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.

Antibody response to the surface envelope of caprine arthritis-encephalitis lentivirus: disease status is predicted by SU antibody isotype.

This study evaluated the hypothesis that the disease status of Saanen goats infected with caprine arthritis-encephalitis lentivirus (CAEV) is associated with the focus of immune responses to viral antigens, particularly the surface envelope glycoprotein (SU). Specifically, we have proposed that Th2 responses promote progressive immune-mediated arthritis, whereas Th1 responses restrict virus replication and development of clinical disease. The present study determined the isotype of SU antibodies associated with progressor and long-term nonprogressor (LTNP) status. We show that chronically infected goats that develop clinical arthritis have predominantly IgG1 antibodies to SU during both preclinical and clinical stages of disease, whereas SU antibodies of LTNP goats are relatively biased toward IgG2. Additional studies determined the isotype of SU antibodies induced initially by CAEV infection. These experiments show that initial IgG1-dominated responses to SU are associated with subsequent development of preclinical inflammatory joint lesions, whereas lack of joint pathology is associated with an IgG2 bias of initial responses to SU. Our results using the CAEV model suggest that isotype bias of SU antibodies is a reliable indicator of clinical disease caused by lentiviruses. Isotype analysis may be a useful method to screen candidate lentiviral vaccines intended to prevent disease progression.

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.

Caprine arthritis-encephalitis virus-infected goats can generate human immunodeficiency virus-gp120 cross-reactive antibodies(1).

Lentiviruses display surprisingly disparate clinical manifestations in their specific hosts, share complex genetic structures, and exhibit extensive diversity, particularly in their envelope genes. The envelope protein, gp135, of caprine arthritis-encephalitis virus (CAEV) has minimal primary sequence homology to gp120, the envelope protein of human immunodeficiency virus (HIV). Nevertheless, they bear certain similarities in that they both possess five variable regions, both are heavily glycosylated, and both share short sequence motifs. We establish a further relationship and demonstrate that some goats, infected with CAEV, possess gp135-specific antibodies which cross-react with gp120 from several HIV strains, provided the protein is expressed in insect cells. We show that, although the cross-reactivity of these immunoglobulins depends on the level of glycosylation, nevertheless, some antibodies recognize the protein epitopes on gp120, at least some of which are linear in character. Further characterization of this unexpected cross-reaction will define its potential therapeutic utility.

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.

Detection of serum antibodies to ovine progressive pneumonia virus in sheep by using a caprine arthritis-encephalitis virus competitive-inhibition enzyme-linked immunosorbent assay.

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [35S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity.

Competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to caprine arthritis-encephalitis virus: diagnostic tool for successful eradication.

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was evaluated for the detection of serum antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) in goats. This assay utilized 96-well microtiter plates containing CAEV-63 SU captured by monoclonal antibody (MAb) F7-299 and measured the competitive displacement of horseradish peroxidase-conjugated MAb GPB 74A binding by undiluted goat sera (F. Ozyörük, W. P. Cheevers, G. A. Hullinger, T. C. McGuire, M. Hutton, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 8:44-51, 2001). Two hundred serum samples from goats in the United States were used to determine the sensitivity and specificity of cELISA based on the immunoprecipitation (IP) of [(35)S]methionine-labeled viral antigens as a standard of comparison. A positive cELISA was defined as >33.2% inhibition of MAb 74A binding based on 2 standard deviations above the mean percent inhibition of 140 IP-negative serum samples. At this cutoff value, there were 0 of 60 false-negative sera (100% sensitivity) and 5 of 140 false-positive sera (96.4% specificity). Additional studies utilized IP-monitored cELISA to establish a CAEV-free herd of 1,640 dairy goats.

CD21-positive follicular dendritic cells: A possible source of PrPSc in lymph node macrophages of scrapie-infected sheep.

Natural sheep scrapie is a prion disease characterized by the accumulation of PrP(Sc) in brain and lymphoid tissues. Previous studies suggested that lymph node macrophages and follicular dendritic cells (FDC) accumulate PrP(Sc). In this study, lymph nodes were analyzed for the presence of PrP(Sc) and macrophage or FDC markers using dual immunohistochemistry. A monoclonal antibody (mAb) to the C-terminus of PrP reacted with CD172a+ macrophages and CD21+ FDC processes in secondary follicles. However, a PrP N-terminus-specific mAb reacted with CD21+ FDC processes but not CD172a+ macrophages in secondary follicles. Neither the PrP N-terminus nor C-terminus-specific mAb reacted with CD172a+ macrophages in the medulla. These results indicate that lymph node follicular macrophages acquire PrP(Sc) by phagocytosis of CD21+ FDC processes. The results also suggest that follicular macrophages have proteases that process full-length PrP(Sc) to N-terminally truncated PrP(Sc).

A maedi-visna virus strain K1514 receptor gene is located in sheep chromosome 3p and the syntenic region of human chromosome 2.

The maedi-visna lentivirus (MVV) induces encephalitis, interstitial pneumonia, arthritis and mastitis in sheep. While some MVV strains can enter cells of ruminant species only, others can enter cells from many species, including human, but not Chinese hamster cells. However, the identity of the receptor(s) used by MVV for entry is unknown. The MVV-K1514 receptor gene was localized in sheep and human chromosomes using hamster x sheep and hamster x human hybrid cell lines. Based on entry by a vector pseudotyped with the MVV-K1514 envelope, the MVV-K1514 receptor gene was mapped to sheep chromosome 3p and to a region of human chromosome 2 (2p25>q13), which has conserved synteny with sheep chromosome 3p. These regions do not include any known lentivirus receptor or coreceptor gene, indicating that MVV-K1514 uses a new lentivirus receptor to infect human cells.

Pregnancy status and fetal prion genetics determine PrPSc accumulation in placentomes of scrapie-infected sheep.

Ovine scrapie is a fatal neurodegenerative disorder that may be transmitted through exposure to infected uterine and placental tissues. Susceptibility to scrapie is primarily controlled by polymorphisms in the prion protein (PrP) gene. Scrapie in the U.S. Suffolk breed and in many breeds in Europe occurs in sheep homozygous for glutamine (171QQ), but rarely in sheep heterozygous for glutamine and arginine (171QR) or homozygous for arginine (171RR) at codon 171 of the PrP gene. This study demonstrated that accumulation of PrP(Sc) in uterine-placental epithelial cells in the placentome was determined by fetal PrP genotype and the pregnancy status of scrapie-infected ewes. PrP(Sc) was detected in 171QQ placentomes of infected ewes, but not in placentomes of infected ewes pregnant with 171QR conceptuses or in the non-pregnant uterus of infected ewes. The distribution of PrP(Sc) plaques in placentomes was temporally associated with stage of gestation. There was a tendency toward increased size and number of placentomal PrP(Sc) plaques from the endometrial stalk (maternal side) to chorionic plate (fetal side). These results indicate that accumulation of PrP(Sc) is eliminated or reduced to undetectable levels in reproductive and placental tissues if infected ewes are not pregnant or conceive conceptuses with a resistant PrP genotype.

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.

PrP(Sc) is not detected in peripheral blood leukocytes of scrapie-infected sheep: determining the limit of sensitivity by immunohistochemistry.

Peripheral blood leukocytes (PBLs) from scrapie-infected sheep were evaluated for the presence of PrP(Sc) by using dissociated retropharyngeal lymph node (DRLN) cells and immunohistochemistry (IHC). PrP(Sc)-positive cells were detected in 2.05% +/- 0.28% of 3 x 10(6) DRLN cells, but were not detected in 3 x 10(6) PBLs from scrapie-infected sheep. Titration of DRLN cells mixed with PBLs showed that IHC detects a minimum of 0.00205% or 60 PrP(Sc)-positive cells in 3 x 10(6) PBLs.

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.