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.

Species-Specific Humoral Immune Responses in Sheep and Goats upon Small Ruminant Lentivirus Infections Inversely Correlate with Protection against Virus Replication and Pathological Lesions.

Maedi-Visna-like genotype A strains and Caprine arthritis encephaltis-like genotype B strains are small ruminant lentiviruses (SRLV) which, for incompletely understood reasons, appear to be more virulent in sheep and goats, respectively. A 9-month in vivo infection experiment using Belgian genotype A and B SRLV strains showed that almost all homologous (genotype A in sheep; genotype B in goats) and heterologous (genotype A in goats; genotype B in sheep) intratracheal inoculations resulted in productive infection. No differences in viremia and time to seroconversion were observed between homologous and heterologous infections. Higher viral loads and more severe lesions in the mammary gland and lung were however detected at 9 months post homologous compared to heterologous infection which coincided with strongly increased IFN-γ mRNA expression levels upon homologous infection. Pepscan analysis revealed a strong antibody response against immune-dominant regions of the capsid and surface proteins upon homologous infection, which was absent after heterologous infection. These results inversely correlated with protection against virus replication in target organs and observed histopathological lesions, and thus require an in-depth evaluation of a potential role of antibody dependent enhancement in SRLV infection. Finally, no horizontal intra- and cross-species SRLV transmission to contact animals was detected.

Expression analysis of lung miRNAs responding to ovine VM virus infection by RNA-seq.

BACKGROUND: MicroRNAs (miRNAs) are short endogenous, single-stranded, noncoding small RNA molecules of approximately 22 nucleotides in length. They regulate gene expression posttranscriptionally by silencing mRNA expression, thus orchestrating many physiological processes. The Small Ruminant Lentiviruses (SRLV) group includes the Visna Maedi Virus (VMV) and Caprine Arthritis Encephalitis (CAEV) viruses, which cause a disease in sheep and goats characterized by pneumonia, mastitis, arthritis and encephalitis. Their main target cells are from the monocyte/macrophage lineage. To date, there are no studies on the role of miRNAs in this viral disease. RESULTS: Using RNA-seq technology and bioinformatics analysis, the expression levels of miRNAs during different clinical stages of infection were studied. A total of 212 miRNAs were identified, of which 46 were conserved sequences in other species but found for the first time in sheep, and 12 were completely novel. Differential expression analysis comparing the uninfected and seropositive groups showed changes in several miRNAs; however, no significant differences were detected between seropositive asymptomatic and diseased sheep. The robust increase in the expression level of oar-miR-21 is consistent with its increased expression in other viral diseases. Furthermore, the target prediction of the dysregulated miRNAs revealed that they control genes involved in proliferation-related signalling pathways, such as the PI3K-Akt, AMPK and ErbB pathways. CONCLUSIONS: To the best of our knowledge, this is the first study reporting miRNA profiling in sheep in response to SRLV infection. The known functions of oar-miR-21 as a regulator of inflammation and proliferation appear to be a possible cause of the lesions caused in the sheep's lungs. This miRNA could be an indicator for the severity of the lung lesions, or a putative target for therapeutic intervention.

Diagnostic response to a cross-border challenge for the Swiss caprine arthritis encephalitis virus eradication program.

INTRODUCTION: Animal trading between countries with different small ruminant lentivirus infectious status is a potential danger for the reintroduction of eradicated genotypes. This was the case in 2017 with the importation of a large flock of seropositive goats into Switzerland. The handling of this case permitted us to test the preventive measures in place. The coordination between the local veterinarian and the cantonal and federal veterinary authorities worked efficiently and rapidly involved the national reference center in the investigations. This case posed a challenge for the reference center and enabled scrutiny of the applied diagnostic tests. ELISA and western blot provided consistent results and pointed to an unusually high infection rate in the flock. This was confirmed by the isolation of several viruses from different organs and cells, demonstrating that the spleen is particularly well suited for isolation of small ruminant lentiviruses. The SU5-ELISA, designed to predict the subtype of the infecting virus, correctly pointed to a B1 subtype as the infectious agent. We confirmed that with this test it is necessary to analyze a representative number of samples from a flock and not just individual sera to obtain reliable results. This analysis permitted us to identify particular amino acid residues in the SU5 peptides that may be crucial in determining the subtype specificity of antibody binding. Different gag-pol and env regions were amplified by PCR using primers designed for this purpose. The phylogenetic analysis revealed a surprisingly high heterogeneity of the sequences, pointing to multiple infections within single animals and the entire flock. In conclusion, this case showed that the defense of the CAEV negative status of the Swiss goat population with respect to the virulent, prototypic B1 subtype of small ruminant lentiviruses, requires, among other measures, a diagnostic facility capable of performing a thorough analysis of the collected samples.

INTRODUCTION: Le commerce d'animaux entre pays où le statut infectieux des lentivirus des petits ruminants est différent constitue un danger potentiel pour la réintroduction de génotypes éradiqués. Ce fut le cas en 2017 avec l'importation d'un grand troupeau de chèvres séropositives en Suisse. Le traitement de cette affaire nous a permis de tester les mesures préventives mises en place. La coordination entre le vétérinaire local et les autorités vétérinaires cantonales et fédérales a été efficace et a impliqué rapidement le centre de référence national dans les enquêtes. Ce cas a constitué un défi pour le centre de référence et a permis d'examiner de près les tests de diagnostic appliqués. Les tests ELISA et Western blot ont fourni des résultats cohérents et ont mis en évidence un taux d'infection anormalement élevé dans le troupeau. Cela a été confirmé par l'isolement de plusieurs virus provenant d'organes et de cellules différents, démontrant que la rate est particulièrement bien adaptée à l'isolement des lentivirus des petits ruminants. Le SU5-ELISA, conçu pour prédire le sous-type du virus infectant, désignait correctement un sous-type B1 en tant qu'agent infectieux. Nous avons confirmé qu'avec ce test, il était nécessaire d'analyser un nombre représentatif d'échantillons d'un troupeau et pas seulement des sérums individuels pour obtenir des résultats fiables. Cette analyse nous a permis d'identifier des résidus d'acides aminés particuliers dans les peptides SU5 qui pourraient jouer un rôle crucial dans la détermination de la spécificité de sous-type de la liaison à l'anticorps. Différentes régions gag-pol et env ont été amplifiées par PCR en utilisant des amorces conçues à cet effet. L'analyse phylogénétique a révélé une hétérogénéité étonnamment élevée des séquences, indiquant de multiples infections chez les animaux isolés et dans l'ensemble du troupeau. En conclusion, cette affaire a montré que la défense du statut négatif CAEV de la population de chèvres suisses vis-à-vis du virus virulent, sous-type B1 des lentivirus des petits ruminants, nécessite, entre autres mesures, un système de diagnostic capable d'effectuer une analyse approfondie des échantillons collectés.

Small ruminant lentivirus-induced arthritis: clinicopathologic findings in sheep infected by a highly replicative SRLV B2 genotype.

We describe the clinicopathologic features of an arthritis outbreak in sheep induced by small ruminant lentivirus (SRLV), linked to the presence of a new SRLV isolate phylogenetically assigned to caprine arthritis encephalitis virus-like subgroup B2. Thirteen SRLV seropositive Rasa Aragonesa adult ewes were selected from 5 SRLV highly infected flocks (mean seroprevalence, 90.7%) for presenting uni- or bilateral chronic arthritis in the carpal joint. A complete study was performed, including symptomatology, histopathology, immunocytochemistry, immunohistochemistry, in situ hybridization, and microbiology. The carpus was the joint almost exclusively affected, with 10 sheep (76%) showing a moderate increase in carpal joint size (diameter range, 18-20 cm; normal range, 15-16 cm) without signs of locomotion problems and with 3 ewes (23%) showing severe inflammation with marked increase in diameter (21-24 cm), pain at palpation, and abnormal standing position. Grossly, chronic proliferative arthritis was observed in affected joints characterized by an increased thickness of the synovial capsule and synovial membrane proliferation. Microscopically, synovial membrane inflammation and proliferation and hyperplasia of synoviocytes were observed. More positive cases of SLRV infection were detected by immunocytochemistry of articular fluid than of bronchoalveolar lavage fluid. Immunohistochemistry and in situ hybridization also detected positive cells in the subsynovial connective tissue, lung, mediastinal lymph node, mammary gland, and mammary lymph node. All animals were negative for the presence of Mycoplasma or other bacteria in the articular space. The present outbreak likely represents an adaptation of a caprine virus to sheep. Our results underline the importance of the arthritis induced by SRLV in sheep, a clinical form that might be underestimated.

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.

Caprine arthritis encephalitis virus-induced apoptosis associated with brain lesions in naturally infected kids.

Acute demyelinating leucoencephalomyelitis was the most conspicuous microscopic change in the brain and spinal cord of kids infected with caprine arthritis encephalitis virus (CAEV). TUNEL positivity and labelling of anti-bax and anti-caspases-3, -8 and -9 were found in a distinct population of glial cells, mainly at the edges of the demyelinated plaques and perivascular areas and, to a lesser extent, in neurons. Double labelling revealed that most of these apoptotic cells in the demyelinated plaques were astrocytes and a few were oligodendroglia. In contrast, expression of bcl-2, an anti-apoptotic protein, was found mainly in neurons of the brainstem and cerebellum and motor neurons of the spinal cord, but was restricted in glial cells. These results suggest that apoptosis plays an important role in the pathogenesis of CAE demyelinating encephalitis.

Influence of caprine arthritis-encephalitis virus infection on the activity of peripheral blood lymphocytes.

Non-specific lymphocyte transformation assay using phytohemagglutinin (PHA) as a mitogen was applied to evaluate influence of caprine arthritis-encephalitis virus (CAEV) infection on activity of peripheral blood lymphocytes. Animals were selected for the CAEV-infected and CAEV-non-infected groups according to the results of two serological surveys carried out at one year interval, with the use of an ELISA test. In goats which were not infected with CAEV, lymphocytes stimulation index (SI) revealed a high diversity of the results with an mean value equal to 5.86 (minimum = 0.45, maximum = 40.00, SD = 8.40). SI values for infected goats reached the average of 1.10 (minimum = 0.46, maximum = 1.85, SD = 0.26). The difference between the average lymphocyte stimulation indices was statistically highly significant in both groups (p = 0.002) which could be an evidence of CAEV infection influence on lymphocyte reactivity. Regarding ELISA test as a "golden standard" the application of lymphocyte transformation assay in diagnosis of CAEV infection was assessed. The ROC curve was drawn. The area under the curve was only 0.324, which indicates very low accuracy of this method and limits its use for the diagnosis of the disease.

Eradication of caprine arthritis encephalitis virus in the goat population of South Tyrol, Italy: analysis of the tailing phenomenon during the 2016-2017 campaign.

Since 2007, the Autonomous Province of Bolzano-South Tyrol (Italy) has carried out a compulsory eradication program against caprine arthritis encephalitis virus (CAEV) in goats. A drastic seroprevalence reduction was achieved during the initial phase (2007-2011); however, a tailing phenomenon has been observed during the latest years, hampering the achievement of the final goal. CAEV belongs to a group of lentiviruses, called small ruminant lentiviruses (SRLVs), which are antigenically related and can infect both goats and sheep. We investigated the possible link between the tailing phenomenon in goats and the role of sheep as a virus reservoir by comparing serologic results between multispecies farms (where goats and sheep coexist) and monospecies farms (goats only). Goats on multispecies farms had a higher prevalence and seroconversion rate (even if to a rather moderate extent), higher antibody titers, and a higher probability of conclusive results in the genotyping analysis, with more frequent identification of SRLV genotype A (sheep-related) infections. Sheep can serve as a SRLV reservoir, thus contributing to scattered positive tests in goats, causing the tailing phenomenon.

Seroprevalence of small ruminant lentivirus (SRLV) infection in wild cervids in Poland.

Small ruminant lentiviruses (SRLV) are widespread amongst domesticated sheep and goats worldwide. Infection of wild ruminants in close contact with affected domesticated small ruminants has been proposed as an actor in SRLV epidemiology, but studies are limited. The aim of this study was to estimate the apparent (AP) and estimated prevalence (EP) of exposure to SRLV infection in wild ruminants from Poland. Samples originating from 198 free-living cervids comprising 142 European red deer and 56 roe deer were serologically tested using a multi-epitope recombinant antigen ELISA representing subtypes A1, A13, B1, and B2 of SRLV and a commercial ELISA test. The estimated prevalence of SRLV infection was estimated using the Bayesian approach with models that adjusted for the misclassification of animals because of a small population and lack of sampling method, the imperfect performance of the ELISAs and because sera of different species were tested. The calculated estimated prevalence ranged from 5.3 % (95 % CI 0.3, 12.5) to 24.6 % (95 % CI 3.3, 38.5) for the ELISA with multi-epitope antigens while estimated prevalence using the commercial ELISA was 2.5 % (95 % CI 0.2, 6.6). These results may suggest the existence of a new SRLV reservoir in Poland and highlight the importance of surveilling and controlling SRLV infection in domestic and wild ruminants sharing pasture areas.

Milk production losses in Latxa dairy sheep associated with small ruminant lentivirus infection.

Visna/Maedi is a disease of sheep caused by small ruminant lentivirus (SRLV) infection that is widespread throughout the world and that has been recognized to be present in the Basque Country (Spain) since the early 1980's. Nearly seven decades of studies have improved the knowledge on its clinical signs and epidemiology. However, its slow progressive nature, subclinical most of the time, makes difficult to assess its real impact on productive traits, a question of critical importance to balance out the economic costs it causes and the benefits of designing and deploying an eradication program. Development of a dairy breeding program since the 90 s in the local Latxa sheep population has provided data on milk productivity in several flocks where SRLV infection prevalence has been continuously monitored. This study analyses retrospectively the association between SRLV prevalence and production variables during ten yearly lactations in three Latxa dairy flocks with medium-high SRLV seroprevalence. Our results indicate that average standard lactation of seropositive sheep was 6.7 % lower than controls. The largest differences (p < 0.001) were observed at the ewe lifetime peak of production between second and fourth lactations. Lifelong milk and lamb production data indicated even a higher impact, with costs rising up to nearly 50 €/ewe/year. This substantial production decrease associated with subclinical SRLV infection in Latxa dairy sheep supports the benefit of establishing a SRLV control program. A rough cost-benefit analysis indicated that even in a medium-yielding breed, testing expenses would be largely covered by milk production improvement.

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.

Seroprevalence and risk factors related to small ruminant lentivirus infections in Belgian sheep and goats.

Maedi-Visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are two prototype members of the group of small ruminant lentiviruses (SRLVs). Both result in progressive and persistent infections of sheep and goats that impact animal health and cause economic losses. In Belgium, the sheep and goat sector is small and consists mostly of hobbyist farmers keeping few animals. A voluntary control program however exists, but less than 2% of the farmers participate to the program. The current lack of SRLV seroprevalence data and knowledge on risk factors related to SRLV seropositivity in this hobbyist sector makes it difficult to evaluate the risk of SRLV transmission from non-certified to SRLV free certified farms. We performed a nationwide SRLV seroprevalence study based on a stratified sampling proportional to the number of sheep and goat holders per province. Randomly selected sheep and goat owners were invited to participate and subject to a short questionnaire to collect information about flock size, animal health condition, age, flock constitution and housing conditions. Samples were collected from maximum 7 animals per farm and tested in a commercial ELISA. In total, we received samples from 87 sheep and 76 goat farms. Sheep flocks showed an overall seroprevalence of 9% (CI 95%: 5-15) and a between-herd seroprevalence of 17% (CI 95%:11-27). Seroprevalence at animal level in goat flocks was 6% (CI 95%: 3-12) and the between-herd seroprevalence was 13% (CI 95%: 7-23). Multiple sheep and goat breeds were found SRLV seropositive. Answers provided during the questionnaire confirmed the mostly hobbyist nature of the sector and showed that more than 65% of sheep and goat farmers had never heard of the disease. The only risk factor found to be related to SRLV seroprevalence was flock size. Herds of more than 10 goats had significantly higher chance to harbor seropositive animals (OR: 4.36; CI: 1.07; 17.73). In conclusion, it was shown that participants to the SRLV free certification program are at risk for reintroduction of the disease in their herds since SRLVs are present on about 15%-20% of non-certified farms. Except from flock size, no clear risk factors were found that are helpfull to identify flocks at risk. Greater effort should be made to inform sheep and goat farmers about the existence and consequences of this disease in order to promote the voluntary control program and further reduce the disease prevalence.

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.

High prevalence of caprine arthritis encephalitis virus (CAEV) in Taiwan revealed by large-scale serological survey.

In this study, a large-scale serological survey of caprine arthritis encephalitis virus (CAEV) infection was conducted between March 2011 and October 2012. 3,437 goat blood or milk samples were collected from 65 goat farms throughout Taiwan. A commercial ELISA kit was used to detect antibodies against CAEV. The overall seropositive rate was 61.7% (2,120/3,437) in goats and in 98.5% (64/65) of goat farms. These results provide the first large-scale serological evidence for the presence of CAEV infection, indicating that the disease is widespread in Taiwan.

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.

Efficient replication of caprine arthritis-encephalitis virus in goat granulosa cells.

Recent reports demonstrated the susceptibility of epithelial cells from different organs to caprine arthritis-encephalitis virus (CAEV) both in vitro and in vivo. Since granulosa cells (GC) are of epithelial origin and currently used for in vitro oocyte maturation, we addressed the question whether these cells are susceptible or resistant to CAEV infection. GC were isolated from goats from certified CAEV-free herds. PCR analysis on GC DNA using CAEV specific primers confirmed the absence of CAEV infection and immunocytochemistry using specific K813 anti-cytokeratin monoclonal antibodies confirmed the epithelial nature of GC. These cells were then inoculated with CAEV using two strains: the CAEV-pBSCA molecular clone and the CAEV-3112 French field isolate. 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 an hyperimmune serum. Supernatant of infected cells were shown to contain high titers (ranging 10(5) tissue culture infectious doses 50 per ml: TCID(50) per ml) of infectious cytopathic viruses when assayed onto the indicator goat synovial membrane (GSM) cells. Our findings demonstrate the large cell tropism of CAEV and suggest that GC could serve as a reservoir for the virus during the sub-clinical phase of infection. Furthermore, given the high seroprevalence of CAEV in the all industrialised countries and the large number of ovaries derived from unknown serological status animals used for in vitro goat embryo production, one can conclude that these feeder cell cultures might be a potential source of early transmission of CAEV to goat embryos.

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.

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.

Establishment and characterization of a goat synovial membrane cell line susceptible to small ruminant lentivirus infection.

Primary goat synovial membrane (GSM) cells are widely used to study small ruminant lentiviruses (SRLV), i.e. maedi visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV), but their limited life-span of 15-20 passages in vitro is problematic. Here, we report that ectopic expression of the catalytic subunit of human telomerase (hTERT) was sufficient to immortalize primary GSM cells. Cultures of hTERT-transfected GSM cells have been passaged for 2 years without showing any phenotypic difference from the original primary GSM cells. The hTERT-transfected cells continued to grow beyond a population doubling number of 250, while no net telomere lengthening was observed for these cells. Moreover, the immortalized GSM cells were susceptible to infection by both CAEV and MVV and were able to propagate theses viruses. Such cell line provides a useful source of standard and robust cells for both research and veterinary purposes.