Antigenic variation of neutralization-sensitive epitopes of caprine arthritis-encephalitis lentivirus during persistent arthritis.

Caprine arthritis-encephalitis virus (CAEV), a naturally occurring lentivirus of goats, causes disease characterized by virus persistence and recurrent arthritis. These studies demonstrate in vitro neutralization of CAEV infectivity by serum from goats infected with CAEV. Serum neutralizing activity was not detectable until 10 to 36 months postinfection, and titers were relatively low (less than or equal to 1:8). Serum neutralization was caused by antibody and was virus specific. Antigenic variants of CAEV were isolated from cell-free joint fluid of arthritic goats 9 to 18 months postinfection. The delayed appearance of neutralizing antibody and the subsequent development of antigenic variants may promote CAEV persistence in vivo and provide a stimulus for recurrent arthritis.     

Sialic acids on the surface of caprine arthritis-encephalitis virus define the biological properties of the virus.

The lentivirus caprine arthritis-encephalitis virus (CAEV) is a pathogen of goats. It is transmitted in milk and causes a persistent infection in goats, which often fail to produce neutralizing antibodies to the virus. Native CAEV particles are remarkably resistant to digestion with proteinase K and are neutralized extremely slowly by immune sera. Our studies showed that the virus particles are heavily sialylated. Studies with highly specific sialyltransferase enzymes identified penultimate carbohydrate linkages typical of O- and N-linked oligosaccharides on the virus and suggested that the virus may be more heavily sialylated on O-linked than on N-linked oligosaccharides. Removal of sialic acids from the virus by neuraminidase treatment did not reduce infectivity of the particles. However, desialylation rendered the virus more susceptible to proteolysis by proteinase K. Desialylation also enhanced the kinetics of neutralization of the virus by goat antibodies. These results suggest that the carbohydrates on the viral surface are important both in protecting viral proteins from digestion by proteases and in protecting the virus from rapid neutralization by antibodies.     

Antibody reactivity to the immunodominant epitopes of the caprine arthritis-encephalitis virus gp38 transmembrane protein associates with the development of arthritis.

High titers of antibodies to caprine arthritis-encephalitis virus (CAEV) envelope (Env) glycoproteins are found in infected goats developing a progressive arthritis. In order to identify linear B epitopes of the CAEV Env, which may be involved in the immunopathology of arthritis, we constructed a lambda gt11 Env expression library. By combining library screening with sera from naturally infected Swiss goats with an enzyme immunoassay with overlapping peptides (pepscan), four group-specific epitopes could be precisely defined in the transmembrane envelope proteins: TM1 to TM4, including a conserved structure (TM3) that corresponds to the immunodominant epitope of human immunodeficiency virus type 1 and other lentiviruses. A panel of 190 CAEV naturally infected goat serum samples, obtained from animals with defined clinical status, was tested for reactivity to synthetic peptides corresponding to the TM epitopes in an enzyme-linked immunosorbent assay. Antibody reactivity to two epitopes was highly associated (TM3, P = 0.002, and TM4, P < 0.001) with the presence of clinically detectable arthritis. Such an association is absent for anti-Gag antibody. Antibodies to the immunodominant structures of the TM glycoprotein could thus have an important role in the immunopathogenic process leading to disease.     

Early embryonic cells from in vivo-produced goat embryos transmit the caprine arthritis-encephalitis virus (CAEV)

The aim of this study was to investigate whether cells of early goat embryos isolated from in vivo-fertilized goats interact with the caprine arthritis-encephalitis virus (CAEV) in vitro and whether the embryonic zona pellucida (ZP) protects early embryo cells from CAEV infection. ZP-free and ZP-intact 8-16 cell embryos were inoculated for 2 h with CAEVat the 10(4) tissue culture infectious dose 50 (TCID50)/ml. Infected embryos were incubated for 72 h over feeder monolayer containing caprine oviduct epithelial cells (COECs) and CAEV indicator goat synovial membrane (GSM) cells. Noninoculated ZP-free and ZP-intact embryos were submitted to similar treatments and used as controls. Six days postinoculation, infectious virus assay of the wash fluids of inoculated early goat embryos showed typical CAEV-induced cytopathic effects (CPE) on indicator GSM monolayers, with fluids of the first two washes only. The mixed cell monolayer (COEC + GSM) used as feeder cells for CAEV inoculated ZP-free embryos showed CPE. In contrast, none of the feeder monolayers, used for culture of CAEV inoculated ZP-intact embryos or the noninoculated controls, developed any CPE. CAEV exposure apparently did not interfere with development of ZP-free embryos in vitro during the 72 h study period when compared with untreated controls (34.6 and 36% blastocysts, respectively, P > 0.05). From these results one can conclude that the transmission of infectious molecularly cloned CAEV-pBSCA (plasmid binding site CAEV) by embryonic cells from in vivo-produced embryos at the 8-16 cell stages is possible with ZP-free embryos. The absence of interactions between ZP-intact embryos and CAEV in vitro suggests that the ZP is an efficient protective embryo barrier.     

Morphological and immunological comparison of caprine arthritis encephalitis and ovine progressive pneumonia viruses.

Caprine arthritis encephalitis virus (CAEV) causes a variety of pathological conditions ranging from mild to very severe and from acute to chronic, depending upon the age of initial infection and other variables. Although the virus has been reported to have properties of characteristic of retroviruses and to be related to maedi-visna virus (also called progressive pneumonia virus [PPV]), relatively little information about its morphological and immunological characteristics has been reported. We describe the morphological features of CAEV replicating in cultured caprine cells. Although the virus replicates slowly and very little virus is released from productively infected cells, it is apparent that the morphogenesis of CAEV is strikingly similar to that of maedi-visna. After the transmission of CAEV to a more permissive permanent cell line derived from Himalayan tahr ovary, it was possible to grow and purify enough virus to initiate biochemical characterization. The structural proteins of CAEV are generally very similar to those of PPV, suggesting that the two viruses are closely related but not identical. This was substantiated by showing that serum from a CAEV-infected goat immunoprecipitated both CAEV and PPV virion structural antigens from extracts of radiolabeled virus and also precipitated putative nonstructural viral antigens from extracts of both CAEV- and PPV-infected cells.     

Presence of caprine arthritis-encephalitis virus (CAEV) infected cells in flushing media following oviductal-stage embryo collection

To improve the knowledge on the risk of transmission of the caprine arthritis-encephalitis virus (CAEV) during embryo manipulations, we conducted a double-nested polymerase chain reaction (PCR) for CAEV proviral-DNA on flushing media recovered from the oviducts 48 h after the beginning of estrus and on blood from 89 donor does. Sixty-four does had negative blood and flushing media by PCR. Among the 25 CAEV infected goats (blood PCR positive), 11 were PCR flushing media positive (P < 0.01). Cell lysate from flushing media samples that were PCR positive were serially diluted 10 times at 1:100. Starting with the second 1:100 dilution all the cell lysate samples were PCR negative. The mean number of embryos recovered was not significantly different between goats with flushing media PCR positive and goats with flushing media PCR negative (6.0 +/- 5.4 versus 7.8 +/- 4.4, respectively; mean +/- S.D.) nor between goats with blood PCR positive and goats with blood PCR negative (7.0 +/- 5.0 versus 5.9 +/- 5.3; mean +/- S.D.). The presence of CAEV infected cells in oviductal flushing media from infected donor does was indicated for the first time during this study. The absence of flushing media PCR positive for goat blood PCR negative seemed to allow the use of the blood PCR test to confidently predict the absence of CAEV provirus in the oviductal fluid.     

Presence of caprine arthritis-encephalitis virus (CAEV) proviral DNA in genital tract tissues of superovulated dairy goat does

Transmission of caprine arthritis-encephalitis virus (CAEV) is not completely understood and the vertical route of infection from the goat to the embryo or to the fetus needs to be investigated. This route of infection involves the presence of CAEV in the genital tract tissues. Prior studies have detected CAEV-infected cells in genital secretions and in flushing media recovered during embryo collection from infected goats. To specify the origin of these cells, we conducted a double-nested polymerase chain reaction (PCR) test on embryo flushing media and on mammary gland, mammary lymph node, synovial membrane, pelvic lymph node, uterus and oviduct tissues from 25 CAEV-infected (blood PCR positive) embryo donor goats for the presence of CAEV proviral DNA.The presence of proviral DNA was found in 22 of 25 mammary gland samples, 14 of 25 uterus samples, and in 16 of 25 oviduct samples. Nineteen of 25 goats had at least one positive genital tract sample. Flushing media from 11 goats were PCR positive. All goats with positive-flushing media were oviduct positive. Of this group of does, except for 1 of the 11, infection of flushing media correlated with infection of almost all the other tissues examined. The frequency of positive tissues for flushing media-positive goats (61/66; 92%) was significantly higher than that for flushing media-negative goats (50/84; 60%) (P<0.01).This study demonstrated the presence of CAEV-infected cells in the goat genital tract. The presence of CAEV-infected cells in the uterus and oviducts suggests potential for vertical transmission of CAEV from doe to embryo or fetus.     

Epidemiological studies on caprine arthritis-encephalitis virus infection in Jordan

To investigate the seroprevalence of, and risk factors for, caprine arthritis-encephalitis virus (CAEV) infection in different breeds of goats in Jordan, sera from 1100 goats from three different geographical regions in Jordan were analyzed. Prevalence of antibodies to CAEV was determined using a competitive ELISA test. A semi-structured questionnaire was used to collect information on herd health and management. Questionnaire data were tested in a multivariable logistic regression model to elucidate risk factors associated with CAEV seropositivity. In addition, the incidence of CAEV antibodies was investigated in six goat herds located in the northern part of Jordan. Out of the 69 goat herds investigated, 16 (23.2%) had antibodies against CAEV. Individual goat true seroprevalence to CAEV was 8.9%. The highest CAEV seroprevalence was observed in goats older than 3-years and younger than 6-years of age. The seroprevalence of CAEV in goats was significantly higher (P < 0.05) in the northern part of Jordan than that in central or southern parts of Jordan. The multivariable logistic regression model identified large herd size (OD = 2.0; 95% CI: 1.1, 2.7), addition of new animals to the herd (OD = 1.3; 95% CI: 0.3, 1.6) and contact with other goat herds (OD = 1.1; 95% CI: 0.9, 2.0) as risk factors for CAEV seropositivity. The incidence of CAEV seropositivity in the six herds monitored in the northern part of Jordan ranged from 2.4 to 5.3%.     

Caprine arthritis encephalitis virus dysregulates the expression of cytokines in macrophages.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that leads to chronic mononuclear infiltration of various tissues, in particular, the radiocarpal joints. Cells of the monocyte/macrophage lineage are the major host cells of CAEV in vivo. We have shown that infection of cultured goat macrophages with CAEV results in an alteration of cytokine expression in vitro. Constitutive expression of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) was increased in infected macrophages, whereas transforming growth factor beta1 (TGF-beta1) mRNA was down-regulated. When macrophages were infected with a CAEV clone lacking the trans-acting nuclear regulatory gene tat, IL-8 and MCP-1 were also increased. No significant differences from cells infected with the wild-type clone were observed, suggesting that Tat is not required for the increased expression of IL-8 and MCP-1 in infected macrophages. Furthermore, infection with CAEV led to an altered pattern of cytokine expression in response to lipopolysaccharide (LPS), heat-killed Listeria monocytogenes plus gamma interferon, or fixed cells of Staphylococcus aureus Cowan I. In infected macrophages, tumor necrosis factor alpha, IL-1beta, IL-6, and IL-12 p40 mRNA expression was reduced in response to all stimuli tested whereas changes in expression of granulocyte-macrophage colony-stimulating factor depended on the stimulating agent. Electrophoretic mobility shift assays demonstrated that, in contrast to effects of human immunodeficiency virus infection of macrophages, CAEV infection had no effect on the level of constitutive nuclear factor-kappaB (NF-kappaB) activity or on the level of LPS-stimulated NF-kappaB activity, suggesting that NF-kappaB is not involved in altered regulation of cytokine expression in CAEV-infected cells. In contrast, activator protein 1 (AP-1) binding activity was decreased in infected macrophages. These data show that CAEV infection may result in a dysregulation of expression of cytokines in macrophages. This finding suggests that CAEV may modulate the accessory functions of infected macrophages and the antiviral immune response in vivo.     

Sequence homology between cloned caprine arthritis encephalitis virus and visna virus, two neurotropic lentiviruses.

Caprine arthritis encephalitis virus (CAEV) is an exogenous, nononcogenic retrovirus which causes neurological disease and crippling arthritis in goats. A complete CAEV genome was cloned from unintegrated viral DNA in two fragments of 9.4 and 0.4 kilobases in length, respectively. The biological activity of these clones was tested by ligation of the fragments followed by transfection onto goat synovial membrane cells; infectious virus was recovered. Cloned CAEV and visna virus, a related neurotropic virus of sheep, were compared by heteroduplex and molecular hybridization analyses. These data demonstrated that the greatest overall conservation of nucleotide sequences occurred in the gag and pol gene regions and two smaller regions, sor and the putative tat gene. The region of greatest divergence occurred in the env gene and, in particular, was localized primarily in the region coding for the glycosylated outer membrane protein. These findings and the recently demonstrated genetic relationship of visna virus, CAEV, and human T-cell lymphotropic virus type III, the etiologic agent of the acquired immune deficiency syndrome, may have important implications concerning the biological properties of these related viruses for human and veterinary medicine.     

Evidence for linkage between the caprine leucocyte antigen (CLA) system and susceptibility to CAE virus-induced arthritis in goats

The distribution of caprine leucocyte antigens (CLA) in goats from four different breeds (n=546) affected by caprine arthritis-encephalitis virus (CAEV)-induced arthritis were determined and compared breed for breed with those of infected but clinically healthy controls (n=402). Differences in frequencies of some of the CLA specificities between the affected and control groups were found, but after correction of the ordinary P values for number of observed alleles, only the CLA Be7 specificity in the Saanen breed showed a significant deviation at the 0.05 probability level. Animals of the Saanen breed carrying this specificity are less prone to develop arthritis after CAE virus infection than goats lacking this specificity. Eleven groups (multiple-case families or halfsibling groups with at least two informative diseased offspring/group) were analyzed for manifestation of the disease and segregation of the parental haplotypes. The results of the maximum likelihood test of association (P<0.005) and the calculated high lod score value of 5.70 give evidence for linkage between the locus encoding the determined class I CLA alleles and a hypothetical locus (i) coding for genes responsible for arthritis resistance/susceptibility. The particular class I CLA allele associated with the disease susceptibility varied from family to family, however. These data provide the first evidence that CAE virus-induced arthritis in the goat is genetically influenced by the MHC system; they also suggest that susceptibility/resistance genes are not directly associated with the determined class I gene products but rather are in close genetic linkage.     

Complement-mediated, infection-enhancing antibodies in plasma from vaccinated macaques before and after inoculation with live simian immunodeficiency virus.

Rhesus monkeys vaccinated against infection with simian immunodeficiency virus (SIV) were examined, in retrospect, for the presence of infection-enhancing antibodies and possible consequences associated with the presence of these antibodies. At the time of experimental inoculation with live virus, complement-mediated, infection-enhancing antibodies were detected in plasma specimens from six of six animals vaccinated with detergent-inactivated whole virus, from nine of nine animals vaccinated with Formalin-inactivated whole virus, and from seven of eight animals immunized with two SIV subunit preparations. The presence of infection-enhancing antibodies at the time of viral challenge gave no indication of predicting vaccine success or failure. After live-virus challenge, titers of infection-enhancing antibodies, like enzyme-linked immunosorbent assay titers, increased in unprotected animals and decreased or became undetectable in animals protected by vaccination. Thus, vaccine protection against SIV infection can still be achieved in the presence of detectable complement-mediated, infection-enhancing antibodies.     

Prevalence and risk factors of caprine arthritis encephalitis virus infection (CAEV) in Northern Somalia

The aim of the present study was to determine seroprevalence and the risk factors associated with caprine arthritis encephalitis virus infection (CAEV) in Northern Somalia (Somaliland). A total of 1198 serum samples were collected from 34 randomly selected goat herds of two major districts of Somaliland namely, Togdheer (Burao) and Waqoyi Galbed (Hergiesa). Serum samples were examined for CAEV antibodies using competitive enzyme linked immunosorbent assay (cELISA). A total of 72 goats were found seropositive with overall seroprevalence of 6.0%. On animal level, multivariate logistic regression showed that there was a significant association between CAEV infection and age (P < 0.01; OR: 16.282; CI 95%, 5.87–25.17), rearing with sheep (P < 0.001; OR: 4.158; CI 95%, 1.47–11.715), and herd size (P < 0.05; OR: 1.22; CI 95%, 0.93–2.17). However, on the herd level, herd size (P < 0.05; OR: 1.96; CI 95%, 0.51–1.76) and rearing with sheep (P < 0.05; OR: 13.31; CI 95%, 1.30–13.24) were the risk factors.The result of the present study indicates that CAEV infection exists in the goat herds in Somaliland. It also provides an overview about the risk factors associated with the disease at the examined localities.     

Evidence for interference, coinfections, and intertypic virus enhancement of infection by ovine-caprine lentiviruses.

The ovine-caprine lentiviruses share nucleotide homology and serological properties in their gag-pol genes and gene products but constitute two distinct biological groups represented by ovine visna virus of Icelandic origin and by caprine arthritis-encephalitis and ovine progressive pneumonia viruses of U.S. origin. Two members of each group, visna 1514 and its antigenic variant LV1-1 in the first group and CAEV/CO and S93, a field isolate virus from a local arthritic sheep, in the second group, were examined in the present study in competitive-binding studies in fibroblast and macrophage cell cultures. The cultures were preinoculated with each of the four viruses and then reinoculated with either 1514 virus or CAEV/CO, labeled with [35S]methionine. Both 1514 and CAEV/CO caused homologous interference. LV1-1 and S93 viruses shared the interference patterns of 1514 and CAEV/CO, respectively. 1514 and LV1-1 did not interfere with binding of CAEV/CO. Similarly, CAEV/CO and S93 did not interfere with binding of 1514. Remarkably, certain combinations, such as S93 plus 1514, resulted in enhanced binding of the second virus. Other experiments showed that the enhancement in binding extended to enhancement in replication of the second virus. These latter data suggested that individual cells supported replication of both viruses. Further testing of this phenomenon showed that goats could be doubly infected with two noninterfering viruses, 1514 and CAEV/CO. The ability of noninterfering related lentiviruses to infect the same cell and also the same host animal may be important in the natural history of these viruses in providing ideal conditions for the development of new recombinant viruses.     

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM.     

Severity of arthritis is predicted by antibody response to gp135 in chronic infection with caprine arthritis-encephalitis virus.

Antibody titers to caprine arthritis-encephalitis virus surface glycoprotein gp135 and core protein p28 in synovial fluid and serum from 35 goats infected for 3 years were compared with the histologic severity of arthritis in these animals. Anti-gp135 antibody titers in synovial fluid and serum directly reflect the severity of carpal arthritis in chronically infected goats.     

Glycosylation of immunodominant linear epitopes in the carboxy-terminal region of the caprine arthritis-encephalitis virus surface envelope enhances vaccine-induced type-specific and cross-reactive neutralizing antibody responses

This study evaluated type-specific and cross-reactive neutralizing antibodies induced by immunization with modified surface glycoproteins (SU) of the 63 isolate of caprine arthritis-encephalitis lentivirus (CAEV-63). Epitope mapping of sera from CAEV-infected goats localized immunodominant linear epitopes in the carboxy terminus of SU. Two modified SU (SU-M and SU-T) and wild-type CAEV-63 SU (SU-W) were produced in vaccinia virus and utilized to evaluate the effects of glycosylation or the deletion of immunodominant linear epitopes on neutralizing antibody responses induced by immunization. SU-M contained two N-linked glycosylation sites inserted into the target epitopes by R539S and E542N mutations. SU-T was truncated at 518A, upstream from the target epitopes, by introduction of termination codons at 519Y and 521Y. Six yearling Saanen goats were immunized subcutaneously with 30 microg of SU-W, SU-M, or SU-T in Quil A adjuvant and boosted at 3, 7, and 16 weeks. SU antibody titers determined by indirect enzyme-linked immunosorbent assay demonstrated anamnestic responses after each boost. Wild-type and modified SU-induced type-specific CAEV-63 neutralizing antibodies and cross-reactive neutralizing antibodies against CAEV-Co, a virus isolate closely related to CAEV-63, and CAEV-1g5, an isolate geographically distinct from CAEV-63, were determined. Immunization with SU-T resulted in altered recognition of SU linear epitopes and a 2.8- to 4.6-fold decrease in neutralizing antibody titers against CAEV-63, CAEV-Co, and CAEV-1g5 compared to titers of SU-W-immunized goats. In contrast, immunization with SU-M resulted in reduced recognition of glycosylated epitopes and a 2.4- to 2.7-fold increase in neutralizing antibody titers compared to titers of SU-W-immunized goats. Thus, the glycosylation of linear immunodominant nonneutralization epitopes, but not epitope deletion, is an effective strategy to enhance neutralizing antibody responses by immunization.