Seven new ovine progressive pneumonia virus (OPPV) field isolates from Dubois Idaho sheep comprise part of OPPV clade II based on surface envelope glycoprotein (SU) sequences

Seven new ovine progressive pneumonia virus (OPPV) field isolates were derived from colostrum and milk of 10 naturally OPPV-infected sheep from the US Sheep Experiment Station in Dubois, Idaho, USA. Sixteen sequences of the surface envelope glycoprotein (SU) from these seven Dubois OPPV field isolates and SU sequence from OPPV WLC1 were obtained, aligned with published SRLV SU sequences, and analyzed using phylogenetic analysis using parsimony (PAUP). Percent nucleotide identity in SU was greater than 95.8% among clones from individual Dubois OPPVs and ranged from 85.5 to 93.8% between different Dubois OPPV clones. SU sequences from Dubois OPPVs and WLC1 OPPV had significantly higher percent nucleotide identity to SU sequences from the North American OPPVs (85/34 and S93) than caprine-arthritis encephalitis virus (CAEVs) or MVVs. PAUP analysis also showed that SU sequences from the Dubois OPPVs and OPPV WLC1 grouped with other North American OPPVs (85/34 and S93) with a bootstrap value of 100 and formed one OPPV clade II group. In addition, Dubois and WLC1 SU amino acid sequences had significantly higher identity to SU sequences from North American OPPVs than CAEV or MVV. These data indicate that the seven new Dubois OPPV field isolates along with WLC1 OPPV are part of the OPPV clade II and are distinct from CAEVs and MVVs.     

Natural transmission and comparative analysis of small ruminant lentiviruses in the Norwegian sheep and goat populations

Serological surveys for small ruminant lentivirus (SRLV) infections have revealed seropositive sheep in several mixed herds, where sheep are kept together with seropositive goats. Here we have examined the genetic relationships in LTR, pol and env surface unit (SU) and the growth patterns in goat (GSM) and sheep (FOS) synovial membrane cell cultures of SRLV isolates obtained from both mixed and single species herds. Phylogenetic analyses of pol and env SU revealed that Norwegian SRLVs derived from both goat and sheep in mixed herds are distributed into group C, while isolates obtained from unmixed sheep flocks cluster in group A, together with maedi-visna-like representatives of the A1 subtype. In this study, the direction of group C virus transmission is proposed to be from goat to sheep. The replication efficiency in GSM and FOS cultures and the cytopathic phenotype induced by the SRLV isolates gave no indication of any species-specific characteristics. No particular nucleotide sequences of the LTR-U3 region or env SU were identified that could be related to cytopathic phenotype. This study shows that sheep in Norway harbour SRLVs belonging to phylogenetic groups A and C, and this provides further evidence for cross-species infection being a regular characteristic of SRLVs, which may represent an important source for viral persistence.     

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.     

Evaluation of five enzyme-linked immunosorbent assays and an agar gel immunodiffusion test for detection of antibodies to small ruminant lentiviruses

In the framework of the Dutch control program for small ruminant lentiviral (SRLV) infections, too many drawbacks were encountered with respect to serological testing. To improve the quality of testing, five enzyme-linked immunosorbent assays (ELISAs) and an agar gel immunodiffusion test (AGIDT) were evaluated. The focus was on the sensitivity, specificity, and variances of the commercially available tests. Clear differences were found among the tests in analytical and diagnostic sensitivity and overall diagnostic performance, whereas no significant differences in specificity were found. For serodiagnosis of sheep with clinical symptoms of maedi-visna virus (MVV) (histopathologically confirmed), one ELISA was significantly more sensitive than the other ELISAs and than the AGIDT, while for asymptomatic sheep originating from infected flocks, three ELISAs and the AGIDT demonstrated similar performance. The diagnostic performance appeared to be related to animal species and virus infection (MVV or caprine arthritis encephalitis virus [CAEV]) as well as the phase of infection/progression of disease. Receiver operating characteristic analysis, demonstrating the diagnostic potential of tests irrespective of defined cutoffs, again revealed clear differences between tests with respect to diagnostic performance for detection of antibodies against CAEV or MVV. An indirect ELISA, of which the solid phase is sensitized with a combination of the core protein p27 of MVV produced in Escherichia coli and a peptide derived from the transmembrane protein gp46, appeared to be the test of choice for serodiagnosis of SRLV infections in sheep and goats.     

Phylogenetic analysis of small-ruminant lentivirus subtype B1 in mixed flocks: evidence for natural transmission from goats to sheep

Small-ruminant lentiviruses (SRLV), consisting of the caprine arthritis-encephalitis virus (CAEV) and the maedi-visna virus (MVV), cause chronic multisystemic infections in goats and sheep. The SRLV subtype B1, characterized by the prototypic strain CAEV-CO, has a worldwide distribution and, remarkably, has been isolated exclusively from goats, suggesting potential host specificity. To test this hypothesis, SRLV pol sequences were obtained by PCR amplification from blood samples of seropositive dairy goats and sheep living in mixed flocks. Phylogenetic analysis of these sequences demonstrates that SRLV subtype B1 does cross the species barrier under field conditions through direct contact between adult animals. This implies that SRLV control programs targeting only sheep or goats can no longer be proposed (based on a putative species specificity of the SRLV subtype B1).     

Small ruminant lentivirus Tat protein induces apoptosis in caprine cells in vitro by the intrinsic pathway

The small ruminant lentiviruses, caprine arthritis-encephalitis virus (CAEV) and maedi visna virus (MVV) naturally cause inflammatory disease in goats and sheep, provoking chronic lesions in several different organs. We have previously demonstrated that in vitro infection of caprine cells by CAEV induces apoptosis through the intrinsic pathway (Rea-Boutrois, A., Pontini, G., Greenland, T., Mehlen, P., Chebloune, Y., Verdier, G. and Legras-Lachuer, C. 2008). In the present study, we used Tat deleted viruses and SLRV Tat-expression vectors to show that the SRLV Tat proteins are responsible for this apoptosis. We have also studied the activation of caspases-3, -8 and -9 by fluorescent assays in caprine cells expressing SRLV Tat proteins, and the effects of transfected dominant negative variants of these caspases, to show that Tat-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of the mitochondrial pathway.     

Development of a semi-nested PCR using degenerate primers for the generic detection of small ruminant lentivirus proviral DNA

A PCR assay was developed for the reliable detection of small ruminant lentivirus (SRLV) proviral DNA. The method involved the use of degenerate deoxyinosine-substituted primers and a second semi-nested PCR step that increased the polyvalency and sensitivity of the detection, respectively. Primers were designed from the pol gene conserved motifs of 85 SRLV isolates and were evaluated using different SRLV isolates together with Maedi-Visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) reference strains. The method successfully detected SRLV proviral DNA in total DNA extracts originating from whole blood samples, separated peripheral blood mononuclear cells (PBMCs) and tissue cultures. The semi-nested PCR was compared with the agar gel immunodiffusion test and proved to be highly sensitive, specific and capable of detecting many SRLV variants in infected or suspect animals. Therefore, it would be useful in the diagnosis of natural SRLV infections, in eradication programs and epidemiological studies. Whole blood samples can be used directly, thus alleviating the need for PBMC separation, and thereby enables a simple, fast and cost-effective analysis of a large number of samples.     

Evaluation of a Caprine Arthritis-Encephalitis Virus/Maedi-Visna Virus Indirect Enzyme-Linked Immunosorbent Assay in the Serological Diagnosis of Ovine Progressive Pneumonia Virus in U.S. Sheep

A caprine arthritis-encephalitis virus (CAEV)/maedi-visna virus (MVV) indirect enzyme-linked immunosorbent assay (iELISA) was validated with samples from U.S. sheep and by the use of radioimmunoprecipitation as the standard for comparison. The sensitivity and the specificity were 86.0% (±5.8%) and 95.9% (±2.9%), respectively. The iELISA format and phylogenetic differences based on the MVV gag sequence contribute to the reduced sensitivity.The diagnosis of small ruminant lentivirus (SRLV) infections in sheep and goats is most commonly determined by the detection of anti-SRLV antibodies in serum by an enzyme-linked immunosorbent assay (ELISA) that is typically created by the use of maedi-visna virus (MVV) or caprine arthritis-encephalitis virus (CAEV) isolates from sheep or goats of a given region or country (1). ELISA formats are typically validated against reference standard tests, including the agar gel immunodiffusion (AGID) assay, the radioimmunoprecipitation (IP) assay, or Western blot analysis. Although most seropositive sheep and goats do not show clinical signs of SRLV disease, they are persistent and potential reservoirs for transmission. Therefore, highly specific and sensitive serological diagnostic assays are essential for the early detection of SRLV.Three hundred ten of 332 serum samples from U.S. sheep from a previous CAEV competitive ELISA (cELISA) validation study (4) were tested in duplicate by using a Chekit CAEV/MVV antibody test kit (IDEXX Laboratories, The Netherlands), according to the manufacturer''s instructions. The CAEV/MVV indirect ELISA (iELISA) results were compared with those of the ovine progressive pneumonia virus (OPPV) WLC1 radio-IP assay, which has been described previously (4). The CAEV/MVV iELISA utilizes whole virus from Swiss MVV strain OLV as the antigen (15, 16). With a value of ≥60% being defined as a CAEV/MVV iELISA-positive serum sample, the sensitivity and the specificity of the CAEV/MVV iELISA were 74.0% (±7.6%) (95% confidence interval) and 98.3% (±2.0%), respectively, compared to the results of the radio-IP assay. Since the sensitivity was less than adequate, we reassessed the cutoff by calculating the mean value (in percent) ± 2 standard deviations for the radio-IP assay-negative serum samples. The results of that analysis placed the cutoff mean value at 33.1%. By using the new cutoff value, the sensitivity of the iELISA improved to 86.0% (±5.8%) and the specificity decreased slightly to 95.9% (±2.9%) compared to the results of the radio-IP assay. However, compared to the CAEV cELISA, which has a sensitivity of 98.6% and a specificity of 96.9% when the results of the radio-IP assay are used as the reference standard, the iELISA had a reduced sensitivity.Since the sera were taken from a number of different U.S. sheep kept under different husbandry and management conditions, we also wanted to test the performance of the CAEV/MVV iELISA with sera from one flock in which the sheep are exposed to the same husbandry and management conditions. Sera from an Idaho sheep flock (n = 405) consisting of sheep of the Rambouillet, Polypay, and Columbia breeds ages 3, 4, 5, and 6 years were tested by the iELISA. The results were compared to those of the CAEV cELISA by using the new iELISA cutoff value of 33.1%, and the discrepant samples were analyzed by Western blotting with OPPV WLC1 and by previously published methods (2). The positive and negative concordances of the CAEV cELISA and the CAEV/MVV iELISA were 92.5% (±3.1%) and 99.3% (±1.4%), respectively. Eighteen of 20 CAEV/MVV iELISA-negative and CAEV cELISA-positive serum samples tested positive by Western blot analysis, and the 2 remaining discrepant serum samples tested negative by Western blot analysis. One CAEV/MVV iELISA-positive and CAEV cELISA-negative sample tested negative by Western blot analysis. The 95% confidence interval for the positive and the negative concordances of the results of the CAEV/MVV iELISA relative to those of the CAEV cELISA for Idaho sheep and U.S. sheep overlapped (data not shown).A difference in the limit of detection between the CAEV/MVV iELISA and the CAEV cELISA may be a major reason for the reduced sensitivity of the CAEV/MVV iELISA (86%) compared to that of the CAEV cELISA (98.6%) with sera from U.S. sheep. Sera require dilution 1:10 for testing by the CAEV/MVV iELISA, whereas undiluted sera are used for the CAEV cELISA. To test whether the limit of detection is greater for the CAEV cELISA than the CAEV/MVV iELISA, 15 Western blot analysis-positive, CAEV cELISA-positive, and CAEV/MVV iELISA-negative serum samples from the Idaho flock were diluted 1:10 and 1:50 with 1× phosphate-buffered saline, pH 7.5, and retested by the CAEV cELISA. Twelve of these 15 serum samples tested positive by the CAEV cELISA at a 1:10 dilution, and 7 of 15 tested positive by the CAEV cELISA at a 1:50 dilution. This indicates that the CAEV cELISA has a higher dilution limit for the detection of anti-SRLV antibodies than the CAEV/MVV iELISA with sera from U.S. sheep. This high dilution limit of detection is likely due to the format of the CAEV cELISA, in which this assay is reliant upon anti-OPPV serum antibodies to inhibit the binding of a peroxidase-labeled monoclonal antibody (monoclonal antibody 74A) to a single, specific epitope on the CAEV-63 surface envelope glycoprotein.A previous report of 95.5% sensitivity and 97.2% specificity has been reported for the iELISA with sera from Swiss sheep (15). Large SRLV strain differences between Swiss MVV and U.S. OPPV strains may account for the lower sensitivity of the iELISA with sera from U.S. sheep. Therefore, gag, which encodes the capsid protein, was evaluated because it is a B-cell-immunodominant viral antigen in sheep naturally infected with MVV and OPPV and is more conserved than other viral genes (2, 7, 17). Unfortunately, no gag sequence is available for the Swiss MVV OLV (the strain used in the iELISA); however, several representative gag sequences from SRLV strains from Swiss goats and sheep that were previously reported for SRLV clades A1, A3, A4, A5, B1, and B2 were utilized (13). Genomic DNA was isolated from peripheral blood leukocytes from nine cELISA- and iELISA-positive Idaho sheep by previously described methods (6). The gag gene encoding the capsid was amplified by PCR with the following primers: primer GAGPSf (5′-TGG-CGA-CGC-AAG-GCT-CAA-A-3′) and primer GAGPSr (5′-GCG-GAC-GGC-ACC-ACA-CG-3′) (Integrated DNA Technology, Coralville, IA). The PCR mixture consisted of 100 ng of genomic DNA from sheep peripheral blood leukocytes or goat synovial membrane cells infected with WLC1, 2.5 mM_f (final concentration) MgCl₂, 0.2 mM_f deoxynucleoside triphosphates, and 1 U of Taq polymerase (Fisher Scientific, Pittsburg, PA). Primers GAGPSf and GAGPSr bind to nucleotides 734 to 752 and 2038 to 2054, respectively, of the sequence with GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY101611","term_id":"21539308","term_text":"AY101611"}}AY101611. The amplification conditions for the gag PCR were as follows: 95°C for 4 min, followed by 25 cycles of 95°C for 30 s, 58.1°C for 30 s, and 72°C for 2 min, followed by 72°C for 7 min and 4°C indefinitely. The gag PCR products were analyzed, cloned, and sequenced by previously described methods (5). The gag sequences were further refined by hand by using the Se-Al (version 2.0) program (http://tree.bio.edu.ac.uk/software/seal/), and a consensus gag sequence was generated from gag sequences from four sheep and OPPV WLC1.For phylogenetic analysis, the appropriate model of nucleotide substitution was selected by using hierarchical likelihood ratio testing with the MrModelTest2 command block (11) executed in the PAUP program (14). A general time-reversible model (also called the GTR or REV model) (12) with invariant sites and a measure of the rate of heterogeneity determined by use of the gamma distribution was chosen for full Bayesian analysis by using the program MrBayes (8). A 50%-majority-rule consensus tree based on the gag sequences was constructed by using the Mesquite software package (10). The resulting Bayesian phylogenetic tree showed that the sequences from the Idaho sheep formed one clade with OPPV WLC1, OPPV 85/34, and a single Swiss goat SRLV strain (strain 5692 A3) with high posterior probabilities (0.99 to 1.0) (Fig. ​(Fig.1).1). The finding that three gag sequences from Swiss sheep are found in other clades aside from the U.S. sheep OPPV clade (6247 A1, 5720 B2, and 5776 B1) suggests that Swiss sheep SRLV strains are quite different from U.S. OPPV strains, and these differences contribute to the reduced sensitivity of the iELISA with sera from U.S. sheep compared to that achieved with sera from Swiss sheep.Open in a separate windowFIG. 1.A 50%-majority-rule consensus tree of SRLV gag nucleotide sequences obtained by Bayesian phylogenetic analyses with the general time-reversible model with invariant sites and a measure of the rate of heterogeneity. Consensus gag nucleotide sequences from Idaho sheep (sheep 28Oct76, 29Oct10, 28Oct101, 28Oct27, 28Oct83, 28Oct18, 28Oct89, 28Oct68, and 28Oct99) and a consensus gag nucleotide sequence for WLC1 were utilized and compared to the gag nucleotide sequences from other U.S. OPPV strains (85-34), MVV strains, and CAEV strains. The GenBank accession numbers are given in parentheses next to the name or identification number given in previous studies. The numbers on the branches represent the Bayesian posterior probabilities.This combination of information suggests that U.S. sheep maintain strains of small ruminant lentiviruses phylogenetically different (determined on the basis of the gag sequences) from those from most other parts of the world. This suggests that the creation and validation of one serological assay that is based on one viral strain and that is accepted worldwide might not be successful due to the diversity of SRLV strains worldwide (3, 9).     

Genomic heterogeneity of small ruminant lentiviruses: existenceof heterogeneous populations in sheep and of the samelentiviral genotypes in sheep and goats

Summary.  We have recently shown that French small ruminant lentiviruses (SRLV) from sheep are more similar to Caprine Arthritis Encephalitis Virus (CAEV) than to visna maedi virus (VMV) in a conserved region of the pol gene. To extend these results, we have examined sequences from a variable region of the env gene in French SRLV. We found that they were nearly equally distant from both CAEV and VMV strains, suggesting a considerable divergence since the initial introduction of the virus. Analysis of separate clones from individual animals showed that some carry a population of variant viruses. The study of further pol gene sequences from both goats and sheep suggests that viral variants show little or no host species specificity. A phylogenetic tree of pol gene sequences confirmed the presence of a novel genotype of SRLV in France. Accepted January 10, 1997 Received August 12, 1996     

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.     

Caprine Toll‐like receptor 8 gene sequence characterization reveals close relationships among ruminant species

TLR8 mediates antiviral immunity by recognizing ssRNA viruses and triggers potent antiviral and antitumor immune responses. In this study, approximately 3.5 Kb nucleotide sequence data of caprine TLR8 gene were generated from one sample each of twelve different Indian goat breeds belonging to different geographical regions. Cloning and characterization of cDNA synthesized from RNA purified from goat spleen revealed TLR8 ORF to be of 3102 nucleotides long coding for 1033 amino acids similar to other ruminant species, that is sheep, buffalo and cattle. The sequence analysis at nucleotide level revealed goat TLR8 to be closer to buffalo sharing 99.6% homology, followed by cattle and sheep. Simple Modular Architecture Research Tool (SMART) used for the structural analysis of goat TLR8 showed the presence of 16 leucine‐rich repeats (LRRs) along with single Toll/interleukin‐1 receptor (TIR) domain. TIR domain when compared with other livestock species was found to be conserved in ruminant species goat, sheep, cattle and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to buffalo followed by cattle and sheep. Total 4 polymorphic sites were observed in TLR8 gene of one specimen goat representing each of 12 different breeds studied, all of which were synonymous and present within the coding region. Of these 4 SNPs, two were in ectodomains, one in TIR domain and one was found to be present in transmembrane domain. PCR‐RFLP genotyping of two of the SNPs indicated variations in allele frequencies among different goat breeds. The expression profiling in 13 tissues of goat showed maximum expression of TLR8 gene in kidney followed by spleen, lung and lymph node. Overall, our results indicate conservation of TLR8 gene among the ruminant species and low variation within Indian goat breeds.     

Genetic and antigenic characterization of small ruminant lentiviruses circulating in Poland

Small ruminant lentivirus (SRLV) infections are widespread in Poland, but the genetic features of sheep viruses are still lacking and limited to partial gag sequences for goat viruses. In this study, segments from the gag and env genes of Polish SRLV strains screened by heteroduplex mobility assay were subjected to genetic analyses. Subtype A1 was found in both sheep and goats, while subtypes B1 and B2 were found in goats and sheep, respectively. In addition, two novel subtypes (named A12 and A13) were found in sheep. Their close phylogenetic relatedness with SRLV strains previously isolated from Polish goats indicated that these new subtypes are predominant and circulate in both species. The antigenic relationships of subtypes A12 and A13 with other SRLV subtypes were tested in an ELISA assay based on recombinant antigens carrying the immunodominant domains of structural proteins (MA, CA and SU). Antigenic cross-reactivity in the Gag epitopes was evident among genotype A subtypes and, to a lower extent, between genotypes A and B. In contrast, a subtype-specific immunoresponse was detected in the SU epitopes. These results emphasize the broad genetic and antigenic diversity of SRLV strains circulating in Europe and confirmed the need to consider all viral genotypes to choose the antigens in serological tests in order to avoid misdiagnosis in control and eradication programs.     

Mutations increasing exposure of a receptor binding site epitope in the soluble and oligomeric forms of the caprine arthritis-encephalitis lentivirus envelope glycoprotein

The caprine arthritis-encephalitis (CAEV) and ovine maedi-visna (MVV) viruses are resistant to antibody neutralization, a feature shared with all other lentiviruses. Whether the CAEV gp135 receptor binding site(s) (RBS) in the functional surface envelope glycoprotein (Env) is protected from antibody binding, allowing the virus to resist neutralization, is not known. Two CAEV gp135 regions were identified by extrapolating a gp135 structural model that could affect binding of antibodies to the RBS: the V1 region and a short sequence analogous in position to the human immunodeficiency virus type 1 gp120 loop B postulated to be located between two major domains of CAEV gp135. Mutation of isoleucine-166 to alanine in the putative loop B of gp135 increased the affinity of soluble gp135 for the CAEV receptor(s) and goat monoclonal antibody (Mab) F7-299 which recognizes an epitope overlapping the gp135 RBS. The I166A mutation also stabilized or exposed the F7-299 epitope in anionic detergent buffers, indicating that the I166A mutation induces conformational changes and stabilizes the RBS of soluble gp135 and enhances Mab F7-299 binding. In contrast, the affinity of a V1 deletion mutant of gp135 for the receptor and Mab F7-299 and its structural stability did not differ from that of the wild-type gp135. However, both the I166A mutation and the V1 deletion of gp135 increased cell-to-cell fusion activity and binding of Mab F7-299 to the oligomeric Env. Therefore, the CAEV gp135 RBS is protected from antibody binding by mechanisms both dependent and independent of Env oligomerization which are disrupted by the V1 deletion and the I166A mutation, respectively. In addition, we found a correlation between side-chain beta-branching at amino acid position 166 and binding of Mab F7-299 to oligomeric Env and cell-to-cell fusion, suggesting local secondary structure constraints in the region around isoleucine-166 as one determinant of gp135 RBS exposure and antibody binding.     

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.     

Adaptation of wild-type measles virus to CD46 receptor usage

Summary.  Vaccine strains of measles virus (MV) use CD46 as receptor and downregulate CD46 from the surface of infected cells. MVs isolated and passaged on B-lymphoid cells (wild-type MVs) seem to use another receptor and do not downregulate CD46. In the present study, we found that isolation of MV on human or marmoset B-lymphoid cells did not alter the MV haemagglutinin (H) protein relative to that in the patient. The wild-type isolates were adapted to the human epithelial HEp-2 cell line or the monkey fibroblast Vero cell line. All HEp-2 cell adapted viruses and 1 out of 4 Vero cell adapted viruses acquired the capacity to use CD46 as receptor, as measured by their ability to infect murine cells expressing human CD46. Adaptation to CD46 receptor usage was coupled to substitution of amino acid 481 of the MV H protein from asparagine to tyrosine but not to CD46 downregulation. The present study demonstrates that CD46 receptor usage can be induced by adaptation of wild-type MV to cells that do not express a wild-type receptor and suggests that a similar mechanism acted on the progenitor viruses of the present MV vaccine strains during their isolation and attenuation. Received June 5, 2000 Accepted October 5, 2000