Replication of Small Ruminant Lentiviruses in Aluminum Hydroxide-Induced Granulomas in Sheep: a Potential New Factor for Viral Dissemination.

Aluminum (Al)-based salts are widely used adjuvants in ruminants and other species to strengthen the immune response elicited against vaccine antigen(s). However, they can lead to the formation of long-lasting granulomas composed of abundant activated macrophages. Small ruminant lentiviruses (SRLV) are widely distributed macrophage-tropic retroviruses that cause persistent infections in sheep and goats. Infected monocytes/macrophages and dendritic cells establish an inflammatory microenvironment that eventually leads to clinical manifestations. The aim of this work was to study the effect of Al-induced granulomas in the replication and pathogenesis of SRLV. Eleven adult, naturally SRLV-infected sheep showing clinical arthritis were distributed in vaccine (n = 6), adjuvant-only (n = 3), and control (n = 2) groups and inoculated with commercial Al-based vaccines, Al hydroxide adjuvant alone, or phosphate-buffered saline, respectively. In vitro studies demonstrated viral replication in Al-induced granulomas in 5 out of 10 sheep. Immunohistochemistry (IHC) evinced granular, intracytoplasmic SRLV presence in macrophages within granulomas. Viral sequences obtained from granulomas, blood monocytes, and other tissues were highly similar in most animals, suggesting virus circulation among body compartments. However, notable differences between isolated strains in granulomas and other tissues in specific animals were also noted. Interestingly, the B2 subtype was the most commonly found SRLV genotype, reaching a wider body distribution than previously described. Recombination events between genotypes B2 and A3 along the gag region were identified in two sheep. Our results indicate that Al-hydroxide-derived granulomas may represent an ideal compartment for SRLV replication, perhaps altering natural SRLV infection by providing a new, suitable target tissue.IMPORTANCE Granulomas are inflammation-derived structures elicited by foreign bodies or certain infections. Aluminum adjuvants included in vaccines induce granulomas in many species. In sheep, these are persistent and consist of activated macrophages. Small ruminant lentiviruses (SRLV), which are macrophage-tropic lentiviruses, cause a chronic wasting disease affecting animal welfare and production. Here, we studied the occurrence of SRLV in postvaccination granulomas retrieved from naturally infected ewes after vaccination or inoculation with aluminum only. SRLV infection was confirmed in granulomas by identification of viral proteins, genomic fragments, and enzymatic activity. The infecting SRLV strain, previously found exclusively in carpal joints, reached the central nervous system, suggesting that occurrence of SRLV in postvaccination granulomas may broaden tissue tropism. SRLV recombination was detected in inoculated animals, a rare event in sheep lentiviruses. Potentially, virus-host interactions within granulomas may modify viral pathogenesis and lead to more widespread infection.

The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis: application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus.

The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N=13) and infected with CAEV (N=13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of ß-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and - according to the geNorm results - the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80°C was also proved.

Diverse host-virus interactions following caprine arthritis-encephalitis virus infection in sheep and goats.

Interspecies transmissions substantially contribute to the epidemiology of small ruminant lentiviruses (SRLVs), including caprine arthritis encephalitis virus (CAEV) and visna-maëdi virus. However, comprehensive studies of host-virus interactions during SRLV adaptation to the new host are lacking. In this study, virological and serological features were analysed over a 6 month period in five sheep and three goats experimentally infected with a CAEV strain. Provirus load at the early stage of infection was significantly higher in sheep than in goats. A broad antibody reactivity against the matrix and capsid proteins was detected in goats, whereas the response to these antigens was mostly type-specific in sheep. The humoral response to the major immunodominant domain of the surface unit glycoprotein was type-specific, regardless of the host species. These species-specific immune responses were then confirmed in naturally infected sheep and goats using sera from mixed flocks in which interspecies transmissions were reported. Taken together, these results provide evidence that SRLV infections evolve in a host-dependent manner, with distinct host-virus interactions in sheep and goats, and highlight the need to consider both SRLV genotypes in diagnosis, particularly in sheep.

Small ruminant lentiviruses: immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus.

The small ruminant lentiviruses include the prototype for the genus, visna-maedi virus (VMV) as well as caprine arthritis encephalitis virus (CAEV). Infection of sheep or goats with these viruses causes slow, progressive, inflammatory pathology in many tissues, but the most common clinical signs result from pathology in the lung, mammary gland, central nervous system and joints. This review examines replication, immunity to and pathogenesis of these viruses and highlights major differences from and similarities to some of the other lentiviruses.

Caprine arthritis-encephalitis virus induces apoptosis in infected cells in vitro through the intrinsic pathway.

Caprine arthritis-encephalitis virus (CAEV) is a lentivirus that causes natural inflammatory disease in goats, with chronic lesions in several different organs. CAEV infection of in vitro cultured cells is accompanied by apoptosis, but the involvement of the intrinsic and extrinsic pathways has not previously been elucidated. We have studied the activation of caspases-3, -8 and -9 by fluorescent assays in various goat cells infected in vitro by CAEV, and the effects of transfected dominant negative variants of theses caspases, to show that CAEV-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of mitochondrial pathway.

Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein.

BACKGROUND: Lentiviral genomes encode multiple structural and regulatory proteins. Expression of the full complement of viral proteins is accomplished in part by alternative splicing of the genomic RNA. Caprine arthritis encephalitis virus (CAEV) and maedi-visna virus (MVV) are two highly related small-ruminant lentiviruses (SRLVs) that infect goats and sheep. Their genome seems to be less complex than those of primate lentiviruses since SRLVs encode only three auxiliary proteins, namely, Tat, Rev, and Vif, in addition to the products of gag, pol, and env genes common to all retroviruses. Here, we investigated the central part of the SRLV genome to identify new splice elements and their relevance in viral mRNA and protein expression. RESULTS: We demonstrated the existence of a new 5' splice (SD) site located within the central part of CAEV genome, 17 nucleotides downstream from the SD site used for the rev mRNA synthesis, and perfectly conserved among SRLV strains. This new SD site was found to be functional in both transfected and infected cells, leading to the production of a transcript containing an open reading frame generated by the splice junction with the 3' splice site used for the rev mRNA synthesis. This open reading frame encodes two major protein isoforms of 18- and 17-kDa, named Rtm, in which the N-terminal domain shared by the Env precursor and Rev proteins is fused to the entire cytoplasmic tail of the transmembrane glycoprotein. Immunoprecipitations using monospecific antibodies provided evidence for the expression of the Rtm isoforms in infected cells. The Rtm protein interacts specifically with the cytoplasmic domain of the transmembrane glycoprotein in vitro, and its expression impairs the fusion activity of the Env protein. CONCLUSION: The characterization of a novel CAEV protein, named Rtm, which is produced by an additional multiply-spliced mRNA, indicated that the splicing pattern of CAEV genome is more complex than previously reported, generating greater protein diversity. The high conservation of the SD site used for the rtm mRNA synthesis among CAEV and MVV strains strongly suggests that the Rtm protein plays a role in SRLV propagation in vivo, likely by competing with Env protein functions.

Activation/proliferation and apoptosis of bystander goat lymphocytes induced by a macrophage-tropic chimeric caprine arthritis encephalitis virus expressing SIV Nef.

Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats, well known for its tropism for macrophages and its inability to cause infection in lymphocytes. The viral genome lacks nef, tat, vpu and vpx coding sequences. To test the hypothesis that when nef is expressed by the viral genome, the virus became toxic for lymphocytes during replication in macrophages, we inserted the SIVsmm PBj14 nef coding sequences into the genome of CAEV thereby generating CAEV-nef. This recombinant virus is not infectious for lymphocytes but is fully replication competent in goat macrophages in which it constitutively expresses the SIV Nef. We found that goat lymphocytes cocultured with CAEV-nef-infected macrophages became activated, showing increased expression of the interleukin-2 receptor (IL-2R). Activation correlated with increased proliferation of the cells. Interestingly, a dual effect in terms of apoptosis regulation was observed in exposed goat lymphocytes. Nef was found first to induce a protection of lymphocytes from apoptosis during the first few days following exposure to infected macrophages, but later it induced increased apoptosis in the activated lymphocytes. This new recombinant virus provides a model to study the functions of Nef in the context of infection of macrophages, but in absence of infection of T lymphocytes and brings new insights into the biological effects of Nef on lymphocytes.

In vitro cross-species infections using a caprine arthritis encephalitis lentivirus carrying the GFP marker gene.

A caprine arthritis encephalitis virus (CAEV), carrying the green fluorescent protein (GFP) into the tat region was recently reported [Mselli-Lakhal, L., Guiguen, F., Greenland, T., Mornex, J.F., Chebloune, Y., 2006. Gene transfer system derived from the caprine arthritis-encephalitis lentivirus. J. Virol. Meth. 136, 177-184]. This construct, called pK2EGFPH replicated to titres up to 10(5)IU/ml on infection of caprine cells, and could be concentrated to 10(6)IU/ml by ultracentrifugation. In the present study, the pK2EGFPH construct was characterized better and used in cross-species infection studies. The pK2EGFPH virus could transduce GFP protein expression both to goat synovial membrane cells and to an immortalized goat milk epithelial cell line. The pK2EGFPH infected cells were demonstrated to express both GFP protein and CAEV viral proteins, as demonstrated by radioimmunoprecipitation and multinucleated cell formation. However GFP expression could not be maintained over passages. This vector was used to investigate cross-species infectious potential of CAEV. The bovine cell lines MDBK and GBK were found to be sensitive to infection while the human cell lines Hela, A431 and THP-1 were not. The pK2EGFPH vector should prove useful in studies of CAEV tropism both in vitro and in vivo.

Establishment and partial characterization of an ovine synovial membrane cell line obtained by transformation with Simian Virus 40 T antigen.

The small ruminant lentiviruses, namely caprine arthritis encephalitis virus (CAEV) and Maedi Visna virus (MVV) are grown currently in secondary synovial membrane cells. Primary and secondary cell cultures are sometimes difficult to obtain and support a low number of passages and, therefore, permissive cell lines are needed. A transformed cell line was obtained by transfection of ovine synovial membrane secondary cell culture with a plasmid containing the SV40 large T antigen gene. The transformed cell culture described in this paper showed a higher growth rate and a more homogenous population of fibroblast-like cells when compared to the original ovine synovial membrane secondary cell cultures. Karyotype analysis has indicated the induction of many random chromosome changes, leading to a decrease in chromosome number. The SV40 DNA was detected in the nucleus and in the cytoplasm of transformed cells. The putative expression of large T antigen was presumed by the detection of the corresponding mRNA by PCR. Finally, the transformed ovine synovial membrane cells were shown to be permissive to small ruminant lentiviruses, and these are suggested as a cell line for in vitro isolation and propagation of these viruses.

Lack of enhancement of caprine arthritis encephalitis virus infection in monocyte-derived macrophage cultures by sera from goats that developed severe arthritis after vaccination and virus challenge.

We examined sera from goats that developed more rapid and severe clinical disease after vaccination with inactivated caprine arthritis encephalitis virus (CAEV) and virus challenge for CAEV infection-enhancing antibodies. Sera from one control and two vaccinated goats were examined for neutralization or enhancement of virus infection in caprine macrophages. Macrophage cultures were incubated with virus-serum mixtures, then washed and fed with fresh media and incubated. Culture fluid was collected at days 2,4 and 8 post-infection and assayed for reverse transcriptase (RT) activity. Serum from one of the vaccinated goats neutralized virus at 10(-2) and 10(-3) dilutions (p = 0.045 and p = 0.020, respectively). The neutralizing effect was lost at higher dilutions (10(-4) and 10(-5)) of the serum, but no enhancement of infection was seen. Serum from the other vaccinated goat did not show any significant neutralizing effect at either 10(-2) or 10(-3) dilutions and increased infection (40% or greater) at higher dilutions, but the increases were not statistically significant. Therefore, there was no evidence of virus infection-enhancing activity in these sera that would suggest that the severe disease experienced by the vaccinated animals was due to serum enhancement of infection. Alternately, the severe arthritis observed could have resulted from the pro-inflammatory activities of cytokines and chemokines produced by macrophages upon phagocytosis, or receptor-mediated uptake of CAEV-antibody complexes.

Caprine arthritis-encephalitis virus envelope surface glycoprotein regions interacting with the transmembrane glycoprotein: structural and functional parallels with human immunodeficiency virus type 1 gp120.

A sequence similarity between surface envelope glycoprotein (SU) gp135 of the lentiviruses maedi-visna virus and caprine arthritis-encephalitis virus (CAEV) and human immunodeficiency virus type 1 (HIV-1) gp120 has been described. The regions of sequence similarity are in the second and fifth conserved regions of gp120, and the similarity is highest in sequences coinciding with beta-strands 4 to 8 and 25, which are located in the most virion-proximal region of the gp120 inner domain. A subset of this structure, formed by gp120 beta-strands 4, 5, and 25, is conserved in most or all lentiviruses. Because of the orientation of gp120 on the virion, this highly conserved virion-proximal region of the gp120 core may interact with the transmembrane glycoprotein (TM) together with the amino and carboxy termini of full-length gp120. Therefore, interactions between SU and TM of lentiviruses may be structurally related. Here we tested whether the amino acid residues in the putative virion-proximal region of CAEV gp135 comprising putative beta-strands 4, 5, and 25, as well as its amino and carboxy termini, are important for stable interactions with TM. An amino acid change at gp135 position 119 or 521, located in the turn between putative beta-strands 4 and 5 and near beta-strand 25, respectively, specifically disrupted the epitope recognized by monoclonal antibody 29A. Thus, similar to the corresponding gp120 regions, these gp135 residues are located in close proximity to each other in the folded protein, supporting the hypothesis of a structural similarity between the gp120 virion-proximal inner domain and gp135. Amino acid changes in the amino- and carboxy-terminal and putative virion-proximal regions of gp135 increased gp135 shedding from the cell surface, indicating that these gp135 regions are involved in interactions with TM. Our results indicate structural and functional parallels between CAEV gp135 and HIV-1 gp120 that may be more broadly applicable to the SU of other lentiviruses.

Analysis of caprine arthritis encephalitis virus (CAEV) temporal gene expression in infected cells.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus that is closely related to visna virus and more distantly related to the human lentivirus, Human Immunodeficiency Virus type 1 (HIV-1). The CAEV genome contains several small open reading frames (ORFs) that encode viral regulatory proteins. One of these non-structural proteins, Rev-C, is required for cytoplasmic transport of viral un/incompletely spliced mRNAs and efficient viral replication. In HIV-1 and visna virus, Rev is responsible for the temporal shift from non-structural protein synthesis to synthesis of structural proteins that is observed during the viral infectious cycle. Since it encodes a Rev protein, CAEV would be predicted to exhibit a similar temporal shift in gene expression during its replicative cycle. Immunoprecipitation analysis of 35S-pulse labeled, CAEV-infected goat synovial membrane (GSM) cells indicates that Rev-C is more abundant than is Gag at 12 h post-infection (PI); at later times PI Gag predominates. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) experiments using nuclear and cytoplasmic RNA from CAEV-infected GSM cells indicates that the viral unspliced gag mRNA accumulates significantly in the cytoplasm only after Rev is detected. These data indicate that a temporal shift from viral non-structural to structural gene expression occurs in CAEV infected GSM cells.

Differential receptor usage of small ruminant lentiviruses in ovine and caprine cells: host range but not cytopathic phenotype is determined by receptor usage.

The ovine maedi-visna (MVV) and caprine arthritis-encephalitis (CAEV) small ruminant lentiviruses (SRLV) exhibit differential species tropism and cytopathic effects in vitro. Icelandic MVV-K1514 is a lytic SRLV which can infect cells from many species in addition to ruminants, whereas a lytic North American MVV strain (85/34) as well as nonlytic MVV strain S93 and CAEV can infect only ruminant cells. In the present study, we determined if differential receptor usage in sheep and goat cells is the basis of differential species tropism or cytopathic phenotype of SRLV. Infection interference assays in sheep and goat synovial membrane cells using pseudotyped CAEV vectors showed that North American MVV strains 85/34 and S93 and CAEV use a common receptor (SRLV receptor A), whereas MVV-K1514 uses a different receptor (SRLV receptor B). In addition, human 293T cells expressing CAEV but not MVV-K1514 envelope glycoproteins fused with a goat cell line persistently infected with MVV-K1514, indicating that MVV-K1514 does not use SRLV receptor A for cell-to-cell fusion. Therefore, our results indicate that the differential species tropism of SRLV is determined by receptor usage. However, receptor usage is unrelated to cytopathic phenotype.

Rapid evolution of two discrete regions of the caprine arthritis-encephalitis virus envelope surface glycoprotein during persistent infection.

Five major regions of sequence diversity between strains (V1-V5) have been described in the caprine arthritis-encephalitis lentivirus (CAEV) envelope surface unit glycoprotein (SU). To determine which of these variable regions is important in persistent infection in vivo, we evaluated SU sequence diversity in five neutralization variants from two goats and proviral DNA from five additional goats infected with CAEV-63 for up to 7 years. Overall amino acid sequence divergence in the SU encoded by provirus and neutralization variants compared to parental CAEV-63 ranged from 1.1 to 4%. However, most of the amino acid substitutions and all of the deletions and insertions were present in two discrete regions designated HV1 and HV2. The HV2 region was variable in all neutralization variants and provirus sequences from most animals. This region overlapped the V4 domain of CAEV SU and the neutralization domain of the closely related ovine maedi-visna lentivirus. HV1 was located in a region of SU strictly conserved in all small ruminant lentivirus strains except CAEV-63. This region only varied in a subset of neutralization variants and proviruses, all derived from goats with arthritis. In contrast, sequences in the V1,V2,V3, and V5 regions were stable in neutralization variants and proviruses from infected goats, indicating that sequence diversity between strains in these regions is not due to selection of variants in persistently infected animals. Our results define two discrete regions of CAEV SU that undergo rapid sequence variation in persistently infected goats which may have important roles in virus-host interactions.

Immortalized goat milk epithelial cell lines replicate CAEV at high level.

Primary milk epithelial cells were isolated from CAEV-uninfected goats and three cell lines designated TIGMEC-1, TIGMEC-2 and TIGMEC-3 were established. The three cell lines retained their morphological characteristics of epithelial cells and expressed specific epithelial cytokeratin marker as well as the immortalizing SV40 large T antigen. The kinetics of growth of TIGMEC1, TIGMEC2 and TIGMEC3 cell lines showed a doubling time of 24-48 hours while the parental cell lines became senescent after the passage 6 in cell culture. Like the parental primary cells, the three cell lines were found to be highly sensitive to CAEV-pBSCA, an infectious molecular clone of CAEV-CO strain, and to a French isolate CAEV-3112. TIGMEC cell lines infected with CAEV-pBSCA became chronically infected producing high virus titers in absence of cytopathic effects. These cell lines may be useful for study of the possible physiological alterations in mammary epithelial cells infected with small ruminant lentiviruses and their consequences on milk quality. On an other hand, these cell lines can be used to study their role in virus transmission and pathogenesis.

Caprine arthritis encephalitis virus: evidence for a B/D-type assembly pathway in a C-type lentivirus replication.

Lentiviruses, among which is caprine arthritis encephalitis virus (CAEV), are known to concomitantly assemble and bud at the plasma membrane of infected cells, in a C-type defined pathway. Electron microscopy analysis of CAEV-infected cells demonstrated viral particles budding at the plasma membrane and into intracellular membrane-surrounded vesicles. Furthermore, nonenveloped immature virus-like particles, resembling intracytoplasmic type-A particles (ICAPs), accumulated within the cytoplasm of those cells. Fractionation on sucrose density gradients of cytoplasmic lysates from CAEV-infected cells revealed that enveloped immature or mature viral particles had a density of 1.16--1.17 g/ml, whereas ICAPs sedimented at a density of 1.2--1.27 g/ml. Endogenous reverse transcriptase activity was only associated with the 1.16--1.17 g/ml density particles despite the presence of viral RNA in both populations. The intracellular enveloped particles were found to be infectious. The CAEV Gag precursor by itself was shown to direct assembly, budding, and release of immature virus-like particles when expressed in goat primary synovial membrane cells using the same pathways of assembly and budding as observed in CAEV-infected cells. These data suggest that CAEV assembly, driven by the Gag precursor, could unusually proceed via two simultaneous pathways characteristic of type-C and type-B/D retroviruses.

Tryptophan 95, an amino acid residue of the Caprine arthritis encephalitis virus vif protein which is essential for virus replication.

The Caprine arthritis encephalitis virus (CAEV) vif gene was demonstrated to be essential for efficient virus replication. CAEV Vif deletion mutants demonstrated an attenuated replication phenotype in primary goat cell cultures and resulted in abortive infection when inoculated into goats. In this study, we determined the in vitro replication phenotype of five CAEV Vif point mutant infectious molecular clones and the ability of the corresponding in vitro translated Vif proteins to interact with the CAEV Pr55(gag) in the glutathione S--transferase (GST) binding assay. Here we show that (i) three of the mutants (S170E, S170G, S197G) behaved as the wild-type CAEV according to virus replication and Vif--Gag interactions; (ii) one mutant (Vif 6mut) was replication incompetent and bound weakly to GST-Gag fusion proteins; and (iii) one mutant (Vif RG) was impaired for replication while retaining its interaction properties. This mutant points out the critical importance of the CAEV Vif tryptophan residue at position 95 for efficient virus replication, defining for this lentivirus a functional domain unrelated to the Gag binding region.

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.

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

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.

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

OBJECTIVE: 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. ANIMALS: 5 Mouflon hybrids. PROCEDURE: Macrophage monolayers were inoculated with virus in vitro. Three animals were inoculated with virus intratracheally. RESULTS: 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. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that Mouflon-domestic sheep hybrids are susceptible to infection with isolates of CAEV that cause infection in domestic small ruminants.