In vivo replicative status and envelope heterogeneity of equine infectious anemia virus in an inapparent carrier.

The distribution and replicative status of equine infectious anemia virus (EIAV) DNA in the tissues of a well-characterized inapparent carrier horse were established by using the PCR technique. The EIAV pol region could be amplified in all of the tissues tested, including the cerebellum and periventricular tissue, at concentrations approximately 10(5)-fold less than in the same tissue from an acutely infected horse. Further analysis of the EIAV genome, with primer pairs diagnostic for sequential stages of reverse transcription, suggests that EIAV DNA in the brain, liver, and lymph nodes was incompletely synthesized. The products of reverse transcription were found to diminish progressively during first-strand synthesis, while products indicative of second-strand synthesis were observed only in kidney and spleen DNA samples. Sequences specific for different regions of the envelope could not be amplified from any of the tissues of the inapparent carrier, suggesting that the envelope is highly variable and may be subject to extensive drift. Together, the data suggest that low levels of EIAV replication persist without causing clinical disease in an inapparent carrier.

Enhancement of EIAV replication and disease by immunization with a baculovirus-expressed recombinant envelope surface glycoprotein.

The potential for antibody-dependent enhancement of replication of macrophage/monocyte tropic viruses has posed a significant problem in the development of vaccines for several animal and human viruses and has raised significant concern in the design of potential AIDS vaccines. Using the previously described equine infectious anemia virus/Shetland pony system as a model for HIV-1 vaccine development, we have evaluated the efficacy of a recombinant subunit vaccine containing a baculovirus-expressed envelope surface glycoprotein (gp90) of EIAV. The results of these trials demonstrate not only that the recombinant vaccine failed to protect against infection by standard homologous and heterologous EIAV challenge strains, but that it resulted in a marked enhancement of virus replication and exacerbation of disease in immunized ponies exposed to the heterologous virus strain. Thus, the recombinant EIAV gp90 vaccine provides a novel in vivo model for examining in detail the mechanisms of immune enhancement of a lentivirus infection and for evaluating strategies to avoid the production of deleterious immune responses in AIDS vaccine design.

Equine infectious anemia.

The ability of EIAV to persistently infect horses in the face of a profound immune response by the host makes it a potentially devastating disease for the horse population of the United States. Its ability to evade host immune defenses by lying dormant in apparently healthy animals and by rapidly changing its antigenic determinants is proving to be a major obstacle to vaccine development. Because most infected horses appear clinically normal and a large proportion of horses in this country remain untested, the virus is not likely to be eradicated in the near future. Yet, for the same reason, because most horses infected with EIAV appear clinically normal, there is a tendency for the horse industry to become complacent in its efforts to control the virus. The cooperation of horse owners, veterinarians, and regulatory officials is necessary to keep the threat of EIA in check in the United States.

The surface envelope protein gene region of equine infectious anemia virus is not an important determinant of tropism in vitro.

Virulent, wild-type equine infectious anemia virus (EIAV) is restricted in one or more early steps in replication in equine skin fibroblast cells compared with cell culture-adapted virus, which is fully competent for replication in this cell type. We compared the sequences of wild-type EIAV and a full-length infectious proviral clone of the cell culture-adapted EIAV and found that the genomes were relatively well conserved with the exception of the envelope gene region, which showed extensive sequence differences. We therefore constructed several wild-type and cell culture-adapted virus chimeras to examine the role of the envelope gene in replication in different cell types in vitro. Unlike wild-type virus, which is restricted by an early event(s) for replication in equine dermis cells, the wild-type outer envelope gene chimeras are replication competent in this cell type. We conclude that even though there are extensive sequence differences between wild-type and cell culture-adapted viruses in the surface envelope gene region, this domain is not a determinant of the differing in vitro cell tropisms.

Detailed mapping of the antigenicity of the surface unit glycoprotein of equine infectious anemia virus by using synthetic peptide strategies.

We describe here a detailed analysis of the antigenic determinants of the surface unit glycoprotein (gp90) of equine infectious anemia virus (EIAV), using a comprehensive panel of synthetic peptides in enzyme-linked immunosorbent assays with immune serum from naturally and experimentally infected horses and with a panel of gp90-specific neutralizing and nonneutralizing monoclonal antibodies. The results of these studies identify immunoreactive segments throughout the conserved and variable domains of gp90 but localize immunodominant (100% reactivity) determinants to the amino and carboxyl termini of the glycoprotein molecule. Analysis of peptide reactivities with longitudinal serum samples taken from experimentally infected ponies revealed that antibody responses to conserved B-cell determinants appeared earlier and at higher titers than do antibodies specific for determinants contained in the variable domain of gp90. These observations suggest an evolution of antibody responses in EIAV-infected ponies that may correspond to the establishment of immunological control of virus replication and disease routinely observed in EIAV infections. In addition, the mapping of monoclonal antibody epitopes to peptides of 9 to 12 amino acids demonstrated that all of the neutralizing epitopes are located in the variable domain of gp90. The arrangement of neutralizing epitopes and critical structural considerations suggest that EIAV gp90 contains a principal neutralizing domain similar to the V3 loop of human immunodeficiency virus type 1. These antigenic analyses provide an important foundation for further analyzing the protective immune response generated during persistent EIAV infections and also provide potential peptide substrates for diagnostic assays and for vaccine strategies.

Immune-mediated thrombocytopenia in horses infected with equine infectious anemia virus.

An adult horse infected with a virulent, cell culture-adapted strain of equine infectious anemia virus (EIAV) developed cyclical thrombocytopenia in which the nadir of platelet counts coincided with peak febrile responses. In order to investigate the mechanism of thrombocytopenia during acute febrile episodes, four adult horses were experimentally infected with the wild-type Wyoming strain of EIAV. Platelet counts decreased from baseline as rectal temperature increased. Serum reverse transcriptase activity increased above background levels in all horses, coincident with increase in rectal temperature. All horses developed an EIAV-specific immune response detectable by Western immunoblot by postinfection day 10. Increases in platelet-associated immunoglobulins G and M were detectable by direct fluorescent-antibody test and flow cytometric assay. Viral replication in bone marrow megakaryocytes was not detectable by in situ hybridization. Results suggest an immune-mediated mechanism of thrombocytopenia in horses infected with EIAV. Despite an inability to identify virion particles in association with platelet-bound antibody, the cyclical nature of the thrombocytopenia and the occurrence of a marked cell-free viremia concomitant with fever and thrombocytopenia suggest immune complex deposition on platelets. We propose that clearance of virus and antibody-coated platelets from the peripheral circulation by hepatic Kupffer cells and splenic macrophages may target infectious virus particles, in the form of immune complexes, to host cells most permissive for in vivo viral replication.

Proviral sequences detected by polymerase chain reaction in peripheral blood cells of horses with equine infectious anemia lentivirus.

Proviral sequences in the peripheral blood mononuclear cells of 3 horses with acute equine infectious anemia virus were monitored using the polymerase chain reaction. Provirus was detected during the initial viremic episode in each horse and during each of 3 relapsing viremic cycles, although the appearance of provirus lagged behind the onset of viremia. Following each viremic episode, provirus levels in the peripheral monocytes decreased to less than 1 copy in 5 x 10(6) cells.

Equine infectious anemia virus derived from a molecular clone persistently infects horses.

A full-length molecular clone of equine infectious anemia virus (EIAV) was isolated from a persistently infected canine fetal thymus cell line (Cf2Th). Upon transfection of equine dermis cells, the clone, designated CL22, yielded infectious EIAV particles (CL22-V) that replicated in vitro in both Cf2Th cells and an equine dermis cell strain. Horses infected with CL22-V developed an antibody response to viral proteins and possessed viral DNA in peripheral blood mononuclear cells, as determined by polymerase chain reaction assays. In addition, horses infected with CL22-V became persistently infected and were capable of transmitting the infection by transfer of whole blood to uninfected horses. However, CL22-V, like the parental canine cell-adapted virus, did not cause clinical signs in infected horses. Reverse transcriptase assays of CL22-V- and virulent EIAV-infected equine mononuclear cell cultures indicated that the lack of virulence of CL22-V was not due to an inability to infect and replicate in equine mononuclear cells in vitro.

Animal diseases caused by retroviruses: enzootic bovine leukosis, equine infectious anaemia and caprine arthritis-encephalitis.

This article presents the essential features of three retroviral infections of animals: enzootic bovine leukosis, equine infectious anaemia and caprine arthritis-encephalitis. A unique feature of these diseases is persistent infection, maintained throughout the life of the host animal by the presence of a provirus integrated into the cells of the host, thereby making an infected animal a continuous source of the pathogen. Information currently available on the epidemiology and detection of these diseases is sufficient to institute effective disease control measures.

Immunopathogenesis of equine infectious anemia lentivirus disease.

Virus replication and subsequent viremia are clearly correlated with clinical disease in EIAV infected horses. Termination of viremia is the result of specific immune responses. Recurrences of viremia are associated with antigenic variation of neutralization-sensitive epitopes. Immunosuppression experiments indicate that the eventual control of EIAV and development of carriers is mediated by the immune system. Even though the immune response to EIAV has a protective effect, immune responses also cause some of the lesions. At least one part of the anemia, erythrocyte destruction, is caused by the immune response. Not all of the mechanisms of decreased erythropoiesis are known, but EIAV infection of monocyte/macrophages results in altered iron metabolism and functional iron deficiency. Viral antigen-antibody-C3 complexes cause glomerulitis and the combination of antigen-antibody reactions results in significant reductions in plasma C3. Lesions in the liver and other organs are infiltrations of lymphocytes and monocytes/macrophages in the interstitial areas and it is assumed that these lesions are initiated by specific immune responses to viral antigens. The observations on kinetics of EIAV infection, immune control by the horse, and immunopathologic basis of most of the lesions lead to the conclusion that mechanisms of lentivirus control and disease can be determined by study of EIAV.

Equine infectious anemia virus (EIAV) humoral responses of recipient ponies and antigenic variation during persistent infection.

Three ponies were inoculated with plasma containing 10(4.8) TCID50 of equine infectious anemia virus (EIAV) and observed for 165 to 440 days. Each pony developed a febrile response within 3 weeks of infection during which a plasma viremia greater than or equal to 10(3.5) TCID50/ml was observed. Analyses of four isolates from sequential febrile episodes in a single pony were conducted by two-dimensional tryptic peptide maps and with monoclonal antibodies in immunoblots. Structural and antigenic alterations were observed in the envelope glycoproteins gp90 and gp45, with greatest variation in gp90. Specific IgG to EIAV gp90, gp45, and p26 of homologous and heterologous isolates was detectable by immunoblots within one month after infection although IgG levels to gp45 at this time were relatively low. The group-specific determinants of gp90 and gp45 were more antigenic than those of p26; however, binding of IgG to these determinants did not correlate with neutralization of EIAV as assayed in fetal equine kidney cells. Neutralizing antibodies were first detectable within two months of infection and only neutralized viruses isolated prior to serum collection. Neutralizing activity of sera collected later in the infection was broadly reactive regardless of the number of clinical episodes the donor had suffered.

Viral DNA in horses infected with equine infectious anemia virus.

The amount and distribution of viral DNA were established in a horse acutely infected with the Wyoming strain of equine infectious anemia virus (EIAV). The highest concentration of viral DNA were found in the liver, lymph nodes, bone marrow, and spleen. The kidney, choroid plexus, and peripheral blood leukocytes also contained viral DNA, but at a lower level. It is estimated that at day 16 postinoculation, almost all of the viral DNA was located in the tissues, with the liver alone containing about 90 times more EIAV DNA than the peripheral blood leukocytes did. Assuming a monocyte-macrophage target, each infected cell contained multiple copies of viral DNA (between 6 and 60 copies in liver Kupffer cells). At day 16 postinoculation, most of the EIAV DNA was not integrated into host DNA, but existed in both linear and circular unintegrated forms. In contrast to acute infection, viral DNA was not detectable in tissues from asymptomatic horses with circulating antibody to EIAV.

Development of an enzyme-linked immunosorbent assay for equine infectious anemia virus detection using recombinant Pr55gag.

To provide more sensitive and convenient methods for the detection of equine infectious anemia virus (EIAV), we developed an enzyme-linked immunosorbent assay (ELISA) employing the EIAV gag precursor (Pr55gag) produced by using recombinant DNA techniques. The antigenic reactivity of the recombinant EIAV Pr55gag was found to be equivalent to that of the virion p24gag and elicited high-titered antiserum in rabbits. When a large number of horse sera were analyzed for the presence of antibodies to EIAV by this ELISA, a radioimmunoassay for EIAV p15gag, or the standard agar gel immunodiffusion test, there was 98.7% concordance among the assays. By using the ELISA it was possible to specifically detect antibodies earlier after experimental infection of horses with EIAV than with the other two tests. A competition ELISA developed in order to detect EIAV gag antigens was found to be approximately 15 times more sensitive than the radioimmunoassay for EIAV p15gag. Antigens of other animal lentiviruses as well as those of the prototype oncovirus failed to compete in this assay.

Immune responses are required to terminate viremia in equine infectious anemia lentivirus infection.

Six normal and four immunodeficient horses were injected with a cloned variant of equine infectious anemia virus (EIAV). The six normal horses had detectable EIAV in their plasma by 7 days postinjection. During their primary viremic episode, which was accompanied by fever and anemia, maximum titers of EIAV in plasma ranged from 10(3.8) to 10(4.8) 50% tissue culture infective doses per ml. All six normal horses cleared detectable virus from their plasma by 21 to 35 days after injection. Horses with combined immunodeficiency became viremic by 9 days postinjection and also developed anemia. In contrast to normal horses, foals with combined immunodeficiency did not eliminate the virus from their plasma.

Role of the host immune response in selection of equine infectious anemia virus variants.

Equine infectious anemia virus was isolated from peripheral blood leukocytes collected during two early febrile cycles of an experimentally infected horse. RNase T1-resistant oligonucleotide fingerprint analyses indicated that the nucleotide sequences of the isolates differed by approximately 0.25% and that the differences appeared randomly distributed throughout the genome. Serum collected in the interval between virus isolations was able to distinguish the isolates by membrane immunofluorescence on live cells. However, no neutralizing antibody was detected in the interval between virus isolations. In fact, multiple clinical cycles occurred before the development of a neutralizing antibody response, indicating that viral neutralization might not be the mechanism for selection of antigenic variants. The ability of early immune sera to recognize variant specific antigens on the surface of infected cells suggested that immune selection occurs through recognition and elimination of certain virus-infected cells. Alternately, the random distribution of the genomic differences observed between the two isolates may indicate that equine infectious anemia virus variants emerge as a result of nonimmunological selection processes.

Antigenic analysis of equine infectious anemia virus (EIAV) variants by using monoclonal antibodies: epitopes of glycoprotein gp90 of EIAV stimulate neutralizing antibodies.

Monoclonal antibodies produced against the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV), a lentivirus, were studied for reactivity with the homologous prototype and 16 heterologous isolates. Eighteen hybridomas producing monoclonal antibodies (MAbs) were isolated. Western blot (immunoblot) analyses indicated that 10 were specific for the major envelope glycoprotein (gp90) and 8 for the transmembrane glycoprotein (gp45). Four MAbs specific to epitopes of gp90 neutralized prototype EIAV infectivity. These neutralizing MAbs apparently reacted with variable regions of the envelope gp90, as evidenced by their unique reactivity with the panel of isolates, suggesting recognition of at least three different neutralization epitopes. The conformation of these epitopes appears to be continuous, as they resisted treatment with sodium dodecyl sulfate and reducing reagents. Monoclonal antibodies that reacted with conserved epitopes on gp90 or gp45 failed to neutralize EIAV. Our data also demonstrated that there was a large spectrum of possible EIAV serotypes and confirmed that antigenic variation occurs with high frequency in EIAV. Moreover, the data showed that variation is a rapid and random process, as no pattern of variant evolution was evident by comparison of 13 isolates from parallel infections. These results represent the first production of neutralizing MAbs specific for a lentivirus glycoprotein and document alterations in one or more neutralization epitopes of the major surface glycoprotein among sequential isolates of EIAV recovered during persistent infection.

Course and extent of variation of equine infectious anemia virus during parallel persistent infections.

Comparisons of peptide and oligonucleotide maps of glycoproteins and RNA from nine isolates of equine infectious anemia virus (EIAV) that were generated during parallel infections of two Shetland ponies revealed that each isolate was structurally unique. Each EIAV isolate contained a unique subset of variant peptides, oligonucleotides, or both, indicating that structural variation in EIAV is a random and noncumulative process and that a large spectrum of possible EIAV variants can be generated in infected animals.

Rapid emergence of novel antigenic and genetic variants of equine infectious anemia virus during persistent infection.

Previous results from our laboratory have demonstrated that equine infectious anemia virus displays structural variations in its surface glycoproteins and RNA genome during passage and chronic infections in experimentally infected Shetland ponies (Montelaro et al., J. Biol. Chem. 259:10539-10544, 1984; Payne et al., J. Gen. Virol. 65:1395-1399, 1984). The present study was undertaken to obtain an antigenic and biochemical characterization of equine infectious anemia virus isolates recovered from an experimentally infected pony during sequential disease episodes, each separated by intervals of only 4 to 8 weeks. The virus isolates could be distinguished antigenically by neutralization assays with serum from the infected pony and by Western blot analysis with a monoclonal antibody against the major surface glycoprotein gp90, thus demonstrating that novel antigenic variants of equine infectious anemia virus predominate during each clinical episode. The respective virion glycoproteins displayed different electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels, indicating structural variation. Tryptic peptide and glycopeptide maps of the viral proteins of each virus isolate revealed biochemical alterations involving amino acid sequence and glycosylation patterns in the virion surface glycoproteins gp90 and gp45. In contrast, no structural variation was observed in the internal viral proteins pp15, p26, and p9 from any of the four virus isolates. Oligonucleotide mapping experiments revealed similar but unique RNase T1-resistant oligonucleotide fingerprints of the RNA genomes of each of the virus isolates. Localization of altered oligonucleotides for one virus isolate placed two of three unique oligonucleotides within the predicted env gene region of the genome, perhaps correlating with the structural variation observed in the envelope glycoproteins. Thus these results support the concept that equine infectious anemia virus is indeed capable of relatively rapid genomic variations during replication, some of which result in altered glycoprotein structures and antigenic variants which are responsible for the unique periodic disease nature observed in persistently infected animals. The findings of envelope specific differences in isolates of visna virus and of human T-cell lymphotropic virus III (acquired immune deficiency syndrome-related virus) suggest that this variation may be a common characteristic of the subfamily Lentivirinae.