Goat milk epithelial cells are highly permissive to CAEV infection in vitro. (1/95)

The main route of small ruminant lentivirus dissemination is the ingestion of infected cells present in colostrum and milk from infected animals. However, whether only macrophages or other cell subtypes are involved in this transmission is unknown. We derived epithelial cell cultures, 100% cytokeratin positive, from milk of naturally infected and noninfected goats. One such culture, derived from a naturally infected goat, constitutively produced a high titer of virus in the absence of any cytopathic effect. The other cultures, negative for natural lentivirus infection, were tested for their susceptibility to infection with the CAEV-CO strain and a French field isolate CAEV-3112. We showed that milk epithelial cells are easily infected by either virus and produce viruses at titers as high as those obtained in permissive goat synovial membrane cells. The CAEV-CO strain replicated in milk epithelial cells in absence of any cytopathic effect, whereas the CAEV-3112 field isolate induced both cell fusion and cell lysis. Our results suggest that CAEV-infected milk epithelial cells of small ruminants may play an important role in virus transmission and pathogenesis.  (+info)

Inducible nitric oxide synthase is expressed in joints of goats in the late stage of infection with caprine arthritis encephalitis virus. (2/95)

We have studied the expression of the inducible form of nitric oxide synthase (iNOS) in joints of goats infected with the caprine arthritis encephalitis virus (CAEV). Nitric oxide generated by iNOS is thought to play an important role in the pathogenesis of various types of arthritis, especially rheumatoid arthritis (RA) in humans. Surprisingly, iNOS immunoreactivity was found only in joints of long-term infected goats with severe clinical arthritis, whereas-despite the presence of high numbers of inflammatory cells in the synovial tissue-no iNOS immunoreactivity was detected in mildly arthritic and in short-term experimentally infected goats. Most iNOS-positive cells expressed neither MHC class II nor CD68, which suggests that they were fibroblast-like synoviocytes. In situ hybridization studies showed that there was no correlation between iNOS immunoreactivity and detectable virus expression in the joint. In addition, infection of macrophages in vitro-the major host cells of CAEV in vivo-did not lead to increased iNOS mRNA expression. In response to stimulation, similar levels of iNOS expression were observed in infected and in uninfected macrophages. These findings suggest that the expression of iNOS is a feature of late-stage chronic arthritis and is not involved in the development of the inflammatory lesions. Both the lack of co-localization of iNOS protein and viral transcripts in the joint and the finding that CAEV does not stimulate the expression of iNOS in vitro further suggest that iNOS is not directly induced by the virus or the anti-viral immune response in the joint, that it may well, however, be involved in tissue remodelling or scar formation.  (+info)

Conserved sequence motifs involving the tat reading frame of Brazilian caprine lentiviruses indicate affiliations to both caprine arthritis-encephalitis virus and visna-maedi virus. (3/95)

The sequence variation in small ruminant lentiviruses from Brazilian herds of milking goats was sampled in a representative region of the pol gene and in a region including the entire tat open reading frame. Clones were amplified from cDNA derived from virus produced in vitro using primers targetting conserved sequences of the pol gene. Iterative sequencing of clones indicated that animals from two herds in the Minas Gerais area were infected by a caprine arthritis-encephalitis virus (CAEV)-like virus and that individual animals carried variant virus populations. Sequences derived from an infected goat from a herd in Pernambuco showed no nucleic acid variation and were distant from the CAEV-type sequence but marginally closer to ovine visna-maedi virus (VMV) sequences. Sequences amplified from a region including the tat gene, amplified with a common upstream primer within the vif coding region and different downstream primers because of the local divergence between CAEV- and VMV-type sequences, confirmed the affiliation of the Minas Gerais sequences to CAEV and indicated that the Pernambuco isolate was indeed related to VMV, which had not previously been reported to cause natural caprine infection. The overlap between the vif and tat open reading frames clearly distinguished between CAEV-like small ruminant lentiviruses, which shared eight common nucleotides, and the VMV group, where the overlap was reduced to a single base; the final adenine of the vif terminator (TAA) is the initial adenine of the presumptive tat initiator codon. This may be useful for epizoological tracing of the origin of outbreaks.  (+info)

A new sensitive serological assay for detection of lentivirus infections in small ruminants. (4/95)

Lentivirus infections in small ruminants represent an economic problem affecting several European countries with important sheep-breeding industries. Programs for control and eradication of these infections are being initiated and require reliable screening assays. This communication describes the construction and evaluation of a new serological screening enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (MVV) in sheep and to caprine arthritis encephalitis virus (CAEV) in goats. The solid phase is sensitized with a combination of the major core protein p25 of MVV produced in Escherichia coli and a peptide derived from the immunodominant region of the viral transmembrane protein gp46. The peptide carries an N-terminal biotin residue and is complexed with streptavidin prior to being coated. The new assay was evaluated with 2,336 sheep serum samples from different European countries with large differences in the levels of prevalence of MVV infections, and the results have been compared to those of the standard agar gel immunodiffusion test. Discrepant samples were analyzed by Western blotting with viral lysate, and most sera could be classified unambiguously. The estimated overall sensitivity of the new ELISA was 99.4% (95% confidence interval [CI], 98.4 to 99. 8%) and the specificity was 99.3% (95% CI, 98.7 to 99.6%). A limited set of goat sera (n = 212) was also analyzed, with similar results. These data indicate that the new assay is a reliable tool that can be used in control and eradication programs for small ruminant lentivirus infections.  (+info)

Isolation of caprine arthritis encephalitis virus from goats in Mexico. (5/95)

A lentivirus was isolated from 2 goats in Mexico that were seropositive to caprine arthritis encephalitis virus (CAEV) by the agar gel immunodiffusion (AGID) test. The lentivirus was identified as CAEV by the observation of giant multinucleated cells (syncytia) in goat synovial membrane (GSM) monolayers co-cultivated with blood mononuclear (BMN) cells from the seropositive goats, and by amplifying a DNA segment of the CAEV gag gene using the polymerase chain reaction (PCR) technique. Subsequently, cell supernatants from the GSM cells co-cultivated with BMN cells were used to infect 2 CAEV-seronegative goats. These goats seroconverted to CAEV as determined by the AGID test, and CAEV was re-isolated from these goats. One of the goats developed polyarthritis 8 mo after inoculation. Previous serological surveys indicate that infection with CAEV is prevalent among goats in Mexico. To our knowledge this is the first report of CAEV isolation in Mexico. Because of globalization of markets and increased trading among nations, the rapid identification and reporting of diseases such as CAEV are important to prevent the dissemination of these diseases.  (+info)

Lack of functional receptors is the only barrier that prevents caprine arthritis-encephalitis virus from infecting human cells. (6/95)

Barriers to replication of viruses in potential host cells may occur at several levels. Lack of suitable and functional receptors on the host cell surface, thereby precluding entry of the virus, is a frequent reason for noninfectivity, as long as no alternative way of entry (e.g., pinocytosis, antibody-dependent adsorption) can be exploited by the virus. Other barriers can intervene at later stages of the virus life cycle, with restrictions on transcription of the viral genome, incorrect translation and posttranslational processing of viral proteins, inefficient viral assembly, and release or efficient early induction of apoptosis in the infected cell. The data we present here demonstrate that replication of caprine arthritis-encephalitis virus (CAEV) is restricted in a variety of human cell lines and primary tissue cultures. This barrier was efficiently overcome by transfection of a novel infectious complete-proviral CAEV construct into the same cells. The successful infection of human cells with a vesicular stomatitis virus (VSV) G-pseudotyped Env-defective CAEV confirmed that viral entry is the major obstacle to CAEV infection of human cells. The fully efficient productive infection obtained with the VSV-G-protein-pseudotyped infectious CAEV strengthened the evidence that lack of viral entry is the only practical barrier to CAEV replication in human cells. The virus thus produced retained its original host cell specificity and acquired no propensity to propagate further in human cultures.  (+info)

B-cell epitopes of the envelope glycoprotein of caprine arthritis-encephalitis virus and antibody response in infected goats. (7/95)

Goats infected with caprine arthritis-encephalitis virus (CAEV) develop high titres of antibodies to Env. Not only is no consistent neutralizing response found but anti-Env antibodies have even been associated with disease in infected goats. To identify the continuous antigenic determinants involved in this atypical anti-Env response, we mapped CAEV-CO Env by screening an epitope expression library with infected goat sera. In addition to the four previously described epitopes, seven novel antigenic sites were identified, of which five were located on the surface (SU) and two in the transmembrane (TM) subunits of Env. The SU antibody-binding domains located in the variable regions of the C-terminal part of the molecule (SU3 to SU5) showed the strongest reactivity and induced a rapid seroconversion in six experimentally infected goats. However, the response to these immunodominant epitopes did not appear to be associated with any neutralizing activity. The pattern of serum reactivity of naturally infected goats with these epitopes was restricted, suggesting a type-specific reaction. Interestingly, the reactivity of peptides representing SU5 sequences derived from CAEV field isolates varied with the geographical and/or breeding origin of the animals. This suggests that peptides corresponding to the immunodominant SU epitopes may well be useful in the serotyping of CAEV isolates. Furthermore, the identification of the CAEV Env epitopes will permit us to functionally dissect the antibody response and to address the role of anti-Env antibodies either in the protection from or in the pathogenesis of CAEV infection.  (+info)

Characterization of chimeric enzymes between caprine arthritis--encephalitis virus, maedi--visna virus and human immunodeficiency virus type 1 integrases expressed in Escherichia coli. (8/95)

In order to investigate the functions of the three putative lentiviral integrase (IN) protein domains on viral DNA specificity and target site selection, enzymatically active chimeric enzymes were constructed using the three wild-type IN proteins of caprine arthritis-encephalitis virus (CAEV), maedi-visna virus (MVV) and human immunodeficiency virus type 1 (HIV-1). The chimeric enzymes were expressed in Escherichia coli, purified by affinity chromatography and analysed in vitro for IN-specific endonuclease and integration activities on various DNA substrates. Of the 21 purified chimeric IN proteins constructed, 20 showed distinct site-specific cleavage activity with at least one substrate and six were able to catalyse an efficient integration reaction. Analysis of the chimeric IN proteins revealed that the central domain together with the C terminus determines the activity and substrate specificity of the enzyme. The N terminus appears to have no considerable influence. Furthermore, an efficient integration activity of CAEV wild-type IN was successfully demonstrated after detailed characterization of the reaction conditions that support optimal enzyme activities of CAEV IN. Also, under the same in vitro assay conditions, MVV and HIV-1 IN proteins exhibited endonuclease and integration activities, an indispensable prerequisite of domain-swapping experiments. Thus, the following report presents a detailed characterization of the activities of CAEV IN in vitro as well as the analysis of functional chimeric lentiviral IN proteins.  (+info)

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