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Serial passage of the prototype (PR) cell-adapted Wyoming strain of equine infectious anemia virus (EIAV) in fetal donkey dermal (FDD) rather than fetal horse (designated fetal equine kidney [FEK]) cell cultures resulted in the generation of a variant virus strain which produced accelerated cytopathic effects in FDD cells and was 100- to 1,000-fold more sensitive to neutralizing antibodies than its parent. This neutralization-sensitive variant was designated the FDD strain. Although there were differences in glycosylation between the PR and FDD strains, passage of the FDD virus in FEK cells did not reduce its sensitivity to neutralizing antibody. Nucleotide sequencing of the region encoding the surface unit (SU) protein from the FDD strain revealed nine amino acid substitutions compared with the PR strain. Two of these substitutions resulted in changes in the polarity of charge, four caused the introduction of a charged residue, and three had no net change in charge. Nucleotide sequence analysis was extended to the region of the FDD virus genome encoding the extracellular domain of the transmembrane envelope glycoprotein (TM). Unlike the situation with the FDD virus coding region, there were minor variations in nucleotide sequence between individual molecular clones containing this region of the TM gene. Although each clone contained three nucleotide substitutions compared with the PR strain, only one of these was common to all, and this did not affect the amino acid content. Of the remaining two nucleotide substitutions, only one resulted in an amino acid change, and in each case, this change appeared to be conservative. To determine if amino acid substitutions in the SU protein of FDD cell-grown viruses were responsible for the enhanced sensitivity to neutralizing antibodies, chimeric viruses were constructed by using an infectious molecular clone of EIAV. These chimeric viruses contained all of the amino acid substitutions found in the FDD virus strain and were significantly more sensitive to neutralizing antibodies than viruses from the parental (PR) molecular clone. These results demonstrated that sensitivity to neutralizing antibodies in EIAV can be conferred by amino acid residues in the SU protein. However, such amino acid substitutions were not sufficient to enhance cytopathogenicity, as the chimeric viruses did not cause excessive degenererative effects in FDD cells, as was observed with the parental FDD virus strain.

In this study we used immune sera from equine infectious anaemia virus (EIAV)-infected horses which uniquely display broad reactivity with different lentivirus capsid proteins (CA) to characterize the cross-reactive determinants of lentivirus CA proteins. In particular, the role of the major homology region (MHR) of lentivirus CA proteins in this serological cross-reactivity was evaluated using both equine immune serum and murine monoclonal antibodies (MAbs) directed against the MHR segment of different lentiviruses. The results of our studies indicate that about 80% of sera from long-term experimentally infected ponies or naturally infected horses react with human immunodeficiency virus type 1 CA in Western immunoblot assays. In addition, the cross-reactive determinants on the EIAV CA were localized within the immunodominant carboxyl terminus of the protein (residues 277 to 367). However, the cross-reactive determinants recognized by the equine sera do not appear to correlate with linear peptides from the carboxyl terminus of the EIAV CA, including the MHR. These results suggest cross-reactivity between more distant lentiviruses is associated with non-linear determinants. In contrast, MHR-specific MAbs did react with linear peptides by ELISA and distinguished the primate lentiviruses from EIAV and feline immunodeficiency virus. These data support the concept of a highly conserved structural and antigenic organization among the CA proteins of lentiviruses from different species.

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.

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.

A sensitive specific enzyme-linked immunosorbent assay utilizing purified p26 antigen was developed for the detection of antibodies to equine infectious anemia virus in naturally and experimentally infected horses. Generally, antibodies to the virus could be detected by the enzyme-linked immunosorbent assay 3 to 4 days earlier than by the standard agar gel immunodiffusion test, and they could be detected more reliably in horses with weak or equivocal agar gel immunodiffusion test reactions. The enzyme-linked immunosorbent assay was also successfully applied to the detection of p26 antigen in tissue culture fluids.

Monoclonal antibodies (MCAbs) were used to dissect the antigenic sites of the surface glycoproteins of the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV). Serologic reactivities of these MCAbs were determined by ELISA, additive ELISA, competitive ELISA, and Western blot assays. The results indicated that antigenic reactivity of gp90 was localized on at least four distinct epitopes, two of which were important in neutralization. Our studies also revealed that these epitopes were localized on overlapping antigenic sites on gp90. On the other hand, only two distinct non-overlapping epitopes were identified on gp45. Competitive binding studies of neutralizing MCAbs and reference EIA-positive horse serum delineated the presence of a neutralization domain on gp90 that appears to be immunodominant both in naturally infected horses and in mice immunized with EIAV. Limited proteolytic fragmentation of the gp90 component of several serologically distinct EIAV isolates produced common 12K immunoreactive fragments that contained a conserved epitope. These results indicate the occurrence of conserved antigenic regions on EIAV glycoproteins as well as a neutralization domain on gp90, which can be used as potential targets for vaccine development.

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.

We describe here a one step HPLC technique for purifying the four gag proteins (p26, p15, p11 and p9) and two env glycoproteins (gp90 and gp45) from purified equine infectious anemia virus (EIAV), a member of the lentivirus subfamily of retroviruses. The purification procedure employs a reverse-phase phenyl Radial-pak cartridge contained in a high pressure radial compression chamber in which a shallow, multistep acetonitrile gradient is applied at ambient temperatures. The purified proteins are recovered at an efficiency of 60-70%. Moreover, the isolated components retain their antigenicity and are suitable for a variety of biochemical analyses including protein sequencing. The purification of EIAV gp90 and gp45 represents the first successful isolation of a lentivirus glycoprotein from purified virus preparations. The availability of these separated proteins permitted direct protein sequencing which confirmed the previously reported env gene sequence and provides important antigens for the development of diagnostic immunoassays and subunit vaccines. The procedures described appear applicable to other lentiviruses, including human immunodeficiency virus (HIV), and perhaps to hydrophobic membrane proteins in general.

The recurrent nature of equine infectious anemia has been attributed to relatively rapid antigenic variations in equine infectious anemia virus (EIAV) during persistent infection under selective immune pressures. This model was tested by serological and biochemical analysis of virus isolates recovered from separate febrile episodes in two experimentally infected ponies. Neutralization assays employing immune sera from the experimentally infected ponies demonstrated that distinct antigenic strains of virus predominate during sequential febrile episodes in a single pony. Analysis of the test strains of EIAV by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed varying electrophoretic mobilities for the respective virion glycoproteins gp90 and gp45. Furthermore, peptide mapping comparisons demonstrated structural variations between the gp90 components of the various strains. In contrast, the respective internal proteins of the virus strains displayed identical electrophoretic mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and, in general, produced identical tryptic peptide maps. The observed differences in glycoprotein electrophoretic mobility and peptide maps were highly reproducible and did not vary with repeated passage of the virus strains in cell culture. Thus, these results demonstrate the occurrence of glycoprotein-specific structural variations during persistent infection by EIAV and support the concept of antigenic variation in this retrovirus. This capacity to alter envelope glycoprotein structure, previously reported for visna virus, may represent an important mechanism of retrovirus persistence in the presence of immune responses by the animal host.

The extent and nature of genomic variation among nine antigenically distinct EIAV isolates recovered during sequential clinical episodes from two experimentally infected ponies were examined by restriction fragment analysis and nucleotide sequencing. Only minor variations in restriction enzyme patterns were observed among the viral genomes. In contrast, env gene sequences of four isolates from one pony revealed numerous clustered base substitutions. Divergence in env gene nucleotide and deduced amino acid sequences between pairs of virus isolates ranged from 0.62 to 3.4% env gene mutation rates for isolates recovered during sequential febrile episodes were calculated to be greater than 10(-2) base substitutions per site per year. The degree and nature of env gene variation in EIAV is remarkably similar to the human immunodeficiency virus, suggesting common mechanisms for env gene variation among lentiviruses.

Previous characterizations of equine infectious anemia virus (EIAV) glycoprotein variation by DNA sequence analysis and epitope mapping using monoclonal antibodies (MAbs) have revealed the presence of conserved and variable regions within the EIAV env gene. To extend these studies, fragments of the EIAV envelope proteins gp90 and gp45 were expressed in Escherichia coli and used in Western blot analysis with a diverse panel of equine immune sera to identify antigenic segments. All sera from EIAV-infected animals reacted with the carboxyl terminal portion of gp90 and the amino terminal portion of gp45, indicating the highly conserved and immunodominant nature of these regions. Other gp90 segments, both from conserved and variable env sequences, displayed variable reactivities with the panel of equine sera. A panel of MAbs was also used in Western blot assays with the recombinant protein fragments for physical localization of previously identified MAb epitopes. The binding sites of two neutralizing MAbs were localized to a highly variable region of gp90, while nonneutralizing epitopes were localized to conserved and variable regions of the envelope glycoproteins. These results, in addition to localizing important antigenic sites on EIAV glycoproteins, indicate that previously defined conserved and variable env nucleotide sequences indeed encode protein sequences constituting conserved and variable immunogens during persistent infection by EIAV.