Evaluation of a kinetic enzyme-linked immunosorbent assay for detection of caprine arthritis-encephalitis virus-specific antibodies.
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A kinetic indirect enzyme-linked immunosorbent assay (k-ELISA) was evaluated for detection of antibody to caprine arthritis-encephalitis virus (CAEV), using sodium dodecyl sulfate-treated CAEV-63 as antigen. Two hundred fifteen caprine sera submitted to the diagnostic laboratory were tested for CAEV antibody by the k-ELISA and by immunoprecipitation of [35S]-methionine-labeled CAEV. A k-ELISA positive cutoff point of 80 yielded a sensitivity of 94.4% and a specificity of 100%, as compared with immunoprecipitation. A k-ELISA cutoff point of 50 resulted in a sensitivity of 100%, with 95.6% specificity. When sera with k-ELISA scores between 50 and 80 were considered suspect, testing of 1,001 diagnostic sera resulted in < 1.5% suspect reactions. Using the 80 cutoff point, the CAEV k-ELISA had good sensitivity and specificity, with the added advantages of quick turn-around time, few suspect reactions, and adaptability to large numbers of samples (+info)
Evaluation of agar gel immunodiffusion serology using caprine and ovine lentiviral antigens for detection of antibody to caprine arthritis-encephalitis virus.
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The sensitivity of the agar gel immunodiffusion (AGID) test for the detection of antibody to caprine arthritis-encephalitis virus (CAEV) was investigated with CAEV or ovine progressive pneumonia virus (OPPV) as the source of antigen. A total of 218 goat serum specimens were tested for anti-CAEV antibody by AGID and immunoprecipitation of [35S]methionine-labeled CAEV. In comparison with that of immunoprecipitation, the sensitivity of the CAEV AGID test was 0.91, and that of the OPPV AGID test was 0.56. The AGID test with either antigen was 100% specific. The lower sensitivity of the OPPV AGID test in detecting caprine antibody to CAEV indicates that OPPV antigen is of limited value for use in CAEV diagnosis and control programs. (+info)
CD8+ cytotoxic T lymphocytes against antigenic variants of caprine arthritis-encephalitis virus.
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Cytotoxic T lymphocytes (CTL) specific for caprine arthritis-encephalitis virus (CAEV) were characterized using a colorimetric immunocytochemistry assay to measure surviving target cells. Peripheral blood lymphocytes from a CAEV-infected goat were cytotoxic to autologous, CAEV-infected dermal fibroblast target cells following in vitro stimulation of the lymphocytes with CAEV antigen. The lymphocytes were not cytotoxic to infected allogeneic target cells or to mock-infected autologous or allogeneic target cells. This CAEV antigen-specific, major histocompatibility complex-restricted cytotoxicity was mediated by CD8+ lymphocytes as demonstrated by selective depletion with anti-CD8 antibody and complement. CTL primed with one isolate of CAEV (CAEV-Co) had no detectable activity against target cells infected with either of two neutralization variants and diminished activity against target cells infected with three other neutralization variants. This apparent variability of CTL-sensitive epitopes among CAEV isolates may contribute to CAEV escaping immune control and may complicate vaccine design. (+info)
Activation of caprine arthritis-encephalitis virus long terminal repeat by gamma interferon.
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Caprine arthritis-encephalitis virus (CAEV) is a lymphotropic lentivirus whose replication increases during monocyte maturation. We examined gene expression directed by the CAEV long terminal repeat (LTR) in a promonocytic cell line stimulated with several agents. Our results demonstrate that the CAEV LTR is activated by treatment of immature monocytes with gamma interferon (IFN-gamma) or a phorbol ester but not with tumor necrosis factor alpha or lipopolysaccharide. The cis-acting element in the LTR for the IFN-gamma response localizes to a duplicated 70-bp motif that contains an IFN-gamma response element, the gamma-activated site. One copy of the motif is necessary and sufficient for the response to IFN-gamma. Multiple copies contribute to basal transcriptional activity in the context of a heterologous promoter. This IFN-gamma response element in the CAEV LTR differs from the element required for the response to phorbol esters. Thus, activation of the CAEV LTR in monocytes that are stimulated by IFN-gamma, a cytokine that is secreted in response to viral infections, could contribute to conversion from latent to high-level viral replication in infected hosts. (+info)
Replication properties of dUTPase-deficient mutants of caprine and ovine lentiviruses.
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The virion-associated dUTPase activities of caprine arthritis-encephalitis virus (CAEV) and visna virus were determined by using an assay which measure the actual ability of the dUTPase to prevent the dUTP misincorporations into cDNA during reverse transcription. We showed that the CAEV molecular clone from the Cork isolate was dUTPase defective as a result of a single amino acid substitution. Using this point mutant and deletion mutants of CAEV as well as a deletion mutant of visna virus, we demonstrated that dUTPase-deficient viruses replicate similarly to wild-type viruses in dividing cells but show delayed replication in nondividing primary macrophages. (+info)
Two species of Rev proteins, with distinct N termini, are expressed by caprine arthritis encephalitis virus.
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Several cDNA clones representing alternatively spliced Rev-specific transcripts were isolated from a cDNA library prepared from Himalayan tahr cells infected with caprine arthritis encephalitis virus (CAEV). We previously characterized two rev-like cDNA species, d1 and d2, and a tat e1 cDNA containing the rev coding sequence downstream to the tat. In these cDNAs, the rev coding domain derives its amino terminus from the N terminus of env, which is spliced to the 3' open reading frame encoding the putative Rev protein. In this study, we report the genetic structure of a fourth rev-like cDNA (designated g1), which lacks the 5' env-derived sequences. All of these rev transcripts, including cDNA g1, increased the level of chloramphenicol acetyltransferase expression when cotransfected with a reporter plasmid containing the CAEV Rev-response element-spanning region downstream of the cat coding sequences. Western blot (immunoblot) analysis showed that each transfected cDNA species gave rise to a 16-kDa protein lacking env-encoded amino-terminal epitopes. In contrast, CAEV-infected Himalayan tahr cells expressed only a 20-kDa protein, whose N terminus, in contrast, is derived from the env. Moreover, only the 20-kDa protein was also detected in the mature CAEV virions. These observations suggest that the transcripts d1, d2, and e1 can potentially, in appropriate cellular context, encode two Rev isoforms differing in their N termini, whereas the g1 transcript encodes only the 16-kDa species. Elucidation of the significance of the 16-kDa Rev protein in CAEV biology must await further studies. (+info)
Restrictive type of replication of ovine/caprine lentiviruses in ovine fibroblast cell cultures.
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Caprine arthritis-encephalitis virus (CAEV) is a natural lentivirus pathogen of goats. CAEV, like all members of the ovine/ caprine lentivirus family, has an in vivo tropism for cells of the monocyte/macrophage cell lineage and activation of viral gene expression is observed only following differentiation of monocytes to macrophages. In addition to cells of the monocyte/ macrophage lineage, CAEV and the closely related maedi visna virus of sheep (MVV) can also replicate productively in fibro-epithelial cells derived from synovial membrane of goats (GSM). However, these viruses varied greatly in their ability to replicate in fibroblasts. We studied the biological and biochemical properties of CAEV and maedi-visna virus (MVV) of sheep following inoculation into the three ovine/caprine cell types. Our data showed no substantial differences in virus titers, viral protein biosynthesis, or processing of the viral proteins between CAEV and MVV following inoculation into primary macrophages and GSM cells. However, unlike MVV, CAEV failed to replicate productively in ovine fibroblasts (sheep choroid plexus cells). This correlated with a specific but abnormal proteolytic cleavage of the envelope glycoprotein of the virus. This abnormal proteolytic cleavage represents a novel type of host cell restriction of lentivirus replication. (+info)
Requirement of caprine arthritis encephalitis virus vif gene for in vivo replication.
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Replication of vif-caprine arthritis encephalitis virus (CAEV) is highly attenuated in primary goat synovial membrane cells and blood-derived macrophages compared to the wild-type (wt) virus. We investigated the requirement for CAEV Vif for in vivo replication and pathogenicity in goats by intra-articular injection of either infectious proviral DNA or viral supernatants. Wild-type CAEV DNA or virus inoculation induced persistent infection resulting in severe inflammatory arthritic lesions in the joints. We were unable to detect any sign of virus replication in vif- CAEV DNA inoculated goats, while vif- CAEV virus inoculation resulted in the seroconversion of the goats. However, virus isolation and RT-PCR analyses on blood-derived macrophage cultures remained negative throughout the experiment as well as in joint or lymphoid tissues taken at necropsy. No pathologic lesions could be observed in joint tissue sections examined at necropsy. Goats inoculated with the vif- virus demonstrated no protection against a pathogenic virus challenge. These results demonstrate that CAEV Vif is absolutely required for efficient in vivo virus replication and pathogenicity and provide additional evidence that live attenuated lentiviruses have to establish a persistent infection to induce efficient protective immunity. (+info)