examine the in vitro cell tropism of these nondomestic cat lentiviruses, prototypical puma and lion lentiviruses (FIV-Pco and FIV-Ple) were cultured in a variety of feline cell cultures. A domestic cat T lymphoma cell line, 3201, best supported the replication of both FIV-Pco and FIV-Ple. Moreover, FIV-Ple was lytic for these cells. RT-PCR amplification of a conserved pol gene region demonstrated species-specific primer homology. Sequence and phylogenetic analyses of this amplification product confirmed the identity of the replicating viruses and classified two previously uncharacterized viruses within predictable lion and puma clades. Sequence analysis of a conserved pol region demonstrated homology with previously characterized FIV-Ple and FIV-Pco. Western blot analysis using domestic cat anti-FIV-Fca sera showed that both FIV-Pco and FIV-Ple were antigenically related, to differing degrees, to three serotypes of FIV-Fca. These studies demonstrate that though nondomestic cat lentiviruses differ significantly from FIV-Fca and that a viral-specific protocol may be necessary for sensitive viral detection, these viruses can replicate in cells of domestic cats. suggesting the potential for cross-species transmission.

Development of the immunofluorescent antibody test for detection of feline leukemia virus infection in cats.

Studies of the immunodetection of various microorganisms by various assay systems indicated that the most specific and sensitive assays are immunofluorescence, radioimmunoassay, and immunoblot analysis (western blot), followed by sensitive but less specific ELISA and agglutination assays and, finally, by even less sensitive but very specific virus isolation and double immunodiffusion techniques. The first test for the clinical detection of FeLV infection in pet cats was the immunofluorescent antibody (IFA) test, which was introduced in 1972. The FeLV test is used for detection for FeLV infection and not as a test for leukemia or any other feline disease. The IFA test was compared with an immunodiffusion (ID) test and with tissue culture isolation (TCI) of the virus in 26 cats to establish a standard for FeLV tests. Excellent correlation was observed between the IFA and the ID tests (100%).

Ten-year study comparing enzyme-linked immunosorbent assay with the immunofluorescent antibody test for detection of feline leukemia virus infection in cats.

Immunodetection tests for feline retroviruses are powerful tools used in modern veterinary practice. Veterinarians must fully understand the characteristics--strengths and weaknesses--of the FeLV tests so that the information gained from them can be used properly. Any FeLV ELISA or immunofluorescent antibody (IFA) test is a method for detection of FeLV infection (the virus) and is not a diagnostic test for leukemia or other feline disease. From previous studies, it was determined that the most accurate test for detection of persistent FeLV infection is the IFA test, which detects FeLV antigens in cytoplasm of leukocytes in the blood of infected cats. In the study reported here, 1,142,600 FeLV IFA tests were performed between June 1972 and December 1990. During this period 19.8% of the IFA test results were positive and 78% were negative. Evaluation was not possible for the remaining 2.2% of the tests because of lack of enough leukocytes in the smears to evaluate, or nonspecific staining reactions. In 1979, 7 years after introduction of the IFA test, in-hospital FeLV ELISA were introduced, which enabled veterinarians to test for FeLV in their hospitals. Ever since that time, continual discrepancies have been reported between results of FeLV ELISA and IFA tests, particularly between positive ELISA results and their IFA test confirmation. A 10-year comparison was made between practitioner-performed in-hospital FeLV ELISA (n = 20, 240 tests) results and FeLV IFA test performed by a commercial laboratory. All samples tested by ELISA were submitted (for confirmation of results) by veterinarians from the United States, Canada, Europe, Japan, and Australia.(ABSTRACT TRUNCATED AT 250 WORDS)

A new acute transforming feline retrovirus with fms homology specifies a C-terminally truncated version of the c-fms protein that is different from SM-feline sarcoma virus v-fms protein.

The HZ5-feline sarcoma virus (FeSV) is a new acute transforming feline retrovirus which was isolated from a multicentric fibrosarcoma of a domestic cat. The HZ5-FeSV transforms fibroblasts in vitro and is replication defective. A biologically active integrated HZ5-FeSV provirus was molecularly cloned from cellular DNA of HZ5-FeSV-infected FRE-3A rat cells. The HZ5-FeSV has oncogene homology with the fms sequences of the SM-FeSV. The genome organization of the 8.6-kilobase HZ5-FeSV provirus is 5' delta gag-fms-delta pol-delta env 3'. The HZ5-and SM-FeSVs display indistinguishable in vitro transformation characteristics, and the structures of the gag-fms transforming genes in the two viruses are very similar. In the HZ5-FeSV and the SM-FeSV, identical c-fms and feline leukemia virus p10 sequences form the 5' gag-fms junction. With regard to v-fms the two viruses are homologous up to 11 amino acids before the C terminus of the SM-FeSV v-fms protein. In HZ5-FeSV a segment of 362 nucleotides then follows before the 3' recombination site with feline leukemia virus pol. The new 3' v-fms sequence encodes 27 amino acids before reaching a TGA termination signal. The relationship of this sequence with the recently characterized human c-fms sequence has been examined. The 3' HZ5-FeSV v-fms sequence is homologous with 3' c-fms sequences. A frameshift mutation (11-base-pair deletion) was found in the C-terminal fms coding sequence of the HZ5-FeSV. As a result, the HZ5-FeSV v-fms protein is predicted to be a C-terminally truncated version of c-fms. This frameshift mutation may determine the oncogenic properties of v-fms in the HZ5-FeSV.

A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family.

A new acute transforming feline retrovirus, the Hardy-Zuckerman 4 feline sarcoma virus (HZ4-FeSV), has been isolated from a feline fibrosarcoma. The viral genome of HZ4-FeSV contains a new oncogene designated v-kit, has the structure 5' delta gag-kit-delta pol-delta env 3' and specifies a gag-kit polyprotein of relative molecular mass 80,000. The predicted kit amino-acid sequence displays partial homology with tyrosine-specific protein kinase oncogenes. HZ4-FeSV appears to have been generated by transduction of feline c-kit sequences with feline leukaemia virus.

Immunological and biochemical characterization of HZ2 feline sarcoma virus and Abelson murine leukaemia virus translation products.

The extent of homology between the translation products of the HZ2 strain of feline sarcoma virus (HZ2-FeSV) and the Abelson murine leukaemia virus (A-MuLV) was examined immunologically and biochemically. Antiserum prepared against the v-abl-encoded determinants of the A-MuLV polyprotein P120gag-abl was also found to precipitate specifically the 98K mol. wt. HZ2-FeSV protein (P98gag-abl). The basis for this immunological crossreactivity was indicated by the findings that the two proteins had at least six [35S]methionine-containing tryptic peptides and at least eight [35S]methionine-containing chymotryptic peptides in common. Each of the two proteins also had tryptic and chymotryptic peptides which were unique. Both proteins were associated with tyrosyl kinase activities which exhibited some similar biochemical properties in vitro. However, the HZ2-FeSV-associated activity was much more sensitive to competitive inhibition by nucleoside and deoxynucleoside diphosphates than was the A-MuLV-associated activity. These results suggest that, while the gag-abl translation products of these two independent isolates of transforming retrovirus are highly related structurally and functionally, the differences in structure contribute to differences in enzyme activity. Further comparative studies of these two proteins should play an important role in determining their roles in induction of two different types of malignancy: lymphosarcoma in the case of the A-MuLV protein and fibrosarcoma in the case of the HZ2-FeSV protein.

Isolation of a new feline sarcoma virus (HZ1-FeSV): biochemical and immunological characterization of its translation product.

A new strain of feline sarcoma virus, designated HZ1-FeSV, was isolated from a 4-year-old domestic cat with multicentric fibrosarcoma. A primary tumor cell line was established and virus produced from that line was found to induce foci in feline embryonic lung fibroblasts (FLF3) and mink lung fibroblasts (CCL64) in tissue culture and fibrosarcomas in inoculated 10-week-old kittens. The derivation of transformed nonproducer clones of FLF3 and CCL64 cells containing helper virus-rescuable, focus-forming activity indicated that HZ1-FeSV was defective for replication. The only discernible translation product of the HZ1-FeSV genome in cultured cells was a 100,000-Da polyprotein (P100) which contained amino-terminal sequences of the FeLV gag gene precursor protein covalently linked to a sarcoma virus-specific domain. Immunoprecipitates containing P100 exhibited a protein kinase activity capable of phosphorylating tyrosine residues of P100. Immunologically, P100 was highly cross-reactive with gag-fes polyproteins encoded by two previously characterized strains of FeSV, the GA- and the ST-FeSV. By comparison of methionine-containing tryptic peptides, the HZ1-FeSV protein was shown to be more closely related to the GA-FeSV protein than to the ST-FeSV protein, but to be distinguishable from both other proteins.

The feline oncornavirus-associated cell membrane antigen (FOCMA) is related to, but distinguishable from, FeLV-C gp70.

The feline oncornavirus-associated cell membrane antigen (FOCMA) on the surface of feline lymphosarcoma (LSA) cells is defined as the target(s) recognized in immunofluorescence (IFA) tests by antibody in sera of cats relatively resistant to development of FeLV (feline leukemia virus) LSA and FeSV (feline sarcoma virus) fibrosarcoma. The specificities of antibodies in cat FOCMA-typing sera and the nature of the LSA antigens recognized were investigated in the present study. FOCMA sera obtained from viremic cats were separable into at least two classes : those which contained antibodies against the envelope glycoprotein (gp70) of subgroup C FeLV and those which did not contain antibodies against any subgroup of FeLV. The first class of sera could be further subdivided into three groups: those whose FOCMA reactivity could be completely absorbed, partially absorbed, or not absorbed by FeLV-C antigens. The second class of sera could be further subdivided into two groups: those whose FOCMA reactivity could be partially absorbed and those whose activity could not be absorbed by FeLV-C. The results indicate that the FOCMA reactivity exhibited by some viremic cat sera can be partially, if not entirely, attributed to antibodies not crossreactive with FeLV virion antigens. A consistent property of all FOCMA sera in this study is the ability to bind to 70-kDa proteins on the surface of LSA cells. Staphylococcus aureus V8 protease partial digest maps of 70-kDa proteins purified from 12 primary feline LSAs (five FeLV positive and seven FeLV negative) all showed 18-, 14-, and 10-kDa fragments. V8 maps of FeLV-C gp70 showed similarly sized fragments while the maps of the RD114, FeLV-A, and FeLV-B gp70s were distinct. However, in a subgroup-specific radioimmunoassay for FeLV-C gp70-related antigens, the LSA 70-kDa proteins were found to be serologically related to, but distinct from, FeLV-C gp70. The results on the antigenic variations among LSA 70-kDa proteins and the antibodies which bind them are entirely consistent with previous studies indicating heterogeneity among FOCMA determinants.

The Hardy-Zuckerman 2-FeSV, a new feline retrovirus with oncogene homology to Abelson-MuLV.

There is substantial evidence that oncogenes (v-onc) of acute transforming retroviruses have been acquired by transduction of cellular genes (c-onc) with retroviruses. Feline leukaemia virus (FeLV)-associated feline fibrosarcomas have proven to be extremely useful for the isolation of acute transforming retroviruses of a mammalian species. Three different v-onc genes have been identified in five acute transforming feline retroviruses. The Susan McDonough feline sarcoma virus (SMFeSV) contains the oncogene fms (ref. 4). The Snyder-Theilen (ST) and Gardner-Arnstein (GA) FeSVs contain the oncogene fes (ref. 4), which is homologous to the oncogene fps of the avian sarcoma viruses FSV, RRCII, PRCIV and 16L (refs 7, 8). The v-onc sequences of the Parodi-Irgens (PI) FeSV have recently been found to be homologous with the v-sis sequences of the simian sarcoma virus. We report here the isolation of another acute transforming feline retrovirus from a naturally occurring feline fibrosarcoma, designated the Hardy-Zuckerman 2 feline sarcoma virus (HZ2-FeSV) and demonstrate that the HZ2-FeSV and Abelson murine leukaemia virus (A-MuLV) have homologous oncogenes.

Development of virus non-producer lymphosarcomas in pet cats exposed to FeLv.

Naturally occurring oncoviruses of several species are transmitted contagiously and cause lymphosarcoma (LSA) or leukaemia in their hosts. All naturally occurring oncoviruses replicate in vivo in the tumours they induce or, as with bovine leukaemia virus, can be isolated from tumour cells grown in short-term cell culture. However, we have shown that feline leukemia virus (FeLV) is not present in a significant minority of pet cats that develop LSA. Unlike experimentally induced virus-negative leukaemias and sarcomas of other species, LSA cells from FeLV-negative LSA cats lack any FeLV proteins, including p15 or p12, and complete functional copies of FeLV provirus and thus do not produce FeLV when grown in cell culture. Thus, except for FeLV, the naturally occurring animal leukaemogenic oncoviruses seem to induce only virus-producing lymphoid tumours. Our earlier findings prompted a study to determine the frequency of occurrence of FeLV non-producer (NP) LSA in pet cats and whether NP LSAs develop in cats exposed to FeLV. We report here epidemiological data which indicate that development of NP LSAs in pet cats is associated with exposure to FeLV and suggest that FeLV may be the aetiological agent for FeLV NP feline LSAs. Thus, feline NP LSAs may be suitable for studying the potential viral aetiology and mechanism of leukaemogenesis of human lymphoid tumours in which no oncoviruses have, as yet, been proved to cause the disease.

The epidemiology of the feline leukemia virus (FeLV).

Clustering of cases of feline lymphosarcoma (LSA) has been observed by veterinarians for many years. In 1964 it was discovered that feline LSA was caused by an oncornavirus, the feline leukemia virus (FeLV). In 1970, a simple, indirect immunoflourescent antibody (IFA) test for FeLV was developed which enabled large numbers of cats, living in their natural (household) environments, to be tested for the virus. In one study, over 2,000 cats were tested and the results showed conclusively that FeLV is a contagious agent for cats. This finding was independently confirmed by several other investigators using different testing procedures. After discovering the contagious nature of FeLV a test and removal program was devised which successfully prevents the spread of FeLV and the development of FeLV diseases in the pet cat population. There is, at present, no evidence that FeLV infects humans living with FeLV infected cats.

Biology of feline leukemia virus in the natural environment.

The feline leukemia virus (FeLV) was discovered in 1964 in a cluster of cats with lymphosarcoma. The observed clustering of cases of feline lymphosarcoma suggested that FeLV was an infectious agent for cats. The development of a simple immunofluorescent test for FeLV permitted a seroepidemiological study to be undertaken on the distribution of the virus in cats living in their natural environment. Over 2000 cats were tested, and the results showed conclusively that FeLV is an infectious agent for cats. This finding has now been independently confirmed using three different test procedures. After the infectious nature of FeLV was discovered, a simple FeLV test and removal program was devised to control the spread of the virus in the natural environment. The spread of FeLV was controlled in 45 households by removing the FeLV-infected cats, while in 25 households, where the infected cats were left in contact with the uninfected cats, 12% of the uninfected cats became infected. The ultimate control of FeLV awaits the development of an effective FeLV vaccine, which now seems feasible since we have already experimentally immunized some cats with attenuated FeLV. Although FeLV is infectious for cats there is no evidence that FeLV can infect humans.