Isolates Of Caprine Arthritis-encephalitis Virus Research Articles

Caprine arthritis encephalitis virus (CAEV) replicates productively in cultured epididymal cells from goats

The transmission of CAEV from male goats has not been well studied and the target cells that support viral replication are not well characterized. Epididymal epithelial cells (EECs) are important and play a key role in the fertility and motility of spermatozoa. During their transit, spermatozoa incorporate several EEC-produced proteins into their plasma membranes to stabilize them and prevent premature acrosomal reaction. This intimate interaction between spermatozoa and EECs may increase the likelihood of the infection of semen with CAEV if epididymal tissue is productively infected and sheds the virus into the duct. The aim of this study was to examine whether goat EECs are susceptible to CAEV infection in tissue culture. Cells were isolated from epididymides obtained from goats that were sampled from a certified-CAEV-free herd. Cultured cells were then inoculated with a molecularly-cloned isolate of CAEV (CAEV-pBSCA). Inoculated cells developed cytopathic effects (CPE), showing numerous multinucleated giant cells (MGC) in cell-culture monolayers. Expression of CAEV proteins was detected by immunofluorescence using an anti-p28, Gag-specific antibody. The culture medium of inoculated cells was shown to contain high titers (10(6) tissue culture infectious doses 50 per ml (TCID50/ml)) of infectious, cytopathic virus when assayed using indicator goat synovial membrane (GSM) cells. Our findings clearly demonstrate that cells of the buck genital tract are targets of CAEV and are thus a potential reservoir that sheds infectious CAEV into the semen of infected animals. These data suggest the use of sperm from CAEV-free goat males for artificial insemination in genetic selection programs to minimize CAEV dissemination.

Diversity of caprine arthritis–encephalitis virus promoters isolated from goat milk and passaged in vitro

Transcriptional regulation in retroviruses resides in the U3 region of the proviral long terminal repeat (LTR). Transcription binding sites (TBS) in the U3 region of proviral sequences derived from the milk of 17 goats infected with caprine arthritis–encephalitis virus (CAEV) were analysed by nested PCR and sequencing. U3 sequences shared a high degree of homology (86–99%) and were closely related to isolates previously ascribed to small ruminant lentivirus subtype B1. Multiple putative AP-1, AP-4, Ets-1, Stat-1 and TATA binding protein (TBP) sites were highly conserved (>85% of isolates), as were single AML(vis), GAS, IRF-1, NFAT and TAS sites. A 10 nucleotide insertion of undetermined relevance was identified in the U3 region of two isolates. To study the stability of TBS within the CAEV U3 region through in vitro passage, milk-derived isolates of CAEV from three infected dams were cultured in goat synovial membrane (GSM) cells; in one isolate the viral U3 region was completely stable during in vitro passage, in a second isolate the viral U3 region accumulated multiple deletions, single nucleotide polymorphisms and insertions, while a third isolate had an intermediate degree of promoter stability. Promoter mutations arising during in vitro passage did not affect most of the conserved putative TBS identified in CAEV.

Phage display identifies two Caprine Arthritis Encephalitis Virus env epitopes

Using phage display and IgG of a goat infected with Caprine Arthritis Encephalitis Virus (CAEV) we obtained families of 7 mer constrained peptides with consensus motifs LxSDPF/Y and SWN/KHWSY and mapped the epitopes mimicked by them at the Env 6-LISDPY-11 and 67-WNTYHW-72 sites of the mature gp135 amino acid sequence. The first epitope fell into the N-terminal immunogenic aa1-EDYTLISDPYGFS- aa14 site identified previously with a synthetic peptide approach; the second epitope has not been described previously. The first epitope is mostly conserved across CAEV isolates whereas the second newly described epitope is extremely conserved in Small Ruminant Lentiviruses env sequences. As being immunodominant, the epitopes are candidate targets for mimotope-mediated diagnosis and/or neutralization.

Sequence comparison on gag gene of caprine arthritis encephalitis virus from Korea

Caprine arthritis encephalitis (CAE) is a lentiviral infection in goats characterized by arthritis. Although CAE has been identified in most goat-rearing countries around the world, Korea had been considered to be free from this disease. Here, we determine the partial sequence on the gag gene of caprine arthritis encephalitis virus (CAEV) suspect from Korea using nested PCR. Genetic analysis of our isolate showed 93.5 and 90.9% sequence homology with the standard strain CAEV Cork and lentivirus strain, respectively. Our results show that the virus is closely related to other CAEV isolates, and therefore, suggest that the causative agent was CAEV. This is the first outbreak of CAE to be ever reported in a local farm in South Korea.

Genetic diversity of small-ruminant lentiviruses: characterization of Norwegian isolates of Caprine arthritis encephalitis virus

Small-ruminant lentiviruses (SRLVs), including Caprine arthritis encephalitis virus (CAEV) in goats and maedi-visna virus (MVV) in sheep, are lentiviruses that, despite overall similarities, show considerable genetic variation in regions of the SRLV genome. To gain further knowledge about the genetic diversity and phylogenetic relationships among field isolates of SRLVs occurring in geographically distinct areas, the full-length genomic sequence of a CAEV isolate (CAEV-1GA) and partial env sequences obtained from Norwegian CAEV-infected goats were determined. The genome of CAEV-1GA consisted of 8,919 bp. Alignment studies indicated significant diversity from published SRLV sequences. Deletions and hypervariability in the 5' part of the env gene have implications for the size of the proposed CAEV-1GA Rev protein and the encoded surface glycoprotein (SU). The variable regions in the C-terminal part of SU obtained from Norwegian CAEV isolates demonstrate higher sequence divergence than has been described previously for SRLVs. Phylogenetic analysis based on SU sequences gives further support for a unique group designation. The results described here reveal a distant genetic relationship between Norwegian CAEV and other SRLVs and demonstrate that there is more geographical heterogeneity among SRLVs than reported previously.

Epithelial cells from goat oviduct are highly permissive for productive infection with caprine arthritis–encephalitis virus (CAEV)

Caprine oviduct epithelial cells (COEC) are commonly used in in vitro goat embryo production protocols to stimulate early embryonic development. These feeder cells are usually collected from slaughterhouses from unknown serological status animals for caprine arthritis–encephalitis virus (CAEV) infection which is frequent in many regions of the world. Tissues derived from this source may be contaminated with CAEV and the use of such material in in vitro fertilisation systems may contribute to transmission of this pathogen to the cultured embryos and dissemination via embryo transfer (ET). The aim of this study was to determine the permissiveness of COEC to CAEV replication in vitro. Cells were isolated from goats from certified CAEV-free herds and then were inoculated with two CAEV strains: the molecularly-cloned isolate of CAEV (CAEV-pBSCA) and the French field isolate (CAEV-3112). Cytopathic effects (CPE) were observed on cell culture monolayers inoculated with both CAEV strains. Expression of CAEV proteins was shown both by immunocytochemistry using anti-p24 gag specific antibodies and by immunoprecipitation using a hyperimmune serum. The CAEV proteins were correctly and properly processed by artificially-infected COEC and the titers of virus released into the supernatant reached 10 6 TCID 50/ml 6 days post-inoculation. Although the macrophage lineage cells are the main centre of infection in the virus-positive animal, these findings suggest that epithelial cells may be important in the viral life cycle probably as a reservoir allowing the viral persistence, dissemination and pathogenesis. These results suggest also that the use in in vitro fertilisation systems of co-culture feeder cells that support efficient replication of CAEV to high titers could represent a serious risk for permanent transmission of virus to the cultured embryos and to the surrogate dam involved.

Monoclonal antibodies to conformational epitopes of the surface glycoprotein of caprine arthritis-encephalitis virus: potential application to competitive-inhibition enzyme-linked immunosorbent assay for detecting antibodies in goat sera.

Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79-63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-N-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection.

B-cell epitopes of the envelope glycoprotein of caprine arthritis-encephalitis virus and antibody response in infected goats.

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.

Variability and immunogenicity of caprine arthritis-encephalitis virus surface glycoprotein.

The complete surface glycoprotein (SU) nucleotide sequences of three French isolates of caprine arthritis-encephalitis virus (CAEV) were determined and compared with those of previously described isolates: three American isolates and one French isolate. Phylogenetic analyses revealed the existence of four distinct and roughly equidistant evolutionary CAEV subtypes. Four conserved and five variable domains were identified in the SU. The fine specificities of antibodies produced against these domains during natural infection were examined using a pepscan analysis. Nine immunogenic segments were delineated throughout the conserved and variable domains of SU, two of them corresponding to conserved immunodominant epitopes. Antigenic determinants which may be involved in the immunopathogenic process induced by CAEV were identified. These results also provide sensitive and specific antigen peptides for the serological detection and differentiation of CAEV and visna/maedi virus infections.

Experimental infection of Mouflon-domestic sheep hybrids with caprine arthritis-encephalitis virus.

To determine whether monocyte-derived macrophages from Mouflon-domestic sheep hybrids (Ovis musimon X Ovis spp) were susceptible to productive infection with caprine arthritis-encephalitis virus (CAEV) in vitro and whether experimental inoculation of Mouflon-domestic sheep hybrids with a molecularly cloned CAEV would result in persistent infection. 5 Mouflon hybrids. Macrophage monolayers were inoculated with virus in vitro. Three animals were inoculated with virus intratracheally. Productive replication of CAEV was demonstrated in monocyte-derived macrophages following in vitro and in vivo inoculation. Titer of infectious cytopathic CAEV produced by macrophages from the Mouflon hybrids was similar to titers produced by macrophages from an infected goat or by synovial membrane cells. Isolation of virus from monocyte-derived macrophages and use of a semiquantitative polymerase chain reaction assay to amplify a portion of the viral genome demonstrated persistent virus replication in all 3 inoculated animals. Two weeks after inoculation of sheep, approximately 1 of 5,000 monocytes was harboring the virus. Results indicate that Mouflon-domestic sheep hybrids are susceptible to infection with isolates of CAEV that cause infection in domestic small ruminants.

CD8+ cytotoxic T lymphocytes against antigenic variants of caprine arthritis-encephalitis virus.

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.

Neutralization-resistant antigenic variants of caprine arthritis-encephalitis lentivirus associated with progressive arthritis.

Fifteen newborn Saanen goats were orally infected with a biologically cloned isolate of caprine arthritis-encephalitis virus (CAEV-63). At 33.5 months after infection, 8 of 12 seropositive goats had CAEV-63-specific neutralizing antibody. Five neutralization-positive goats developed clinically apparent arthritis of carpal joints, three of which had periarticular swelling indicating severe synovitis. Arthritis was not evident in age-matched controls or infected goats without neutralizing antibody. Multiple viral isolates were obtained from synovial fluid or synovial fluid cells of arthritic joints between 36.4 and 44.9 months after infection, and successive isolates from individual joints were defined as antigenic variants expressing type-specific neutralization epitopes. Thus, the evolution of neutralizing antibody does not arrest viral replication or development of progressive inflammatory lesions. Rather, some antigenic variants produced in the presence of neutralizing antibody are clonally expanded within carpal joints, and severe joint inflammation is specifically associated with these variants.

Genetic structure of the pol-env region of the caprine arthritis-encephalitis lentivirus genome

The nucleotide sequence of the pol-env intergenic region of two isolates of caprine arthritis-encephalitis virus (CAEV) was determined. Two open reading frames (orfs) were identified, designated Q and S by homology with visna virus. CAEV orf S is a single exon encoding a deduced 87-amino acid gene product sharing 36 amino acid identities with the visna trans-acting transcriptional activator (Tat). Ten of these identities comprise a conserved CGCRLCNPGW sequence similar to a cysteine-rich domain essential for trans-activation by human immunodeficiency virus Tat. To determine if transcription promoted by the CAEV long terminal repeat (LTR) could be stimulated in CAEV-infected goat synovial membrane cells, a plasmid (pCAE-CAT) expressing bacterial chloramphenicol acetyltransferase (CAT) under control of the CAEV LTR was transfected into uninfected and infected cells. Sixfold enhancement of CAT activity was observed in infected cells using 100 ng of transfected plasmid. To determine if the pol-env region encodes a gene product which trans-activates the CAEV LTR, goat synovial membrane cells were cotransfected with pCAE-CAT and pRSV-1.9, a plasmid expressing the pol-env region under control of the Rous sarcoma virus LTR. Results indicated that the CAEV genome encodes a tat gene product attributable to orf S.

Virus-specific polypeptides of caprine arthritis-encephalitis virus recognized by monoclonal antibodies to virion proteins p24 and p14.

The proteins of an isolate of caprine arthritis-encephalitis virus (CAEV) were analysed by SDS-PAGE and Western blotting. Monoclonal antibodies (MAbs) produced to the main core protein p24 and the small structural protein p14 also recognized two major polypeptides of Mr 41K and 55K in infected cell material, consistent with a precursor role for these gag polypeptides. In addition, the p24 MAbs detected a 33K polypeptide in extracellular virus preparations, while the p14 MAbs reacted strongly with a polypeptide of 18K (corresponding to structural protein p18) and weakly with another of 21K. The use of these MAbs in an indirect fluorescent antibody method revealed an intracytoplasmic location of these viral antigens in both mononucleated and multinucleated (syncytial) infected cells. Cross-reactivity with several other isolates indicated that these MAbs may be useful for diagnosis of CAEV infection.

Antigenic variation of caprine arthritis-encephalitis virus during persistent infection of goats.

Six caprine arthritis-encephalitis virus (CAEV)-free goats kept in strict isolation were inoculated intravenously with a cloned CAEV isolate (virus 020). At 78 weeks post-infection a virus (virus 095) isolated from one of the goats was shown to have the characteristics of CAEV, but was antigenically distinct from virus 020 and two other CAEV isolates by serum neutralization tests. Serum from the goat that had the variant virus neutralized the inoculum virus and the variant virus but serum from other inoculated goats neutralized only the inoculum virus. The variant virus and the inoculum virus were shown to co-exist in the infected goat, but the presence of the antigenic variant did not appear to be associated with an increase in severity of lesions compared with other inoculated goats.

Antigenic and Structural Variation of the p28 Core Polypeptide of Goat and Sheep Retroviruses

The p28 core polypeptides of four isolates of caprine arthritis-encephalitis virus (CAEV) from goats was compared with those of visna virus (VV) and progressive pneumonia virus (PPV) from sheep. Monoclonal antibodies recognized p28 epitopes common to all six retrovirus isolates, a p28 epitope on four CAEV isolates, but not VV and PPV isolates, a p28 epitope on four CAEV isolates and VV, but not PPV and a p28 epitope unique to the CAEV isolate used for immunizing the mouse spleen donor. Comparison of two-dimensional maps of tyrosine containing tryptic peptides of p28 demonstrated that three CAEV isolates had similar maps while a fourth CAEV isolate, VV and PPV had several different from the three closely related CAEV p28s and from each other.

Biochemical characterisation of caprine arthritis-encephalitis virus

Abstract Extract Sir:– Recently, caprine arthritis-encephalitis virus (CAEV) was isolated in New Zealand.(5) The virus induced syncytial cell formation in cell culture, budded from the cytoplasmic membrane of infected cells, was 80-100 nm in diameter, and reacted with both CAEV- and maedi/visna-positive antisera. We wish to present evidence that this isolate of CAEV has other biochemical characteristics of the Retroviridae. The two biochemical techniques used were the detection of 3H uridine-Iabelled virus and the assay for viral RNA-directed DNA polymerase (RDDP).

Experimental Infection of Sheep and Goats with Caprine Arthritis-Encephalitis Virus

Y Sir, — The isolation of caprine arthritis-encephalitis virus (CAEV) from the carpal joint of a yearling goat with chronic interstitial pneumonia is reported in this issue of the Journal.(3) We wish to report on the experimental infection of sheep and goats with this isolate of CAEV. Two 20-month-old Romney lambs (483, 485) and two 2-month-old feral goat kids (43, 45) were inoculated with CAEV suspension (104.0 median tissue culture infective doses [TCID50ml]). Each animal was inoculated with 1.0 ml of virus suspension by each of the following routes: intravenous, intracerebral into the right cerebral hemisphere and intra-articularly into the right radio-carpal joint.(4) An additional control lamb (490) and goat kid (44) were inoculated in identical fashion with cell culture fluid from uninfected goat synovial membrane cells.